Pentacyclic triterpenoid ursolic acid interferes with mast cell activation via a lipid-centric mechanism affecting FcεRI signalosome functions.

Pentacyclic triterpenoids, including ursolic acid (UA), are bioactive compounds with multiple biological activities involving anti-inflammatory effects. However, the mode of their action on mast cells, key players in the early stages of allergic inflammation, and underlying molecular mechanisms remain enigmatic. To better understand the effect of UA on mast cell signaling, here we examined the consequences of short-term treatment of mouse bone marrow-derived mast cells with UA. Using IgE-sensitized and antigen- or thapsigargin-activated cells, we found that 15 min exposure to UA inhibited high affinity IgE receptor (FcεRI)-mediated degranulation, calcium response, and extracellular calcium uptake. We also found that UA inhibited migration of mouse bone marrow-derived mast cells toward antigen but not toward prostaglandin E2 and stem cell factor. Compared to control antigen-activated cells, UA enhanced the production of tumor necrosis factor-α at the mRNA and protein levels. However, secretion of this cytokine was inhibited. Further analysis showed that UA enhanced tyrosine phosphorylation of the SYK kinase and several other proteins involved in the early stages of FcεRI signaling, even in the absence of antigen activation, but inhibited or reduced their further phosphorylation at later stages. In addition, we show that UA induced changes in the properties of detergent-resistant plasma membrane microdomains and reduced antibody-mediated clustering of the FcεRI and glycosylphosphatidylinositol-anchored protein Thy-1. Finally, UA inhibited mobility of the FcεRI and cholesterol. These combined data suggest that UA exerts its effects, at least in part, via lipid-centric plasma membrane perturbations, hence affecting the functions of the FcεRI signalosome.

Simplified PCR-Based Quantification of Proteins with DNA Aptamers and Methylcellulose as a Blocking Agent.

Due to their unique three-dimensional structure, DNA or RNA oligonucleotide aptamers bind to various molecules with high affinity and specificity. Aptamers, alone or in combination with antibodies, can be used to sensitively quantify target molecules by quantitative real-time polymerase chain reaction (qPCR). However, the assays are often complicated and unreliable. In this study, we explored the feasibility of performing the entire assay on wells of routinely used polypropylene PCR plates. We found that polypropylene wells efficiently bind proteins. This allows the entire assay to be run in a single well. To minimize nonspecific binding of the assay components to the polypropylene wells, we tested various blocking agents and identified methylcellulose as an effective alternative to the commonly used BSA. Methylcellulose not only demonstrates comparable or superior blocking capabilities but also offers the advantage of a well-defined composition and non-animal origin. Our findings support the utilization of aptamers, either alone or in combination with antibodies, for sensitive quantification of selected molecules immobilized in polypropylene PCR wells in a streamlined one-well qPCR assay under well-defined conditions.

Changing the threshold-Signals and mechanisms of mast cell priming.

Mast cells play a key role in allergy and other inflammatory diseases involving engagement of multivalent antigen with IgE bound to high-affinity IgE receptors (FcεRIs). Aggregation of FcεRIs on mast cells initiates a cascade of signaling events that eventually lead to degranulation, secretion of leukotrienes and prostaglandins, and cytokine and chemokine production contributing to the inflammatory response. Exposure to pro-inflammatory cytokines, chemokines, bacterial and viral products, as well as some other biological products and drugs, induces mast cell transition from the basal state into a primed one, which leads to enhanced response to IgE-antigen complexes. Mast cell priming changes the threshold for antigen-mediated activation by various mechanisms, depending on the priming agent used, which alone usually do not induce mast cell degranulation. In this review, we describe the priming processes induced in mast cells by various cytokines (stem cell factor, interleukins-4, -6 and -33), chemokines, other agents acting through G protein-coupled receptors (adenosine, prostaglandin E2 , sphingosine-1-phosphate, and ß-2-adrenergic receptor agonists), toll-like receptors, and various drugs affecting the cytoskeleton. We will review the current knowledge about the molecular mechanisms behind priming of mast cells leading to degranulation and cytokine production and discuss the biological effects of mast cell priming induced by several cytokines.

Crosstalk between ORMDL3, serine palmitoyltransferase, and 5-lipoxygenase in the sphingolipid and eicosanoid metabolic pathways.

