Effects of a new nutraceutical ingredient on allergen-induced sulphidoleukotrienes production and CD63 expression in allergic subjects.

Allergic diseases represent conditions affecting millions of individuals across the world. The objective of this study was to investigate the potential anti-allergic effects of a new nutraceutical ingredient, Pantescal (Bionap, Italy), contained in different food supplements. Pantescal is a mixture of plant extracts, such as Capparis spinosa, Olea europaea, Panax Ginseng and Ribes nigrum. The study was a randomized, double-blind, placebo controlled design. 60 patients allergic to common aeroallergens were chosen. Allergic patients were divided into two groups: one group was supplemented by Pantescal and the other, using a placebo formulation. Two in vitro tests were performed on blood samples taken from patients before and at 2 h, 2, 3 and 10 days after supplementation: cellular antigen stimulation test (CAST) was used to analyze the amount of sulphidoleukotrienes (SLT) production and flow-cytometric antigen stimulation test (FAST) to measure expression of basophil degranulation marker (CD63) was also performed. CAST showed that after 2 and 3 days, a slight decrease of SLT production was evident but only after 10 days did it become significant with a percentage of inhibition (P.I)=43.3%. FAST revealed that there were no statistical differences for the first 2 days after supplementation although there was an inhibitory trend in the supplemented patients. CD63 expression was significantly reduced after 10 days (P.I.=64.8%). This study suggests that Pantescal is effective in reducing allergic biomarkers such as CD63 protein and SLT in atopic subjects. The higher inhibitory effect on CD63 expression compared to SLT production allows us to hypothesize cell membrane stabilization as the main potential mechanism to explain the observed Pantescal protective effects.

Interleukin-3 promotes the expression of E-NPP3/CD203C on human blood basophils in healthy subjects and in patients with birch pollen allergy.

We recently identified the ectoenzyme CD203c as a novel basophil activation antigen that is upregulated in response to FcepsilonRI cross-linkage. We investigated the effects of various interleukins (ILs) on expression of CD203c on blood basophils using an antibody against CD203c and flow cytometry. Of all cytokines tested, only IL-3 was found to upregulate expression of CD203c on basophils above baseline levels. The effects of IL-3 were dose- and time-dependent (EC(50): 0.1-1 ng/ml) without differences observed between healthy and allergic donors. Whereas anti-IgE induced maximum upregulation of CD203c within 15 minutes, the IL-3-induced upregulation showed a maximum after 180 minutes. IgE-receptor cross-linking resulted in enhanced expression of both CD63 and CD203c, whereas IL-3 enhanced the levels of CD203c without promoting expression of CD63. The IL-3-induced upregulation of CD203c was also observed in highly enriched basophils and was counteracted by a blocking antibody against the alpha chain of the IL-3 receptor (CD123). The IL-3-induced upregulation of CD203c was also found to depend on the presence of calcium. To analyze signaling pathways involved in IL-3-induced upregulation of CD203c, pharmacologic inhibitors were applied. The PI3-kinase inhibitors, wortmannin and LY294002 counteracted the IL-3-induced expression of CD203c, whereas MEK- and PKC inhibitors showed no effects. In conclusion, IL-3 upregulates expression of CD203c on basophils through a specific receptor and via a PI3-kinase-dependent signaling-pathway. Compared to FcepsilonRI-mediated cell activation, IL-3-induced upregulation of CD203c is a late(r) event and is not accompanied by upregulation of CD63.

Platelet-activating factor in the enteric nervous system of the guinea pig small intestine.

Platelet-activating factor (PAF) is a proinflammatory mediator that may influence neuronal activity in the enteric nervous system (ENS). Electrophysiology, immunofluorescence, Western blot analysis, and RT-PCR were used to study the action of PAF and the expression of PAF receptor (PAFR) in the ENS. PAFR immunoreactivity (IR) was expressed by 6.9% of the neurons in the myenteric plexus and 14.5% of the neurons in the submucosal plexus in all segments of the guinea pig intestinal tract as determined by double staining with anti-human neuronal protein antibody. PAFR IR was found in 6.1% of the neurons with IR for calbindin, 35.8% of the neurons with IR for neuropeptide Y (NPY), 30.6% of the neurons with IR for choline acetyltransferase (ChAT), and 1.96% of the neurons with IR for vasoactive intestinal peptide (VIP) in the submucosal plexus. PAFR IR was also found in 1.5% of the neurons with IR for calbindin, 51.1% of the neurons with IR for NPY, and 32.9% of the neurons with IR for ChAT in the myenteric plexus. In the submucosal plexus, exposure to PAF (200-600 nM) evoked depolarizing responses (8.2 +/- 3.8 mV) in 12.4% of the neurons with S-type electrophysiological behavior and uniaxonal morphology and in 12.5% of the neurons with AH-type electrophysiological behavior and Dogiel II morphology, whereas in the myenteric preparations, depolarizing responses were elicited by a similar concentration of PAF in 9.5% of the neurons with S-type electrophysiological behavior and uniaxonal morphology and in 12.0% of the neurons with AH-type electrophysiological behavior and Dogiel II morphology. The results suggest that subgroups of secreto- and musculomotor neurons in the submucosal and myenteric plexuses express PAFR. Coexpression of PAFR IR with ChAT IR in the myenteric plexus and ChAT IR and VIP IR in the submucosal plexus suggests that PAF, after release in the inflamed bowel, might act to elevate the excitability of submucosal secretomotor and myenteric musculomotor neurons. Enhanced excitability of motor neurons might lead to a state of neurogenic secretory diarrhea.

