BACKGROUND: Chronic lymphocytic leukemia (CLL) is a common adult leukemia characterized by the accumulation of neoplastic mature B cells in blood, bone marrow, lymph nodes, and spleen. The disease biology remains unresolved in many aspects, including the processes underlying the disease progression and relapses. However, studying CLL in vitro poses a considerable challenge due to its complexity and dependency on the microenvironment. Several approaches are utilized to overcome this issue, such as co-culture of CLL cells with other cell types, supplementing culture media with growth factors, or setting up a three-dimensional (3D) culture. Previous studies have shown that 3D cultures, compared to conventional ones, can lead to enhanced cell survival and altered gene expression. 3D cultures can also give valuable information while testing treatment response in vitro since they mimic the cell spatial organization more accurately than conventional culture. METHODS: In our study, we investigated the behavior of CLL cells in two types of material: (i) solid porous collagen scaffolds and (ii) gel composed of carboxymethyl cellulose and polyethylene glycol (CMC-PEG). We studied CLL cells' distribution, morphology, and viability in these materials by a transmitted-light and confocal microscopy. We also measured the metabolic activity of cultured cells. Additionally, the expression levels of MYC, VCAM1, MCL1, CXCR4, and CCL4 genes in CLL cells were studied by qPCR to observe whether our novel culture approaches lead to increased adhesion, lower apoptotic rates, or activation of cell signaling in relation to the enhanced contact with co-cultured cells. RESULTS: Both materials were biocompatible, translucent, and permeable, as assessed by metabolic assays, cell staining, and microscopy. While collagen scaffolds featured easy manipulation, washability, transferability, and biodegradability, CMC-PEG was advantageous for its easy preparation process and low variability in the number of accommodated cells. Both materials promoted cell-to-cell and cell-to-matrix interactions due to the scaffold structure and generation of cell aggregates. The metabolic activity of CLL cells cultured in CMC-PEG gel was similar to or higher than in conventional culture. Compared to the conventional culture, there was (i) a lower expression of VCAM1 in both materials, (ii) a higher expression of CCL4 in collagen scaffolds, and (iii) a lower expression of CXCR4 and MCL1 (transcript variant 2) in collagen scaffolds, while it was higher in a CMC-PEG gel. Hence, culture in the material can suppress the expression of a pro-apoptotic gene (MCL1 in collagen scaffolds) or replicate certain gene expression patterns attributed to CLL cells in lymphoid organs (low CXCR4, high CCL4 in collagen scaffolds) or blood (high CXCR4 in CMC-PEG).

• MeSH
• buněčné kultury metody   MeSH
• chronická lymfatická leukemie * patologie   metabolismus   MeSH
• gely chemie   MeSH
• kolagen * chemie   farmakologie   MeSH
• lidé MeSH
• polyethylenglykoly * chemie   MeSH
• receptory CXCR4 metabolismus   MeSH
• sodná sůl karboxymethylcelulosy * chemie   farmakologie   MeSH
• techniky 3D buněčné kultury metody   MeSH
• tkáňové podpůrné struktury * chemie   MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The increase in resistance of microorganisms is a current worldwide problem, not only in hospitalized patients, but also in patients treated on an outpatient basis. Therefore, it is necessary to look for innovative therapeutic alternatives that could reduce antibiotic selection pressure on given microorganisms and increase chances of their eradication. Currently, at the forefront of interest in wound management and local application are natural products. Based on our preliminary experiments, one of the most promising materials that meets modern requirements for temporal dressing (epithelialization support, antimicrobial activity, exudate absorption etc.) is undoubtedly Gum Karaya (GK). In the project, we will focus on a modern approach to wound healing by using GK in different forms (hydrogel, porous film, fiber dressing) to control antibacterial biogenic system release (bacteriophages, antibacterial proteins and nanoparticles) targeted at multidrug resistant bacterial strains. Dressing efficacy against bacteria will be evaluated preclinically on pig animal model.

Nárůst rezistence mikroorganismů je současný celosvětový problém nejen u hospitalizovaných pacientů, ale také u pacientů léčených ambulantně. Je proto nutné hledat terapeutické alternativy, které by mohly snížit selekční tlak antibiotik na dané mikroorganismy a zvýšily by šanci na jejich eradikaci. V současné době se dostávají do popředí zájmu ve wound managementu i v lokálním podání přírodní produkty. Jedním z velmi nadějných materiálů je přírodní pryskyřice Gum Karaya (GK), která na základě našich předběžných výsledků splňuje současné požadavky na dočasný kryt ran (podpora epitelizace, antimikrobiální aktivita, absorpce exudátu aj.). V projektu se zaměříme na moderní přístup k hojení infikované rány, kdy využijeme pryskyřici GK v různých formách (hydrogel, porézní film a vlákenný kryt) na řízené uvolňování antibakteriálních biogenních systémů (bakteriofágů, antibakteriálních proteinů a nanočástic) zaměřených proti multirezistentním kmenům bakterií. Účinnost krytů vůči bakteriím bude testována preklinicky na animálním modelu prasete.

