Schmallenberg virus, an emerging viral pathogen of cattle and sheep and a potential contaminant of raw materials, is detectable by classical in-vitro adventitious virus assays.

Emerging viruses, as potential contaminants of raw materials used in the manufacture of biologicals represent a challenge in the safety testing of biopharmaceutical products intended for human or veterinary use. Here, we report the challenge of an in vitro adventitious virus platform used in safety testing of biologicals, where a broad panel of detector cell lines was challenged to provide evidence that Schmallenberg virus is detectable by a classical reporting endpoint of cytopathic effect with Vero, BHK-21 and CHO-K1 detector cells, within typical in vitro assay timescales. We conclude that Schmallenberg virus is robustly detectable by classical in vitro viral biosafety assays.

Ovalbumin modified with pyrraline, a Maillard reaction product, shows enhanced T-cell immunogenicity.

The Maillard reaction (also referred to as "glycation") takes place between reducing sugars and compounds with free amino groups during thermal processing of foods. In the final stage of the complex reaction cascade, the so-called advanced glycation end products (AGEs) are formed, including proteins with various glycation structures. It has been suggested that some AGEs could have immunostimulatory effects. Here, we aimed to identify specific glycation structure(s) that could influence the T-cell immunogenicity and potential allergenicity of food allergens, using ovalbumin (OVA, an egg white allergen) as a model allergen. OVA was specifically modified with representative glycation structures: N(ε)-carboxymethyl lysine (CM-OVA), N(ε)-carboxyethyl lysine (CE-OVA), pyrraline (Pyr-OVA), or methylglyoxal-derived arginine derivatives (MGO-OVA). As well as AGE-OVA, a crude glycation product in thermal incubation of OVA with glucose, only Pyr-OVA, and not other modified OVAs, was efficiently taken up by bone marrow-derived murine dendritic cells (BMDCs). The uptake of Pyr-OVA was reduced in scavenger receptor class A (SR-A)-deficient BMDCs, but not in cells treated with inhibitors of scavenger receptor class B, galectin-3, or blocking antibodies against CD36, suggesting that pyrraline binds to SR-A. Compared with other modified OVAs, Pyr-OVA induced higher activation of OVA-specific CD4(+) T-cells in co-culture with BMDCs. Furthermore, compared with native OVA, AGE-OVA and Pyr-OVA induced higher IgE production in mice. Pyrraline could induce better allergen uptake by DCs via association with SR-A and subsequently enhance CD4(+) T-cell activation and IgE production. Our findings help us to understand how Maillard reaction enhances the potential allergenicity of food allergens.

The Maillard reaction and food allergies: is there a link?

Food allergies are abnormal responses to a food triggered by the immune system. The majority of allergenic foods are often subjected to thermal processing before consumption. The Maillard reaction is a non-enzymatic reaction between reducing sugars and compounds with free amino groups such as amino acids and proteins, and takes place during thermal processing and storage of foods. Among many other effects the reaction leads to modification of proteins with various types of glycation structures such as Nε-(carboxymethyl-)lysine (CML), pentosidine, pyrraline and methylglyoxal-H1, which are collectively called advanced glycation end-products (AGEs). Notably, evidence has accumulated that some glycation structures of AGEs function as immune epitopes. Here we discuss the possible involvement of food allergen AGEs in the pathogenesis of food allergies.

Combination peptide immunotherapy based on T-cell epitope mapping reduces allergen-specific IgE and eosinophilia in allergic airway inflammation.

Peptide immunotherapy using soluble peptides containing allergen-derived immunodominant T-cell epitopes holds therapeutic promise for allergic asthma. Previous studies in BALB/c mice using the immunodominant peptide epitope of chicken ovalbumin (p323-339) have been unable to demonstrate therapeutic effects in ovalbumin-induced allergic airway inflammation. We have previously shown that intravenous application of p323-339 can effectively tolerise p323-339-reactive T cells in a non-allergic model in C57BL/6 mice. This study aimed to assess the effects of using p323-339 immunotherapy in a C57BL/6 model of ovalbumin-induced allergic airway inflammation, identify any additional epitopes recognized by the ovalbumin-responsive T-cell repertoire in C57BL/6 mice and assess the effects of combination peptide immunotherapy in this model. Ovalbumin-reactive T-cell lines were generated from ovalbumin-immunized C57BL/6 mice and proliferative responses to a panel of overlapping peptides covering the ovalbumin sequence were assessed. Soluble peptides (singly or combined) were administered intravenously to C57BL/6 mice before the induction of ovalbumin-induced allergic airway inflammation. Peptide immunotherapy using the 323-339 peptide alone did not reduce the severity of allergic airway inflammation. An additional immunodominant T-cell epitope in ovalbumin was identified within the 263-278 sequence. Combination peptide immunotherapy, using the 323-339 and 263-278 peptides together, reduced eosinophilia in the bronchoalveolar lavage and ovalbumin-specific IgE, with apparent reductions in interleukin-5 and interleukin-13. Characterization of the T-cell response to a model allergen has allowed the development of combination peptide immunotherapy with improved efficacy in allergic airway inflammation. This model holds important potential for future mechanistic studies using peptide immunotherapy in allergy.

