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Sample records for lysate preparation rich

  1. Comparative Analysis of Different Platelet Lysates and Platelet Rich Preparations to Stimulate Tendon Cell Biology: An In Vitro Study

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    Franka Klatte-Schulz

    2018-01-01

    Full Text Available The poor healing potential of tendons is still a clinical problem, and the use of Platelet Rich Plasma (PRP was hypothesized to stimulate healing. As the efficacy of PRPs remains unproven, platelet lysate (PL could be an alternative with its main advantages of storage and characterization before use. Five different blood products were prepared from 16 male donors: human serum, two PRPs (Arthrex, (PRP-ACP; RegenLab (PRP-BCT, platelet concentrate (apheresis, PC, and PL (freezing-thawing destruction of PC. Additionally, ten commercial allogenic PLs (AlloPL from pooled donors were tested. The highest concentration of most growth factors was found in AlloPL, whereas the release of growth factors lasted longer in the other products. PRP-ACP, PRP-BCT, and PC significantly increased cell viability of human tenocyte-like cells, whereas PC and AlloPL increased Col1A1 expression and PRP-BCT increased Col3A1 expression. MMP-1, IL-1β, and HGF expression was significantly increased and Scleraxis expression decreased by most blood products. COX1 expression significantly decreased by PC and AlloPL. No clear positive effects on tendon cell biology could be shown, which might partially explain the weak outcome results in clinical practice. Pooled PL seemed to have the most beneficial effects and might be the future in using blood products for tendon tissue regeneration.

  2. Comparative analyses of industrial-scale human platelet lysate preparations.

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    Pierce, Jan; Benedetti, Eric; Preslar, Amber; Jacobson, Pam; Jin, Ping; Stroncek, David F; Reems, Jo-Anna

    2017-12-01

    Efforts are underway to eliminate fetal bovine serum from mammalian cell cultures for clinical use. An emerging, viable replacement option for fetal bovine serum is human platelet lysate (PL) as either a plasma-based or serum-based product. Nine industrial-scale, serum-based PL manufacturing runs (i.e., lots) were performed, consisting of an average ± standard deviation volume of 24.6 ± 2.2 liters of pooled, platelet-rich plasma units that were obtained from apheresis donors. Manufactured lots were compared by evaluating various biochemical and functional test results. Comprehensive cytokine profiles of PL lots and product stability tests were performed. Global gene expression profiles of mesenchymal stromal cells (MSCs) cultured with plasma-based or serum-based PL were compared to MSCs cultured with fetal bovine serum. Electrolyte and protein levels were relatively consistent among all serum-based PL lots, with only slight variations in glucose and calcium levels. All nine lots were as good as or better than fetal bovine serum in expanding MSCs. Serum-based PL stored at -80°C remained stable over 2 years. Quantitative cytokine arrays showed similarities as well as dissimilarities in the proteins present in serum-based PL. Greater differences in MSC gene expression profiles were attributable to the starting cell source rather than with the use of either PL or fetal bovine serum as a culture supplement. Using a large-scale, standardized method, lot-to-lot variations were noted for industrial-scale preparations of serum-based PL products. However, all lots performed as well as or better than fetal bovine serum in supporting MSC growth. Together, these data indicate that off-the-shelf PL is a feasible substitute for fetal bovine serum in MSC cultures. © 2017 AABB.

  3. Comparative Effects of Platelet-Rich Plasma, Platelet Lysate, and Fetal Calf Serum on Mesenchymal Stem Cells.

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    Lykov, A P; Bondarenko, N A; Surovtseva, M A; Kim, I I; Poveshchenko, O V; Pokushalov, E A; Konenkov, V I

    2017-10-01

    We studied the effects of human platelet-rich plasma and platelet lysate on proliferation, migration, and colony-forming properties of rat mesenchymal stem cells. Platelet-rich plasma and platelet lysate stimulated the proliferation, migration, and colony formation of mesenchymal stem cells. A real-time study showed that platelet-rich plasma produces the most potent stimulatory effect, while both platelet-rich plasma and platelet lysate stimulated migration of cells.

  4. Platelet-Rich Fibrin Lysate Can Ameliorate Dysfunction of Chronically UVA-Irradiated Human Dermal Fibroblasts.

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    Wirohadidjojo, Yohanes Widodo; Budiyanto, Arief; Soebono, Hardyanto

    2016-09-01

    To determine whether platelet-rich fibrin lysate (PRF-L) could restore the function of chronically ultraviolet-A (UVA)-irradiated human dermal fibroblasts (HDFs), we isolated and sub-cultured HDFs from six different human foreskins. HDFs were divided into two groups: those that received chronic UVA irradiation (total dosages of 10 J cm⁻²) and those that were not irradiated. We compared the proliferation rates, collagen deposition, and migration rates between the groups and between chronically UVA-irradiated HDFs in control and PRF-L-treated media. Our experiment showed that chronic UVA irradiation significantly decreased (p<0.05) the proliferation rates, migration rates, and collagen deposition of HDFs, compared to controls. Compared to control media, chronically UVA-irradiated HDFs in 50% PRF-L had significantly increased proliferation rates, migration rates, and collagen deposition (p<0.05), and the migration rates and collagen deposition of chronically UVA-irradiated HDFs in 50% PRF-L were equal to those of normal fibroblasts. Based on this experiment, we concluded that PRF-L is a good candidate material for treating UVA-induced photoaging of skin, although the best method for its clinical application remains to be determined.

  5. Mineralization by mesenchymal stromal cells is variously modulated depending on commercial platelet lysate preparations.

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    Boraldi, Federica; Burns, Jorge S; Bartolomeo, Angelica; Dominici, Massimo; Quaglino, Daniela

    2018-03-01

    Numerous cellular models have been developed to investigate calcification for regenerative medicine applications and for the identification of therapeutic targets in various complications associated with age-related diseases. However, results have often been contradictory due to specific culture conditions, cell type ontogeny and aging status. Human platelet lysate (hPL) has been recently investigated as valuable alternative to fetal bovine serum (FBS) in cell culture and bone regeneration. A parallel comparison of how all these multiple factors may converge to influence mineralization has yet to be reported. To compare mineralization of human mesenchymal cell types known to differ in extracellular matrix calcification potency, bone marrow-derived mesenchymal stromal cells and dermal fibroblasts from neonatal and adult donors, at both low and high passages, were investigated in an ex vivo experimental model by supplementing the osteogenic induction medium with FBS or with hPL. Four commercial hPL preparations were profiled by liquid chromatography/electrospray ionization quadrupole time-of-flight spectrometry, and mineralization was visualized by von Kossa staining and quantified by morphometric evaluations after 9, 14 and 21 days of culture. Data demonstrate that (i) commercial hPL preparations differ according to mass spectra profiles, (ii) hPL variously influences mineral deposition depending on cell line and possibly on platelet product preparation methods, (iii) donor age modifies mineral deposition in the presence of the same hPL and (iv) reduced in vitro proliferative capacity affects osteogenic induction and response to hPL. Despite the standardized procedures applied to obtain commercial hPL, this study highlights the divergent effects of different preparations and emphasizes the importance of cellular ontology, donor age and cell proliferative capacity to optimize the osteogenic induction capabilities of mesenchymal stromal cells and design more effective

  6. PDGF-AB rich-trombocyte lysate supplementation from breast cancer patients increased the proliferation of breast cancer stem cells

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    Wiwi A. Kartolo

    2018-05-01

    Full Text Available Background: Thrombocytosis in breast cancer (BC patient was thought to play a role in the invasiveness of breast cancer stem cells (BCSCs. Modification of tumor microenvironment was proposed to increase the efficacy of anticancer therapy. This study was aimed to analyze the effect of platelet lysate (PL as well as its PDGF-AB content as a tumor microenvironment on (CD24-/CD44+ BCSC proliferation.Methods: This was an experimental study that treated culture of BCSCs with PL from breast cancer (BC patients or healthy donors. Venous blood from all subjects were subjected to prior hematology test and then processed to obtain platelet rich plasma  (PRP. Platelet counts in PRP were determined. PRP was processed to obtain PL. PDGF-AB contents in PL were measured. PL at concentrations of 0.01% (v/v was supplemented into DMEM-F12 medium and used for culturing BCSCs (CD24-/CD44+ cells. After 48 hours, total cell count, population doubling time (PDT, and cell viability were calculated and their correlation with platelet count and PDGF-AB levels were analyzed.Results: BC patients (n=5 had higher platelet counts and PDGF-AB levels in PL compared to healthy donors (n=15, (p=0.02. PL from BC patients could stimulate the proliferation of BCSCs higher than healthy donors (p<0.001 and showed lower PDT value (p=0.001. Cell proliferation and PDT showed strong correlation with PDGF-AB level. This observation suggests that PDGF-AB has a role on BCSCs proliferation. PL showed no effect on BCSCs viability.Conclusion: Breast cancer patient platelet lysate stimulated BCSC proliferation.

  7. A contribution to the study of pyrogenic substances in radiopharmaceutical preparations. Comparison between methods using Rabbit and those using Limulus amebocytes lysate

    International Nuclear Information System (INIS)

    Bruneau, Jacky.

    1982-10-01

    We have studied two methods for pyrogenic substances detection. We used: the hyperthermic action of these substances after injection in Rabbit, and the gelation reaction of a Limulus amebocytes lysate. To apply these two methods of pyrogenic substances detection to the radiopharmaceutical preparations, we have conceived and designed a material allowing their handling in compliance with the radioactive safety norms. We have compared the sensitivity, reliability and reproducibility of these methods, one based on gelation of Limulus amebocytes lysate in presence of endotoxins, the other on the hyperthermic action of these same endotoxins in the rabbit when injected intravenously or through the suboccipital route. The discussion of the results obtained, shows that the method using the Limulus amebocytes lysate is more sensitive, less expansive and less dangerous. This method particulary well adapted to the control of radiopharmaceutical preparations, brings an additional security to the patients for whom these products are destined [fr

  8. A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis.

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    Nitzan Krinsky

    Full Text Available Cell-free protein synthesis (CFPS systems are important laboratory tools that are used for various synthetic biology applications. Here, we present a simple and inexpensive laboratory-scale method for preparing a CFPS system from E. coli. The procedure uses basic lab equipment, a minimal set of reagents, and requires less than one hour to process the bacterial cell mass into a functional S30-T7 extract. BL21(DE3 and MRE600 E. coli strains were used to prepare the S30-T7 extract. The CFPS system was used to produce a set of fluorescent and therapeutic proteins of different molecular weights (up to 66 kDa. This system was able to produce 40-150 μg-protein/ml, with variations depending on the plasmid type, expressed protein and E. coli strain. Interestingly, the BL21-based CFPS exhibited stability and increased activity at 40 and 45°C. To the best of our knowledge, this is the most rapid and affordable lab-scale protocol for preparing a cell-free protein synthesis system, with high thermal stability and efficacy in producing therapeutic proteins.

  9. A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis

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    Kaduri, Maya; Shainsky-Roitman, Janna; Goldfeder, Mor; Ivanir, Eran; Benhar, Itai; Shoham, Yuval; Schroeder, Avi

    2016-01-01

    Cell-free protein synthesis (CFPS) systems are important laboratory tools that are used for various synthetic biology applications. Here, we present a simple and inexpensive laboratory-scale method for preparing a CFPS system from E. coli. The procedure uses basic lab equipment, a minimal set of reagents, and requires less than one hour to process the bacterial cell mass into a functional S30-T7 extract. BL21(DE3) and MRE600 E. coli strains were used to prepare the S30-T7 extract. The CFPS system was used to produce a set of fluorescent and therapeutic proteins of different molecular weights (up to 66 kDa). This system was able to produce 40–150 μg-protein/ml, with variations depending on the plasmid type, expressed protein and E. coli strain. Interestingly, the BL21-based CFPS exhibited stability and increased activity at 40 and 45°C. To the best of our knowledge, this is the most rapid and affordable lab-scale protocol for preparing a cell-free protein synthesis system, with high thermal stability and efficacy in producing therapeutic proteins. PMID:27768741

  10. Allogeneic effector/memory Th-1 cells impair FoxP3+ regulatory T lymphocytes and synergize with chaperone-rich cell lysate vaccine to treat leukemia.

