Colorectal mucosal histamine release by mucosa oxygenation in comparison with other established clinical tests in patients with gastrointestinally mediated allergy

AIM： This study evaluated colorectal mucosal histamine release in response to blinded food challenge-positive and-negative food antigens as a new diagnostic procedure. METHODS： 19 patients suffering from gastrointestinally mediated allergy confirmed by blinded oral provocation were investigated on grounds of their case history, skin prick tests, serum IgE detection and colorectal mucosal histamine release by ex vivo mucosa oxygenation. Intact tissue particles were incubated/stimulated in an oxygenated culture with different food antigens for 30 min. Specimens challenged with anti-human immunoglobulin E and without any stimulus served as positive and negative controls, respectively. Mucosal histamine release （% of total biopsy histamine content） was considered successful （positive）, when the rate of histamine release from biopsies in response to antigens reached more than twice that of the spontaneous release. Histamine measurement was performed by radioimmunoassay. RESULTS： The median （range） of spontaneous histamine release from colorectal mucosa was found to be 3.2 （0.1%-25.8%） of the total biopsy histamine content. Food antigens tolerated by oral provocation did not elicit mast cell degranulation 3.4 （0.4%-20.7%, P=0.4）, while anti-IgE and causative food allergens induced a significant histamine release of 5.4 （1.1%-25.6%, P = 0.04） and 8.1 （1.5%-57.9%, P = 0.008）, respectively. 12 of 19 patients （63.1%） showed positive colorectal mucosal histamine release in accordance with the blinded oral challenge responding to the same antigen （s）, while the specificity of the functional histamine release to accurately recognise tolerated foodstuffs was found to be 78.6%. In comparison with the outcome of blinded food challenge tests, sensitivity and specificity of history （30.8% and 57.1%）, skin tests （47.4% and 78.6%） or antigen-specific serum IgE determinations （57.9% and 50%） were found to be of lower diagnostic accuracy in gastrointestinally mediated allergy. CONCLUSION： Functional testing of the reactivity of colorectal mucosa upon antigenic stimulation in patients with gastrointestinally mediated allergy is of higher diagnostic efficacy.

Redox regulation of mast cell histamine release in thioredoxin-1 （TRX） transgenic mice

Thioredoxin-1 （TRX） is a stress-inducible redox-regulatory protein with antioxidative and anti-inflammatory effects. Here we show that the release of histamine from mast cells elicited by cross-linking of high-affinity receptor for IgE （FcεRI） was significantly suppressed in TRX transgenic （TRX-tg） mice compared to wild type （WT） mice. Intracellular reactive oxygen species （ROS） of mast cells stimulated by IgE and antigen was also reduced in TRX-tg mice compared to WT mice. Whereas there was no difference in the production ofcytokines （IL-6 and TNF-α） from mast cells in response to 2,4-dinitrophenylated bovine serum albumin （DNP-BSA） stimulation in TRX-tg and WT mice. Immunological status of TRX-tg mice inclined to T helper （Th） 2 dominant in primary immune response, although there was no difference in the population of dendritic cells （DCs） and regulatory T cells. We conclude that the histamine release from mast cells in TRX-tg mice is suppressed by inhibition of ROS generation. As ROS are involved in mast cell activation and facilitate mediator release, TRX may be a key signaling molecule regulating the early events in the IgE signaling in mast cells and the allergic inflammation.

INFLUENCE OF SP AND CON A ON HISTAMINE RELEASE FROM CULTURED MAST CELLS OF RAT SKIN

The mast cells from enzymatically dispersed rat skin were incubated in DMEM mediumcontaining 10% fetal bovine serum. 1 day after culture, the various concentrations of substance P (SP),SP+Ca2+ or concanavalin A (Con A) were added into the media to stimulate mast cells for different periods of time. The media were rapidly seperated from the cultured tells using the micropore filter. The contents of histamine in the media were determined by spectrofluorimetry and the release rates of histaminewere caculated. The results showed that SP and Con A could stimulate in vitro mast cells tO release histamine in a time--and dose-dependent pattern, and that the effect of SP was significantly influenced by theconcentration of Ca2+.

Signaling transduction pathways involved in basophil adhesion and histamine release

Background Little is known about basophil with respect to the different signaling transduction pathways involved in spontaneous, cytokine or anti-IgE induced adhesion and how this compares to IgE-dependent and IgE-independent mediator secretion. The purpose of the present study was to investigate the roles of β1 and β2 integrins in basophil adhesion as well as hosphatidylinositol 3-kinase （PI3K）, src-kinases and extracellular signal regulated kinase （ERK）1/2 in basophil adhesion and histamine release （HR）. Methods Basophils （purity of 10%-50%） were preincubated with anti-CD29 or anti-CD 18 blocking antibodies before used for adhesion study. Basophils were preincubated with the pharmacological inhibitors wortmannin, PP1, PD98059 before used for adhesion and HR study. Cell adherence to bovine serum albumin （BSA） or fibronectin （Fn） was monitored using cell associated histamine as a basophil marker and the histamine was measured by the glass fiber assay. Results Basophil spontaneous adhesion to Fn was inhibited by anti-CD29. Interleukin （IL）-3, granulocyte/macrophage colony stimulating factor （GM-CSF） induced adhesion to BSA was inhibited by anti-CD18. Wortmannin at 1 μmol/L and PP1 at 20 μmol/L strongly interfered with, whereas PD98059 at 50μmol/L weakly inhibited basophil spontaneous adhesion to Fn. One μmol/L wortmannin strongly inhibited IL-3, IL-5, GM-CSF and anti-IgE induced adhesion to BSA. PP1 at 20 μmol/L partly inhibited anti-IgE induced adhesion. Fifty μmol/L PD98059 marginally inhibited IL-5, weakly inhibited anti-IgE, partly inhibited GM-CSF induced adhesion. Wortmannin, PP1 and PD98059 inhibited anti-IgE （1：100 or 1：1000） induced basophil HR in a dose dependent manner. They inhibited calcium ionophore A23187 （10 μmol/L, 5 μmol/L） induced basophil HR in a dose dependent manner, but to different extend with PP1 being the most efficient. Conclusions Basophil spontaneous adhesion to Fn is mediated by β 1-integrins whereas cytokine induced adhesion to BSA is mediated by β 2-integrins. PI3K, src-kinases and ERK1/2 play distinct signaling roles in basophil adhesion and HR. PI3K is the key player while ERK1/2 is the weakest participant.

Baicalin and rutin are major constituents in Shuanghuanglian injection involving anaphylactoid reaction

OBJECTIVE: To identify the constituents in Shuanghuanglian injection(SHLI) that correlate with anaphylactoid reaction.METHODS: Chemical fingerprints of 10 batches SHLI samples were determined by High Performance Liquid Chromatography(HPLC), and further investigated by similarity analysis. Combined with optical microscopy, both anaphylactoid experiments and confirmatory assay were displayed in Rat basophil leukemia cells(RBL-2H3) to obtain the histamine release inducing by SHLI. The content of histamine was tested by Enzyme-Linked Immuno Sorbent As-say method. Partial least squares regression(PLSR)method and HPLC-DAD-ESI-MSntechnology were conducted to analyze constituents in SHLI involving anaphylactoid reaction.RESULTS: The results of spectrum and effect relationships showed that the eight constituents were positively correlated with anaphylactoid reaction.Among which, nearly 90% of them were identified as baicalin and rutin with PLSR and HPLC-DAD-ESI-MSn. This result was in accordance with confirmatory assay on RBL-2H3 cells.CONCLUSION: Baicalin and rutin from SHLI were the main constituents involving anaphylactoid reaction.