Pediatric Allergic Diseases, Food Allergy, and Oral Tolerance.

Pediatric allergic disease is a significant health concern worldwide, and the prevalence of childhood eczema, asthma, allergic rhinitis, and food allergy continues to increase. Evidence to support specific interventions for the prevention of eczema, asthma, and allergic rhinitis is limited, and no consensus on prevention strategies has been reached. Randomized controlled trials investigating the prevention of food allergy via oral tolerance induction and the early introduction of allergenic foods have been successful in reducing peanut and egg allergy prevalence. Infant weaning guidelines in the United Sates were recently amended to actively encourage the introduction of peanut for prevention of peanut allergy.

Effect of Avoidance on Peanut Allergy after Early Peanut Consumption.

BACKGROUND: In a randomized trial, the early introduction of peanuts in infants at high risk for allergy was shown to prevent peanut allergy. In this follow-up study, we investigated whether the rate of peanut allergy remained low after 12 months of peanut avoidance among participants who had consumed peanuts during the primary trial (peanut-consumption group), as compared with those who had avoided peanuts (peanut-avoidance group). METHODS: At the end of the primary trial, we instructed all the participants to avoid peanuts for 12 months. The primary outcome was the percentage of participants with peanut allergy at the end of the 12-month period, when the participants were 72 months of age. RESULTS: We enrolled 556 of 628 eligible participants (88.5%) from the primary trial; 550 participants (98.9%) had complete primary-outcome data. The rate of adherence to avoidance in the follow-up study was high (90.4% in the peanut-avoidance group and 69.3% in the peanut-consumption group). Peanut allergy at 72 months was significantly more prevalent among participants in the peanut-avoidance group than among those in the peanut-consumption group (18.6% [52 of 280 participants] vs. 4.8% [13 of 270], P<0.001). Three new cases of allergy developed in each group, but after 12 months of avoidance there was no significant increase in the prevalence of allergy among participants in the consumption group (3.6% [10 of 274 participants] at 60 months and 4.8% [13 of 270] at 72 months, P=0.25). Fewer participants in the peanut-consumption group than in the peanut-avoidance group had high levels of Ara h2 (a component of peanut protein)-specific IgE and peanut-specific IgE; in addition, participants in the peanut-consumption group continued to have a higher level of peanut-specific IgG4 and a higher peanut-specific IgG4:IgE ratio. CONCLUSIONS: Among children at high risk for allergy in whom peanuts had been introduced in the first year of life and continued until 5 years of age, a 12-month period of peanut avoidance was not associated with an increase in the prevalence of peanut allergy. Longer-term effects are not known. (Funded by the National Institute of Allergy and Infectious Diseases and others; LEAP-On ClinicalTrials.gov number, NCT01366846.).

Randomized trial of peanut consumption in infants at risk for peanut allergy.

BACKGROUND: The prevalence of peanut allergy among children in Western countries has doubled in the past 10 years, and peanut allergy is becoming apparent in Africa and Asia. We evaluated strategies of peanut consumption and avoidance to determine which strategy is most effective in preventing the development of peanut allergy in infants at high risk for the allergy. METHODS: We randomly assigned 640 infants with severe eczema, egg allergy, or both to consume or avoid peanuts until 60 months of age. Participants, who were at least 4 months but younger than 11 months of age at randomization, were assigned to separate study cohorts on the basis of preexisting sensitivity to peanut extract, which was determined with the use of a skin-prick test--one consisting of participants with no measurable wheal after testing and the other consisting of those with a wheal measuring 1 to 4 mm in diameter. The primary outcome, which was assessed independently in each cohort, was the proportion of participants with peanut allergy at 60 months of age. RESULTS: Among the 530 infants in the intention-to-treat population who initially had negative results on the skin-prick test, the prevalence of peanut allergy at 60 months of age was 13.7% in the avoidance group and 1.9% in the consumption group (P<0.001). Among the 98 participants in the intention-to-treat population who initially had positive test results, the prevalence of peanut allergy was 35.3% in the avoidance group and 10.6% in the consumption group (P=0.004). There was no significant between-group difference in the incidence of serious adverse events. Increases in levels of peanut-specific IgG4 antibody occurred predominantly in the consumption group; a greater percentage of participants in the avoidance group had elevated titers of peanut-specific IgE antibody. A larger wheal on the skin-prick test and a lower ratio of peanut-specific IgG4:IgE were associated with peanut allergy. CONCLUSIONS: The early introduction of peanuts significantly decreased the frequency of the development of peanut allergy among children at high risk for this allergy and modulated immune responses to peanuts. (Funded by the National Institute of Allergy and Infectious Diseases and others; ClinicalTrials.gov number, NCT00329784.).