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    Janikashvili, Nona; LaCasse, Collin J; Larmonier, Claire; Trad, Malika; Herrell, Amanda; Bustamante, Sara; Bonnotte, Bernard; Har-Noy, Michael; Larmonier, Nicolas; Katsanis, Emmanuel

    2011-02-03

    Therapeutic strategies combining the induction of effective antitumor immunity with the inhibition of the mechanisms of tumor-induced immunosuppression represent a key objective in cancer immunotherapy. Herein we demonstrate that effector/memory CD4(+) T helper-1 (Th-1) lymphocytes, in addition to polarizing type-1 antitumor immune responses, impair tumor-induced CD4(+)CD25(+)FoxP3(+) regulatory T lymphocyte (Treg) immunosuppressive function in vitro and in vivo. Th-1 cells also inhibit the generation of FoxP3(+) Tregs from naive CD4(+)CD25(-)FoxP3(-) T cells by an interferon-γ-dependent mechanism. In addition, in an aggressive mouse leukemia model (12B1), Th-1 lymphocytes act synergistically with a chaperone-rich cell lysate (CRCL) vaccine, leading to improved survival and long-lasting protection against leukemia. The combination of CRCL as a source of tumor-specific antigens and Th-1 lymphocytes as an adjuvant has the potential to stimulate efficient specific antitumor immunity while restraining Treg-induced suppression.

  11. Analysis of Reparative Activity of Platelet Lysate: Effect on Cell Monolayer Recovery In Vitro and Skin Wound Healing In Vivo.

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    Sergeeva, N S; Shanskii, Ya D; Sviridova, I K; Karalkin, P A; Kirsanova, V A; Akhmedova, S A; Kaprin, A D

    2016-11-01

    Platelet lysate prepared from donor platelet concentrate and pooled according to a developed technique stimulates migration of multipotent mesenchymal stromal cells of the human adipose tissue and promotes healing of the monolayer defect in cultures of human fibroblasts and multipotent mesenchymal stromal cells in vitro in concentrations close those of fetal calf serum (5-10%). Lysate of platelets from platelet-rich rat blood plasma stimulated healing of the skin defect by promoting epithelialization and granulation tissue formation. The regenerative properties of platelet lysate in vivo increased with increasing its concentration.

  12. Preparing Mathematics Teachers for Technology-Rich Environments

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    Sturdivant, Rodney X.; Dunham, Penelope; Jardine, Richard

    2009-01-01

    This article describes key elements for faculty development programs to prepare mathematics teachers for technology-rich environments. We offer practical examples from our experiences in teaching mathematics with technology and in teaching others to incorporate technology-based pedagogies. We address challenges faced by faculty using technology,…

  13. Autologous blood preparations rich in platelets, fibrin and growth factors.

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    Fioravanti, C; Frustaci, I; Armellin, E; Condò, R; Arcuri, C; Cerroni, L

    2015-01-01

    Bone regeneration is often needed prior to dental implant treatment due to the lack of adequate quantity and quality after infectious diseases. The greatest regenerative power was obtained with autologous tissue, primarily the bone alive, taken from the same site or adjacent sites, up to the use centrifugation of blood with the selection of the parts with the greatest potential regenerative. In fact, various techniques and technologies were chronologically successive to cope with an ever better preparation of these concentrates of blood. Our aim is to review these advances and discuss the ways in which platelet concentrates may provide such unexpected beneficial therapeutic effects. The research has been carried out in the MEDLINE and Cochrane Central Register of Controlled Trials database by choosing keywords as "platelet rich plasma", "platelet rich fibrin", "platelet growth factors", and "bone regeneration" and "dentistry". Autologous platelet rich plasma is a safe and low cost procedure to deliver growth factors for bone and soft tissue healing. The great heterogeneity of clinical outcomes can be explained by the different PRP products with qualitative and quantitative difference among substance.

  14. Detection of endotoxins in radiopharmaceutical preparations. II. Comparison of the sensitivity of methods using the rabbit and the Limulus amoebocyte lysate for the detection of endotoxins

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    Bruneau, J; Cohen, Y; Merlin, L; Peysson, S

    1986-01-01

    The rise of the rabbit internal temperature after i.v. injection of an endotoxin solution is proportional to concentration. Gelation of Limulus amoebocyte, when in presence of an endotoxin solution, is also related to concentration. We compared the sensitivity of these two methods. With our experimental procedure, the rabbit is sensitive to a 0.40 ng/mL solution and the Limulus amoebocyte lysate to a 0.14 ng/mL solution. The rabbit sensitivity increase is related to the per kilogramme injected volume, whereas sensitivity is not related to the volume to check in the case of the lysate.

  15. Effect of Titanium-prepared Platelet-rich Fibrin Treatment on the ...

    African Journals Online (AJOL)

    Effect of Titanium-prepared Platelet-rich Fibrin Treatment on the Angiogenic Biomarkers in Gingival Crevicular Fluid in Infrabony Defects of Patients with Chronic Periodontitis: A Randomized Controlled Clinical Trial.

  16. Preparation of proton rich radionuclides in support of radiochemical analysis

    International Nuclear Information System (INIS)

    Jerome, Simon; Larijani, Cyrus; Parker, David

    2012-01-01

    The production of proton rich radionuclides supports a wide range of radiochemical analyses via radioactive yield tracers ( 95m Tc and 236 Pu). In recent years, NPL and the University of Birmingham cyclotron have collaborated to produce these, and other, radionuclides. - Highlights: ► In this paper we options for the production of Tc and Pu tracers. ► The irradiation and measurement of targets producing Tc-95 m and Pu-236 are described. ► Options for production are discussed. ► The results of this study and future work needed are described.

  17. The potential impact of the preparation rich in growth factors (PRGF) in different medical fields.

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    Anitua, Eduardo; Sánchez, Mikel; Orive, Gorka; Andía, Isabel

    2007-11-01

    Platelet-rich preparations constitute a relatively new biotechnology for the stimulation and acceleration of tissue healing and bone regeneration. The versatility and biocompatibility of this approach has stimulated its therapeutic use in numerous medical and scientific fields including dentistry, oral implantology, orthopaedics, ulcer treatment, tissue engineering among others. Here we discuss the important progress that has been accomplished in the field of platelet-rich preparations in the last few years. Some of the most interesting therapeutic applications of this technology are discussed as are some of the limitations, future challenges and directions in the field.

  18. Preparing Content-Rich Learning Environments with VPython and Excel, Controlled by Visual Basic for Applications

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    Prayaga, Chandra

    2008-01-01

    A simple interface between VPython and Microsoft (MS) Office products such as Word and Excel, controlled by Visual Basic for Applications, is described. The interface allows the preparation of content-rich, interactive learning environments by taking advantage of the three-dimensional (3D) visualization capabilities of VPython and the GUI…

  19. Anti-Atherosclerotic Effects of a Phytoestrogen-Rich Herbal Preparation in Postmenopausal Women

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    Veronika A. Myasoedova

    2016-08-01

    Full Text Available The risk of cardiovascular disease and atherosclerosis progression is significantly increased after menopause, probably due to the decrease of estrogen levels. The use of hormone replacement therapy (HRT for prevention of cardiovascular disease in older postmenopausal failed to meet expectations. Phytoestrogens may induce some improvements in climacteric symptoms, but their effect on the progression of atherosclerosis remains unclear. The reduction of cholesterol accumulation at the cellular level should lead to inhibition of the atherosclerotic process in the arterial wall. The inhibition of intracellular lipid deposition with isoflavonoids was suggested as the effective way for the prevention of plaque formation in the arterial wall. The aim of this double-blind, placebo-controlled clinical study was to investigate the effect of an isoflavonoid-rich herbal preparation on atherosclerosis progression in postmenopausal women free of overt cardiovascular disease. One hundred fifty-seven healthy postmenopausal women (age 65 ± 6 were randomized to a 500 mg isoflavonoid-rich herbal preparation containing tannins from grape seeds, green tea leaves, hop cone powder, and garlic powder, or placebo. Conventional cardiovascular risk factors and intima-media thickness of common carotid arteries (cIMT were evaluated at the baseline and after 12 months of treatment. After 12-months follow-up, total cholesterol decreased by 6.3% in isoflavonoid-rich herbal preparation recipients (p = 0.011 and by 5.2% in placebo recipients (p = 0.020; low density lipoprotein (LDL cholesterol decreased by 7.6% in isoflavonoid-rich herbal preparation recipients (p = 0.040 and by 5.2% in placebo recipients (non-significant, NS; high density lipoprotein (HDL cholesterol decreased by 3.4% in isoflavonoid-rich herbal preparation recipients (NS and by 4.5% in placebo recipients (p = 0.038; triglycerides decreased by 6.0% in isoflavonoid-rich herbal preparation recipients (NS and by

  20. A Novel Platelet Concentrate: Titanium-Prepared Platelet-Rich Fibrin

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    Mustafa Tunalı; Hakan Özdemir; Zafer Küçükodacı; Serhan Akman; Emre Yaprak; Hülya Toker; Erhan Fıratlı

    2014-01-01

    We developed a new product called titanium-prepared platelet-rich fibrin (T-PRF). The T-PRF method is based on the hypothesis that titanium may be more effective in activating platelets than the silica activators used with glass tubes in Chouckroun's leukocyte- and platelet-rich fibrin (L-PRF) method. In this study, we aimed to define the structural characteristics of T-PRF and compare it with L-PRF. Blood samples were collected from 10 healthy male volunteers. The blood samples were drawn us...

  1. Effect of platelet lysate on human cells involved in different phases of wound healing.

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    Barsotti, Maria Chiara; Chiara Barsotti, Maria; Losi, Paola; Briganti, Enrica; Sanguinetti, Elena; Magera, Angela; Al Kayal, Tamer; Feriani, Roberto; Di Stefano, Rossella; Soldani, Giorgio

    2013-01-01

    Platelets are rich in mediators able to positively affect cell activity in wound healing. Aim of this study was to characterize the effect of different concentrations of human pooled allogeneic platelet lysate on human cells involved in the different phases of wound healing (inflammatory phase, angiogenesis, extracellular matrix secretion and epithelialization). Platelet lysate effect was studied on endothelial cells, monocytes, fibroblasts and keratinocytes, in terms of viability and proliferation, migration, angiogenesis, tissue repair pathway activation (ERK1/2) and inflammatory response evaluation (NFκB). Results were compared both with basal medium and with a positive control containing serum and growth factors. Platelet lysate induced viability and proliferation at the highest concentrations tested (10% and 20% v/v). Whereas both platelet lysate concentrations increased cell migration, only 20% platelet lysate was able to significantly promote angiogenic activity (pplatelet lysate concentrations activated important inflammatory pathways such as ERK1/2 and NFκB with the same early kinetics, whereas the effect was different for later time-points. These data suggest the possibility of using allogeneic platelet lysate as both an alternative to growth factors commonly used for cell culture and as a tool for clinical regenerative application for wound healing.

