P2X7 Receptor-Induced Human Mast Cell Degranulation Is Enhanced by Interleukin 33.

MCs are tissue-resident immune cells that strategically reside in barrier organs and respond effectively to a wide range of stimuli, such as IL-33, a mediator released upon epithelial damage. Adenosine triphosphate (ATP) accumulates at sites of tissue injury and is known to modulate MC activities. This study investigated how an inflammatory tissue environment rich in IL-33 modulates the ATP-mediated activation of MCs. Human primary MCs primed with IL-33 displayed a strongly increased response to ATP but not ADP. This resulted in increased degranulation, IL-8 release, and pERK1/2 signalling. Such effects are unique to IL-33 stimulation and not shared by the epithelial alarmin, TSLP. MC exposure to IL-33 also increased membrane expression of purinergic and ATP-binding P2X receptors. The use of selective P2X receptor inhibitors identified P2X7 receptor as the key mediator of the enhanced ATP-induced ERK1/2 signalling and degranulation in IL-33-primed MCs. Whilst the inhibition of P2X1 and P2X4 receptors had no effect on MC degranulation, inhibiting these receptors together with P2X7 resulted in further decreased MC-mediated degranulation. These data therefore point toward the potential mechanisms by which IL-33 contributes to the modulation of ATP-mediated activation in human MCs.

Development of an environmentally sensitive fluorescent peptide probe for MrgX2 and application in ligand screening of peptide antibiotics.

Mast cells (MCs) are primary effector cells involved in immediate allergic reactions. Mas-related G protein-coupled receptor-X2 (MrgX2), which is highly expressed on MCs, is involved in receptor-mediated drug-induced pseudo-anaphylaxis. Many small-molecule drugs and peptides activate MrgX2, resulting in MC activation and allergic reactions. Although small-molecule drugs can be identified using existing MrgX2 ligand-screening systems, there is still a lack of effective means to screen peptide ligands. In this study, to screen for peptide drugs, the MrgX2 high-affinity endogenous peptide ligand substance P (SP) was used as a recognition group to design a fluorescent peptide probe. Spectroscopic properties and fluorescence imaging of the probe were assessed. The probe was then used to screen for MrgX2 agonists among peptide antibiotics. In addition, the effects of peptide antibiotics on MrgX2 activation were investigated in vivo and in vitro. The environment-sensitive property of the probe was revealed by the dramatic increase in fluorescence intensity after binding to the hydrophobic ligand-binding domain of MrgX2. Based on these characteristics, it can be used for in situ selective visualization of MrgX2 in live cells. The probe was used to screen ten types of peptide antibiotics, and we found that caspofungin and bacitracin could compete with the probe and are hence potential ligands of MrgX2. Pharmacological experiments confirmed this hypothesis; caspofungin and bacitracin activated MCs via MrgX2 in vitro and induced local anaphylaxis in mice. Our research can be expected to provide new ideas for screening MrgX2 peptide ligands and reveal the mechanisms of adverse reactions caused by peptide drugs, thereby laying the foundation for improving their clinical safety.

Effects of Simvastatin on RBL-2H3 Cell Degranulation.

Hypercholesterolemia is a major complication of arteriosclerosis. Mast cells in arteriosclerosis plaques induce inflammatory reactions and promote arterial sclerosis. In this study, we evaluated the pharmacological effects of simvastatin (SV)-3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitors on the degranulation of rat basophilic leukemia (RBL)-2H3 cells, which are commonly used as mast cell models. SV significantly decreased the degranulation induced by three types of stimulation: antigen antibody reaction (Ag-Ab), thapsigargin (Tg) serosal endoplasmic reticulum calcium ATPase (SERCA) inhibitor, and A23187 calcium ionophore. SV had a stronger inhibitory effect on degranulation induced by Ag-Ab stimulation than the other two stimulations. However, SV did not inhibit increase of intracellular Ca2+ concentrations. Mevalonate or geranylgeraniol co-treatment with SV completely prevented the inhibitory effect of SV on the degranulation induced by these stimulations. Immunoblotting results showed that SV inhibited protein kinase C (PKC) delta translocation induced by Ag-Ab but not by Tg or A23187. SV induced a reduction in active Rac1, and actin filament rearrangement. In conclusion, SV inhibits RBL-2H3 cell degranulation by inhibiting downstream signaling pathways, including the sequential degranulation pathway. These inhibitory effects were completely reversed by the addition of geranylgeraniol and might be induced by changes in the translocation of the small guanosine 5'-triphosphatase (GTPase) families Rab and Rho, which are related to vesicular transport PKC delta translocation and actin filament formation, respectively. These changes are caused by the inhibition of HMG-CoA reductase by SV following the synthesis of geranylgeranyl pyrophosphates, which play important roles in the activation of small GTPases, Rab.

