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Notes: Eighteen children with perennial asthma and allergy to house-dust mite (HDM) underwent a bronchial challenge with HDM. Before and 24 h after the test, a venous blood sample was taken to determine levels of eosinophils, eosinophil cationic protein (ECP), soluble interleukin-2 receptor (IL-2R), and interleukin-6 (IL-6). A histamine challenge was performed before and 24 h after the HDM challenge. All subjects showed an immediate asthmatic reaction (IAR). A definite late asthmatic reaction (LAR) was observed in 15 children, a probable LAR in two, and no LAR in one. Because of persistent bronchial obstruction (FEV1〉70%), eight children were unable to perform a histamine challenge 24 h after the allergen challenge. These were the children with the lowest prechallenge provocation dose (PD20) of histamine. In the other 10 children, the mean PD20 histamine decreased after the HDM challenge (mean PD20 before was 0.56 mg/ml; after challenge it was 0.14 mg/ml; P= 0.007). After the HDM challenge, an increase was detected in the mean values of blood eosinophils (mean before was 446/mm3; mean after was 733/mm3; P= 0.002), ECP (mean before was 26.3 μg/1; mean after was 34.3 μg/1; P〈0.040), and IL-2R (mean before was 116.35 U/ml; mean after was 128.52 U/ml; P〈0.040). On the other hand, IL-6 remained unchanged after the HDM challenge (mean before was 9.47 pg/1; mean after was 9.70 pg/1; P= 0.360). Furthermore, as compared with a group of normal, age-matched children (n =18), asthmatic children were found to have higher prechallenge levels of ECP (mean: 10.3 μg/1 compared with 26.3 μg/1) (P〉0.001) and IL-2R (mean: 80.30 U/ml compared with 116.35 U/ml) (P =0.009), but not of IL-6 (mean: 11.34 pg/1 compared with 9.47 pg/1) (P = 0.436).A correlation was found between the duration of asthma and the severity of the LAR expressed as area under the curve (AUCLAR) (r = 0.50; P〈0.040). Furthermore, a correlation was detected between the level of total IgE and the level of ECP (r = 0.51; P〈0.030). The decrease in FEV1 during the LAR tended to correlate with the increase of IL-2R (r = 0.48; P = 0.050). This tendency was not found with the increase of eosinophils, nor with the increase of ECP. We conclude that both lymphocytes and eosinophils are activated by an allergen challenge, but that only the activation of lymphocytes tends to correlate with the LAR, suggesting that lymphocytes are also closely involved in the pathogenesis of the allergen-induced LAR.

Notes: With the conventional, discrete RAST various tests are required to detect IgE of different specificities in the same serum. To overcome this problem and to reduce the costs, a multiple RAST with seven different mixtures was compared with the individual mixture constituents and with the 12 individual allergens currently in use in our department. One grass pollen mixture (gx3), two weed pollen mixtures (wx3, wx4), two tree pollen mixtures (tx5, tx6), one mould mixture (mx1) and one epithelial mixture (ex1) were used. A mixture of mites was not evaluated as there is only one important pathogenic organism in our regions (Dermatophagoides pteronyssinus or House dust mite). For grasses the gx3 mixture offered no advantage over the discrete RAST. The weed mixture wx3 was more sensitive than the most common discrete RAST's but at the cost of specificity. The wx4 mixture should not be used because the specificity is too low. The tree mixtures were not significantly more sensitive than the most common individual tree allergens, and were less specific. Mould mixtures should not be used because there is little cross-reactivity between the individual allergens, thus using a mixture would necessitate the subsequent determination of individual allergens, and the number of tests and the cost would be even higher. Neither should a mixture be used for epithelia because one wants to detect allergies to individual allergens. Moreover, the sensitivity of the epithelial mixture was too low. In general, we suggest the use of a limited panel of discrete RAST's instead of mixtures.

Notes: In patients with allergic asthma and rhinitis high numbers of hypodense eosinophils (HE) have been demonstrated. In a previous study we reported that asthmatic and healthy children had more HE than their adult counterparts. We assumed that this might, in part, he due to the presence of immature eosinophils in children. To distinguish between immature and activated eosinophils, determination of eosinophil cationic protein (ECP) might be interesting as it is known that high serum levels of ECP are associated with increased activation of eosinophiis. In this study we determined (he levels of ECP in scrum in asthmatic and healthy children and adults trying to distinguish activated from immature eosinophils. We found that ECP levels were not increased in children (healthy and asthmatic) compared to adults (healthy and asthmatic). This supports the hypothesis that increased numbers of HE in childhood are, at least in part, immature eosinophils. Nevertheless, we could confirm that inflammation was present in children because soluble interleukin-2-receptor (slL-2R), a marker of lymphocyte activation, was higher in asthmatic children as compared to healthy children. IL-6, a marker of macrophage/monocyte activation, was not different in the different patient groups. We conclude that although signs of inflammation are present in childhood asthma, the increased numbers of HE in children are in part due to the presence of immature eosinophils.

