OBJECTIVE: A new interleukin-6 (IL-6)-dependent plasma cell leukemia cell line UHKT-944 was established from bone marrow cells derived from a 55-yr-old man with plasma cell leukemia. RESULTS: The cell line possesses phenotypic characteristics of plasma cells including the production of a monoclonal immunoglobulin IgA1-kappa. VH3-9 region of IgVH genes was rearranged and somatically hypermutated. The UHKT-944 cells were found to be negative for most of tested B-cell, T-cell, and myeloid markers. According to cytogenetic analysis, the cells were classified as near tetraploid with several numerical and structural abnormalities including the t(14;20) involving IgH locus. CONCLUSION: The established permanent plasma cell leukemia cell line is a suitable model for the study of cellular and molecular mechanisms of pathogenesis of this rare malignant disease.
- MeSH
- biologické markery MeSH
- cytogenetické vyšetření MeSH
- imunofenotypizace MeSH
- imunoglobuliny genetika metabolismus MeSH
- lidé středního věku MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- plazmocelulární leukemie diagnóza metabolismus patologie MeSH
- proliferace buněk MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
- práce podpořená grantem MeSH
We established and characterized a new IL-6 dependent multiple myeloma (MM) cell line UHKT-893 from the bone marrow of a relapsed 57-year-old woman. RESULTS: Using nephelometry, cells with plasma cell phenotype and morphology were found to secrete IgG and free kappa (κ)-light chain of immunoglobulin. κ-Light chain was also recognized intracellularly by flow cytometry and by mass spectrometry. VH4-39 region of IgVH genes was rearranged and somatically hypermutated. Cytogenetic analysis of cells revealed new chromosome abnormalities in all breakpoints unique in both MM patients and cell lines - t(1;6), t(1;11), t(5;15), t(5;21), +der(11;15) and der(16). IL-6 independent subline UHKT-893a was established by adaptation to descending IL-6 concentration, while the original cell line keeps on maintaining its IL-6 dependency. CONCLUSION: The cell line provides a suitable material for cellular and molecular studies of tumor abnormalities, with potentially unique mutagenic features of myeloma disease. It may be utilized for human hybridoma construction and vaccine development. Both IL-6 dependent and independent cell clones represent an important model for studies of myeloma cell growth and resistance emerging during targeted therapy.
- MeSH
- buňky kostní dřeně účinky léků patologie fyziologie MeSH
- cytogenetické vyšetření MeSH
- interleukin-6 farmakologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- mnohočetný myelom patologie MeSH
- nádorové buněčné linie MeSH
- primární buněčná kultura * metody MeSH
- proliferace buněk účinky léků MeSH
- recidiva MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Celkem jsme v roce 2007 vyšetřili 1048 jedinců klíštěte Ixodes ricinus z toho 935 ze sběrů vlajkováním v pražských parcích a 114 jedinců ze zákusu na člověku. V roce 2008 jsme vyšetřili 364 jedinců z toho 277 ze sběru a 86 sejmutých z lidí. Promořenost klíšťat původcem lymeské borreliózy v roce 2007 byla 9,7 %, zjištěná v temném poli a v 13 % byla prokázána DNA pomocí PCR. V roce 2008 byla promořenost klíšťat vyšší, 14 % v temném poli a 20 % pomocí PCR. Klíšťata sejmutá po zákusu na lidech byla infikována v 7 %. Přítomnost Anaplasma phagocytophilum, Bartonella sp. a Rickettsia sp byla prokázána pomocí polymerázové řetězové reakce a kontrolována sekvencí geonomu u 11,3 - 11,8 % klíšťat. Spiroplasma rodu Babesia byla zjištěna v 7,4 % u samic I. ricinus. Klíšťata sejmutá z lidí byla infikovaná borrelií v 6,5 % a anaplasmou v 1,5 %. Pravidelnými sběry v jednotlivých měsících r. 2007 a 2008 na jedné lokalitě v Praze 10 jsme ověřili změny výskytu ruzných stádií klíštěte a jejich měnící se nákazu ruznými agens v závislosti na teplotě a vývoji. K růstu bakterií Borrelia a Anaplasma sp. ve střevě klíštěte je zapotřebí určité doby s vyšší teplotou. Nejmenší počet infikovaných klíšťat byl v srpnu 2007 a 2008, kdy se z velké většiny vyskytovaly pouze larvy.