• Klíčová slova
• Bakteriofágy, Bacteriophages, antimikrobiální peptidy, antimicrobial peptides, fágy, phages, Biopolymerní hydrogel, Multirezistentní kmeny bakterií, Komplikovaná infekce kůže a měkkých tkání, Nanočástice selenu, Biopolymer hyrogel, Multidrug-resistant bacterial strains, Complicated skin and soft tissue infection, Selenium nanoparticles,

• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR

Several in vitro models have been developed to mimic chronic lymphocytic leukemia (CLL) proliferation in immune niches; however, they typically do not induce robust proliferation. We prepared a novel model based on mimicking T-cell signals in vitro and in patient-derived xenografts (PDXs). Six supportive cell lines were prepared by engineering HS5 stromal cells with stable expression of human CD40L, IL4, IL21, and their combinations. Co-culture with HS5 expressing CD40L and IL4 in combination led to mild CLL cell proliferation (median 7% at day 7), while the HS5 expressing CD40L, IL4, and IL21 led to unprecedented proliferation rate (median 44%). The co-cultures mimicked the gene expression fingerprint of lymph node CLL cells (MYC, NFκB, and E2F signatures) and revealed novel vulnerabilities in CLL-T-cell-induced proliferation. Drug testing in co-cultures revealed for the first time that pan-RAF inhibitors fully block CLL proliferation. The co-culture model can be downscaled to five microliter volume for large drug screening purposes or upscaled to CLL PDXs by HS5-CD40L-IL4 ± IL21 co-transplantation. Co-transplanting NSG mice with purified CLL cells and HS5-CD40L-IL4 or HS5-CD40L-IL4-IL21 cells on collagen-based scaffold led to 47% or 82% engraftment efficacy, respectively, with ~20% of PDXs being clonally related to CLL, potentially overcoming the need to co-transplant autologous T-cells in PDXs.

• MeSH
• buňky stromatu * metabolismus   patologie   MeSH
• chronická lymfatická leukemie * patologie   genetika   farmakoterapie   MeSH
• inhibitory proteinkinas farmakologie   MeSH
• interleukiny genetika   metabolismus   MeSH
• kokultivační techniky * MeSH
• lidé MeSH
• ligand CD40 * metabolismus   genetika   MeSH
• myši MeSH
• proliferace buněk * MeSH
• T-lymfocyty imunologie   metabolismus   MeSH
• xenogenní modely - testy antitumorózní aktivity MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

• Publikační typ
• abstrakt z konference MeSH

BACKGROUND: The purpose of dermal substitutes is to mimic the basic properties of the extracellular matrix of human skin. The application of dermal substitutes to the defect reduces the formation of hypertrophic scars and improves the scar quality. This study aims to develop an original dermal substitute enriched with stable fibroblast growth factor 2 (FGF2-STAB®) and test it in an animal model. METHODS: Dermal substitutes based on collagen/chitosan scaffolds or collagen/chitosan scaffolds with nanofibrous layer were prepared and enriched with FGF2-STAB® at concentrations of 0, 0.1, 1.0, and 10.0 μg ‧ cm-2. The performance of these dermal substitutes was tested in vivo on artificially formed skin defects in female swine. The outcomes were evaluated using cutometry at 3 and 6 months. In addition, visual appearance was assessed based on photos of the scars at 1-month, 3-month and 6-month follow-ups using Yeong scale and Visual Analog Scale. RESULTS: The dermal substitute was fully integrated into all defects and all wounds healed successfully. FGF2-STAB®-enriched matrices yielded better results in cutometry compared to scaffolds without FGF2. Visual evaluation at 1, 3, and 6 months follow-ups detected no significant differences among groups. The FGF2-STAB® effectiveness in improving the elasticity of scar tissues was confirmed in the swine model. This effect was independently observed in the scaffolds with nanofibres as well as in the scaffolds without nanofibres. CONCLUSION: The formation of scars with the best elasticity was exhibited by addition 1.0 μg ‧ cm-2of FGF2-STAB® into the scaffolds, although it had no significant effect on visual appearance at longer follow-ups. This study creates the basis for further translational studies of the developed product and its progression into the clinical phase of the research.

