Measurement of Gluten in Food Products: Proficiency‐Testing Rounds as a Measure of Precision and Applicability

In 2008, Codex Alimentarius endorsed the R5 Enzyme‐Linked Immunosorbent Assay (ELISA) method as Method Type 1 for gluten measurement in gluten‐free foods. The most recognized R5 ELISA test kit is the RIDASCREEEN® Gliadin (R7001; manufacturer R‐Biopharm). Beside collaborative tests that led to several international approved methods of this test kit, proficiency‐testing (PT) rounds are regularly performed in Europe by different PT providers. Results from these rounds were analyzed regarding the number of participating labs with acceptable results for the RIDASCREEN® Gliadin. All PT rounds document the excellent consistency and comparability of results. The data show that the RIDASCREEN® Gliadin R5 ELISA is also applicable to cake mix, oat‐based foodstuff, infant soya formula, cookies, canned boiled sausage, gravy thickener, pasta, and potato dumpling. These rounds also included the analysis of blank matrices. It was found that more than 95% of all participating laboratories correctly detected these samples as negative. Other gluten test kit manufacturers were analyzed as well, but due to the low number of participants using these test kits results were often only analyzed in a qualitative manner questioning the comparability of these kits to the RIDASCREEN® Gliadin R5 ELISA

Epicutanoeus immunotherapy as a novel prophylactic and therapeutic strategy for birch pollen allergy

