In vitro basophil activation using CD63 expression in patients with bee and wasp venom allergy.

The diagnosis of insect venom allergy and the indication for specific immunotherapy is based on history, skin tests and demonstration of hymenoptera venom-specific IgE-antibodies. Cellular tests can add useful information but the role of basophil activation tests for the different venoms has to be elucidated further. We evaluated positive reactions in a basophil activation test using CD63 expression as marker independently for bee or wasp venom in patients with hymenoptera allergy. Fifty-seven patients with a history of insect venom anaphylaxis were examined (12 x bee venom, 39 x wasp venom, 6 x bee plus wasp venom). Skin tests and determination of specific IgE-antibodies were performed. Basophil activation test (BAT) using CD63 expression was performed after stimulation with different concentrations of bee and wasp venom. The BAT is based on double staining with anti-IgE antibodies and anti-CD63 and subsequent determination of the percentage of activated basophils by flow cytometry. In patients with bee venom allergy, BAT was positive in 100% to bee venom and 75% to wasp venom. In patients with bee and wasp venom allergy, positive reactions for both venoms were found in 100%. In patients with wasp venom allergy, 97% reacted positive to wasp venom and only 56% to bee venom. These results show the reliability of the basophil activation test as a cellular test in the in vitro diagnosis in patients with bee and wasp venom allergy. They also show that positive reactions in the basophil activation test reflect both sensitization status and cross-reactivity between venom species.

Quantitative patch and repeated open application testing in methyldibromo glutaronitrile-sensitive patients.

Contact allergy to methyldibromo glutaronitrile (MDBGN), often combined with phenoxyethanol (PE) (e.g., Euxyl K 400), increased throughout the 1990s in Europe. Consequently, in 2003, the European Commission banned its use in leave-on products, where its use concentration was considered too high and the non-sensitizing use concentration as yet unknown. The 2 objectives of the study are (a) to find a maximum non-eliciting concentration in a leave-on product in MDBGN/PE-sensitized patients, which could possibly also be considered safe regarding induction and (b) to find the best patch test concentration for MDBGN. We, therefore, performed a use-related test (ROAT) in patients sensitized to MDBGN/PE (n = 39) with 3 concentrations of MDBGN/PE (50, 100 and 250 p.p.m. MDBGN, respectively). A subset of these patients (n = 24) was later patch-tested with various concentrations (0.1, 0.2, 0.3 and 0.5% MDBGN, respectively). 15 patients (38%, 95% confidence interval (CI) = 23-55%) had a negative and 24 (62%; 95% CI = 45-77%) a positive overall repeated open application test (ROAT) result. 13 reacted to the lowest (50 p.p.m.), 8 to the middle (100 p.p.m.) and 3 to the highest concentration (250 p.p.m.) only. In those 13 reacting to the lowest ROAT concentration, dermatitis developed within a few days (1-7). The strength of the initial and the confirmatory patch test result, respectively, and the outcome of the ROAT were positively associated. Of the 24 patients with a use and confirmatory patch test, 15 reacted to 0.1% MDBGN, 16 to 0.2%, 17 to 0.3% and 22 to 0.5%. With the patch test concentration of 0.5%, the number of ROAT-negative patients but patch-test-positive patients increases considerably, particularly due to + reactions. A maximum sensitivity of 94% (95% CI = 70-100%) is reached with a patch test concentration of 0.2%, and is not further improved by increasing the concentration. However, the specificity decreases dramatically from 88 (95% CI = 47-100%) with 0.2% to a mere 12.5% (95% CI = 0-53%) with 0.5%. It can be concluded (a) that for MDBGN 0.2% is very likely the best patch test concentration and (b) that 50 p.p.m. in a leave-on product can elicit contact dermatitis in sensitized persons. We were, therefore, unable to find a safe, still microbicidal, concentration for leave-on products. By contrast, with other contact allergens, dose-response use tests may be able to identify a non-eliciting concentration, which could give valuable clues to a non-inducing (i.e., safe) concentration in products.

Use of CD63 expression as marker of in vitro basophil activation in identifying the culprit in insect venom allergy.

