Sequential engagement of adhesion molecules and cytokine receptors impacts both piecemeal and anaphylactic degranulation of human basophils.

Basophils are rare granulocytes in circulation which home to tissues in a process depending on rolling, adhesion and cytokine exposure. However, it is still unclear how these steps affect basophil degranulation. Our aim was to imitate these processes associated with homing by sequential crosslinking of adhesion molecules and cytokine exposure and evaluate the effect on basophil piecemeal (PMD) and anaphylactic degranulation (AND). Blood donors with or without allergic asthma were recruited from an ongoing cohort study. Basophils were subjected to CD62L-, CD49d- or CD11b crosslinking and IL-3 or IL-33 stimulation in different orders followed by anti-IgE and fMLP stimulation. Basophil CD203c and CD63 expression were analysed by flow cytometry to determine PMD and AND, respectively. IL-3 induced PMD in basophils and combined with CD62L- or CD11b crosslinking, IL-3 potentiated the degranulation regardless of sequential order. IL-3 priming followed by adhesion molecule crosslinking induced AND and potentiated the effect of anti-IgE. CD62L- and CD11b crosslinking did not further potentiate this effect. CD49d crosslinking followed by IL-3 increased CD63 expression following anti-IgE. IL-3 potentiated the effect of fMLP on AND while adhesion molecule crosslinking did not. IL-33 had impact on PMD only when followed by adhesion molecule crosslinking but did not potentiate neither IgE-dependent nor IgE-independent degranulation. Our data indicate that sequential interactions between basophils, cytokines and adhesion molecule ligands have a decisive effect on basophil degranulation and that these interactions are operational for fine-tuning the activity of tissue dwelling basophils. These data should be considered when the effect of different pharmaceutical on basophil function is studied.

Allergy in Young Adults Associates with Elevated Fractional Exhaled Nitric Oxide Levels and IgE-Verified Parental Allergy but Is Confounded by Self-Reported Symptoms.

INTRODUCTION: Knowledge of IgE-verified allergy in young adults is limited as most studies are based on self-reported data. Allergic heredity is important in allergy development in early life, but less is known about the hereditary component later in life. The aim was to investigate IgE-verified and self-reported allergy and asthma at 20 years of age in association to parental allergy and environmental factors. METHODS: In total, 281 individuals born into the cohort of well-characterized parents regarding allergic disease were followed to 20 years of age. The participants were categorized by parental allergy and examined regarding allergic diseases (IgE sensitization and allergic symptoms) at 2, 5, 10, and 20 years of age. FeNO was measured at 10 and 20 years. RESULTS: In total, 45% of the study participants were allergic, with twice as many self-reported cases at age 20. Rhinitis was key to distinguishing confirmed allergy from self-reported. Having two allergic parents and increased FeNO were associated with an increased prevalence of allergic disease at 20 years. From a longitudinal perspective, rhinitis increased from childhood to young adulthood, in all heredity groups. CONCLUSION: In this longitudinal study, we have shown that two allergic parents as well as increased FeNO levels seem to be of importance for being allergic at 20 years old. Self-reported allergy was overreported - a result that should be considered in future survey-based reports on allergic diseases.

Adhesion molecule cross-linking and cytokine exposure modulate IgE- and non-IgE-dependent basophil activation.

Basophils are known for their role in allergic inflammation, which makes them suitable targets in allergy diagnostics such as the basophil activation test (BAT) and the microfluidic immunoaffinity basophil activation test (miBAT). Beside their role in allergy, basophils have an immune modulatory role in both innate immunity and adaptive immunity. To accomplish this mission, basophils depend on the capability to migrate from blood to extravascular tissues, which includes interactions with endothelial cells, extracellular matrix and soluble mediators. Their receptor repertoire is well known, but less is known how these receptor-ligand interactions impact the degranulation process and the responsiveness to subsequent activation. As the consequences of these interactions are crucial to fully appreciate the role of basophils in immune modulation and to enable optimization of the miBAT, we explored how basophil activation status is regulated by cytokines and cross-linking of adhesion molecules. The expression of adhesion molecules and activation markers on basophils from healthy blood donors was analysed by flow cytometry. Cross-linking of CD203c, CD62L, CD11b and CD49d induced a significant upregulation of CD63 and CD203c. To mimic in vivo conditions, valid also for miBAT, CD62L and CD49d were cross-linked followed by IgE-dependent activation (anti-IgE), which caused a reduced CD63 expression compared with anti-IgE activation only. IL-3 and IL-33 priming caused increased CD63 expression after IgE-independent activation (fMLP). Together, our data suggest that mechanisms operational both in the microfluidic chip and in vivo during basophil adhesion may impact basophil anaphylactic and piecemeal degranulation procedures and hence their immune regulatory function.