Leukotrienes (LTs) and sphingolipids are critical lipid mediators participating in numerous cellular signal transduction events and developing various disorders, such as bronchial hyperactivity leading to asthma. Enzymatic reactions initiating production of these lipid mediators involve 5-lipoxygenase (5-LO)-mediated conversion of arachidonic acid to LTs and serine palmitoyltransferase (SPT)-mediated de novo synthesis of sphingolipids. Previous studies have shown that endoplasmic reticulum membrane protein ORM1-like protein 3 (ORMDL3) inhibits the activity of SPT and subsequent sphingolipid synthesis. However, the role of ORMDL3 in the synthesis of LTs is not known. In this study, we used peritoneal-derived mast cells isolated from ORMDL3 KO or control mice and examined their calcium mobilization, degranulation, NF-κB inhibitor-α phosphorylation, and TNF-α production. We found that peritoneal-derived mast cells with ORMDL3 KO exhibited increased responsiveness to antigen. Detailed lipid analysis showed that compared with WT cells, ORMDL3-deficient cells exhibited not only enhanced production of sphingolipids but also of LT signaling mediators LTB4, 6t-LTB4, LTC4, LTB5, and 6t-LTB5. The crosstalk between ORMDL3 and 5-LO metabolic pathways was supported by the finding that endogenous ORMDL3 and 5-LO are localized in similar endoplasmic reticulum domains in human mast cells and that ORMDL3 physically interacts with 5-LO. Further experiments showed that 5-LO also interacts with the long-chain 1 and long-chain 2 subunits of SPT. In agreement with these findings, 5-LO knockdown increased ceramide levels, and silencing of SPTLC1 decreased arachidonic acid metabolism to LTs to levels observed upon 5-LO knockdown. These results demonstrate functional crosstalk between the LT and sphingolipid metabolic pathways, leading to the production of lipid signaling mediators.

Carboxymethylcellulose enhances the production of single-stranded DNA aptamers generated by asymmetric PCR.

Nucleic acid aptamers are single-stranded (ss)DNA or RNA oligonucleotides that can take various conformations and bind specifically and with high affinity to selected targets. While the introduction of SELEX (systematic evolution of ligands by exponential enrichment) revolutionized the production of the aptamers, this procedure is impeded by the formation of undesirable by-products reflecting hybridization among complementary oligonucleotides in the ssDNA libraries during asymmetric PCR. To reduce nonspecific amplification we tested cellulose-derived compounds and found that sodium carboxymethylcellulose (CMC) at a concentration 0.05%-0.2% efficiently suppressed production of undesirable large DNA amplicons during asymmetric PCR in the course of SELEX. Formation of the PCR by-products was reduced by CMCs of low and medium viscosity more than by CMCs of high viscosity, and all of them bound to DNA oligonucleotides as determined by electrophoresis in agarose gels. In contrast to CMC, methylcellulose did not reduce the formation of the PCR by-products and did not bind to DNA. DNA aptamers selected in the presence of CMC could be used directly in enzyme-linked immunosorbent-like assay. The combined data suggest that CMC binds weekly to DNA oligonucleotides through hydroxyl groups and in this way inhibits low-affinity DNA-DNA hybridization and enhances the production of specific amplicons in asymmetric PCR.

Positive and Negative Signals in Mast Cell Activation.

Mast cells are powerful immune modulators of the tissue microenvironment. Within seconds of activation, these cells release a variety of preformed biologically active products, followed by a wave of mediator synthesis and secretion. Increasing evidence suggests that an intricate network of inhibitory and activating receptors, specific signaling pathways, and adaptor proteins governs mast cell responsiveness to stimuli. Here, we discuss the biological and clinical relevance of negative and positive signaling modalities that control mast cell activation, with an emphasis on novel FcεRI regulators, immunoglobulin E (IgE)-independent pathways [e.g., Mas-related G protein-coupled receptor X2 (MRGPRX2)], tetraspanins, and the CD300 family of inhibitory and activating receptors.

Tetraspanins in the regulation of mast cell function.