Distinct parameters of the basophil activation test reflect the severity and threshold of allergic reactions to peanut.

BACKGROUND: The management of peanut allergy relies on allergen avoidance and epinephrine autoinjector for rescue treatment in patients at risk of anaphylaxis. Biomarkers of severity and threshold of allergic reactions to peanut could significantly improve the care for patients with peanut allergy. OBJECTIVE: We sought to assess the utility of the basophil activation test (BAT) to predict the severity and threshold of reactivity to peanut during oral food challenges (OFCs). METHODS: The severity of the allergic reaction and the threshold dose during OFCs to peanut were determined. Skin prick tests, measurements of specific IgE to peanut and its components, and BATs to peanut were performed on the day of the challenge. RESULTS: Of the 124 children submitted to OFCs to peanut, 52 (median age, 5 years) reacted with clinical symptoms that ranged from mild oral symptoms to anaphylaxis. Severe reactions occurred in 41% of cases, and 57% reacted to 0.1 g or less of peanut protein. The ratio of the percentage of CD63(+) basophils after stimulation with peanut and after stimulation with anti-IgE (CD63 peanut/anti-IgE) was independently associated with severity (P = .001), whereas the basophil allergen threshold sensitivity CD-sens (1/EC50 × 100, where EC50 is half maximal effective concentration) value was independently associated with the threshold (P = .020) of allergic reactions to peanut during OFCs. Patients with CD63 peanut/anti-IgE levels of 1.3 or greater had an increased risk of severe reactions (relative risk, 3.4; 95% CI, 1.8-6.2). Patients with a CD-sens value of 84 or greater had an increased risk of reacting to 0.1 g or less of peanut protein (relative risk, 1.9; 95% CI, 1.3-2.8). CONCLUSIONS: Basophil reactivity is associated with severity and basophil sensitivity is associated with the threshold of allergic reactions to peanut. CD63 peanut/anti-IgE and CD-sens values can be used to estimate the severity and threshold of allergic reactions during OFCs.

Atopic dermatitis increases the effect of exposure to peanut antigen in dust on peanut sensitization and likely peanut allergy.

BACKGROUND: History and severity of atopic dermatitis (AD) are risk factors for peanut allergy. Recent evidence suggests that children can become sensitized to food allergens through an impaired skin barrier. Household peanut consumption, which correlates strongly with peanut protein levels in household dust, is a risk factor for peanut allergy. OBJECTIVE: We sought to assess whether environmental peanut exposure (EPE) is a risk for peanut sensitization and allergy and whether markers of an impaired skin barrier modify this risk. METHODS: Peanut protein in household dust (in micrograms per gram) was assessed in highly atopic children (age, 3-15 months) recruited to the Consortium of Food Allergy Research Observational Study. History and severity of AD, peanut sensitization, and likely allergy (peanut-specific IgE, ≥5 kUA/mL) were assessed at recruitment into the Consortium of Food Allergy Research study. RESULTS: There was an exposure-response relationship between peanut protein levels in household dust and peanut skin prick test (SPT) sensitization and likely allergy. In the final multivariate model an increase in 4 log2 EPE units increased the odds of peanut SPT sensitization (1.71-fold; 95% CI, 1.13- to 2.59-fold; P = .01) and likely peanut allergy (PA; 2.10-fold; 95% CI, 1.20- to 3.67-fold; P < .01). The effect of EPE on peanut SPT sensitization was augmented in children with a history of AD (OR, 1.97; 95% CI, 1.26-3.09; P < .01) and augmented even further in children with a history of severe AD (OR, 2.41; 95% CI, 1.30-4.47; P < .01); the effect of EPE on PA was also augmented in children with a history of AD (OR, 2.34; 95% CI, 1.31-4.18; P < .01). CONCLUSION: Exposure to peanut antigen in dust through an impaired skin barrier in atopically inflamed skin is a plausible route for peanut SPT sensitization and PA.

IgG4 inhibits peanut-induced basophil and mast cell activation in peanut-tolerant children sensitized to peanut major allergens.