Notes: In the first part of this study the proliferative response of lymphocytes (lymphocyte transformation test) to house dust mite (HDM) stimulation in cultures was studied in normal children (n= 16), asthmatic children who never received hyposensitization (HS) (n = 50) and asthmatic children receiving HS with HDM for at least 6 months (n = 20). The results are expressed as disintegrations per minute (d.p.m.) and as stimulation index (SI = d.p.m. in the presence of the allergen/d.p.m. in the control culture). A positive SI (〉 2) was found in 54% of the asthmatic children who never received HS, in 30% of the asthmatics receiving HS and in none of the normal children. Furthermore, between asthmatics with and without HS, the SI was not statistically different, although asthmatics without HS tended to have a higher SI (median value: 2.13 vs 1.38) (P= 0.10). In a second series of experiments the effect of adding interleukin-2 (IL-2) to the lymphocyte cell culture was studied in asthmatic children with and without HS. Interleukin-2 induced an additional stimulatory effect on the lymphoproliferative response to HDM and to phytohaemagglutinin in patients who never received HS, but had no effect in patients receiving HS. We conclude that HS treatment seems to have an inhibiting effect upon this proliferative response, not only inhibiting the degree of the allergen-induced lymphocyte proliferation, but also inhibiting the sensitivity of proliferating lymphocytes for IL-2. These inhibiting effects upon lymphocytic activation could be responsible for the anti-inflammatory effects (i.e. suppression of the late asthmatic reaction) of HS.

Notes: Beside lymphocytes and neutrophils, eosinophils are also involved in the inflammatory reaction in rheumatoid arthritis (RA). In this study, adhesion characteristics of peripheral blood eosinophils were studied in 43 RA patients and 19 controls, together with the expression of the β2-integrin Mac-1 (CD11b/CD18). In addition, the production of oxygen radicals of isolated peripheral blood eosinophils and serum levels of eosinophil cationic protein (ECP) were measured in order to evaluate eosinophil activation. Adhesion of eosinophils to unstimulated human vascular endothelium was significantly higher in RA patients with active disease (n= 4) compared with controls (n= 14) (P 〈 0.005) and compared with patients with less active RA (n= 16) (P 〈 0.05). Nevertheless, the expression of the adhesion molecule Mac-1 (CD11b/CD18) was not increased in RA patients. ECP levels were higher in RA patients with active disease (P 〈 0.01). Release of oxygen radicals in response to phorbol stimulation was significantly elevated in active RA compared with controls (P 〈 0.05) and to less active RA (P 〈 0.05). We conclude that eosinophils of RA patients, especially those with active disease, are activated or at least primed and are involved in the inflammatory process in RA, analogous to the inflammation in asthma. The higher adhesion to inflamed endothelium is indicative of a higher infiltration in the joints, where tissue damage can be caused by toxic oxygen radicals and by granular proteins, such as ECP.

Notes: Objective To evaluate the influence of perinatal environmental factors on early sensitization, atopic dermatitis and wheezing during the first year.Methods Information on pregnancy-related factors, parental atopic history, environmental factors and the clinical course of the infant until age one was gathered by questionnaires, as part of a prospective birth cohort study (Prospective study on the Influence of Perinatal factors on the Occurrence of asthma and allergies [PIPO-study]). Quantification of total and specific IgE was performed in 810 children and their parents.Results Early sensitization was found in 107/810 (13%) of the infants. Multiple regression analysis showed that specific IgE in fathers was a risk factor for early sensitization in their daughters (adjusted odds ratios (ORadj) 2.21 (95% confidence interval (CI) 1.10–4.49); P=0.03), whereas in boys, day care attendance was shown to be protective for early sensitization (ORadj 0.38 (95% CI 0.20–0.71); P=0.001). Atopic dermatitis occurred in 195/792 infants (25%). Specific IgE in the mother (ORadj 1.52 (95% CI 1.06–2.19); P=0.02) and in the infant (ORadj 4.20 (95% CI 2.63–6.68); P〈0.001) were both risk factors for the occurence of atopic dermatitis, whereas postnatal exposure to cats was negatively associated with atopic dermatitis (ORadj 0.68 (0.47–0.97); P=0.03).Postnatal exposure to cigarette smoke (ORadj 3.31 (95% CI 1.79–6.09); P〈0.001) and day care attendance (ORadj 1.96 (95% CI 1.18–3.23); P=0.009) were significantly associated with early wheezing, which occurred in 25% (197/795) of the infants.Conclusion The effect of paternal sensitization and day care attendance on sensitization is gender dependent. Maternal sensitization predisposes for atopic dermatitis, whereas postnatal exposure to cats had a protective effect.

