Basophil markers

So far a suitable test for the detection of PPI-specific IgE antibodies does not appear to be available. There are at least two reports of a positive basophil activation test on patients allergic to omeprazole—one utilizing the CD63 basophil marker that was positive to omeprazole but negative to pantoprazole and the other detected by the flow-cytometric cellular allergen simulation test (FAST). 

The detection of reactions mediated by specific IgE to agents triggering anaphylaxis may be achieved by means of serological methods serum-specific IgE, or by means of cellular tests which determine the release of basophil mediators (leukotrienes and histamine) or by means of the analysis of basophil expression markers, a technique known as the basophil activation test (BAT). 

The principle underlying the BAT is that the attachment of the antigen to the IgE present on the surface of the basophil leads to the activation of the basophil and the release of its mediators (histamine, leukotrienes, prostaglandins, etc.) and the expression on its membrane of molecules such as CD63, CD203c or others which are markers of basophil activation. The basophils are identified with monoclonal antibodies marked with fluorochromes and anti-IgE and anti-CD63 receptors [for a complete review, we suggest readers read references 19-22]. 

Gamboa PM. Sanz ML. Caballero MR. Antepara I. Urrutia I. Gonzalez G. Dieguez I. De Week AL Use of CD63 expression as a marker of in vitro basophil activation and leukotriene determination in methi- 46 mazole allergic patients. Allergy 2003 58 312-317. 

Eberlein-Konig B. Rakoski J, Behrendt H, Ring J Use of CD63 expression as marker of in vitro basophil activation in identifying the culprit in insect venom allergy. J Investig Allergol Chn Immunol 2004 14 10-16. 

Of their many preformed intracellular mediators, histamine, not only plays an important physiological role, but can be used as a marker for these cells. In the human peripheral blood, the basophil is the only leukocyte that contains histamine. As a result, histamine release from human blood leukocyte preparations can be used to monitor basophil degranulation. In this chapter we will describe the histamine release assay, alternative techniques to measure the concentration of histamine and, finally, a method to purify human basophils to confirm that the chemokines are acting directly on the basophil. 

Additional biological tests have been suggested to improve the accuracy further. The leukocyte histamine release test has been found helpful (75) but it is expensive and time-consuming. Several surface molecules have been studied as markers, for example CD63. IgE-mediated degranulation of basophils after incubation of the patient s serum with a neuromuscular blocking agent can... 

The lack of a specific marker for basophils has hampered studying them in allergic diseases, and their relative... 

Fig. 1. Pertussis toxin-mediated ADP ribosylation of membrane G proteins. Isolated cell membranes (50 ng of protein) from N1E 115 cells (mouse neuroblastoma cell line), N2A cells (mouse neuroblastoma cell line), S49-1 eye cells (S49(-) mutated mouse lymphoma cell line deficient in Ga ), 549 wt cells (wild-type mouse lymphoma cell line), RBL (RBL 2H3 rat basophilic leukemia cell line), GH3 cells (GH3 rat hypophyseal tumor cell line), PC-12 (rat pheochromocytoma cell line), HIT-T15 cells (hamster insulinoma cell line), Y-1 cells (mouse adrenal cortex tumor cell line), 108 cc 15 cells (mouse/rat neuroblastoma x glioma hybrid cell line), HL-60 cells (DMSO-differentiated human leukemia cell line), HL-60 (+PT) cells (HL-60 cells pretreated with 25 ng/ml of pertussis toxin for 24 h prior to preparation of membranes), RINm5F cells (rat insulinoma cell line), and C6-2 cells (rat glioma cell line) were subjected to P-ADP-ribosylation as described in section 4.3.3. Samples were precipitated as outlined in section 4.3.5 and subjected to SDS-PAGE with separating gels containing 8% acrylamide (w/v). An autoradiogram of the dried gel is shown. Molecular masses of marker proteins are indicated (kDa). Modified Ga proteins migrate at approximately 40 kDa. Radioactivity running in front of the 30 kDa marker protein comigrates with the dye front...

The actions of chemicals that cause disease or death occur at the cellular level therefore, it is necessary to conduct an array of in vitro cell culture tests to determine cytotoxicity. The bulk absorbable material is tested also, hydrolyzed products as well as extracts are assessed. The most obvious marker of toxicity is cell death. Necrosis, as compared with normal cellular apoptosis, is characterized by cellular and organelle swelling followed by rupture. Apoptosis, in contrast, is programmed cell death, which is characterized by a reduction in cell volume and basophilic staining of the chromatin. There are three common in vitro screens for toxicity — agar, direct contact, and elution assays. 

In contrast to the above results, the basophil activation test was positive to the implicated quinolone in all five subjects with immediate reactions to a quinolone who were tested [19 ]. However, this assay quantified CD203C, which may be a more sensitive marker than CD63 for detecting IgE-mediated allergy. 

Incubation of cells with the stimulation buffer provides the all-important negative control, fliat is, the spontaneous expression of the activation marker. In general, negative controls remain below 5 % in 80 % of cases. For the positive control, anti-IgE, eiflier as a monoclonal or polyclonal antibody, is employed although the latter is generally superior since monoclonal anti-IgE antibodies are often poor activators of basophils. A monoclonal antibody to flie high affinity IgE receptor FceRI... 

From the study, one can conclude overall that flow cytometry can be used to examine histamine and its release along with the simultaneous quantification of basophil activation markers. This methodology has been termed HistaFlow by the authors. Results demonstrated that the appearance of CD63 indicates anaphylactic degranulation and significant histamine release whereas... 

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