Gluten proteins from wheat, rye, barley and, in rare cases, oats, are responsible for triggering hypersensitivity reactions such as celiac disease, non-celiac gluten sensitivity and wheat allergy. ...Well-defined reference materials (RM) are essential for clinical studies, diagnostics, elucidation of disease mechanisms and food analyses to ensure the safety of gluten-free foods. Various RM are currently used, but a thorough characterization of the gluten source, content and composition is often missing. However, this characterization is essential due to the complexity and heterogeneity of gluten to avoid ambiguous results caused by differences in the RM used. A comprehensive strategy to isolate gluten protein fractions and gluten protein types (GPT) from wheat, rye, barley and oat flours was developed to obtain well-defined RM for clinical assays and gluten-free compliance testing. All isolated GPT (ω5-gliadins, ω1,2-gliadins, α-gliadins, γ-gliadins and high- and low-molecular-weight glutenin subunits from wheat, ω-secalins, γ-75k-secalins, γ-40k-secalins and high-molecular-weight secalins from rye, C-hordeins, γ-hordeins, B-hordeins and D-hordeins from barley and avenins from oats) were fully characterized using analytical reversed-phase high-performance liquid chromatography (RP-HPLC), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), N-terminal sequencing, electrospray-ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF-MS) and untargeted LC-MS/MS of chymotryptic hydrolyzates of the single GPT. Taken together, the analytical methods confirmed that all GPT were reproducibly isolated in high purity from the flours and were suitable to be used as RM, e.g., for calibration of LC-MS/MS methods or enzyme-linked immunosorbent assays (ELISAs).
Celiac disease (CD) is an inflammatory disorder of the upper small intestine caused by the ingestion of storage proteins (prolamins and glutelins) from wheat, barley, rye, and, in rare cases, oats. ...CD patients need to follow a gluten-free diet by consuming gluten-free products with gluten contents of less than 20 mg/kg. Currently, the recommended method for the quantitative determination of gluten is an enzyme-linked immunosorbent assay (ELISA) based on the R5 monoclonal antibody. Because the R5 ELISA mostly detects the prolamin fraction of gluten, a new independent method is required to detect prolamins as well as glutelins. This paper presents the development of a method to quantitate 16 wheat marker peptides derived from all wheat gluten protein types by liquid chromatography tandem mass spectrometry (LC-MS/MS) in the multiple reaction monitoring mode. The quantitation of each marker peptide in the chymotryptic digest of a defined amount of the respective reference wheat protein type resulted in peptide-specific yields. This enabled the conversion of peptide into protein type concentrations. Gluten contents were expressed as sum of all determined protein type concentrations. This new method was applied to quantitate gluten in wheat starches and compared to R5 ELISA and gel-permeation high-performance liquid chromatography with fluorescence detection (GP-HPLC-FLD), which resulted in a strong correlation between LC-MS/MS and the other two methods.
Celiac disease is triggered by the ingestion of gluten from wheat, barley, rye, and possibly oats. Gluten is quantitated by DNA-based methods or enzyme-linked immunosorbent assays (ELISAs). ELISAs ...mostly detect the prolamin fraction and potentially over- or underestimate gluten contents. Therefore, a new independent method is required to comprehensively detect gluten. A targeted liquid chromatography–tandem mass spectrometry method was developed to quantitate seven barley, seven rye, and three oat marker peptides derived from each gluten protein fraction (prolamin and glutelin) and type (barley, B-, C-, D-, and γ-hordeins; rye, γ-75k-, γ-40k-, ω-, and HMW-secalins). The quantitation of each marker peptide in the chymotryptic digest of a defined amount of the respective reference gluten protein type resulted in peptide-specific yields, which enabled the conversion of peptide into protein concentrations. This method was applied to quantitate gluten in samples from the brewing process, in raw materials for sourdough fermentation, and in dried sourdoughs.
Certain cereals like wheat, rye or barley contain gluten, a protein mixture that can trigger celiac disease (CD). To make gluten-free diets available for affected individuals the gluten content of ...foodstuff must be monitored. For this purpose, antibody-based assays exist which rely on the recognition of certain linear gluten sequence motifs. Yet, not all CD-active gluten constituents and fragments formed during food processing/fermentation may be covered by those tests. In this study, we therefore assayed the coverage of reportedly CD-active gluten components by currently available detection antibodies and determined the antibody-inducing capacity of wheat gluten constituents in order to provide novel diagnostic targets for comprehensive gluten quantitation. Immunizations of outbred mice with purified gliadins and glutenins were conducted and the linear target recognition profile of the sera was recorded using synthetic peptide arrays that covered the sequence space of gluten constituents present in those preparations. The resulting murine immunorecognition profile of gluten demonstrated that further linear binding sites beyond those recognized by the monoclonal antibodies α20, R5 and G12 exist and may be exploitable as diagnostic targets. We conclude that the safety of foodstuffs for CD patients can be further improved by complementing current tests with antibodies directed against additional CD-active gluten components. Currently unrepresented linear gluten detection sites in glutenins and α-gliadins suggest sequences QQQYPS, PQQSFP, QPGQGQQG and QQPPFS as novel targets for antibody generation.