Notes: Background Different types of circulating dendritic cells have been described. Dendritic cells influence differentiation of naive T lymphocytes into T helper type 1 (Th1) and Th2 effector cells.Objective The purpose of this study was to evaluate the number of circulating DC subtypes in peripheral blood of allergic and healthy children and in cord blood of neonates from allergic and non-allergic parents.Methods Circulating dendritic cells were flow cytometrically identified in whole blood samples as lineage (CD3, CD14, CD16, CD19, CD20, CD56) negative, CD34 negative and HLA-DR-positive cells. According to the expression of CD123 and CD11c, different DC subtypes were identified.Results Apart from DC1 (CD11c+ CD123dim+) and DC2 (CD11c– CD123high+), a third DC population was described with less differentiated phenotypic characteristics, namely CD11c– CD123dim+, and therefore defined here as less differentiated DC (ldDC). These ldDC represented the major DC population in cord blood and showed an age-depended decrease. The highest level of ldDC was detected in children with atopic dermatitis, whereas asthmatic children showed the lowest ldDC counts. Furthermore, high-dose inhaled corticosteroid treatment in asthmatic children was related to a decreased ldDC number. The number of circulating DC2 was significantly lower in allergic children, especially in asthmatics, compared to healthy children. In cord blood, no differences in DC subtypes were detectable between neonates at low and high risk for allergic disorders.Conclusion These results indicate that, apart from DC1 and DC2, a third population of dendritic cells, identified as CD11c– CD123dim+ cells and defined as less differentiated DC, must be considered in the evaluation of circulating DC. Furthermore, DC2 counts were decreased in allergic children, especially in asthmatics, which might be the consequence of an increased recruitment to the target organs.

Notes: During the last 5 years, an increasing number of studies have demonstrated that flow cytometric quantification of in vitro basophil activation can be a quite performant and reliable tool to measure IgE-dependent allergen-specific responses in allergic patients. So far, most assays have used CD63 as a basophil activation marker and native allergen extracts for stimulation. However, other basophil markers and recombinant allergens have recently been introduced. The technique has been applied for the diagnosis of allergy to pollen, house dust mite, food, natural rubber latex, hymenoptera venom and drugs. In addition, the technique has proven to be useful in non-IgE-mediated reactions such as hypersensitivity to drugs as well as detection of auto-antibodies in chronic urticaria. This review will focus on some specific issues: (1) principles of flow cytometric analysis of in vitro-activated basophils, (2) general technical aspects of the technique (including passive sensitization), (3) clinical applications and (4) recommendations for further development and evaluation of the technique.

Notes: Background During the last decade, evidence has been provided for profilins and cross-reactive carbohydrate determinants (CCDs) to be capable of inducing cross-reactive IgE antibodies with little clinical relevance.Objective To investigate the prevalence of sensitization to CCD and profilin in isolated allergies (birch, timothy grass, house dust mite, pets (cat and/or dog), natural rubber latex (NRL) and hymenoptera venom). To study the contribution of anti-CCD and anti-profilin IgE antibodies as a cause of clinically irrelevant IgE for NRL and apple.Methods For the first part of the study, 100 patients with inhalant allergy, 17 patients with NRL allergy and 40 patients with venom anaphylaxis were enrolled. Diagnosis was based on a questionnaire and a positive IgE determination and skin test for relevant allergen. Patients were identified as sensitized to CCD if they had a negative prick test and positive IgE for the glycoprotein bromelain. Sensitization to profilin was assessed by IgE for rBet v 2 (recombinant birch profilin). For the second part of the study, sera containing IgE against apple (n=82) or NRL (n=38) were classified as true-negative or false-positive according to the presence or absence of an oral allergy syndrome (OAS) or NRL-induced anaphylaxis. In these patients, sensitization to CCD and profilin was evaluated as described above.Results No sensitization to bromelain-type CCD and profilin was found in isolated birch pollen or NRL allergy. In contrast, sensitization to bromelain-type CCD was found in 4/17 patients with isolated grass pollinosis, 5/24 patients with combined pollinosis (birch, timothy, mugwort) and 7/33 patients with venom anaphylaxis. Sensitization to profilin was almost restricted to patients with combined pollen allergy (5/24). In pollen-allergic individuals with a false-positive IgE against NRL the prevalence of sensitization to bromelain-type CCD and profilin IgE was higher than in NRL-allergic patients (P〈0.00001 and P=0.0006, respectively). In pollen-allergic individuals with a false-positive IgE to apple, the frequency of sensitization to bromelain-type CCD was higher than in OAS patients (P=0.004). Clinically irrelevant NRL and apple were also found in four and five out of the seven patients sensitized to venom CCD, respectively. In pollinosis, clinically irrelevant NRL and apple IgE antibodies were inhibited by bromelain and recombinant birch profilin, whereas in isolated venom anaphylaxis these antibodies were inhibited by bromelain.Conclusions Patients monoallergic to NRL or birch pollen showed no sensitization to bromelain-type CCD or profilin. Sensitization to profilin and/or bromelain-type CCD, caused by pollen (timothy grass, mugwort) or hymenoptera venom allergens, can elicit false-positive IgE antibodies against NRL and apple.