Mycobacterium tuberculosis, the etiologic agent of tuberculosis, is an intracellular pathogen of alveolar macrophages. These cells avidly take up nanoparticles, even without the use of specific targeting ligands, making the use of nanotherapeutics ideal for the treatment of such infections. Methoxy poly(ethylene oxide)- block-poly(ε-caprolactone) nanoparticles of several different polymer blocks' molecular weights and sizes (20-110 nm) were developed and critically compared as carriers for rifampicin, a cornerstone in tuberculosis therapy. The polymeric nanoparticles' uptake, consequent organelle targeting and intracellular degradation were shown to be highly dependent on the nanoparticles' physicochemical properties (the cell uptake half-lives 2.4-21 min, the degradation half-lives 51.6 min-ca. 20 h after the internalization). We show that the nanoparticles are efficiently taken up by macrophages and are able to effectively neutralize the persisting bacilli. Finally, we demonstrate, using a zebrafish model of tuberculosis, that the nanoparticles are well tolerated, have a curative effect, and are significantly more efficient compared to a free form of rifampicin. Hence, these findings demonstrate that this system shows great promise, both in vitro and in vivo, for the treatment of tuberculosis.

• MeSH
• dánio pruhované MeSH
• lidé MeSH
• makrofágy * metabolismus   mikrobiologie   MeSH
• modely nemocí na zvířatech MeSH
• Mycobacterium tuberculosis růst a vývoj   MeSH
• myši MeSH
• nanočástice * chemie   terapeutické užití   MeSH
• nosiče léků * chemie   farmakokinetika   farmakologie   MeSH
• RAW 264.7 buňky MeSH
• rifampin * chemie   farmakokinetika   farmakologie   MeSH
• tuberkulóza farmakoterapie   metabolismus   patologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Polyester-based nanostructures are widely studied as drug-delivery systems due to their biocompatibility and biodegradability. They are already used in the clinic. In this work, we describe a new and simple biodegradable and biocompatible system as the Food and Drug Administration approved polyesters (poly-ε-caprolactone, polylactic acid, and poly(lactic- co-glycolic acid)) for the delivery of the anticancer drug paclitaxel (PTX) as a model drug. A hydrophobic polyester, poly(propylene succinate) (PPS), was prepared from a nontoxic alcohol (propylene glycol) and monomer from the Krebs's cycle (succinic acid) in two steps via esterification and melt polycondensation. Furthermore, their amphiphilic block copolyester, poly(ethylene oxide monomethyl ether)- block-poly(propylene succinate) (mPEO- b-PPS), was prepared by three steps via esterification followed by melt polycondensation and the addition of mPEO to the PPS macromolecules. Analysis of the in vitro cellular behavior of the prepared nanoparticle carriers (NPs) (enzymatic degradation, uptake, localization, and fluorescence resonance energy-transfer pair degradation studies) was performed by fluorescence studies. PTX was loaded to the NPs of variable sizes (30, 70, and 150 nm), and their in vitro release was evaluated in different cell models and compared with commercial PTX formulations. The mPEO- b-PPS copolymer analysis displays glass transition temperature < body temperature < melting temperature, lower toxicity (including the toxicity of their degradation products), drug solubilization efficacy, stability against spontaneous hydrolysis during transport in bloodstream, and simultaneous enzymatic degradability after uptake into the cells. The detailed cytotoxicity in vitro and in vivo tumor efficacy studies have shown the superior efficacy of the NPs compared with PTX and PTX commercial formulations.

• MeSH
• antitumorózní látky aplikace a dávkování   farmakokinetika   MeSH
• micely MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nanočástice škodlivé účinky   chemie   metabolismus   MeSH
• paclitaxel aplikace a dávkování   farmakokinetika   MeSH
• polyestery chemická syntéza   chemie   MeSH
• polyethylenglykoly chemie   MeSH
• polypropyleny chemie   MeSH
• sukcináty chemie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

We have developed a biodegradable, biocompatible system for the delivery of the antituberculotic antibiotic rifampicin with a built-in drug release and nanoparticle degradation fluorescence sensor. Polymer nanoparticles based on poly(ethylene oxide) monomethyl ether-block-poly(ε-caprolactone) were noncovalently loaded with rifampicin, a combination that, to best of our knowledge, was not previously described in the literature, which showed significant benefits. The nanoparticles contain a Förster resonance energy transfer (FRET) system that allows real-time assessment of drug release not only in vitro, but also in living macrophages where the mycobacteria typically reside as hard-to-kill intracellular parasites. The fluorophore also enables in situ monitoring of the enzymatic nanoparticle degradation in the macrophages. We show that the nanoparticles are efficiently taken up by macrophages, where they are very quickly associated with the lysosomal compartment. After drug release, the nanoparticles in the cmacrophages are enzymatically degraded, with half-life 88±11 min.

