METHODS AND KITS FOR DETECTING BASOPHIL ACTIVATION

Abstract

The present invention relates to methods and kits for detecting basophil cells activation. The inventors showed that fluorescent avidin binds to basophil cell surface upon degranulation and that this probe can be used to monitor basophil degranulation More specifically the present invention relates to methods for monitoring of basophil degranulation using avidin-based probes. The method of the invention described here allows to measure direct basophil degranulation following FcRI crosslinking with allergen. This method provides a direct measurement of degranulation by staining exteriorized granules and unambiguously detects activated basophils degranulated. The extent of the degranulation can be directly deduced from the intensity of fluorescence of fluorochrome-labelled avidin measured on basophils. When applied to allergic patient samples the avidin-based method detected efficiently specific basophil responses.

Claims

1. A method for detecting/monitoring basophil activation in a fluid sample comprising the steps of i) adding an allergen extract to the fluid sample and ii) detecting the cell surface expression of CD123 and FcRI markers or CD123 and CD203c markers or CD123 and HLA-DR-negative markers in the cell population contained in the fluid sample iii) detecting degranulation of the cell population contained in the fluid sample using an avidin-based fluorescent probe and iv) concluding that the cells expressing CD123 and FcRI markers or CD123 and CD203c markers or CD123 and HLA-DR-negative markers which are bound by the avidin-based fluorescent probe are activated basophils.

2. The method according to claim 1 wherein step ii) of detecting the cell surface expression of markers and step iii) of detecting degranulation of the cell population are reversed.

3. The method according to claim 1 wherein the fluid sample is selected from the group consisting of blood samples, bone marrow samples, WBC (white blood cells) samples and samples of basophils in suspension.

4. The method of claim 1 wherein the detection of the cell surface expression of CD123 and FcRI markers or CD123 and CD203c markers or CD123 and HLA-DR-negative markers on the cell population contained in the fluid sample is performed with a set of antibodies specific for the markers.

5. The method of claim 4 wherein the antibodies specific for the markers are fluorochrome-labelled antibodies.

6. The method of claim 1 wherein the detection of the cell surface expression of CD123 and FcRI markers or CD123 and CD203c markers or CD123 and HLA-DR-negative markers on the cell population contained in the fluid sample and the detection of the degranulation of the said cell population is performed by flow cytometry.

7. The method of claim 1 which further comprises a step of determining the level of activated basophils present in the sample.

8. (canceled)

9. An in vitro method for diagnosing an allergic disease to a given allergen in a subject, comprising the steps of determining in a fluid sample obtained from the subject the level of activated basophils by performing the method of claim 7, ii) comparing the level determined in step i) with a reference value and iii) concluding that the subject suffers from an allergic reaction to the tested allergen when the level determined at step i) is higher than the reference value.

10. An in vitro method for monitoring an allergic disease comprising the steps of i) determining the level of activated basophils in a sample obtained from the subject at a first specific time of the disease by performing the method of claim 7, ii) determining the level of activated basophil cells in a sample obtained from the subject at a second specific time of the disease by performing the method of claim 7, iii) comparing the level determined at step i) with the level determined at step ii) and iv) concluding that the allergic disease has evolved in a worse manner when the level determined at step ii) is higher than the level determined at step i).

11. An in vitro method for monitoring the treatment of an allergic disease comprising the steps of i) determining the level of activated basophil cells in a sample obtained from the subject before the treatment by performing the method of claim 7, ii) determining the level of activated basophil cells in a sample obtained from the subject after the treatment by performing the method of claim 7, iii) comparing the level determined at step i) with the level determined at step ii) and iv) concluding that the treatment is efficient when the level determined at step ii) is lower than the level determined at step i).

12. The method of claim 11 wherein the treatment is done with an anti-allergic agent or by allergen specific immunotherapy.

13-15. (canceled)

Description

FIGURES

[0061] FIG. 1. Fluorescent avidin allows to monitor basophil degranulation. A-C, flow cytometry analysis of basophil stimulated with anti-IgE Abs. Gating strategy and representative FACS profiles (A). Percentages of av.A488+ or CD63+ basophils (B) and gMFI of gated basophils following avidin or CD63 stainings (C). Percentages in the FACS profiles indicate the frequency of gated cells. Each point represents a donor, bars represent median. Two-tailed paired t-test, ns P>0.05, *P<0.05, ***<0.001, ****<0.0001.