  2. Effect of platelet lysate on human cells involved in different phases of wound healing.

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    Maria Chiara Barsotti

    Full Text Available BACKGROUND: Platelets are rich in mediators able to positively affect cell activity in wound healing. Aim of this study was to characterize the effect of different concentrations of human pooled allogeneic platelet lysate on human cells involved in the different phases of wound healing (inflammatory phase, angiogenesis, extracellular matrix secretion and epithelialization. METHODOLOGY/PRINCIPAL FINDINGS: Platelet lysate effect was studied on endothelial cells, monocytes, fibroblasts and keratinocytes, in terms of viability and proliferation, migration, angiogenesis, tissue repair pathway activation (ERK1/2 and inflammatory response evaluation (NFκB. Results were compared both with basal medium and with a positive control containing serum and growth factors. Platelet lysate induced viability and proliferation at the highest concentrations tested (10% and 20% v/v. Whereas both platelet lysate concentrations increased cell migration, only 20% platelet lysate was able to significantly promote angiogenic activity (p<0.05 vs. control, comparably to the positive control. Both platelet lysate concentrations activated important inflammatory pathways such as ERK1/2 and NFκB with the same early kinetics, whereas the effect was different for later time-points. CONCLUSION/SIGNIFICANCE: These data suggest the possibility of using allogeneic platelet lysate as both an alternative to growth factors commonly used for cell culture and as a tool for clinical regenerative application for wound healing.

  3. Platelet-rich plasma differs according to preparation method and human variability.

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    Mazzocca, Augustus D; McCarthy, Mary Beth R; Chowaniec, David M; Cote, Mark P; Romeo, Anthony A; Bradley, James P; Arciero, Robert A; Beitzel, Knut

    2012-02-15

    Varying concentrations of blood components in platelet-rich plasma preparations may contribute to the variable results seen in recently published clinical studies. The purposes of this investigation were (1) to quantify the level of platelets, growth factors, red blood cells, and white blood cells in so-called one-step (clinically used commercial devices) and two-step separation systems and (2) to determine the influence of three separate blood draws on the resulting components of platelet-rich plasma. Three different platelet-rich plasma (PRP) separation methods (on blood samples from eight subjects with a mean age [and standard deviation] of 31.6 ± 10.9 years) were used: two single-spin processes (PRPLP and PRPHP) and a double-spin process (PRPDS) were evaluated for concentrations of platelets, red and white blood cells, and growth factors. Additionally, the effect of three repetitive blood draws on platelet-rich plasma components was evaluated. The content and concentrations of platelets, white blood cells, and growth factors for each method of separation differed significantly. All separation techniques resulted in a significant increase in platelet concentration compared with native blood. Platelet and white blood-cell concentrations of the PRPHP procedure were significantly higher than platelet and white blood-cell concentrations produced by the so-called single-step PRPLP and the so-called two-step PRPDS procedures, although significant differences between PRPLP and PRPDS were not observed. Comparing the results of the three blood draws with regard to the reliability of platelet number and cell counts, wide variations of intra-individual numbers were observed. Single-step procedures are capable of producing sufficient amounts of platelets for clinical usage. Within the evaluated procedures, platelet numbers and numbers of white blood cells differ significantly. The intra-individual results of platelet-rich plasma separations showed wide variations in

  4. Platelet-rich fibrin prepared from stored whole-blood samples.

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    Isobe, Kazushige; Suzuki, Masashi; Watanabe, Taisuke; Kitamura, Yutaka; Suzuki, Taiji; Kawabata, Hideo; Nakamura, Masayuki; Okudera, Toshimitsu; Okudera, Hajime; Uematsu, Kohya; Nakata, Koh; Tanaka, Takaaki; Kawase, Tomoyuki

    2017-12-01

    In regenerative therapy, self-clotted platelet concentrates, such as platelet-rich fibrin (PRF), are generally prepared on-site and are immediately used for treatment. If blood samples or prepared clots can be preserved for several days, their clinical applicability will expand. Here, we prepared PRF from stored whole-blood samples and examined their characteristics. Blood samples were collected from non-smoking, healthy male donors (aged 27-67 years, N = 6), and PRF clots were prepared immediately or after storage for 1-2 days. Fibrin fiber was examined by scanning electron microscopy. Bioactivity was evaluated by means of a bioassay system involving human periosteal cells, whereas PDGF-BB concentrations were determined by an enzyme-linked immunosorbent assay. Addition of optimal amounts of a 10% CaCl 2 solution restored the coagulative ability of whole-blood samples that contained an anticoagulant (acid citrate dextrose) and were stored for up to 2 days at ambient temperature. In PRF clots prepared from the stored whole-blood samples, the thickness and cross-links of fibrin fibers were almost identical to those of freshly prepared PRF clots. PDGF-BB concentrations in the PRF extract were significantly lower in stored whole-blood samples than in fresh samples; however, both extracts had similar stimulatory effects on periosteal-cell proliferation. Quality of PRF clots prepared from stored whole-blood samples is not reduced significantly and can be ensured for use in regenerative therapy. Therefore, the proposed method enables a more flexible treatment schedule and choice of a more suitable platelet concentrate immediately before treatment, not after blood collection.

  5. A novel platelet concentrate: titanium-prepared platelet-rich fibrin.

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    Tunalı, Mustafa; Özdemir, Hakan; Küçükodacı, Zafer; Akman, Serhan; Yaprak, Emre; Toker, Hülya; Fıratlı, Erhan

    2014-01-01

    We developed a new product called titanium-prepared platelet-rich fibrin (T-PRF). The T-PRF method is based on the hypothesis that titanium may be more effective in activating platelets than the silica activators used with glass tubes in Chouckroun's leukocyte- and platelet-rich fibrin (L-PRF) method. In this study, we aimed to define the structural characteristics of T-PRF and compare it with L-PRF. Blood samples were collected from 10 healthy male volunteers. The blood samples were drawn using a syringe. Nine milliliters was transferred to a dry glass tube, and 9 mL was transferred to a titanium tube. Half of each clot (i.e., the blood that was clotted using T-PRF or L-PRF) was processed with a scanning electron microscope (SEM). The other half of each clot was processed for fluorescence microscopy analysis and light microscopy analysis. The T-PRF samples seemed to have a highly organized network with continuous integrity compared to the other L-PRF samples. Histomorphometric analysis showed that T-PRF fibrin network covers larger area than L-PRF fibrin network; also fibrin seemed thicker in the T-PRF samples. This is the first human study to define T-PRF as an autogenous leukocyte- and platelet-rich fibrin product. The platelet activation by titanium seems to offer some high characteristics to T-PRF.

  6. A Novel Platelet Concentrate: Titanium-Prepared Platelet-Rich Fibrin

    Directory of Open Access Journals (Sweden)

    Mustafa Tunalı

    2014-01-01

    Full Text Available We developed a new product called titanium-prepared platelet-rich fibrin (T-PRF. The T-PRF method is based on the hypothesis that titanium may be more effective in activating platelets than the silica activators used with glass tubes in Chouckroun’s leukocyte- and platelet-rich fibrin (L-PRF method. In this study, we aimed to define the structural characteristics of T-PRF and compare it with L-PRF. Blood samples were collected from 10 healthy male volunteers. The blood samples were drawn using a syringe. Nine milliliters was transferred to a dry glass tube, and 9 mL was transferred to a titanium tube. Half of each clot (i.e., the blood that was clotted using T-PRF or L-PRF was processed with a scanning electron microscope (SEM. The other half of each clot was processed for fluorescence microscopy analysis and light microscopy analysis. The T-PRF samples seemed to have a highly organized network with continuous integrity compared to the other L-PRF samples. Histomorphometric analysis showed that T-PRF fibrin network covers larger area than L-PRF fibrin network; also fibrin seemed thicker in the T-PRF samples. This is the first human study to define T-PRF as an autogenous leukocyte- and platelet-rich fibrin product. The platelet activation by titanium seems to offer some high characteristics to T-PRF.

  7. Platelet-rich preparations to improve healing. Part II: platelet activation and enrichment, leukocyte inclusion, and other selection criteria.

    Science.gov (United States)

    Davis, Vicki L; Abukabda, Alaeddin B; Radio, Nicholas M; Witt-Enderby, Paula A; Clafshenkel, William P; Cairone, J Vito; Rutkowski, James L

    2014-08-01

    Multiple platelet-rich preparations have been reported to improve wound and bone healing, such as platelet-rich plasma (PRP) and platelet rich fibrin (PRF). The different methods employed during their preparation are important, as they influence the quality of the product applied to a wound or surgical site. Besides the general protocol for preparing the platelet-rich product (discussed in Part 1 of this review), multiple choices need to be considered during its preparation. For example, activation of the platelets is required for the release and enmeshment of growth factors, but the method of activation may influence the resulting matrix, growth factor availability, and healing. Additionally, some methods enrich leukocytes as well as platelets, but others are designed to be leukocyte-poor. Leukocytes have many important roles in healing and their inclusion in PRP results in increased platelet concentrations. Platelet and growth factor enrichment reported for the different types of platelet-rich preparations are also compared. Generally, TGF-β1 and PDGF levels were higher in preparations that contain leukocytes compared to leukocyte-poor PRP. However, platelet concentration may be the most reliable criterion for comparing different preparations. These and other criteria are described to help guide dental and medical professionals, in large and small practices, in selecting the best procedures for their patients. The healing benefits of platelet-rich preparations along with the low risk and availability of simple preparation procedures should encourage more clinicians to incorporate platelet-rich products in their practice to accelerate healing, reduce adverse events, and improve patient outcomes.

  8. Platelet-rich Preparation may serve as a Powerful Tool for Therapeutic Dental Pulp Regeneration

    Directory of Open Access Journals (Sweden)

    Hai-Hua Sun

    2011-09-01

    Full Text Available Introduction: Regeneration of dental pulp tissues presents one of the most challenging issues in regenerative dentistry due to their extremely poor intrinsic ability for self-healing and re-growth.The hypothesis: We hypothes-ize that patient-derived platelet-rich preparation can be used in clinical endodontic regenerative procedure, serving as a powerful tool for therapeutic dental pulp regeneration.Evaluation of the hypothesis: The cell transplantation does not always obtain the good result because of the low survival rate of transplanted cells. In addition, the use of ex vivo manipulated cell products faces many translational hurdles in treating non-vital disease. Recently, the body cells are focused as a potential source for therapeutics. Some researchers have demonstrated that endogenous stem cells may be recruited to a desired anatomic site pharma-cologically. This is spurring interest in developing new generation of biomaterials that incorporate and release selected powerful extracellular influences in a near-physiological fashion, and subsequently capture endogenous stem cells and influence their fates for regene-ration. The use of patient-derived products such as platelet-rich preparations that contain a multitude of endogenous growth factors and proteins is a clinically translatable biotechnology for this proposes. These simple and cost efficient procedures may have a potential impact in reducing the economic costs for standard medical treatments in regenerative endodontics.

  9. Platelet collection efficiencies of three different platelet-rich plasma preparation systems.

    Science.gov (United States)

    Aydin, Fatma; Pancar Yuksel, Esra; Albayrak, Davut

    2015-06-01

    Different systems have been used for the preparation of platelet-rich plasma (PRP), but platelet collection efficiencies of these systems are not clear. To evaluate the platelet collection efficiencies of three different PRP preparation systems. Blood samples were obtained from the same 16 volunteers for each system. The samples were centrifuged and PRP was prepared by three systems. The ratio of the total number of platelets in PRP to the total number of platelets of the venous blood sample of the patient expressed in percentage was named as platelet collection efficiency and calculated for each system. Mean platelet collection efficiencies were 66.6 (min: 56.9, max: 76.9), 58.3 (min: 27.3, max: 102.8), 50.8 (min: 27.2, max: 73) for top and bottom bag system, system using citrated tube, and the system using tube with Ficoll and cell extraction kit, respectively. Statistically significant difference was found only between the platelet collection efficiencies of systems using the tube with ficoll and cell extraction kit and the top and bottom bag system (p = 0.002). All three systems could be used for PRP preparation, but top and bottom bag system offers a slight advantage over the system using Ficoll and cell extraction kit regarding the platelet collection efficiency.