• MeSH
• antituberkulotika aplikace a dávkování   MeSH
• biokompatibilní materiály chemie   MeSH
• makrofágy účinky léků   metabolismus   MeSH
• myši MeSH
• nanočástice chemie   MeSH
• polyestery chemie   MeSH
• polyethylenglykoly chemie   MeSH
• RAW 264.7 buňky MeSH
• rezonanční přenos fluorescenční energie MeSH
• rifampin aplikace a dávkování   MeSH
• systémy cílené aplikace léků * MeSH
• uvolňování léčiv * MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

CYP3A4 is the most important drug-metabolizing enzyme that is involved in biotransformation of more than 50% of drugs. Pregnane X receptor (PXR) dominantly controls CYP3A4 inducibility in the liver, whereas vitamin D receptor (VDR) transactivates CYP3A4 in the intestine by secondary bile acids. Four major functional PXR-binding response elements of CYP3A4 have been discovered and their cooperation was found to be crucial for maximal up-regulation of the gene in hepatocytes. VDR and PXR recognize similar response element motifs and share DR3(XREM) and proximal ER6 (prER6) response elements of the CYP3A4 gene. In this work, we tested whether the recently discovered PXR response elements DR4(eNR3A4) in the XREM module and the distal ER6 element in the CLEM4 module (CLEM4-ER6) bind VDR/RXRalpha heterodimer, whether the elements are involved in the intestinal transactivation, and whether their cooperation with other elements is essential for maximal intestinal expression of CYP3A4. Employing a series of gene reporter plasmids with various combinations of response element mutations transiently transfected into four intestinal cell lines, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP), we found that the CLEM4-ER6 motif interacts with VDR/RXRalpha heterodimer and partially cooperates with DR3(XREM) and prER6 in both basal and VDR-mediated inducible CYP3A4 regulation in intestinal cells. In contrast, eNR3A4 is involved only in the basal transactivation in intestinal cells and in the PXR-mediated rifampicin-induced transactivation of CYP3A4 in LS174T intestinal cells. We thus describe a specific ligand-induced VDR-mediated transactivation of the CYP3A4 gene in intestinal cells that differs from PXR-mediated CYP3A4 regulation in hepatocytes.

• MeSH
• chromatinová imunoprecipitace MeSH
• cytochrom P-450 CYP3A genetika   MeSH
• DNA primery MeSH
• genetická transkripce fyziologie   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• receptory kalcitriolu fyziologie   MeSH
• regulace genové exprese enzymů fyziologie   MeSH
• sekvence nukleotidů MeSH
• střeva enzymologie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

Class C G-protein coupled receptors form obligatory dimers. Metabotropic glutamate receptors (mGluRs) are found commonly as homodimers. Alternative splicing of mGluR1 gene results in vivo in the expression of a long variant mGluR1a and at least two short variants mGluR1b and d. The amino acid sequences diverge within their carboxyl-termini six amino acid residues following RRKK motif. This four basic residue sequence was shown to have pronounced impact on function and trafficking of the short variants, while for mGluR1a the long C-terminus reduces the effects caused by presence of the RRKK motif. Here we investigated consequences of interactions between long mGluR1a and short mGluR1b variants. Our results show that mGluR1a interferes with mGluR1b trafficking to the cell surface in HEK293 transfected cells. Expression of a mGlu1a mutant incapable of activating G-proteins with mGluR1b mutated in the glutamate binding site led to the formation of a functional heterodimer. Moreover, we show that swapping long mGluR1a and/or short mGluR1b C-termini with corresponding regions in chimerical GB1 and GB2 gamma-amino butyric acid b (GABAb) receptor subunits do not exclude heterodimerization. These data reveal that the C-terminal ends of mGluR1 do not control subunit association, such that mGluR1 dimers with two distinct C-termini can form and function properly.

• MeSH
• alternativní sestřih genetika   MeSH
• exprese genu fyziologie   MeSH
• fosfatasy metabolismus   MeSH
• imunoprecipitace metody   MeSH
• lidé MeSH
• mutageneze fyziologie   MeSH
• receptory metabotropního glutamátu genetika   klasifikace   metabolismus   MeSH
• terciární struktura proteinů fyziologie   MeSH
• transfekce metody   MeSH
• transformované buněčné linie fyziologie   MeSH
• transport proteinů fyziologie   MeSH
• vápník metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

Congenital abnormalities, various diseases and injuries may result in the degeneration of articular cartilage. Recently, stem cell therapy has offered new treatment possibilities for this condition. The aim of our study was to verify the chondrogenic differentiation potential of human bone marrow mesenchymal stem cells (BMSCs) and adipose tissue-derived mesenchymal stem cells (AMSCs) in vitro in the presence or absence of transforming growth factor beta (TGF-beta1). Human BMSCs and AMSCs from healthy donors were collected during orthopaedic surgeries and expanded in vitro to obtain a sufficient quantity of cells; their chondrogenic differentiation was studied in the pellet culture system. Spontaneous chondrogenesis occurred in both BMSC and AMSC pellet cultures and was similar in both TGF-beta1 treated and untreated pellet cultures. BMSC pellets contained more cells with a chondrogenic phenotype. The presence of TGF-beta1 led to a decrease in the levels of collagen type I mRNA and to increased levels of collagen type II mRNA only in the BMSC pellet culture. Our results demonstrate that although both mesenchymal cell types can be used in cartilage tissue engineering, the chondrogenic potential of human BMSCs is higher than that of AMSCs.

• MeSH
• finanční podpora výzkumu jako téma MeSH
• imunohistochemie metody   využití   MeSH
• kloubní chrupavka abnormality   patologie   růst a vývoj   MeSH
• kolagen typ II fyziologie   genetika   MeSH
• kostní dřeň fyziologie   chirurgie   MeSH
• kultivované buňky fyziologie   transplantace   MeSH
• lidé MeSH
• mezenchymální kmenové buňky fyziologie   MeSH
• polymerázová řetězová reakce s reverzní transkripcí metody   využití   MeSH
• transformující růstový faktor beta1 terapeutické užití   MeSH
• tuková tkáň fyziologie   chirurgie   MeSH
• Check Tag
• lidé MeSH