Glycation of a food allergen by the Maillard reaction enhances its T-cell immunogenicity: role of macrophage scavenger receptor class A type I and II.

BACKGROUND: The Maillard reaction occurs between reducing sugars and proteins during thermal processing of foods. It produces chemically glycated proteins termed advanced glycation end products (AGEs). The glycation structures of AGEs are suggested to function as pathogenesis-related immune epitopes in food allergy. OBJECTIVE: This study aimed at defining the T-cell immunogenicity of food AGEs by using ovalbumin (OVA) as a model allergen. METHODS: AGE-OVA was prepared by means of thermal processing of OVA in the presence of glucose. Activation of OVA-specific CD4(+) T cells by AGE-OVA was evaluated in cocultures with bone marrow-derived murine myeloid dendritic cells (mDCs) as antigen-presenting cells. The uptake mechanisms of mDCs for AGE-OVA were investigated by using inhibitors of putative cell-surface receptors for AGEs, as well as mDCs deficient for these receptors. RESULTS: Compared with the controls (native OVA and OVA thermally processed without glucose), AGE-OVA enhanced the activation of OVA-specific CD4(+) T cells on coculture with mDCs, indicating that the glycation of OVA enhanced the T-cell immunogenicity of the allergen. The mDC uptake of AGE-OVA was significantly higher than that of the controls. We identified scavenger receptor class A type I and II (SR-AI/II) as a mediator of the AGE-OVA uptake, whereas the receptor for AGEs and galectin-3 were not responsible. Importantly, the activation of OVA-specific CD4(+) T cells by AGE-OVA was attenuated on coculture with SR-AI/II-deficient mDCs. CONCLUSION: SR-AI/II targets AGE-OVA to the MHC class II loading pathway in mDCs, leading to an enhanced CD4(+) T-cell activation. The Maillard reaction might thus play an important role in the T-cell immunogenicity of food allergens.

Effects of glycation of the model food allergen ovalbumin on antigen uptake and presentation by human dendritic cells.

Advanced glycation endproducts (AGEs) of food proteins resulting from the Maillard reaction after cooking or heating may have particular importance in food allergy. The underlying immunological mechanisms are only poorly understood. The aim of the study was to examine the effects of AGE derived from the model food allergen ovalbumin (AGE-OVA) on dendritic cells (DCs), their immunostimulatory capacity and the T-cell response compared with regular OVA. For this purpose, human immature DCs were exposed to fluorescein isothiocyanate (FITC)-labelled AGE-OVA and FITC-labelled regular OVA and uptake was analysed by flow cytometry and fluorescence microscopy. Furthermore, autologous CD4(+) T-cell proliferation and cytokine production induced by mature DCs loaded with AGE-OVA were compared with those induced by mature DCs loaded with OVA. Finally, expression of the receptor for advanced glycation endproducts (RAGE) and activation of the transcription factor nuclear factor (NF)-kappaB by AGE were investigated. Internalization of FITC-AGE-OVA by immature DCs was significantly increased compared with FITC-OVA. Blocking the mannose receptor, macropinocytosis or the scavenger receptor strongly reduced uptake of both FITC-OVA and FITC-AGE-OVA. In a comparison of CD4(+) T cells co-cultured with AGE-OVA-loaded mature DCs versus those co-cultured with OVA-loaded mature DCs, AGE-OVA DCs were found to produce more interleukin (IL)-6 and to induce a stronger T helper type 2 (Th2) and a weaker Th1 cytokine response, while there was no difference in proliferation of CD4(+) T cells. The expression of RAGE was higher on immature DCs compared with mature DCs. AGE-OVA-exposed immature DCs showed a stronger expression of RAGE and activation of the transcription factor NF-kappaB compared with OVA-loaded immature DCs. Our data indicate that AGE-OVA may be more immunogenic/allergenic than regular OVA.