  10. Preparation and characterization of polymer-derived amorphous silicon carbide with silicon-rich stoichiometry

    Energy Technology Data Exchange (ETDEWEB)

    Masuda, Takashi, E-mail: mtakashi@jaist.ac.jp [School of Material and Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292 (Japan); Iwasaka, Akira [School of Material and Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292 (Japan); Takagishi, Hideyuki [Faculty of Symbiotic System Science, Fukushima University, 1 Kanayagawa, Fukushima-shi, Fukushima 960-1296 (Japan); Shimoda, Tatsuya [School of Material and Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292 (Japan)

    2016-08-01

    Polydihydrosilane with pendant hexyl groups was synthesized to obtain silicon-rich amorphous silicon carbide (a-SiC) films via the solution route. Unlike conventional polymeric precursors, this polymer requires neither catalysts nor oxidation for its synthesis and cross-linkage. Therefore, the polymer provides sufficient purity for the fabrication of semiconducting a-SiC. Here, we investigated the correlation of Si/C stoichiometry between the polymer and the resultant a-SiC film. The structural, optical, and electrical properties of the films with various carbon contents were also explored. Experimental results suggested that the excess carbon that did not participate in Si−C configurations was decomposed and was evaporated during polymer-to-SiC conversion. Consequently, the upper limit of the carbon in resultant a-SiC film was < 50 at.%; namely, the polymer provided silicon-rich a-SiC, whereas the conventionally used polycarbosilane inevitably provides carbon-rich one. These features of this unusual polymer open up a frontier of polymer-derived SiC and solution-processed SiC electronics. - Highlights: • Polymeric precursor solution for silicon carbide (SiC) is synthesized. • Semiconducting amorphous SiC is prepared via solution route. • The excess carbon is decomposed during cross-linking resulting in Si-rich SiC films. • The grown SiC films contain substantial amount of hydrogen atoms as SiH{sub n}/CH{sub n} entities. • Presence of CH{sub n} entities induces dangling bonds, causing poor electrical properties.

  11. Preparing Platelet-Rich Plasma with Whole Blood Harvested Intraoperatively During Spinal Fusion.

    Science.gov (United States)

    Shen, Bin; Zhang, Zheng; Zhou, Ning-Feng; Huang, Yu-Feng; Bao, Yu-Jie; Wu, De-Sheng; Zhang, Ya-Dong

    2017-07-22

    BACKGROUND Platelet-rich plasma (PRP) has gained growing popularity in use in spinal fusion procedures in the last decade. Substantial intraoperative blood loss is frequently accompanied with spinal fusion, and it is unknown whether blood harvested intraoperatively qualifies for PRP preparation. MATERIAL AND METHODS Whole blood was harvested intraoperatively and venous blood was collected by venipuncture. Then, we investigated the platelet concentrations in whole blood and PRP, the concentration of growth factors in PRP, and the effects of PRP on the proliferation and viability of human bone marrow-derived mesenchymal stem cells (HBMSCs). RESULTS Our results revealed that intraoperatively harvested whole blood and whole blood collected by venipuncture were similar in platelet concentration. In addition, PRP formulations prepared from both kinds of whole blood were similar in concentration of platelet and growth factors. Additional analysis showed that the similar concentrations of growth factors resulted from the similar platelet concentrations of whole blood and PRP between the two groups. Moreover, these two kinds of PRP formulations had similar effects on promoting cell proliferation and enhancing cell viability. CONCLUSIONS Therefore, intraoperatively harvested whole blood may be a potential option for preparing PRP spinal fusion.

  12. Optimization of dendritic cell loading with tumor cell lysates for cancer immunotherapy.

    Science.gov (United States)

    Hatfield, Paul; Merrick, Alison E; West, Emma; O'Donnell, Dearbhaile; Selby, Peter; Vile, Richard; Melcher, Alan A

    2008-09-01

    The immune response to cancer is critically determined by the way in which tumor cells die. As necrotic, stress-associated death can be associated with activation of antitumor immunity, whole tumor cell antigen loading strategies for dendritic cell (DC)-based vaccination have commonly used freeze-thaw "necrotic" lysates as an immunogenic source of tumor-associated antigens. In this study, the effect of such lysates on the ability of DCs to mature in response to well-established maturation stimuli was examined, and methods to enhance lysate-induced DC activation explored. Freeze-thaw lysates were prepared from murine tumor cell lines and their effects on bone marrow-derived DC maturation and function examined. Unmodified freeze-thaw tumor cell lysates inhibited the toll-like receptor-induced maturation and function of bone marrow-derived DCs, preventing up-regulation of CD40, CD86, and major histocompatibility complex class II, and reducing secretion of inflammatory cytokines [interleukin (IL)-12 p70, tumor necrosis factor-alpha, and IL-6]. Although IL-10 secretion was increased by lysate-pulsed DCs, this was not responsible for the observed suppression of IL-12. Although activation of the nuclear factor-kappaB pathway remained intact, the kinase activity of phosphorylated p38 mitogen-activated protein kinase was inhibited in lysate-pulsed DCs. Lysate-induced DC suppression was partially reversed in vitro by induction of tumor cell stress before lysis, and only DCs loaded with stressed lysates afforded protection against tumor challenge in vivo. These data suggest that ex vivo freeze-thaw of tumor cells does not effectively mimic in vivo immunogenic necrosis, and advocates careful characterization and optimization of tumor cell-derived vaccine sources for cancer immunotherapy.

  13. Preparation and Hydrogen Storage Properties of Mg-Rich Mg-Ni Ultrafine Particles

    Directory of Open Access Journals (Sweden)

    Jianxin Zou

    2012-01-01

    Full Text Available In the present work, Mg-rich Mg-Ni ultrafine powders were prepared through an arc plasma method. The phase components, microstructure, and hydrogen storage properties of the powders were carefully investigated. It is found that Mg2Ni and MgNi2 could be obtained directly from the vapor state reactions between Mg and Ni, depending on the local vapor content in the reaction chamber. A nanostructured MgH2 + Mg2NiH4 hydrogen storage composite could be generated after hydrogenation of the Mg-Ni ultrafine powders. After dehydrogenation, MgH2 and Mg2NiH4 decomposed into nanograined Mg and Mg2Ni, respectively. Thermogravimetry/differential scanning calorimetry (TG/DSC analyses showed that Mg2NiH4 phase may play a catalytic role in the dehydriding process of the hydrogenated Mg ultrafine particles.

  14. Crystallization characteristics of iron-rich glass ceramics prepared from nickel slag and blast furnace slag

    Science.gov (United States)

    Wang, Zhong-Jie; Ni, Wen; Li, Ke-Qing; Huang, Xiao-Yan; Zhu, Li-Ping

    2011-08-01

    The crystallization process of iron-rich glass-ceramics prepared from the mixture of nickel slag (NS) and blast furnace slag (BFS) with a small amount of quartz sand was investigated. A modified melting method which was more energy-saving than the traditional methods was used to control the crystallization process. The results show that the iron-rich system has much lower melting temperature, glass transition temperature ( T g), and glass crystallization temperature ( T c), which can result in a further energy-saving process. The results also show that the system has a quick but controllable crystallization process with its peak crystallization temperature at 918°C. The crystallization of augite crystals begins from the edge of the sample and invades into the whole sample. The crystallization process can be completed in a few minutes. A distinct boundary between the crystallized part and the non-crystallized part exists during the process. In the non-crystallized part showing a black colour, some sphere-shaped augite crystals already exist in the glass matrix before samples are heated to T c. In the crystallized part showing a khaki colour, a compact structure is formed by augite crystals.

  15. Optimisation of a double-centrifugation method for preparation of canine platelet-rich plasma.

    Science.gov (United States)

    Shin, Hyeok-Soo; Woo, Heung-Myong; Kang, Byung-Jae

    2017-06-26

    Platelet-rich plasma (PRP) has been expected for regenerative medicine because of its growth factors. However, there is considerable variability in the recovery and yield of platelets and the concentration of growth factors in PRP preparations. The aim of this study was to identify optimal relative centrifugal force and spin time for the preparation of PRP from canine blood using a double-centrifugation tube method. Whole blood samples were collected in citrate blood collection tubes from 12 healthy beagles. For the first centrifugation step, 10 different run conditions were compared to determine which condition produced optimal recovery of platelets. Once the optimal condition was identified, platelet-containing plasma prepared using that condition was subjected to a second centrifugation to pellet platelets. For the second centrifugation, 12 different run conditions were compared to identify the centrifugal force and spin time to produce maximal pellet recovery and concentration increase. Growth factor levels were estimated by using ELISA to measure platelet-derived growth factor-BB (PDGF-BB) concentrations in optimised CaCl 2 -activated platelet fractions. The highest platelet recovery rate and yield were obtained by first centrifuging whole blood at 1000 g for 5 min and then centrifuging the recovered platelet-enriched plasma at 1500 g for 15 min. This protocol recovered 80% of platelets from whole blood and increased platelet concentration six-fold and produced the highest concentration of PDGF-BB in activated fractions. We have described an optimised double-centrifugation tube method for the preparation of PRP from canine blood. This optimised method does not require particularly expensive equipment or high technical ability and can readily be carried out in a veterinary clinical setting.

  16. The effect of platelet lysate supplementation of a dextran-based hydrogel on cartilage formation.

    Science.gov (United States)

    Moreira Teixeira, Liliana S; Leijten, Jeroen C H; Wennink, Jos W H; Chatterjea, Anindita G; Feijen, Jan; van Blitterswijk, Clemens A; Dijkstra, Pieter J; Karperien, Marcel

    2012-05-01

    In situ gelating dextran-tyramine (Dex-TA) injectable hydrogels have previously shown promising features for cartilage repair. Yet, despite suitable mechanical properties, this system lacks intrinsic biological signals. In contrast, platelet lysate-derived hydrogels are rich in growth factors and anti-inflammatory cytokines, but mechanically unstable. We hypothesized that the advantages of these systems may be combined in one hydrogel, which can be easily translated into clinical settings. Platelet lysate was successfully incorporated into Dex-TA polymer solution prior to gelation. After enzymatic crosslinking, rheological and morphological evaluations were performed. Subsequently, the effect of platelet lysate on cell migration, adhesion, proliferation and multi-lineage differentiation was determined. Finally, we evaluated the integration potential of this gel onto osteoarthritis-affected cartilage. The mechanical properties and covalent attachment of Dex-TA to cartilage tissue during in situ gel formation were successfully combined with the advantages of platelet lysate, revealing the potential of this enhanced hydrogel as a cell-free approach. The addition of platelet lysate did not affect the mechanical properties and porosity of Dex-TA hydrogels. Furthermore, platelet lysate derived anabolic growth factors promoted proliferation and triggered chondrogenic differentiation of mesenchymal stromal cells. Copyright © 2012 Elsevier Ltd. All rights reserved.

  17. A Systematic Review of Autologous Platelet-Rich Plasma and Fat Graft Preparation Methods.

    Science.gov (United States)

    Luck, Joshua; Smith, Oliver J; Mosahebi, Afshin

    2017-12-01

    The addition of platelet-rich plasma (PRP) to adipose tissue may improve fat graft survival, although graft retention rates vary markedly between studies. To what extent this outcome heterogeneity reflects differing methodological factors remains unknown. This systematic review aims to synthesize and critically review methodological approaches to autologous PRP and fat cotransplantation in both human and animal studies. In accordance with PRISMA guidelines, Ovid MEDLINE, Scopus, and Cochrane Library databases were searched from inception to April 2017. Data were extracted from all in vivo studies involving autologous PRP and fat cotransplantation. A secondary aim was to assess reporting of technical detail; authors were not contacted to provide missing data. From 335 articles, 23 studies were included in the qualitative synthesis. Some 21 were performed in humans and 2 in rabbits. Six studies were randomized control trials; the remainder reported on observational data. Methods of PRP extraction and activation varied markedly between studies. Fat graft preparation was comparatively more consistent. Methods of PRP and fat mixing differed significantly, especially with regards to relative volume/volume ratios. Our study represents the first systematic review of methodological factors in autologous PRP and fat cotransplantation. It demonstrates that technical factors in graft preparation and administration vary significantly between in vivo studies. Such methodological heterogeneity may explain observed differences in experimental and clinical outcomes. Reporting of key procedural information is inconsistent and often inadequate. These issues make meaningful evaluation of the PRP-enhanced fat grafting literature difficult and may limit its translation into clinical practice.

