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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: Background Venom immunotherapy (VIT) has proven to be very effective in hymenoptera venom anaphylaxis. However, the underlying immunoregulatory mechanisms of venom immunotherapy remain poorly understood. Recent studies measured the total amount of cytokine in culture supernatans, suggesting a shift in cytokine production from Th2 to a Th1 cytokine profile during VIT. We wanted to examine the contribution of specific T lymphocyte subpopulations, which is impossible using an extracellular method to determine cytokines in supernatants.Objective The present study was designed to evaluate the effect of VIT on the percentages of type 1 (IL-2, interferon-gamma (IFN-γ)) and type 2 (IL-4) CD4+ and CD8+ T lymphocytes of patients with wasp venom anaphylaxis during immunotherapy.Methods Peripheral blood mononuclear cells of 20 individuals with a history of wasp sting anaphylaxis and a positive serum wasp venom specific IgE were isolated and in vitro stimulated with phorbol-12-myristate-13-acetate and ionomycin before VIT, at the end of a 5-day semirush VIT and at 6 months during VIT. Three-colour flow cytometric analysis was used for intracellular cytokine (IL-2, IFN-γ, IL-4) detection in CD4+ (CD3+CD8−) T lymphocytes and CD8+ (CD3+CD8+) T lymphocytes.Results At the end of a 5-day semirush VIT, there was a significant decrease in percentage of IL-4-producing CD4+ and CD8+ T cells, compared with cytokine-producing cells before VIT (P = 0.0002 and 0.004). After 6 months of VIT, patients showed an increased number of IL-2-producing stimulated CD4+ and CD8+ lymphocytes compared with values before VIT (P = 0.002 and P = 0.0003). A higher amount of IFN-γ-producing stimulated CD4+ and CD8+ cells was found after 6 months of VIT (P = 0.001 and P = 0.0006). There was no correlation between cytokine-producing cells and specific IgE for wasp.Conclusion Venom immunotherapy induced a shift from IL-4-producing towards IFN-γ-producing CD4+ as well as CD8+ T lymphocytes.

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.

Notes: Background:  IgE-dependent triggering of basophils not only elicits the release of different mediators but also the up-regulation of certain markers, e.g. CD63, which can be detected by flow cytometry. We intended to investigate if flow cytometric analysis of basophil activation could be a valuable tool in the diagnosis of latex allergy, and to evaluate if the basophil activation test (BAT) could be helpful in determining the clinical significance of a positive latex IgE in individuals with negative history and negative latex skin test. Additionally we aimed to determine the role of cross-reactive carbohydrate determinants (CCDs) in causing positive latex IgE without apparent clinical significance. Methods:  Twelve healthy controls without a history of latex hypersensitivity with a negative latex IgE and skin test (group 1), 24 individuals without a history of latex hypersensitivity with a negative latex IgE and skin test but with other inhalant allergies (group 2), and 29 latex allergic patients with a compelling history of latex allergy with a positive latex IgE and prick test (group 3) were enrolled. The diagnostic performances of the BAT were further evaluated in 13 individuals with a history of latex allergy but with negative specific IgE and/or skin test (group 4). Twenty-four individuals with positive latex IgE without apparent clinical relevance, i.e. without history of latex hypersensitivity and negative latex skin tests, were also analyzed (group 5). The putative role of CCDs causing positive latex IgE results without apparent clinical significance was evaluated by quantification of IgE for bromelain. Results:  According, to the receiver operating characteristics(ROC)-generated threshold value of 17% between latex allergic patients and the pooled group of nonlatex allergic individuals, the sensitivity and specificity of the basophil activation test was 93.1% and 91.7%, respectively. In healthy controls, allergic patients without latex hypersensitivity and latex allergic patients the number of positive BATs was 0/12, 3/24 and 27/29, respectively. In the individuals with an evocative history of latex allergy but with negative latex IgE and/or skin test the BAT was positive in all 13 cases. Twenty of 24 individuals without apparent latex allergy but with positive latex IgE had a negative BAT. IgE for bromelain was positive in 1/19 sera from group 2, 1/24 sera from group 3, none of the 8 sera from group 4, but in 16/18 sera from group 5, respectively. Conclusion:  Flow cytometric analysis of activated basophils seems a highly sensitive and specific tool for diagnosing latex allergy. In addition, the technique might help to determine the clinical relevance of positive IgE quantification in the absence of overt latex allergy. CCDs of natural rubber latex allergens were confirmed to mimic latex sensitization.

Notes: Abstract: The clinical manifestations of hypoparathyroidism are mainly characterised by increased neuromuscular irritability as a consequence of hypocalcaemia. Occasionally, elevation of the muscle enzymes may mimic polymyositis. Reduced parathyroid hormone production, but also vitamin D treatment and calcium supplementation, may contribute to the increased bone mass found in patients with postsurgical hypoparathyroidism. We report the case of a 36-year-old woman with untreated idiopathic hypoparathyroidism and a high bone mass despite severe muscle impairment due to hypocalcaemic myopathy.