Specific Avenin Cross-Reactivity with G12 Antibody in a Wide Range of Current Oat Cultivars.

Current clinical studies confirm that the consumption of oats for people suffering from celiac disease is safe. Some studies have confirmed different levels of immunoreactive gluten epitopes of oats in different cultivars, while others explain these differences due to contamination with gluten-rich species or as random cross-reactivity ELISA of homologous oat epitopes with anti-wheat gliadin antibodies. The aim of our two-year study was therefore to map cross-reactive oat epitopes in a set of 132 oat cultivars using a G12-based ELISA kit. The results were focused on the varietal and annual level of cross-reactivity (interference) of avenin epitopes with the G12 antibody on the identification of potential cultivars with significantly different interferences and assessing the degree of risk of possible false-contamination with external gluten. Although repeated evaluations confirmed high year-to-year variability (RSD ≥ 30%) in approximately 2/3 of the cultivars, the content of interfering avenin epitopes with G12 did not exceed the considered safe limit (20 mg·kg-1) for celiacs. At the same time, not only annual but, above all, significant cultivar dependences in the interference of avenins to the G12 antibody were demonstrated. Genetic dependence was further confirmed in connection with the proven avenin polymorphism as well as immunoblotting with the identification of interfering peptides with the G12 antibody in the 25 and 30 kDa regions. It was the occurrence of two bands around 30 kDa that predominantly occurred in oat cultivars with a relatively higher content of cross-reactive avenins (12-16 mg·kg-1). Due to the fact that the contents of interfering avenins ranged in several cultivars even over 16 mg·kg-1, the choice of a suitable oat cultivar may be crucial for gluten-free food producers, as it reduces the risk of a possible false-response of the commercial ELISA kits when checking the real-gluten contamination.

Avenanthramides: Unique Bioactive Substances of Oat Grain in the Context of Cultivar, Cropping System, Weather Conditions and Other Grain Parameters.

Our study was focused on the evaluation of the content of a wider spectrum of eight avenanthramides (AVNs) as unique components of oat grain under the effects of four selected factors (cultivar, locality, cropping system, and year). The weather effects on changes in the AVN content and their relationship to other important parameters of oat grain were further evaluated in more detail. A sensitive UHPLC system coupled with a QExactive Orbitrap mass spectrometer was used for AVN quantification. AVNs confirmed a high variability (RDS = 72.7-113.5%), which was dominantly influenced by the locality and year factors. While most AVN types confirmed mutually high correlations (r = 0.7-0.9), their correlations with the other 10 grain parameters were lower (r ≤ 0.48). Their significant correlations (0.27-0.46) with ß-D-glucan could be used in perspective in breeding programs for the synergetic increase of both parameters. PCA analysis and Spearman correlations based on individual cultivars confirmed a significant effect of June and July precipitation on the increase of Σ AVNs. However, the results also indicated that higher precipitation can generate favorable conditions for related factors, such as preharvest sprouting evoking a direct increase of AVNs synthesis in oat grain.

DNA methylation and mRNA expression of HLA-DQA1 alleles in type 1 diabetes mellitus.

Type 1 diabetes (T1D) belongs among polygenic multifactorial autoimmune diseases. The highest risk is associated with human leucocyte antigen (HLA) class II genes, including HLA-DQA1 gene. Our aim was to investigate DNA methylation of HLA-DQA1 promoter alleles (QAP) and correlate methylation status with individual HLA-DQA1 allele expression of patients with T1D and healthy controls. DNA methylation is one of the epigenetic modifications that regulate gene expression and is known to be shaped by the environment.Sixty one patients with T1D and 39 healthy controls were involved in this study. Isolated DNA was treated with sodium bisulphite and HLA-DQA1 promoter sequence was amplified using nested PCR. After sequencing, DNA methylation of HLA-DQA1 promoter alleles was analysed. Individual mRNA HLA-DQA1 relative allele expression was assessed using two different endogenous controls (PPIA, DRA). We have found statistically significant differences in HLA-DQA1 allele 02:01 expression (PPIA normalization, Pcorr = 0·041; DRA normalization, Pcorr = 0·052) between healthy controls and patients with T1D. The complete methylation profile of the HLA-DQA1 promoter was gained with the most methylated allele DQA1*02:01 and the least methylated DQA1*05:01 in both studied groups. Methylation profile observed in patients with T1D and healthy controls was similar, and no correlation between HLA-DQA1 allele expression and DNA methylation was found. Although we have not proved significant methylation differences between the two groups, detailed DNA methylation status and its correlation with expression of each HLA-DQA1 allele in patients with T1D have been described for the first time.