Enhancement of Mast Cell Degranulation Mediated by Purinergic Receptors' Activation and PI3K Type δ.

Mast cells express multiple metabotropic purinergic P2Y receptor (P2YR) subtypes. Few studies have evaluated their role in human mast cell (HMC) allergic response as quantified by degranulation induced by cross-linking the high-affinity IgE receptor (FcεRI). We have previously shown that extracellular nucleotides modify the FcεRI activation-dependent degranulation in HMCs derived from human lungs, but the mechanism of this action has not been fully delineated. This study was undertaken to determine the mechanism of activation of P2YRs on the degranulation of HMCs and elucidate the specific postreceptor pathways involved. Sensitized LAD2 cells, a human-derived mast cell line, were subjected to a weak allergic stimulation (WAS) using a low concentration of Ag in the absence and presence of P2YR agonists. Only the metabotropic purinergic P2Y11 receptor (P2Y11R) agonist, adenosine 5'-(3-thio)triphosphate (ATPγS), enhanced WAS-induced degranulation resulting in a net 7-fold increase in release (n = 4; p < 0.01). None of the P2YR agonists tested, including high concentrations of ATPγS (1000 µM), enhanced WAS-induced intracellular Ca2+ mobilization, an essential component of activated FcεRI-induced degranulation. Both a PI3K inhibitor and the relevant gene knockout decreased the ATPγS-induced enhancement. The effect of ATPγS was associated with enhanced phosphorylation of PI3K type δ and protein kinase B, but not the phosphoinositide-dependent kinase-1. The effects of ATPγS were dose dependently inhibited by NF157, a P2Y11R antagonist. To our knowledge, these data indicate for the first time that P2YR is linked to enhancement of allergic degranulation in HMC via the PI3K/protein kinase B pathway.

TLR2 Regulates Mast Cell IL-6 and IL-13 Production During Listeria monocytogenes Infection.

Listeria monocytogenes (L.m) is efficiently controlled by several cells of the innate immunity, including the Mast Cell (MC). MC is activated by L.m inducing its degranulation, cytokine production and microbicidal mechanisms. TLR2 is required for the optimal control of L.m infection by different cells of the immune system. However, little is known about the MC receptors involved in recognizing this bacterium and whether these interactions mediate MC activation. In this study, we analyzed whether TLR2 is involved in mediating different MC activation responses during L.m infection. We found that despite MC were infected with L.m, they were able to clear the bacterial load. In addition, MC degranulated and produced ROS, TNF-α, IL-1ß, IL-6, IL-13 and MCP-1 in response to bacterial infection. Interestingly, L.m induced the activation of signaling proteins: ERK, p38 and NF-κB. When TLR2 was blocked, L.m endocytosis, bactericidal activity, ROS production and mast cell degranulation were not affected. Interestingly, only IL-6 and IL-13 production were affected when TLR2 was inhibited in response to L.m infection. Furthermore, p38 activation depended on TLR2, but not ERK or NF-κB activation. These results indicate that TLR2 mediates only some MC activation pathways during L.m infection, mainly those related to IL-6 and IL-13 production.

Inherited and acquired determinants of serum tryptase levels in humans.

