Margo M. Hagendorens

Flow cytometric analysis of in vitro activated basophils, specific IgE and skin tests in the diagnosis of pollen-associated food allergy

Specific immunoglobulin E (IgE) and commercially available skin prick tests have been demonstrated to be unreliable methods to diagnose pollen‐associated food allergy. To evaluate the predictive value of the basophil activation test (BAT) in pollen‐associated food allergy, the apple‐mediated oral allergy syndrome (OAS) in patients with birch pollinosis was chosen as a representative model.

Lung function and bronchodilator response in 4-year-old children with different wheezing phenotypes

Persistent wheeze is a common chronic disease in early childhood and later may progress to asthma. However, the association between pre- and post-bronchodilator lung function and the wheezing phenotype in preschool children is not known. Children 4 yrs of age involved in a prospective birth cohort study (in Antwerp, Belgium) concerning perinatal factors and the occurrence of asthma and allergies, were invited to participate in lung function measurements with the forced oscillation technique. The wheezing phenotype was assessed via (bi)annual questionnaires. Wheezing phenotype and baseline respiratory impedance data were available for 325 children, 96% of whom underwent bronchodilation tests. The baseline resistance at 4 Hz was higher in children with early transient (11.0 hPa·s·L−1, n = 127) or persistent wheeze (11.9 hPa·s·L−1, n = 54) than in children who never wheezed (10.3 hPa·s·L−1, n = 144). After bronchodilation, the resistance decreased on average by 22%. The decrease was greater among the persistent wheezers than among those who never wheezed (3.4 versus 2.3 hPa·s·L−1). The baseline lung function was poorer and the bronchodilator response was greater in 4-yr-old children with persistent wheeze than in those who never wheeze or who had early transient wheeze, implying a higher bronchomotor tone in the former group.

Analyzing histamine release by flow cytometry (HistaFlow): A novel instrument to study the degranulation patterns of basophils

BACKGROUND Stimulated human basophils exhibit different degranulation patterns with release of mediators and appearance of activation markers such as CD63 and CD203c. Traditionally, released mediators are quantified in the supernatant of activated cells, whereas the expression of activation markers by individual cells is analyzed by flow cytometry. Alternatively, intracellular histamine and its release by basophils and mast cells have been repeatedly studied applying an enzyme-affinity-gold method based on the affinity of the histaminase diamine oxidase for its substrate histamine. OBJECTIVE To develop a flow cytometric technique enabling to study histamine release by individual basophils in combination with the expression of activation markers. To elucidate the principles of basophil degranulation on a single cell level. METHODS Intracellular histamine and its release is analyzed flow cytometrically by an enzyme-affinity method using diamine oxidase conjugated to laser-excitable fluorochromes. Phenotyping of cells implied flow cytometric quantification of CD63 and CD203c. Stimuli such as allergen, anti-IgE, N-formyl-met-leu-phe (fMLP), phorbol 12-myristate 13-acetate (PMA), ionomycin and interleukin (IL-)3 are applied to obtain different degranulation profiles. RESULTS Stimulation with anti-IgE, allergen, fMLP and PMA±ionomycin induces a rapid release of histamine that can be analyzed flow cytometrically. Analyses on a single cell level reveal that histamine release is not restricted to cells showing significant up-regulation of CD63. Alternatively, up-regulation of CD203c does not per se indicate histamine release. In some patients, priming of cells with IL-3 not only facilitates basophil responsiveness but also implies an increased ability of DAO to label the cells. CONCLUSION This study provides the proof-of-concept that histamine and its release can be studied by multicolor flow cytometry on a single cell level (HistaFlow). Coupling the data to simultaneous phenotyping of activated basophils confirms that histamine release principally results from anaphylactic degranulation and in a lesser extent from piecemeal degranulation.

Early exposure to solid foods and the development of eczema in children up to 4 years of age.

Sariachvili M, Droste J, Dom S, Wieringa M, Hagendorens M, Stevens W, van Sprundel M, Desager K, Weyler J. Early exposure to solid foods and the development of eczema in children up to 4 years of age.
Pediatr Allergy Immunol 2010: 21: 74–81.
© 2009 John Wiley & Sons A/S

Young infants with atopic dermatitis can display sensitization to Cor a 9, an 11S legumin‐like seed‐storage protein from hazelnut (Corylus avellana)

To cite this article: Verweij MM, Hagendorens MM, De Knop KJ, Bridts CH, De Clerck LS, Stevens WJ, Ebo DG. Young infants with atopic dermatitis can display sensitization to Cor a 9, an 11S legumin‐like seed‐storage protein from hazelnut (Corylus avellana). Pediatr Allergy Immunol 2011; 22: 196–201.

Prenatal exposure to house dust mite allergen (Der p 1), cord blood T cell phenotype and cytokine production and atopic dermatitis during the first year of life