BACKGROUND: Most children with detectable peanut-specific IgE (P-sIgE) are not allergic to peanut. We addressed 2 non-mutually exclusive hypotheses for the discrepancy between allergy and sensitization: (1) differences in P-sIgE levels between children with peanut allergy (PA) and peanut-sensitized but tolerant (PS) children and (2) the presence of an IgE inhibitor, such as peanut-specific IgG4 (P-sIgG4), in PS patients. METHODS: Two hundred twenty-eight children (108 patients with PA, 77 PS patients, and 43 nonsensitized nonallergic subjects) were studied. Levels of specific IgE and IgG4 to peanut and its components were determined. IgE-stripped basophils or a mast cell line were used in passive sensitization activation and inhibition assays. Plasma of PS subjects and patients submitted to peanut oral immunotherapy (POIT) were depleted of IgG4 and retested in inhibition assays. RESULTS: Basophils and mast cells sensitized with plasma from patients with PA but not PS patients showed dose-dependent activation in response to peanut. Levels of sIgE to peanut and its components could only partially explain differences in clinical reactivity between patients with PA and PS patients. P-sIgG4 levels (P = .023) and P-sIgG4/P-sIgE (P < .001), Ara h 1-sIgG4/Ara h 1-sIgE (P = .050), Ara h 2-sIgG4/Ara h 2-sIgE (P = .004), and Ara h 3-sIgG4/Ara h 3-sIgE (P = .016) ratios were greater in PS children compared with those in children with PA. Peanut-induced activation was inhibited in the presence of plasma from PS children with detectable P-sIgG4 levels and POIT but not from nonsensitized nonallergic children. Depletion of IgG4 from plasma of children with PS (and POIT) sensitized to Ara h 1 to Ara h 3 partially restored peanut-induced mast cell activation (P = .007). CONCLUSIONS: Differences in sIgE levels and allergen specificity could not justify the clinical phenotype in all children with PA and PS children. Blocking IgG4 antibodies provide an additional explanation for the absence of clinical reactivity in PS patients sensitized to major peanut allergens.

IL-9 is a key component of memory TH cell peanut-specific responses from children with peanut allergy.

BACKGROUND: Differentiation between patients with peanut allergy (PA) and those with peanut sensitization (PS) who tolerate peanut but have peanut-specific IgE, positive skin prick test responses, or both represents a significant diagnostic difficulty. Previously, gene expression microarrays were successfully used to identify biomarkers and explore immune responses during PA immunotherapy. OBJECTIVE: We aimed to characterize peanut-specific responses from patients with PA, subjects with PS, and atopic children without peanut allergy (NA children). METHODS: A preliminary exploratory microarray investigation of gene expression in peanut-activated memory TH subsets from 3 children with PA and 3 NA children identified potential PA diagnostic biomarkers. Microarray findings were confirmed by using real-time quantitative PCR in 30 subjects (12 children with PA, 12 children with PS, and 6 NA children). Flow cytometry was used to identify the TH subsets involved. RESULTS: Among 12,257 differentially expressed genes, IL9 showed the greatest difference between children with PA and NA children (45.59-fold change, P < .001), followed by IL5 and then IL13. Notably, IL9 allowed the most accurate classification of children with PA and NA children by using a machine-learning approach with recursive feature elimination and the random forest algorithm. Skin- and gut-homing TH cells from donors with PA expressed similar TH2- and TH9-associated genes. Real-time quantitative PCR confirmed that IL9 was the highest differentially expressed gene between children with PA and NA children (23.3-fold change, P < .01) and children with PS (18.5-fold change, P < .05). Intracellular cytokine staining showed that IL-9 and the TH2-specific cytokine IL-5 are produced by distinct TH populations. CONCLUSION: In this study IL9 best differentiated between children with PA and children with PS (and atopic NA children). Mutually exclusive production of IL-9 and the TH2-specific cytokine IL-5 suggests that the IL-9-producing cells belong to the recently described TH9 subset.

Basophil activation test discriminates between allergy and tolerance in peanut-sensitized children.