Celiac disease (CD) is an inflammatory disorder of the small intestine. Gluten peptides are supposed to be responsible for the reaction, the best-researched of which is the so-called ‘33-mer’. ...Analogous peptides in secalins (rye) and hordeins (barley) have been described. This study presents the degradation of gliadins, glutenins, hordeins and secalins purified from the respective flours using a prolyl endopeptidase from the Basidiomycete Flammulina velutipes (FvpP). The flour fractions were incubated with the enzyme, and the cleavage sites were determined using high-resolution nLC-qTOF-MS/MS. For the wheat samples, eight cleavage sites in the 33-mer peptide were shown, and all of the six described epitopes were successfully cleaved. For the commercially available prolyl-specific endopeptidase from Aspergillus niger (An-Pep), which was used as a control, only two cleavage sites that cleaved three of the six epitopes were identified. For the secalins, four prolyl-specific cleavage sites in the CD-active peptide QPFPQPQQPIPQ were found for the FvpP but none for the An-Pep. The CD-active peptide QPFPQPEQPFPW in C-hordein was cleaved at three prolyl-specific positions by the FvpP. The study proves the usability of FvpP to degrade CD-inducing peptides in real-grain flour samples and indicates its higher effectiveness compared with An-Pep. A clinical study would be required to assess the therapeutic or preventive potential of FvpP for CD.
Coeliac disease (CD) is triggered by the ingestion of gluten proteins from wheat, rye, and barley. The 33-mer peptide from α2-gliadin has frequently been described as the most important ...CD-immunogenic sequence within gluten. However, from more than 890 published amino acid sequences of α-gliadins, only 19 sequences contain the 33-mer. In order to make a precise assessment of the importance of the 33-mer, it is necessary to elucidate which wheat species and cultivars contain the peptide and at which concentrations. This paper presents the development of a stable isotope dilution assay followed by liquid chromatography tandem mass spectrometry to quantitate the 33-mer in flours of 23 hexaploid modern and 15 old common (bread) wheat as well as two spelt cultivars. All flours contained the 33-mer peptide at levels ranging from 91-603 μg/g flour. In contrast, the 33-mer was absent (<limit of detection) from tetra- and diploid species (durum wheat, emmer, einkorn), most likely because of the absence of the D-genome, which encodes α2-gliadins. Due to the presence of the 33-mer in all common wheat and spelt flours analysed here, the special focus in the literature on this most immunodominant peptide seems to be justified.
Coeliac disease (CD) is triggered by the ingestion of gluten proteins from wheat, rye, and barley. The 33-mer peptide from α2-gliadin has frequently been described as the most important ...CD-immunogenic sequence within gluten. However, from more than 890 published amino acid sequences of α-gliadins, only 19 sequences contain the 33-mer. In order to make a precise assessment of the importance of the 33-mer, it is necessary to elucidate which wheat species and cultivars contain the peptide and at which concentrations. This paper presents the development of a stable isotope dilution assay followed by liquid chromatography tandem mass spectrometry to quantitate the 33-mer in flours of 23 hexaploid modern and 15 old common (bread) wheat as well as two spelt cultivars. All flours contained the 33-mer peptide at levels ranging from 91-603 μg/g flour. In contrast, the 33-mer was absent (
Cell-to-cell variability in apoptosis signaling contributes to heterogenic responses to cytotoxic stress in clinically heterogeneous neoplasia, such as acute myeloid leukemia (AML). The BCL-2 ...proteins BAX and BAK can commit mammalian cells to apoptosis and are inhibited by retrotranslocation from the mitochondria into the cytosol. The subcellular localization of BAX and BAK could determine the cellular predisposition to apoptotic death.
The relative localization of BAX and BAK was determined by fractionation of AML cell lines and patient samples of a test cohort and a validation cohort.
This study shows that relative BAX localization determines the predisposition of different AML cell lines to apoptosis. Human AML displays a surprising variety of relative BAX localizations. In a test cohort of 48 patients with AML, mitochondria-shifted BAX correlated with improved patient survival, FLT3-ITD status, and leukocytosis. Analysis of a validation cohort of 80 elderly patients treated with myelosuppressive chemotherapy confirmed that relative BAX localization correlates with probability of disease progression, FLT3-ITD status, and leukocytosis. Relative BAX localization could therefore be helpful to identify elderly or frail patients who may benefit from cytotoxic therapy.
In this retrospective analysis of two independent AML cohorts, our data suggest that Bax localization may predict prognosis of patients with AML and cellular predisposition to apoptosis, combining the actual contribution of known and unknown factors to a final "common path."
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Abstract Introduction Traditional risk factors for cardiovascular diseases have been shown to have an even higher impact in the HIV infected population. Cardiovascular risk factors amongst people ...living with HIV treated in doctor's offices in Austria have not been documented before. Our study aimed to close this gap, focusing on dyslipidemia, diabetes mellitus and diabetes risk. Patients and methods After ethics approval, consecutive patients who visited their treating physicians for routine checks were enrolled. The lipid profile was assessed by measuring total cholesterol, triglycerides, HDL and apolipoprotein B and calculating LDL and non-HDL-cholesterol. The diabetes risk was calculated by measuring insulin and blood glucose levels and assessing insulin resistance and beta cell function using the HOMA-IR model. Results 522 patients were included in the analysis. 90.2% of the participants were on antiretroviral therapy. Two third had an impaired lipid profile, but dyslipidemia had been diagnosed only in 46.3% of the patients. There was a clear correlation between protease inhibitor use and pathologic blood lipids. Of the persons with dyslipidemia, 18.4% received lipid lowering drugs. 8 persons (1.6%) fulfilled the criteria for diabetes mellitus. Of those, 4 patients already had a diagnosed diabetes mellitus. 50.1% of the study participants showed an increased insulin resistance. Patients on nucleoside reverse transcriptase inhibitors had significantly higher markers for impaired glucose metabolism. Discussion We found a high percentage of increased insulin resistance, of impaired lipid profile and in contrast to this a low treatment rate with lipid lowering drugs in this cohort of people living with HIV.
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