Die Entwicklung von komfortablen, effektiven und sicheren Allergen-spezifischen Immuntherapien(SIT) für die Behandlung von Birkenpollenallergie, einer der häufigsten Formen von pollen-assoziierten Allergien in Nordeuropa, Nordamerika und Nordjapan, ist besonders wichtig. Epikutane Immuntherapie (EPIT) wird als sichere und nicht invasive Alternative für die konventionelle subkutane Immuntherapie (SCIT) gehandelt. Jedoch zeigen klinische Studien eine limitierte Effektivität der EPIT. Aus diesem Grund besteht die Notwendigkeit der Verbesserung dieser Therapie-Methode. In der vorliegenden Arbeit wurde die Hypothese aufgestellt, dass eine Strategie bestehend aus der Kombination von einem Hypoallergen und einem geeigneten Adjuvant zur Verbesserung der EPIT führen könnte. Um diese Hypothese zu verifizieren wurde die Effektivität von einer epikutanen Behandlung mit rBet v 1, dem Major Allergen der Birke zusammen mit einem Toll-like Rezeptor (TLR) Agonisten untersucht. Dabei wurde die Kombination in einem prä-klinischen Mausmodell eines Birkenpollen-induziertem Asthmas in einem prophylaktischen und einem therapeutischen Versuchsansatz eingesetzt. Des Weiteren wurde die Effektivität von rBet v 1B2, einer hypoallergenen rekombinaten Variante des Wild-Typ Allergens Bet v 1, als therapeutisches Allergen in der epikutanen Immunisierung (EPI) im Mausmodell untersucht. TLR erkennen konservierte mikrobielle Moleküle (wie PAMPs) und sind bekannt für die Gegenregulation von TH2 Antworten durch die Induktion von TH1-Typ und/oder regulatorischen Zytokinen durch Immunzellen. Das Hypoallergen Bet v 1B2 ist eine Faltungsvariante des Wild-Typ Allergens Bet v 1 mit reduzierter Allergenität, jedoch ist dieT-Zell Immunogenität erhalten geblieben. Die geringe Allergenität könnte es erlauben, das Hypoallergen in höheren Dosen während der Immuntherapie zu verwenden, da es weniger negative Seiteneffekte auslösen sollte. Höhere Dosen erlauben zudem eine effektivere Behandlung um T-Zell Antworten zu regulieren. In dieser Arbeit wurde zuerst die Proteinexpression der rekombinanten Moleküle Bet v 1 und Bet v 1B2 optimiert. Verglichen mit natürlichen Proteinen bieten rekombinante Proteine die Möglichkeit, genau charakterisierte Moleküle mit einer konsistenten pharmazeutischen Qualität in der Immuntherapie zu verwenden. Durch einen Vergleich verschiedener Escherichia coli-Stämme war es möglich ein optimales Expressionssystem zu entwickeln. Eine Kombination mit einer immobilisierten Metal-Chelat Affinitäts-Chromatographie (IMAC)und einer Größenaufschluss Chromatographie (SEC) erlaubte die Produktion von großen Mengen der Proteine Bet v 1 und Bet v 1B2 mit einer hohen Reinheit. Das allergene Potenzial von rBet v 1 und die hypoallergenen Eigenschaften von rBet v 1B2 wurden durch die Bestimmung der IgE-Reaktivität nach Immunisierung von Mäusen in einem ELISA bestätigt. Zudem wurden beide Parameter mit der Mediator-Freisetzung in Ratten-basophilen und in einem humanen Basophil-Aktivierungs-Test verifiziert. In einem zweiten Teil der vorliegenden Arbeit wurde ein murines Modell des Birkenpolleninduziertem Asthmas etabliert. Es konnte gezeigt werden, dass eine Kombination aus intraperetonealer Sensibilisierung mit einer optimalen Dosis von rBet v 1 und die intranasale Provokation mit Birkenpollen-Extrakt zu erhöhten IgE-Leveln, Atemwegs-Eosinophilie und pulmonaler Entzündung in BALB/c-Mäusen führte. Die klinische Ausprägung dieser Symptome ist vergleichbar mit denen eines Patienten mit allergischem Asthma. Diese Symptome im Mausmodell bestärken die prä-klinische Anwendung des Mausmodells zu Untersuchung der Effektivität bei der Behandlung von Birkenpollen-Allergie. Als nächstes wurde der Effekt der Adjuvantien Polyadentilic:Polyuridylic Säure (Poly(A:U)), einem TLR3 Agonisten und R848 (Resiquimod), einem TLR7 Agonisten in einer prophylaktischen Behandlung von Birkenpollen-Allergie mit Bet v 1 in EPI untersucht. Hier wurde die Hypothese aufgestellt, dass TLR3 und TLR7 potenzielle Ziel-Rezeptoren für die Steigerung von positiven Effekten in EPI durch die Adjuvant-Wirkung darstellen. Diese Rezeptoren werden in Langerhans-Zellen und dermalen Dendritischen Zellen, persistente Antigen-präsentierende Zellen in Hautgewebe, exprimiert. BALB/c Mäuse werden mit einer EPI über acht Wochen behandelt. Den Tieren wurde ein mal pro Woche ein Pflaster mit rBet v 1 oder einer Kombination aus rBet v 1 und Poly(A:U) oder R848 auf den enthaarten Rücken geklebt. Die epikutan behandelten Mäuse wurden danach mit rBet v 1 in ALUM über drei Wochen intraperetoneal sensibilisiert gefolgt von einer intranasalen Provokation mit Birkenpollen-Extrakt an drei aufeinander folgenden Tagen. Die prophylaktische Behandlung mit EPI bestehend aus rBet v 1 und R848 inhibierte die Produktion von Bet v 1-spezifischen IgE Antikörpern nach Sensibilisierung, unterdrückte die pulmonale Entzündung und die Atemwegshyperreagibilität nach der Provokation. Im Unterschied zu R848, zeigte das Adjuvant Poly(A:U) keine Wirkung auf die Unterdrückung von asthmatischen Symptomen. Die Ergebnisse weisen darauf hin, dass R848, jedoch nicht Poly(A:U) als potentielles Adjuvant in einer prophylaktischen Behandlung mit EPI verwendet werden kann um Birkenpolleninduziertes Asthma zu behandeln. In einem finalen Experiment wurde das therapeutische Potenzial einer EPI mit rBet v 1, rBet v 1B2 allein oder in Kombination mit R848 untersucht. Nach Sensibilisierung mit Bet v 1 und Provokation mit Birkenpollen-Extrakt wurden die Mäuse mit einer 8-wöchigen therapeutischen EPI mit rBet v 1, rBet v 1B2 allein oder in Kombination mit R84 behandelt und danach wieder mit Birkenpollen-Extrakt provoziert. Die Tiere wurden nach der ersten Provokation auf Entwicklung von Bet v 1-spezifischem IgE und die Entwicklung einer Atemwegs-Hyperreagibilität untersucht. Alle Mäuse zeigten einen signifikant erhöhten Level an IgE und eine signifikant erhöhte Atemwegsreagibilität verglichen zu unbehandelten Kontrolltieren. Nach der therapeutischen Behandlung mit Bet v 1B2 konnten der etablierte Level an Bet v 1-spezifischem IgE unterdrückt werden, die Atemwegshyperreagibilität wurde signifikant reduziert. Auch die pulmonale Entzündung konnte verglichen mit Positiv-Kontrolltieren reduziert werden. Eine therapeutische Behandlung mit dem rekombinanten Wild-Typ Allergen Bet v 1 zeigte keinen Einfluss auf diese