Measurement Of Histamine Release Research Articles

Genetically encoded sensors for measuring histamine release both in vitro and in vivo

Histamine (HA) is a key biogenic monoamine involved in a wide range of physiological and pathological processes in both the central and peripheral nervous systems. Because the ability to directly measure extracellular HA in real time will provide important insights into the functional role of HA in complex circuits under a variety of conditions, we developed a series of genetically encoded G-protein-coupled receptor-activation-based (GRAB) HA (GRABHA) sensors with good photostability, sub-second kinetics, nanomolar affinity, and high specificity. Using these GRABHA sensors, we measured electrical-stimulation-evoked HA release in acute brain slices with high spatiotemporal resolution. Moreover, we recorded HA release in the preoptic area of the hypothalamus and prefrontal cortex during the sleep-wake cycle in freely moving mice, finding distinct patterns of HA dynamics between these specific brain regions. Thus, GRABHA sensors are robust tools for measuring extracellular HA transmission in both physiological and pathological processes.

Developing an in Vitro Functional Model of Allergic Transfusion Reactions

AbstractAbstract 3426 Background:Allergic transfusion reactions (ATRs) occur in ∼2–3% of platelet transfusions, but their etiology remains largely unclear. Both donor and recipient factors have been shown to contribute to ATR risk. An in vitro model of ATRs would enable mechanistic studies. Objective:To develop a basophil activation test as a model of ATRs to apheresis platelets (AP) by measuring histamine release (HR) and leukotriene C4 (LTC4) production after exposing basophils to AP supernatants. Methods:Basophil-enriched suspensions (∼5–15% basophils) from healthy donors were prepared using double-Percoll density centrifugation. After washing and counting basophil numbers using Alcian blue staining, cells were resuspended in buffer containing 2 ng/mL IL-3 and 5mM Ca++/Mg++ such that 20–30×104 basophils were added in 0.025mL aliquots. IL-3 is a potent basophil priming agent and was used to model the increased recipient susceptibility to allergic reactions that has been observed clinically. Reaction tubes (1.5 mL) were set-up containing 0.025mL of 2× (twice the final conc.) of ATR/control supernatant (final titer 1:2–1:8), negative (buffer alone), and positive control reagents (100 ng/mL anti-IgE antibody, 50 ng/mL C5a, 1 μM FMLP). Cells and reaction tubes were pre-warmed separately to 37°C for 15’ before aliquoting 0.025mL cell suspension to reaction tubes for a final volume of 0.050mL. After 30′ incubation at 37°C, 0.950mL of cold buffer (without IL-3 or Ca++/Mg++) was added to each reaction tube. Tubes were centrifuged 10" at ∼300g (4°C) before removing 0.900mL for histamine detection by automated fluorimetry and LTC4 detection by ELISA. AP supernatants that were (n=10) or were not (n=10) associated with ATRs were tested. HR values were determined as a percentage of the total histamine content by lysis with perchloric acid (1.6%). Results used in analysis are net HR or LTC4 production (AP sample minus IL-3 primed negative control) normalized to HR or LTC4 production observed with positive control. Results:IL-3 priming of basophils was necessary in order to evoke a response after exposure to AP supernatant. HR in response to AP incubation with unprimed basophils was <3% of positive controls. Maximal HR and LTC4 production in response to AP supernatants occurred at a final titer of 1:4 using IL-3 primed basophils.HR and LTC4 production within individual reactions were correlated (Spearman rho=0.83, P<0.0001). Mean net HR among all AP supernatant samples was 55±28%, 60±49%, and 53±29% of c5a, anti-IgE, and FMLP positive controls, respectively. LTC4 production was 19±26%, 14±22%, and 15±20% of c5a, anti-IgE, and FMLP positive controls.No statistically significant differences were observed in HR or LTC4 production when ATR and control groups were compared (P>0.1). ATRs were stratified into clinical categories of mild (pruritus/urticaria, n=3), moderate (angioedema/dyspnea, n=5), and severe (any symptom with hypotension, n=2). There were no differences or trends in HR and LTC4 production among the categories (P>0.2). There was no correlation between pre/post transfusion tryptase elevations in recipient plasma (n=4) and HR or LTC4 production. HR and LTC4 production was similar among blood types of AP products (A: n=12, O: n=5, AB: n=3), (P>0.7).We previously reported that concentrations of C5a are higher in AP products associated with ATRs than controls. C5a content in AP supernatant modestly correlated with basophil HR release (rho: 0.47, P=0.04), but less with LTC4 production (rho: 0.27, P=0.3). Summary:An in vitro basophil activation test that measures HR and LTC4 production was developed to study ATRs. We find that 1) IL-3 priming of basophils was necessary to elicit responses to AP supernatants; 2) LTC4 and HR are correlated within each reaction; 3) there are no differences in HR and LTC4 production between ATR and control AP supernatants were observed, regardless of clinical ATR severity; and 4) HR is associated with C5a content in the AP supernatant. Conclusions:The observations of 1) an overall requirement for basophil priming and 2) the lack of correlation between the clinical severity of ATRs and basophil HR and LTC4 release implicate recipient atopic priming as a risk factor for ATRs. However, the association of AP C5a content with HR suggests a donor/product role in ATRs, as well. Studies focusing on the nature of recipient susceptibility may further elucidate mechanisms of ATRs. Disclosures:No relevant conflicts of interest to declare.

