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

• Publikační typ
• abstrakty MeSH

• 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

BACKGROUND: Autoreactive T cells have a crucial role in type 1 diabetes (T1D) pathogenesis. OBJECTIVES: The aim of our study was to monitor the in vitro production of cytokines by peripheral blood mononuclear cells (PBMCs) after stimulation with diabetogenic autoantigens. Subjects: Ten T1D patients (tested at the time of diagnosis and 6 and 12 months later), 10 first-degree relatives of the T1D patients, and 10 controls underwent the study. METHODS: PBMCs were stimulated with glutamic acid decarboxylase 65 (GAD65) amino acids (a.a.) 247-279, 509-528, and 524-543; proinsulin a.a. 9-23; and tyrosine phosphatase (islet antigen-2)/R2 a.a. 853-872. Interleukin (IL)-2, IL-4, IL-5, IL-6, IL-10, IL-13, interferon (IFN)-gamma, tumor necrosis factor beta, transforming growth factor beta1, and granulocyte colony-stimulating factor (GCSF) were analyzed by protein microarray. RESULTS: Differences in cytokine(s) poststimulatory and mainly in basal production were observed in all groups. The most prominent findings were in controls, the higher basal levels of IL-2, IL-4, IL-5, IL-13, and GCSF were observed when compared with relatives (p < 0.05, for all). After stimulation in controls, there was a significant decrease in IL-2, IL-13, GCSF, and IFN-gamma (p < 0.05, for all). The group of relatives was the most variable in poststimulatory production. A strong correlation between cytokines production was found but groups differed in this aspect. CONCLUSION: By multiplex analysis, it may be possible, for example, to define the risk immunological response pattern among relatives or to monitor the immune response in patients on immune modulation therapy.

• MeSH
• autoprotilátky genetika   MeSH
• čipová analýza proteinů MeSH
• cytokiny chemická syntéza   MeSH
• diabetes mellitus 1. typu genetika   imunologie   MeSH
• dítě MeSH
• financování organizované MeSH
• glutamát dekarboxyláza genetika   chemie   MeSH
• interleukiny genetika   MeSH
• lidé MeSH
• mladiství MeSH
• molekulární sekvence - údaje MeSH
• předškolní dítě MeSH
• rodina MeSH
• sekvence aminokyselin MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH

Type 1 diabetes (T1D) is a great medical challenge and its incidence rises rapidly. T lymphocytes and their cytokine production are supposed to play a major role in T1D development. So far, there is no potent tool to recognize the early signs of cellular auto-reactivity which leads to beta-cell damage. The naive immune system of the newborn (not yet influenced by external factors) can be used as an important model for T1D pathogenesis studies. Cord blood samples of 22 healthy neonates born at term to a diabetic parent (T1DR) and 15 newborns with no family history of any autoimmune disease (controls) were collected. Determination of 23 cytokines was performed before and after the stimulation with diabetogenic autoantigens using protein microarray. We observed lower basal production of all detected cytokines in the T1DR group - granulocyte/macrophage colony-stimulating factor (GM-CSF) (P = 0.025), growth regulated protein (GRO) (P = 0.002), GRO-alpha (P = 0.027), interleukin (IL)-1-alpha (P = 0.051), IL-3 (P = 0.008), IL-7 (P = 0.027), IL-8 (P = 0.042), monocyte chemoattractant proteins (MCP)-3 (P = 0.022), monokine-induced by IFN-gamma (MIG) (P = 0.034) and regulated upon activation normal T-cell express sequence (RANTES) (P = 0.004). Exclusively lower post-stimulative levels of G-CSF (P = 0.030) and GRO-alpha (P = 0.04) were observed in controls in comparison with the basal levels. A significant post-stimulative decrease in G-CSF (P = 0.030) and MCP-2 (P = 0.009) levels was observed in controls in comparison with T1DR neonates. We also observed the interesting impact of the risky genotype on the protein microarray results. Protein microarray seems to be a useful tool to characterize a risk pattern of the immune response for T1D also in newborns.

• MeSH
• autoantigeny imunologie   MeSH
• autoimunita MeSH
• čipová analýza proteinů MeSH
• cytokiny analýza   biosyntéza   MeSH
• diabetes mellitus 1. typu imunologie   krev   MeSH
• fetální krev chemie   imunologie   metabolismus   MeSH
• leukocyty mononukleární imunologie   metabolismus   MeSH
• lidé MeSH
• novorozenec MeSH
• polymerázová řetězová reakce MeSH
• rodokmen MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• novorozenec MeSH
• ženské pohlaví MeSH