Allergic Donors Research Articles

IL-10-modulated dendritic cells from birch pollen- and hazelnut-allergic patients facilitate Treg-mediated allergen-specific and cross-reactive tolerance.

Approximately 70% of individuals allergic to birch pollen (Bet v 1.01 [Bet]) develop a secondary food allergy (e.g., hazelnut: Cor a 1.04 [Cor]), due to allergen cross-reactivity. However, standard immunotherapy for type I allergies often does not improve the food allergy sufficiently. We analyzed the allergen-specific and cross-reactive suppressive capacity of primary human regulatory T cells (Treg) induced by autologous IL-10-modulated dendritic cells (IL-10 DC) invitro and invivo. CD4+ T cells of patients with birch pollen and associated hazelnut allergies were differentiated into Bet-specific or non-specific induced Treg (iTreg). After Bet- or Cor-specific restimulation the phenotype, proliferation, and suppressive capacity of iTreg subsets were analyzed. iTreg function was further investigated in humanized mouse models of airway and intestinal allergy, generated by engraftment of peripheral blood mononuclear cells from allergic donors into immunodeficient animals. After IL-10 DC priming and allergen-specific restimulation (Bet or Cor), non-specific control iTreg remained anergic, whereas Bet-specific iTreg proliferated extensively and exhibited a regulatory phenotype (enhanced expression of CTLA-4, PD-1, TNFR2, IL-10). Accordingly, activated Bet-specific iTreg displayed a high capacity to suppress Bet- and Cor-induced responder Th2 cell responses invitro, indicating induction of both allergen-specific (birch) and cross-reactive tolerance (hazelnut). Invivo, the beneficial effect of Bet-specific iTreg was verified in humanized mouse models of allergic airway and intestinal inflammation, resulting in reduced allergen-induced clinical symptoms, and immune responses. Human IL-10 DC-induced iTreg facilitate allergen-specific and cross-reactive tolerance. Therefore, they are potential candidates for regulatory cell therapy in allergic and autoimmune diseases.

The disrupted molecular circadian clock of monocytes and macrophages in allergic inflammation.

Macrophage dysfunction is a common feature of inflammatory disorders such as asthma, which is characterized by a strong circadian rhythm. We monitored the protein expression pattern of the molecular circadian clock in human peripheral blood monocytes from healthy, allergic, and asthmatic donors during a whole day. Monocytes cultured of these donors allowed us to examine circadian protein expression in human monocyte-derived macrophages, M1- and M2- polarized macrophages. In monocytes, particularly from allergic asthmatics, the oscillating expression of circadian proteins CLOCK, BMAL, REV ERBs, and RORs was significantly altered. Similar changes in BMAL1 were observed in polarized macrophages from allergic donors and in tissue-resident macrophages from activated precision cut lung slices. We confirmed clock modulating, anti-inflammatory, and lung-protective properties of the inverse ROR agonist SR1001 by reduced secretion of macrophage inflammatory protein and increase in phagocytosis. Using a house dust mite model, we verified the therapeutic effect of SR1001 in vivo. Overall, our data suggest an interaction between the molecular circadian clock and monocytes/macrophages effector function in inflammatory lung diseases. The use of SR1001 leads to inflammatory resolution in vitro and in vivo and represents a promising clock-based therapeutic approach for chronic pulmonary diseases such as asthma.

Targeting inhibitory Siglec-3 to suppress IgE-mediated human basophil degranulation

