Digestomics of walnut and its nsLTPs allergens reveals their ultimate resistance to gastric digestion

Background: Sensitization to non-specific lipid transfer protein (ns-LTPs) in plant foods is regarded as a risk factor for generalized allergic reactions. Stability to gastric digestion represents very important parameter of food proteins allergenicity. Usually studies of digestion were carried out on purified proteins, but has never been examined the influence of different food matrices on different allergens. Allergens from the nsLTP family are known to share a characteristic structure which is highly resistant to proteolysis, and therefore, IgE cross-reactivity of nsLTPs needs to be investigated in the environment such as complex food matrix. Objective: The aim of this research project is to reveal how proteins are digested (by Minekus protocol) within the natural food matrix and possible consequences on their allergenicity, with the special focus on ns-LTP. Methods: Pure nsLTPs from walnut were labelled with Alexa 633 and added to whole grain of grounded raw walnuts, incubated with human α-amylase, and pepsin, therefore mimicking the effects of oral and gastric digestion, in total duration of 2h. Proteins extracted from the mixture were analyzed by one-dimensional (1D) and two-dimensional SDS-PAGe, and respective 1D and 2D immunoblots. Results: Most proteins from pepsin digested walnut sample were more resistant to digestion according to 1D SDS PAGE. Pepsin digested raw walnut sample with nsLTP were assessed by 2D PAGE to compare profiles of the digested and control sample (no pepsin added). 2D SDSPAGE of digested and control walnut samples showed almost identical profiles, especially in the context of fluorescently labelled nsLTP allergens. These results demonstrate substantial resistance of nsLTP allergens to gastric digestion since they remained mostly intact after 2 h of gastric (pepsin) digestion. Conclusion: Further research is needed to be able to grade stability/resistance of selected food allergens to gastric digestion as a consequence of food matrix modulating effects. We propose that certain combinations of foods and allergens could provide additional protection or on the contrary ease the digestion, by comparing trends between control and digested samples and between different digested combinations as well

Refined analytical methods for improved component resolved diagnosis in food allergy