Mast cells (MCs) are long-living immune cells highly specialized in the storage and release of different biologically active compounds and are involved in the regulation of innate and adaptive immunity. MC degranulation and replacement of MC granules are accompanied by active membrane remodelling. Tetraspanins represent an evolutionary conserved family of transmembrane proteins. By interacting with lipids and other membrane and intracellular proteins, they are involved in organisation of membrane protein complexes and act as "molecular facilitators" connecting extracellular and cytoplasmic signaling elements. MCs express different tetraspanins and MC degranulation is accompanied by changes in membrane organisation. Therefore, tetraspanins are very likely involved in the regulation of MC exocytosis and membrane reorganisation after degranulation. Antiviral response and production of exosomes are further aspects of MC function characterized by dynamic changes of membrane organization. In this review, we pay a particular attention to tetraspanin gene expression in different human and murine MC populations, discuss tetraspanin involvement in regulation of key MC signaling complexes, and analyze the potential contribution of tetraspanins to MC antiviral response and exosome production. In-depth knowledge of tetraspanin-mediated molecular mechanisms involved in different aspects of the regulation of MC response will be beneficial for patients with allergies, characterized by overwhelming MC reactions.

Negative regulatory roles of ORMDL3 in the FcεRI-triggered expression of proinflammatory mediators and chemotactic response in murine mast cells.

Single-nucleotide polymorphism studies have linked the chromosome 17q12-q21 region, where the human orosomucoid-like (ORMDL)3 gene is localized, to the risk of asthma and several other inflammatory diseases. Although mast cells are involved in the development of these diseases, the contribution of ORMDL3 to the mast cell physiology is unknown. In this study, we examined the role of ORMDL3 in antigen-induced activation of murine mast cells with reduced or enhanced ORMDL3 expression. Our data show that in antigen-activated mast cells, reduced expression of the ORMDL3 protein had no effect on degranulation and calcium response, but significantly enhanced phosphorylation of AKT kinase at Ser 473 followed by enhanced phosphorylation and degradation of IκBα and translocation of the NF-κB p65 subunit into the nucleus. These events were associated with an increased expression of proinflammatory cytokines (TNF-α, IL-6, and IL-13), chemokines (CCL3 and CCL4), and cyclooxygenase-2 dependent synthesis of prostaglandin D2. Antigen-mediated chemotaxis was also enhanced in ORMDL3-deficient cells, whereas spreading on fibronectin was decreased. On the other hand, increased expression of ORMDL3 had no significant effect on the studied signaling events, except for reduced antigen-mediated chemotaxis. These data were corroborated by increased IgE-antigen-dependent passive cutaneous anaphylaxis in mice with locally silenced ORMDL3 using short interfering RNAs. Our data also show that antigen triggers suppression of ORMDL3 expression in the mast cells. In summary, we provide evidence that downregulation of ORMDL3 expression in mast cells enhances AKT and NF-κB-directed signaling pathways and chemotaxis and contributes to the development of mast cell-mediated local inflammation in vivo.

Molecular targets on mast cells and basophils for novel therapies.

Mast cells and basophils (MCs/Bs) play a crucial role in type I allergy, as well as in innate and adaptive immune responses. These cells mediate their actions through soluble mediators, some of which are targeted therapeutically by, for example, H1- and H2-antihistamines or cysteinyl leukotriene receptor antagonists. Recently, considerable progress has been made in developing new drugs that target additional MC/B mediators or receptors, such as serine proteinases, histamine 4-receptor, 5-lipoxygenase-activating protein, 15-lipoxygenase-1, prostaglandin D2, and proinflammatory cytokines. Mediator production can be abrogated by the use of inhibitors directed against key intracellular enzymes, some of which have been used in clinical trials (eg, inhibitors of spleen tyrosine kinase, phosphatidylinositol 3-kinase, Bruton tyrosine kinase, and the protein tyrosine kinase KIT). Reduced MC/B function can also be achieved by enhancing Src homology 2 domain-containing inositol 5' phosphatase 1 activity or by blocking sphingosine-1-phosphate. Therapeutic interventions in mast cell-associated diseases potentially include drugs that either block ion channels and adhesion molecules or antagonize antiapoptotic effects on B-cell lymphoma 2 family members. MCs/Bs express high-affinity IgE receptors, and blocking their interactions with IgE has been a prime goal in antiallergic therapy. Surface-activating receptors, such as CD48 and thymic stromal lymphopoietin receptors, as well as inhibitory receptors, such as CD300a, FcγRIIb, and endocannabinoid receptors, hold promising therapeutic possibilities based on preclinical studies. The inhibition of activating receptors might help prevent allergic reactions from developing, although most of the candidate drugs are not sufficiently cell specific. In this review recent advances in the development of novel therapeutics toward different molecules of MCs/Bs are presented.