Enamel matrix protein interactions.

UNLABELLED: The recognized structural proteins of the enamel matrix are amelogenin, ameloblastin, and enamelin. While a large volume of data exists showing that amelogenin self-assembles into multimeric units referred to as nanospheres, other reports of enamel matrix protein-protein interactions are scant. We believe that each of these enamel matrix proteins must interact with other organic components of ameloblasts and the enamel matrix. Likely protein partners would include integral membrane proteins and additional secreted proteins. INTRODUCTION: The purpose of this study was to identify and catalog additional proteins that play a significant role in enamel formation. MATERIALS AND METHODS: We used the yeast two-hybrid assay to identify protein partners for amelogenin, ameloblastin, and enamelin. Once identified, RT-PCR was used to assess gene transcription of these newly identified and potential "enamel" proteins in ameloblast-like LS8 cells. RESULTS: In the context of this yeast assay, we identified a number of secreted proteins and integral membrane proteins that interact with amelogenin, ameloblastin, and enamelin. Additionally, proteins whose functions range from the inhibition of soft tissue mineralization, calcium ion transport, and phosphorylation events have been identified as protein partners to these enamel matrix proteins. For each protein identified using this screening strategy, future studies are planned to confirm this physiological relationship to biomineralization in vivo. CONCLUSION: Identifying integral membrane proteins of the secretory surface of ameloblast cells (Tomes' processes) and additional enamel matrix proteins, based on their abilities to interact with the most abundant enamel matrix proteins, will better define the molecular mechanisms of enamel formation at its most rudimentary level.

In vitro analysis of birch-pollen-associated food allergy by use of recombinant allergens in the basophil activation test.

BACKGROUND: Basophil activation is associated with the expression of CD63. In birch-pollen-associated food allergy to celery, carrot and apple, Bet v 1, Api g 1, Dau c 1 and Mal d 1 are major allergens. Recombinant allergens have not yet been used in the CD63-based basophil activation test (BAT). OBJECTIVE: To evaluate the feasibility of using recombinant allergens in the BAT in the diagnosis of allergy to apple, carrot and celery and to compare results with routine tests, i.e. skin prick tests (SPTs) and specific IgE. METHODS: Thirty-two patients with an oral allergy syndrome induced by apple, carrot or celery and 22 controls were studied. SPTs were performed with native foods. Specific IgE was determined by the CAP method and basophil activation by flowcytometry upon double staining with anti-IgE/anti-CD63 monoclonal antibodies after incubating with purified recombinant Bet v 1, Bet v 2, Api g 1, Dau c 1 and Mal d 1. RESULTS: By the combined use of the BAT and the CAP method, sensitization to Bet v 1 and Bet v 2 was detected in 100 and 25% of all subjects, respectively. Sensitivity of specific IgE for apple, carrot and celery was 60, 70 and 75% with corresponding specificities of 64, 86 and 82%. Sensitivity of the BAT for Mal d 1, Dau c 1 and Api g 1 was 75, 65 and 75% with corresponding specificities of 68, 100 and 77%. CONCLUSIONS: The BAT using recombinant allergens provides a valuable new in vitro method for the detection of sensitization to foods. Although double-blind placebo-controlled food challenges remain the gold standard to confirm food allergy, the CD63-based BAT with recombinant allergens may supplement routine tests for allergy diagnosis.

[Effect of danshen injection on expression of platelet membrane glycoproteins in patients with type II diabetes mellitus].

OBJECTIVE: To investigate the effect of Danshen Injection on platelet membrane glycoprotein expression and fibrinogen binding in patients with type II diabetes mellitus (DM). METHODS: 82 patients and 30 normal individuals were enrolled into this study. Platelet glycoprotein (GP) Ib, Gp IIb, GP IIIa, GP IIb-IIIa complex and p-selectin expression as well as fibrinogen binding were analyzed by flowcytomery. RESULTS: The platelet membrane GP IIb-IIIa complex, p-selectin expression and fibrinogen binding were higher in patients with vascular diseases than those of normal subjects and the patients without vascular diseases. Platelet surface GP Ib expression in patients with vascular diseases was lower than the other two groups. On the other hand, the GP IIb and GP IIIa were not significantly changed. There was no difference between the patients without vascular disease and the normal. Danshen Injection may improve above-mentioned the marks. CONCLUSION: Dan Shen Injection may reduce the activity of platelet membrane glycoproteins and improve the vascular disease.