Komponenten-basierte Tests tragen zu einer verbesserten Diagnosestellung für Nahrungsmittelallergien. Dies ist umso wichtiger, da es derzeit keine zugelassene Immuntherapie bei Nahrungsmittelallergien gibt. Daher ist die Identifizierung der allergieauslösenden Nahrungsmittel wichtig, um geeignete Diätpläne zu erstellen. In der jüngsten Vergangenheit hat sich die Allergieforschung zusätzlich zur Identifizierung einzelner Nahrungsmittelallergene auch mit der Zusammensetzung der Nahrungsmittelmatrix und deren Einfluss auf die Allergenizität der einzelnen Proteine beschäftigt. Diese Untersuchungen helfen das Wissen über „natürliche“ Adjuvantien, die in Nahrungsmitteln vorkommen, zu vertiefen. Im ersten Teil der PhD Thesis wurde eine umfassende Studie über die Interaktion des Hauptallergens aus Pfirsich, Pru p 3 (nichtspezifisches Lipid-transfer-protein), und Lipiden durchgeführt um die Rolle der Nahrungsmittelmatrix bei Pfirsichallergie zu verstehen. Die Lipidbindung von gereinigtem Pru p 3 wurde im „ANS Test“, untersucht. Weiters wurde die Lipidbindung mittels NMR „Water-Ligand Observed Gradient NMR Spektroskopie“ analysiert. Parallel zu den experimentell erhobenen Daten wurde eine „in-silico“ Analyse mittels „Molecular Dynamics“ durchgeführt. Anschließend wurde der Einfluss der Lipid-Allergen Interaktion auf die IgE-Bindungsfähigkeit von Pru p 3 in „ELISA“ und „BAT“ Tests mit Hilfe von Seren von Pfirsichallergikern untersucht. Die Resultate des ANS Tests und der NMR-Experimente ergaben, dass Pru p 3 präferentiell ungesättigte freie Fettsäuren, wie Ölsäure bindet. Diese Interaktion verändert die dreidimensionale Struktur von Pru p 3, wie auch mit Hilfe von Molecular Dynamics errechnet wurde. Die „C-terminale Loop“ wird nach außen, Richtung Oberfläche gedreht und ändert damit die Zugänglichkeit eines bereits bekannten IgE-Epitops. Damit erhöht diese Konformationsänderung die IgE-Bindung signifikant im Vergleich zu Pru p 3 ohne Ligandenbindung. Diese Konformationsänderung ist aber abhängig vom Liganden und nicht alle Bindungen führen zu einer erhöhten IgE Bindungsaktivität von Pru p 3. Walnüsse wie auch andere Baumnüsse sind eine der häufigsten Verursacher von schweren, anaphylaktischen Reaktionen. Daher wurden im zweiten Teil der PhD Thesis Allergene aus Walnuss biochemisch untersucht. Mittels spezifischer Hochleistungs-Spektrometrie Methoden wurden einzelne Allergene detailliert charakterisiert. Ein Protein aus der Familie der Viciline wurde aus Walnusskernen gereinigt und anschließend mittels Massenspektrometrie charakterisiert. Die allergenen und kreuzreaktiven Eigenschaften des Proteins wurden im Immunblot und ELISA an 77 Seren von Allergikern mit Walnussallergie getestet. Im Proteingel erscheint das gereinigte Vicilin als eine Bande in der Höhe von ca. 50kDa und wurde von IgE Antikörpern aus Seren von allergischen Patienten erkannt. MALDI-TOF und ESI-TOF Analysen ergaben eine Masse von 47.1kDa und 48.8kDa. Tandem-MS und daran gekoppelte Flüssigkeitschromatographie ermöglichte die Sequenzierung des Proteins. Die Sequenz dieses Proteins stimmte jedoch nicht mit der vom bereits bekannten Vicilin aus Walnuss, Jug r 2, überein. Daher wurde für dieses neue Protein der Allergenname Jug r 6 von der IUIS Allergendatenbank zuerkannt. Weiters wurde für Jug r 6 Kreuzreaktivität zu den homologen Allergenen aus Haselnuss, Sesam und Pistazie gefunden, die auch der Sequenzhomologie mit diesen Proteinen entspricht. Zusammenfassend konnte in dieser Arbeit gezeigt werden, dass die Anwendung von speziellen hochsensitiven Analysemethoden die Identifizierung von einzelnen Allergenen verbessert. Diese gut charakterisierten Allergene können damit die Sensitivität und Spezifität der molekülbasierten Allergiediagnostik verbessern, die im weiteren dazu beitragen, dass die entsprechenden therapeutischen Maßnahmen für den Patienten mit Nahrungsmittelallergien optimiert werden können.Component resolved tests contribute to improved diagnosis for food allergies and facilitate an individual management of patients. This is of utmost importance since there is no approved immunotherapy for food allergies available. The identification of causative allergens and allergenic foods is essential in order to formulate individual exclusion diets. In the recent past allergy research has focused on the identification of food allergens and in addition started to investigate the composition of food matrices and its impact on the allergenicity of individual allergens. These studies contribute to further expand our knowledge on “natural” adjuvants from food sources. In the first study of the PhD Thesis a comprehensive study on the interaction of the major peach allergen Pru p 3 (non-specific lipid transfer protein) and lipids was performed to better understand the role of lipid-food matrix in peach allergy. Lipid-binding activity of purified Pru p 3 was monitored by fluorescence-based ANS assay and Water-Ligand Observed via Gradient NMR Spectroscopy. In addition to the experimental approach an in silico assessment was performed, applying Molecular Dynamics. Subsequently, the impact of this interaction on the IgE binding activity of Pru p 3 was investigated using sera from peach allergic patients in ELISA and BAT assays. Our results showed that Pru p 3 preferentially binds unsaturated free fatty acids such as oleic acid as shown by ANS assay and verified by NMR experiments. This interaction between oleic acid and Pru p 3 affects its 3D structure as indicated by molecular dynamic analysis. The C-terminal loop is moved out towards the surface of the molecule thus changing accessibility of a known IgE epitope. Thus this conformational change significantly increases the IgE binding capacity of Pru p 3. However, this conformational change is also dependent on the type of ligand and not all lipids tested increased the IgE binding activity of Pru p 3. Among those walnuts like other tree nuts are ranked high in the list of the culprit foods inducing severe reactions including anaphylaxis. Therefore allergens from walnuts were characterized in the second part of the PhD Thesis applying high throughput mass spectrometry. A member of the vicilin protein family was purified from walnut kernels and further characterized by mass spectrometry. Heat and enzymatic stability of the purified protein was investigated by CD-spectroscopy and gastrointestinal digestion simulation, respectively. Allergenicity and cross-reactivity of the target protein was verified by immunoblots and ELISA using sera from 77 walnut allergic patients. In SDS-PAGE purified vicilin migrated as a single band of about 50 kDa and was recognized by specific IgE from allergic patients sera in Western blots. MALDI-TOF and ESI-TOF analyses provided mass data of 47.1 kDa and 48.8 kDa, respectively. Tandem mass spectrometry coupled with liquid chromatography allowed the identification of the protein sequence. However, the obtained amino acid sequence was not in accordance with the previously identified walnut vicilin, Jug r 2. Therefore, this vicilin obtained the allergen designation Jug r 6 from the IUIS allergen nomenclature database. Jug r 6 displays a remarkable level of cross reactivity when tested with homologues from hazelnut, sesame seed and pistachio, which is also reflected by the sequence similarity. Summarizing we showed that the application of refined analytical methods can improve the identification and characterization of individual allergens. These well-defined allergenic molecules contribute to increase the sensitivity and specificity of component resolved diagnosis, which in turn improves patient tailored management. Therefore, up to date analytical methods should be included for in depth allergen characterization prior to their application in food allergy component resolved diagnosis.submitted by Pawel DubielaAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersMedizinische Universität Wien, Diss., 2018(VLID)256578