BACKGROUND: The diagnosis of insect venom allergy and the indication for specific immunotherapy is based on history, skin tests and demonstration of hymenoptera venom specific IgE-antibodies. In cases with contradictory test results additional cellular tests are recommended. OBJECTIVE: We evaluated the usefulness of a newly introduced test based on basophil CD63 expression as marker of activation in comparison with the basophil histamine release test (BHR) and the cellular antigen stimulation test (CAST) measuring leukotriene release. METHODS: In 14 patients (10 males, 4 females; age; 12 to 67 years, mean: 42.5 +/- 15.1 years) with systemic reactions to hymenoptera stings in their history skin tests and determination of specific IgE-antibodies (CAP-RAST-FEIA) had shown inconsistent results: No demonstration of specific IgE-antibodies (n = 4), one sting by an unknown insect together with positive skin test and/or positive RAST to both bee and wasp venom (n = 4), several stings of partly unknown insects with positive skin test and/or demonstrable specific IgE-antibodies to more than one insect venom (n = 4), uncertain history and divergent results in skin test and/or RAST (n = 2). BHR, CAST and basophil activation test (BAT) were done according to the manufacturers with negative and positive controls and different concentrations of bee and wasp venom. The BAT is based on double staining with anti-IgE antibodies and anti-CD63 and subsequent determination of the percentage of activated basophils by flow cytometry. RESULTS: BAT and skin test were concordant in 42.9%, BAT and RAST in 57.1%. Concordance of all three cellular tests was seen in 57.1%, of BAT and BHR in 69.1%, of BAT and CAST in 78.6% and of BHR and CAST in 64.3%. In 6 cases where the three cellular tests (BHR, CAST, BAT) were not in accordance the addition of BAT led to a more reliable diagnostic result concerning the relevant insect in 3 cases and added no further information in 3 cases. BAT in controls always was negative. Correlation between CAST and BAT was higher than between CAST and BHR. CONCLUSIONS: In difficult cases of hymenoptera allergy, where history, skin tests and determination of specific antibodies do not allow a clear decision regarding the relevant insect species for immunotherapy, the additional performance of cellular tests (CAST and BAT) may be helpful.

[Allergen immunotherapy - a position paper of the German society for allergology and clinical immunology]. / Die spezifische Immuntherapie (Hyposensibilisierung) mit Allergenen1 - Positionspapier der Deutschen Gesellschaft für Allergologie und klinische Immunologieinhaltlich abgestimmt mit dem Arzteverband Deutscher Allergologen -

Mechanisms of allergen immunotherapy (AIT) are complex inducing numerous immunological effects. Successful AIT is most likely based on a functional switch of and tolerance induction in specific T cells downregulating allergic hypersensitivity and inflammation. Subcutaneous AIT for allergic rhinoconjunctivitis and allergic asthma has been successfully assessed in controlled studies with several clinically important allergens (i. e. birch-, grass- and mugwortpollen, dust mites, animal dander) and has shown convincing clinical efficacy. Considered as the only causal treatment besides allergen avoidance at present, AIT can alter the natural course of allergic diseases. Hymenopteravenom hypersensitivity (to bee- and wasp venom) treated with AIT gives the best results compared to AIT with other allergens. AIT is indicated in patients with IgE-mediated sensitizations and corresponding clinical symptoms to allergens, which do not or hardly permit allergen avoidance and which are available as suitable extracts. Decisions about indication and allergen selection should only be made by a physician with certified training or qualified knowledge and skills in allergology. AIT is administered by physicians experienced in this therapy. After addressing tolerability and present status of health the recommended or individually adjusted does is injected and precisely documented, followed by a mandatory waiting period of 30 minutes. Indication for and application of AIT in children are quite similar compared to the treatment of adults. Children tolerate AIT very well and benefit especially from its immunomodulatory effects. Risk factors for and results of unwanted systemic effects can effectively be minimized by training of the staff members involved, adhering to safety standards and immediate emergency treatment. Modified allergens, recombinant proteins and immunomodulatory adjuvants created by basic research are promises for an improved efficacy of AIT with reduced unwanted effects in the future.

Immunohistochemical characterization of the perivascular infiltrate cells in tissues adjacent to stainless steel implants compared with titanium implants.