Peanuts in the air - clinical and experimental studies.

BACKGROUND: Allergic reactions to food allergens usually occur after ingestion. However, fear of reactions to airborne peanut is a common concern for people with peanut allergy. There are no scientific reports on severe reactions with airborne peanut allergen. OBJECTIVE: To investigate the occurrence of allergic reactions in peanut-allergic children undergoing airborne peanut challenge and to determine levels of airborne peanut protein in a separate experimental evaluation. METHODS: Eighty-four children with peanut allergy underwent an airborne peanut challenge, 0.5 m from a bowl of peanuts for 30 min under controlled conditions. In a separate experiment, airborne peanut proteins from roasted and dry-roasted peanuts were collected at varying distances and at varying times with an electret SensAbues filter connected to an air pump. Collected airborne peanut proteins were extracted, dissolved and detected by ELISA. Basophil activation test was used to confirm biological activity. RESULTS: No moderate/severe allergic reactions to airborne peanut allergens were observed. Two children (2%) had mild rhino-conjunctivitis which required no treatment. The IgE-antibodies to peanut or Ara h 2 did not predict a reaction. In the experimental set-up, biological active peanut proteins were detected, in a very low amount, in median 166 ng/ml for dry-roasted and 33 ng/ml for roasted peanuts and decreased dramatically when the collection occurred at a greater distance (0.5-2 m) from the peanut source. Increased exposure time did affect the amount of collected peanut protein at 0 m, and the highest median was obtained after 60 min (p = .012); for time trend p = .0006. CONCLUSIONS AND CLINICAL RELEVANCE: Allergic reactions to airborne peanut proteins are rare and cannot be predicted by high levels of IgE-antibodies to peanut or Ara h 2. Only small amounts of biologically active peanut proteins were detected in the air and seem unlikely to trigger moderate/severe allergic reactions.

A pilot study towards the immunological effects of omalizumab treatment used to facilitate oral immunotherapy in peanut-allergic adolescents.

Anti-IgE treatments, such as omalizumab, have shown promising effects in allergy treatment. Our previous work has shown that individualized omalizumab treatment (OT) allows a safe initiation and rapid up-dosing of peanut oral immunotherapy (OIT) in peanut-allergic adolescents. However, the broader immunological effects of this OT are incompletely understood. In this pilot study, we longitudinally followed the total B- and T-cell immunity during OT, using flow cytometry, ELISpot and ELISA. Peripheral blood mononuclear cells (PBMCs) and plasma were collected from participants (n = 17) at several timepoints during treatment, before starting OT (baseline), prior to starting OIT during OT (start OIT) and at maintenance dose OIT prior to OT reduction (maintenance). OT did not affect the total B-cell compartment over treatment time, but our results suggest an association between the OT dosage scheme and the B-cell compartment. Further, in vitro polyclonal T-cell activation at the different timepoints suggests a cytokine skewing towards the Th1 phenotype at the expense of Th2- and Th9-related cytokines during treatment. No differences in the frequencies or phenotype of regulatory T cells (Tregs) over treatment time were observed. Finally, plasma chemokine levels were stable over treatment time, but suggest elevated gut homing immune responses in treatment successes during the treatment as compared to treatment failures. The novel and explorative results of this pilot study help to improve our understanding on the immunological effects of OT used to facilitate OIT and provide guidance for future immunological investigation in large clinical trials.

A novel tool for clinical diagnosis of allergy operating a microfluidic immunoaffinity basophil activation test technique.

The Basophil Activation Test (BAT) is a valuable allergy diagnostic tool but is time-consuming and requires skilled personnel and cumbersome processing, which has limited its clinical use. We therefore investigated if a microfluidic immunoaffinity BAT (miBAT) technique can be a reliable diagnostic method. Blood was collected from allergic patients and healthy controls. Basophils were challenged with negative control, positive control (anti-FcεRI), and two concentrations of a relevant and non-relevant allergen. CD203c and CD63 expression was detected by fluorescent microscopy and flow cytometry. In basophils from allergic patients the CD63% was significantly higher after allergen activation as compared to the negative control (p<.0001-p=.0004). Activation with non-relevant allergen showed equivalent CD63% expression as the negative control. Further, the miBAT data were comparable to flow cytometry. Our results demonstrate the capacity of the miBAT technology to measure different degrees of basophil allergen activation by quantifying the CD63% expression on captured basophils.

