Recommendations from the COST action CA17116 (SPRINT) for the standardization of perinatal derivative preparation and in vitro testing.

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.

Human Amniotic Membrane Enriched with Urinary Bladder Fibroblasts Promote the Re-Epithelization of Urothelial Injury.

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.

REGENERATION OF CHRONIC WOUNDS WITH ALLOGENEIC PLATELET GEL VERSUS HYDROGEL TREATMENT: A PROSPECTIVE STUDY.

- 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.

Amniotic membrane properties and current practice of amniotic membrane use in ophthalmology in Slovenia.

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.

Selective disruption of lysosomes in HeLa cells triggers apoptosis mediated by cleavage of Bid by multiple papain-like lysosomal cathepsins.

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 microM L-trans-epoxysuccinyl(OEt)-Leu-3-methylbutylamide, an inhibitor of papain-like cysteine proteases. Incubation of cells with 15 microM 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.

Apoptotic pathways: involvement of lysosomal proteases.

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.