Metallic orthopaedics implants are composed of elements that are known to be skin sensitizers in the general population. In this study, we analyzed the cells of perivascular infiltration in the tissue adjacent to titanium (n = 23) and steel (n = 8) implants after explantation of the metals by immunohistochemical methods. The following panel of monoclonal antibodies were used as parameters: CD 1a (Langerhans cells), CD 4 (T-helper cells), CD 8 (T-suppressor cells), CD 11c (monocytes and macrophages), CD 45 RO (memory cells), CD 45 RA (naive cells), eosinophil cationic proteins (ECP), neutrophil elastase, and HLA-DR. The number of perivascular total cells did not differ significantly. All cells were identified in both metal subgroups, but a statistical difference was not seen in the above-mentioned parameters. We conclude that sensitization to metals is possible in the tissue adjacent to steel and titanium implants, because all cells which play an important role in allergic delayed-type hypersensitivity (type IV) reactions are present. This phenomenon may be called a 'pre-sensitization' phase, because no sensitization or allergic reactions were seen in our cases. Second, in the present study, a statistical difference was not seen in the number of infiltrate cells in the tissue adjacent to steel compared with titanium implants.

Immunohistochemical investigation of the cellular infiltrates at the sites of allergoid-induced late-phase cutaneous reactions associated with pollen allergen-specific immunotherapy.

BACKGROUND: Reduction in the size of the allergen-induced late-phase reaction (LPR) is seen as a consequence of successful allergen specific immunotherapy. OBJECTIVE: It was the aim of this study to characterize the cellular infiltrate at the sites of cutaneous LPR that may occur following injection of a depot pollen allergoid (Allergovit(R)) during immunotherapy and thereby determine the immunological nature of the response. METHODS: Punch biopsies were taken 24 h after subcutaneous injection of a depot pollen allergoid from eight patients that showed LPR and a further five patients that did not. Additional biopsies taken 24 h after injection of allergoid-free depot in the same patients served as controls. Immunoenzymatic labelling of the cryostat sections with different antibodies was performed with the APAAP technique. Results were expressed as cells/field (400 x magnification). RESULTS: Similar dermal cellular infiltrations were seen following depot allergoid injections in patients both with and without LPR. Patients with LPR showed statistically significant increases in total cells, CD4+ cells, CD11c+ cells, CD45RO+ cells, CD45RB+ cells and activated eosinophils at the reactions sites as compared with control sites. In patients without LPR CD11c+ cells, HLA-DR+ cells and CD45RA+ T cells increased significantly. CD8+, CD1a+, NP57+, CD23+ and CD25+ cells did not differ significantly in either group. CONCLUSION: These results indicate that activation of T cells, monocytes/macrophages and eosinophils at the sites of LPR following injection of depot allergoid are comparable with those following injection of allergen. Even in the absence of a cutaneous LPR, subsets of T cells and monocytes/macrophages increased. These cell activations may reflect events associated with the mechanisms of allergoid-based specific immunotherapy, and suggest that at least part of the late-phase reaction may be independent of IgE.

Suppression of immediate-type hypersensitivity elicitation in the skin prick test by ultraviolet B irradiation.

Reproducibility of skin prick testing (SPT) and its modulation by ultraviolet B (UVB) radiation is of clinical interest. Sensitized atopic volunteers (groups A and B, n=21) were prick tested with common commercial allergen solutions (undiluted, diluted 1:10 and diluted 1:100) before, 24 h after one and 24 h after three suberythematous UVB irradiations. Volunteers in group A (n=8) received local UVB irradiation of prick test areas, whereas volunteers in group B (n=13) received whole body UVB irradiation, with prick test areas covered. In group A, the wheal intensities, expressed as the ratio allergen wheal size to histamine wheal size, were decreased by 28% (1:10 dilution) (P=0.01) and 45% (1:100 dilution) (P=0.02) after one UVB irradiation. Flare intensities were decreased by 48% (1:10 dilution) (P=0.03) after three UVB irradiations. In group B, the wheal and flare responses tended to decrease. Possible mechanisms of this short-term suppressive effect of UVB irradiation on SPT reactions include a direct effect on mast cells. It is concluded that UV irradiation, even a single exposure, prior to skin testing may compromise the validity of SPT testing.

National rates and regional differences in sensitization to allergens of the standard series. Population-adjusted frequencies of sensitization (PAFS) in 40,000 patients from a multicenter study (IVDK).

