The release of neutrophil extracellular traps (NETs) is one of the weapons neutrophils have in their armory. NETs consist of extracellular chromatin fibers decorated with a plethora of cytoplasmic and granular proteins, such as the antimicrobial serine protease neutrophil elastase (NE). Because the first description of NETs as beneficial to the host, reports on their double-faced role in health and disease have considerably increased recently. On one hand, NETs reportedly trap and kill bacteria and also participate in the resolution of the acute inflammation associated with infection and with tissue damage. On the other hand, numerous negative aspects of NETs contribute to the etiopathogenesis of autoimmune disorders. Employing soluble and solid fluorescent substrates, we demonstrate the interaction of NE with aggregated NETs (aggNETs), the limitation of its enzymatic activity and the containment of the enzyme from surrounding tissues. These events prevent the spread of inflammation and tissue damage. The detection of DNase 1-dependent elevation of NE activity attests the continuous presence of patrolling neutrophils forming NETs and aggNETs even under conditions physiologic conditions.

• MeSH
• aktivace enzymů MeSH
• deoxyribonukleasa I metabolismus   MeSH
• deoxyribonukleasy metabolismus   MeSH
• extracelulární pasti imunologie   metabolismus   MeSH
• leukocytární elastasa metabolismus   MeSH
• lidé MeSH
• myši MeSH
• neutrofily imunologie   metabolismus   MeSH
• tělesné tekutiny metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

DNase I hypersensitive sites (DHS) are abundant in regulatory elements, such as promoter, enhancer and transcription factor binding sites. Many studies have revealed that disease-associated variants were concentrated in DHS-related regions. However, limited studies are available on the roles of DHS-related variants in lung cancer. In this study, we performed a large-scale case-control study with 20 871 lung cancer cases and 15 971 controls to evaluate the associations between regulatory genetic variants in DHS and lung cancer susceptibility. The expression quantitative trait loci (eQTL) analysis and pathway-enrichment analysis were performed to identify the possible target genes and pathways. In addition, we performed motif-based analysis to explore the lung-cancer-related motifs using sequence kernel association test. Two novel variants, rs186332 in 20q13.3 (C>T, odds ratio [OR] = 1.17, 95% confidence interval [95% CI]: 1.10-1.24, P = 8.45 × 10-7) and rs4839323 in 1p13.2 (T>C, OR = 0.92, 95% CI: 0.89-0.95, P = 1.02 × 10-6) showed significant association with lung cancer risk. The eQTL analysis suggested that these two SNPs might regulate the expression of MRGBP and SLC16A1, respectively. What's more, the expression of both MRGBP and SLC16A1 was aberrantly elevated in lung tumor tissues. The motif-based analysis identified 10 motifs related to the risk of lung cancer (P < 1.71 × 10-4). Our findings suggested that variants in DHS might modify lung cancer susceptibility through regulating the expression of surrounding genes. This study provided us a deeper insight into the roles of DHS-related genetic variants for lung cancer.

• MeSH
• deoxyribonukleasa I metabolismus   MeSH
• genetická predispozice k nemoci * MeSH
• jednonukleotidový polymorfismus MeSH
• lidé středního věku MeSH
• lidé MeSH
• lokus kvantitativního znaku MeSH
• nádory plic genetika   MeSH
• senioři MeSH
• studie případů a kontrol MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Monitoring immune responses to solid cancers may be a better prognostic tool than conventional staging criteria, and it can also serve as an important criterion for the selection of individualized therapy. Multiparametric phenotyping by mass cytometry extended possibilities for immunoprofiling. However, careful optimization of each step of such method is necessary for obtaining reliable results. Also, with respect to procedure length and costs, sample preparation, staining, and storage should be optimized. Here, we designed a panel of 31 antibodies which allows for identification of several subpopulations of lymphoid and myeloid cells in a solid tumor and peripheral blood simultaneously. For sample preparation, disaggregation of tumor tissue with two different collagenases combined with DNase I was compared, and removal of dead or tumor cells by magnetic separation was evaluated. Two possible procedures of barcoding for single-tube staining of several samples were examined. While the palladium-based barcoding affected the stability of several antigens, the staining with two differently labeled CD45 antibodies was suitable for cells isolated from a patient's blood and tumor. The storage of samples in the intercalation solution for up to two weeks did not influence results of the analysis, which allowed the measurement of samples collected within this interval on the same day. This procedure optimized on samples from patients with head and neck squamous cell carcinoma enabled identification of various immune cells including rare subpopulations.