International Journal of Molecular Sciences / Dendritic Cells and Their Role in Allergy: Uptake, Proteolytic Processing and Presentation of Allergens

Dendritic cells (DCs) are the most important antigen presenting cells to activate naïve T cells, which results in the case of Type 1 allergies in a Type 2 helper T cell (Th2)-driven specific immune response towards allergens. So far, a number of different subsets of specialized DCs in different organs have been identified. In the recent past methods to study the interaction of DCs with allergenic proteins, their different uptake and processing mechanisms followed by the presentation to T cells were developed. The following review aims to summarize the most important characteristics of DC subsets in the context of allergic diseases, and highlights the recent findings. These detailed studies can contribute to a better understanding of the pathomechanisms of allergic diseases and contribute to the identification of key factors to be addressed for therapeutic interventions.(VLID)486716

Steroid-Resistant Nephrotic Syndrome Caused by <i>NUP93</i> Pathogenic Variants

Background: Although steroid therapy is a standard of care for nephrotic syndrome treatment, 15–20% of patients do not respond to it. Finding the genetic background is possible in >10% of steroid-resistant nephrotic syndrome (SRNS) cases. Variants in genes encoding nuclear pore complex proteins are a novel cause of paediatric steroid-resistant nephrotic syndrome (SRNS). Recent studies suggest NUP93 variants to be a significant cause of paediatric onset SRNS. The clinical data on certain variants and disease history are still very limited. Methods and results: We report the SRNS case of a 12-year-old boy with two detected NUP93 variants, which are pathogenic and possibly pathogenic. The onset of the disease was early and severe. The patient was admitted to the paediatric nephrology department due to nephrotic-range proteinuria and hypoalbuminemia with a long medical history of steroid and non-steroid immunosuppressive treatment. The genetic panel targeting 50 genes, clinically relevant for nephrotic syndrome, was performed. The only gene which was found to be affected by mutations, namely c.2326C>T and c.1162C>T, respectively, was NUP93. Conclusions: NUP93 variants are rarely identified as causes of SRNS. Clinical data are of utmost importance to establish the standard of care for SRNS patients suffering from this genetic disfunction. This is the first case of a heterozygous patient with the c.2326C>T and c.1162C>T variants and confirmed clinical history of the SRNS described so far. Our data suggest the clinical relevance of the c.1162C>T variant