OBJECTIVE: To aid the clinician in correctly interpreting serum tryptase levels. DATA SOURCES: Primary peer-reviewed literature. STUDY SELECTIONS: Clinical and basic science peer-reviewed studies characterizing the genetic and physiological bases for tryptase generation, secretion, and elevation, including those describing serum tryptase levels in population-based cohort studies. RESULTS: Clinically measured basal serum tryptase (BST) consists of ostensibly inactive alpha- and beta-tryptase precursors. The autosomal dominant genetic trait hereditary alpha-tryptasemia is the most often cause for elevated BST levels, with other acquired causes, such as renal failure and clonal myeloid diseases being far less common. Acute increases in serum tryptase levels resulting from release of mature tryptase from secretory granules is specific to mast cell degranulation but is not detected in all cases of systemic anaphylaxis. CONCLUSION: Understanding the differences and distinguishing between acute increases in serum tryptase and chronic elevations in BST owing to inherited or acquired conditions is critical in the correct interpretation of this useful clinical biomarker.

The Role of Skin Mast Cells in Acupuncture Induced Analgesia in Animals: A Preclinical Systematic Review and Meta-analysis.

While mast cells (MCs) are previously well-known as a pathological indicator of pain, their role in alleviating pain is recently emerged in acupuncture research. Thus, this study systematically reviews the role of MC in acupuncture analgesia. Animal studies on MC changes associated with the acupuncture analgesia were searched in PubMed and EMBASE. The MC number, degranulation ratio and pain threshold changes were collected as outcome measures for meta-analyses. Twenty studies were included with 13 suitable for meta-analysis, most with a moderate risk of bias. A significant MC degranulation after acupuncture was indicated in the normal and was significantly higher in the pain model. In the subgroup analysis by acupuncture type, manual (MA) and electrical (EA, each P < .00001) but not sham acupuncture had significant MC degranulation. Meta-regression revealed the linear proportionality between MC degranulation and acupuncture-induced analgesia (P < .001), which was found essential in MA (P < .00001), but not in EA (P = .45). MC mediators, such as adenosine and histamine, are involved in its mechanism. Taken together, skin MC is an essential factor for acupuncture-induced analgesia, which reveals a new aspect of MC as a pain alleviator. However, its molecular mechanism requires further study. PERSPECTIVE: This systematic review synthesizes data from studies that examined the contribution of skin MC in acupuncture analgesia. Current reports suggest a new role for skin MC and its mediators in pain alleviation and explain a peripheral mechanism of acupuncture analgesia, with suggesting the need of further studies to confirm these findings.

GABAergic signaling in human and murine NK cells upon challenge with Toxoplasma gondii.

Protective cytotoxic and proinflammatory cytokine responses by NK cells impact the outcome of infections by Toxoplasma gondii, a common parasite in humans and other vertebrates. However, T. gondii can also sequester within NK cells and downmodulate their effector functions. Recently, the implication of GABA signaling in infection and inflammation-related responses of mononuclear phagocytes and T cells has become evident. Yet, the role of GABAergic signaling in NK cells has remained unknown. Here, we report that human and murine NK cells synthesize and secrete GABA in response to infection challenge. Parasitized NK cells secreted GABA, whereas activation stimuli, such as IL-12/IL-18 or parasite lysates, failed to induce GABA secretion. GABA secretion by NK cells was associated to a transcriptional up-regulation of GABA synthesis enzymes (glutamate decarboxylases [GAD65/67]) and was abrogated by GAD inhibition. Further, NK cells expressed GABA-A receptor subunits and GABA signaling regulators, with transcriptional modulations taking place upon challenge with T. gondii. Exogenous GABA and GABA-containing supernatants from parasitized dendritic cells (DCs) impacted NK cell function by reducing the degranulation and cytotoxicity of NK cells. Conversely, GABA-containing supernatants from NK cells enhanced the migratory responses of parasitized DCs. This enhanced DC migration was abolished by GABA-A receptor antagonism or GAD inhibition and was reconstituted by exogenous GABA. Jointly, the data show that NK cells are GABAergic cells and that GABA hampers NK cell cytotoxicity in vitro. We hypothesize that GABA secreted by parasitized immune cells modulates the immune responses to T. gondii infection.