• MeSH
• analýza jednotlivých buněk MeSH
• antigeny CD45 imunologie   MeSH
• deoxyribonukleasa I metabolismus   MeSH
• imunofenotypizace metody   MeSH
• kolagenasy metabolismus   MeSH
• lidé MeSH
• lymfocyty fyziologie   MeSH
• monoklonální protilátky metabolismus   MeSH
• myeloidní buňky fyziologie   MeSH
• nádory diagnóza   imunologie   MeSH
• palladium metabolismus   MeSH
• průtoková cytometrie MeSH
• separace buněk MeSH
• taxonomické DNA čárové kódování MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Kidney and uropoetic system diseases represent a major social, economic and health burden. This is mainly because early diagnosis of kidney dysfunction is currently unavailable, since the current markers are often reliably increased only after advanced progression of the renal diseases. Recently, circulating nucleosomes, DNA and numerous forms of RNA have been detected in human biological fluids, such as plasma, urine, saliva, and breast milk. Although their biological functions remain mostly unknown, they are attractive as potential biomarkers of various diseases. In urine, many of the circulating nucleic acids originate from the cells of the kidney and the urinary tract making these non-invasive and easily obtained new biomarkers in the nephrology or urology. This review focuses on cell free nucleic acids in urine and its potential in human studies. Although, there are some technical and biological limitations, the urinary circulating nucleic acids hold a great potential as new biomarkers of renal diseases.

• Klíčová slova
• transrenální DNA, postrenální DNA,
• MeSH
• biologické markery * MeSH
• deoxyribonukleasa I MeSH
• extracelulární vezikuly MeSH
• lidé MeSH
• mitochondriální DNA moč   MeSH
• nádory diagnóza   MeSH
• nemoci ledvin * diagnóza   MeSH
• volné cirkulující nukleové kyseliny * krev   moč   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH
• přehledy MeSH

The aim of this study was to investigate the potential of extracellular DNA as a prognostic and/or therapeutic target in inflammatory bowel disease. Fifty male C57BL/6J mice were used in the experiment. Acute colitis was induced by intake of 2% dextran sulphate sodium (DSS) for seven days followed by three days of water intake. DNase I was injected intravenously on days 3 and 7. Plasmatic levels of extracellular DNA (ecDNA) were measured on days 6 and 10. Weight loss, stool consistency and liquid intake were monitored throughout the experiment. Colon length and weight, myeloperoxidase activity and tumour necrosis factor α (TNF-α) levels were measured at sacrifice. DSS-treated mice displayed severe colitis, as shown by disease activity parameters. Both groups with colitis (DNase treated and untreated) had significantly poorer weight loss, colon length and stool consistency compared with control groups on water. No differences between the DNasetreated and untreated DSS groups were recorded. Myeloperoxidase activity and levels of TNF-α in colonic tissue were notably greater in both groups with colitis compared to controls. In addition, both biochemical markers were improved in the DNasetreated group with colitis compared to the untreated group. Although the disease activity was proved by several independent parameters in both groups with colitis, levels of ecDNA did not show any difference between the groups throughout or at the end of experiment. The role of ecDNA in experimental colitis has not been confirmed. However, DNase I injection resulted in some improvement, and thus should be studied in more detail.