  18. Platelet lysate embedded scaffolds for skin regeneration.

    Science.gov (United States)

    Sandri, Giuseppina; Bonferoni, Maria Cristina; Rossi, Silvia; Ferrari, Franca; Mori, Michela; Cervio, Marila; Riva, Federica; Liakos, Ioannis; Athanassiou, Athanassia; Saporito, Francesca; Marini, Lara; Caramella, Carla

    2015-04-01

    The work presents the development of acellular scaffolds extemporaneously embedded with platelet lysate (PL), as an innovative approach in the field of tissue regeneration/reparation. PL embedded scaffolds should have a tridimensional architecture to support cell migration and growth, in order to restore skin integrity. For this reason, chondroitin sulfate (CS) was associated with sodium alginate (SA) to prepare highly porous systems. The developed scaffolds were characterized for chemical stability to γ-radiation, morphology, hydration and mechanical properties. Moreover, the capability of fibroblasts and endothelial cells to populate the scaffold was evaluated by means of proliferation test 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and confocal laser scanning microscopy study. The scaffolds, not altered by sterilization, were characterized by limited swelling and high flexibility, by foam-like structure with bubbles that formed a high surface area and irregular texture suitable for cell adhesion. Cell growth and scaffold population were evident on the bubble surface, where the cells appeared anchored to the scaffold structure. Scaffold network based on CS and SA demonstrated to be an effective support to enhance and to allow fibroblasts and endothelial cells (human umbilical vein endothelial cells, HUVEC) adhesion and proliferation. In particular, it could be hypothesized that cell adhesion was facilitated by the synergic effect of PL and CS. Although further in vivo evaluation is needed, on the basis of in vitro results, PL embedded scaffolds seem promising systems for skin wound healing.

  19. Effect of two different preparations of platelet-rich plasma on synoviocytes.

    Science.gov (United States)

    Assirelli, Elisa; Filardo, Giuseppe; Mariani, Erminia; Kon, Elizaveta; Roffi, Alice; Vaccaro, Franca; Marcacci, Maurilio; Facchini, Andrea; Pulsatelli, Lia

    2015-09-01

    To analyse the modifications induced by two different platelet-rich plasma (PRP) preparations on osteoarthritis (OA) synoviocytes, by documenting changes in gene expression of factors involved in joint physiopathology. OA synoviocytes were cultured for 7 days in medium with different concentrations of either P-PRP (a pure platelet concentrate without leucocytes but with a limited number of platelets), L-PRP (a higher platelet concentrate containing leucocytes) or platelet-poor plasma (PPP). Gene expression of interleukin (IL)-1beta, IL-6, IL-8/CXCL8, tumour necrosis factor alpha, IL-10, IL-4, IL-13, metalloproteinase-13, tissue inhibitor of metalloproteinase (TIMP)-1, (TIMP)-3, (TIMP)-4, vascular endothelial growth factor, transforming growth factor beta1, fibroblast growth factor (FGF)-2, hepatocyte growth factor (HGF), hyaluronic acid (HA) synthases (HAS)-1, (HAS)-2, and (HAS)-3 was analysed by RT-PCR. HA production was determined in culture supernatants by ELISA. IL-1β, IL-8 and FGF-2 were significantly induced by L-PRP compared to both P-PRP and PPP; HGF was down-modulated by L-PRP versus both P-PRP and PPP, and an inverse dose-response influence was shown for all preparations. Expression level of TIMP-4 was lower in the presence of L-PRP compared with P-PRP. HA production and HAS gene expression did not seem to be modulated by PRP. L-PRP is able to sustain the up-regulation of proinflammatory factors, (IL-1beta, IL-8 and FGF-2), together with a down-modulation of HGF and TIMP-4 expression, two factors that have been recognized as anti-catabolic mediators in cartilage, thus supporting the need to further optimize the PRP preparations to be applied in clinical practice.

  20. Platelet-rich plasma preparation for regenerative medicine: optimization and quantification of cytokines and growth factors

    Science.gov (United States)

    2013-01-01

    Introduction Platelet-rich plasma (PRP) is nowadays widely applied in different clinical scenarios, such as orthopedics, ophthalmology and healing therapies, as a growth factor pool for improving tissue regeneration. Studies into its clinical efficiency are not conclusive and one of the main reasons for this is that different PRP preparations are used, eliciting different responses that cannot be compared. Platelet quantification and the growth factor content definition must be defined in order to understand molecular mechanisms behind PRP regenerative strength. Standardization of PRP preparations is thus urgently needed. Methods PRP was prepared by centrifugation varying the relative centrifugal force, temperature, and time. Having quantified platelet recovery and yield, the two-step procedure that rendered the highest output was chosen and further analyzed. Cytokine content was determined in different fractions obtained throughout the whole centrifugation procedure. Results Our method showed reproducibility when applied to different blood donors. We recovered 46.9 to 69.5% of total initial platelets and the procedure resulted in a 5.4-fold to 7.3-fold increase in platelet concentration (1.4 × 106 to 1.9 × 106 platelets/μl). Platelets were highly purified, because only blood cells and leukocytes was present in the final PRP preparation. We also quantified growth factors, cytokines and chemokines secreted by the concentrated platelets after activation with calcium and calcium/thrombin. High concentrations of platelet-derived growth factor, endothelial growth factor and transforming growth factor (TGF) were secreted, together with the anti-inflammatory and proinflammatory cytokines interleukin (IL)-4, IL-8, IL-13, IL-17, tumor necrosis factor (TNF)-α and interferon (IFN)-α. No cytokines were secreted before platelet activation. TGF-β3 and IFNγ were not detected in any studied fraction. Clots obtained after platelet coagulation retained a high concentration of

  1. Comparison of point-of-care methods for preparation of platelet concentrate (platelet-rich plasma).

    Science.gov (United States)

    Weibrich, Gernot; Kleis, Wilfried K G; Streckbein, Philipp; Moergel, Maximilian; Hitzler, Walter E; Hafner, Gerd

    2012-01-01

    This study analyzed the concentrations of platelets and growth factors in platelet-rich plasma (PRP), which are likely to depend on the method used for its production. The cellular composition and growth factor content of platelet concentrates (platelet-rich plasma) produced by six different procedures were quantitatively analyzed and compared. Platelet and leukocyte counts were determined on an automatic cell counter, and analysis of growth factors was performed using enzyme-linked immunosorbent assay. The principal differences between the analyzed PRP production methods (blood bank method of intermittent flow centrifuge system/platelet apheresis and by the five point-of-care methods) and the resulting platelet concentrates were evaluated with regard to resulting platelet, leukocyte, and growth factor levels. The platelet counts in both whole blood and PRP were generally higher in women than in men; no differences were observed with regard to age. Statistical analysis of platelet-derived growth factor AB (PDGF-AB) and transforming growth factor β1 (TGF-β1) showed no differences with regard to age or gender. Platelet counts and TGF-β1 concentration correlated closely, as did platelet counts and PDGF-AB levels. There were only rare correlations between leukocyte counts and PDGF-AB levels, but comparison of leukocyte counts and PDGF-AB levels demonstrated certain parallel tendencies. TGF-β1 levels derive in substantial part from platelets and emphasize the role of leukocytes, in addition to that of platelets, as a source of growth factors in PRP. All methods of producing PRP showed high variability in platelet counts and growth factor levels. The highest growth factor levels were found in the PRP prepared using the Platelet Concentrate Collection System manufactured by Biomet 3i.

  2. The effect of platelet lysate supplementation of a dextran-based hydrogel on cartilage formation

    NARCIS (Netherlands)

    Moreira Teixeira, Liliana; Leijten, Jeroen Christianus Hermanus; Wennink, J.W.H.; Ganguly, Anindita; Feijen, Jan; van Blitterswijk, Clemens; Dijkstra, Pieter J.; Karperien, Hermanus Bernardus Johannes

    2012-01-01

    In situ gelating dextran-tyramine (Dex-TA) injectable hydrogels have previously shown promising features for cartilage repair. Yet, despite suitable mechanical properties, this system lacks intrinsic biological signals. In contrast, platelet lysate-derived hydrogels are rich in growth factors and

  3. Platelet-rich plasma preparation for regenerative medicine: optimization and quantification of cytokines and growth factors.

    Science.gov (United States)

    Amable, Paola Romina; Carias, Rosana Bizon Vieira; Teixeira, Marcus Vinicius Telles; da Cruz Pacheco, Italo; Corrêa do Amaral, Ronaldo José Farias; Granjeiro, José Mauro; Borojevic, Radovan

    2013-06-07

    Platelet-rich plasma (PRP) is nowadays widely applied in different clinical scenarios, such as orthopedics, ophthalmology and healing therapies, as a growth factor pool for improving tissue regeneration. Studies into its clinical efficiency are not conclusive and one of the main reasons for this is that different PRP preparations are used, eliciting different responses that cannot be compared. Platelet quantification and the growth factor content definition must be defined in order to understand molecular mechanisms behind PRP regenerative strength. Standardization of PRP preparations is thus urgently needed. PRP was prepared by centrifugation varying the relative centrifugal force, temperature, and time. Having quantified platelet recovery and yield, the two-step procedure that rendered the highest output was chosen and further analyzed. Cytokine content was determined in different fractions obtained throughout the whole centrifugation procedure. Our method showed reproducibility when applied to different blood donors. We recovered 46.9 to 69.5% of total initial platelets and the procedure resulted in a 5.4-fold to 7.3-fold increase in platelet concentration (1.4 × 10(6) to 1.9 × 10(6) platelets/μl). Platelets were highly purified, because only platelets after activation with calcium and calcium/thrombin. High concentrations of platelet-derived growth factor, endothelial growth factor and transforming growth factor (TGF) were secreted, together with the anti-inflammatory and proinflammatory cytokines interleukin (IL)-4, IL-8, IL-13, IL-17, tumor necrosis factor (TNF)-α and interferon (IFN)-α. No cytokines were secreted before platelet activation. TGF-β3 and IFNγ were not detected in any studied fraction. Clots obtained after platelet coagulation retained a high concentration of several growth factors, including platelet-derived growth factor and TGF. Our study resulted in a consistent PRP preparation method that yielded a cytokine and growth factor pool

  4. Platelet lysates produced from expired platelet concentrates support growth and osteogenic differentiation of mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    Sandra Mjoll Jonsdottir-Buch

    Full Text Available BACKGROUND: Mesenchymal stem cells are promising candidates in regenerative cell therapy. Conventional culture methods involve the use of animal substances, specifically fetal bovine serum as growth supplement. Since the use of animal-derived products is undesirable for human applications, platelet lysates produced from human platelets are an attractive alternative. This is especially true if platelet lysates from already approved transfusion units at blood banks can be utilized. The purpose of this study was to produce human platelet lysates from expired, blood bank-approved platelet concentrates and evaluate their use as growth supplement in the culture of mesenchymal stem cells. METHODOLOGY/PRINCIPAL FINDINGS: In this study, bone marrow-derived mesenchymal stem cells were cultured with one of three culture supplements; fetal bovine serum, lysates from freshly prepared human platelet concentrates, or lysates from expired human platelet concentrates. The effects of these platelet-derived culture supplements on basic mesenchymal stem cell characteristics were evaluated. All cultures maintained the typical mesenchymal stem cell surface marker expression, trilineage differentiation potential, and the ability to suppress in vitro immune responses. However, mesenchymal stem cells supplemented with platelet lysates proliferated faster than traditionally cultured cells and increased the expression of the osteogenic marker gene RUNX-2; yet no difference between the use of fresh and expired platelet concentrates was observed. CONCLUSION/SIGNIFICANCE: Our findings suggest that human platelet lysates produced from expired platelet concentrates can be used as an alternative to fetal bovine serum for mesenchymal stem cell culture to the same extent as lysates from fresh platelets.

  5. Enzymatic preparation of "functional oil" rich in feruloylated structured lipids with solvent-free ultrasound pretreatment.