Authentic and Ectopically Expressed MRGPRX2 Elicit Similar Mechanisms to Stimulate Degranulation of Mast Cells.

The identification of the Mas-related G-protein-coupled receptors (Mrgpr) as targets of diverse stimuli of mast cells (MCs), including neuropeptides and pseudo-allergy causing drugs, has placed these receptors at a prime position in MC research. However, the species-dependent diversity of these receptors raises the need for an adequate model for investigating the human MRGPRX2 receptor. RBL-2H3 cells, stably transfected with MRGPRX2 (RBL-MRGPRX2), are increasingly used for this purpose. Therefore, we investigated whether ectopically expressed MRGPRX2, in rat MCs, recapitulates its authentic signaling. To this purpose, we performed a broad comparative study of the responses of human LAD-2 MCs that express MRGPRX2 endogenously, and RBL-MRGPRX2 cells to compound 48/80, substance P and vancomycin, three proto-type ligands of MRGPRX2. We demonstrate that both models share similar dose-response relationships, kinetics and sensitivities to a wide range of signaling targeting drugs. Therefore, our results indicate that ectopically expressed MRGPRX2 preserves the signaling pathways employed to evoke human MC degranulation, which we show to rely on ERK1/2 MAP kinases, phospholipase C (PLC) and autophagy-related signaling. Importantly, we also show that the underlying mechanisms of MRGPRX2-triggered MC degranulation in either LAD-2 or RBL-MRGPRX2 cells are different from those elicited by its rodent orthologs.

Revisiting the Schistosoma japonicum life cycle transcriptome for new insights into lung schistosomula development.

Schistosome parasites are complex trematode blood flukes responsible for the disease schistosomiasis; a global health concern prevalent in many tropical and sub-tropical countries. While established transcriptomic databases are accessed ad hoc to facilitate studies characterising specific genes or gene families, a more comprehensive systematic updating of gene annotation and survey of the literature to aid in annotation and context is rarely addressed. We have reanalysed an online transcriptomic dataset originally published in 2009, where seven life cycle stages of Schistosoma japonicum were examined. Using the online pathway analysis tool Reactome, we have revisited key data from the original study. A key focus of this study was to improve the interpretation of the gene expression profile of the developmental lung-stage schistosomula, since it is one of the principle targets for worm elimination. Highly enriched transcripts, associated with lung schistosomula, were related to a number of important biological pathways including host immune evasion, energy metabolism and parasitic development. Revisiting large transcriptomic databases should be considered in the context of substantial new literature. This approach could aid in the improved understanding of the molecular basis of parasite biology. This may lead to the identification of new targets for diagnosis and therapies for schistosomes, and other helminths.

Inhibitory activity of narirutin on RBL-2H3 cells degranulation.

Context: It is an efficient strategy to apply inhibition of mast cell degranulation for evaluating anti-allergic effects of compounds. Previous works confirmed that narirutin had anti-allergic activity in OVA induced allergic asthma murine model. However, the mechanism is not clear. Objective: Here, inhibitory mechanism of narirutin on RBL-2H3 cells degranulation was investigated. Materials and methods: Cell viability was analyzed by CCK-8 kits, cell degranulation was analyzed by ELISA methods, morphology and ultrastructure of cells was observed by atomic force microscopy, intracellular Ca 2+ concentration was measured by fluorescence microscopre, mRNA expression were measured by PCR, and signaling pathways were measured by WB. Results: The results showed that narirutin have no direct effects on mRNA expression of FcεRI subunit. However, it inhibited Ca2+ influx by suppressing the phosphorylation of Syk, LAT and PLCγ1 signaling pathway transduction. Subsequently, the inhibition of Ca2+ influx directly leads to NF-κB signaling pathway transduction decreased. Narirutin can also suppress the phosphorylation of MAPK signaling pathways by decreasing the expression of P-p38, P-ERK and P-JNK, inhibit the synergistic effect for Ca2+ influx, and then reduce the release of IL-4, TNF-α, histamine and ß-HEX. Conclusion: Our study suggested that the inhibitory mechanism of narirutin on RBL-2H3 cells degranulation could be related to regulate MAPK, NF-κB and Tyrosine kinase signaling pathway.