HLA-DRB1, -DQA1 and -DQB1 genotyping of 180 Czech individuals from the Czech Republic pop 3.

One hundred and eighty Czech individuals from the Czech Republic pop 3 were genotyped at the HLA-DRB1, -DQA1 and -DQB1 loci using sequence-specific primers PCR methods. HLA-DRB1, -DQA1 and -DQB1 genotypes are consistent with expected Hardy-Weinberg (HW) proportions. These genotype data are available in the Allele Frequencies Net Database under identifier AFND.

Differences in promoter DNA methylation and mRNA expression of individual alleles of the HLA class II DQA1 gene.

OBJECTIVES: Extensive polymorphism of HLA class II genes is not restricted to the coding region of the gene. It extends also to the linked promoter region, where it forms the basis for different levels of individual allele's expression. Differential expression of HLA class II alleles can shape an immune response and influence the risk of developing autoimmune disease. In addition to genetic variability, variation in epigenetic modifications, including DNA methylation, can be another cause of the uneven expression of individual alleles. We aimed to analyze the DNA methylation of promoter sequences and the levels of expression of individual DQA1 gene alleles, interallelic variation of these two characteristics and the relationship between them. METHODS: The 60 healthy donors included into study were HLA-DRB1, HLA-DQB1 and HLA-DQA1 genotyped using PCR-SSP. Genomic DNA was treated by sodium bisulfite and the target segment in the HLA-DQA1 gene promoter was PCR amplified. PCR product was cloned into Escherichia coli and individual clones were sequenced. Transcripts of individual DQA1 alleles in peripheral blood leukocytes were quantified by Real-Time PCR. RESULTS: In this study, we have described detailed DNA methylation profile of promoter area of DQA1 gene alleles. The overall promoter methylation is increased for DQA1*02:01 and DQA1*04:01 alleles, on the other side, DQA1*05:01 allele shows decreased methylation level. Our results suggest that there are only minor interindividual differences in DRA-normalized expression level of specific allele. Furthermore, expression levels of individual alleles followed DQA1*03>*01:03 (in DRB1*13-DQA1*01:03-DQB1*06:03 haplotype)>*01:01,*05:05, and DQA1*03>*02:01>*05:05 hierarchy. The statistically significantly most expressed allele, DQA1*03, comprises part of DQ8 molecule, which is commonly linked to autoimmune diseases. A clear relationship between promoter DNA methylation and mRNA expression level of the DQA1 gene could not be identified.

The intron 4 polymorphism in the calcium-sensing receptor gene in diabetes mellitus and its chronic complications, diabetic nephropathy and non-diabetic renal disease.

BACKGROUND/AIMS: Calcium-Sensing Receptor (CaSR) significantly affects calcium-phosphate metabolism in kidneys, and it is implicated in the pathogenesis of diabetes mellitus (DM) due to its expression in pancreatic ß-cells. The role of CaSR as one of the players in pathogenesis of chronic kidney disease (CKD) has been speculated. METHODS: 158 Type 2 diabetic patients divided into three groups according to occurrence and type of kidney complications, 66 nondiabetic patients CKD, and 93 healthy subjects were enrolled into the study to analyze the role of two CaSR polymorphisms (in the codon 990 and in the intron 4) in ethiopathogenesis of DM and CKD. The Type 2 diabetic groups consisted of 48 patients without any kidney abnormalities, 58 patients with diabetic nephropathy (DN), and 52 patients with nondiabetic renal disease (NDRD). The distribution of genotype and allele frequencies was studied using PCR with the TaqMan Discrimination Assay or followed by the Restriction Fragment Length Polymorphism method, respectively. RESULTS: We have found that the intron 4 polymorphism is a risk factor for the development of DM and CKD, except DN, while the codon 990 does not show any disease association. CONCLUSION: We conclude that CaSR is a general factor in pancreas and kidney pathologies.

Pepsin digest of wheat gliadin fraction increases production of IL-1ß via TLR4/MyD88/TRIF/MAPK/NF-κB signaling pathway and an NLRP3 inflammasome activation.