[Relationship between syndrome differentiation-typing and expression of platelet-activation molecule CD62P and CD63 on platelets in psoriatic patients].

OBJECTIVE: To understand the relationship between Syndrome Differentiation- Typing and platelet activation in psoriatic patients. METHODS: Expression of platelet-activation molecules CD62P (alpha-granule membrane glycoprotein) and CD63 (lysosomal integral membrane protein) on platelets from 36 psoriatic patients with Syndrome Differentiation-Typing in TCM and 31 health subjects were investigated by using flow cytometry and specific monoclonal antibodies against activated platelet. RESULTS: (1) Increased expression of CD62P and CD63 on platelets from psoriatic patients was observed (P < 0.001); (2) The expression of CD62P and CD63 was in following order: The group of Blood Stasis > Blood Dryness > Blood Heat; (3) Compared with the group of Blood Heat, increased expression of CD62P and CD63 was observed in the group of Blood Dryness (P < 0.001); (4) Compared with the group of Blood Dryness, increased expression of CD62P and CD63 was observed in the group of Blood Stasis (P < 0.001). CONCLUSION: The platelet activation might play an important role in higher blood viscosity, endothelial cell injury, abnormal microcirculation, which was correlated with Blood Stasis, found in psoriasis.

Could coenzyme Q10 affect hemostasis by inhibiting platelet vitronectin (CD51/CD61) receptor?

Improved cardiovascular morbidity and mortality have been observed in several clinical studies of dietary supplementation with coenzyme Q10 (CoQ10, ubiquinone). Several mechanisms have been proposed to explain the effects of CoQ10. One attractive theory links ubiquinone with the inhibition of platelets. The effect of CoQ10 intake on platelet surface antigens, and certain hemostatic parameters was examined in 15 humans and 10 swine. Study participants received 100 mg of CoQ10 twice daily in addition to their usual diet for 20 days resulting in a three-fold increase of total serum CoQ10 level. We observed a decline in plasma fibronectin (-20.2%), thromboxane B2 (-20.6%), prostacyclin (-23.2%), and endothelin-1 (-17.9%) level. Significant inhibition of vitronectin receptor expression was observed consistently throughout ubiquinone treatment. Inhibition of the platelet vitronectin receptor is a direct evidence of a link between dietary CoQ10 intake, platelets, and hemostasis. These findings may contribute to the observed clinical benefits by a diminished incidence of thrombotic complications in such patients.

Characterization of platelet-activating factor binding to human airway epithelial cells: modulation by fatty acids and ion-channel blockers.

Radioligand-binding studies were performed in primary cultured human airway epithelial cells with [3H]PAF to determine whether these cells express platelet-activating factor (PAF) receptors. Scatchard analysis of PAF binding data revealed a single class of PAF binding sites with Kd 1.8 +/- 0.2 nM and Bmax. 21.0 +/- 2.1 fmol/10(6) cells (13,000 receptors/cell). PAF binding increased the intracellular free Ca2+ concentration ([Ca2+]i), indicating functional PAF receptors. Palmitate (C16:0), linoleic acid (C18:2 omega 6) or eicosapentaenoic acid (C20:5 omega 3) was incubated with the cells to test the effect on PAF binding. Incorporation of each fatty acid into cellular phospholipid occurred. [3H]PAF (1 nM) binding decreased in cells supplemented with C20:5 omega 3, but increased in the cells supplemented with C16:0. Scatchard analysis revealed that the inhibition of PAF binding by supplementation with C20:5 omega 3 was due to a decrease in both affinity and number of PAF receptors. PAF-stimulated increase in [Ca2+]i was also decreased by 60% in cells supplemented with C20:5 omega 3. Verapamil, a Ca(2+)-channel blocker, and amiloride, a Na(+)-channel blocker, inhibited specific binding of [3H]PAF to the cells, with IC50 4-5 microM and 0.2 mM respectively. Diphenylamine-2-carboxylate (DPC), a Cl(-)-channel blocker, dramatically increased PAF binding to the cell in a dose-dependent manner. Scatchard analysis revealed that verapamil and amiloride decreased both binding affinity and number of PAF receptors, whereas DPC increased PAF binding sites without affecting binding affinity. These results demonstrate that human airway epithelial cells have a functional receptor for PAF and that PAF receptor binding can be modulated by exogenous fatty acids and by ion-channel blockers.