Search Results (5)

Hazelnuts are frequently involved in IgE-mediated reactions and are the main cause of nut allergies in Europe. Most food products are processed before human consumption. Food processing can modify the structure, properties, and function of proteins, and as a result, the IgE-binding capacity of allergens can be affected. In this study, we aimed to investigate epitope changes caused by the roasting of hazelnuts using epitope fingerprinting. Rabbit sera were raised against hazelnut proteins, and their epitopes were characterized. Immunoassays using specific polyclonal antibodies from rabbits targeting the main allergens in hazelnuts revealed marked reductions in the levels of Cor a 1 (PR-10), Cor a 11 (7S globulin), and Cor a 14 (2S albumin). However, rabbit antibodies can recognize different epitopes. Using antibodies that are different and characterized could help establish reliable methods for estimating the effects of treatments on the allergenicity of foods. In this work, we provide the first practical application that could lead to sets of peptide epitopes to compare and standardize immune diagnostics, even for complex protein preparations. Full article

The pathophysiology of many neuropsychiatric disorders is still poorly understood. Identification of biomarkers for these diseases could benefit patients due to better classification and stratification. Exosomes excreted into the circulatory system can cross the blood–brain barrier and carry a cell type-specific set of molecules. Thus, exosomes are a source of potential biomarkers for many diseases, including neuropsychiatric disorders. Here, we investigated exosomal proteins produced from human-induced pluripotent stem cells (iPSCs) and iPSC-derived neural stem cells, neural progenitors, neurons, astrocytes, microglia-like cells, and brain capillary endothelial cells. Of the 31 exosome surface markers analyzed, a subset of biomarkers were significantly enriched in astrocytes (CD29, CD44, and CD49e), microglia-like cells (CD44), and neural stem cells (SSEA4). To identify molecular fingerprints associated with disease, circulating exosomes derived from healthy control (HC) individuals were compared against schizophrenia (SCZ) patients and late-onset Alzheimer’s disease (LOAD) patients. A significant epitope pattern was identified for LOAD (CD1c and CD2) but not for SCZ compared to HC. Thus, analysis of cell type- and disease-specific exosome signatures of iPSC-derived cell cultures may provide a valuable model system to explore proteomic biomarkers for the identification of novel disease profiles. Full article

The production and purification of recombinant proteins are crucial to acquiring pure MPT64 protein. Due to the fact that protein epitopes may undergo conformational changes during purification, this study, therefore, investigated an effective rapid purification method to produce highly intracellular pure MPT64 protein without causing conformational changes in the epitope under denaturing conditions. MPT64 was isolated from E. coli and electrophoresed using gel SDS-PAGE. Then, the desired protein bands were excised and purified with two methods: electroelution and passive elution. The isolated protein was identified via peptide mass fingerprinting using MALDI-TOF MS and reacted with IgG anti-MPT64, and the cross-reactivity of the isolated protein with IgY anti-MPT64 was confirmed using Western blot. The results show that both of these methods produced pure MPT64 protein, and the MPT64 protein was confirmed based on the MALDI-TOF MS results. Neither of these two methods resulted in epitope changes in the MPT64 protein so it could react specifically with both antibodies. The yield of MPT64 protein was higher with electroelution (2030 ± 41 µg/mL) than with passive elution (179.5 ± 7.5 µg/mL). Thus, it can be inferred that the electroelution method is a more effective method of purifying MPT64 protein and maintaining its epitope than the passive elution method. Full article

Acute myeloid leukemia (AML) is characterized by a dysregulated expansion of poorly differentiated myeloid cells. Although patients are usually treated effectively by chemotherapy, a high rate of relapsed or refractory disease poses a major hurdle in its treatment. Recently, several studies have proposed implications of protein glycosylation in the pathobiology of AML including chemoresistance. Accordingly, associations have been found between specific glycan epitopes and the outcome of the disease. To advance this poorly studied field, we performed an exploratory glycomics study characterizing 21 widely used AML cell lines. Exploiting the benefits of porous graphitized carbon chromatography coupled to tandem mass spectrometry (PGC nano-LC-MS²), we qualitatively and quantitatively profiled N- and O-linked glycans. AML cell lines exhibited distinct glycan fingerprints differing in relevant glycan traits correlating with their cellular phenotype as classified by the FAB system. By implementing transcriptomics data, specific glycosyltransferases and hematopoietic transcription factors were identified, which are candidate drivers of the glycan phenotype of these cells. In conclusion, we report the varying expression of glycan structures across a high number of AML cell lines, including those associated with poor prognosis, identified underlying glycosyltransferases and transcription factors, and provide insights into the regulation of the AML glycan repertoire. Full article

The search for epitopes that will effectively trigger an immune response remains the “El Dorado” for immunologists. The development of promising immunotherapeutic approaches requires the appropriate targets to elicit a proper immune response. Considering the high degree of HLA/TCR diversity, as well as the heterogeneity of viral and tumor proteins, this number will invariably be higher than ideal to test. It is known that the recognition of a peptide-MHC (pMHC) by the T-cell receptor is performed entirely in a structural fashion, where the atomic interactions of both structures, pMHC and TCR, dictate the fate of the process. However, epitopes with a similar composition of amino acids can produce dissimilar surfaces. Conversely, sequences with no conspicuous similarities can exhibit similar TCR interaction surfaces. In the last decade, our group developed a database and in silico structural methods to extract molecular fingerprints that trigger T-cell immune responses, mainly referring to physicochemical similarities, which could explain the immunogenic differences presented by different pMHC-I complexes. Here, we propose an immunoinformatic approach that considers a structural level of information, combined with an experimental technology that simulates the presentation of epitopes for a T cell, to improve vaccine production and immunotherapy efficacy. Full article