Schlüssel-Symptome des allergischen Asthmas. Im Kontrast zu den Ergebnissen der prophylaktischen Behandlung mit rBet v 1 in Kombination mit R848 konnte hier kein therapeutischer Vorteil von R848 gefunden werden. Dieser Effekt könnte an der hohen Anzahl an Behandlungen liegen, eine Reduktion der Anzahl könnte den vorteilhaften Effekt herausarbeiten. Jedoch zeigen die Resultate, dass rBet v 1B2 als potenzielles therapeutisches Allergen bei der Behandlung von Birkenpollen-Allergie eingesetzt werden kann.The development of a convenient, effective and safe allergen-specific immunotherapy (SIT) for birch pollen allergy, one of the most prevalent allergic diseases in Northern Europe, North America and Northern Japan, is of crucial importance. Epicutaneous immunotherapy (EPIT) has gained attention as a safe and non-invasive alternative for subcutaneous immunotherapy, a conventional SIT. However, clinical studies showed a limited effcacy of EPIT, indicating the necessity of improvement of the treatment regime. In this study, we hypothesized that a combination of a hypoallergen with an appropriate adjuvant could be a strategy to improve EPIT. To verify this hypothesis, we aimed at investigating the efficacy of epicutaneous treatment with rBet v 1, the major birch pollen allergen, plus Toll-like receptor (TLR) agonists for prophylaxis and therapy of birch pollen allergy using a murine model of birch pollen-induced allergic asthma. Furthermore, the efficacy of rBet v 1B2, a hypoallergenic variant of Bet v 1, as a therapeutic allergen in EPI was pre-clinically investigated. TLRs recognize conserved microbial molecules (like PAMPs), and are known to promote the counter-regulation of TH2 responses by the induction of TH1-type and/or regulatory cytokines by immune cells. The hypoallergen Bet v 1B2 is a folding-variant of the wild-type allergen rBet v 1 with reduced allergenicity, but retained T-cell immunogenicity. The low allergenicity, could allow the application of hypoallergens in higher doses, and therefore provide a safer and more effective treatment to regulate T-cell immune responses. First, the expression and purification of recombinant Bet v 1 and Bet v 1B2 was optimized. Compared to natural proteins, recombinant proteins offer the possibility to use well-defined molecules with a consistent pharmaceutical quality. Using optimal Escherichia coli expression strains in combination with immobilized metal chelate affinity chromatography (IMAC) and size exclusion chromatography (SEC), we successfully prepared a large amount of rBet v 1 and rBet v 1B2 with a high purity. The allergenic potency of rBet v 1 and the hypoallergenic characteristics of rBet v 1B2 were confirmed by measurement of IgE reactivity and mediator release capacity using ELISA and basophil activation tests, respectively. In a second part, a murine model of birch pollen-induced allergic asthma was established. It was shown that intraperetoneal sensitization with an optimal dose of rBet v 1 and intranasal challenge with birch pollen extract induced elevated IgE levels, airway eosinophilia and pulmonary inflammation in BALB/c mice. The clinical features are comparable to those in patients with allergic asthma, indicating that sensitized and challenged mice could be used for a pre-clinical study to assess the efficacy of the treatment for birch pollen allergy. Next, we investigated the adjuvant effects of Polyadenylic:polyuridylic acid (Poly(A:U)), a TLR3 agonist, and R848 (resiquimod), a TLR7 agonist, in prophylactic EPI with rBet v 1 to intervene with birch pollen allergy. Here, we hypothesized that TLR3 and TLR7 could be possible target receptors to induce adjuvant effects in EPI, since these receptors are expressed in Langerhans cells and dermal dendritic cells, persistent antigen presenting cells in the cutaneous tissues. BALB/c mice received EPI with rBet v 1 alone, or plus Poly(A:U), or R848 on their depilated back using patches. Mice treated epicutaneously were then sensitized with rBet v 1 plus ALUM and intranasally challenged with birch pollen extract. We found that prophylactic EPI with rBet v 1 plus R848 inhibited the production of Bet v 1-specific IgE antibodies in sensitization, suppressed pulmonary inflammation and airway hyperreactivity upon challenge. In contrast to R848, no adjuvant effect of Poly(A:U) on suppression of asthmatic features was observed. Our results indicated that R848, but not Poly(A:U), could be a potential adjuvant for prophylactic EPI of birch pollen induced allergic asthma. Finally, the therapeutic potency of EPI with rBet v 1, or rBet v 1B2 alone, or plus R848 was assessed. After sensitization and challenge, mice received therapeutic EPI with rBet v 1 alone, or plus R848, and re-challenge with birch pollen extract. We found that therapeutic treatment with Bet v 1B2 reduced established Bet v 1-specific IgE antibodies, pulmonary inflammation and airway hyperreactivity upon re-challenge. Therapeutic treatment with the recombinant wild-type allergen does not influence these key characteristics of allergic asthma. In contrast to the findings in the prophylactic treatment with rBet v 1 plus R848,no therapeutic benefit was found upon combination with R848. This could be due to the high number of treatment days. Reduction of this number may lead to a beneficial effect. However, these findings indicate that Bet v 1B2 could be a potential therapeutic agent for the treatment of established birch pollen induced allergic asthma. In conclusion, this study demonstrates for the first time that prophylactic EPI with the recombinant form of Bet v 1 in combination with R848 could prevent and suppress asthmatic features in an established birch pollen allergy. Not only therapeutic, but also prophylactic applications of EPI could be of importance to prevent allergic sensitization, considering the high prevalence of allergic diseases. R848 could be a potential adjuvant for enhancing the prophylactic potential of EPI for the treatment of birch pollen allergy. Furthermore, the beneficial use of the hypoallergen Bet v 1B2 in therapeutic EPI was demonstrated by intervention of established asthmatic features. In the future, a combination of hypoallergens alone or together with adjuvants in EPIT could lead to a more convenient and effective therapeutic treatment of established birch pollen induced allergic asthma