[0062] FIG. 2 Analysis of basophil degranulation in allergic patients. A-D, 1106 WBC were stimulated or not with anti-IgE mAb (2.5 g/mL) or with indicated allergen preparations. Basophils were gated as in FIG. 1, representative FACS profiles (A), Avidin+ basophil frequency (B), CD63+ basophil frequency (C) and CD203c or avidin fluorescence rMFI following stimulation (D). E, avidin rMFI and Prick test results from tested patients.

[0063] FIG. 3 Avidin staining is not always correlated with the CD203c upregulation-based assay. 1106 Human WBC were stimulated or not with anti-IgE mAb (2.5 g/mL). Basophils were gated as in FIG. 1. A, Representative FACS profiles of donor #7 and #1. B-C, gMFI of Av. A488 (B) and CD203c (C) staining on basophils (pooled data, n=18), arrows indicate discordant results for donors #1 and #6 (yellow circles). D, rMFI of Av. A488+ (red) and CD203c (green) (pooled data, n=18). Threshold (rMFI=0.1, dashed line) was determined using non responder donors (grey circles). Percentages in the FACS profiles indicate the frequency of gated cells. Each point represents a donor, Arrows indicate donor #13. Two-tailed paired t-test, **<0.01 ***<0.001.

[0064] FIG. 4 pDC and FcRI.sup.low monocyte are not stained with avidin upon stimulation. 110.sup.6 Human PWBC were stimulated or not with either PMA/Ionomycin or anti-IgE mAb (2.5 g/mL). (A) Gating strategy used to isolate pDC and FcRI.sup.low monocyte. Doublets were excluded using FSC-A and FSC-H parameters, then low SSC-A and FSC-A cells were gated; FcRI.sup.low monocyte and CD123.sup.+ cells were selected. To further separate pDC from basophil, pDC were gated as CD203c.sup. cells. Representative FACS profiles of gated CD123+ pDCs (B) or FcRI.sup.low monocytes (D) for Avidin-A488 staining following stimulation with PMA/Ionomycin or anti-IgE. Percentages of Av.488+ pDCs (C) or FcRI.sup.low monocytes (E) from pooled data n=11. Percentages in the FACS profiles indicate the frequency of gated cells. Each point represents a donor, bars represent median. Two-tailed paired t-test, ns P>0.05.

[0065] FIG. 5A small fraction of eosinophils or neutrophils stained dimly positive for avidin following stimulation. 110.sup.6 Human PWBC were stimulated or not with either PMA/Ionomycin or anti-IgE mAb (2.5 g/mL). (A) Gating strategy used to isolate neutrophils and eosinophils. Doublets were excluded using FSC-A and FSC-H parameters, CD16.sup.high/FcRI.sup. neutrophils and CD16.sup.+/FcRI.sup.+ eosinophils were gated from high SSC-A and FSC-A cells. Representative FACS profiles of gated neutrophils (B) or eosinophil (D) for Avidin-A488 staining following stimulation with PMA/Ionomycin or anti-IgE. Percentages of Av.488+ neutrophils (C) or eosinophils (E) from pooled data n=14. Percentages in the FACS profiles indicate the frequency of gated cells. Each point represents a donor, bars represent median. Two-tailed paired t-test, ns P>0.05, ***<0.001.

EXAMPLE 1: (INSERM)

Material & Methods

[0066] Reagents. Primary antibodies used for immunostaining: anti-FcRI efluor 450 (clone AER-37, eBioscience), anti-CD203c BV510 (2.5 L per test, clone NP4D6, BD Biosciences), anti-CD123 PE-Cy5 (clone 9F5, BD Biosciences), anti-CD16 Alexa 700 (clone 3G8, Beckman Coulter). Reagents used to stimulate peripheral blood cells were as follows: anti-IgE (clone MH25-1, Santa Cruz), PMA, phorbol 12-myristate 13-acetate and ionomycin (Sigma-Aldrich). Allergens extracts: bermuda grass (BAG-G2), orchard grass (BAG-G3), perennial rye grass (BAG-G5), timothy grass (BAG-G6), 6-grass mix (BAG-GX1: Orchard grass, perennial rye grass, Timothy grass, meadow fescue, meadow grass, velvet grass), ragweed mix (BAG-WX1: common ragweed, giant ragweed), all from Bhlmann laboratories and 5-grass mix (Alyostal: Phleum pretense, Dactylis glomerata, Anthoxanthum odoratum, Lolium perenne, Poa pratensis, Stallergenes). Avidin-Alexa 488 (Invitrogen) or anti-CD63 PE (clone H5C6 BD Biosciences) were used to monitor basophils degranulation. Basophils were purified using EasySep Human Basophil Enrichment Kit (#19069, Stemcell) according to the manufacturer's recommendations.