Histamine release and surface CD200R1 staining as sensitive methods for assessing murine mast cell activation

Mast cells are important effector cells of allergy and are involved in the pathology of many other diseases. Measurement of β-hexosaminidase activity, the most commonly used method for evaluation of murine mast cell activity, requires a large number of cells and thus is of limited utility for studying mast cells in mouse models of disease. In this study we evaluated the sensitivity of histamine release as compared to β-hexosaminidase activity in the measurement of mast cell activation. Whereas a minimum of 6×104 mast cells per ml were required to detect slight increases in β-hexosaminidase activity after anti-IgE and ionomycin stimulation, substantial increases in histamine release could be detected under the same activating conditions with as few as 480 mast cells per ml. These findings demonstrate that measurement of histamine release is substantially more sensitive than assessment of β-hexosaminidase activity for detecting mast cell activation. Additionally, we describe a novel flow cytometric method for detecting murine mast cell activation. When using 7.5×105 peritoneal cells per condition and gating on IgE+c-kit+cells, mast cell expression of surface CD200R1 increased after both IgE and non IgE-mediated activation. This flow cytometric procedure was uncomplicated and rapid, with increases in surface CD200R1 expression appearing after as little as 30min of stimulation time. Measuring histamine release and surface CD200R1 expression are sensitive approaches for detection of murine mast cell activation. Further, both approaches can be done on unpurified peritoneal cell populations. By requiring low numbers of cells, these approaches are ideal for investigating mast cell activation in murine models of disease.

Potent effect of brown algae (Ishige okamurae) on suppression of allergic inflammation in human basophilic KU812F cells

The effect of solvent fractions from brown algae (Ishige okamurae) on allergic inflammatory reactions was investigated via measuring histamine release and cytokine generation in human basophilic KU812F cells induced by calcium ionophore A23187. Moreover, polyunsaturated fatty acids (PUFAs) profiles were identied and quantied in the most active fraction. It was found that n-hexane and ethyl acetate (EtOAc) fraction at 50 μg/mL significantly reduce histamine release with the release rates of 24 and 20%, respectively. Likewise, the suppressive effect of n-hexane and EtOAc fraction on production as well as expression of interleukin (IL)-4 and IL-13 was determined. Notably, EtOAc fraction exhibited more effective inhibition than n-hexane in all assays. Furthermore, GC analysis revealed that EtOAc fraction contains various anti-inflammatory PUFAs such as eicosapentaenoic acid, eicosatrienoic acid, linolenic acid, and γ-linolenic acid. Collectively, EtOAc fraction from brown algae could be a potential inhibitor of allergic inflammatory reactions in basophils that is suggested to be due to PUFAs.