    Science.gov (United States)

    Zhang, Haiping; Zheng, Mingming; Shi, Jie; Tang, Hu; Deng, Qianchun; Huang, Fenghong; Luo, Dan

    2018-05-15

    In this study, a series of functional oils rich in feruloylated structured lipids (FSLs) was prepared by enzymatic transesterification of ethyl ferulate (EF) with triglycerides under ultrasound pretreatment. A conversion of more than 92.7% and controllable FSLs (3.1%-26.3%) can be obtained under the following conditions: 16% enzyme, substrate ratio 1:5 (oil/EF, mol/mol), 85 °C, ultrasound 1 h, pulse mode 3 s/3s (working/waiting), and 17.0 W/mL. Compared to conventional mechanical stirring, the activation energy decreased from 50.0 kJ/mol to 40.7 kJ/mol. The apparent kinetic constant increased by more than 13 times, and the time required for the maximum conversion reduced sharply from 20-60 h to 4-6h, which was the fastest rate for enzymatic synthesis of FSLs. The antioxidant activities of the functional oil significantly increased 1.0- to 8.1-fold more than that of the raw oil. The functional oil could be widely applied in various fields of functional foods. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Characteristics of canine platelet-rich plasma prepared with five commercially available systems.

    Science.gov (United States)

    Franklin, Samuel P; Garner, Bridget C; Cook, James L

    2015-09-01

    To characterize platelet-rich plasma (PRP) products obtained from canine blood by use of a variety of commercially available devices. Blood samples from 15 dogs between 18 months and 9 years of age with no concurrent disease, except for osteoarthritis in some dogs. PRP products were produced from blood obtained from each of the 15 dogs by use of each of 5 commercially available PRP-concentrating systems. Complete blood counts were performed on each whole blood sample and PRP product. The degree of platelet, leukocyte, and erythrocyte concentration or reduction for PRP, compared with results for the whole blood sample, was quantified for each dog and summarized for each concentrating system. The various PRP-concentrating systems differed substantially in the amount of blood processed, method of PRP preparation, amount of PRP produced, and platelet, leukocyte, and erythrocyte concentrations or reductions for PRP relative to results for whole blood. The characteristics of PRP products differed considerably. Investigators evaluating the efficacy of PRPs need to specify the characteristics of the product they are assessing. Clinicians should be aware of the data (or lack of data) supporting use of a particular PRP for a specific medical condition.

  7. The preparation and composition design of boron-rich lanthanum hexaboride target for sputtering

    International Nuclear Information System (INIS)

    Chen, Defang; Min, Guanghui; Wu, Yan; Yu, Huashun; Zhang, Lin

    2015-01-01

    Highlights: • High-purity LaB 6 powder was prepared due to significant reduction of residual B 4 C and effective purification process. • The effects of raw materials ratio on the size, morphology, phase structure and crystalline size of LaB 6 were studied. • The correlation of component between LaB 6 films and boron-rich targets was established. • The variation of densities of LaB 6 targets with sintering time and sintering temperature was investigated. - Abstract: Lanthanum Hexaboride (LaB 6 ) nano-film has been proved to be promising transparent thermal insulation material, while its properties are limited on purity and composition. High-purity LaB 6 polycrystalline powder was prepared through boron carbide reduction method in this work. A series of techniques such as scanning electron microscopy, X-ray diffraction, laser particle analyzer and inductively coupled plasma emission spectrometer were employed to characterize LaB 6 powder. As raising the content of La 2 O 3 in reactants, more uniform, finer (2.686 μm) and purer (99.5139 wt%) LaB 6 powder is prepared, with only 0.4434 wt% residual B 4 C. The density of targets increases with the rise of sintering temperature and the extension of sintering time, while crystallite size increases simultaneously with the extension of sintering time. The introduction of B powder in target is conductive to sintering process, increasing hardness and flexural strength of targets. X-ray photoelectron spectrometer was used to characterize the composition and microstructure of LaB 6 nano-film which is tentatively considered to be composed of LaB 6 nanocrystalline and amorphous microstructure of La and B atoms. The film LaB 6.0627±0.02 was obtained when the ratio of B and La of sputtering target reached 12.5. The thickness and deposition rate decrease with the increase of B content in targets

  8. Compaction agent clarification of microbial lysates

    Science.gov (United States)

    DeWalt, Brad W.; Murphy, Jason C.; Fox, George E.; Willson, Richard C.

    2003-01-01

    Recombinant proteins are often purified from microbial lysates containing high concentrations of nucleic acids. Pre-purification steps such as nuclease addition or precipitation with polyethyleneimine or ammonium sulfate are normally required to reduce viscosity and to eliminate competing polyanions before anion exchange chromatography. We report that small polycationic compaction agents such as spermine selectively precipitate nucleic acids during or after Escherichia coli lysis, allowing DNA and RNA to be pelleted with the insoluble cell debris. Analysis by spectrophotometry and protein assay confirmed a significant reduction in the concentration of nucleic acids present, with preservation of protein. Lysate viscosity is greatly reduced, facilitating subsequent processing. We have used 5mM spermine to remove nucleic acids from E. coli lysate in the purification of a hexahistidine-tagged HIV reverse transcriptase.

  9. [Assessment study on a set of platelet-rich plasma preparation].

    Science.gov (United States)

    Li, Ming; Zhang, Changqing; Yuan, Ting; Chen, Shengbao; Lü, Ruju

    2011-01-01

    To calculate the recovery rate and enrichment factor and to analyse the correlation by measuring the concentrations of platelets, leukocyte, and growth factors in platelet-rich plasma (PRP) so as to evaluate the feasibility and stability of a set of PRP preparation. The peripheral blood (40 mL) was collected from 30 volunteers accorded with the inclusion criteria, and then 4 mL PRP was prepared using the package produced by Shandong Weigao Group Medical Polymer Company Limited. Automatic hematology analyzer was used to count the concentrations of platelets and leukocyte in whole blood and PRP. The enrichment factor and recovery rate of platelets or leukocyte were calculated; the platelet and leukocyte concentrations of male and female volunteers were measured, respectively. The concentrations of platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-beta), and vascular endothelial growth factor (VEGF) were assayed by ELISA. The platelet concentrations of whole blood and PRP were (131.40 +/- 29.44) x 10(9)/L and (819.47 +/- 136.32) x 10(9)/L, respectively, showing significant difference (t = 27.020, P = 0.000). The recovery rate of platelets was 60.85% +/- 8.97%, and the enrichment factor was 6.40 +/- 1.06. The leukocyte concentrations of whole blood and PRP were (5.57 +/- 1.91) x 10(12)/L and (32.20 +/- 10.42) x 10(12)/L, respectively, showing significant difference (t = 13.780, P = 0.000). The recovery rate of leukocyte was 58.30% +/- 19.24%, and the enrichment factor was 6.10 +/- 1.93. The concentrations of platelets and leukocyte in PRP were positively correlated with the platelet concentration (r = 0.652, P = 0.000) and leukocyte concentration (r = 0.460, P = 0.011) in whole blood. The concentrations of platelet and leukocyte in PRP between male and female were not significantly different (P > 0.05). The concentrations of PDGF, TGF-beta, and VEGF in PRP were (698.15 +/- 64.48), (681.36 +/- 65.90), and (1071.55 +/- 106.04) ng/mL, which were

  10. Effect of Titanium-prepared Platelet-rich Fibrin Treatment on the ...

    African Journals Online (AJOL)

    2018-02-07

    Feb 7, 2018 ... Defects of Patients with Chronic Periodontitis: A Randomized. Controlled ... Faculty of Dentistry Department of Periodontology, Kirikkale. University ..... using leukocyte and platelet-rich fibrin: A report of a case. Case. Rep Dent ...

  11. Platelet lysate formulations based on mucoadhesive polymers for the treatment of corneal lesions.

    Science.gov (United States)

    Sandri, Giuseppina; Bonferoni, Maria Cristina; Rossi, Silvia; Ferrari, Franca; Mori, Michela; Del Fante, Claudia; Perotti, Cesare; Scudeller, Luigia; Caramella, Carla

    2011-02-01

    Growth factors contained in platelet α-granules initiate and modulate tissue repair and are proposed for the treatment of soft and hard-tissue surgical conditions and in the management of non-healing wounds. Platelet lysate is a hemoderivative obtained from platelet-rich plasma and is capable of releasing a pool of growth factors. Many medical and surgical techniques have been proposed for the treatment of corneal lesions; management of these conditions remains problematic and healing with standard protocols is unattainable. The aim of this study was to develop formulations suitable for prolonging the contact of platelet lysate with the damaged cornea for the time necessary to exert a therapeutic effect. Two vehicles, one based on polyacrylic acid and one based on chitosan, were autoclaved and loaded with platelet lysate and the resultant formulations were characterized for rheology, mucoadhesion, vehicle compatibility and stability. The proliferation effect was tested on two cell culture types (rabbit corneal epithelial cells and fibroblasts). An in-vitro wound-healing test was performed on fibroblasts. In both cases the formulations were compared with platelet lysate diluted with saline at the same concentration. Both formulations maintained the rheological and mucoadhesive properties of the vehicles and the proliferative activity of platelet lysate. The chitosan formulation was able to significantly enhance epithelial cell growth even after storage of up to 2 weeks (in-use conditions), while the polyacrylic acid formulation was less efficient, probably due to the characteristics of the cell model used. The in-vitro wound-healing test performed on fibroblasts confirmed the differences between the two vehicles. The effect induced by the platelet lysate and chitosan formulation was faster than that of the polyacrylic acid formulation and complete in-vitro wound repair was achieved within 48 h. © 2010 The Authors. JPP © 2010 Royal Pharmaceutical Society.

  12. Study on the preparation of boron-rich film by magnetron sputtering in oxygen atmosphere

    Energy Technology Data Exchange (ETDEWEB)

    Pan, Zhangmin; Yang, Yiming; Huang, Jian; Ren, Bing; Yu, Hongze; Xu, Run; Ji, Huanhuan; Wang, Lin; Wang, Linjun, E-mail: ljwang@shu.edu.cn

    2016-12-01

    Highlights: • Boron ({sup 10}B) oxide films were successfully grown using RF magnetron sputtering. • Effects of oxygen partial pressure on the property of the films were studied. • Substrates were covered with B-rich film and film surface was covered with B{sub 2}O{sub 3}. • The growth mechanism of films in oxygen atmosphere was analyzed using XPS. - Abstract: In this paper, the growth of boron ({sup 10}B) oxide films on (1 0 0) silicon substrate were achieved by radio frequency (r.f.) magnetron sputtering under the different oxygen partial pressure with a target of boron and boron oxide. The structure and properties of deposited films were characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy spectrometer (FTIR), X-ray photoelectron spectroscopy (XPS), respectively. The results showed that the substrate was covered with boron-rich films tightly and the surface of films was covered with B{sub 2}O{sub 3}. And the growth mechanism of boron-rich film in oxygen atmosphere was also analyzed.

  13. Comparison of different procedures to prepare platelet-rich plasma for studies of platelet aggregation by light transmission aggregometry.

    Science.gov (United States)

    Femia, Eti Alessandra; Pugliano, Mariateresa; Podda, Gianmarco; Cattaneo, Marco

    2012-01-01

    Light transmission aggregometry (LTA), the gold standard for the study of patients with defects of platelet function, is a poorly standardized technique. The guidelines that have been produced so far are largely based on consensus of experts, due to the absence of studies directly comparing different procedures. Therefore, ad hoc studies are needed to gather scientific evidence on how to choose the most appropriate procedures for LTA measurement. In this study, we aimed at evaluating the most appropriate conditions for preparing samples of platelet-rich plasma (PRP) for studies of platelet aggregation by LTA. Citrate-anticoagulated blood from 32 individuals was centrifuged at 150, 200, 250 or 300×g at room temperature for 10 min. Red blood cells contamination was highest in PRP prepared at 150×g; mean platelet volume (MPV) was lowest in PRP prepared at 300×g. The extent of platelet aggregation measured by LTA was lower and more variable in PRP prepared at 300×g. Therefore, centrifugation of blood at 200×g or 250×g for 10 min appears to be the best condition for preparing PRP for LTA studies.