[0067] Blood donors. Blood from randomly selected anonymous donors (allergy status unknown) was obtained from buffy coats or fresh heparined blood (Etablissement Franais du Sang). Seven patients (3 men, 4 women, at the age of 19 to 57 years, mean age: 36.6 years, median age: 29 years) with a confirmed grass pollen allergy (positive history of rhinoconjunctivitis and positive skin prick test to grass pollen, 5-grass mix, Stallergenes) were included in this study. Venous blood was collected in 10 mL EDTA tubes. The study was approved by the INSERM national ethics committee (#16-280).

[0068] White blood cell preparation. Blood was centrifugated, the plasma discarded and red blood cells were lysed using red blood cells lysis buffer (15 mmol/L NH.sub.4Cl, 1 mmol/L KHCO.sub.3, 10 mmol/L EDTA). After centrifugation, white blood cells were washed in PBS and distributed in 96-well V-bottom plate in Tyrode's buffer (110.sup.6 cells in 100 L). The cells were adapted to 37 C. for 15 minutes before stimulation.

[0069] Cell stimulation for confocal analysis. 110.sup.4 purified basophils were sensitized with human IgE for two hours. Then cells were plated on Poly-D-Lysine (Sigma)-coated Lab-Tek chambered coverglass (Nunc) in Tyrode's buffer supplemented with 8 g mL.sup.1 avidin-sulforhodamine 101 (Av.SRho) and warmed at 37 C. for 20 min. Cells were stimulated with 2.5 g/mL goat anti-human IgE at time=0. Fluorescence was acquired every 2.3 seconds using Zeiss LSM 710 confocal microscope and ZEN software, environmental chamber (37 C. and 5% CO2), 63 Plan-Apochromat objective (1.4 oil). For some experiments, z-stacks image were acquired with an interval of 0.4 m.

[0070] Cell stimulation for FACS analysis. Cells were stimulated with 100 L of Tyrode's buffer containing either anti-IgE mAb (final concentration 2.5 g/ml) or a combination of phorbol 12-myristate 13-acetate and ionomycin (final concentration, 50 ng/ml and 1 g/ml respectively) or allergen extract (recommended concentration or 1/1000 for 5-grass mix) for 20 minutes at 37 C. Cells were washed and stained with a combination of anti-FcRI efluor 450, anti-CD203c BV510, anti-CD123 PE-Cy5, anti-CD16 Alexa 700, avidin-Alexa 488 and anti-CD63 PE diluted in 50 L FACS buffer (PBS plus 0.1% bovine serum albumin) for 30 minutes at 4 C. data were acquired by flow cytometry on a BD LSR-II cytometer and were analyzed by using FlowJo software (Tree Star, Inc, Ashland, Ore.).

[0071] Statistical analysis. Relative MFI was calculated as follow: rMFI=(gMFI of stimulated conditiongMFI of unstimulated condition)/gMFI of unstimulated condition. Positive rMFI threshold was determined using non responder donors (Av. A488.sup., no CD203c or CD63 upregulation). Two-tailed paired Student's t-tests were used for comparing two groups (Prism 5, GraphPad Software). P-value range is indicated: ns>0.05 *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Shown flow cytometry profiles and microscopy images are representative of repeated experiments.

Results

[0072] IgE-sensitized basophils were stimulated with anti-IgE Abs. Avidin-sulforhodamine (Av.SRho) was added to the incubation medium to monitor degranulation dynamics by time-lapse confocal laser scanning microscopy. Five minutes after stimulation, basophils underwent morphologic changes (e.g. increase of cell perimeter and cell spreading) while exteriorized granules were detected on the cell surface. The Av.SRho fluorescence intensity (FI) of the degranulating basophil augmented progressively to reach a plateau. Taken together, these results show that the addition of Av.SRho to the culture medium allows to monitor basophil degranulation.

