LD-PCR Dotaz Zobrazit nápovědu
- MeSH
- histokompatibilita - antigeny genetika metabolismus MeSH
- HLA antigeny genetika metabolismus MeSH
- leukemie metabolismus MeSH
- lidé MeSH
- monoklonální protilátky analýza MeSH
- nádorové buňky kultivované MeSH
- polymerázová řetězová reakce s reverzní transkripcí metody MeSH
- regulace genové exprese MeSH
- techniky in vitro MeSH
- Check Tag
- lidé MeSH
Cílem práce bylo zavedení screeningové PCR do diagnostiky Borrelia burgdorferi sensu lato vevektoru, výběr nejvhodnějšího primeru derivovaného z chromosomální DNA a detekce jednotlivýchgenomospecies.Citlivost primerů popsaných v literatuře (LD, 16S, Wk, 5S-23S) byla testována pomocí různéhomnožství DNA kmenů borelií. Nejcitlivější primer, LD, byl použit pro detekci borelií ve vektoru.Klíšťata byla sbírána v městských parcích od roku 1995 do roku 1997. Celkem bylo vyšetřeno 635klíšťat. Pozitivita souboru se v různých letech lišila: 9,2 % v roce 1995, 3,4 % v roce 1996 a 4,5 % v roce1997. Dospělá klíšťata byla výrazně více infikována než nymfy. Borrelia garinii na lokalitě převládá,Borrelia burgdorferi sensu stricto nebyla dosud detekována. Smíšená infekce Borrelia garinii/Bor-relia afzelii byla nalezena v roce 1997 v jednom vzorku (samice klíštěte).Polymerázová řetězová reakce (PCR) je citlivá a specifická metoda, která je vhodná ke zjištěnípromořenosti klíšťat boreliemi. S poměrně vysokou citlivostí umožňuje rozlišit jednotlivé genomo-species Borrelia burgdorferi ve vektoru. Před jejím použitím je nutno testovat citlivost reakce, a toi v přítomnosti klíšťové DNA.
The objective of the work to introduce screening PCR into the diagnosis of Borrelia burgdorferisensu lato in the vector selection of the most suitable primer, derived from chromosomal DNA anddetection of different genome species.The sensitivity of primers, described in the literature (LD, 16S, Wk, 5S-23S) was tested by differentamounts of DNA strains of borrelias. The most sensitive primer – LD was used for detection ofborrelias in the vector. Ticks were collected in municipal parks from 1995 – 1997. A total of 635 tickswere examined. The positivity of the group differs in individual years: 9.2% in 1995, 3.4% in 1996,and 4.5% in 1997. Adult ticks were markedly more infected than nymphs. Borrelia garinii prevailsat the site, Borrelia burgdorferi sensu stricto was not detected so far. Mixed infection with Borreliagarinii/Borrelia afzelii was found in 1997 in one sample (female ticks).PCR is a sensitive and specific method suitable for assessment of the herd immunity of ticks withborrelias. It makes it possible to differentiate with a relatively high sensitivity individual genomespecies of Borrelia burgdorferi in the vector. Before its use the sensitivity of the reaction must betested in the presence of tick DNA.
Lyme disease (LD) spirochetes are well known to be able to disseminate into the tissues of infected hosts, including humans. The diverse strategies used by spirochetes to avoid the host immune system and persist in the host include active immune suppression, induction of immune tolerance, phase and antigenic variation, intracellular seclusion, changing of morphological and physiological state in varying environments, formation of biofilms and persistent forms, and, importantly, incursion into immune-privileged sites such as the brain. Invasion of immune-privileged sites allows the spirochetes to not only escape from the host immune system but can also reduce the efficacy of antibiotic therapy. Here we present a case of the detection of spirochetal DNA in multiple loci in a LD patient's post-mortem brain. The presence of co-infection with Borrelia burgdorferi sensu stricto and Borrelia garinii in this LD patient's brain was confirmed by PCR. Even though both spirochete species were simultaneously present in human brain tissue, the brain regions where the two species were detected were different and non-overlapping. The presence of atypical spirochete morphology was noted by immunohistochemistry of the brain samples. Atypical morphology was also found in the tissues of experimentally infected mice, which were used as a control.
- MeSH
- Borrelia burgdorferi komplex * genetika MeSH
- Borrelia burgdorferi * genetika MeSH
- Borrelia * genetika MeSH
- lidé MeSH
- lymeská nemoc * MeSH
- mozek MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
Hyalinizing clear cell carcinoma (HCCC) is a rare salivary gland carcinoma with a generally indolent behavior, characterized by recurrent chromosomal translocation involving EWSR1 (22q12.2) leading to two fusion genes EWSR1::ATF1 or EWSR1::CREM. We report one case of HCCC with a novel SMARCA2::CREM fusion, identified by targeted RNA next generation sequencing by LD-RT-PCR, which has until now never been described in salivary glands. The exon 4 of SMARCA2 is fused to exon 5 of CREM. This fusion has been described previously in only one tumor, a central nervous system tumor (intracranial mesenchymal tumor) but not in other FET::CREB fused tumors. This fusion was confirmed by CREM break-apart FISH and reverse transcriptase polymerase chain reaction (RT-PCR). The tumor cells showed retained expression of INI1, SMARCA2, and SMARCA4 by immunohistochemistry. We compare its clinical, histopathological, immunophenotypic, genetic features with those previously described in HCCC, FET::CREB fusion-positive. Our results added data suggesting that different histomolecular tumor subtypes seem to be included within the terminology "HCCC, FET::CREB fusion-positive," and that further series of cases are needed to better characterize them.