Sensitization rates to contact allergens vary between centers and are influenced by sex and age. Eliminating the latter 2 factors by standardization of data by age and sex, the present analysis addresses possible differences between centers remaining after elimination of these confounders, and analyzes other factors which might influence rates, e.g., the MOAHL index. Overall standardized rates were well within the range reported in previous studies and may be regarded as representing the rates of the "patch test population" in Central Europe (e.g., nickel sulfate 12.9%, fragrance mix 10.5%, balsam of Peru 7.3%, thimerosal 5.6%). For this analysis, data of those departments which contributed more than 2000 patients, or of those with extreme proportions concerning sex, age and occupational cases were selected. Patients from these 10 departments differed considerably with regard to the items of the MOAHL index and with regard to standardized rates. The items of the MOAHL index proved to be suitable for describing different patch test populations and for explaining some differences between centers. Only 'atopic dermatitis' seems to have little influence on (standardized) rates. Face dermatitis is not yet represented in the MOAHL index, but should be included, together with age > 40 years, in an extended index (acronym: MOAHLFA). Regional allergen exposure (with striking differences between East Germany, West Germany and, to a lesser extent, Austria) seems to have a great influence on the sensitization pattern observed in a department. In addition, sociological factors may influence sensitization rates, which is exemplified by high rates of nickel allergy in a socially defined subgroup. Future studies should focus on these factors, as well as on factors concerning patch test practices and quality control.

Immunohistochemical analysis of late local skin reactions during rush venom immunotherapy.

During rush venom immunotherapy (VIT), about 65% of patients develop large local reactions (LLR) at the application site that last for at least 24 h. However, LLR subside during long-term treatment. To learn more about the provenance of infiltrating cells in late, local skin reactions during VIT, we analyzed the skin infiltrates of 23 Hymenoptera venom (HV)-allergic patients. Punch biopsies were obtained 24 h after s.c. injection of HV allergens from 23 HV-allergic patients and five nonallergic controls. Seven patients did not show LLR at the beginning of VIT. Ten patients had LLR when the dose of HV allergens was increased. Six patients showed reduced LLR after long-term treatment. Immunoenzymatic labeling of the cryostal sections with a panel of monoclonal antibodies was performed by the APAAP method. S.c. application of HV allergens induced a perivascular and periadnexial cutaneous mononuclear cell infiltrate consisting mainly of CD4+, CD45RO+; and HLA-DR+ cells in patients without clinically apparent LLR. In contrast, LLR were associated with a significant increase in total cells, CD4+ cells, CD8+ cells, CD11c+ cells, EG2+ cells, NP57+ cells, HLA-DR+ cells, CD45RO+ cells, CD45RA+ cells, CD23+ cells and CD25+ cells (P < 0.001). Decreased LLR after long-term VIT was correlated with a significantly reduced recruitment of CD4+ cells, EG2+ cells, and CD23+ cells as compared to LLR in the course of dose increases (P < 0.05), whereas the number of CD8+ cells, CD11c+ cells, NP57+ cells, and CD25+ cells remained high. Our data suggest that s.c. injections of HV allergens attract CD4+ helper T cells, of both the naive (CD45RA+) and memory (CD45RO+) phenotypes, to the allergen application site. LLR represent delayed allergic rather than toxic reactions to HV components and might be relevant to the development of clinical protection during VIT.

[Reversible increase in photosensitivity to UV-B caused by St. John's wort extract]. / Reversible Erhöhung der Photosensitivität im UV-B-Bereich durch Johanniskrautextrakt-Präparate.

A 61-year-old woman with depression developed recurring elevated itching erythematous lesions in light-exposed areas after taking St. John's Wort-extract for three years. Routine patchtesting did not reveal any relevant reactions and photopatch testing was negative. Using a systemic oral photoprovocation test with St. John's Wort, we were able to demonstrate a decrease of the MED-UVB which was reversible after withdrawal of the medication.

Successful treatment of dyshidrotic hand eczema using tap water iontophoresis with pulsed direct current.

The efficacy of tap water iontophoresis in treating palmoplantar hyperhidrosis has been sufficiently documented and has led to its extensive use in clinical practice. In order to test the efficacy of this treatment modality in cases of dyshidrotic hand eczema, 20 patients were treated with tap water iontophoresis in addition to two-sided steroid-free topical therapy in a randomized half-side-study. A special score for dyshidrotic eczema including objective and subjective criteria was developed to document the success of the therapy. Only those sides treated with tap water iontophoresis showed significant improvement. This significant effect of iontophoresis indicates the efficacy of this treatment in cases of dyshidrotic hand eczema.