BackgroundSiglec-3 (CD33) is a major Siglec expressed on human mast cells and basophils and engagement of CD33 leads to inhibition of cellular signaling via immunoreceptor tyrosine-based inhibitory motifs (ITIMs). ObjectiveWe sought to inhibit human basophil degranulation by simultaneously recruiting inhibitory CD33 to the IgE-FcεRI complex using monoclonal anti-IgE directly conjugated to CD33 ligand (CD33L). MethodsDirect and indirect basophil activation tests (BAT) were used to assess both antigen-specific (peanut) and antigen non-specific (polyclonal anti-IgE) stimulation. Whole blood from allergic donors was used for direct BAT, whereas non-food allergic donor blood was passively sensitized with peanut-allergic plasma in the indirect BAT. Blood was incubated with anti-IgE-CD33L or controls for one hour or overnight, then stimulated with peanut, polyclonal anti-IgE, or N-formylmethionyl-leucyl-phenylalanine (fMLP) for 30 minutes. Degranulation was determined by measuring CD63 expression on the basophil surface by flow cytometry. ResultsIncubation for one hour with anti-IgE-CD33L significantly reduced basophil degranulation after both allergen-induced (peanut) and polyclonal anti-IgE stimulation, with further suppression after overnight incubation with anti-IgE-CD33L. As expected, anti-IgE-CD33L did not block basophil degranulation due to fMLP, providing evidence that this inhibition is IgE-pathway specific. Finally, CD33L is necessary for this suppression, as monoclonal anti-IgE without CD33L was unable to reduce basophil degranulation. ConclusionsPre-treating human basophils with anti-IgE-CD33L significantly suppressed basophil degranulation through the IgE-FcεRI complex. The ability to abrogate IgE-mediated basophil degranulation is of particular interest, as treatment with anti-IgE-CD33L prior to antigen exposure could have broad implications for the treatment of food, drug, and environmental allergies.

RBet v 1a-BanLecwt induce upregulation of IL-10 and IFN-γ gene expression in Caco-2/THP-1 co-culture and secretion of IL-10 and IFN-γ/IL-4 levels in PBMCs of birch pollen allergic donors

Novel allergen immunotherapy (AIT) approaches necessitate the use of more effective and safe therapeutics, which can be accomplished by employing novel adjuvants for improved innate immune cell activation, as well as hypoallergenic allergen forms. In this study, we investigate the immunomodulatory effects of a chimera rBet v 1a-BanLecwt (rBv1a-BLwt; Cwt) composed of the major birch pollen allergen Bet v 1a and banana lectin (BanLecwt; BLwt) and two novel chimeras, rBv1l-BLH84T (rBet v 1l-BanLecH84T; C1) and rBLH84T-Bv1l (rBanLecH84T-Bet v 1l; C2), both composed of BLH84T and hypoallergenic birch pollen allergen Bv1l in the co-culture model Caco-2/THP-1, and PBMCs from donors with birch pollen allergy. The chimeric molecules rBv1l-BLH84T (C1) and rBLH84T-Bv1l (C2) were created in silico and then produced in E. coli using recombinant DNA technology. Real-time PCR analysis of gene expression following compound treatment in the co-culture model revealed that all three chimeras have the potential to induce the anti-inflammatory cytokine IL-10 gene expression in Caco-2 cells and IFN-γ gene expression in THP-1 cells. Sandwich ELISA revealed that Cwt increased IL-10 secretion and IFN-/IL-4 levels in PBMCs from birch pollen allergic donors, whereas C1 and C2 were less effective. The findings suggest that Cwt should be analyzed further due to its potential benefit in AIT.

Assessing IgE sensitization profiles to birch and timothy grass pollen allergens in birch pollen allergic blood donors using an oligoplex specific IgE assay

IgE sensitization profiles to single birch allergens in birch-sensitized patients differ among European countries. The aim of this study was to determine the distribution of specific IgE antibodies to major and minor birch pollen allergens in a population of allergic Norwegian individuals by using a birch allergic blood donor population as a surrogate sample. Sixty blood donors were recruited and sampled based on birch allergy symptoms such as rhinitis, rhinoconjunctivitis and/or mild asthma in previous seasons. All sera were collected before start of the pollen season and tested using a line blot assay (Euroimmun AG, Lübeck, Germany) for IgE to birch and timothy pollen. Both extracts, single allergens, and cross-reacting carbohydrate determinants (CCD) were analysed. Only donors with specific IgE to birch and/or timothy grass were further evaluated. Specific IgE to birch pollen extract was found in 52 sera, and sensitization to timothy grass in 40 sera. Specific IgE to Bet v 1 was predominant in contrast to Bet v 4 which was absent. However, sensitization to the minor allergens Bet v 2 and 6 was always found together with high levels of IgE to Bet v 1. Subjects sensitized to the profilin Bet v 2 from birch were also sensitized to Phl p 12 from timothy grass. In conclusion, there was predominantly Bet v 1 sensitization in this cohort and low sensitization to minor allergens and cross-reactive allergens (Bet v 2, Bet v 4, Phl p 7 and Phl p 12).