Syk-kinase inhibition prevents mast cell activation in nasal polyps

Mast cells are crucial effector cells in the allergic cascade. The cross-linking of the high affinity IgE receptor (FcεRI) activates mast cells and basophils. Spleen tyrosine kinase (Syk) is positioned upstream of the IgE receptor signal transducing pathway and may represent an important target for the treatment of nasal inflammatory diseases. We measured effects of a specific Syk inhibitor in the release of mast cell mediators in human cord blood-derived mast cells (CBDMCs) (in-vitro) and in human nasal tissue (ex-vivo). Surgical samples were collected from patients with nasal polyposis who underwent sinus surgery. Tissue cubes of +- 0.9 mm3 were primed with myeloma IgE (1 microg/ml), preincubated with Syk inhibitor NVP-QAB205 in different concentrations and then stimulated with tissue culture medium, anti-IgE 10 microg/ml and anti-IgE 30 microg/ml. Supernatants were analysed for concentrations of histamine, LTC4/LTD4/LTE4 and PGD2. CBDMCs were likewise pre-incubated with compound, prior to stimulation with anti-IgE at 10 microg/ml. In CBDMCs, the Syk inhibitor prevented degranulation assessed by measurement of histamine release and the production of LTC4/LTD4/LTE4 and PGD2. Furthermore, the Syk inhibitor was similarly able to significantly inhibit the release of these granules and newly synthesized mediators by nasal polyp mast cells in a dose dependent manner. Although the critical role of Syk in the IgE receptor signal transduction pathway has been well documented in vitro, this study supports the importance of Syk in IgE receptor-mediated degranulation of mast cells ex-vivo within nasal tissue. Thus, inhibition of Syk may represent an important therapeutic strategy for the treatment of upper airway disease with mast cell involvement, such as allergic rhinitis.

Cyclic guanosine monophosphate dependent pathway contributes to human mast cell inhibitory actions of the nitric oxide donor, diethylamine NONOate

We have previously demonstrated that exogenous nitric oxide (NO) inhibited anti-IgE-mediated histamine release from human cultured mast cells. In the current study, we further investigated if syntheses of eicosanoids and cytokines were also suppressed by NO donors and evaluated if activation of soluble guanylyl cyclase (sGC) was an underlying mechanism. The effects of the NO donor diethylamine NONOate (DEA/NO) on IgE-dependent syntheses of eicosanoids (prostaglandin D 2 and cysteinyl leukotrienes) and cytokines (tumor necrosis factor-α and interleukin-8) from buffy coat derived human cultured mast cells were examined. The effects of sGC related agents on human mast cell activation were studied by measuring histamine release. DEA/NO (10 − 7 –10 − 4 M) dose-dependently inhibited anti-IgE induced release of histamine, eicosanoids and cytokines. It could also significantly increase intracellular cyclic guanosine monophosphate (cGMP) but reduce anti-IgE induced activation of ERK1/2, JNK1/2 and NF-κB. The inhibition of anti-IgE induced histamine release by DEA/NO was reversed by the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 − 7 M) and the cGMP-dependent protein kinase (PKG) inhibitor, Rp-8-(4-Chlorophenylthio)-guanosine-3′,5′-cyclic monophosphorothioate (Rp-8-pCPT-cGMPS, 10 − 5 M). The current study confirmed the inhibitory action of exogenous NO on immunological activation of human mast cells. We also provided evidence for the first time that the activation of the sGC–cGMP–PKG pathways together with the suppression of phosphorylation of MAPKs and NF-κB contributed to the mast cell modulating action of NO in human.

Inhibition of anti‐IgE mediated human mast cell activation by NO donors is dependent on their NO release kinetics

