BACKGROUND & OBJECTIVES: Trisomy 21 is the most common chromosomal aneuploidy in live born infants. Recently, the over expression of chromosome 21-derived microRNAs (miR-99a, let-7c, miR-125b-2, miR-155 and miR-802) in human fetal hippocampus and heart samples from individuals with Down syndrome was observed. Therefore, concentrations and expression profile of extracellular chromosome 21-derived microRNAs were studied to verify their ability to distinguish noninvasively between pregnancies bearing euploid fetuses and those affected with Down syndrome. METHODS: RNA enriched for small RNAs was isolated from plasma samples of 12 pregnant women with high risk of bearing Down syndrome foetuses (median gestation 18.5 wk), 12 women with normal course of gestation and 10 non-pregnant women. MicroRNA transcribed into cDNA using specific stem-loop primer was detected using real-time PCR assay. Simulation experiments using RNA pools of healthy non-pregnant individuals and aneuploid amniotic fluid samples in descending dilution ratio ranging from 1:1 to 1000:1 were used to test the detection limit of the technique for overexpressed chromosome 21-derived microRNAs specific for Down syndrome. The expression profile of the gene encoding microRNA was studied through the relative gene expression using the comparative Ct (threshold cycle) method. Concentrations of individual microRNAs were subtracted from the calibration curves in the course of analyses and expressed as pg of total RNA per milliliter of plasma. RESULTS: Four of the five extracellular chromosome 21-derived microRNAs (miR-99a, let-7c, miR-125b-2 and miR-155) were reliably detected in plasma samples. Simulation experiments revealed the detection limit of aneuploidy at a ratio 100:1 for let-7c, miR-125b-2 and miR-155, and a ratio of 1000:1 for miR-99a. Overexpression of extracellular miR-99a, miR-125b-2 and miR-155 was observed in pregnant women compared to non-pregnant women. Similarly, increased concentrations of extracellular miR-99a and miR-125b-2 were detected in pregnant women than in non-pregnant women. The concentrations and relative gene expression of extracellular chromosome 21-derived microRNAs did not differ between the cohorts of pregnancies bearing euploid foetuses and those affected with Down syndrome. INTERPRETATION & CONCLUSIONS: Analysis of extracellular chromosome 21-derived microRNAs has no benefit for screening programmes and non-invasive diagnosis of Down syndrome.
- MeSH
- aneuploidie MeSH
- diploidie MeSH
- dospělí MeSH
- Downův syndrom genetika patologie MeSH
- lidé MeSH
- lidské chromozomy, pár 21 genetika MeSH
- mikro RNA biosyntéza genetika MeSH
- plod MeSH
- těhotenství MeSH
- vývojová regulace genové exprese * MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cíl studie: Cílem této studie bylo zjistit, zda je možné detekovat v mateřské cirkulaci expresi genů přítomných na chromozomu 21, které kódují mikroRNA (miR-99a, let-7c, miR-125b-2, miR-155 a miR-802). Následně bylo cílem této studie zhodnotit, zda se budou plazmatické koncentrace a expresní profil chromozom 21 specifických mikroRNA lišit mezi těhotenstvím s euploidním a aneuploidním plodem (Downův syndrom). Typ studie: Pilotní studie. Název a sídlo pracoviště: Oddělení molekulární biologie a patologie buňky, Gynekologicko-porodnická klinika, 3. lékařská fakulta, Univerzita Karlova v Praze. Metodika: Celkem bylo retrospektivně testováno 12 těhotných žen s fyziologickým průběhem gravidity (průměr 16,4 týdne, medián 16. týden), 12 těhotných žen s potvrzeným Downovým syndromem u plodu (průměr 18,2 týdne, medián 18,5 týdne) a 6 zdravých žen bez známek těhotenství. Z 1 ml plazmy byla izolována RNA obohacená o frakci malých RNA (včetně mikroRNA). Následně byla příslušná mikroRNA přepsána do cDNA pomocí specifického stem-loop primeru a detekována pomocí PCR v reálném čase. Výsledky: Komerční systémy umožnily spolehlivě detekovat 4 z 5 extracelulárních chromozom 21 specifických mikroRNA (miR-99a, let-7c, miR-125b-2 a miR-155). Expresní profil extracelulárních miR-99a, miR-125b-2 a miR-155 se významně lišil mezi souborem těhotných a netěhotných žen. Také plazmatické hladiny miR-99a a miR-125b-2 byly významně vyšší u těhotných žen. Plazmatické koncentrace a genová exprese extracelulárních chromozom 21 specifických mikroRNA (miR-99a, let-7c, miR-125b-2 a miR-155) se nelišily mezi souborem těhotných žen s euploidním a aneuploidním plodem. Závěr: Analýza extracelulárních chromozom 21 specifických mikroRNA neumožňuje rozlišení mezi těhotenstvími s euploidním a aneuploidním plodem, a nemá tudíž žádný přínos pro screeningový program.