Biological activity of human IgE monoclonal antibodies targeting Der p 2, Fel d 1, Ara h 2 in basophil mediator release assays.

Human Immunoglobulin E monoclonal antibodies (hIgE mAb) are unique tools for investigating IgE responses. Here, the biological activity of hIgE mAb, derived from immortalized B cells harvested from the blood of allergic donors, targeting three allergens (Der p 2, Fel d 1 and Ara h 2) was investigated. Three Der p 2-, three Fel d 1- and five Ara h 2-specific hIgE mAb produced by human B cell hybridomas, were combined in pairs and used to passively sensitize humanized rat basophilic leukemia cells and compared with sensitization using serum pools. Sensitized cells were stimulated with corresponding allergens (recombinant or purified), allergen extracts or structural homologs, having 40-88% sequence similarity, and compared for mediator (β-hexosaminidase) release. One, two and eight pairs of Der p 2-, Fel d 1- and Ara h 2-specific hIgE mAb, respectively, produced significant mediator release (>50%). A minimum hIgE mAb concentration of 15-30 kU/L and a minimum antigen concentration between 0.01-0.1 µg/mL were sufficient to induce a pronounced mediator release. Individual sensitization with one Ara h 2-specific hIgE mAb was able to induce crosslinking independently of a second specific hIgE mAb. Der p 2- and Ara h 2-specific mAb showed a high allergen specificity when compared to homologs. Mediator release from cells sensitized with hIgE mAb was comparable to serum sensitization. The biological activity of hIgE mAb reported here provides the foundation for novel methods of standardization and quality control of allergen products and for mechanistic studies of IgE-mediated allergic diseases, using hIgE mAb.

Precision cut intestinal slices, a novel model of acute food allergic reactions.

Food allergy affects up to 10% of the pediatric population. Despite ongoing efforts, treatment options remain limited. Novel models of food allergy are needed to study response patterns downstream of IgE-crosslinking and evaluate drugs modifying acute events. Here, we report a novel human ex vivo model that displays acute, allergen-specific, IgE-mediated smooth muscle contractions using precision cut intestinal slices (PCIS). PCIS were generated using gut tissue samples from children who underwent clinically indicated surgery. Viability and metabolic activity were assessed from 0 to 24 h. Distribution of relevant cell subsets was confirmed using single nucleus RNA sequencing. PCIS were passively sensitized using plasma from peanut allergic donors or peanut-sensitized non-allergic donors, and exposed to various stimuli including serotonin, histamine, FcɛRI-crosslinker, and food allergens. Smooth muscle contractions and mediator release functioned as readouts. A novel program designed to measure contractions was developed to quantify responses. The ability to demonstrate the impact of antihistamines and immunomodulation from peanut oral immunotherapy (OIT) was assessed. PCIS viability was maintained for 24 h. Cellular distribution confirmed the presence of key cell subsets including mast cells. The video analysis tool reliably quantified responses to different stimulatory conditions. Smooth muscle contractions were allergen-specific and reflected the clinical phenotype of the plasma donor. Tryptase measurement confirmed IgE-dependent mast cell-derived mediator release. Antihistamines suppressed histamine-induced contraction and plasma from successful peanut OIT suppressed peanut-specific PCIS contraction. PCIS represent a novel human tissue-based model to study acute, IgE-mediated food allergy and pharmaceutical impacts on allergic responses in the gut.

Ex vivo assays show human gamma-delta T cells specific for common allergens are Th1-polarized in allergic donors

Gamma-delta (γδ) Tcells contribute to the pathology of many immune-related diseases; however, no exvivo assays to study their activities are currently available. Here, we established a methodology to characterize human allergen-reactive γδ Tcells in peripheral blood using an activation-induced marker assay targeting upregulated 4-1BB and CD69 expression. Broad and reproducible exvivo allergen-reactive γδ Tcell responses were detected in donors sensitized to mouse, cockroach, house dust mite, and timothy grass, but the response did not differ from that in non-allergic participants. The reactivity to 4 different allergen extracts was readily detected in 54.2%-100% of allergic subjects in a donor- and allergen-specific pattern and was abrogated by Tcell receptor (TCR) blocking. Analysis of CD40L upregulation and intracellular cytokine staining revealed a T helper type 1 (Th1)-polarized response against mouse and cockroach extract stimulation. These results support the existence of allergen-reactive γδ Tcells and their potential use in rebalancing dysregulated Th2 responses in allergic diseases.

