Hansjoerg Schild

Analysis of Protein Composition of Red Wine in Comparison with Rosé and White Wines by Electrophoresis and High-Pressure Liquid Chromatography−Mass Spectrometry (HPLC-MS)

Wine proteins not only influence wine stability but are also being discussed as potential allergens. Proteins from red, rosé, and white wines were enriched by dialysis and lyophilization followed by separation by SDS-PAGE. Significant differences were detected in the protein compositions of the analyzed wine varieties, and the major protein bands were identified by mass spectrometry after in-gel digestion with trypsin. In German Portugieser red wine, a total of 121 tryptic peptides were identified, which were attributed to 12 grape proteins and 6 proteins derived from yeast. Among the identified constituents are several proteins considered to influence wine stability and previously described potential grape allergens. The pathogenesis-related proteins represent the main proteins in all of the wines, but only some red wines show a band with a molecular mass of 12 kDa, identified as a lipid transfer protein (LTP). The occurrence and distribution of LTP depend on the wine variety.

Distinct molecular mechanisms leading to deficient expression of ER-resident aminopeptidases in melanoma

Immune surveillance of tumour cells by CD8+ cytotoxic T cells plays a key role in the establishment and control of an anti-tumour response. This process requires the generation of antigenic peptides, which are largely produced by the proteasome in combination with other proteases located in either the cytoplasm and/or the endoplasmic reticulum (ER). The ER-resident aminopeptidases ERAP1 and ERAP2 trim or even destroy HLA class I-binding peptides thereby shaping the peptide repertoire presented for T cell recognition. So far there exists limited information about the expression pattern of ERAP1 and/or ERAP2 in human tumours of distinct histotypes. Therefore, the expression profiles and modes of regulation of both aminopeptidases were determined in a large series of melanoma cell lines. A heterogeneous expression ranging from high to reduced or even total loss of ERAP1 and/or ERAP2 mRNA and/or protein expression was detected, which often could be induced/upregulated by interferon-γ treatment. The observed altered ERAP1 and/or ERAP2 expression and activity levels were either mediated by sequence alterations affecting the promoter or enzymatic activities, leading to either transcriptional and/or post-transcriptional downregulation mechanisms or limited or excessive processing activities, which both might have an impact on the antigenic peptide repertoire presented on HLA class I molecules.

Cockroach allergens Per a 3 are oligomers.

Allergens from cockroaches cause major asthma-related health problems worldwide. Among them Per a 3 belongs to the most potent allergens. Although the sequences of some members of the Per a 3-family are known, their biochemical and biophysical properties have not been investigated. Here we present for the first time a thorough structural characterization of these allergens, which have recently been tested to induce an increase of allergy specific indicators in blood of Europeans. We isolated two Per a 3 isoforms, which occur freely dissolved in the hemolymph as hexamers with molecular masses of 465+/-25kDa (P II) and 512+/-25kDa (P I). Their sedimentation coefficients (S(20,W)) were determined to be 17.4+/-0.7 S (P II) and 19.0+/-0.9 S (P I), respectively. Sequence analysis revealed that P II consists of two subunit types known as allergens Per a 3.01 and Per a 3.0201, while PI consists of a new allergenic subunit type designated as Per a 3.03. A 3D model of the hexameric allergen Per a 3 was obtained by homology modelling. Almost all of the recently predicted 11 putative antigenic peptides and reported IgE-epitopes could be located on the surface of the hexamer, thus being freely accessible in the hexameric structure of the native molecules. We propose this might contribute to their allergic potential as well as their extreme stability with respect to temperature.

A trifunctional dextran-based nanovaccine targets and activates murine dendritic cells, and induces potent cellular and humoral immune responses in vivo.

Dendritic cells (DCs) constitute an attractive target for specific delivery of nanovaccines for immunotherapeutic applications. Here we tested nano-sized dextran (DEX) particles to serve as a DC-addressing nanocarrier platform. Non-functionalized DEX particles had no immunomodulatory effect on bone marrow (BM)-derived murine DCs in vitro. However, when adsorbed with ovalbumine (OVA), DEX particles were efficiently engulfed by BM-DCs in a mannose receptor-dependent manner. A DEX-based nanovaccine containing OVA and lipopolysaccharide (LPS) as a DC stimulus induced strong OVA peptide-specific CD4+ and CD8+ T cell proliferation both in vitro and upon systemic application in mice, as well as a robust OVA-specific humoral immune response (IgG1>IgG2a) in vivo. Accordingly, this nanovaccine also raised both a more pronounced delayed-type hypersensitivity response and a stronger induction of cytotoxic CD8+ T cells than obtained upon administration of OVA and LPS in soluble form. Therefore, DEX-based nanoparticles constitute a potent, versatile and easy to prepare nanovaccine platform for immunotherapeutic approaches.

Purification and structural characterisation of lipid transfer protein from red wine and grapes

Lipid transfer proteins (LTP) play a major role in plant defence and are of particular interest due to their known ability to cause allergic reactions. These proteins are expressed in grapes and also remain detectable after vinification, especially in red wine. However, it remains unknown whether the protein undergoes any changes during the vinification process. Here, we present a purification method for LTPs from Dornfelder grapes and wine. By liquid-chromatography-mass spectroscopy (LC-MS/MS) we identified LTPs from two different species (Vitis vinifera and Vitis aestivalis). Additionally, the purified LTPs were characterised using spectrometric methods, confirming their high purity and structural stability during vinification. We conclude that LTPs are resistant to the alcohol content (13.5 vol%), acidic milieu of wine and other ingredients present during the vinification process, indicating that the allergenic potential of grape LTP is not diminished by the vinification process.

Tumor immunoevasion via acidosis-dependent induction of regulatory tumor-associated macrophages

Many tumors evolve sophisticated strategies to evade the immune system, and these represent major obstacles for efficient antitumor immune responses. Here we explored a molecular mechanism of metabolic communication deployed by highly glycolytic tumors for immunoevasion. In contrast to colon adenocarcinomas, melanomas showed comparatively high glycolytic activity, which resulted in high acidification of the tumor microenvironment. This tumor acidosis induced Gprotein–coupled receptor–dependent expression of the transcriptional repressor ICER in tumor-associated macrophages that led to their functional polarization toward a non-inflammatory phenotype and promoted tumor growth. Collectively, our findings identify a molecular mechanism of metabolic communication between non-lymphoid tissue and the immune system that was exploited by high-glycolytic-rate tumors for evasion of the immune system.Tumors can vary in both their control by immunosurveillance and their glycolytic activity. Bopp and colleagues demonstrate that highly glycolytic tumors acidify their microenvironment and use this to initiate a mechanism of localized immunosuppression.