Cross-talk between tetraspanin CD9 and transmembrane adaptor protein non-T cell activation linker (NTAL) in mast cell activation and chemotaxis.

Chemotaxis, a process leading to movement of cells toward increasing concentrations of chemoattractants, is essential, among others, for recruitment of mast cells within target tissues where they play an important role in innate and adaptive immunity. Chemotaxis is driven by chemoattractants, produced by various cell types, as well as by intrinsic cellular regulators, which are poorly understood. In this study we prepared a new mAb specific for the tetraspanin CD9. Binding of the antibody to bone marrow-derived mast cells triggered activation events that included cell degranulation, Ca(2+) response, dephosphorylation of ezrin/radixin/moesin (ERM) family proteins, and potent tyrosine phosphorylation of the non-T cell activation linker (NTAL) but only weak phosphorylation of the linker for activation of T cells (LAT). Phosphorylation of the NTAL was observed with whole antibody but not with its F(ab)(2) or Fab fragments. This indicated involvement of the Fcγ receptors. As documented by electron microscopy of isolated plasma membrane sheets, CD9 colocalized with the high-affinity IgE receptor (FcεRI) and NTAL but not with LAT. Further tests showed that both anti-CD9 antibody and its F(ab)(2) fragment inhibited mast cell chemotaxis toward antigen. Experiments with bone marrow-derived mast cells deficient in NTAL and/or LAT revealed different roles of these two adaptors in antigen-driven chemotaxis. The combined data indicate that chemotaxis toward antigen is controlled in mast cells by a cross-talk among FcεRI, tetraspanin CD9, transmembrane adaptor proteins NTAL and LAT, and cytoskeleton-regulatory proteins of the ERM family.

Simultaneous deletion of ORMDL1 and ORMDL3 proteins disrupts immune cell homeostasis.

ORMDL3 is a prominent member of a family of highly conserved endoplasmic reticulum resident proteins, ORMs (ORM1 and ORM2) in yeast, dORMDL in Drosophila and ORMDLs (ORMDL1, ORMDL2, and ORMDL3) in mammals. ORMDL3 mediates feedback inhibition of de novo sphingolipid synthesis. Expression levels of ORMDL3 are associated with the development of inflammatory and autoimmune diseases including asthma, systemic lupus erythematosus, type 1 diabetes mellitus and others. It has been shown that simultaneous deletions of other ORMDL family members could potentiate ORMDL3-induced phenotypes. To understand the complex function of ORMDL proteins in immunity in vivo, we analyzed mice with single or double deletions of Ormdl genes. In contrast to other single and double knockouts, simultaneous deletion of ORMDL1 and ORMDL3 proteins disrupted blood homeostasis and reduced immune cell content in peripheral blood and spleens of mice. The reduced number of splenocytes was not caused by aberrant immune cell homing. A competitive bone marrow transplantation assay showed that the development of Ormdl1-/-/Ormdl3-/- B cells was dependent on lymphocyte intrinsic factors. Highly increased sphingolipid production was observed in the spleens and bone marrow of Ormdl1-/-/Ormdl3-/- mice. Slight, yet significant, increase in some sphingolipid species was also observed in the spleens of Ormdl3-/- mice and in the bone marrow of both, Ormdl1-/- and Ormdl3-/- single knockout mice. Taken together, our results demonstrate that the physiological expression of ORMDL proteins is critical for the proper development and circulation of lymphocytes. We also show cell-type specific roles of individual ORMDL family members in the production of different sphingolipid species.

Novel mechanism for Fc{epsilon}RI-mediated signal transducer and activator of transcription 5 (STAT5) tyrosine phosphorylation and the selective influence of STAT5B over mast cell cytokine production.