BACKGROUND: Most of the peanut-sensitized children do not have clinical peanut allergy. In equivocal cases, oral food challenges (OFCs) are required. However, OFCs are laborious and not without risk; thus, a test that could accurately diagnose peanut allergy and reduce the need for OFCs is desirable. OBJECTIVE: To assess the performance of basophil activation test (BAT) as a diagnostic marker for peanut allergy. METHODS: Peanut-allergic (n = 43), peanut-sensitized but tolerant (n = 36) and non-peanut-sensitized nonallergic (n = 25) children underwent skin prick test (SPT) and specific IgE (sIgE) to peanut and its components. BAT was performed using flow cytometry, and its diagnostic performance was evaluated in relation to allergy versus tolerance to peanut and validated in an independent population (n = 65). RESULTS: BAT in peanut-allergic children showed a peanut dose-dependent upregulation of CD63 and CD203c while there was no significant response to peanut in peanut-sensitized but tolerant (P < .001) and non-peanut-sensitized nonallergic children (P < .001). BAT optimal diagnostic cutoffs showed 97% accuracy, 95% positive predictive value, and 98% negative predictive value. BAT allowed reducing the number of required OFCs by two-thirds. BAT proved particularly useful in cases in which specialists could not accurately diagnose peanut allergy with SPT and sIgE to peanut and to Arah2. Using a 2-step diagnostic approach in which BAT was performed only after equivocal SPT or Arah2-sIgE, BAT had a major effect (97% reduction) on the number of OFCs required. CONCLUSIONS: BAT proved to be superior to other diagnostic tests in discriminating between peanut allergy and tolerance, particularly in difficult cases, and reduced the need for OFCs.

Peanut allergy: effect of environmental peanut exposure in children with filaggrin loss-of-function mutations.

BACKGROUND: Filaggrin (FLG) loss-of-function mutations lead to an impaired skin barrier associated with peanut allergy. Household peanut consumption is associated with peanut allergy, and peanut allergen in household dust correlates with household peanut consumption. OBJECTIVE: We sought to determine whether environmental peanut exposure increases the odds of peanut allergy and whether FLG mutations modulate these odds. METHODS: Exposure to peanut antigen in dust within the first year of life was measured in a population-based birth cohort. Peanut sensitization and peanut allergy (defined by using oral food challenges or component-resolved diagnostics [CRD]) were assessed at 8 and 11 years. Genotyping was performed for 6 FLG mutations. RESULTS: After adjustment for infantile atopic dermatitis and preceding egg skin prick test (SPT) sensitization, we found a strong and significant interaction between natural log (ln [loge]) peanut dust levels and FLG mutations on peanut sensitization and peanut allergy. Among children with FLG mutations, for each ln unit increase in the house dust peanut protein level, there was a more than 6-fold increased odds of peanut SPT sensitization, CRD sensitization, or both in children at ages 8 years, 11 years, or both and a greater than 3-fold increased odds of peanut allergy compared with odds seen in children with wild-type FLG. There was no significant effect of exposure in children without FLG mutations. In children carrying an FLG mutation, the threshold level for peanut SPT sensitization was 0.92 µg of peanut protein per gram (95% CI, 0.70-1.22 µg/g), that for CRD sensitization was 1.03 µg/g (95% CI, 0.90-1.82 µg/g), and that for peanut allergy was 1.17 µg/g (95% CI, 0.01-163.83 µg/g). CONCLUSION: Early-life environmental peanut exposure is associated with an increased risk of peanut sensitization and allergy in children who carry an FLG mutation. These data support the hypothesis that peanut allergy develops through transcutaneous sensitization in children with an impaired skin barrier.

Identifying infants at high risk of peanut allergy: the Learning Early About Peanut Allergy (LEAP) screening study.

BACKGROUND: Peanut allergy (PA) is rare in countries in which peanuts are introduced early into infants' diets. Learning Early About Peanut Allergy (LEAP) is an interventional study aiming to assess whether PA can be prevented by oral tolerance induction. OBJECTIVE: We sought to characterize a population screened for the risk of PA. METHODS: Subjects screened for the LEAP interventional trial comprise the LEAP screening study cohort. Infants were aged 4 to 10 months and passed a prescreening questionnaire. RESULTS: This analysis includes 834 infants (mean age, 7.8 months). They were split into the following: group I, patients with mild eczema and no egg allergy (n = 118); group II, patients with severe eczema, egg allergy, or both but 0-mm peanut skin prick test (SPT) wheal responses (n = 542); group III, patients with severe eczema, egg allergy, or both and 1- to 4-mm peanut wheal responses (n = 98); and group IV, patients with greater than 4-mm peanut wheal responses (n = 76). Unexpectedly, many (17%) in group II had peanut-specific IgE sensitization (≥ 0.35 kU/L); 56% of group III were similarly sensitized. In contrast, none of the patients in group I and 91% of those in group IV had peanut-specific IgE sensitization. Sensitization on skin testing to peanut (SPT response of 1-4 mm vs 0 mm) was associated with egg allergy and severe eczema (odds ratio [OR], 2.31 [95% CI, 1.39-3.86] and 2.47 [95% CI, 1.14-5.34], respectively). Similar associations were observed with specific IgE sensitization. Black race was associated with a significantly higher risk of peanut-specific IgE sensitization (OR, 5.30 [95% CI, 2.85-9.86]). Paradoxically, for a given specific IgE level, black race was protective against cutaneous sensitization (OR, 0.15 [95% CI, 0.04-0.61]). CONCLUSION: Egg allergy, severe eczema, or both appear to be useful criteria for identifying high-risk infants with an intermediate level of peanut sensitization for entry into a PA prevention study. The relationship between specific IgE level and SPT sensitization needs to be considered within the context of race.