[0073] To directly measure basophil specific activation among white blood cells (WBCs), we analyzed fluorescent avidin binding by flow cytometry. Freshly isolated peripheral WBCs from healthy donors were stimulated with anti-IgE Abs. 20 minutes after stimulation, cells were stained with avidin-A488, anti-CD203c, anti-FcRI, anti-CD123 and anti-CD63 mAbs and analyzed by flow cytometry. Basophils were identified as CD203c.sup.+ FcRI.sup.+ cells. Degranulated basophils stained positive for Av.A488 (FIG. 1A). Analysis of the degranulation as assessed by measurement of CD63 exposure or Av.A488 binding provided similar results, by showing that a substantial fraction of the basophil population degranulated for 10 donors in 11 (FIG. 1B-C). We next compared av.A488 staining to CD203c upregulation on basophil surface following stimulation. Basophils from two donors in 18 did not respond to anti-IgE stimulation as measured by both methods (FIG. 3). Noticeably, we observed a discrepancy among the two methods in two donors (donor #1 and #6) with an increase of avidin staining and no difference in CD203c staining before and after stimulation (FIG. 3). To analyze the relative increase of MFI for the two staining procedures, we took advantage of the two non-responder donors (donors #2 and #17) to set the threshold of these tests to rMFI=0.1. We plotted the relative MFI (rMFI) obtained for all the donors (FIG. 3D). This analysis showed that the rMFI calculated using the avidin-based assay did not correlate with rMFI calculated using the CD203c-based assay. In other words, the two methods do not identify the same donors as high responder donors. The discrepancies observed when comparing CD203c and avidin-based assays could be explained by the fact that avidin staining is strictly associated to degranulation whereas CD203c is a more generic marker of activation.

[0074] Since other immune cells might be activated following stimulus application to the whole WBC population, we initially investigated whether other cells might bind avidin. In the same set of experiments, we analyzed FcRI.sup.+ monocytes and plasmacytoid dendritic cells (pDC) that are present in the low SSC gate. We observed no avidin-A488 staining on FcRI.sup.low monocytes and on CD203c.sup. CD123.sup.+ FcRI.sup.+ pDC (FIG. 4). We in a second approach, we analyzed avidin binding on the two other granulocyte populations, i.e. neutrophils (FSC.sup.high SSC.sup.high CD16.sup.high) and eosinophils (FSC.sup.high SSC.sup.high CD16.sup.+ FcRI.sup.+) (FIG. 5). We did not detect avidin-A488.sup.+ neutrophils following stimulation for 9 donors in 14 and a very low percentage of slightly avidin-A488.sup.+ neutrophils for 5 donors in 14 (FIG. 5B-C). Nevertheless we observed that a small fraction of the eosinophils stained dimly for avidin-A488 for all the donors upon PMA/ionomycin stimulation only (FIG. 5D-E).

[0075] We next tested the avidin-based method on blood samples from patients with confirmed grass pollen allergy. Patients' WBC were challenged with an array of allergens and analyzed using the three methods (Avidin, CD63 or CD203c staining). The avidin-based method allowed to efficiently detect patients' response to allergens and provided results comparable to those obtained using CD63 and CD203c staining (FIG. 2B-D). Interestingly, when compared to CD203c staining, avidin binding appeared to be a more sensitive method to evaluate the extent of degranulation in response to different allergens (FIG. 2D). The analysis of avidin rMFI upon different allergen challenges (either single allergens or allergen mixtures) allowed us to establish the response pattern of each patient (FIG. 2E). Prick-tests were carried out using the five grasses mixture and correlated with avidin-based assays (FIG. 2E).

[0076] In conclusion, we described a new test easy to perform that provides similar results when compared to routinely used CD63-based test. It has been reported that CD63 expression does not always correlate with histamine release (5, 13, 14). Accordingly we observed, in mast cells, that degranulation as detected by avidin binding (but not by CD63 staining) correlates with -hexosaminidase release 12. Additional caveats might apply to CD63-based assays. First, CD63 exposure upon stimulation is not restricted to basophils, underlying the necessity of accurate gating strategies. Second, platelets express CD63 and can bind to basophils upon activation, thus providing a source of potential artifact (4).

[0077] Finally, the avidin-based method is a suitable alternative to current methods. Its advantage resides in the fact that avidin directly stains cell-bound granules upon degranulation and that the Av.SRho FI provide a measure of the degranulation magnitude.

EXAMPLE 2: (STANFORD)

Methods

Blood Specimens

[0078] Blood from randomly selected anonymous blood donors (allergy status unknown) was obtained from the Stanford Blood Center (Palo Alto, Calif., USA) and blood from 22 peanut allergic patients (Table 1) was obtained as part of their enrollment into two IRB-approved clinical trials (16 from POISED ClinicalTrials.gov Identifier: NCT02103270 and 6 from MAP-X ClinicalTrials.gov Identifier: NCT02643862). All blood samples were collected in heparin tubes and stored at 4 C. for 24 hours with gentle agitation before analysis (9). Peanut allergy was defined as having a clinical reaction to peanut during a double-blind, placebo-controlled food challenge to peanut (up to 500 mg total of peanut protein) and a positive skin prick test to peanut (>= to 5 mm).

