Boiling down the cysteine-stabilized LTP fold - loss of structural and immunological integrity of allergenic Art v 3 and Pru p 3 as a consequence of irreversible lanthionine formation.

BACKGROUND: Non-specific lipid transfer proteins (LTPs) are important allergens in fruits, pollen, vegetables, nuts and latex. Due to their compact structure, LTPs are highly resistant to heat treatment. Here, Art v 3 from mugwort pollen and Pru p 3 from peach were used as model allergens to in-depth investigate structural and immunological properties upon thermal treatment at different buffer conditions. METHODS: Recombinant Art v 3 and Pru p 3 were purified from E. coli and incubated at 95 °C up to 120 min using sodium phosphate buffer pH 3.4 or 7.3. Physicochemical properties of allergens were analyzed in circular dichroism spectroscopy, Fourier transform infrared spectroscopy, dynamic light scattering, size exclusion chromatography, and mass spectrometry. The crystal structure of Art v 3.0201 was determined to 1.9 Šresolution. IgG and IgE binding was investigated in ELISA using murine and LTP allergic patients' sera. RESULTS: Highly pure and homogenous recombinant allergens were obtained from bacterial production. The crystal structure of Art v 3.0201 revealed an antiparallel four helix bundle with a C-terminal extension mediating an asymmetric, transient dimer interface and differently sized cavities. Both allergens showed high thermal stability at acidic conditions. In contrast, extensive heat treatment in neutral buffer induced irreversible structural changes due to lanthionine-based cysteine rearrangement. This fostered loss of the typical α-helical structure, increased molecular size and abrogation of IgG and IgE binding epitopes. Pru p 3 lost its structural integrity at shorter heat stress duration than Art v 3, which did however only partially affect the molecule's IgE binding epitopes. CONCLUSION: During thermal treatment, susceptibility to structural changes of the LTP-fold is highly dependent on the surrounding environment but also on intrinsic features of individual LTPs. This is a crucial fact to consider when processing LTP-containing food or food products as this will directly influence their allergenic potential.

Do Plantago lanceolata Skin Prick Test-Positive Patients Display IgE to Genuine Plantain Pollen Allergens? Investigation of Pollen Allergic Patients from the North-East of France.

BACKGROUND: English plantain (Plantago lanceolata) is an important weed pollen allergen source triggering allergic symptoms during summer. To elucidate genuine versus cross-reactive sensitization, we investigated IgE reactivity patterns and inhibition capacities of plantain-sensitized patients. METHODS: Sera of 35 rhinoconjunctivitis patients from the north-east of France with positive skin prick tests (SPT) to Plantago lanceolata pollen were tested with clinically relevant allergen sources using ELISA, ImmunoCAP, and immunoblot inhibition. RESULTS: The patients were multisensitized with additional reactivity to grass (94.3%), ash (74.3%), birch (71.4%), and mugwort (55.2%) pollen in SPT. Sensitization prevalence to allergen molecules was 34.3% (Pla l 1), 94.3% (Phl p 1/5), 60.0% (Ole e 1), 65.7% (Bet v 1), 37.1% (profilin), and 40.0% (CCD). In immunoblot, IgE reactivity to plantain pollen was inhibited with relevant pollen extracts and purified rPla l 1. Two sera did not reveal any IgE cross-reactivity, while reactivity to plantain was efficiently inhibited by grass pollen in the sera of 10 patients. The sera from 17 different patients could be inhibited by grass, birch, or ash pollen to varying degrees. Thus, only 37.1% of our patients demonstrated true plantain pollen sensitization, while 62.9% were solely positive due to IgE cross-reactive molecules from other clinically relevant pollen. CONCLUSIONS: Plantain pollen-sensitized patients are multi-reactors demonstrating varying and complex IgE-reactivity profiles. In vivo and in vitro tests using extracts are typically blurred due to the presence of homologous allergens or CCD in grass, birch, or ash pollen. So far, Pla l 1 represents the only indicative marker allergen for the diagnosis of genuine plantain pollen sensitization.

Endolysosomal Degradation of Allergenic Ole e 1-Like Proteins: Analysis of Proteolytic Cleavage Sites Revealing T Cell Epitope-Containing Peptides.

Knowledge of the susceptibility of proteins to endolysosomal proteases provides valuable information on immunogenicity. Though Ole e 1-like proteins are considered relevant allergens, little is known about their immunogenic properties and T cell epitopes. Thus, six representative molecules, i.e., Ole e 1, Fra e 1, Sal k 5, Che a 1, Phl p 11 and Pla l 1, were investigated. Endolysosomal degradation and peptide generation were simulated using microsomal fractions of JAWS II dendritic cells. Kinetics and peptide patterns were evaluated by gel electrophoresis and mass spectrometry. In silico MHC (major histocompatibility complex) class II binding prediction was performed with ProPred. Cleavage sites were assigned to the primary and secondary structure, and in silico docking experiments between the protease cathepsin S and Ole e 1 were performed. Different kinetics during endolysosomal degradation were observed while similar peptide profiles especially at the C-termini were detected. Typically, the identified peptide clusters comprised the previously-reported T cell epitopes of Ole e 1, consistent with an in silico analysis of the T cell epitopes. The results emphasize the importance of the fold on allergen processing, as also reflected by conserved cleavage sites located within the large flexible loop. In silico docking and mass spectrometry results suggest that one of the first Ole e 1 cleavages might occur at positions 107-108. Our results provided kinetic and structural information on endolysosomal processing of Ole e 1-like proteins.