MRGPRX2 is critical for clozapine induced pseudo-allergic reactions.

BACKGROUND: Clozapine is one of the most widely used second-generation antipsychotics in clinic. However, allergy-like symptoms such as rash and angioedema have been reported frequently, and the mechanism is still not clear. Mas-related G protein-coupled receptor X2 (MRGPRX2) expressed on mast cells is a crucial receptor for drug induced pseudo-allergic reactions. Therefore, we explored whether the symptoms induced by clozapine were associated with allergic reaction through MRGPRX2. METHODS: The effects of clozapine on pseudo-allergic reactions were evaluated by mast cells degranulation and calcium mobilization assay in vitro, and mice hindpaw swelling, serum histamine detection, avidin and H&E staining assay in vivo. The overexpressed MRGPRX2 cells membrane chromatography (MRGPRX2-HEK293/CMC), MRGPRX2-HEK293 cells calcium mobilization assay and molecular docking were applied to research the correlation between clozapine and MRGPRX2. RESULTS: The study showed that clozapine induced the release of ß-hexosaminidase, histamine and monocyte chemoattractant protein-1 (MCP-1), and trigged calcium mobilization in mast cells. In vivo, clozapine induced paw swelling, degranulation and vasodilation. Furthermore, clozapine could activate the calcium mobilization obviously in MRGPRX2-HEK293 cells, not in NC-HEK293 cells. Clozapine also had a good retention characteristic on MRGPRX2-HEK293/CMC column and the K D value is (2.33 ± 0.21)×10-01M. CONCLUSIONS: Our findings demonstrated that clozapine could induce pseudo-allergic reactions and MRGPRX2 might be the critical receptor for it.

The Role of Platelet Homeostasis in a Novel Topical PRP Formulation.

BACKGROUND: Topical platelet-rich plasma (PRP) must demonstrate stability to insure biologic activity in aesthetic medicine. OBJECTIVE: The objective of this research was to evaluate the role of platelet homeostasis in a novel PRP topical cosmetic formulation to provide facial appearance improvement. METHODS: The stability of the topical PRP formulation was evaluated in vitro followed by clinical in vivo testing. The in vitro evaluation examined platelet stability and morphology over a 90-day period within the preservative cosmetic base utilizing ELISA and light microscopy (LM)/scanning electron microscopy (SEM). The in vivo clinical study enrolled 20 subjects in a 120-day double blind split face study to evaluate the effect of 5–7x concentrated PRP compared to 2–3x concentrated PRP on facial photoaging. Cosmetic effect was evaluated by the subject and the dermatologist investigator on a 5-point ordinal scale at baseline, week 8, and week 16. RESULTS: 90-day stability for the topical PRP formulation was verified via ELISA and LM/SEM. ELISA showed the PRP was more inactive than control conditions via analyte concentration curves (PDGF-AB, EGF, and P-Selectin). LM/SEM demonstrated the PRP had less aggregation/activation over time within the cosmetic base and that refrigeration is superior to room-temperature storage thus delaying full platelet degranulation. The in vivo clinical study demonstrated parity between 20ml and 60ml PRP in terms of clinical efficacy. CONCLUSION: Platelets remain viable for up to 90 days in a refrigerated cosmetic vehicle with demonstrated topical clinical PRP facial benefits. PRP kits of 20ml and 60ml volumes for topical PRP are equally efficacious. J Drugs Dermatol. 2020;19(12): doi:10.36849/JDD.2020.5495.

Zinc Supplementation Modulates NETs Release and Neutrophils' Degranulation.