In Vitro Assays for Diagnosis of Drug-Induced Nonsevere Exanthemas: A Systematic Review and Meta-Analysis

Frequent mislabelled causal relationship between drug hypersensitivity reactions and culprit drugs reinforces the need for an accurate diagnosis. The systematic reviews and meta-analyses of in vitro assays published so far focused on immediate reactions and the most severe delayed reactions, while the most frequent drug-induced delayed reactions—nonsevere exanthemas—have been underestimated. We aim to fill this gap. A systematic review of studies on in vitro assays used in the diagnosis of nonsevere drug-induced delayed reactions was conducted following the methodology of Preferred Reporting Items for a Systematic Review and Meta-analysis of Diagnostic Test Accuracy Studies Statement. The EMBASE and PubMed databases were searched. We have included 33 studies from which we extracted the data, then performed meta-analysis where possible, or synthesised the evidence narratively. The quality of the analysed studies was assessed with the QUADAS-2 tool. The tests identified the most frequently were lymphocyte transformation test (LTT), ELISpot, and ELISA. In the meta-analysis carried out for LTT in reactions induce by beta-lactams, the pool estimate of sensitivity and specificity amounted to 49.1% (95% CI: 14.0%, 85.0%) and 94.6% (95% CI: 81.7%, 98.6%), respectively. The studies showed heterogeneity in study design and laboratory settings, which resulted in a wide range of specificity and sensitivity of testing

The role of rradykinin receptors in hereditary angioedema due to C1-Inhibitor deficiency

Background: Hereditary angioedema (HAE) is a rare, genetic disease caused by the decreased level or function of the C1 inhibitor. The primary mediator of symptoms in HAE is bradykinin acting through its two receptors, namely receptors 1 (BR1) and 2 (BR2). Although BR2 is well characterized, the role of BR1 remains unclear. Objective: To study the role of bradykinin receptors 1 (BR1) in the etiopathogenesis of HAE. Methods: A total of 70 individuals, 40 patients with HAE, and 30 healthy subjects were recruited to the study. HAE was diagnosed in accordance with the international guideline. The level of bradykinin receptors was determined in populations of CD3(+), CD4(+), CD8(+), and CD14(++)CD16(−), CD14(++)CD16(+) monocytes. In addition, the level of disease activity-specific markers was measured. Results: There were statistically significant differences in the subpopulation of lymphocytes and monocytes between patients with HAE compared to healthy subjects. The level of BR1 and BR2 on PBMCs was comparable in healthy subjects and HAE patients during remission with significant overexpression of both receptors, triggered by HAE attack. Moreover, a significant increase in TNF-alpha and IL-1 plasma levels was observed among HAE patients. Conclusions: BR1 expression may play an important role in the pathomechanism of HAE

Scientific Reports / Jug r 6 is the allergenic vicilin present in walnut responsible for IgE cross-reactivities to other tree nuts and seeds

Walnuts are ranked high in the list of the culprit foods inducing severe allergic reactions. Jug r 2 has been identified as a major allergen in common walnut by cDNA cloning from a somatic cell line. So far, studies were performed on the allergenic activity of recombinant Jug r 2, yet there is still no evidence about the physicochemical characteristics of the natural allergen. Therefore, we aimed to purify and deeply characterize natural Jug r 2 and to assess IgE cross-reactivity among vicilins from different tree nuts. Extensive mass spectrometry analysis of the obtained purified vicilin allowed identification of the protein sequence that displayed only 44% identity to Jug r 2. The newly identified vicilin (Jug r 6) was recognized by IgE of 26% in walnut allergic patients sera tested. In contrast to Jug r 2, Jug r 6 displayed a remarkable level of cross-reactivity when tested with homologues from hazelnut, sesame and pistachio. It is the first report showing the necessity of proteomic studies to improve allergy component resolved diagnosis.(VLID)464408