• MeSH
• biologické markery metabolismus   MeSH
• cílená molekulární terapie * MeSH
• deoxyribonukleasa I terapeutické užití   MeSH
• DNA metabolismus   MeSH
• kolitida farmakoterapie   patologie   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• prognóza MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Biologická léčba Crohnovy choroby a ulcerózní kolitidy je u části nemocných spojena s výskytem nežádoucích účinků, jejichž etiologie nebyla dosud objasněna. U 10-20% nemocných se objevují kožní změny a dosud přesně neurčená část pacientů trpí dalšími imunopatologickými komplikacemi, zejména kloubními a svalovými. Již dříve jsme identifikovali jako rizikový faktor kožních komplikací sníženou aktivitu deoxyribonukleázy I. Na rozsáhlém souboru nemocných budeme provádět systematický výzkum epidemiologie, etiopatogeneze, klinického obrazu a rizikových faktorů kožních a imunopatologicky indukovaných nežádoucích účinků biologické terapie. Předpokládáme spolupráci klinických výzkumníků a specialistů v oblasti imunologie, histopatologie a laboratorní diagnostiky, včetně metod molekulární biologie. Výsledky výzkumu mohou mít bezprostřední přínos pro bezpečnost biologické léčby střevních zánětů.; Biological therapy of CD and UC is associated with occurrence of adverse events of unknown aetiology in a proportion of patients. From 10 to 20% of patients experience various skin complications, while others suffer from other immunopathological complications, mainly arthral and muscular. We have previously identified decreased activity of deoxyribonuclease I (DNase I) enzyme as a risk factor of skin complications. The current project will be focused on systematic research of epidemiology, etiopathogenesis, clinical course and risk factors of skin and other immunopathological adverse events of biological therapy and based on collaboration of clinical researchers and specialists in the field of immunology, histopathology and laboratory diagnostics including methods of molecular biology. The results of the project can have a direct implication in clinical practice providing a better safety of biological therapy in IBD.

• MeSH
• biologická terapie škodlivé účinky   MeSH
• Crohnova nemoc farmakoterapie   MeSH
• deoxyribonukleasa I účinky léků   MeSH
• idiopatické střevní záněty farmakoterapie   MeSH
• kožní manifestace MeSH
• nemoci imunitního systému MeSH
• nežádoucí účinky léčiv etiologie   patofyziologie   MeSH
• regresní analýza MeSH
• TNF-alfa antagonisté a inhibitory   MeSH
• tumor nekrotizující faktory MeSH
• ulcerózní kolitida farmakoterapie   MeSH

• Konspekt
• Farmacie. Farmakologie
• NLK Obory
• farmacie a farmakologie
• dermatovenerologie
• gastroenterologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu IGA MZ ČR

Although physiological steroid levels are often pulsatile (ultradian), the genomic effects of this pulsatility are poorly understood. By utilizing glucocorticoid receptor (GR) signaling as a model system, we uncovered striking spatiotemporal relationships between receptor loading, lifetimes of the DNase I hypersensitivity sites (DHSs), long-range interactions, and gene regulation. We found that hormone-induced DHSs were enriched within ± 50 kb of GR-responsive genes and displayed a broad spectrum of lifetimes upon hormone withdrawal. These lifetimes dictate the strength of the DHS interactions with gene targets and contribute to gene regulation from a distance. Our results demonstrate that pulsatile and constant hormone stimulations induce unique, treatment-specific patterns of gene and regulatory element activation. These modes of activation have implications for corticosteroid function in vivo and for steroid therapies in various clinical settings.

• MeSH
• buněčné linie MeSH
• chromatin genetika   metabolismus   MeSH
• chromatinová imunoprecipitace MeSH
• deoxyribonukleasa I genetika   metabolismus   MeSH
• glukokortikoidy farmakologie   MeSH
• membránové proteiny genetika   metabolismus   MeSH
• myši MeSH
• receptory glukokortikoidů genetika   metabolismus   MeSH
• regulace genové exprese MeSH
• responzivní elementy * MeSH
• sekvenční analýza DNA MeSH
• transportní proteiny genetika   metabolismus   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, N.I.H., Intramural MeSH

OTC encodes ornithine carbamoyltransferase, mitochondrial matrix enzyme involved in the synthesis of urea. The tissue-specific expression of OTC in the liver and intestine is dependent on the interaction of OTC promoter with an upstream enhancer. HNF-4 and C/EBPβ are crucial for this interaction in the rat and mouse. In the present study we focused on characterization of elements involved in the regulation of OTC transcription in human. Using a set of 5'-deleted promoter mutants in a reporter assay we identified two positive cis-acting regulatory elements located at c.-105 and c.-136 within the human OTC promoter. Both are essential for the transcriptional activity of the promoter itself and for the interaction with the enhancer. Protein binding at the corresponding sites was confirmed by DNase I footprinting. Electromobility shift assay with a specific competitor and anti-HNF-4α antibody identified the DNA-protein binding sites as HNF-4α recognition motifs. A third HNF-4α binding site has been found at the position c.-187. All three HNF-4α binding sites are located within 35 bp upstream of the transcription start sites at positions c.-95, c.-119 (major) and c.-169 (minor). A series of C/EBPβ recognition motifs was identified within the enhancer. Involvement of C/EBPβ and HNF-4α in the promoter-enhancer interaction is further supported by a massive DNAprotein interaction observed in the footprinting and EMSA assays. Since the OTC promoter lacks general core promoter elements such as TATA-box or initiators in standard positions, HNF-4α most likely plays an essential role in the initiation of OTC transcription in human.