• MeSH
• chitosan * MeSH
• fibroblastový růstový faktor 2 * MeSH
• hojení ran účinky léků   MeSH
• jizva hypertrofická MeSH
• kolagen MeSH
• kůže MeSH
• modely nemocí na zvířatech MeSH
• nanovlákna terapeutické užití   MeSH
• popálení MeSH
• prasata MeSH
• pružnost * MeSH
• tkáňové podpůrné struktury MeSH
• umělá kůže * MeSH
• viskozita MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Příručka a směrnice, které se zaměřují na klinické laboratorní diagnostické metody. Určeno odborné veřejnosti, praktickým lékařům.

• MeSH
• klinické laboratorní techniky MeSH
• praktické lékařství MeSH
• Publikační typ
• příručky MeSH
• směrnice pro lékařskou praxi MeSH

• Konspekt
• Patologie. Klinická medicína
• NLK Obory
• diagnostika
• všeobecné lékařství
• NLK Publikační typ
• brožury

OBJECTIVES: Resistance to antibiotics among bacteria of clinical importance, including Staphylococcus aureus, is a serious problem worldwide and the search for alternatives is needed. Some metal complexes have antibacterial properties and when combined with antibiotics, they may increase bacterial sensitivity to antimicrobials. In this study, we synthesized the iron complex and tested it in combination with ampicillin (Fe16 + AMP) against S. aureus. METHODS: An iron complex (Fe16) was synthesized and characterized using spectroscopy methods. Confirmation of the synergistic effect between the iron complex (Fe16) and ampicillin (AMP) was performed using ζ-potential, infrared spectra and FICI index calculated from the minimum inhibitory concentration (MIC) from the checkerboard assay. Cytotoxic properties of combination Fe16 + AMP was evaluated on eukaryotic cell line. Impact of combination Fe16 + AMP on chosen genes of S. aureus were performed by Quantitative Real-Time PCR. RESULTS: The MIC of Fe16 + AMP was significantly lower than that of AMP and Fe16 alone. Furthermore, the infrared spectroscopy revealed the change in the ζ-potential of Fe16 + AMP. We demonstrated the ability of Fe16 + AMP to disrupt the bacterial membrane of S. aureus and that likely allowed for better absorption of AMP. In addition, the change in gene expression of bacterial efflux pumps at the sub-inhibitory concentration of AMP suggests an insufficient import of iron into the bacterial cell. At the same time, Fe16 + AMP did not have any cytotoxic effects on keratinocytes. CONCLUSIONS: Combined Fe16 + AMP therapy demonstrated significant synergistic and antimicrobial effects against S. aureus. This study supports the potential of combination therapy and further research.

• MeSH
• ampicilin farmakologie   MeSH
• antibakteriální látky farmakologie   MeSH
• lidé MeSH
• methicilin rezistentní Staphylococcus aureus * MeSH
• mikrobiální testy citlivosti MeSH
• stafylokokové infekce * farmakoterapie   MeSH
• Staphylococcus aureus MeSH
• synergismus léků MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Solution of bone defects is not always easy medical procedure, especially in the field of osteomyelitis, or near joint replacements on the surface of the attritor granuloma. In these cases, the "dead space" occurs, which not only locally weakens the bone with the risk of fracture, but as well becomes a suitable place to form or exacerbate osteomyelitis. In the present project we will newly utilize resorbable, injectable self-hardening bone substitute material consisting of calcium phosphate, thermosensitive thixotropic copolymer and short biodegradable nanofibers improving rheology and ductility of osteoinductive cement. The advantage of this cement is the choice of porosity enabling fixation of the implants and the selection of bone substitute according to the type and location of bone defect in the body. This porous cement will be saturated with antibiotics and tested in-vitro, ex-vivo and in-vivo on animal models for the treatment or prevention of osteomyelitis. The bioactive cement is expected to resorb within the healing process along with gradual remodelling by new bone.