Prevention of intestinal allergy in mice by rflaA:Ova is associated with enforced antigen processing and TLR5-dependent IL-10 secretion by mDC

Conjugated vaccines consisting of flagellin and antigen activate TLR5 and induce strong innate and adaptive immune responses. Objective of the present study was to gain further insight into the mechanisms by which flagellin fusion proteins mediate their immune modulating effects. In a mouse model of Ova-induced intestinal allergy a fusion protein of flagellin and Ova (rflaA:Ova) was used for intranasal and intraperitoneal vaccination. Aggregation status of flaA, Ova and flaA:Ova were compared by light scattering, uptake of fluorescence labeled proteins into mDC was analyzed, processing was investigated by microsomal digestion experiments. Mechanism of DC-activation was investigated using proteasome and inflammasome inhibitors. Immune responses of wildtype, IL-10−/−, TLR5−/− mDCs and Ova-transgenic T cells were investigated. Mucosal and i.p.-application of rflaA:Ova were able to prevent allergic sensitization, suppress disease-related symptoms, prevent body weight loss and reduction in food uptake. Intranasal vaccination resulted in strongest suppression of Ova-specific IgE production. These protective effects were associated with increased aggregation of rflaA:Ova and accompanied by tenfold higher uptake rates into mDC compared to the mixture of both proteins. Microsomal digestion showed that stimulation with rflaA:Ova resulted in faster degradation and the generation of different peptides compared to rOva. rflaA:Ova-mediated activation of mDC could be suppressed in a dose-dependent manner by the application of both inflammasome and proteasome inhibitors. Using TLR5−/− mDC the rflaA:Ova induced IL-10 secretion was shown to be TLR5 dependent. In co-cultures of IL-10−/− mDC with DO11.10 T cells the lack of rflaA:Ova-mediated IL-10 secretion resulted in enhanced levels of both TH2 (IL-4, IL-5) and TH1 (IL-2 and IFN-y) cytokines. In summary, mucosal vaccination with flaA:Ova showed strongest preventive effect. Stimulation with rflaA:Ova results in strong immune modulation mediated by enhanced uptake of the aggregated fusion protein, likely resulting in a different processing by DC as well as stronger TLR5 mediated cell activation

