gliadin
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Celiakie je onemocněním autoimunního charakteru s geneticky podmíněnou vazbou HLA třídy II DQ2 nebo DQ8 charakterizovanou reakcemi střevních T buněk na proteiny pšeničného lepku v potravě. Unikátní peptidový fragment α2-gliadinu, gliadin-33mer, je považován za nejdůležitější imunogenní sekvenci v glutenu, peptid je zcela rezistentní na gastrointestinální peptidázy, a je zcela specifický pro prolaminy. Gliadin 33-mer je stimulátorem CD4-T buněk po deamidaci tkáňovou transglutaminázou. Jedinou ověřenou léčbou celiakie je celoživotní bezlepková dieta a v současné době se vyvíjí několik nových terapeutických přístupů. Enzymatické štěpení lepku pomocí glutenáz se zaměřením na cytotoxický gliadin 33-mer bylo ověřeno v řadě klinických studií. Detekce glutenových imunogenních peptidů, gliadin 33-meru, ve stolici a moči se stává novým neinvazivním biomarkerem a nabízí nový jednoduchý a objektivní způsob hodnocení příjmu lepku a ověření souladu s dodržováním bezlepkové diety u pacientů s celiakií. V diagnostice celiakie umožňuje spolehlivě ověřit non-responzibilní celiakii.
Celiac disease is an autoimmune disease with genetically determined HLA class II binding DQ2 or DQ8 characterized by intestinal T cell responses to wheat gluten proteins in the diet. The unique α2-gliadin peptide fragment, gliadin-33mer, is considered to be the most important immunogenic sequence in gluten, this peptide is completely resistant to gastrointestinal peptidases and is completely specific for prolamins. Gliadin 33-mer is a stimulator of CD4-T cells after deamidation by tissue transglutaminase. The only proven treatment for celiac disease is a lifelong gluten-free diet, and several new therapeutic approaches are currently being developed. The enzymatic cleavage of gluten by glutenases with a focus on the cytotoxic gliadin 33-mer has been verified in a number of clinical studies. Detection of gluten immunogenic peptides, gliadin 33-mer, in faeces and urine is becoming a new non-invasive biomarker and offers a new simple and objective way to assess gluten intake and verify compliance with a gluten-free diet in patients with celiac disease. In the diagnosis of celiac disease allows you to reliably verify non-responsive celiac disease.
- MeSH
- adherence a compliance při léčbě MeSH
- bezlepková dieta MeSH
- biologické markery MeSH
- celiakie * diagnóza dietoterapie terapie MeSH
- gliadin * analýza moč MeSH
- lidé MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
A simple modification of the horizontal starch-gel electrophoresis of gliadin proteins with the use of LKB Multiphor System has been developed. The thin-layer starch gel is prepared by using the original gel-moulding cassette and the procedure on the whole is easier. The sensitivity is comparable with that of the original method. Our modification moreover widens the field of application of the LKB Multiphor System.
The tendency to form a beta-turn in alpha-gliadin was estimated using the B-cell determinant prediction program based on the Chou and Fasman probability of beta-turn formation. Six sequences possessing a high probability of beta-turn formation were found. A statistically high agreement was found between these six sequences and three areas in alpha-gliadin with the occurrence of Pro-Ser-Gln-Gln sequence which has recently been considered responsible for toxicity in coeliac disease. By means of solid-phase synthesis seven peptides were obtained covering the above-mentioned regions. Their toxicity was tested using the fetal chick duodenum. The results support the suggestion that peptides containing the sequences Pro-Ser-Gln-Gln and Gln-Gln-Gln-Pro may be involved in the pathogenesis of coeliac disease.
A gluten-free diet (GFD) is the sole effective long-lasting treatment of celiac disease. Four monoclonal antibodies (Abs) were prepared by immunization of animals kept on GFD with gliadin. The specificity of these Abs to decapeptides of alpha- and gamma-gliadin and omega-secalin was analyzed by the PEPSCAN technique. Repetitive sequences of alpha- and gamma-gliadin and omega-secalin containing the motifs QPFPXQ (X = Q, L, P) were recognized by all Abs tested. These Abs also frequently reacted with peptides containing the sequences QQSFPQQ, QQTFPQP, and QPFRPQ. On the basis of PEPSCAN results two Abs--8D4 and 7C6--were selected for the construction of a new ELISA kit for the detection of gliadin in food. The comparison of data obtained using the newly developed ELISA kit and commercially available ones indicated that Abs selection on the basis of their fine specificity to gliadin is useful for sensitive detection of gliadin in foods.
- MeSH
- analýza potravin metody MeSH
- celiakie dietoterapie MeSH
- ELISA metody MeSH
- financování organizované MeSH
- gliadin analýza chemie imunologie MeSH
- gluteny analýza MeSH
- monoklonální protilátky imunologie MeSH
- myši MeSH
- sekvence aminokyselin MeSH
- specificita protilátek MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- MeSH
- absorpce MeSH
- buněčné linie MeSH
- gliadin fyziologie MeSH
- senzitivita a specificita MeSH
- vazba proteinů MeSH
- Publikační typ
- srovnávací studie MeSH
Monoclonal antibodies reactive with gliadin were prepared by fusion of spleen cells isolated from gliadin-immunized BALB/c mice with myeloma cells. The reactivity of mAbs with different preparations of gliadin and their enzymatic digest were measured using ELISA method. The mAb produced by GL 1 clone was shown to react preferentially with alpha-gliadin and its enzymatic digest.
Peptidic fragments of alpha-gliadin were obtained by peptic-tryptic-pancreatic (PTP) digestion of the alpha-gliadin fraction isolated by ion-exchange chromatography on a sulphopropyl-Sephadex C-50 column. The proteolytic digest was fractionated by ultrafiltration into three subfractions, PTPa1-PTPa3. The subfraction PTPa2 was then analysed and individual peaks were separated using reversed-phase high-performance liquid chromatography (RP-HPLC) using a gradient of acetonitrile in 0.1% trifluoroacetic acid and a Separon SGX-C18 sorbent. A 100-mg amount of the PTPa2 subfraction was separated in a single analysis by preparative RP-HPLC and twenty peaks were obtained for further characterization. The molecular mass in range 300-3000 was established for individual peptidic fragments by gel-permeation chromatography on a TSK-G2000 SW column.