Impact of culture medium on maturation of bone marrow-derived murine dendritic cells via the aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AhR) plays a role in modulating dendritic cell (DC) immunity. Iscove's modified Dulbecco's medium (IMDM) contains higher amounts of AhR ligands than RPMI1640 medium. Here, we examined the influence of AhR ligand-containing medium on the maturation and T-cell stimulatory capacity of bone marrow-derived murine dendritic cells (BMDCs). BMDCs generated in IMDM (BMDCs/IMDM) expressed higher levels of co-stimulatory and MHC class II molecules, and lower levels of pattern-recognition receptors, especially toll-like receptor (TLR) 2, TLR4, and scavenger receptor class A (SR-A), compared to BMDCs generated in RPMI1640 medium (BMDCs/RPMI). Cytokine responses against ligands of TLRs and antigen uptake mediated by SR-A were remarkably reduced in BMDCs/IMDM, whereas the T-cell stimulatory capacity of the cells was enhanced, compared to BMDCs/RPMI. The enhanced maturation of BMDCs/IMDM was attenuated in the presence of an AhR antagonist, indicating involvement of AhR in the maturation. Interestingly, BMDCs/IMDM induced Th2 and Th17 differentiation at low and high concentrations of antigen respectively, when co-cultured with CD4(+) T-cells from antigen-specific T-cell receptor transgenic mice. In contrast, BMDCs/RPMI induced Th1 differentiation predominantly in the co-culture. Taken together, optimal selection of medium seems necessary when studying BMDCs, depending on the target receptors on the cell surface of DCs and type of helper T-cells for the co-culture.

Oral tolerance induction does not resolve gastrointestinal inflammation in a mouse model of food allergy.

SCOPE: Oral immunotherapy (OIT) involving continuous oral administration of allergenic foods has gained attention as a therapy for food allergies. To study the influence of oral administration of allergenic foods on gastrointestinal symptoms including inflammation, we established a mouse model of food-induced intestinal allergy. METHODS AND RESULTS: BALB/c mice were fed an egg white (EW) diet containing ovalbumin (OVA, a major EW allergen) after intraperitoneal sensitisation with OVA and Alum. The mice on the EW diet for one wk presented gastrointestinal symptoms (i.e. weight loss and soft stools) and inflammation in the small intestines (i.e. duodenum, jejunum and ileum). Further continuous EW diet resolved the weight loss but not the soft stools. Splenic CD4(+) T-cells of EW diet-fed mice on the continuous diet showed less proliferation and cytokine production compared with those of control mice, suggesting tolerance induction by the diet. The continuous EW diet reduced levels of OVA-specific IgE antibodies, but significantly aggravated the inflammation in the jejunum. CONCLUSION: Our mouse model would be useful to investigate inflammatory and regulatory mechanisms in food-induced intestinal allergies. Our results suggest potential gastrointestinal inflammation in patients undergoing OIT as continuous administration of allergenic foods, even though the therapy may induce clinical tolerance.

Characterization of native and reconstituted exosome complexes from the hyperthermophilic archaeon Sulfolobus solfataricus.

The eukaryotic exosome is a protein complex with essential functions in processing and degradation of RNA. Exosome-like complexes were recently found in Archaea. Here we characterize the exosome of Sulfolobus solfataricus. Two exosome fractions can be discriminated by density gradient centrifugation. We show that the Cdc48 protein is associated with the exosome from the 30S-50S fraction but not with the exosome of the 11.3S fraction. While only some complexes contain Cdc48, the archaeal DnaG-like protein was found to be a core exosome subunit in addition to Rrp4, Rrp41, Rrp42 and Csl4. Assays with depleted extracts revealed that the exosome is responsible for major ribonucleolytic activity in S. solfataricus. Various complexes consisting of the Rrp41-Rrp42 hexameric ring and Rrp4, Csl4 and DnaG were reconstituted. Dependent on their composition, different complexes showed variations in RNase activity indicating functional interdependence of the subunits. The catalytic activity of these complexes and of the native exosome can be ascribed to the Rrp41-Rrp42 ring, which degrades RNA phosphorolytically. Rrp4 and Csl4 do not exhibit any hydrolytic RNase activity, either when assayed alone or in context of the complex, but influence the activity of the archaeal exosome.