Although the mast cell is a source of nitric oxide (NO), the effect of NO on human mast cells has not been defined. This study investigated if exogenous NO could affect human mast cell activation. Effects of different NO donors on immunoglobulin E (IgE)-dependent activation of human-cultured mast cells (HCMC) derived from precursors in buffy coat were investigated by measuring histamine release. Intracellular NO in HCMC was monitored with confocal microscopy using the fluorescent NO indicator 4-amino-5-methylamino-2', 7'-difluorofluorescein. Diethylamine NONOate (DEA/NO) and MAHMA NONOate (NOC-9), both have rapid NO release rates, only inhibited anti-IgE-induced histamine release when added to HCMC at the time of activation. NO donors with slower NO release kinetics were ineffective even after 30 min incubation. Confocal microscopy revealed that the effectiveness of NO donors was dependent on the availability of adequate NO inside HCMC during activation. The inhibitory action of DEA/NO was diminished by the NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl but potentiated by the anti-oxidant, N-acetylcysteine (NAC). Furthermore, co-incubation with NAC allowed previously ineffective NO donors to suppress HCMC activation and thus suggested that NAC could increase the availability of NO from NO donors. Our results demonstrated that NO was able to modulate human mast cell activation but only when enough NO was present at the time of cell activation. Our findings explain the controversy over the effectiveness of NO on mast cell degranulation and supports the possibility that NO donors could be beneficial for treating allergic inflammation.

Development of an LC-ESI–MS/MS method for the determination of histamine: Application to the quantitative measurement of histamine degranulation by KU812 cells

A rapid, simple, and sensitive liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI–MS/MS) method was developed for the identification and quantification of histamine without a previous derivatization step or the addition of general ion-pairing reagents to the mobile phase. This method was used to measure histamine release following degranulation of KU812 human basophilic cells, using pyrazol as an internal standard. Analyses were performed on an LC system employing a Cosmosil 5C 18 PAQ column and an isocratic elution with methanol–0.005% trifluoroacetic acid (1:1) at a flow rate of 0.2 mL/min. A triple-quadrupole mass spectrometer, equipped with an electrospray ionization interface was employed, operating in the positive ion mode. The retention time of histamine and the internal standard were 4.0 and 5.0 min, respectively. The relative standard deviations (R.S.D.s) of the retention time and peak area were between 0.47% and 2.03%. Micropipette tip solid-phase extraction (SPE) using LooseTip C 18 allowed for not only rapid sample preparation, but also decreased suppression effects, improving peak shape. This method was used to evaluate the anti-allergic effects of compounds contained in Taxus yunnanensis extracts. Four constituents that were isolated from the wood extracts of T. yunnanensis and sodium cromoglicate, which is used as a first line anti-allergic drug, were tested in an in vitro histamine release inhibition assay. Of these compounds, taxiresinol and isotaxiresinol were more inhibitory than sodium cromoglicate.

Bacterial peptidoglycan breaks down intestinal tolerance via mast cell activation: The role of TLR2 and NOD2

Intestinal microbes are believed to be involved in the pathogenesis of inflammatory bowel disease. Microbes and their products are generally well tolerated by intestinal epithelial cells in the intestinal tract of healthy individuals. It is of significance to understand what breaks down the established tolerance leading to intestinal barrier dysfunction and intestinal inflammation. T84 monolayer transported peptidoglycan (PGN) was determined by enzyme-linked immune assay. Mast cell line HMC-1 cell activation in response to PGN stimulation was observed with electron microscopy and measurement of histamine release. T84 monolayer barrier function was determined by recording the transepithelial electric resistance (TER) and measuring the permeability in response to PGN-induced HMC-1 cell activation. Expression of Toll-like receptor (TLR) 2 and nucleotide-binding oligomerization domain (NOD) 2 were determined by immunocytochemistry, real-time reverse transcription (RT)-PCR and Western blot. Exposure to PGN alone did not alter TER and permeability of T84 monolayers. T84 monolayers transported PGN from the apical chamber to the basal chamber of transwell system. TLR2 expressed on the surface of HMC-1 cells. HMC-1 cells absorbed PGN. HMC-1 cells released histamine in response to the PGN stimulation, which was blocked by pretreatment with antibodies or small interfering RNA against TLR2 or NOD2. In a co-culture system, T84 monolayer transported PGN activated HMC-1 cells and increased the horseradish peroxidase flux. TLR2 mediated the PGN-absorption in HMC-1 cells. Blockade of TLR2 or NOD2 abolished PGN-induced HMC-1 cell activation and T84 monolayer barrier dysfunction. T84 monolayer transported PGN activates HMC-1 cells to release chemical mediators to induce T84 monolayer dysfunction that are mediated by TLR2 and NOD2.