TABLE-US-00001 TABLE 1 Demographic characteristics of patients with peanut allergy Age (y) Range 4-26; median = 10, mean = 11 Sex 82% Male Ethnicity 95% Not Hispanic/Latino Race* 68% Caucasian, 27% Asian and 5% multi-racial *Subjects self-identified ethnicity and race.

Reagents

[0079] RPMI-1640 medium was purchased from Gibco, Grand Island, N.Y., USA. Polyclonal rabbit anti-human IgE (Bethyl Laboratories, Montgomery, Tex., USA) was used for BATs. The antibody cocktail for surface staining for BATs consisted of FITC-conjugated anti-CD63 mAb (clone: H5C6 from BD Bioscience, San Jose, Calif., USA), PE-conjugated anti-HLA-DR mAb (clone: G46-6 from BD Bioscience), and PerCP-conjugated anti-CD123 mAb (clone: 7G3 from BD Bioscience). Alexa488-conjugated avidin was purchased from ThermoFisher Scientific (Carlsbad, Calif. USA). Calcium/magnesium-free PBS (CMF-PBS) was purchased from Corning Cellgro, Mediatech, Manassas, Va., USA. 0.5 M EDTA was purchased from Invitrogen Life Technologies, Carlsbad, Calif., USA. Bovine serum albumin (BSA) was purchased from Sigma, St. Louis, Mo., USA. Round bottomed tubes (352058) were purchased from BD Falcon, San Jose, Calif., USA. Fixation/Permeabilization Concentrate and Diluent, and Permeabilization Buffer (10), were purchased from eBioscience, San Diego, Calif., USA. Staining buffer refers to 5% BSA and 2 mM EDTA in CMF-PBS. All reagents were kept sterile at 4 C.

Basophil Activation Tests

[0080] Blood specimens were gently rotated at 4 C. for 24 h after blood collection. Immediately before starting BAT assays, samples were put into a water bath at 37 C. for 30 seconds. 100 L of whole blood were mixed with 100 L of RPMI or anti-IgE (final concentration: 1 g/mL) in 100 L of RPMI in round-bottomed tubes with loose lids as described in Ref (9). After a 30-min incubation at 37 C. in a 5% CO2 incubator (Panasonic, Osaka, Japan), reactions were stopped by adding 900 L, of cold 2.5 mM EDTA/CMF-PBS, followed by centrifuging for 5 min at 4 C. (all centrifuge runs were done with these conditions). After removal of supernatants, the antibody cocktail for surface staining (5 L, of each antibody mentioned above, total 20 L), or avidin-Alexa488 (final concentration: 1 g/mL) instead of FITC-labeled CD63 antibody, was added and mixed with the cell pellets, then incubated on ice for 20 min. After incubation, 3 mL of staining buffer was added and the tubes were centrifuged, the supernatant was removed, and 1 mL of Fix/Perm solution was added and mixed, and incubated for 30 min on ice. 2 mL of permeabilization buffer was added after the incubation and the tubes were centrifuged, supernatants were removed, and 150 L, of staining buffer was added, and then flow cytometry was performed with a FACS Canto II (BD Bioscience). Data were analyzed with FlowJo (TreeStar, Ashland, Ore., USA) by gating basophils as CD123 (IL-3 receptor alpha chain) positive and HLA-DR negative cells, and then measuring expression of and CD63 or avidin positive populations (data not shown).

Basophil Analysis by Confocal Microscopy

[0081] Human basophil enrichment kit (STEMCELL Technologies, Vancouver, Canada) was used for purification of basophils as per manufacturer's instructions. 510.sup.4 Purified basophils were placed into poly-D-Lysine-coated (5 g/ml in water, #P6407, Sigma Aldrich, USA) Nunc Lab-Tek 1.0 borosilicate cover glass system 8 chambers (#155411, Thermoscientific, USA) in RPMI medium supplemented with 1 g/ml of Av.A488 in a controlled atmosphere (using a Zeiss stage-top incubation system with objective heater, 37 C. and 5% humidified CO2) (8). Stimuli were added as described above and 30 minutes later single cell images were taken using a Zeiss LSM780 Meta inverted confocal laser-scanning microscope and a 63/1.40 Oil DIC M27 objective and electronic zoom 3 (dimension x:512 y:512, scaling x=0.264 m and y=0.264 m) (8).