In 2007, 1048 Ixodes ricinus ticks were investigated: 935 of these were collected by flagging in Prague parks and 113 were ticks attached to the human skin. In 2008, 364 ticks were investigated, i.e. 277 and 87 ticks collected by flagging and from humans, respectively. In 2007, the causative agent of lyme borreliosis was detected in 9.7% of ticks in dark field and in 13% of ticks in DNA by PCR. In 2008, higher positivity rates were found, i.e. 14% and 20%, respectively. Seven percent of ticks obtained from humans were infected. Anaplasma phagocytophilum, Bartonella sp. and Rickettsia sp. were detected by PCR and sequence analysis in 11.3% - 11.8% of ticks. Spiroplasma of the genus Babesia was detected in 7.4% of I. ricinus females. The ticks collected from humans were infected by Borrelia in 6.5% and by Anaplasma in 1.5%. Regular monthly flagging was performed in one locality in Prague 10 in 2007 and 2008 to obtain data on the incidence of different stages of ticks and rates of infection by different agents depending on temperature and season. To grow in the tick intestine, Borrelia and Anaplasma need higher temperature for a certain period of time. The lowest numbers of infected ticks were found in August 2007 and 2008 when mostly tick larvae were collected.
- MeSH
- Anaplasma phagocytophilum genetika izolace a purifikace MeSH
- Babesia genetika izolace a purifikace MeSH
- Bartonella genetika izolace a purifikace MeSH
- Borrelia burgdorferi genetika izolace a purifikace MeSH
- infekce přenášené vektorem MeSH
- klíště mikrobiologie MeSH
- lymeská nemoc epidemiologie přenos MeSH
- polymerázová řetězová reakce využití MeSH
- Publikační typ
- grafy a diagramy MeSH
Anaplasma phagocytophilum has been first isolated from the blood of two Czech patients simultaneously with a cultivation of Borrelia burgdorferi sensu lato from their erythema migrans lesions. Cultivation of different Borrelia spp. from 12 erythema migrans biopsies, from 2 blood, one liquor and one placenta sample in BSK-H medium was successful. Adapted conventional methods targeting 16S rRNA and OspA genes for real-time polymerase chain reaction (PCR) and partial sequencing of these genes together with microscopical examinations of the blood smears provided a direct detection of the B. afzelii, B. burgdorferi, B. garinii, B. valaisiana and B. bissettii in the skin, B. garinii in the blood, placenta and liquor in 24 (36.3 %) patients, and A. phagocytophilum in 10 (15 %) patients with erythema migrans. Positive indirect IgM immunofluorescence against Anaplasma sp. was obtained in 7 cases, specific IgG antibodies were detected in 12 patients. Three women suffering from erythema migrans in the first trimester had positive PCR for Anaplasma and/or for Borrelia in the blood and two of them, later, in the placenta. Interpretation of laboratory data can bring important contribution to establishing the role of Anaplasma sp. in erythema migrans and forming the principle of precaution with laboratory diagnosis during pregnancy which always should be reflected in the resistance of Anaplasma sp. toward penicillins.