Effects of polyunsaturated fatty acids on calcium response and degranulation from RBL-2H3 cells

To investigate the biological activity of various polyunsaturated fatty acids (PUFAs) on the allergic reaction, we examined the effects of six PUFAs and two saturated fatty acids on calcium response and degranulation from rat basophilic leukemia (RBL-2H3) cells. Between 20 and 40 μM of six PUFAs (ω-6 series: arachidonic acid [AA, C20:4], γ-linolenic acid [γ-LN, C18:3] and linoleic acid [LA, C18:2]; ω-3 series: α-linolenic acids [α-LN, C18:3] and eicosapentaenoic acid [EPA, C20:5]; and ω-9 series: oleic acid [OLE, C18:1]), or two saturated fatty acids (stearic acid [STA, C18:0] and arachidic acid [AD, C20:0]) were used to examine the effects on calcium response and degranulation from RBL-2H3 cells. Calcium response was monitored using the fluorescent calcium indicator fura-2, while degranulation was monitored by measuring histamine release from the cells. Three ω-6 PUFAs (AA, α-LN and LA) dose-dependently increased the cytosolic free-calcium concentration and histamine release from RBL-2H3 cells. This phenomenon was specific to the ω-6 PUFAs, the ω-3 PUFAs (α-LA and EPA), ω-9 PUFA (OLE) and the saturated fatty acids (STA and AD) had no effect. The increase in the cytosolic free-calcium concentration caused by the ω-6 PUFAs depended on the existence of external calcium, cell viability and the cellular IP 3 levels remained unchanged throughout the experiment. These results suggest that ω-6 PUFAs work as direct mediators of calcium signaling pathways in RBL-2H3 cells.

Design of tomato fruits with reduced allergenicity by dsRNAi‐mediated inhibition of ns‐LTP (Lyc e 3) expression

Plant genetic engineering has the potential to introduce new allergenic proteins into foods but, at the same time, it can be used to remove established allergens. Here, we report the molecular characterization of Lyc e 3, a new tomato (Lycopersicon esculentum) allergen, and the efficient down-regulation of its expression in transgenic tomato plants. Following the identification of an immunoglobulin E (IgE)-binding 9-kDa polypeptide in tomato peel, designated Lyc e 3, its partial amino acid sequence was determined by N-terminal protein sequencing. Sequence comparison revealed that Lyc e 3 encodes a nonspecific lipid transfer protein (ns-LTP). In plants, ns-LTPs are encoded by large gene families which differ in primary amino acid sequence, expression and proposed cellular function. To identify Lyc e 3 encoding complementary DNAs (cDNAs), public tomato expressed sequence tag (EST) databases were screened for ns-LTP sequences. Following this strategy, two cDNAs, LTPG1 and LTPG2, with high homology to the N-terminal sequence of Lyc e 3, were identified. Ectopic expression of LTPG1 and LTPG2 in Escherichia coli, followed by immunoblotting, verified their IgE reactivity. Subsequently, transgenic tomato plants constitutively expressing LTPG1- or LTPG2-specific double-stranded RNA interference (dsRNAi) constructs were created and tested for the suppression of Lyc e 3 accumulation. Efficient silencing of Lyc e 3 was documented by Northern and Western blotting. In both cases, Lyc e 3 accumulation was decreased to levels below the detection limit (less than 0.5% of the wild-type protein). The allergenic potential of Lyc e 3-deficient tomato fruits was tested by measuring histamine release from sensitized human basophils stimulated with transgenic and parental lines. These assays revealed a strong (10- to 100-fold) decrease in histamine release of human basophils challenged with transgenic fruit extracts when compared with control extracts. These results demonstrate the feasibility of creating low allergenic tomato fruits by means of dsRNAi inhibition.