Zinc plays an important physiological role in the entire body, especially in the immune system. It is one of the most abundant microelements in our organism and an essential component of enzymes and antibacterial proteins. Zinc levels were reported to be correlated with the intensity of innate immunity responses, especially those triggered by neutrophils. However, as the results are fragmentary, the phenomenon is still not fully understood and requires further research. In this study, we aimed to perform a comprehensive assessment and study the impact of zinc on several basic neutrophils' functions in various experimental setups. Human and murine neutrophils were preincubated in vitro with zinc, and then phagocytosis, oxidative burst, degranulation and release of neutrophil extracellular traps (NETs) were analyzed. Moreover, a murine model of zinc deficiency and zinc supplementation was introduced in the study and the functions of isolated cells were thoroughly studied. We showed that zinc inhibits NETs release as well as degranulation in both human and murine neutrophils. Our study revealed that zinc decreases NETs release by inhibiting citrullination of histone H3. On the other hand, studies performed in zinc-deficient mice demonstrated that low zinc levels result in increased release of NETs and enhanced neutrophils degranulation. Overall, it was shown that zinc affects neutrophils' functions in vivo and in vitro. Proper zinc level is necessary to maintain efficient functioning of the innate immune response.

Taurine chloramine selectively regulates neutrophil degranulation through the inhibition of myeloperoxidase and upregulation of lactoferrin.

Taurine is a free amino acid rich in neutrophils, and neutrophils play an important role in the forefront defense against infection. Upon neutrophil activation, taurine reacts with hypochlorous acid (HOCl/OCl-) produced by the myeloperoxidase (MPO) system and gets converted to taurine chloramine (Tau-Cl). Neutrophils have three types of granules, of which the primary granule MPO, secondary granule lactoferrin, and tertiary granule matrix metalloproteinase (MMP)-9 are released into the extracellular space by a process called degranulation. MPO produces hypochlorous acid to kill microorganisms, and the released MPO forms neutrophil extracellular traps (NETs) with released chromatin. Excessive secretion of MPO causes oxidative damage to the surrounding tissues. Lactoferrin exerts antioxidant activity, prevents pro-inflammatory pathway activation, sepsis, and tissue damages, and delays neutrophil apoptosis. Our experimental results show that neutrophils released small amount of granules in an inactive state, and phorbol 12-myristate 13-acetate (PMA) and N-formyl-methionine-leucyl-phenylalanine induced neutrophil degranulation. Tau-Cl inhibited the PMA-induced degranulation of MPO and formation of NETs. While Tau-Cl increased the degranulation of lactoferrin, it had no effect on MMP-9 degranulation. MPO negatively regulated the production of macrophage inflammatory protein (MIP)-2, which stimulates the degranulation and migration of neutrophils. Tau-Cl abrogated MIP-2 expression, suggestive of its inhibitory effect on MPO release. The increase in the intracellular level of MPO may negatively regulates MIP-2 expression, thereby contributing to the further regulation of neutrophil degranulation and migration. Here, we suggest that Tau-Cl selectively inhibits MPO degranulation and stimulates lactoferrin degranulation from neutrophils, thereby protecting inflamed tissues from oxidative damage induced by excessively released MPO.

Filling the Antibody Pipeline in Allergy: PIPE Cloning of IgE, IgG1 and IgG4 against the Major Birch Pollen Allergen Bet v 1.

Birch pollen allergy is among the most prevalent pollen allergies in Northern and Central Europe. This IgE-mediated disease can be treated with allergen immunotherapy (AIT), which typically gives rise to IgG antibodies inducing tolerance. Although the main mechanisms of allergen immunotherapy (AIT) are known, questions regarding possible Fc-mediated effects of IgG antibodies remain unanswered. This can mainly be attributed to the unavailability of appropriate tools, i.e., well-characterised recombinant antibodies (rAbs). We hereby aimed at providing human rAbs of several classes for mechanistic studies and as possible candidates for passive immunotherapy. We engineered IgE, IgG1, and IgG4 sharing the same variable region against the major birch pollen allergen Bet v 1 using Polymerase Incomplete Primer Extension (PIPE) cloning. We tested IgE functionality and IgG blocking capabilities using appropriate model cell lines. In vitro studies showed IgE engagement with FcεRI and CD23 and Bet v 1-dependent degranulation. Overall, we hereby present fully functional, human IgE, IgG1, and IgG4 sharing the same variable region against Bet v 1 and showcase possible applications in first mechanistic studies. Furthermore, our IgG antibodies might be useful candidates for passive immunotherapy of birch pollen allergy.