Distribution of peanut protein in the home environment.

BACKGROUND: To halt the increase in peanut allergy, we must determine how children become sensitized to peanut. High household peanut consumption used as an indirect marker of environmental peanut exposure is associated with the development of peanut allergy. OBJECTIVE: We sought to validate a method to quantify environmental peanut exposure, to determine how peanut is transferred into the environment after peanut consumption, and to determine whether environmental peanut persists despite cleaning. METHODS: After initial comparative studies among 3 ELISA kits, we validated and used the Veratox polyclonal peanut ELISA to assess peanut protein concentrations in dust and air and on household surfaces, bedding, furnishings, hand wipes, and saliva. RESULTS: The Veratox polyclonal peanut ELISA had the best rate of recovery of an independent peanut standard. We demonstrated 100% sensitivity and specificity and a less than 15% coefficient of variation for intra-assay, interassay, and interoperator variability. There was high within-home correlation for peanut protein levels in dust and household surface wipes. Airborne peanut levels were lower than the limit of quantitation for the Veratox polyclonal peanut ELISA in a number of simulated scenarios, except for a brief period directly above peanuts being deshelled. Peanut protein persisted on hands and in saliva 3 hours after peanut consumption. Peanut protein was completely removed from granite tables after cleaning with detergent, and levels were reduced but still present after detergent cleaning of laminate and wooden table surfaces, pillows, and sofa covers. CONCLUSIONS: Peanut spread easily around the home and might be resistant to usual cleaning methods. Peanut protein can be transferred into the environment by means of hand transfer and saliva but is unlikely to be aerosolized.

Peanut protein in household dust is related to household peanut consumption and is biologically active.

BACKGROUND: Peanut allergy is an important public health concern. To understand the pathogenesis of peanut allergy, we need to determine the route by which children become sensitized. A dose-response between household peanut consumption (HPC; used as an indirect marker of environmental peanut exposure) and the development of peanut allergy has been observed; however, environmental peanut exposure was not directly quantified. OBJECTIVE: We sought to explore the relationship between reported HPC and peanut protein levels in an infant's home environment and to determine the biological activity of environmental peanut. METHODS: Peanut protein was quantified in wipe and dust samples collected from 45 homes with infants by using a polyclonal peanut ELISA. Environmental peanut protein levels were compared with peanut consumption assessed by using a validated peanut food frequency questionnaire and other clinical and household factors. Biological activity of peanut protein in dust was assessed with a basophil activation assay. RESULTS: There was a positive correlation between peanut protein levels in the infant's bed, crib rail, and play area and reported HPC over 1 and 6 months. On multivariate regression analysis, HPC was the most important variable associated with peanut protein levels in the infant's bed sheet and play area. Dust samples containing high peanut protein levels induced dose-dependent activation of basophils in children with peanut allergy. CONCLUSIONS: We have shown that an infant's environmental exposure to peanut is most likely to be due to HPC. Peanut protein in dust is biologically active and should be assessed as a route of possible early peanut sensitization in infants.

Early consumption of peanuts in infancy is associated with a low prevalence of peanut allergy.