Flow cytometric investigation of peri‐anaesthetic anaphylaxis using CD63 and CD203c

The investigation of anaphylactic reactions in the peri-operative period is difficult. Elevation of serum tryptase levels is a good indicator of an anaphylactic event but the ability of subsequent investigations to identify the drug(s) responsible for the reaction is still potentially unreliable. The aim of this study was to examine basophil activation as an investigative tool. We performed flow cytometric analysis of the expression on the cell surface of the basophil activation markers CD63 and CD203c and measured histamine release in 21 patients who were referred with possible peri-operative anaphylaxis. The sensitivity of CD63, CD203c, basophil histamine release and skin prick for the muscle relaxants was found to be 79%, 36%, 36% and 64%, respectively; the specificity was found to be 100%. These results demonstrate the difficulty in investigating the cause of an unexpected clinical event following drug administration, but the higher sensitivity of neo-expression on the cell surface of CD63 suggests that flow cytometric analysis of its neo-expression on basophils in vitro may be a diagnostic aid.

Elucidation of Anti-allergic Activities of Curcumin-Related Compounds with a Special Reference to Their Anti-oxidative Activities

The anti-allergic and anti-oxidative activities of curcumin-related compounds (glycosides, reductants and bis-demethoxy analogs) were investigated to elucidate the underlying active mechanisms and structural features of curcumin in exerting these activities. The anti-allergic activities were assessed by measurement of histamine release from rat basophilic leukemia cells, RBL-2H3. Curcumin and tetrahydrocurcumin (THC) caused a marked decrease in histamine release. Glycosides of curcumin, bis-demethoxycurcumin and THC also inhibited the release of histamine, though less potently than curcumin did. The anti-oxidative activities were assessed by measurement of cell-free or cellular radical scavenging. All compounds but diglycosides or bis-demethoxycurcumin analogs distinctly exerted anti-oxidative effects. The relationship between both of these activities revealed that all compounds with potent radical scavenging activities caused a definite decrease in histamine release, but some compounds with non-potent radical scavenging activities also inhibited the histamine release. These results suggest that the hydroxy groups of curcumin play a significant role in exerting both the anti-oxidative and anti-allergic activities, and that most of the compounds develop the anti-allergic activities through mechanisms related to anti-oxidative activities, but some through mechanisms unrelated to anti-oxidation activity.

Histamine dilutions modulate basophil activation.

In order to demonstrate that high dilutions of histamine are able to inhibit basophil activation in a reproducible fashion, several techniques were used in different research laboratories. The aim of the study was to investigate the action of histamine dilutions on basophil activation. Basophil activation was assessed by alcian blue staining, measurement of histamine release and CD63 expression. Study 1 used a blinded multi-centre approach in 4 centres. Study 2, related to the confirmation of the multi-centre study by flow cytometry, was performed independently in 3 laboratories. Study 3 examined the histamine release (one laboratory) and the activity of H(2) receptor antagonists and structural analogues (two laboratories). High dilutions of histamine (10(-30)-10(-38) M) influence the activation of human basophils measured by alcian blue staining. The degree of inhibition depends on the initial level of anti-IgE induced stimulation, with the greatest inhibitory effects seen at lower levels of stimulation. This multicentre study was confirmed in the three laboratories by using flow cytometry and in one laboratory by histamine release. Inhibition of CD63 expression by histamine high dilutions was reversed by cimetidine (effect observed in two laboratories) and not by ranitidine (one laboratory). Histidine tested in parallel with histamine showed no activity on this model. In 3 different types of experiment, it has been shown that high dilutions of histamine may indeed exert an effect on basophil activity. This activity observed by staining basophils with alcian blue was confirmed by flow cytometry. Inhibition by histamine was reversed by anti-H2 and was not observed with histidine these results being in favour of the specificity of this effect We are however unable to explain our findings and are reporting them to encourage others to investigate this phenomenon.

Relevance of Ara h1, Ara h2 and Ara h3 in peanut-allergic patients, as determined by immunoglobulin E Western blotting, basophil-histamine release and intracutaneous testing: Ara h2 is the most important peanut allergen.