Mast Cell Activation Test (MAT).

The mast cell (MC) activation assay is a robust in vitro tool for exploring MC reactivity in allergy. Here we describe the use of the mast cell activation test (MAT) that makes use of human primary MCs generated from peripheral blood progenitors, sensitized overnight with patients' sera and activated with allergens. Flow cytometry is used to assess the changes in expression of the activation marker CD63, and the percentage of cell degranulation is defined as the percentage of CD63+-positive MCs.

Basophil Stimulation and Signaling Pathways.

Despite the growing use of flow cytometry to analyze the functional characteristics of basophils, the intracellular signaling cascades that control their ability to elaborate various pro-allergic and inflammatory mediators and cytokines remain comparatively obscure. Additionally, some studies require the analysis of pro-allergic and inflammatory mediators, such as histamine, LTC4, and various basophil-derived cytokines (e.g., IL-4 and IL-13). Elucidation of intracellular signaling proteins by Western blotting, cytosolic free calcium concentration by spectrofluorophotometry, and detection of mediator releases, as well as analysis of gene expressions by RT-PCR, generally requires relatively large numbers of purified basophils. In selected assays, flow cytometry enables the analysis of relatively low cell numbers and purity for the expression of intracellular signaling proteins or measurement of cytosolic free calcium concentrations by basophil-specific gating strategies. Unfortunately, many aspects of signal transduction relevant to human basophils cannot be readily extrapolated from the use of basophil or mast cell lines. This chapter therefore focuses on how to employ primary human basophils for studying mediator releases and signaling characteristics.

Dynamic effects of calcium on in vivo and ex vivo platelet behavior after trauma.

BACKGROUND: Mobilization of intra and extracellular calcium is required for platelet activation, aggregation, and degranulation. However, the importance of alterations in the calcium-platelet axis after injury is unknown. We hypothesized that in injured patients, in vivo initial calcium concentrations (pretransfusion) predict ex vivo platelet activation and aggregation, viscoelastic clot strength, and transfusion of blood products. We additionally hypothesized that increasing calcium concentrations ex vivo increases the expression of platelet activation surface receptors and platelet aggregation responses to agonist stimulation in healthy donor blood. METHODS: Blood samples were collected from 538 trauma patients on arrival to the emergency department. Standard assays (including calcium), platelet aggregometry (PA) and thromboelastometry (ROTEM) were performed. In PA, platelet activation (prestimulation impedance [Ω]) and aggregation responses to agonist stimulation (area under the aggregation curve [AUC]) with adenosine diphosphate (ADP), thrombin receptor-activating peptide, arachidonic acid (AA), and collagen (COL) were measured. Multivariable regression tested the associations of calcium with PA, ROTEM, and transfusions. To further examine the calcium-platelet axis, calcium was titrated in healthy blood. Platelet aggregometry and ROTEM were performed, and expression of platelet glycoprotein IIb/IIIa and P-selectin was measured by flow cytometry. RESULTS: The patients were moderately injured with normal calcium and platelet counts. Higher calcium on arrival (pretransfusion) was independently associated with increased platelet activation (prestimulation, Ω; p < 0.001), aggregation (ADP-stimulated, AUC; p = 0.002; thrombin receptor-activating peptide-stimulated, AUC; p = 0.038), and clot strength (ROTEM max clot firmness; p < 0.001), and inversely associated with 24-hour transfusions of blood, plasma, and platelets (all p < 0.005). Up-titrating calcium in healthy blood increased platelet activation (prestimulation, Ω; p < 0.001), aggregation (ADP, AA, COL-stimulated AUCs; p < 0.050), and expression of P-selectin (p = 0.003). CONCLUSION: Initial calcium concentrations (pretransfusion) are independently associated with platelet activation, aggregation, clot-strength, and transfusions after injury. These changes may be mediated by calcium driven expression of surface receptors necessary for platelet activation and aggregation. However, the therapeutic benefit of early, empiric calcium repletion in trauma patients remains undefined. LEVEL OF EVIDENCE: Prognostic, level V.