rflaA:Ova-induced IL-6 and IL-10 secretion from mDC are mediated by different signaling pathways.

<p>C57BL/6 mDC derived from C57BL/6 wt, MyD88^−/−, Trif^−/−, or MyD88^−/−Trif^−/− mice were co-cultured with OT-II CD4⁺ T cells and stimulated with equimolar amounts of rOva, rflaA, rflaA+rOva, or rflaA:Ova, respectively. Levels of IL-6, IL-10, and IFN-y in the culture supernatants were analyzed after 72 h. Results are means ± SD of two independent experiments. Please note that results from C57BL/6 and Myd88^−/− cells were already published in Schülke <i>et al.</i>, 2011 JACI.</p

rflaA:Ova induces strong cytokine secretion from human monocytes.

<p>Monocyte activation test was performed using 5 µl of fresh blood stimulated with the indicated, equimolar protein amounts for 24 h. Supernatants were analysed for IL-1ß and IL-6 cytokine secretion by ELISA. Results are mean values ± SD for four different donors.</p

Intranasal and intraperitoneal vaccination with rflaA:Ova prevent Ova-induced intestinal allergy.

<p>Control (PBS→PBS→NF), Ova-sensitized but unvaccinated animals (PBS→Ova/A→EW), Ova-vaccinated (Ova→Ova/A→EW) and rflaA:Ova-vaccinated (flaA:Ova→Ova/A→EW) animals were continuously challenged with Ova-containing pellets (EW) for 8 days (<b>A</b>). Mean symptom scores (<b>B</b>), body weight normalized to the individual body weight before challenge (<b>C</b>), and mean EW-pellet uptake per mouse and day (<b>D</b>), were analyzed (n = 6 mice per group). NF: normal food.</p

flaA:Ova mediated IL-6 secretion depends on both proteasome and inflammasome activation.

<p>BALB/c mDC were stimulated with the indicated equimolar amounts of rflaA, rflaA+rOva and rflaA:Ova for 24 h and the resulting IL-6 and IL-10 secretion in the culture supernatants was analyzed by ELISA (<b>A</b>). BALB/c mDC were incubated with either the inflammasome inhibitors Z-VAD-FMK and glyburide (or the proteasome inhibitor lactacystin) for 2 h (<b>B</b>). Subsequently mDC were stimulated for 22 h with 4 µg of rflaA:Ova. Supernatants were analyzed for IL-6 secretion via ELISA. Results are mean values of three independent experiments ± SD.</p

rflaA:Ova is differently processed in mDC-derived microsomes.

<p>Microsomes were isolated from unstimulated BALB/c mDC and used for digestion of rOva and rflaA:Ova (<b>A+B</b>). Protein amounts equimolar to 5 µg rOva (5 µg rOva, 8.5 µg rflaA:Ova) were digested for each indicated time point. Subsequently, half of the samples were analyzed by SDS-PAGE (<b>A</b>) and for the resulting peptide pattern by mass spectrometry B). M = molecular weight marker, arrow indicates the MW of the digested target protein. M molecular weight marker, P protein without microsomes, M microsomes without protein, aa# amino acid residue number.</p

flaA:Ova induced IL-10 secretion from mDC is abrogated in TLR5 deficient mice.

<p>C57BL/6 and TLR5^−/− mDC were stimulated with rflaA:Ova. Levels of IL-6 and IL-10 were analyzed by ELISA after 72 h. Results are means ± SD of two independent experiments.</p