Previous studies indicate that STAT5 expression is required for mast cell development, survival, and IgE-mediated function. STAT5 tyrosine phosphorylation is swiftly and transiently induced by activation of the high affinity IgE receptor, FcεRI. However, the mechanism for this mode of activation remains unknown. In this study we observed that STAT5 co-localizes with FcεRI in antigen-stimulated mast cells. This localization was supported by cholesterol depletion of membranes, which ablated STAT5 tyrosine phosphorylation. Through the use of various pharmacological inhibitors and murine knock-out models, we found that IgE-mediated STAT5 activation is dependent upon Fyn kinase, independent of Syk, PI3K, Akt, Bruton's tyrosine kinase, and JAK2, and enhanced in the context of Lyn kinase deficiency. STAT5 immunoprecipitation revealed that unphosphorylated protein preassociates with Fyn and that this association diminishes significantly during mast cell activation. SHP-1 tyrosine phosphatase deficiency modestly enhanced STAT5 phosphorylation. This effect was more apparent in the absence of Gab2, a scaffolding protein that docks with multiple negative regulators, including SHP-1, SHP-2, and Lyn. Targeting of STAT5A or B with specific siRNA pools revealed that IgE-mediated mast cell cytokine production is selectively dependent upon the STAT5B isoform. Altogether, these data implicate Fyn as the major positive mediator of STAT5 after FcεRI engagement and demonstrate importantly distinct roles for STAT5A and STAT5B in mast cell function.

STIM1-directed reorganization of microtubules in activated mast cells.

Activation of mast cells by aggregation of the high-affinity IgE receptors (FcεRI) initiates signaling events leading to the release of inflammatory and allergic mediators stored in cytoplasmic granules. A key role in this process play changes in concentrations of intracellular Ca(2+) controlled by store-operated Ca(2+) entry (SOCE). Although microtubules are also involved in the process leading to degranulation, the molecular mechanisms that control microtubule rearrangement during activation are largely unknown. In this study, we report that activation of bone marrow-derived mast cells (BMMCs) induced by FcεRI aggregation or treatment with pervanadate or thapsigargin results in generation of protrusions containing microtubules (microtubule protrusions). Formation of these protrusions depended on the influx of extracellular Ca(2+). Changes in cytosolic Ca(2+)concentration also affected microtubule plus-end dynamics detected by microtubule plus-end tracking protein EB1. Experiments with knockdown or reexpression of STIM1, the key regulator of SOCE, confirmed the important role of STIM1 in the formation of microtubule protrusions. Although STIM1 in activated cells formed puncta associated with microtubules in protrusions, relocation of STIM1 to a close proximity of cell membrane was independent of growing microtubules. In accordance with the inhibition of Ag-induced Ca(2+) response and decreased formation of microtubule protrusions in BMMCs with reduced STIM1, the cells also exhibited impaired chemotactic response to Ag. We propose that rearrangement of microtubules in activated mast cells depends on STIM1-induced SOCE, and that Ca(2+) plays an important role in the formation of microtubule protrusions in BMMCs.

Enhanced Membrane Fluidization and Cholesterol Displacement by 1-Heptanol Inhibit Mast Cell Effector Functions.

Signal transduction by the high-affinity IgE receptor (FcεRI) depends on membrane lipid and protein compartmentalization. Recently published data show that cells treated with 1-heptanol, a cell membrane fluidizer, exhibit changes in membrane properties. However, the functional consequences of 1-heptanol-induced changes on mast cell signaling are unknown. This study shows that short-term exposure to 1-heptanol reduces membrane thermal stability and dysregulates mast cell signaling at multiple levels. Cells treated with 1-heptanol exhibited increased lateral mobility and decreased internalization of the FcεRI. However, this did not affect the initial phosphorylation of the FcεRI-ß chain and components of the SYK/LAT1/PLCγ1 signaling pathway after antigen activation. In contrast, 1-heptanol inhibited SAPK/JNK phosphorylation and effector functions such as calcium response, degranulation, and cytokine production. Membrane hyperfluidization induced a heat shock-like response via increased expression of the heat shock protein 70, increased lateral diffusion of ORAI1-mCherry, and unsatisfactory performance of STIM1-ORAI1 coupling, as determined by flow-FRET. Furthermore, 1-heptanol inhibited the antigen-induced production of reactive oxygen species and potentiated stress-induced plasma membrane permeability by interfering with heat shock protein 70 activity. The combined data suggest that 1-heptanol-mediated membrane fluidization does not interfere with the earliest biochemical steps of FcεRI signaling, such as phosphorylation of the FcεRI-ß chain and components of the SYK/LAT/PLCγ1 signaling pathway, instead inhibiting the FcεRI internalization and mast cell effector functions, including degranulation and cytokine production.

Simultaneous reduction of all ORMDL proteins decreases the threshold of mast cell activation.