Objective: Initially, we focused on the detection of extracellular microRNAs in maternal circulation, whose genes are located on human chromosome 21 (miR-99a, let-7c, miR-125b-2, miR-155 and miR-802). Subsequently, we studied if plasmatic concentrations and/or expression profile of extracellular chromosome 21-derived microRNAs would distinguish between pregnancies bearing euploid foetuses and those affected with Down syndrome. Design: Pilot study. Setting: Division of Molecular Biology and Cell Pathology, Department of Gynaecology and Obstetrics, Third Faculty of Medicine, Charles University, Prague. Methods: 12 women with normal course of gestation (mean 16.4 weeks, median 16.0 weeks), 12 pregnancies bearing Down syndrome foetus (mean 18.2 weeks, median 18.5 weeks) and 6 non-pregnant individuals were involved in the retrospective study. RNA enriched for small RNAs (including microRNAs) was isolated from 1ml of plasma sample. Consequently relevant microRNA was transcribed into cDNA using specific stem-loop primer and detected by specific real-time PCR assay. Results: Commercial systems enabled reliable detection of 4 out of 5 extracellular chromosome 21-derived microRNAs (miR-99a, let-7c, miR-125b-2 and miR-155). Expression profile of extracellular miR-99a, miR-125b-2 and miR-155 was significantly higher in the cohort of pregnant women than in non-pregnant individuals. Also plasmatic levels of miR-99a and miR-125b-2 were significantly increased in pregnant women. Unfortunately, the concentrations and gene expression of extracellular chromosome 21-derived microRNAs (miR-99a, let-7c, miR-125b-2 and miR-155) did not differ between the cohorts of pregnancies bearing euploid foetuses and those affected with Down syndrome. Conclusion: Analysis of extracellular chromosome 21-derived microRNAs does not distinguish between pregnancies with euploid and aneuploid foetuses and has no benefit for screening programmes.
- Klíčová slova
- PCR v reálném čase,
- MeSH
- diferenciální diagnóza MeSH
- Downův syndrom * diagnóza genetika MeSH
- exprese genu MeSH
- financování organizované MeSH
- lidé MeSH
- lidské chromozomy, pár 21 * genetika MeSH
- mikro RNA * genetika izolace a purifikace krev MeSH
- nemoci plodu * diagnóza genetika MeSH
- pilotní projekty MeSH
- placenta MeSH
- plod * MeSH
- polymerázová řetězová reakce MeSH
- retrospektivní studie MeSH
- statistika jako téma MeSH
- těhotenství MeSH
- Check Tag
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- abstrakt z konference MeSH
The role of infection in autoimmunity is widely discussed. In this study we concentrated on relationship between HELICOBACTER PYLORI as a very important gastroduodenal pathogen and autoimmune thyroiditis (AT). Forty seven AT patients and 34 healthy controls were enrolled. They were split into: THP ( H.PYLORI positive patients, n=17), THN ( H.PYLORI negative patients, n=30), CP ( H.PYLORI positive controls, n=17) and CN groups ( H.PYLORI negative controls, n=17). By protein microarray we analysed production of 23 cytokines and chemokines prior and post stimulation with H.PYLORI lysate and its lipopolysaccharide (LPS). Reactivity to lysate as well as to bacterial LPS differed within groups. The lowest basal cytokine and chemokine production was observed in CN group but these subjects reacted significantly to specific stimulation by increasing IFN-gamma (in comparison with THP p=0.01 for LPS and p=0.004 for H.PYLORI lysate) and TGF-beta production (p=0.015 for LPS). In contrast, IL-10 and IL-5 were decreased in this group. In CP, THN and THP groups, we observed in general higher chemokine response. THP group increased proinflammatory IL-6 after specific stimulation as well (in comparison with CP p<0.0001 for LPS stimulation). We observed different "reactivity pattern" to H.PYLORI within groups with low basal cytokine and chemokine production in healthy H.PYLORI negative controls but with clear specific response in IFN-gamma and TGF-beta production in this group. Adequate immune reaction which is joined to appropriate immunoregulation leads to prevention of the chronic infection and on the other hand may prevent the development of "connected" diseases such as autoimmune. J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart.New York.