- MeSH
- Anaplasma phagocytophilum imunologie izolace a purifikace MeSH
- antigeny povrchové genetika MeSH
- bakteriální vakcíny genetika MeSH
- Borrelia burgdorferi genetika imunologie izolace a purifikace MeSH
- diferenciální diagnóza MeSH
- dospělí MeSH
- ehrlichióza diagnóza krev mikrobiologie MeSH
- erythema chronicum migrans diagnóza krev mikrobiologie MeSH
- financování organizované MeSH
- HL-60 buňky MeSH
- infekční komplikace v těhotenství diagnóza mikrobiologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- lipoproteiny genetika MeSH
- lymeská nemoc diagnóza krev mikrobiologie MeSH
- mladiství MeSH
- placenta mikrobiologie MeSH
- proteiny vnější bakteriální membrány genetika MeSH
- protilátky bakteriální krev MeSH
- senioři MeSH
- těhotenství MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mužské pohlaví MeSH
- senioři MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
The aim of this study is to present molecular, serologic, and clinical findings for dogs that were naturally infected with Anaplasma phagocytophilum or Borrelia burgdorferi sensu lato (s. l.) in the Czech Republic. This data can provide information relevant to human infection. In total, blood samples from 296 dogs and 118 engorged ticks were examined. Samples were tested for A. phagocytophilum using polymerase chain reaction (PCR) amplification, nested PCR, and direct sequencing of the 16S rDNA, and for B. burgdorferi s. l. using PCR amplification of the 16S rDNA and restriction fragment length polymorphism analysis of the 5S-23S rDNA intergenic spacer. In addition, blood samples were screened for antibodies to these bacteria. Ten (3.4%) dogs were PCR-positive for A. phagocytophilum. Morulae of A. phagocytophilum in granulocytes were found in two of these dogs. Nine of the PCR-positive dogs had clinical signs related to anaplasmosis. Statistically significant differences in the PCR detection rates were found between breeds and between symptomatic and asymptomatic dogs. Infection with Borrelia garinii was detected by PCR in a dog with meningoencephalitis. DNA of A. phagocytophilum and B. burgdorferi s. l. (B. garinii or Borrelia afzelii) was detected in 8.5% and 6.8% of ticks, respectively. Immunoglobulin (Ig) G seropositivity to A. phagocytophilum was 26%. Significant differences were found with respect to breed and gender. IgM and IgG antibodies to B. burgdorferi s. l. were detected in 2.4% and 10.3% of dogs, respectively. Our findings suggest that the exposure to B. burgdorferi s. l. exists in dogs in the Czech Republic, and exposure to A. phagocytophilum is common.
- MeSH
- Anaplasma phagocytophilum imunologie izolace a purifikace MeSH
- Borrelia burgdorferi imunologie izolace a purifikace MeSH
- ehrlichióza krev epidemiologie mikrobiologie veterinární MeSH
- imunoglobulin G MeSH
- imunoglobulin M MeSH
- klíšťata mikrobiologie MeSH
- lymeská nemoc krev epidemiologie mikrobiologie veterinární MeSH
- nemoci psů krev epidemiologie mikrobiologie MeSH
- polymerázová řetězová reakce veterinární MeSH
- protilátky bakteriální MeSH
- psi MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- psi MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
To investigate the effect of dehydroepiandrosterone (DHEA) on intracellular mRNA levels of peroxisome proliferator-activated receptors (PPARs) and PPAR-gamma coactivators (PGCs), we conducted a quantitative real-time RT-PCR study using HepG2 cells. Treatment with 100 micromol/l DHEA for 2-20 h caused a time-dependent elevation of mRNA levels in the cells. Upon 20 h, PPAR-alpha, -gamma1, and -gamma2 mRNAs and PGC-1alpha and -1beta mRNAs increased to 157, 161, 155, 656, and 475% of control levels, respectively (p < 0.05 each). Treatment with actinomycin D for 2.5-8 h revealed a significant stabilization effect of DHEA on PPAR-gamma1 and PGC-1alpha mRNAs at both 2.5 and 8 h incubation periods and a mild but significant stabilization effect on PGC-1beta mRNA at the 8 h incubation period suggesting that DHEA can modulate turnover of these mRNA transcripts. Basal mRNA levels of PPAR-alpha and PGC-1alpha were significantly suppressed upon 20 h treatment with cycloheximide, while those of PPAR-gamma1, -gamma2, and PGC-1beta were elevated. Cycloheximide also significantly reduced DHEA-induced accumulation of PPAR-alpha, -gamma1, -gamma2, and PGC-1alpha mRNAs, demonstrating the dependence of the DHEA action on de novo protein synthesis. The findings demonstrate that a supraphysiological concentration of DHEA can substantially influence gene expression of the PPAR signalling machinery at both transcriptional and posttranscriptional levels.