Allergen-Specific IgA Antibodies Block IgE-Mediated Activation of Mast Cells and Basophils.

Mast cells and basophils have long been implicated in the pathogenesis of IgE-mediated hypersensitivity reactions. They express the high-affinity IgE receptor, FcϵRI, on their surface. Antigen-induced crosslinking of IgE antibodies bound to that receptor triggers a signaling cascade that results in activation, leading to the release of an array of preformed vasoactive mediators and rapidly synthesized lipids, as well as the de novo production of inflammatory cytokines. In addition to bearing activating receptors like FcεRI, these effector cells of allergy express inhibitory ones including FcγR2b, an IgG Fc receptor with a cytosolic inhibitory motif that activates protein tyrosine phosphatases that suppress IgE-mediated activation. We and others have shown that food allergen-specific IgG antibodies strongly induced during the course of oral immunotherapy (OIT), signal via FcγR2b to suppress IgE-mediated mast cell and basophil activation triggered by food allergen challenge. However, the potential inhibitory effects of IgA antibodies, which are also produced in response to OIT and are present at high levels at mucosal sites, including the intestine where food allergens are encountered, have not been well studied. Here we uncover an inhibitory function for IgA. We observe that IgA binds mouse bone marrow-derived mast cells (BMMCs) and peritoneal mast cells. Binding to BMMCs is dependent on calcium and sialic acid. We also found that IgA antibodies inhibit IgE-mediated mast cell degranulation in an allergen-specific fashion. Antigen-specific IgA inhibits IgE-mediated mast cell activation early in the signaling cascade, suppressing the phosphorylation of Syk, the proximal protein kinase mediating FcεRI signaling, and suppresses mast cell production of cytokines. Furthermore, using basophils from a peanut allergic donor we found that IgA binds to basophils and that activation by exposure to peanuts is effectively suppressed by IgA. We conclude that IgA serves as a regulator of mast cell and basophil degranulation, suggesting a physiologic role for IgA in the maintenance of immune homeostasis at mucosal sites.

Immunological mechanisms involved in the human response to a dog dander allergoid

BackgroundDog allergens are a common cause of allergic sensitisation and trigger respiratory symptoms worldwide. However, clinical evidence regarding dog immunotherapy is limited. Therefore, the aim of this study was to analyse the immunomodulatory properties of a new allergoid from dog dander, thereby deepening the understanding of the molecular mechanisms involved in the reestablishment of the tolerogenic response. MethodsThree independent batches of dog dander native and allergoid allergen extracts were manufactured and characterised. Allergenic profiles were analysed by the identification of all dog allergens and quantification of the major allergens Can f 1 and Can f 5. The allergenicity profile of the allergoid was studied using biological potency and basophil activation tests. In vitro immunomodulatory parameters was evaluated as the capacity of the allergoid to induce IgG antibodies that block IgE binding to the allergen and cytokine promotion (IFN-γ, IL-4, IL-6, IL-10, IL-13, and TNF-α) in PBMCs from allergic donors. ResultsThe presence of all dog allergens, including Can f 1 and Can f 5, was confirmed in both types of extracts. The new allergoid showed a low IgE binding capacity, which significantly affected the activation of effector cells, such as basophils. The IgG antibodies induced by the allergoid in rabbits blocked human IgE binding epitopes on the dog native extract and induced Th1 and Treg responses by increasing IFN-γ and IL-10 levels in PBMCs from allergic donors. ConclusionThis new dog dander allergoid containing Can f 1 and Can f 5 showed a low capacity to bind IgE and to activate basophils in dog allergic patients. Furthermore, it showed potent activation of Th1 mediators and induction of tolerance through Treg activation. This allergoid could offer a safer profile than the native extract and could be an effective immunotherapy treatment for dog allergic patients.