BACKGROUND: Despite guidelines recommending avoidance of peanuts during infancy in the United Kingdom (UK), Australia, and, until recently, North America, peanut allergy (PA) continues to increase in these countries. OBJECTIVE: We sought to determine the prevalence of PA among Israeli and UK Jewish children and evaluate the relationship of PA to infant and maternal peanut consumption. METHODS: A clinically validated questionnaire determined the prevalence of PA among Jewish schoolchildren (5171 in the UK and 5615 in Israel). A second validated questionnaire assessed peanut consumption and weaning in Jewish infants (77 in the UK and 99 in Israel). RESULTS: The prevalence of PA in the UK was 1.85%, and the prevalence in Israel was 0.17% (P < .001). Despite accounting for atopy, the adjusted risk ratio for PA between countries was 9.8 (95% CI, 3.1-30.5) in primary school children. Peanut is introduced earlier and is eaten more frequently and in larger quantities in Israel than in the UK. The median monthly consumption of peanut in Israeli infants aged 8 to 14 months is 7.1 g of peanut protein, and it is 0 g in the UK (P < .001). The median number of times peanut is eaten per month was 8 in Israel and 0 in the UK (P < .0001). CONCLUSIONS: We demonstrate that Jewish children in the UK have a prevalence of PA that is 10-fold higher than that of Jewish children in Israel. This difference is not accounted for by differences in atopy, social class, genetic background, or peanut allergenicity. Israeli infants consume peanut in high quantities in the first year of life, whereas UK infants avoid peanuts. These findings raise the question of whether early introduction of peanut during infancy, rather than avoidance, will prevent the development of PA.

The Use of Dual-Cell-Tracker Dye Staining for the Identification and Characterization of Peanut-Specific T-Cell Subsets.

Cell-tracker fluorescent dye labeling is widely used for investigating antigen-specific immune responses in vitro and in vivo. Here we describe a development of this technique-the use of dual-cell-tracker dye staining for the identification and characterization of the responses of different T-cell subsets to peanut proteins in vitro.

Characterization of lymphocyte responses to peanuts in normal children, peanut-allergic children, and allergic children who acquired tolerance to peanuts.

Comparing lymphocyte responses to allergenic and nonallergenic foods could reveal the differences between pathogenic and normal immune responses to foods. Defining the cytokine-producing phenotypes of peanut-specific lymphocytes from peanut-allergic children, children who outgrew peanut allergy, and children who have always tolerated peanuts may be useful for understanding the mechanisms of food tolerance. Investigating immune responses against foods is hindered, however, by the fact that circulating food antigen-specific lymphocytes are very rare. In a novel approach we used carboxyfluorescein succinimidyl ester to detect peanut-specific lymphocytes by flow cytometry. We confirmed that these cells are indeed peanut specific by cloning. Peanut-allergic donors show Th2 polarization of cytokine production by peanut-specific cells (IFN-gamma (low), TNF-alpha (low), IL-4 (high), IL-5 (high), IL-13 (high)). Conversely, nonallergic children and children who have outgrown their allergy show Th1 skewing to peanut antigens (IFN-gamma(high), TNF-alpha (high), IL-4 (low), IL-5 (low), IL-13(low)), similarly to nonallergenic food antigens (beta-lactoglobulin, OVA). This finding suggests that peanut antigens do not intrinsically induce Th2 skewing, but that the type of response depends upon the donor's allergic status. In conclusion, food allergic status is characterized by a Th2 response whereas Th1-skewed responses underlie oral tolerance.

Immune mechanisms of food allergy and its prevention by early intervention.

The environmental factors driving the increase in food allergies are unclear and possibly involve dual exposure to allergens, microbiome-driven effects or other mechanisms. Until they can be better understood, early intervention aiming at establishing oral tolerance provides an effective way to decrease the window-of-risk when children may develop allergic sensitisation to foods due to the absence of a protective immune response. Thus, the recent LEAP (Learning Early About Peanut allergy) and LEAP-On studies achieved a high level of peanut allergy prevention by early introduction of peanuts in the infants diet and conveyed more information regarding the evolution of IgE and IgG4 antibody responses to food antigens over time.

The expression of CD123 can decrease with basophil activation: implications for the gating strategy of the basophil activation test.