  14. Preparation and Characterization of Lightweight Mullite-Silica Rich Glass Aggregates

    Directory of Open Access Journals (Sweden)

    Nan Li

    2011-09-01

    Full Text Available Phase compositions, microstructures and properties of four lightweight mullite-silica rich glass aggregates with high strength were investigated by X-ray diffractometry (XRD, scanning electron microscopy (SEM, and FactSage 6.2 software. It was found that the lightweight aggregates with higher Al2O3 content had higher mullite content, porosity and larger mullite crystallites, but lower content and viscosity of melt at elevated temperature. Most of Fe2O3 and TiO2 were incorporated in mullite and most of K2O and Na2O were in glass to reduce viscosity of melt at elevated temperature.

  15. Preparation of boron-rich aluminum boride nanoparticles by RF thermal plasma

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Sooseok [Inha University, Department of Chemical Engineering and Regional Innovation Center for Environmental Technology of Thermal Plasma (Korea, Republic of); Matsuo, Jiro; Cheng, Yingying [Tokyo Institute of Technology, Department of Environmental Chemistry and Engineering (Japan); Watanabe, Takayuki, E-mail: watanabe@chemenv.titech.ac.jp [Kyushu University, Department of Chemical Engineering (Japan)

    2013-08-15

    Boron-rich compounds of AlB{sub 12} and AlB{sub 10} nanoparticles were synthesized by a radiofrequency thermal plasma. Aluminum and boron raw powders were evaporated in virtue of high enthalpy of the thermal plasma in upstream region, followed by the formation of aluminum boride nanoparticles in the tail region of plasma flame with rapid quenching. A high production rate of aluminum boride was confirmed by the X-ray diffraction measurement in the case of high input power, high boron content in raw material and helium inner gas. Polyhedral nanoparticles of 20.8 nm in mean size were observed by a transmission electron microscope. In the raw powder mixture of aluminum, titanium, and boron, titanium-boride nanoparticles were synthesized preferentially, because the Gibbs free energy for the boridation of titanium is lower than that of aluminum. Since the nucleation temperature of boron is higher than that of aluminum, the condensation of metal monomers onto boron nuclei results in the formation of boron-rich aluminum boride nanoparticles.

  16. Highly porous activated carbons prepared from carbon rich Mongolian anthracite by direct NaOH activation

    Energy Technology Data Exchange (ETDEWEB)

    Byamba-Ochir, Narandalai [School of Chemical Engineering, Chonnam National University, 77 Yongbong-Ro, Gwangju 61186 (Korea, Republic of); Shim, Wang Geun [Department of Polymer Science and Engineering, Sunchon National University, 255 Jungang-Ro, Suncheon, Jeollanam-Do 57922 (Korea, Republic of); Balathanigaimani, M.S., E-mail: msbala@rgipt.ac.in [Department of Chemical Engineering, Rajiv Gandhi Institute of Petroleum Technology, Ratapur Chowk, Rae Bareli, 229316 Uttar Pradesh (India); Moon, Hee, E-mail: hmoon@jnu.ac.kr [School of Chemical Engineering, Chonnam National University, 77 Yongbong-Ro, Gwangju 61186 (Korea, Republic of)

    2016-08-30

    Highlights: • Highly porous carbon materials from Mongolian anthracite by chemical activation. • Cheaper and eco-friendly activation process has been employed. • Activated carbons with graphitic structure and energetically heterogeneous surface. • Surface hydrophobicity and porosity of the activated carbons can be controlled. - Abstract: Highly porous activated carbons (ACs) were prepared from Mongolian raw anthracite (MRA) using sodium hydroxide as an activation agent by varying the mass ratio (powdered MRA/NaOH) as well as the mixing method of chemical agent and powdered MRA. The specific BET surface area and total pore volume of the prepared MRA-based activated carbons (MACs) are in the range of 816–2063 m{sup 2}/g and of 0.55–1.61 cm{sup 3}/g, respectively. The pore size distribution of MACs show that most of the pores are in the range from large micropores to small mesopores and their distribution can be controlled by the mass ratio and mixing method of the activating agent. As expected from the intrinsic property of the MRA, the highly graphitic surface morphology of prepared carbons was confirmed from Raman spectra and transmission electron microscopy (TEM) studies. Furthermore the FTIR and XPS results reveal that the preparation of MACs with hydrophobic in nature is highly possible by controlling the mixing conditions of activating agent and powdered MRA. Based on all the results, it is suggested that the prepared MACs could be used for many specific applications, requiring high surface area, optimal pore size distribution, proper surface hydrophobicity as well as strong physical strength.

  17. Quality Assessment of Platelet-Rich Fibrin-Like Matrix Prepared from Whole Blood Samples after Extended Storage

    Directory of Open Access Journals (Sweden)

    Hideo Kawabata

    2017-09-01

    Full Text Available The platelet-rich fibrin–like matrix (PRFM is usually prepared onsite and immediately used for regenerative therapy. Nonetheless, to meet the clinical necessity of preserving the PRFM without quality deterioration, we developed a method for preparation of PRFMs from short-term-stored whole blood (WB samples. In this study, to evaluate the practical expiration date of storage, we extended the storage time of WB samples from 2 to 7 days and assessed the quality of the resulting PRFMs. WB samples collected with acid-citrate-dextrose were stored with gentle agitation at ambient temperature. To prepare PRFMs, the stored WB samples were mixed with CaCl2 in glass tubes and centrifuged. Fibrin fiber networks, CD41 and CD62P expression, and Platelet Derived Growth Factor-BB (PDGF-BB levels were examined by scanning electron microscopy (SEM, flow cytometry, and an Enzyme-Linked ImmunoSorbent Assay (ELISA, respectively. Long-term storage had no significant effect on either blood cell counts or platelet functions tested. The resulting PRFMs were visually identical to freshly prepared ones. PDGF-BB levels did not markedly decrease in a time-dependent manner. However, fibrin fibers gradually became thinner after storage. Although the coagulation activity may diminish, we propose that PRFMs can be prepared—without evident loss of quality—from WB samples stored for up to 7 days by our previously developed method.

  18. New Sample Preparation Method for Quantification of Phenolic Compounds of Tea (Camellia sinensis L. Kuntze: A Polyphenol Rich Plant

    Directory of Open Access Journals (Sweden)

    P. A. Nimal Punyasiri

    2015-01-01

    Full Text Available Chemical analysis of the Sri Lankan tea (Camellia sinensis, L. germplasm would immensely contribute to the success of the tea breeding programme. However, the polyphenols, particularly catechins (flavan-3-ols, are readily prone to oxidation in the conventional method of sample preparation. Therefore, optimization of the present sample preparation methodology for the profiling of metabolites is much important. Two sample preparation methodologies were compared, fresh leaves (as in the conventional procedures and freeze-dried leaves (a new procedure, for quantification of major metabolites by employing two cultivars, one is known to be high quality black tea and the other low quality black tea. The amounts of major metabolites such as catechins, caffeine, gallic acid, and theobromine, recorded in the new sampling procedure via freeze-dried leaves, were significantly higher than those recorded in the conventional sample preparation procedure. Additionally new method required less amount of leaf sample for analysis of major metabolites and facilitates storage of samples until analysis. The freeze-dried method would be useful for high throughput analysis of large number of samples in shorter period without chemical deterioration starting from the point of harvest until usage. Hence, this method is more suitable for metabolite profiling of tea as well as other phenol rich plants.

  19. Pathogen-free, plasma-poor platelet lysate and expansion of human mesenchymal stem cells.

    Science.gov (United States)

    Iudicone, Paola; Fioravanti, Daniela; Bonanno, Giuseppina; Miceli, Michelina; Lavorino, Claudio; Totta, Pierangela; Frati, Luigi; Nuti, Marianna; Pierelli, Luca

    2014-01-27

    Supplements to support clinical-grade cultures of mesenchymal stem cells (MSC) are required to promote growth and expansion of these cells. Platelet lysate (PL) is a human blood component which may replace animal serum in MSC cultures being rich in various growth factors. Here, we describe a plasma poor pathogen-free platelet lysate obtained by pooling 12 platelet (PLT) units, to produce a standardized and safe supplement for clinical-grade expansion of MSC. PL lots were obtained by combining 2 6-unit PLT pools in additive solution (AS) following a transfusional-based procedure including pathogen inactivation (PI) by Intercept technology and 3 cycles of freezing/thawing, followed by membrane removal. Three PI-PL and 3 control PL lots were produced to compare their ability to sustain bone marrow derived MSC selection and expansion. Moreover, two further PL, subjected to PI or not, were also produced starting from the same initial PLT pools to evaluate the impact of PI on growth factor concentration and capacity to sustain cell growth. Additional PI-PL lots were used for comparison with fetal bovine serum (FBS) on MSC expansion. Immunoregulatory properties of PI-PL-generated MSC were documented in vitro by mixed lymphocyte culture (MLC) and peripheral blood mononuclear cells (PBMC) mitogen induced proliferation. PI-PL and PL control lots had similar concentrations of 4 well-described growth factors endowed with MSC stimulating ability. Initial growth and MSC expansion by PI-PL and PL controls were comparable either using different MSC populations or in head to head experiments. Moreover, PI-PL and PL control sustained similar MSC growth of frozen/thawed MSC. Multilineage differentiation of PI-derived and PI-PL-derived MSC were maintained in any MSC cultures as well as their immunoregulatory properties. Finally, no direct impact of PI on growth factor concentration and MSC growth support was observed, whereas the capacity of FBS to sustain MSC expansion in basic

  20. High coercivity microcrystalline Nd-rich Nd–Fe–Co–Al–B bulk magnets prepared by direct copper mold casting

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, L.Z.; Hong, Y. [School of Materials Science and Engineering, South China University of Technology, Guangzhou 510640 (China); Fang, X.G. [Institute for Advanced Materials and Laboratory of Quantum Engineering and Quantum Materials, South China Normal University, Guangzhou 510640 (China); Qiu, Z.G.; Zhong, X.C. [School of Materials Science and Engineering, South China University of Technology, Guangzhou 510640 (China); Gao, X.S. [Institute for Advanced Materials and Laboratory of Quantum Engineering and Quantum Materials, South China Normal University, Guangzhou 510640 (China); Liu, Z.W., E-mail: zwliu@scut.edu.cn [School of Materials Science and Engineering, South China University of Technology, Guangzhou 510640 (China)

    2016-06-15

    High coercivity Nd{sub 25}Fe{sub 40}Co{sub 20}Al{sub 15−x}B{sub x} (x=7–15) hard magnets were prepared by a simple process of injection casting. Different from many previous investigations on nanocomposite compositions, the magnets in this work contain hard magnetic Nd{sub 2}(FeCoAl){sub 14}B, Nd-rich, and Nd{sub 1+ε}(FeCo){sub 4}B{sub 4} phases. The magnetic properties, phase evolution, and microstructure of the as-cast and annealed magnets were investigated. As the boron content increased from 7 to 11 at%, the intrinsic coercivity H{sub cj} of the as-cast magnet increased from 816 to 1140 kA/m. The magnets annealed at 750 °C have shown more regular and smaller grains than the as-cast alloys, especially for the x=11 alloy. The high intrinsic coercivities for the annealed alloys with x=8~11 result from the presence of small-sized grains in the microstructure. The highest H{sub cj} of 1427 kA/m was obtained for the heat treated alloy with x=10. This work provides an alternative approach for preparing fully dense Nd-rich bulk hard magnets with relatively good properties. - Highlights: • 2 mm hard magnetic Nd{sub 25}Fe{sub 40}Co{sub 20}Al{sub 15−x}B{sub x} rods were prepared by direct casting. • High coercivity of 1.78 T was achieved in x=11 sample after heat treatment. • Small grains are responsible for the significant increase in H{sub C} after annealing. • Nd{sub 2}Fe{sub 14}B grains with two different sizes lead to two-step demagnetization process.