Blood from Highly Allergic Donors Yields High-Affinity IgE Antibodies to Food and Non-food Allergens

Individuals with severe forms of allergic disease have evolved potent IgE responses to otherwise innocuous antigens. The B cells that produce these IgE antibodies are rare, however, and the technical challenge of isolating them has impeded progress toward a molecular understanding of allergen recognition and slowed the pace of therapeutic development.

Distinction between peanut allergy and tolerance by characterization of B cell receptor repertoires.

BackgroundSpecific IgE against a peanut 2S albumin (Ara h 2 or 6) is the best predictor of clinically relevant peanut sensitization. However, sIgE levels of peanut allergic and those of peanut sensitized but tolerant patients partly overlap, highlighting the need for improved diagnostics to prevent incorrect diagnosis and consequently unnecessary food restrictions. Thus, we sought to explore differences in V(D)J gene transcripts coding for peanut 2S albumin‐specific monoclonal antibodies (mAbs) from allergic and sensitized but tolerant donors.Methods2S albumin‐binding B‐cells were single‐cell sorted from peripheral blood of peanut allergic (n=6) and tolerant (n=6) donors sensitized to Ara h2 and/or 6 (≥ 0.1 kU/l) and non‐atopic controls (n=5). h 2 and/or 6 (≥ 0.1 kU/l). Corresponding h heavy and light chain gene transcripts were heterologously expressed as mAbs and tested for specificity to native Ara h2 and 6. HCDR3 sequence motifs were identified by Levenshtein distances and hierarchically clustering.ResultsThe frequency of 2S albumin‐binding B cells was increased in allergic (median: 0.01%) compared to tolerant (median: 0.006%) and non‐atopic donors (median: 0.0015%, p = 0.008). The majority of mAbs (74%, 29/39) bound specifically to Ara h 2 and/or 6. Non‐specific mAbs (9/10) were mainly derived from non‐atopic controls. In allergic donors, 89% of heavy chain gene transcripts consisted of VH3 family genes, compared with only 54% in sensitized but tolerant and 63% of non‐atopic donors. Additionally, certain HCDR3 sequence motifs were associated with allergy (n = 4) or tolerance (n = 3) upon hierarchical clustering of their Levenshtein distances.ConclusionsPeanut allergy is associated with dominant VH3 family gene usage and certain public antibody sequences (HCDR3 motifs).

Functional characterization of gamma-delta (γδ) T cells in allergy

Abstract Despite a growing consensus on the involvement of γδ T cells in allergy and other human immunological disorders, the detailed mechanisms remain hypothetical due to lack of investigative tools. Herein, we sought to develop functional assays to study the role of γδ T cells in allergy. Using an Activation-Induced Marker (AIM) assay, based on the upregulation of 4-1BB (CD137) and CD69, we were able to detect ex vivo allergen-specific responses from γδ T cells to multiple allergen extracts in human PBMCs, including mouse, cockroach (CR), house dust mite (HDM) and timothy grass (TG) allergens, which were reproducible and observed in multiple allergic cohorts. The magnitude of allergen-reactive γδ T cells differed between allergic and non-allergic cohorts with HDM and TG allergy, but not mouse or CR allergy. Further characterization showed that the γδ T cells reactive to mouse allergen were mostly the Vδ2 subset and from effector memory (Tem and Temra) compartments. Interestingly, in mouse-specific allergic donors we found differential polarized responses for γδ and ab T cells, with a Th1 and Th2 pattern, respectively. Using a complementary AIM assay for CD40L (CD154) combined with intracellular staining (ICS), we observed that Th1 polarization (INFg and TNFa) in responses to mouse extract was also common in non-allergic donors. Conversely, In the case of CR responses, Th1 polarization of Vδ2 T cells was only observed in the allergic cohort. Overall, these results suggest that allergen-specific Vδ2 T cells are potential to skew dysregulated cytokine responses in immunotherapy. In conclusion, we developed new functional assays that serve as tools for in-depth characterization and deciphering the roles of γδ T cells in allergy.

Natural Killer Cells from Allergic Donors Are Defective in Their Response to CCL18 Chemokine.