- MeSH
- autoimunitní tyreoiditida imunologie mikrobiologie MeSH
- chemokiny biosyntéza MeSH
- čipová analýza proteinů MeSH
- cytokiny biosyntéza MeSH
- dítě MeSH
- dospělí MeSH
- ELISA MeSH
- Helicobacter pylori imunologie MeSH
- infekce vyvolané Helicobacter pylori imunologie mikrobiologie MeSH
- kultivované buňky MeSH
- leukocyty mononukleární imunologie MeSH
- lidé MeSH
- mladiství MeSH
- Check Tag
- dítě MeSH
- dospělí MeSH
- lidé MeSH
- mladiství MeSH
- ženské pohlaví MeSH
- Publikační typ
- práce podpořená grantem MeSH
Perfect maternal diabetes compensation is crucial for the outcome of the baby. However, little is known how hyperglycaemia influences the specific immune response. Furthermore, babies of type 1 diabetes (T1D) mothers have less risk of development T1D than babies with a T1D father. This study aimed to analyze the effect of maternal hyperglycaemia on newborns with focus on the response to diabetes-associated autoantigens. Populations: (1) Newborns of T1D mothers split into groups according to maternal diabetes compensation during the 3rd trimester: perfect (n = 15) or acceptable (n = 25) compensation. (2) newborns with T1D father (n = 12) (3) newborns with a mother treated for either gestational or type 2 diabetes (n = 10) (4) control newborns (n = 25). Spontaneous as well as diabetes-associated autoantigen-stimulated production of 23 cytokines and chemokines were tested using protein microarray. In addition, the influence of glucose on cytokine and chemokine responsiveness was analyzed in vitro. The study groups differed in their spontaneous as well as stimulated cytokine and chemokine spectra. A prominent Th1 response (high IFN-gamma) from autoantigen stimulation was observed especially in babies of T1D fathers (P = 0.001) and also in mothers with perfect diabetes compensation during the 3rd trimester (P = 0.016) in comparison with control newborns. By contrast, cord blood mononuclear cells cultivated in vitro in high glucose concentration decreased the diabetogenic stimulated Th1 cytokine response. Maternal 'sweet' as well as 'autoimmune environment' may both lead to lower occurrence of T1D within their offspring. Further studies will reveal the exact immunological mechanism of this observation.
- MeSH
- autoantigeny imunologie MeSH
- čipová analýza proteinů MeSH
- cytokiny biosyntéza metabolismus účinky léků MeSH
- diabetes mellitus 1. typu imunologie MeSH
- dospělí MeSH
- fetální krev imunologie metabolismus MeSH
- glukosa farmakologie MeSH
- glutamát dekarboxyláza farmakologie MeSH
- hyperglykemie imunologie MeSH
- leukocyty mononukleární imunologie účinky léků MeSH
- lidé MeSH
- novorozenec MeSH
- těhotenství při diabetu imunologie MeSH
- těhotenství MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- novorozenec MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- práce podpořená grantem MeSH
50 stran ; 30 cm
Sborník textů, které se zaměřují na metody molekulární genetické a cytogenetické vyšetřovací metody. Součást projektu Metabolické vzdělávací centrum. Určeno odborné veřejnosti.
- MeSH
- cytogenetické vyšetření MeSH
- cytogenetika MeSH
- diagnostické techniky molekulární MeSH
- genetické testování MeSH
- molekulární biologie MeSH
- Publikační typ
- sborníky MeSH
- Konspekt
- Patologie. Klinická medicína
- NLK Obory
- genetika, lékařská genetika
- molekulární biologie, molekulární medicína
- cytologie, klinická cytologie
- NLK Publikační typ
- brožury
- projekty
- MeSH
- autoprotilátky diagnostické užití MeSH
- chemokiny analýza diagnostické užití sekrece MeSH
- cytokiny analýza diagnostické užití sekrece MeSH
- diabetes mellitus 1. typu diagnóza imunologie MeSH
- diagnostické techniky a postupy MeSH
- ELISA MeSH
- financování organizované MeSH
- interpretace statistických dat MeSH
- lidé MeSH
- mikročipová analýza MeSH
- prediktivní hodnota testů MeSH
- senzitivita a specificita MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- abstrakt z konference MeSH
- Publikační typ
- abstrakt z konference MeSH