The disturbance of epidermal barrier function in atopy patch test reactions in atopic eczema.

Atopic eczema (AE) is a common skin disorder. Eczematous lesions showing macroscopic, microscopic and immunopathological resemblance to lesional AE can be induced by aeroallergens by epicutaneous testing (atopy patch test, APT). Altered epidermal barrier function, as determined by transepidermal water loss (TEWL), is a typical feature of patients with AE. The present investigation was performed to define the differences in the epidermal barrier function between positive APT reactions to aeroallergens and positive patch test reactions to contact allergens in AE patients. Allergen extracts from grass pollen, birch pollen, cat dander and house dust mite (Dermatophagoides pteronyssinus) were applied in large Finn chambers on Scanpor for 48 h on the clinically unaffected and untreated skin of the back, in 11 patients with AE. The same procedure was done with 27 contact allergens of a standard test battery. Test reactions were read and TEWL was measured after 48 and 72 h. Eight of the 11 patients developed positive APT reactions to D. pteronyssinus, two to cat dander and one to birch pollen. Seven of the 11 patients showed positive patch test reactions to nickel sulphate, two to potassium dichromate, one to thiuram-mix and one to paraphenylenediamine. Vehicle controls were negative. The TEWL of the positive APT reactions was significantly higher, both after 48 h (mean +/- standard deviation 10.0 +/- 6.5 g/m2h) and after 72 h (9.7 +/- 5.4 g/m2h) as compared with the control site (48/72h: 4.4 +/- 1.5/4.1 +/- 1.4 g/m2h) (P < 0.01). In contrast, TEWL of the positive patch test reactions to contact allergens (48/72 h: 5.4 +/- 2.2/5.4 +/- 1.9 g/m2h) was similar to that of the control site (48/72 h: 5.2 +/- 2.1/5.0 +/- 1.8 g/m2h) (not significant). The relative TEWL at 48 h and 72 h, expressed as the ratio between the positive patch test and the control site, was significantly higher in the positive APT reactions (48/72 h: 218.8 +/- 80.4%/232.0 +/- 85.9%) compared with positive patch test reactions to contact allergens (48/72 h: 102.1 +/- 12.0%/107.1 +/- 9.5%) (P < 0.01). It is concluded that the epidermal barrier function in AE patients is altered only in positive APT reactions, in contrast to positive patch test reactions to contact allergens. As a consequence of this aeroallergen-induced altered epidermal barrier function, further allergens can more easily penetrate the skin, inducing a vicious circle and perpetuating the eczematous lesions.

Design of an optimally-diagnostic skin test solution for diagnosis of sensitivity to timothy grass (Phleum pratense) pollen.

BACKGROUND: Although most of the common allergen extracts that are used for diagnosis of type 1 hypersensitivity are now well standardized, this gives no assurance that they are within the concentration range that gives the best chance of a true diagnosis. OBJECTIVE: The objective of this study was to identify the most appropriate concentration range of timothy grass pollen Phleum pratense extract to diagnose sensitivity to this pollen correctly through skin-testing. METHODS: Dilutions of a well-standardized extract were made and used to skin test "true' positive and "true' negative populations of subjects as identified by case history, challenge tests and radioallergosorbent test (RAST). Weal diameters were measured and the data were submitted to receiver operating characteristics (ROC) analysis. For any particular weal size cut-off, the optimal diagnostic concentration (ODC) range was thus calculated. RESULTS: A 3 mm weal diameter cut-off was chosen as an appropriate size for routine diagnosis. Therefore the ODC range at this diameter was used to establish a product target concentration and specification for formulation of the diagnostic reagent. This method of allergen extract standardization can lead to a true-biological unitage that can be used for labelling purposes. CONCLUSION: The optimum concentration range at which to formulate an allergen extract, in terms of an in vitro immunologically based assay, can be determined by carrying out ROC analysis of the results of clinical studies as described in this communication. Diagnostic units (DU), are now used by us for labelling of such final formulations which conveys the information that the product is at the most appropriate concentration for diagnosis.