Natural killer (NK) cells were originally described as cytolytic effector cells, but since then have been recognized to possess regulatory functions on immune responses. Chemokines locate NK cells throughout the body in homeostatic and pathological conditions. They may also directly stimulate immune cells. CCL18 is a constitutive and inducible chemokine involved in allergic diseases. The aim of this study was to evaluate CCL18’s effect on NK cells from allergic and nonallergic donors in terms of both chemotactic and immune effects. Results showed that CCL18 was able to induce migration of NK cells from nonallergic donors in a G-protein-dependent manner, suggesting the involvement of a classical chemokine receptor from the family of seven-transmembrane domain G-protein-coupled receptors. In contrast, NK cells from allergic patients were unresponsive. Similarly, CCL18 was able to induce NK cell cytotoxicity only in nonallergic subjects. Purified NK cells did not express CCR8, one of the receptors described to be involved in CCL18 functions. Finally, the defect in CCL18 response by NK cells from allergic patients was unrelated to a defect in CCL18 binding to NK cells. Overall, our results suggest that some NK cell functions may be defective in allergic diseases.

IL-1β promotes immunoregulatory responses in human blood basophils.

Human basophils are essential effector cells of chronic allergic inflammation. IL-1 family cytokines such as interleukin (IL)-33 and IL-1β are elevated in serum and bronchoalveolar lavage fluid of allergic asthmatics. IL-33 is known to be a critical regulator of basophil's T2 immune responses. However, the effect of IL-1β on the function of basophils has not been well investigated. Here, we elucidate whether IL-1β regulates the function of human basophils and compared the effects of IL-1β and IL-33 on basophils of healthy and allergic subjects. We found that IL-1β activates the p38 MAPK signaling pathway and promotes IL-8 release in basophils of healthy donors, while FcεRI-mediated LCT4 and histamine secretion is not affected. Strikingly, in the presence of IL-3, IL-1β shows more potency than IL-33, as evidenced by the enhanced p38 phosphorylation and NF-κB activation, as well as the release of both IL-13 and IL-8. We found that the enhanced basophil responsiveness is achieved through IL-3-induced IL-1RI surface expression. Importantly, basophils of allergic donors release significantly higher amounts of IL-8 compared to those from healthy donors upon IL-33 and IL-1β stimulation. Consistently, we detected increased IL-1RI and decreased IL-3 receptor alpha-chain (CD123) and CCR3 expression on basophils of allergic subjects compared to healthy controls, suggesting an in vivo IL-3 priming in allergic donors. In summary, our results suggest enhanced sensitivity of basophils toward IL-33 and IL-1β in allergic subjects compared to those from healthy controls.

A Report of Fourcases of Severethrombocytopenia with Rh G-CSF Mobilized Allogeneic Peripheral Blood Stem Cell Transplant Donors

Background and Objective: Granulocyte colony-stimulating factor (G-CSF) is used to mobilize progenitor cells into the peripheral bloodin healthy donors. Thrombocytopenia is one of common side effects of G-CSFmobilization, but seriousthrombocytopenia is rare. Herein,we report serious thrombocytopeniain 4 allogeneic peripheral blood stem cell transplantation (Allo-PBSCT) healthy donors induced by recombinant human G-CSF (rhG-CSF) mobilization. Methods: The donor received 10ug/kg/d G-CSF via subcutaneous injection.The blood parameterswere examined every day, Abdominal ultrasound detected the spleen size before, during, and after rhG-CSF mobilization. Results: From April 2015 to May 2020, 1127 donors received hematopoietic stem cell mobilization, the percentage of serious thrombocytopenia in healthy donors was 0.35%.The count of white blood cell increased to the 4-7 timesbasic level of (35.4x109/L)after rhG-CSF mobilization 4 days, the lowestcount ofplatelet was 22x109/Lafter single postharvest. After mobilization, length of spleen increased 0.6-1.3 cm (median 10.1 cm), thickness ofspleenincreased 0-1.3 cm (median 3.4 cm). There were no obvious bleeding symptomsafter mobilization in four cases of donor.The quality of stem cell collection was relatively poor in 3 of the 4 donors, the number of mononuclear cells (MNC) was 7.47-18.58×108/kg, and the value of CD34+ was 0.82-2.42×106/kg (CD34+ proportion: 0.11-0.16%). The quality of stem cell collection in the other 1 donor was satisfactory, the number of MNC was 9.29-11.75×108/kg, and the proportion of CD34+ was 0.26~0.38%. Platelet count of 4 donors recovered to the basal level 3-10 days after drug withdrawal. Conclusion:The severe thrombocytopenia caused by rhG-CSF mobilization healthy donor is very rare.while, the mechanismis unknown. Some studies suggested that it may be related to the insufficient expression of megakaryocytes proliferation-related genes, such as PF4 and PTFN4. Other studies indicated that rhG-CSF may aggravate the immune-mediated thrombocytopenia, which leads to hypersplenism and extensive activation of reticuloendothelial cells, thus increasing the consumption of platelets.In addition, the probability of severe thrombocytopenia was increased in allergic donors after rhG-CSF mobilization.In our report, although there was thrombocytopenia, mobilization was not affected.After the mobilization of rhG-CSF, platelets changes should be closely monitored to keep alert of donor bleeding tendency. While ensuring safe collection of hematopoietic stem cells by donors, patients should not be placed in extreme danger of not being able to receive allogeneic hematopoietic stem cells on time. Key words:Severe thrombocytopenia;Healthy donor; rhG-CSF; Mobilization; Allogeneic peripheral blood stem cell transplantation. Disclosures No relevant conflicts of interest to declare.