This study investigated the influence of prenatal exposure to house dust mite (HDM, D. pteronyssinus) on interleukin (IL)‐2, interferon‐gamma (IFN‐γ) and IL‐4 producing CD4+ and CD8+ T lymphocytes in cord blood as well as on the development of sensitization and occurrence of atopic dermatitis (AD) as the first symptom of allergy during the first year of life. Dust samples (n = 22) were collected by vacuum cleaning the maternal mattress during early to mid‐pregnancy. In these samples, the amount of the major HDM antigen (Der p 1) was assessed by a sensitive enzyme‐linked immunosorbent assay technique (detection limit 0.004 μg/g dust). Flow cytometry was used to determine cord blood lymphocyte subtypes and to quantify the intracellular amounts of IL‐2, IFN‐γ and IL‐4 produced by cord blood CD4+ helper and CD8+ cytotoxic T lymphocytes, both spontaneously and after stimulation with phorbol‐12‐mirystate‐13‐acetate and ionomycin. Children were followed for 1 yr for the presence of symptoms associated with allergy. In addition, at the age of 1 yr specific IgE to different classical inhalant and food allergens was measured. Higher prenatal exposure to Der p 1 (>0.2 μg/g dust) was associated with a significant lower percentage of IFN‐γ producing stimulated CD4+ T lymphocytes, compared with lower prenatal Der p 1 exposure (p = 0.03). The presence of AD during the first year of life (n = 9) was associated with an increased number of naive CD4+ CD45RA+ lymphocytes (p = 0.03), with an increased spontaneous IL‐4 production by CD8+ lymphocytes (p = 0.04) and with a decreased percentage of IFN‐γ producing stimulated CD4+ lymphocytes (p = 0.04). Furthermore, exposure to HDM during pregnancy tended to be higher in mothers of children with AD during the first year of life when compared with those without AD (p = 0.08). This study shows that the level of prenatal exposure to Der p 1 influences the immune profile of cord blood T lymphocytes and the clinical outcome in early life. Therefore, the prenatal environment must be regarded as a possible early risk factors for allergic diseases in children.

Critical evaluation of prognostic factors in childhood asthma

Current knowledge of the natural history of asthma is improving through the establishment of a more precise definition of asthma linked with information from a number of large‐scale longitudinal studies. Risk factors for the development of childhood asthma are now more clearly understood. They include gender, atopic status, genetic and familial factors, respiratory infections, and outdoor and indoor pollution ( 1 ). In the present review two types of asthma and their prognosis will be discussed:

Simultaneous flow cytometric detection of basophil activation marker CD63 and intracellular phosphorylated p38 mitogen-activated protein kinase in birch pollen allergy†

Phosphorylation of p38 MAPK is a crucial step in IgE‐receptor signaling in basophils. The relation of p38 MAPK to the well‐validated diagnostic cell surface marker CD63 has not been evaluated in a clinical allergy model.

Is breast feeding a risk factor for eczema during the first year of life

Breast feeding (BF) provides many advantages to the offspring; however, at present there is an ongoing debate as to whether or not it prevents allergic diseases. The aim of the current study was to investigate the effect of duration of BF on eczema in the first year of life. A birth cohort of 1128 infants was followed prospectively from 5 months of pregnancy. Data were collected using questionnaires, a medical examination and blood tests for allergy at the age of 1 yr. Breast feeding was not statistically significant associated with eczema in the first year of life [adj ORs with 95% CIs: 0.8 (0.4–1.3), 0.8 (0.5–1.3) and 1.0 (0.6–1.5) for BF duration of 1–6 wk, 7–12 wk and ≥13 wk, respectively]. Eczema was positively associated with atopy and educational level of the mother, use of antibiotics in pregnancy and passive smoking by the child during the first 12 months. Regular postnatal contact of the infants with dogs was inversely associated with eczema. Breast feeding was positively associated with eczema among children with non‐atopic parents [adj ORs with 95% CIs: 2.1 (0.4–10.6), 2.2 (0.4–11.3) and 1.9 (0.4–8.5) for BF duration of 1–6 wk, 7–12 wk and ≥13 wk, respectively], whereas an inverse association was found among children with atopic parents [adj ORs with 95% CIs: 0.6 (0.3–1.3), 0.7 (0.3–1.4) and 0.9 (0.5–1.7) for the same BF durations]. However, these associations were not statistically significant. Breast feeding has no significant effect on the prevalence of eczema in the first year of life. The effect of BF on eczema in children depends on parental atopy.

Determination of T-cell subpopulations and intracellular cytokine production (interleukin-2, interleukin-4, and interferon-γ) by cord blood T-lymphocytes of neonates from atopic and non-atopic parents

This report describes the results of a prospective study on immunological markers in cord blood for the prediction of allergic diseases in children. First we evaluated methodological aspects of the flow cytometric technique on cord blood cytokine measurements. Subsequently, the T‐cell subsets and percentage of cytokine‐producing cord blood T‐helper (Th) and T‐suppressor/cytotoxic lymphocytes of neonates from atopic and non‐atopic parents were compared. A group of 33 healthy, full‐term newborn infants of whom 23/33 were at risk for atopy (i.e. having at least one parent with one or more atopic symptoms and positive specific immunoglobulin E [IgE] to at least one common inhalant allergen) was studied. A flow cytometric technique was used to analyze cord blood T‐cell subsets and to determine the percentage of interleukin (IL)‐2‐, IL‐4‐, and interferon‐γ (IFN‐γ)‐producing cord blood Th and T‐suppressor/cytotoxic lymphocytes following stimulation with phorbol 12‐myristate 13‐acetate (PMA) and ionomycin. The percentage of CD3 (T lymphocytes), CD3+ CD4+ (Th lymphocytes), CD3+ CD8+ (T‐suppressor/cytotoxic lymphocytes), CD19+ (B lymphocytes), CD3+ CD4+ CD45RO+ (memory Th lymphocytes), and CD3+ CD4+ CD45RA+ (naive Th lymphocytes) cells was unrelated to parental atopic status. PMA stimulation augmented the percentage of IL‐2‐ and IFN‐γ‐producing Th and T‐suppressor/cytotoxic lymphocytes, whereas the number of IL‐4‐producing T lymphocytes remained very low or undetectable. No differences in the percentage of IL‐2‐, IL‐4‐ and IFN‐γ‐producing Th and T‐suppressor/cytotoxic lymphocytes were found between neonates from atopic and non‐atopic parents. These results will be re‐evaluated when the atopic status of the children at the age of 1 and 2 years can be assessed.