Celiac disease (CD) is a gluten-responsive, chronic inflammatory enteropathy. IL-1 cytokine family members IL-1ß and IL-18 have been associated with the inflammatory conditions in CD patients. However, the mechanisms of IL-1 molecule activation in CD have not yet been elucidated. We show in this study that peripheral blood mononuclear cells (PBMC) and monocytes from celiac patients responded to pepsin digest of wheat gliadin fraction (PDWGF) by a robust secretion of IL-1ß and IL-1α and a slightly elevated production of IL-18. The analysis of the upstream mechanisms underlying PDWGF-induced IL-1ß production in celiac PBMC show that PDWGF-induced de novo pro-IL-1ß synthesis, followed by a caspase-1 dependent processing and the secretion of mature IL-1ß. This was promoted by K+ efflux and oxidative stress, and was independent of P2X7 receptor signaling. The PDWGF-induced IL-1ß release was dependent on Nod-like receptor family containing pyrin domain 3 (NLRP3) and apoptosis-associated speck like protein (ASC) as shown by stimulation of bone marrow derived dendritic cells (BMDC) from NLRP3(-/-) and ASC(-/-) knockout mice. Moreover, treatment of human PBMC as well as MyD88(-/-) and Toll-interleukin-1 receptor domain-containing adaptor-inducing interferon-ß (TRIF)(-/-) BMDC illustrated that prior to the activation of caspase-1, the PDWGF-triggered signal constitutes the activation of the MyD88/TRIF/MAPK/NF-κB pathway. Moreover, our results indicate that the combined action of TLR2 and TLR4 may be required for optimal induction of IL-1ß in response to PDWGF. Thus, innate immune pathways, such as TLR2/4/MyD88/TRIF/MAPK/NF-κB and an NLRP3 inflammasome activation are involved in wheat proteins signaling and may play an important role in the pathogenesis of CD.

Polymorphisms in the vitamin D receptor gene and parathyroid hormone gene in the development and progression of diabetes mellitus and its chronic complications, diabetic nephropathy and non-diabetic renal disease.

BACKGROUND: We chose to study polymorphisms of vitamin D receptor gene (VDR) and parathyroid hormone genes (PTH), whose protein products significantly affect calciumphosphate metabolism in kidneys and are implicated in the pathogenesis of diabetes, which may also involve kidney damage. METHODS: Distribution of genotypes of four polymorphisms in VDR gene, i.e, TaqI (rs731236), BsmI (rs1544410) ApaI (rs7975232), FokI (rs2228570) and two polymorphisms of PTH gene, i.e., DraII (rs6256), BstBI (rs6264), were studied using PCRRFLP. Examined groups consisted of 147 patients with diabetes (DM), 47 patients with nondiabetic renal disease (NDRD), 132 patients with diabetic nephropathy (DN) and 118 healthy subjects. CONCLUSION: Comparison of DN group and healthy subjects identified statistically significant difference for the FokI polymorphism in VDR gene (P<10-4) and also for the BstBI polymorphism in PTH gene (P=0,023). Differences in DraII polymorphism distribution in PTH gene were statistically significant in each group of patients compared to healthy subjects. In DN patients, the BBFFAATt combination of VDR gene was more frequent than in healthy subjects (P=0,046), and the BbFFAaTt variant was more frequent than in DM2 patients (P=0,018). The BBDD haplotype of PTH gene seems to be a predisposing factor for diabetes itself (P=0,019).

Transcriptionally regulated adhA gene encodes alcohol dehydrogenase required for ethanol and n-propanol utilization in Corynebacterium glutamicum R.

Corynebacterium glutamicum R adhA gene encodes a homodimeric, NAD-dependent, 345 amino acid residue alcohol dehydrogenase with two zinc ions per subunit. Chromosomal inactivation of the adhA gene rendered the strain incapable of growth on either ethanol or n-propanol as the sole carbon source. RNA hybridization analysis revealed that adhA transcription was not only induced by these two substrates, but it was also subject to glucose catabolite repression. Accordingly, both induction of AdhA activity and ethanol utilization were detected only after depletion of glucose. Deletion of either or both of potential cyclic adenosine monophosphate (cAMP) receptor binding site and an inverted repeat of sequence 5'-GCAATTGATG-N (8)-CACAATTGC-3' in the promoter region of adhA strongly suggested that IR, which does not share significant similarity with other regulatory DNA elements of C. glutamicum, represents a transcriptional repressor binding site. Purified recombinant AdhA displayed the highest substrate specificities towards ethanol and n-propanol and their corresponding aldehydes.