- MeSH
- DNA-helikasy genetika MeSH
- exony MeSH
- fúzní onkogenní proteiny genetika metabolismus MeSH
- jaderné proteiny genetika metabolismus MeSH
- karcinom * genetika MeSH
- lidé MeSH
- modulátor elementu responzivního pro cyklický AMP genetika metabolismus MeSH
- nádory slinných žláz * genetika patologie MeSH
- protein EWS vázající RNA genetika MeSH
- slinné žlázy metabolismus MeSH
- transkripční faktory genetika metabolismus MeSH
- translokace genetická MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
Identification and quantitative monitoring of mutant BCR-ABL1 subclones displaying resistance to tyrosine kinase inhibitors (TKIs) have become important tasks in patients with Ph-positive leukemias. Different technologies have been established for patient screening. Various next-generation sequencing (NGS) platforms facilitating sensitive detection and quantitative monitoring of mutations in the ABL1-kinase domain (KD) have been introduced recently, and are expected to become the preferred technology in the future. However, broad clinical implementation of NGS methods has been hampered by the limited accessibility at different centers and the current costs of analysis which may not be regarded as readily affordable for routine diagnostic monitoring. It is therefore of interest to determine whether NGS platforms can be adequately substituted by other methodological approaches. We have tested three different techniques including pyrosequencing, LD (ligation-dependent)-PCR and NGS in a series of peripheral blood specimens from chronic myeloid leukemia (CML) patients carrying single or multiple mutations in the BCR-ABL1 KD. The proliferation kinetics of mutant subclones in serial specimens obtained during the course of TKI-treatment revealed similar profiles via all technical approaches, but individual specimens showed statistically significant differences between NGS and the other methods tested. The observations indicate that different approaches to detection and quantification of mutant subclones may be applicable for the monitoring of clonal kinetics, but careful calibration of each method is required for accurate size assessment of mutant subclones at individual time points.
- MeSH
- bcr-abl fúzové proteiny chemie genetika MeSH
- chronická myeloidní leukemie genetika patologie MeSH
- DNA analýza genetika metabolismus MeSH
- lidé MeSH
- polymerázová řetězová reakce * MeSH
- sekvenční analýza DNA * MeSH
- srovnávací genomová hybridizace MeSH
- vysoce účinné nukleotidové sekvenování * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
BACKGROUND: Statins (HMG-CoA reductase inhibitors) represent a major class of compounds for the treatment of hypercholesterolemia due to their ability to inhibit de novo cholesterol synthesis. In addition to their hypolipidemic effects, chemoprotective properties have been attributed to statins as well. These effects involve multiple mechanisms, which, however, are not known in detail. The aim of our study was to assess in non-malignant as well as cancer cells the impact of simvastatin on the amount of cytosolic lipid droplets (LDs) implicated in many biological processes including proliferation, inflammation, carcinogenesis, apoptosis, necrosis or growth arrest. METHODS: Human embryonic kidney cells HEK-293T and human pancreatic cancer cells MiaPaCa-2 were treated with simvastatin (6 and 12 muM) for 24 and 48 hours respectively. Neutral lipid probe Nile Red was used for detection of LDs by fluorescence microscopy. Cellular cholesterol content was determined by HPLC. Changes in expression of genes related to lipid metabolism in simvastatin-treated MiaPaCa-2 cells were examined by DNA microarray analysis. Validation of gene expression changes was performed using quantitative RT-PCR. RESULTS: The treatment of the cells with simvastatin increased their intracellular content of LDs in both non-malignant as well as cancer cells, partially due to the uptake of cholesterol and triacylglyceroles from medium; but in particular, due to enhanced synthesis of triacylglyceroles as proved by significant overexpression of genes related to de novo synthesis of triacylglyceroles and phospholipids. In addition, simvastatin also markedly influenced expression of genes directly affecting cell proliferation and signaling. CONCLUSIONS: Simvastatin treatment led to accumulation of cytosolic LDs within the examined cells, a phenomenon which might contribute to the antiproliferative effects of statins.
- MeSH
- cholesterol metabolismus MeSH
- HEK293 buňky účinky léků MeSH
- lidé MeSH
- lipidová tělíska * metabolismus účinky léků MeSH
- metabolismus lipidů genetika účinky léků MeSH
- nádorové buněčné linie účinky léků MeSH
- nádory slinivky břišní * farmakoterapie metabolismus MeSH
- proliferace buněk genetika účinky léků MeSH
- regulace genové exprese účinky léků MeSH
- simvastatin * farmakologie MeSH
- statiny * farmakologie MeSH
- Check Tag
- lidé MeSH