BACKGROUND: Basophil activation test (BAT) reproduces IgE-mediated allergic reactions in vitro and has been used as a diagnostic test. Different markers can be used to identify basophils in whole blood and have implications for the outcome of the test. We aimed to assess changes in the expression of CD123 and HLA-DR following basophil activation and to select the best gating strategy for BAT using these markers. METHODS: BAT was performed in whole blood from 116 children. Peanut extract, anti-IgE, anti-FcεRI or formyl-methionyl-leucyl-phenylalanin (fMLP) was used for stimulation. Surface expression of CD123, HLA-DR, CD63 and CD203c was evaluated by flow cytometry. RESULTS: In some cases, gating on CD123+/HLA-DR- led to the loss-to-analysis of basophils in conditions where basophils were activated. Adding CD203c as an identification marker restored the cell number. Basophils remained HLA-DR-negative with activation. CD123 expression decreased following stimulation with fMLP (n = 116, p < 0.001), anti-IgE (n = 104, p < 0.001) and peanut (n = 42, p < 0.001). The decrease in the mean fluorescence intensity of CD123 correlated with the up-regulation of basophil activation markers, CD63 (rs = -0.31, p < 0.001) and CD203c (rs = -0.35, p < 0.001). BAT to peanut gating basophils on CD203c+/CD123+/HLA-DR- reduced the false-negatives (1 vs. 5 %) and showed a higher diagnostic accuracy compared to using CD123+/HLA-DR- (97 vs. 91 %). CD203c+ appeared as an alternative gating strategy allowing two-colour BAT. CONCLUSIONS: Basophils of a subset of patients down-regulate CD123 with activation. The use of CD203c before gating on CD123+/HLA-DR- cells or in isolation ensures the identification of the entire basophil population and accurate assessment of basophil activation, with important diagnostic implications.

Novel biomarkers for asthma stratification and personalized therapy.

A stepwise pharmacological treatment is currently recommended for all asthma patients and is personalized mainly on disease severity, aiming for the lowest disease-controlling step. Nevertheless, asthma comprises several related pathologies with similar clinical manifestations resulting from distinct underlying mechanisms. Therefore novel biomarkers could lead to asthma stratification and thus improve upon the current stepwise approach. The aim of this review is to update the reader with regard to different assays proposed in the recent asthma literature for measuring potential biomarkers for patient stratification and treatment personalization. Promising biomarkers are sputum eosinophils, serum periostin and exhaled nitric oxide. Periostin could differentiate between Th2-high and Th2-low asthma (Th2-high patients are more responsive to glucocorticoids) and the less-defined asthma types which often present a therapeutic challenge. Several other biomarkers, mainly cytokines, leukotrienes and exhaled air components, can be quantified in body fluids and exhaled breath and could also be useful for asthma stratification.

Neural respiratory drive measurement for COPD assessment and monitoring.

Currently there is an unmet need for more objective assessments that could determine COPD severity. Ideally such objective assessments could also anticipate COPD exacerbations in order to decrease the need for repeated hospital admissions. In this review we outline how patients' neural respiratory drive (NRD) may be determined using the electromyography of the diaphragm as an objective measurement of COPD severity. Respiratory muscle NRD is indeed less influenced by patients' voluntary effort limitation than for example when testing for exercise tolerance in which case the patients themselves decide when to stop. Exercise tolerance tests are better correlated with muscle weakness rather than COPD severity per se. NRD would also be less dependent upon patients' subjective perception of the severity of their breathlessness. A key further advantage is that recent studies showed that the diaphragm electromyography measurements using electrodes placed on the skin are correlated with those obtained using specific electrodes, therefore this method is non-invasive and more acceptable for routine clinical practice. Thus, NRD measurements could be used in COPD in a similar way as electrocardiography is used to evaluate and monitor ischemic heart disease. NRD measurements could therefore complement more established instruments such as lung function tests, FEV1, exercise tolerance tests, the BODE index etc. in COPD. This could lead to better COPD management and reduce the acute exacerbations which are amongst the most common causes of repeated hospital admissions and consume significant resources.

Molecular diagnosis of peanut allergy.

Peanut allergy prevalence has increased in developed countries over the last few decades in the frame of the allergy epidemics, currently affecting 1-2% of children. While less frequent in developing countries, its prevalence is rising as these countries adopt a more westernized lifestyle. There is no curative treatment for peanut allergy at present so patient management relies on peanut avoidance, which requires an accurate diagnosis. Recent progress in peanut allergy diagnosis was made with the introduction of component resolved diagnosis that allows the assessment of IgE specific to individual peanut allergens. Component-resolved diagnosis needs to be interpreted in the context of clinical data but overall increases the diagnostic accuracy, as described in the typical cases that we present. Novel diagnostic tools have been proposed recently, such as the basophil activation test, mRNA expression and resonance magnetic evaluation of biomarkers.