A number of allergenic proteins in peanut has been described and the relative importance of these allergens is yet to be determined. We have investigated the relevance of previously identified peanut allergens in well-characterized peanut-allergic patients by in vitro, ex vivo and in vivo assays. Thirty-two adult peanut-allergic patients were included based on careful and standardized patient history and the presence of peanut-specific IgE. The diagnosis peanut allergy was confirmed using double-blind placebo-controlled food challenges in 23 patients. Major peanut allergens Ara h1, Ara h2 and Ara h3 were purified from peanuts using ion-exchange chromatography. IgE immunoblotting was performed and IgE-cross-linking capacity was examined by measuring histamine release (HR) after incubating patient basophils as well as passively sensitized basophils with several dilutions of the allergens. Intracutaneous tests (ICTs) using 10-fold dilution steps of the purified allergens and crude peanut extract were performed. Ara h2 was recognized most frequently (26 out of 32) in all tests and induced both positive skin tests and basophil degranulation at low concentrations, whereas Ara h1 and Ara h3 were recognized less frequently and reacted only at 100-fold higher concentrations as analysed with HR and intracutaneous testing (ICT). Next to the three tested allergens, proteins with molecular weights of somewhat smaller than 15 kDa were identified as a IgE-binding proteins on immunoblot in the majority of the patients (20 out of 32). We conclude that Ara h2 is, for our patient group, the most important peanut allergen, and that previously unidentified peanut proteins with molecular weights of somewhat smaller than 15 kDa may be important allergens as well. ICT in combination with basophil-HR and IgE immunoblotting provides insight in the patient specificity towards the individual peanut allergens.

The Basic Research on Physiological Property of Functionalized Hyaluronan (II): Effect of Sulfated Hyaluronan on Histamine Release from the Mast Cell

Hyaluronan (HA) is one of polysaccharides in living body that is found widely in connective tissue of the mammals. It has no sulfate group but high molecular weight in comparison with other glycosaminoglycans (GAGs). We synthesized sulfated HA (S-HA) with various degrees of substitution (D.S.) in order to give HA new biological functions, since GAGs with sulfate groups interact with various biocomponents to express some specific biofunctions. Moreover in order to develop biomedical polymers, immunological response against artificial polymer matrices must be evaluated. So we measured histamine release from the mast cell and discussed the effect of S-HA on the immunological response. It was confirmed that they could be used as biomedical materials with immunological safety. And S-HA inhibited the histamine release that was induced by compound 48/80 from the mast cell. The inhibitory effect of S-HA was seemed to be due to such various factors as D.S., molecular weight, substitution site of the sulfate group and the structure of a polysaccharide chain.

Homogeneous time resolved fluorescence assay to measure histamine release.

Histamine is a biogenic amine synthesized by the enzymatic decarboxylation of histidine. Implication of histamine in allergy is well described but histamine is also found in some specific neurones, functions as a neurotransmitter and regulates sleep/wake cycles, hormonal secretion, cardiovascular control and thermo-regulation. We have developed a TR-FRET histamine assay, based on the competition between sample histamine and allophycocyanine (XL665) labelled histamine for binding to a Europium cryptate (EuK) labelled antibody. As histamine is a small monoamine molecule, high affinity antibodies have been raised against carrier protein conjugated histamine. Therefore, sample histamine needs to be derivatized in the same way as the conjugated histamine, so that the antibody will have a similar affinity for both molecules. This acylation step is performed directly in wells and does not need to be done in separate vials, making handling easier for large numbers of samples. The incubation takes place at room temperature for 3 hours. The assay covers a measurement range of 1.56 to 400 nM and shows an analytical sensitivity of 1.3nM. We have shown that miniaturization of sample and reagents volumes down to 20 micro l does not alter these performances. This histamine release assay provides a particularly well adapted procedure for HTS and secondary screening compared to current heterogeneous methods.

Histamine release by hydrochloric acid is mediated via reactive oxygen species generation and phospholipase D in RBL-2H3 mast cells.