In mammals, the ORMDL family of evolutionarily conserved sphingolipid regulators consists of three highly homologous members, ORMDL1, ORMDL2 and ORMDL3. ORMDL3 gene has been associated with childhood-onset asthma and other inflammatory diseases in which mast cells play an important role. We previously described increased IgE-mediated activation of mast cells with simultaneous deletions of ORMDL2 and ORMDL3 proteins. In this study, we prepared mice with Ormdl1 knockout and thereafter, produced primary mast cells with reduced expression of one, two or all three ORMDL proteins. The lone deletion of ORMDL1, or in combination with ORMDL2, had no effect on sphingolipid metabolism nor IgE-antigen dependent responses in mast cells. Double ORMDL1 and ORMDL3 knockout mast cells displayed enhanced IgE-mediated calcium responses and cytokine production. Silencing of ORMDL3 in mast cells after maturation increased their sensitivity to antigen. Mast cells with reduced levels of all three ORMDL proteins demonstrated pro-inflammatory responses even in the absence of antigen activation. Overall, our results show that reduced levels of ORMDL proteins shift mast cells towards a pro-inflammatory phenotype, which is predominantly dependent on the levels of ORMDL3 expression.

Down-regulation of protein-tyrosine phosphatases activates an immune receptor in the absence of its translocation into lipid rafts.

The earliest known biochemical step that occurs after ligand binding to the multichain immune recognition receptor is tyrosine phosphorylation of the receptor subunits. In mast cells and basophils activated by multivalent antigen-IgE complexes, this step is mediated by Src family kinase Lyn, which phosphorylates the high affinity IgE receptor (Fc epsilonRI). However, the exact molecular mechanism of this phosphorylation step is incompletely understood. In this study, we tested the hypothesis that changes in activity and/or topography of protein-tyrosine phosphatases (PTPs) could play a major role in the Fc epsilonRI triggering. We found that exposure of rat basophilic leukemia cells or mouse bone marrow-derived mast cells to PTP inhibitors, H(2)O(2) or pervanadate, induced phosphorylation of the Fc epsilonRI subunits, similarly as Fc epsilonRI triggering. Interestingly, and in sharp contrast to antigen-induced activation, neither H(2)O(2) nor pervanadate induced any changes in the association of Fc epsilonRI with detergent-resistant membranes and in the topography of Fc epsilonRI detectable by electron microscopy on isolated plasma membrane sheets. In cells stimulated with pervanadate, H(2)O(2) or antigen, enhanced oxidation of active site cysteine of several PTPs was detected. Unexpectedly, most of oxidized phosphatases bound to the plasma membrane were associated with the actin cytoskeleton. Several PTPs (SHP-1, SHP-2, hematopoietic PTP, and PTP-MEG2) showed changes in their enzymatic activity and/or oxidation state during activation. Based on these and other data, we propose that down-regulation of enzymatic activity of PTPs and/or changes in their accessibility to the substrates play a key role in initial tyrosine phosphorylation of the Fc epsilonRI and other multichain immune receptors.

The transmembrane adaptor protein NTAL signals to mast cell cytoskeleton via the small GTPase Rho.

The transmembrane adaptor protein NTAL (non-T-cell activation linker) participates in signalosome assembly in hematopoietic cells, but its exact role in cell physiology remains enigmatic. We report here that BM-derived mast cells from NTAL-deficient mice, responding to Ag alone or in combination with SCF, exhibit reduced spreading on fibronectin, enhanced filamentous actin depolymerization and enhanced migration towards Ag relative to WT cells. No such differences between WT and NTAL(-/-) BM-derived mast cells were observed when SCF alone was used as activator. We have examined the activities of two small GTPases, Rac and Rho, which are important regulators of actin polymerization. Stimulation with Ag and/or SCF enhanced activity of Rac(1,2,3) in both NTAL(-/-) and WT cells. In contrast, RhoA activity decreased and this trend was much faster and more extensive in NTAL(-/-) cells, indicating a positive regulatory role of NTAL in the recovery of RhoA activity. After restoring NTAL into NTAL(-/-) cells, both spreading and actin responses were rescued. This is the first report of a crucial role of NTAL in signaling, via RhoA, to mast cell cytoskeleton.

1,2-propanediol-trehalose mixture as a potent quantitative real-time PCR enhancer.

