During a gluten free diet EMA and AGA disappear. Their presence or absence is therefore an indicator of dietary compliance. After reintroduction of gluten into the diet 110/134 (82%) of the patients who had a flat mucosa at diagnosis relapsed, but 24/134 still had a normal mucosa after 2-15 years of challenge. AU these patients without a morphological relapse were less than 2 years old at diagnosis so we conclude that patients who are young at diagnosis should be challenged. AGA often reappear earlier than EMA. After one month of challenge 93% of patients are AGA and 69% EMA positive. After more than three years of gluten intake the percentage of AGA positive patients decreased to about 50% whereas the percentage of EMA positive sera was then highest (93%). Therefore EMA are more sensitive for the detection of 'silent' relapse after prolonged periods of gluten intake.

BackgroundWe have developed a recombinant B cell epitope-based vaccine (BM32) for allergen-specific immunotherapy (AIT) of grass pollen allergy. The vaccine contains recombinant fusion proteins consisting of allergen-derived peptides and the hepatitis B surface protein domain preS as immunological carrier.MethodsWe conducted a randomized, double-blind, placebo-controlled AIT study to determine safety, clinical efficacy and immunological mechanism of three subcutaneous injections of three BM32 doses adsorbed to aluminum hydroxide versus aluminum hydroxide (placebo) applied monthly to grass pollen allergic patients (n = 70). Primary efficacy endpoint was the difference in total nasal symptom score (TNSS) through grass pollen chamber exposure before treatment and 4 weeks after the last injection. Secondary clinical endpoints were total ocular symptom score (TOSS) and allergen-specific skin response evaluated by titrated skin prick testing (SPT) at the same time points. Treatment-related side effects were evaluated as safety endpoints. Changes in allergen-specific antibody, cellular and cytokine responses were measured in patients before and after treatment.ResultsSixty-eight patients completed the trial. TNSS significantly decreased with mean changes of − 1.41 (BM32/20 μg) (P = 0.03) and − 1.34 (BM32/40 μg) (P = 0.003) whereas mean changes in the BM32/10 μg and placebo group were not significant. TOSS and SPT reactions showed a dose-dependent decrease. No systemic immediate type side effects were observed. Only few grade 1 systemic late phase reactions occurred in BM32 treated patients. The number of local injection site reactions was similar in actively and placebo-treated patients. BM32 induced highly significant allergen-specific IgG responses (P < 0.0001) but no allergen-specific IgE. Allergen-induced basophil activation was reduced in BM32 treated patients and addition of therapy-induced IgG significantly suppressed T cell activation (P = 0.0063).ConclusionThe B cell epitope-based recombinant grass pollen allergy vaccine BM32 is well tolerated and few doses are sufficient to suppress immediate allergic reactions as well as allergen-specific T cell responses via a selective induction of allergen-specific IgG antibodies. (ClinicalTrials.gov number, NCT01445002.)

BackgroundGrass pollen is one of the most important sources of respiratory allergies worldwide.ObjectiveThis study describes the development of a grass pollen allergy vaccine based on recombinant hypoallergenic derivatives of the major timothy grass pollen allergens Phl p 1, Phl p 2, Phl p 5, and Phl p 6 by using a peptide-carrier approach.MethodsFusion proteins consisting of nonallergenic peptides from the 4 major timothy grass pollen allergens and the PreS protein from hepatitis B virus as a carrier were expressed in Escherichia coli and purified by means of chromatography. Recombinant PreS fusion proteins were tested for allergenic activity and T-cell activation by means of IgE serology, basophil activation testing, T-cell proliferation assays, and xMAP Luminex technology in patients with grass pollen allergy. Rabbits were immunized with PreS fusion proteins to characterize their immunogenicity.ResultsTen hypoallergenic PreS fusion proteins were constructed, expressed, and purified. According to immunogenicity and induction of allergen-specific blocking IgG antibodies, 4 hypoallergenic fusion proteins (BM321, BM322, BM325, and BM326) representing Phl p 1, Phl p 2, Phl p 5, and Phl p 6 were included as components in the vaccine termed BM32. BM321, BM322, BM325, and BM326 showed almost completely abolished allergenic activity and induced significantly reduced T-cell proliferation and release of proinflammatory cytokines in patients' PBMCs compared with grass pollen allergens. On immunization, they induced allergen-specific IgG antibodies, which inhibited patients' IgE binding to all 4 major allergens of grass pollen, as well as allergen-induced basophil activation.ConclusionA recombinant hypoallergenic grass pollen allergy vaccine (BM32) consisting of 4 recombinant PreS-fused grass pollen allergen peptides was developed for safe immunotherapy of grass pollen allergy.

The solvent effect on the NMR chemical shielding in liquid water is calculated from a combination of molecular dynamics simulations and quantum chemical calculations for protons and 1 7 0 . The simulations are performed with three different potentials, ab initio as well as empirical ones, to study the influence of the force field. From the liquid configurations obtained in these simulations, molecules are randomly chosen together with neighbouring molecules to give clusters of water typical for the liquid at the selected temperature and density. Different cluster sizes are studied. The clusters are treated as supermolecules in quantum chemical calculations of chemical shifts by sumover-states density functional perturbation theory with individual gauge for localised orbitals. The influence of the quantum chemical method is studied with