Development of a protein microarray-based diagnostic chip mimicking the skin prick test for allergy diagnosis

Protein microarrays have been successfully used for detection of allergen-specific IgE in patient sera. Here, we demonstrate proof-of-concept of a solid-phase technique coupling the high-throughput potential of protein microarrays with the biologically relevant readout provided by IgE reporter cells, creating a novel allergic sensitization detection system. Three proteins (κ-casein, timothy grass pollen extract, polyclonal anti-human IgE) were printed onto three different polymer-coated surfaces (aldehyde-, epoxy- and NHS ester-coated). ToF–SIMs analysis was performed to assess printed protein stability and retention during washing steps. NFAT-DsRed rat basophil leukemia cell attachment and retention during washing steps was assessed after treatment with various extracellular matrix proteins. NFAT-DsRed IgE reporter cells were sensitized with serum of an allergic donor, incubated on the printed slides, and cell activation determined using a microarray laser scanner. NFAT DsRed IgE reporter cell binding was significantly increased on all polymer surfaces after incubation with fibronectin and vitronectin, but not collagen or laminin. All surfaces supported printed protein stability during washing procedure, with epoxy- and NHS ester-coated surfaces showing best protein retention. Cell activation was significantly higher in NHS ester-coated slides after timothy grass pollen extract stimulation appearing a suitable substrate for further development of an automated allergy diagnosis system.

Structural Aspects of the Allergen-Antibody Interaction.

The development of allergic disease involves the production of IgE antibodies upon allergen exposure in a process called sensitization. IgE binds to receptors on the surface of mast cells and basophils, and subsequent allergen exposure leads to cross-linking of IgE antibodies and release of cell mediators that cause allergy symptoms. Although this process is quite well-understood, very little is known about the epitopes on the allergen recognized by IgE, despite the importance of the allergen-antibody interaction for the allergic response to occur. This review discusses efforts to analyze allergen-antibody interactions, from the original epitope mapping studies using linear peptides or recombinant allergen fragments, to more sophisticated technologies, such as X-ray crystallography and nuclear magnetic resonance. These state-of-the-art approaches, combined with site-directed mutagenesis, have led to the identification of conformational IgE epitopes. The first structures of an allergen (egg lysozyme) in complex with Fab fragments from IgG antibodies were determined in the 1980s. Since then, IgG has been used as surrogate for IgE, due to the difficulty of obtaining monoclonal IgE antibodies. Technical developments including phage display libraries have contributed to progress in epitope mapping thanks to the isolation of IgE antibody constructs from combinatorial libraries made from peripheral blood mononuclear cells of allergic donors. Most recently, single B cell antibody sequencing and human hybridomas are new breakthrough technologies for finally obtaining human IgE monoclonal antibodies, ideal for epitope mapping. The information on antigenic determinants will facilitate the design of hypoallergens for immunotherapy and the investigation of the fundamental mechanisms of the IgE response.