Individually dosed omalizumab facilitates peanut oral immunotherapy in peanut allergic adolescents.

BACKGROUND: Peanut oral immunotherapy (pOIT) has showed good short-term outcomes, but allergic reactions may prevent effective up-dosing and is a major cause of stopping OIT. In placebo-controlled trials, omalizumab has been shown to facilitate allergen immunotherapy and increase tolerance to peanut. OBJECTIVE: We hypothesized that by combining omalizumab with pOIT, and monitor treatment effects with basophil allergen threshold sensitivity tests (CD-sens), peanut allergic patients could safely initiate pOIT and thereafter slowly withdraw omalizumab. METHODS: This is the 2nd part of a one-armed open phase-2 study where peanut allergic adolescents (n = 23) started pOIT after an individualized omalizumab treatment. The pOIT dose was increased from 280 to 2800 mg peanut protein in 8 weeks followed by an individualized step-wise withdrawal of omalizumab, based on clinical symptoms and CD-sens levels. pOIT continued for 12 weeks followed by an open peanut challenge. Peanut CD-sens and allergen-binding activity (ABA) and IgE-ab, IgG-ab and IgG4-ab to peanut and its components were measured during the study. RESULTS: All 23 patients successfully reached the 2800 mg maintenance dose. Moderate/systemic allergic reactions were rare while receiving full-dose omalizumab. Eleven of 23 (48%) successfully continued with pOIT after omalizumab was stopped. Compared to treatment failures, median baseline IgE-ab to peanut components Ara h 1-3 and CD-sens to peanut were significantly lower among successfully treated patients and IgG4-ab to peanut, Ara h 2 and 6 increased significantly more during treatment. CONCLUSIONS AND CLINICAL RELEVANCE: This study indicates that omalizumab is an effective adjunctive therapy for initiation and rapid up-dosing of pOIT; however, adverse events from pOIT become more frequent as omalizumab doses are decreased. CLINICAL TRIALS REGISTRATION: ClinicalTrials.gov; NCT02402231. EudraCT; 2012-005625-78.

Altered basophil function in patients with chronic kidney disease on hemodialysis
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AIMS: Chronic kidney disease (CKD) leads to impairment of immune cell function. Given the potential role of basophils in the pathogenesis of CKD, we aimed to study the basophil responsiveness towards microbial antigen exposure, judged as adhesion molecule expression and degranulation, in CKD patients on hemodialysis. MATERIALS AND METHODS: We selected markers linked to two crucial biological phases: the transmigration and degranulation processes, respectively. For the transmigration process, we selected the adhesion molecules CD11b, active CD11b epitope, and CD62L and for the degranulation process CD203c (piecemeal degranulation marker), CD63 (degranulation marker), and CD300a (inhibitory marker of degranulation). We measured basophil responsiveness after stimulation of different activation pathways in basophils using lipopolysaccharide (LPS), peptidoglycan (PGN), formyl-methyinoyl-leucyl-phenylalanine (fMLP), and anti-FcεRI-ab. RESULTS: The expression of CD63 in basophils following activation by fMLP was significantly higher in the patient group compared to matched healthy controls, but no differences were observed after activation by anti-FcɛI. CD300a expression was significantly higher in patients following activation by fMLP and anti-FcɛI, and the active epitope CD11b expression was significantly higher in patients after LPS activation. In addition, we found that CD62L was not shed from the cell surface after activation with LPS and fMLP. A slight downregulation was noted after activation with anti-FcɛI in healthy controls. CONCLUSION: Together, these data demonstrate that basophil functions related to adhesion and degranulation are altered in CKD patients on hemodialysis, which indicates a potential role for the basophil in the pathogenesis of complications related to infections.

Pros and Cons of Clinical Basophil Testing (BAT).

PURPOSE OF REVIEW: We review basophil testing by flow cytometry with an emphasis on advantages and disadvantages. RECENT FINDINGS: There are many tools available to assess the presence and severity of allergic diseases in patients. For 50 years, peripheral blood basophils have been used as tools to study these diseases. It is a very accessible cell that binds IgE antibody and secretes the classical mediators responsible for the symptoms of allergic reactions. In the last decade, an even more accessible methodology, using flow cytometry, has been developed to enhance the ability to use basophils for both mechanistic and clinical diagnostics. Basophil testing has been included in diagnostics for different forms of allergies as well as to monitor disease status. A variety of studies have begun to establish both precise methods and their clinical relevance for disease diagnosis, but there remain some important questions on how to take optimal advantage of the behaviours of basophils.