- MeSH
- cykloheximid farmakologie MeSH
- daktinomycin farmakologie MeSH
- dehydroepiandrosteron farmakologie MeSH
- financování organizované MeSH
- hepatocelulární karcinom genetika MeSH
- lidé MeSH
- messenger RNA genetika metabolismus MeSH
- nádorové buněčné linie MeSH
- protein - isoformy genetika MeSH
- receptory aktivované proliferátory peroxizomů genetika MeSH
- regulace genové exprese u nádorů účinky léků MeSH
- Check Tag
- lidé MeSH
AIMS: Very modest changes in mRNA stability can affect critical points in cellular energy pathways. The aim of this study was to investigate the impact of energy abundant substrates on peroxisome proliferator-activated receptors (PPARs) and PPAR-gamma coactivators (PGCs) mRNA's steady-state levels. METHODS: Quantitative RT-PCR study was performed to assess the effect of zero or normal (5 mmol/l) glucose and/or oleic acid (0.3 mmol/l) on mRNA levels of (PPARs) (PGCs) in HepG2 cells. RESULTS: PGC-1alpha mRNA was significantly upregulated in glucose deprived cells (123 % of the control level; p < 0.05), while PGC-1beta mRNA was significantly enhanced in oleate-fed cells (134 % and 160 % of control levels for zero glucose plus oleate and normal glucose plus oleate, respectively; p < 0.05) during the 0.5 h incubation. Upon the 4 h incubation, PPAR-gamma1 and PGC-1alpha mRNAs were significantly elevated in cells lacking glucose (142 % and 163 % of control levels, respectively; p < 0.05). Oleate significantly suppressed PPAR-alpha and PGC-1beta mRNA levels in glucose-deprived cells (58 % and 49 % of control levels, respectively; p < 0.05). PPAR-gamma1 and -gamma2 mRNAs were significantly superinduced when the cells were treated with cycloheximide, whereas PPAR-alpha and PGC-1alpha and-1beta mRNAs were destabilized. Upon actinomycin D treatment, glucose shortage significantly stabilized PPAR-alpha mRNA, while PGC-1alpha mRNA was destabilized by oleate in glucose-deprived cells. CONCLUSIONS: Our findings provide evidence that transcriptional processes that are under the control of energetic substrates are interconnected with concurrent translational processes that can change stability of mRNAs.
- MeSH
- cykloheximid farmakologie MeSH
- daktinomycin farmakologie MeSH
- glukosa metabolismus farmakologie MeSH
- inhibitory syntézy proteinů farmakologie MeSH
- kyselina olejová farmakologie MeSH
- lidé MeSH
- messenger RNA metabolismus MeSH
- nádorové buněčné linie metabolismus MeSH
- nádory jater metabolismus MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- PPAR gama genetika metabolismus MeSH
- receptory aktivované proliferátory peroxizomů genetika metabolismus MeSH
- techniky in vitro MeSH
- upregulace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cryopreservation offers the potential to maximize the use and availability of biological materials that have a limited supply. This study demonstrates an enhanced technique for the parallel cryopreservation of a series of liver tissue slices using a tray modeled from aluminium foil and low concentrations of a cryoprotectant. Cooling and warming rates of approximately 2000 and 3900 degrees C min(-1), respectively, were achieved as the thermal capacity of the foil-tray was significantly reduced compared to the aluminium sandwich device introduced by Day et al. [S.H. Day, D.A. Nicoll-Griffith, J.M. Silva, Cryopreservation of rat and human liver slices by rapid freezing, Cryobiology 38 (1999) 154-159]. Additionally, the two critical steps involved in the sandwich approach, i.e., clamping the plates and complete filling of the entire space between the plates with liquid, can be omitted using the foil tray. The viability of the slices was verified by measuring tetrazolium salt reduction capacity, cytosolic enzyme lactate dehydrogenase leakage, and ethoxycoumarin metabolism.
- MeSH
- časové faktory MeSH
- financování organizované MeSH
- játra MeSH
- kryoprezervace metody MeSH
- L-laktátdehydrogenasa metabolismus MeSH
- myši MeSH
- tetrazoliové soli metabolismus MeSH
- thiazoly metabolismus MeSH
- uchovávání orgánů MeSH
- umbeliferony metabolismus MeSH
- viabilita buněk MeSH
- zmrazování MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- hodnotící studie MeSH