In order to investigate the underlying mechanism of HCl in oesophagitis, the inflammatory response to HCl was observed in RBL-2H3 mast cells. Rat basophilic leukemia (RBL-2H3) cells were used to measure histamine release, arachidonic acid (AA) release, reactive oxygen species (ROS) and peroxynitrite generation induced by HCl. Exogenous HCl increased the level of histamine release and ROS generation in a dose dependent manner, whereas it decreased the spontaneous release of [3H] AA and the spontaneous production of peroxynitrite. Mepacrine (10 microM), oleyloxyethyl phosphorylcholine (10 microM) and bromoenol lactone (10 microM) did not affect both the level of histamine release and ROS generation induced by HCl. U73122 (1 microM), a specific phospholipase C (PLC) inhibitor did not have any influence on level of histamine release and ROS generation. Propranolol (200 microM), a phospholipase D (PLD) inhibitor, and neomycin (1 mM), a nonspecific PLC and PLD inhibitor, significantly inhibited both histamine release and ROS generation. Diphenyleneiodonium (10 microM), a NADPH oxidase inhibitor, and tiron (5 mM), an intracellular ROS scavenger significantly inhibited the HCl-induced histamine release and ROS generation. These findings suggest that the inflammatory responses to HCl is related to histamine release and ROS generation, and that the ROS generation by HCl may be involved in histamine release via the PLD pathway in RBL-2H3 cells.

Mechanisms of autoimmune activation of basophils in chronic urticaria

Background: Approximately 35% to 40% of patients with chronic urticaria possess a circulating antibody directed to the α subunit of the high-affinity type I IgE receptor (FcϵRI), which is detectable by using histamine release assays or immunoblotting. Prior reports suggest that purified IgG may not directly activate basophils but rather does so through complement activation. Objective: We sought to further elucidate the mechanism by which this antibody causes basophil histamine release, including the role of complement, and to reassess the relationship of functional versus binding assays. Methods: We incubated human basophils with patient serum, patient IgG, or patient IgG plus normal serum as a complement source and measured histamine release for each condition. IgG fractions were neutralized with cloned α subunit to determine whether histamine release decreased proportionately. We also screened sera from 260 patients to compare histamine release with immunoblotting results. Results: We initially tested 35 sera from patients with chronic urticaria by using basophils from 2 atopic donors and one nonreleaser with rabbit anti-IgE. No histamine was released from the nonreleaser, yet all donors responded identically to monocyte chemotactic protein 1, indicating a requirement for IgE or the IgE receptor. Basophil histamine release was markedly augmented by complement if release by IgG alone was low. Incubation of purified IgG with an increasing concentration of cloned α subunit gradually reduced the histamine-releasing capability in patients with positive or negative immunoblot results. Of 260 patients tested, 43% had positive histamine release results, and 47% had positive immunoblot results, yet there was no correlation when individual patients were assessed. Conclusion: A subpopulation of patients with chronic urticaria possess IgG antibody directed to the α subunit of FcϵRI. This IgG activates basophils, which is dependent on or augmented by complement. Binding assays for the FcϵRI α subunit, such as immunoblotting, are not currently feasible as a screening method. A functional assay is required. (J Allergy Clin Immunol 2001;107:1056-62.)

Further Characterization of FcγRII and FcγRIII Expression by Cultured Human Mast Cells

Background: We have reported that resting human mast cells exhibit minimal expression for FcγRI, and that interferon-γ will upregulate this expression. The expression of FcγRII and FcγRIII by human mast cells remains to be fully examined. Methods: To investigate FcγRII and FcγRIII expression, we determined mRNA and protein expression of FcγRII and FcγRIII in human peripheral blood CD34+ derived cultured mast cells by RT-PCR and flow cytometry. The expression of FcγRII and FcγRIII in intact and permeabilized mast cells was also compared. We measured histamine release to monitor mast cell degranulation following cross-linking of FcγRII. Results: We found by RT-PCR that resting human mast cells exhibit mRNA for FcγRIIA, FcγRIIb1, FcγRIIb2 and FcγRIII but not FcγRIIC. FACS analysis of Fcγ receptors in intact versus permeabilized mast cells showed expression of FcγRII to be 42.2 ± 3.9% and this was unchanged by permeabilization. FcγRIII protein expression was minimal and this was also unchanged by permeabilization. Aggregation of FcγRII on human mast cells led to no significant degranulation as evidenced by histamine release. Conclusions: In addition to FcγRI expression, human mast cells express FcγRIIA, FcγRIIb1, FcγRIIb2 and FcγRIII mRNA, and significant surface expression of FcγRII. Aggregation of FcγRII on cultured human mast cells in this model was not followed by histamine release.