Evaluation of basophil allergen threshold sensitivity (CD-sens) to peanut and Ara h 8 in children IgE-sensitized to Ara h 8.

BACKGROUND: Diagnosing peanut allergy properly is important and can be achieved by combining clinical history with various diagnostic methods such as IgE-antibody (IgE-ab) measurements, skin-prick test, basophil allergen threshold sensitivity (CD-sens) and food challenge. We aimed to evaluate CD-sens to peanut, Ara h 8 and Gly m 4 in relation to an oral peanut challenge in children IgE-sensitized to birch, peanut and Ara h 8 avoiding peanuts. METHODS: Twenty children IgE-sensitized to birch pollen and Ara h 8, but not to Ara h 1, Ara h 2 or Ara h 3 were challenged orally with roasted peanuts. Blood samples were drawn for IgE-ab and CD-sens analysis. To measure CD-sens, basophils were stimulated in vitro with decreasing doses of allergens until threshold sensitivity was reached. RESULTS: All children passed challenge without objective symptoms, but mild oral allergy syndrome (OAS) symptoms were reported in 6/20 children. Nineteen of twenty children were negative in CD-sens to peanut but 17/20 were positive to rAra h 8. Eleven of twenty children were positive in CD-sens to rGly m 4. CONCLUSION: Positive CD-sens to rAra h 8 show that the Ara h 8 IgE-ab sensitized basophils can be activated by a rAra h 8 allergen and initiate an allergic inflammation despite a negative challenge. Hence, children sensitized to Ara h 8 but not to peanut storage proteins may be at risk for systemic allergic reaction when eating larger amounts of peanuts but most likely don't have to fear smaller amounts.

Activation of basophils is a new and sensitive marker of biocompatibility in hemodialysis.

The hemodialysis procedure involves contact between peripheral blood and the surface of dialyzer membranes, which may lead to alterations in the pathways of innate and adaptive immunity. We aimed to study the effect of blood-membrane interaction on human peripheral basophils and neutrophils in hemodialysis with high- and low-permeability polysulfone dialyzers. The surface expression of CD203c (basophil selection marker) and CD63 (activation marker) after activation by the bacterial peptide formyl-methionyl-leucyl-phenylalanine (fMLP) or anti-Fcε receptor I (FcεRI) antibody and the absolute number of basophils was investigated before and after hemodialysis with each of the dialyzers. Moreover, the expression on neutrophils of CD11b, the CD11b active epitope, and CD88 was analyzed in the same groups of individuals. The expression of CD63 in basophils following activation by fMLP was significantly higher in the patient group compared with that in healthy controls, but no differences were observed after activation by anti-FcεRI. During the hemodialysis procedure, the low-flux membrane induced up-regulation of CD63 expression on basophils, while passage through the high-flux membrane did not significantly alter the responsiveness. In addition, the absolute number of basophils was unchanged after hemodialysis with either of the dialyzers and compared with healthy controls. We found no significant differences in the expression of the neutrophil activation markers (CD11b, the active epitope of CD11b, and CD88) comparing the two different dialyzers before and after dialysis and healthy controls. Together, these findings suggest that alterations in basophil activity may be a useful marker of membrane bioincompatibility in hemodialysis.

Basophil allergen threshold sensitivity to casein (casein-specific CD-sens) predicts allergic reactions at a milk challenge in most but not all patients.

BACKGROUND: The basophil activation test is an emerging clinical tool in the diagnosis of cow's milk allergy (CMA). The aim was to assess the association between the basophil allergen threshold sensitivity to the major milk protein casein (casein-specific CD-sens), the levels of milk- and casein-specific Immunoglobulin E antibodies (IgE-ab), and the severity of allergic reactions at milk challenges. METHODS: We enrolled 34 patients aged 5-15 (median 9) years who underwent a double-blind placebo-controlled milk-challenge (DBPCMC) as screening before inclusion in an oral immunotherapy study for CMA. The severity of the allergic reaction at the DBPCMC was graded using Sampson's severity score. Venous blood was drawn before the DBPCMC. Milk- and casein-specific IgE-ab were analyzed. Following in vitro stimulation of basophils with casein, casein-specific CD-sens, was determined. RESULTS: Thirty-three patients completed the DBPCMC. There were strong correlations between casein-specific CD-sens and IgE-ab to milk (rs = 0.682, p < .001), and between casein-specific CD-sens and IgE-ab to casein (rs = 0.823, p < .001). There was a correlation between the severity of the allergic reaction and casein-specific CD-sens level (rs = 0.395, p = .041) and an inverse correlation between casein-specific CD-sens level and the cumulative dose of milk protein to which the patient reacted at the DBPCMC (rs = -0.418, p = .027). Among the 30 patients with an allergic reaction at the DBPCMC, 67% had positive casein-specific CD-sens, 23% had negative casein-specific CD-sens, and 10% were declared non-responders. CONCLUSION: Two thirds of those reacting at the DBPMC had positive casein-specific CD-sens, but reactions also occurred despite negative casein-specific CD-sens. The association between casein-specific CD-sens and the severity of the allergic reaction and cumulative dose of milk protein, respectively, was moderate.