Řešení kostních defektů nebývá vždy snadným léčebným postupem, obzvláště v terénu osteomyelitidy nebo v místě kloubních náhrad na podkladě otěrového granulomu. V těchto případech vzniká „mrtvý prostor“, který nejenže v daném místě oslabuje kost s rizikem vzniku zlomeniny, ale také bývá místem vhodným k vytvoření nebo exacerbaci osteomyelitidy. V předloženém projektu nově použijeme resobovatelný, injekčně aplikovatelný, samotuhnoucí materiál náhrady kostní tkáně složený z fosforečnanu vápenatého, termocitlivého tixotropního kopolymeru a krátkých biodegradabilních nanovláken zlepšující reologii a tažnost osteoindukčního cementu. Výhodou tohoto cementu je možnost výběru porozity, umožňující fixaci implantátů a volbu náhrady kostní tkáně podle typu a lokalizace kostního defektu v organizmu. Tento porézní cement bude navíc sycen antibiotiky a testován in-vitro, ex-vivo i in-vivo na zvířecích modelech pro léčbu nebo prevenci osteomyelitidy. Očekává se, že během hojení bude bioaktivní cement resorbován a postupně přestaven novou kostí.

• Klíčová slova
• injektabilní kostní cement, infikované kostní defekty, osteomyelitida, osteointegrace, injectable bone cement, infected bone defects, osteomyelitis, osteointegration,

• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR

Infection of the skin and soft tissues is the most common infectious complications not only in hospitalized patients but also treated outpatient settings. Treatment with local and systemic antimicrobials today is increasingly failing. The dominant cause is the increase in resistance of potentially pathogenic microorganisms to various antimicrobial strategies. Enzybiotics represent a promising group of proteins that may have the ambition to firmly established itself among antimicrobial drugs. The reason is their ability to efficiently split the cell wall of bacteria and the absence of resistant mechanisms to the effect of enzybiotics. During the project we will extend the understanding of the interaction between "biobetter" rLysdermF1, rLysstafS1 and LysbiofP1 lytic enzymes (enzybiotics) and Staphylococcus aureus MRSA gram-positive bacteria resistant to methicillin in laboratory conditions as well as in animal models in simulated skin and soft tissue infections using a skin collagen and cellulosic nanofibers. The main ambition of the project is to prepare the "biobetter" enzybiotics themselves and immobilize them on the skin cover as appropriate forms of use in human medicine in the real situations.

Infekce kůže a měkkých tkání je nejčastější infekční komplikací nejen u hospitalizovaných pacientů, ale také u pacientů léčených v ambulantní sféře. Terapie pomocí lokálních i systémových antimikrobiálních preparátů dnes stále častěji selhává. Dominantní příčinou je nárůst rezistence potenciálně patogenních mikroorganismů vůči nejrůznějším antimikrobiálním strategiím. Enzybiotika představují nadějnou skupinu proteinů, které mohou mít ambici se pevně etablovat mezi antimikrobiální preparáty. Důvodem je jejich schopnost účinně štěpit buněčnou stěnu bakterií a absence rezistentních mechanismů k účinku enzybiotika. V průběhu tohoto projektu budeme posouvat poznání interakce mezi "vylepšenými" lytickými enzymy rLysderm, rLysstaf a Lysbiof (enzybiotika) a kmeny grampozitivní bakterie Staphylococcus aureus jak v laboratorních podmínkách, tak také na animálním modelu v simulované infekci kůže a měkkých tkání za použití kožního krytu tvořeného kolagenem a celulosových nanovláken. Hlavní ambicí projektu bude příprava samotných "vylepšených" enzybiotik a jejich imobilizace na kožní kryt jako vhodné formy použití v humánní medicíně za reálných podmínek.

• Klíčová slova
• MRSA, MRSA, enzymová terapie, S. aureus, enzyme therapy, S. aureus, enzybiotika, vylepšené enzymy, krycí materiály, enzybiotics, biobetters, wound dressing,

• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR

The study investigates the use of fiber carriers, based on biopolymeric gums as potential candidates for cosmetic and dermatological applications, in particular for skin regeneration. Gum arabic (GA), xanthan gum (XA), and gum karaya (GK) were used as the main gum materials for the fibers, which were prepared by centrifugal spinning from an aqueous solution. These solutions of different mass gum ratios were blended with poly (ethylene oxide) (PEO) for better spinnability. Finally, vitamins E and C were added to selected solutions of gums. The resulting fibers were extensively investigated. The morphology and structure of all fibers were investigated by scanning electron microscopy and Fourier transformed infrared spectroscopy. Most importantly, they were characterized by the release of vitamin E loaded in the fibers using UV-VIS spectroscopy. The presentation will show that the newly prepared fibers from GA and PEO represent a very promising material for cosmetic and dermatologic applications.

• MeSH
• arabská guma chemie   MeSH
• guma karaya * chemie   MeSH
• kůže MeSH
• polyethylenglykoly MeSH
• regenerace MeSH
• vitaminy * MeSH
• Publikační typ
• časopisecké články MeSH