Influence of Intrinsic and Lifestyle Factors on the Development of IgE Sensitization.

BACKGROUND: IgE sensitization is a prerequisite for the development of allergic symptoms. The investigation of factors influencing the development of IgE is therefore crucial for understanding the onset of allergic diseases. METHODS: This epidemiological study investigated personal, intrinsic, and lifestyle factors in a nonselected cohort of 501 Austrian adolescents (aged 12-21 years). IgE levels to 112 allergen molecules were analyzed in the serum of participants using the ImmunoCAP ISAC®. Allergic sensitization, IgE levels to single allergens, and ISAC score sums were correlated with results obtained from a questionnaire. RESULTS: In this adolescent cohort, male participants showed a higher sensitization frequency (56.8%) compared to females (50.9%) and significantly increased IgE levels to profilins. Underweight subjects demonstrated a stronger IgE sensitization. Family size inversely correlated with IgE levels to PR-10 allergens, and predominately paternal allergies were a predictive factor for IgE sensitization in the children. Vaccination, breastfeeding, and delivery mode showed no influence, while a highly protective effect was observed for growing up on a farm. Of all of the investigated lifestyle factors, only smoking significantly influenced the risk for IgE development. Participants with moderate frequencies of colds showed increased sensitization levels. CONCLUSION: A hereditary predisposition and lifestyle factors such as a farming environment, smoking, family size, body weight, or frequency of colds significantly influenced the development of allergen-specific IgE in this cohort of adolescents.

Exposure to Indoor Allergens in Different Residential Settings and Its Influence on IgE Sensitization in a Geographically Confined Austrian Cohort.

BACKGROUND: Exposure to indoor allergens is crucial for IgE sensitization and development of allergic symptoms. Residential settings influence the allergen amount in house dust and hence allergic sensitization. Within this study, we investigated allergen exposure and molecule-based IgE levels in a geographically confined region and evaluated the impact of housing, pets and cleaning. METHODS: 501 adolescents from Salzburg, Austria participated in this cross-sectional study. House dust samples were examined regarding major mite, cat, dog, and mold allergens using a multiplex assay. Serum samples of participants were analyzed for specific IgE to Der p 1, Der p 2, Fel d 1, Can f 1 and Alt a 1 using the multiplex array ImmunoCAP ISAC. Information on allergies, living areas, dwelling form (house, flat, farm), pets, and household cleanliness were obtained by a questionnaire. RESULTS: In investigated house dust samples, the concentration of cat allergen was highest while the prevalence of mold allergens was very low. Participants showed IgE sensitization to Der p 1 (13.2%), Der p 2 (18.2%), Fel d 1 (14.4%), Can f 1 (2.4%) and Alt a 1 (2.0%). In alpine regions, lower mite allergen concentrations were detected which correlated with reduced IgE levels. A trend for increased sensitization prevalence from rural to alpine to urban regions was noted. Living on farms resulted in lower sensitization prevalence to mite and cat allergens, even though exposure to mites was significantly elevated. The presence of cats was associated with a lower sensitization rate and IgE levels to cat and mite allergens, and less frequent allergic diseases. Cleaning did not impact allergen concentrations, while IgE reactivity to mites and allergic diseases were more pronounced when living in cleaner homes. CONCLUSION: Allergen exposure to indoor allergens was influenced by setting of homes. Living in a farm environment and having a cat at home showed a protective effect for IgE sensitization and allergies. This cross-sectional study in combination with hereditary and lifestyle factors enables development of risk schemes for a more efficient management and potential prevention of allergic diseases.

Marker allergens of weed pollen - basic considerations and diagnostic benefits in the clinical routine: Part 16 of the Series Molecular Allergology.

The term weed is referring to plants used as culinary herbs and medicinal plants as well as ecologically adaptive and invasive segetal plants. In Europe, pollen of ragweed, mugwort, English plantain and pellitory are the main elicitors of weed pollen allergies. Presently, 35 weed pollen allergens have been identified. The most relevant belong to the protein families of pectate lyases, defensin-like proteins, non-specific lipid transfer proteins, and Ole e 1-like proteins. The sensitization frequency depends on geographic regions and might affect more than 50 % of pollen allergic patients in distinct regions. Due to overlapping flowering seasons, similar habitats, polysensitizations and cross-reactive (pan)-allergens, it is difficult to diagnose genuine weed pollen sensitization using pollen extracts. Marker allergens for component-resolved diagnostics are available for the important weed pollen. These are Amb a 1 (ragweed), Art v 1 (mugwort), Pla l 1 (English plantain) and Par j 2 (pellitory). Molecule-based approaches can be used to identify the primary sensitizer and thus enable selection of the appropriate weed pollen extracts for allergen immunotherapy.