TCRs with segment TRAV9-2 or a CDR3 histidine are overrepresented among nickel-specific CD4+ T cells.

Nickel is the most frequent cause of T cell-mediated allergic contact dermatitis worldwide. In vitro, CD4+ T cells from all donors respond to nickel but the involved αβ Tcell receptor (TCR) repertoire has not been comprehensively analyzed. We introduce CD154 (CD40L) upregulation as a fast, unbiased, and quantitative method to detect nickel-specific CD4+ T cells ex vivo in blood of clinically characterized allergic and non allergic donors. Naïve (CCR7+ CD45RA+) and memory (not naïve) CD154+ CD4+ Tcells were analyzed by flow cytometry after 5hours of stimulation with 200µmol/L NiSO4 ., TCR α- and β-chains of sorted nickel-specific and control cells were studied by high-throughput sequencing. Stimulation of PBMCs with NiSO4 induced CD154 expression on ~0.1% (mean) of naïve and memory CD4+ T cells. In allergic donors with recent positive patch test, memory frequencies further increased ~13-fold and were associated with markers of in vivo activation. CD154 expression was TCR-mediated since single clones could be specifically restimulated. Among nickel-specific CD4+ T cells of allergic and non allergic donors, TCRs expressing the α-chain segment TRAV9-2 or a histidine in their α- or β-chain complementarity determining region 3 (CDR3) were highly overrepresented. Induced CD154 expression represents a reliable method to study nickel-specific CD4+ T cells. TCRs with particular features respond in all donors, while strongly increased blood frequencies indicate nickel allergy for some donors. Our approach may be extended to other contact allergens for the further development of diagnostic and predictive in vitro tests.

Streamlining basophil activation testing to enable assay miniaturization and automation of sample preparation

BackgroundNumerous studies have demonstrated the capabilities of the basophil activation test (BAT) but various parameters such as a lack of standardization and a time consuming and labor intensive workflow continue to hinder the field to fully leverage the capabilities of this technique. When pediatric patients have to be considered, an additional limitation is related to blood volume consumption. ObjectivesThis work aimed at developing and characterizing a simplified and standardized whole-blood based BAT prototype procedure and at further assessing the feasibility of automating and miniaturizing the developed assay into a 96 well plate format. MethodsA dry and room temperature stable reagent technology was used to simplify and standardize BAT. Under optimized conditions, EDTA anticoagulated whole blood samples of non-allergic and allergic donors (<24 h old) together with calcium containing buffer were added to ready-to-use dry reagent tubes or 96 well plates (negative controls, positive controls and allergen tests) containing a 5 color compensation-free antibody panel (CD45-KrO/CD3-PC7/CRTH2-A647/CD203c-PE/CD63-PB). Upon mixing and incubation at 37 °C for 15 min, erythrocytes were lysed and samples were analyzed by flow cytometry without further washing steps. While it is important to precisely control the incubation time to minimize the assay variability, herein, a 15 min incubation time was chosen as it provides a suitable compromise for both the magnitude of basophil activation and the quality of the staining. A Biomek NXP robotic platform (Beckman Coulter) was used for automation and both CD203c and CD63 levels were monitored to characterize basophil reactivity. ResultsThis streamlined BAT protocol is no-wash, compensation free and only requires 4 pipetting steps to be completed. The assessment of assay performance characteristics showed wide applicability, satisfactory repeatability and a high degree of standardization as demonstrated by very low intra-assay and inter-operator variabilities (CVs < 10%). Leveraging these technical foundations, it was then proven that this new BAT procedure can easily be transposed into the 96 well plate format, thereby benefiting from a miniaturized format and full automation capabilities. When considering 8 dilution points to characterize the ex vivo basophil reactivity of a given whole blood sample, we found that as little as 5 μL of blood per point could be used. ConclusionsA whole blood based and simplified procedure for BAT is proposed. It relies on a dry antibody formulation technology and requires only a few manual steps to be completed. This procedure can also be transposed in a 96 well plate format, fully automated and miniaturized, when sample volume reduction, throughput increase or unattended sample preparation is required.