High basophil allergen sensitivity (CD-sens) is associated with severe allergic asthma in children.

Children with problematic severe asthma (PA) have persistent symptoms and/or severe exacerbations despite treatment with several drugs. Classification of asthma severity is currently based on level of treatment and assessment of asthma control, but objective biomarkers of asthma severity are needed. To investigate the clinical relevance of basophil allergen threshold sensitivity (CD-sens) as a measure of allergen sensitivity in a well-characterized cohort of children with different manifestations of persistent allergic asthma. Cat-allergic children (6-18 yr) with problematic severe asthma (n = 11) according to GINA were compared with eleven age-matched children with controlled, but persistent asthma (CA). The protocol included standardized questionnaires, asthma control test (ACT), spirometry, methacholine challenges, measurement of FE(NO,) IgE, cat IgE and IgG antibodies, and analysis of CD-sens (CD63-expression) by flow cytometry. The 11 cat-allergic children with PA had a significantly lower ACT score (p < 0.001), reduced FEV(1) (p = 0.04), and increased numbers of blood eosinophils (p = 0.03) compared with the 11 children with CA. The former exhibited a higher CD-sens to cat (p = 0.02). No significant differences were detected with respect to FE(NO) (p = 0.17), IgE (p = 0.84), cat IgE (p = 0.12), and the major cat-allergen rFel d 1 (p = 0.30). CD-sens significantly correlated with ACT (p = 0.002, r = -0.63) and FE(NO) (p = 0.01, r = 0.55). No significant differences between PA and CA were found regarding IgG antibodies to rFel d 1. Cat-allergic children with problematic severe asthma have higher sensitivity to cat allergen, as measured by CD-sens, compared with children with controlled asthma. This suggests that CD-sens could be used as an additional marker for identifying children with the most severe allergic asthma.

B cell and monocyte phenotyping: A quick asset to investigate the immune status in patients with IgA nephropathy.

BACKGROUND: IgA nephropathy (IgAN) advances from multiple pathogenic "hits" resulting in poorly O-galactosylated IgA1 glycoforms (Gd-IgA1), production of antibodies and glomerular deposition of immune complexes. A sequence of immune responses arising from plasma cells, T cells and antigen presenting cells (APCs), causes glomerular injury. This study was designed to phenotype subsets of B cells, monocytes and T cells in the peripheral circulation and their association with inflammatory cytokines and kidney function in patients with IgAN, healthy controls (HC) and disease controls with autosomal dominant polycystic kidney disease (ADPKD). METHODS: Patients with IgAN (n = 13), median estimated glomerular filtration rate (eGFR) of 57 ml/min/1.73m2 (IQR 42-84), patients with ADPKD (n = 13) matched for kidney function, gender and age and gender and age-matched HC (n = 13) were recruited. CD3+ and CD3- peripheral blood mononuclear cells were isolated and profiled based on their specific surface markers for different subsets of monocytes, B and T cells and analyzed by flow cytometry. Cytokines were analyzed by ELISA. RESULTS: We observed a significant decrease in the proportion of pre-switched B cells and plasmablasts, but an increase in long-lived plasma cells in the peripheral circulation of IgAN patients compared to HC. The proportion of non-classical monocytes was significantly higher in IgAN patients compared to both HC and ADPKD. We also report an association between sCD40L levels and the proportion of pre-switched B cells, as well as sCD40L and MCP-1 levels and albuminuria in IgAN patients. CONCLUSIONS: We applied an easy-access method to analyze subsets of immune cells as well as relevant inflammatory mediators in IgAN patients. Our data demonstrate an altered B cell profile that indicates a pathophysiological role of the B cell lineage and an increased proportion of non-classical monocytes that suggests their role in the disease process.