BACKGROUND: Quantitative real-time PCR (qPCR) is becoming increasingly important for DNA genotyping and gene expression analysis. For continuous monitoring of the production of PCR amplicons DNA-intercalating dyes are widely used. Recently, we have introduced a new qPCR mix which showed improved amplification of medium-size genomic DNA fragments in the presence of DNA dye SYBR green I (SGI). In this study we tested whether the new PCR mix is also suitable for other DNA dyes used for qPCR and whether it can be applied for amplification of DNA fragments which are difficult to amplify. RESULTS: We found that several DNA dyes (SGI, SYTO-9, SYTO-13, SYTO-82, EvaGreen, LCGreen or ResoLight) exhibited optimum qPCR performance in buffers of different salt composition. Fidelity assays demonstrated that the observed differences were not caused by changes in Taq DNA polymerase induced mutation frequencies in PCR mixes of different salt composition or containing different DNA dyes. In search for a PCR mix compatible with all the DNA dyes, and suitable for efficient amplification of difficult-to-amplify DNA templates, such as those in whole blood, of medium size and/or GC-rich, we found excellent performance of a PCR mix supplemented with 1 M 1,2-propanediol and 0.2 M trehalose (PT enhancer). These two additives together decreased DNA melting temperature and efficiently neutralized PCR inhibitors present in blood samples. They also made possible more efficient amplification of GC-rich templates than betaine and other previously described additives. Furthermore, amplification in the presence of PT enhancer increased the robustness and performance of routinely used qPCRs with short amplicons. CONCLUSIONS: The combined data indicate that PCR mixes supplemented with PT enhancer are suitable for DNA amplification in the presence of various DNA dyes and for a variety of templates which otherwise can be amplified with difficulty.

Molecular Mechanisms of Mast Cell Activation by Cholesterol-Dependent Cytolysins.

Mast cells are potent immune sensors of the tissue microenvironment. Within seconds of activation, they release various preformed biologically active products and initiate the process of de novo synthesis of cytokines, chemokines, and other inflammatory mediators. This process is regulated at multiple levels. Besides the extensively studied IgE and IgG receptors, toll-like receptors, MRGPR, and other protein receptor signaling pathways, there is a critical activation pathway based on cholesterol-dependent, pore-forming cytolytic exotoxins produced by Gram-positive bacterial pathogens. This pathway is initiated by binding the exotoxins to the cholesterol-rich membrane, followed by their dimerization, multimerization, pre-pore formation, and pore formation. At low sublytic concentrations, the exotoxins induce mast cell activation, including degranulation, intracellular calcium concentration changes, and transcriptional activation, resulting in production of cytokines and other inflammatory mediators. Higher toxin concentrations lead to cell death. Similar activation events are observed when mast cells are exposed to sublytic concentrations of saponins or some other compounds interfering with the membrane integrity. We review the molecular mechanisms of mast cell activation by pore-forming bacterial exotoxins, and other compounds inducing cholesterol-dependent plasma membrane perturbations. We discuss the importance of these signaling pathways in innate and acquired immunity.

Negative regulation of mast cell signaling and function by the adaptor LAB/NTAL.

Engagement of the Fcepsilon receptor I (FcepsilonRI) on mast cells and basophils initiates signaling pathways leading to degranulation. Early activation events include tyrosine phosphorylation of two transmembrane adaptor proteins, linker for activation of T cells (LAT) and non-T cell activation linker (NTAL; also called LAB; a product of Wbscr5 gene). Previous studies showed that the secretory response was partially inhibited in bone marrow-derived mast cells (BMMCs) from LAT-deficient mice. To clarify the role of NTAL in mast cell degranulation, we compared FcepsilonRI-mediated signaling events in BMMCs from NTAL-deficient and wild-type mice. Although NTAL is structurally similar to LAT, antigen-mediated degranulation responses were unexpectedly increased in NTAL-deficient mast cells. The earliest event affected was enhanced tyrosine phosphorylation of LAT in antigen-activated cells. This was accompanied by enhanced tyrosine phosphorylation and enzymatic activity of phospholipase C gamma1 and phospholipase C gamma2, resulting in elevated levels of inositol 1,4,5-trisphosphate and free intracellular Ca2+. NTAL-deficient BMMCs also exhibited an enhanced activity of phosphatidylinositol 3-OH kinase and Src homology 2 domain-containing protein tyrosine phosphatase-2. Although both LAT and NTAL are considered to be localized in membrane rafts, immunogold electron microscopy on isolated membrane sheets demonstrated their independent clustering. The combined data show that NTAL is functionally and topographically different from LAT.