• MeSH
• 5' přiléhající oblast DNA genetika   MeSH
• buňky Hep G2 MeSH
• deoxyribonukleasa I metabolismus   MeSH
• DNA footprinting MeSH
• DNA metabolismus   MeSH
• genetická transkripce MeSH
• hepatocytární jaderný faktor 4 metabolismus   MeSH
• krysa rodu rattus MeSH
• lidé MeSH
• luciferasy metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• mutace genetika   MeSH
• myši MeSH
• ornithinkarbamoyltransferasa genetika   metabolismus   MeSH
• počítačová simulace MeSH
• promotorové oblasti (genetika) * MeSH
• regulace genové exprese enzymů * MeSH
• reportérové geny MeSH
• retardační test MeSH
• sekvence nukleotidů MeSH
• sekvenční seřazení MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Dendritic poly(L-lysines) (DGL) constitute promising nanomaterials applicable as a nonviral gene-delivery vector. In this study, we evaluate the transfection abilities of four DGL generations with special emphasis on the systematic description of the relationship of how generation (i.e., molecule size) affects the transfection efficacy. Using Hep2 cells, we demonstrated that the capability of unmodified DGL to deliver plasmid is of a magnitude lower than that of jetPEI. On the other hand, employing the Hep2 cell line stably transduced with eGFP, we observed that DGL G5 delivers the siRNA oligonucleotide with the same efficiency as Lipofectamine 2000. In further experiments, it was shown that DGL affords excellent ability to bind DNA, protect it against DNase I attack, and internalize it into cells.

• MeSH
• buněčné linie MeSH
• deoxyribonukleasa I metabolismus   MeSH
• lidé MeSH
• lipidy MeSH
• malá interferující RNA MeSH
• molekulární sekvence - údaje MeSH
• oligonukleotidy metabolismus   farmakokinetika   MeSH
• plazmidy farmakokinetika   MeSH
• polylysin chemie   farmakokinetika   MeSH
• sekvence nukleotidů MeSH
• transfekce metody   MeSH
• viabilita buněk účinky léků   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zelené fluorescenční proteiny genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

A new method of the light microscopy detection of BrdU-labeled DNA in situ is described. It is based on the oxidative attack at the deoxyribose moiety by copper(I) in the presence of oxygen, which leads to the abstraction of hydrogen atom from deoxyribose culminating in the elimination of the nucleobase, scission of the nucleic-acid strand and formation of frequent gaps. The gaps allow the reaction of the antibodies with the commonly used markers of replication (e.g. 5-bromo-2'-deoxyuridine), which are otherwise masked. The method developed makes it possible to detect nuclear and mitochondrial DNA replication efficiently. In most cases, it does not inhibit effective protein detections and in addition enables simultaneous localization of newly-synthesized RNA. The alternative presently-used methods result in protein denaturation and/or extensive DNA cleavage followed by the DNA-bound proteins peeling off.

• MeSH
• barvení a značení MeSH
• bromodeoxyuridin chemie   metabolismus   MeSH
• buněčné jádro metabolismus   MeSH
• deoxyribonukleasa I MeSH
• fluorescenční protilátková technika nepřímá MeSH
• HeLa buňky MeSH
• kyselina askorbová chemie   MeSH
• kyslík chemie   MeSH
• lidé MeSH
• mitochondriální DNA chemie   genetika   MeSH
• oxidace-redukce MeSH
• replikace DNA * MeSH
• síran měďnatý chemie   MeSH
• štěpení DNA * MeSH
• superoxiddismutasa chemie   MeSH
• superoxidy chemie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH