Culturing cells in three-dimensional systems that include extracellular matrix components and different cell types mimic the native tissue and as such provide much more representative results than ...conventional two-dimensional cell cultures. In order to develop biomimetic bladder tissue in vitro, we used human amniotic membrane (AM) extracellular matrix as a scaffold for bladder fibroblasts (BFs) and urothelial cells. Our aims were to evaluate the integration of BFs into the AM stroma, to assess the differentiation of the urothelium on BFs-enriched AM scaffolds, and to evaluate the AM as a urothelial wound dressing. First, to achieve the optimal integration of BFs into AM stroma, different intact and de- epithelialized AM (dAM) scaffolds were tested. BFs secreted matrix metalloproteinase (MMP)-1 and MMP-2 and integrated into the stroma of all types of AM scaffolds. Second, to establish urothelial tissue equivalent, urothelial cells were seeded on dAM scaffolds enriched with BFs. The BFs in the stroma of the AM scaffolds promoted (1) the proliferation of urothelial cells, (2) the attachment of urothelial cells on AM basal lamina with hemidesmosomes, and (3) development of multilayered urothelium with expressed uroplakins and well-developed cell junctions. Third, we established an ex vivo model of the injured bladder to evaluate the dAM as a wound dressing for urothelial full-thickness injury. dAM acted as a promising wound dressing since it enabled rapid re-epithelization of urothelial injury and integrated into the bladder tissue. Herein, the developed urothelial tissue equivalents enable further mechanistic studies of bladder epithelial–mesenchymal interactions, and they could be applied as biomimetic models for preclinical testing of newly developed drugs. Moreover, we could hypothesize that AM may be suitable as a dressing of the wound that occurs during transurethral resection of bladder tumor, since it could diminish the possibility of tumor recurrence, by promoting the rapid re-epithelization of the urothelium.
Increasing evidence suggests that lysosomal proteases are actively involved in apoptosis. Using HeLa cells as the model system,
we show that selective lysosome disruption with l -leucyl- l -leucine ...methyl ester results in apoptosis, characterized by translocation of lysosomal proteases into the cytosol and by
the cleavage of a proapoptotic Bcl-2-family member Bid. Apoptosis and Bid cleavage, but not translocation of lysosomal proteases
to the cytosol, could be prevented by 15 μ m l - trans -epoxysuccinyl(OEt)-Leu-3-methylbutylamide, an inhibitor of papain-like cysteine proteases. Incubation of cells with 15 μ m N -benzoyloxycarbonyl-VAD-fluoromethyl ketone prevented apoptosis but not Bid cleavage, suggesting that cathepsin-mediated apoptosis
in this system is caspase-dependent. In vitro experiments performed at neutral pH showed that papain-like cathepsins B, H, L, S, and K cleave Bid predominantly at Arg 65 or Arg 71 . No Bid cleavage was observed with cathepsins C and X or the aspartic protease cathepsin D. Incubation of full-length Bid
treated with cathepsins B, H, L, and S resulted in rapid cytochrome c release from isolated mitochondria. Thus, Bid may be an important mediator of apoptosis induced by lysosomal disruption.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
- The aim of the study was to evaluate the efficacy and safety of the new method of platelet-rich plasma activation in the form of platelet gel, used in the treatment of non-healing chronic lower leg ...ulcers. The study was prospectively randomized, double blind and placebo controlled. We treated 60 patients (42 males and 18 females, mean age 69.43 years, SD 14.74) with chronic lower leg ulcers of different etiologies. Thirty patients were treated with allogeneic platelet gel and 30 with hydrogel. Both groups were comparable for duration of ulcer and its size. Treatment was repeated once a week for three consecutive weeks and then the last examination was scheduled at 6 months of the first platelet gel application. The t-test was used to analyze independent samples. Healing of chronic wounds with platelet gel was statistically significantly more effective compared to the treatment with hydrogel (p<0.05). At 6 months of platelet gel application, the mean wound area in the experimental group decreased to 35.01% (SD 53.69) of the initial wound size. In the control group, the wound area decreased to 89.95% (SD 71.82) of the initial wound size (p=0.001). The circumference of the wounds diminished to 54.62% (SD 39.85) of the initial value in the experimental group, compared to 91.28% (SD 29.32) in the control group (p<0.001). Allogeneic platelet gel prepared by the new method used in this study was found to be a good treatment option for non-healing chronic wounds when other methods are ineffective.
Many preclinical studies have shown that birth-associated tissues, cells and their secreted factors, otherwise known as perinatal derivatives (PnD), possess various biological properties that make ...them suitable therapeutic candidates for the treatment of numerous pathological conditions. Nevertheless, in the field of PnD research, there is a lack of critical evaluation of the PnD standardization process: from preparation to
in vitro
testing, an issue that may ultimately delay clinical translation. In this paper, we present the PnD e-questionnaire developed to assess the current state of the art of methods used in the published literature for the procurement, isolation, culturing preservation and characterization of PnD
in vitro
. Furthermore, we also propose a consensus for the scientific community on the minimal criteria that should be reported to facilitate standardization, reproducibility and transparency of data in PnD research. Lastly, based on the data from the PnD e-questionnaire, we recommend to provide adequate information on the characterization of the PnD. The PnD e-questionnaire is now freely available to the scientific community in order to guide researchers on the minimal criteria that should be clearly reported in their manuscripts. This review is a collaborative effort from the COST SPRINT action (CA17116), which aims to guide future research to facilitate the translation of basic research findings on PnD into clinical practice.
Izhodišče: Osnovna naloga amnijske membrane je zaščita ploda pred zunanjimi mehanskimi vplivi in izsušitvijo ter zagotavljanje primernega okolja za njegov razvoj. Razumevanje zgradbe in delovanja ...amnijske membrane je pomembno za njeno klinično uporabo, še posebej v regenerativni medicini. V prispevku opisujemo številne, za regenerativno medicino zelo zaželene mehanske in biološke lastnosti amnijske membrane ter predstavljamo njene protirakave lastnosti.Zaključek: Študije na modelih in vitro kot tudi študije na živalskih modelih dokazujejo, da amnijska membrana zavira proliferacijo rakavih celic in sproža njihovo apoptozo, deluje imunozaviralno, zavira energijsko presnovo rakavih celic in angiogenezo. Delo podaja pregled najnovejših spoznanj o protirakavem delovanju amnijske membrane in vrednoti njeno potencialno uporabo v zdravljenju raka in regenerativni medicini.
Izhodišča: Amnijska membrana (AM) je notranja stran posteljice, ki obdaja in ščiti zarodek. AM je večplastna struktura, ki je sestavljena iz amnijskih epitelijskih celic, amnijskih mezenhimskih ...stromalnih celic, bazalne lamine in vezivnega tkiva. Iz njene zgradbe izhajajo tudi lastnosti AM, zaradi katerih se že vrsto let uporablja v terapevtske namene, predvsem v oftalmologiji, saj pospešuje epitelizacijo, deluje kot substrat za celice, zmanjšuje fibrozo in neovaskularizacijo tkiva ter deluje protimikrobno. Zaradi mehanskih lastnosti AM, ki so posledica predvsem molekul zunajceličnega matriksa bazalne lamine in vezivnega tkiva, se AM v zadnjih letih vedno pogosteje uporablja tudi kot biološki nosilec v regenerativni medicini. Zaključki: Regenerativna medicina je interdisciplinarno področje raziskav in kliničnih aplikacij, ki uporablja načela bioloških in inženirskih znanosti za razvoj živih tkivnih ali organskih nadomestkov. V regenerativni medicini ločimo tri pristope: 1) vsaditev funkcionalnih celic, med drugim tudi matičnih celic, v poškodovano ali okvarjeno tkivo, 2) uporaba različnih sintetičnih materialov ali materialov naravnega izvora, ki pomagajo pri ponovnem oblikovanju poškodovanega ali okvarjenega tkiva in 3) tkivno inženirstvo, tj. uporaba ustreznih nosilcev, ki spodbujajo rast tkivno specifičnih celic in oblikovanje novega tkiva. V prispevku predstavljamo tudi uporabo amnijske membrane kot biološkega nosilca v regenerativni medicini v Sloveniji.
Background: The primary role of amniotic membrane is to protect the foetus from drying out and to provide a suitable environment for its development. Understanding of the structure and function of ...amniotic membrane is important in terms of its use in clinical settings, especially in regenerative medicine. This paper describes favourable mechanical and biological properties of amniotic membrane for regenerative medicine uses and emphasizes its anti-cancer properties. Conclusion: Studies on in vitro as well as on animal models demonstrate that amniotic membrane inhibits the proliferation of cancer cells and induces their apoptosis. It also acts immunoinhibitory, and it inhibits the metabolic activity of cancer cells and angiogenesis. This work provides an overview of the latest knowledge about the anticancer role of amniotic membrane and evaluates the potential of the amniotic membrane use for anticancer therapy and regenerative medicine.
We investigated the mechanism of lysosome-mediated cell death using purified recombinant pro-apoptotic proteins, and cell-free extracts from the human neuronal progenitor cell line NT2. Potential ...effectors were either isolated lysosomes or purified lysosomal proteases. Purified lysosomal cathepsins B, H, K, L, S, and X or an extract of mouse lysosomes did not directly activate either recombinant caspase zymogens or caspase zymogens present in an NT2 cytosolic extract to any significant extent. In contrast, a cathepsin L-related protease from the protozoan parasite Trypanosoma cruzi, cruzipain, showed a measurable caspase activation rate. This demonstrated that members of the papain family can directly activate caspases but that mammalian lysosomal members of this family may have been negatively selected for caspase activation to prevent inappropriate induction of apoptosis. Given the lack of evidence for a direct role in caspase activation by lysosomal proteases, we hypothesized that an indirect mode of caspase activation may involve the Bcl-2 family member Bid. In support of this, Bid was cleaved in the presence of lysosomal extracts, at a site six residues downstream from that seen for pathways involving capase 8. Incubation of mitochondria with Bid that had been cleaved by lysosomal extracts resulted in cytochrome c release. Thus, cleavage of Bid may represent a mechanism by which proteases that have leaked from the lysosomes can precipitate cytochrome c release and subsequent caspase activation. This is supported by the finding that cytosolic extracts from mice ablated in the bid gene are impaired in the ability to release cytochrome c in response to lysosome extracts. Together these data suggest that Bid represents a sensor that allows cells to initiate apoptosis in response to widespread adventitious proteolysis.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Amniotic membrane (AM) is the innermost, multilayered part of the placenta. When harvested, processed and stored properly, its properties, stemming from AM biological composition, make it a useful ...tissue for ophthalmic surgery. AM was shown to have several beneficial effects: it promotes epithelization, has antimicrobial effects, decreases inflammation, fibrosis and neovascularization. Many case reports and case series as well as practical experience (e.g. reconstruction of conjunctival and corneal defects, treatment of corneal ulcers) demonstrated the beneficial effect of AM for different ophthalmological indications. The combination of the above mentioned beneficial effects and reasonable mechanical properties are also the reason why AM is used as a substrate for ex vivo expansion of epithelial progenitor cells. Recently, amnion-derived cells, which also have stem cell characteristics, have been proposed as potential contributors to cell-based treatment of ocular surface disease. However, the use of AM remains one of the least standardized methods in ophthalmic surgery. In this review, the various properties of AM and its current clinical use in ophthalmology in Slovenia are discussed.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Apoptosis or programmed cell death is the major mechanism used by multicellular organisms to remove infected, excessive and potentially dangerous cells. Cysteine proteases from the caspase family ...play a crucial role in the process. However, there is increasing evidence that lysosomal proteases are also involved in apoptosis. In this review various lysosomal proteases and their potential contribution to propagation of apoptosis are discussed.
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