Statistical Analysis

[0082] Mann-Whitney U tests were performed (the groups analyzed are described in the figure legends), and the results are reported in figure legends. We considered a P value of less than 0.05 to be statistically significant.

Result

[0083] First, we purified basophils from the whole blood of anonymous blood donors and used single cell confocal imaging to determine whether Av.A488 could stain basophils stimulated by activation with anti-IgE, which results in basophil degranulation. In accord with our previous reports on primary human and mouse MCs, (11, 15) we observed that, compared to RPMI-challenged control basophils, anti-IgE stimulated basophils exhibited enhanced staining with Av.A488, with a pattern of staining consistent with the exteriorization and cell surface association of negatively-charged granule constituents (e.g. chondroitin sulfate- and/or heparan sulfate-containing proteoglycans) (data not shown).

[0084] We recently reported a simple protocol which permits BATs to be performed, either by conventional flow cytometry or by Cytometry by Time-of-Flight mass spectrometry (CyTOF), on whole blood stored at 4 C. for up to 24 h before analysis (16). We used those conditions of blood storage to determine whether Av.A488 staining could be used to monitor basophil activation in whole blood from a group of 11 anonymous blood donors. We tested 100 L of whole blood in which cells were challenged with either 1 g/mL anti-IgE or RPMI (control), then gated on basophils as CD123-positive and HLA-DR-negative cells (two stable markers that, compared to FcRI or CD203c, do not vary upon basophil activation, (data not shown), (16) and compared detection of activated basophils by flow cytometry using either FITC-labeled anti-CD63 antibody or Av.A488 (both using the same detection channel; we defined activated basophils as those with a positive fluorescence signal for FITC-labeled anti-CD63 or for Av.488 as compared to the unstained and non-stimulated conditions) (data not shown). We found that Av.A488 readily detected anti-IgE-induced basophil activation in the whole blood of such subjects, whose allergy status is unknown (data not shown). Importantly, no significant increase in Av.A488 staining was observed after anti-IgE challenge of other granulocyte populations, i.e., neutrophils and eosinophils.

[0085] We next investigated whether Av.A488 could be used to monitor basophil activation in whole blood from 22 peanut allergic patients, drawn from subjects enrolled in one of two IRB-approved clinical trials (16 from POISED ClinicalTrials.gov Identifier: NCT02103270 and 6 from MAP-X ClinicalTrials.gov Identifier: NCT02643862) whose demographic features are shown in Table 1. Peanut allergy in both trials was defined as having a clinical reaction to a double-blind, placebo-controlled food challenge to peanut (up to 500 mg total of peanut protein) and a positive skin prick test to peanut (>= to 5 mm).Math.9

[0086] Using 100 L of whole blood per patient, we compared the ability of FITC-labeled anti-CD63 antibody versus Av.A488 to detect activated whole blood basophils by flow cytometry. Compared to CD63 detection, Av.A488 identified a higher percentage of activated basophils in 20 of 22 patients tested, both at baseline (i.e., after RPMI incubation) and after basophil activation by anti-IgE (data not shown). We also compared the detection of Av.A488+ basophils in the whole blood of anonymous blood donors versus subjects suffering from peanut allergies, both at baseline (i.e., after RPMI incubation) and 30 min after anti-IgE treatment. Both groups showed comparable increases in the percentage of detected Av.A488+ basophils after anti-IgE stimulation (data not shown). However, while anonymous blood donors in general harbored a small percentage of Av.A488+ basophils at baseline (global median 3.4%), this percentage was significantly increased in the blood of allergic subjects (global median 7.1%) (data not shown). Interestingly, in 11 of 22 allergic patients (compared to only 1 of 11 anonymous blood donors), basophils exhibited detectable Av.A488+ structures on their surface without prior in vitro stimulation with anti-IgE. It is therefore tempting to speculate that such Av.A488+ basophils either exhibited low basal levels of activation even without antigen stimulation and/or persistent effects of prior, perhaps subclinical, episodes of activation (e.g., related to the oral immunotherapy treatments). Notably, whatever its origin, this activated status of blood basophils at baseline in these 11 subjects was not observed by assessment of CD63 (data not shown).

[0087] Taken together, these results show that fluorochrome-labelled avidin can detect basophil degranulation and also might represent a more specific and sensitive alternative to CD63 detection to monitor the activation status of blood basophils in the whole blood, either at baseline or after IgE-dependent stimulation ex vivo.

REFERENCES

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