  1. Complexation-Induced Phase Separation: Preparation of Metal-Rich Polymeric Membranes

    KAUST Repository

    Villalobos Vazquez de la Parra, Luis Francisco

    2017-08-01

    The majority of state-of-the-art polymeric membranes for industrial or medical applications are fabricated by phase inversion. Complexation induced phase separation (CIPS)—a surprising variation of this well-known process—allows direct fabrication of hybrid membranes in existing facilities. In the CIPS process, a first step forms the thin metal-rich selective layer of the membrane, and a succeeding step the porous support. Precipitation of the selective layer takes place in the same solvent used to dissolve the polymer and is induced by a small concentration of metal ions. These ions form metal-coordination-based crosslinks leading to the formation of a solid skin floating on top of the liquid polymer film. A subsequent precipitation in a nonsolvent bath leads to the formation of the porous support structure. Forming the dense layer and porous support by different mechanisms while maintaining the simplicity of a phase inversion process, results in unprecedented control over the final structure of the membrane. The thickness and morphology of the dense layer as well as the porosity of the support can be controlled over a wide range by manipulating simple process parameters. CIPS facilitates control over (i) the thickness of the dense layer throughout several orders of magnitude—from less than 15 nm to more than 6 μm, (ii) the type and amount of metal ions loaded in the dense layer, (iii) the morphology of the membrane surface, and (iv) the porosity and structure of the support. The nature of the CIPS process facilitates a precise loading of a high concentration of metal ions that are located in only the top layer of the membrane. Moreover, these metal ions can be converted—during the membrane fabrication process—to nanoparticles or crystals. This simple method opens up fascinating possibilities for the fabrication of metal-rich polymeric membranes with a new set of properties. This dissertation describes the process in depth and explores promising

  2. Platelet-Rich Plasma Preparation Types Show Impact on Chondrogenic Differentiation, Migration, and Proliferation of Human Subchondral Mesenchymal Progenitor Cells.

    Science.gov (United States)

    Kreuz, Peter Cornelius; Krüger, Jan Philipp; Metzlaff, Sebastian; Freymann, Undine; Endres, Michaela; Pruss, Axel; Petersen, Wolf; Kaps, Christian

    2015-10-01

    To evaluate the chondrogenic potential of platelet concentrates on human subchondral mesenchymal progenitor cells (MPCs) as assessed by histomorphometric analysis of proteoglycans and type II collagen. Furthermore, the migratory and proliferative effect of platelet concentrates were assessed. Platelet-rich plasma (PRP) was prepared using preparation kits (Autologous Conditioned Plasma [ACP] Kit [Arthrex, Naples, FL]; Regen ACR-C Kit [Regen Lab, Le Mont-Sur-Lausanne, Switzerland]; and Dr.PRP Kit [Rmedica, Seoul, Republic of Korea]) by apheresis (PRP-A) and by centrifugation (PRP-C). In contrast to clinical application, freeze-and-thaw cycles were subsequently performed to activate platelets and to prevent medium coagulation by residual fibrinogen in vitro. MPCs were harvested from the cortico-spongious bone of femoral heads. Chondrogenic differentiation of MPCs was induced in high-density pellet cultures and evaluated by histochemical staining of typical cartilage matrix components. Migration of MPCs was assessed using a chemotaxis assay, and proliferation activity was measured by DNA content. MPCs cultured in the presence of 5% ACP, Regen, or Dr.PRP formed fibrous tissue, whereas MPCs stimulated with 5% PRP-A or PRP-C developed compact and dense cartilaginous tissue rich in type II collagen and proteoglycans. All platelet concentrates significantly (ACP, P = .00041; Regen, P = .00029; Dr.PRP, P = .00051; PRP-A, P platelet concentrates but one (Dr.PRP, P = .63) showed a proliferative effect on MPCs, as shown by significant increases (ACP, P = .027; Regen, P = .0029; PRP-A, P = .00021; and PRP-C, P = .00069) in DNA content. Platelet concentrates obtained by different preparation methods exhibit different potentials to stimulate chondrogenic differentiation, migration, and proliferation of MPCs. Platelet concentrates obtained by commercially available preparation kits failed to induce chondrogenic differentiation of MPCs, whereas highly standardized PRP

  3. Platelet lysate-based pro-angiogenic nanocoatings.

    Science.gov (United States)

    Oliveira, Sara M; Pirraco, Rogério P; Marques, Alexandra P; Santo, Vítor E; Gomes, Manuela E; Reis, Rui L; Mano, João F

    2016-03-01

    Human platelet lysate (PL) is a cost-effective and human source of autologous multiple and potent pro-angiogenic factors, such as vascular endothelial growth factor A (VEGF A), fibroblast growth factor b (FGF b) and angiopoietin-1. Nanocoatings previously characterized were prepared by layer-by-layer assembling incorporating PL with marine-origin polysaccharides and were shown to activate human umbilical vein endothelial cells (HUVECs). Within 20 h of incubation, the more sulfated coatings induced the HUVECS to the form tube-like structures accompanied by an increased expression of angiogenic-associated genes, such as angiopoietin-1 and VEGF A. This may be a cost-effective approach to modify 2D/3D constructs to instruct angiogenic cells towards the formation of neo-vascularization, driven by multiple and synergistic stimulations from the PL combined with sulfated polysaccharides. The presence, or fast induction, of a stable and mature vasculature inside 3D constructs is crucial for new tissue formation and its viability. This has been one of the major tissue engineering challenges, limiting the dimensions of efficient tissue constructs. Many approaches based on cells, growth factors, 3D bioprinting and channel incorporation have been proposed. Herein, we explored a versatile technique, layer-by-layer assembling in combination with platelet lysate (PL), that is a cost-effective source of many potent pro-angiogenic proteins and growth factors. Results suggest that the combination of PL with sulfated polyelectrolytes might be used to introduce interfaces onto 2D/3D constructs with potential to induce the formation of cell-based tubular structures. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  4. Cat-CVD-prepared oxygen-rich μc-Si:H for wide-bandgap material

    International Nuclear Information System (INIS)

    Matsumoto, Yasuhiro; Ortega, Mauricio; Peza, Juan-Manuel; Reyes, Mario-Alfredo; Escobosa, Arturo

    2005-01-01

    Microcrystalline phase-involved oxygen-rich a-Si:H (hydrogenated amorphous silicon) films have been obtained using catalytic chemical vapor deposition (Cat-CVD) process. Pure SiH 4 (silane), H 2 (hydrogen), and O 2 (oxygen) gases were introduced in the chamber by maintaining a pressure of 0.1 Torr. A tungsten catalyzer was fixed at temperatures of 1750 and 1950 deg. C for film deposition on glass and crystalline silicon substrates at 200 deg. C. As revealed from X-ray diffraction spectra, the microcrystalline phase appears for oxygen-rich a-Si:H samples deposited at a catalyzer temperature of 1950 deg. C. However, this microcrystalline phase tends to disappear for further oxygen incorporation. The oxygen content in the deposited films was corroborated by FTIR analysis revealing Si-O-Si bonds and typical Si-H bonding structures. The optical bandgap of the sample increases from 2.0 to 2.7 eV with oxygen gas flow and oxygen incorporation to the deposited films. In the present thin film deposition conditions, no strong tungsten filament degradation was observed after a number of sample preparations

  5. In vivo evaluation of titanium-prepared platelet-rich fibrin (T-PRF): a new platelet concentrate.

    Science.gov (United States)

    Tunalı, Mustafa; Özdemir, Hakan; Küçükodacı, Zafer; Akman, Serhan; Fıratlı, Erhan

    2013-07-01

    We have developed a new, titanium-prepared, platelet-rich fibrin (T-PRF) together with the protocol for forming it, which is based on the hypothesis that titanium tubes may be more effective at activating platelets than the glass tubes used by Chouckroun in his platelet-rich fibrin (PRF) method. The aim of this study was to find a suitable animal model in which to evaluate the method and to investigate the efficacy of T-PRF for wound healing. Blood samples from 6 rabbits were used to confirm the protocol for formation of T-PRF. We evaluated T-PRF or T-PRF-like clots morphologically using scanning electron microscopy (EM). Blood samples from 5 rabbits were used to develop an experiment in which to evaluate the effects of T-PRF on wound healing. The mucoperiosteal flaps were filled with autologous T-PRF membranes from the vestibule in the anterior mandibular regions. Samples collected from the surgical sites were stained with haematoxylin and eosin. We found a mature fibrin network in T-PRF clots that had been centrifuged for 15 min at 3500 rpm and, 15 days after placement of the membrane, we found newly-forming connective tissue and islets of bony tissue in the T-PRF membrane. These results show that T-PRF could induce the formation of new bone with new connective tissue in a rabbit model of wound healing within 30 days of treatment. Published by Elsevier Ltd.

  6. Human platelet lysate: Replacing fetal bovine serum as a gold standard for human cell propagation?

    Science.gov (United States)

    Burnouf, Thierry; Strunk, Dirk; Koh, Mickey B C; Schallmoser, Katharina

    2016-01-01

    The essential physiological role of platelets in wound healing and tissue repair builds the rationale for the use of human platelet derivatives in regenerative medicine. Abundant growth factors and cytokines stored in platelet granules can be naturally released by thrombin activation and clotting or artificially by freeze/thaw-mediated platelet lysis, sonication or chemical treatment. Human platelet lysate prepared by the various release strategies has been established as a suitable alternative to fetal bovine serum as culture medium supplement, enabling efficient propagation of human cells under animal serum-free conditions for a multiplicity of applications in advanced somatic cell therapy and tissue engineering. The rapidly increasing number of studies using platelet derived products for inducing human cell proliferation and differentiation has also uncovered a considerable variability of human platelet lysate preparations which limits comparability of results. The main variations discussed herein encompass aspects of donor selection, preparation of the starting material, the possibility for pooling in plasma or additive solution, the implementation of pathogen inactivation and consideration of ABO blood groups, all of which can influence applicability. This review outlines the current knowledge about human platelet lysate as a powerful additive for human cell propagation and highlights its role as a prevailing supplement for human cell culture capable to replace animal serum in a growing spectrum of applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Porcine platelet lysate as a supplement for animal cell culture

    Science.gov (United States)

    Aldén, Anna; Gonzalez, Lorena; Persson, Anna; Christensson, Kerstin; Holmqvist, Olov

    2007-01-01

    A novel supplementation of cell growth media based on a porcine platelet lysate was developed for culture of animal-derived cells. The platelet lysate was produced from porcine blood and contained lysate of platelets and plasma components. It showed satisfactory microbiological integrity and it carried only low amount of endotoxins (platelet lysate supported well proliferation of Vero (African green monkey transformed kidney epithelial cells), Chinese hamster ovary (CHO) and hybridoma cells comparable to fetal bovine serum (FBS). Platelet lysate shows promise as a viable choice over FBS as it can be produced in large quantities, high lot-to-lot consistency and with an attractive price structure. Furthermore it is a strong alternative to FBS for ethical reasons. It is expected that it can be used as a general supplementation for most animal cells for research studies on the proliferation of cells and their expression of products. PMID:19002989

  8. Exosomes: novel effectors of human platelet lysate activity

    Directory of Open Access Journals (Sweden)

    E Torreggiani

    2014-09-01

    Full Text Available Despite the popularity of platelet-rich plasma (PRP and platelet lysate (PL in orthopaedic practice, the mechanism of action and the effectiveness of these therapeutic tools are still controversial. So far, the activity of PRP and PL has been associated with different growth factors (GF released during platelet degranulation. This study, for the first time, identifies exosomes, nanosized vesicles released in the extracellular compartment by a number of elements, including platelets, as one of the effectors of PL activity. Exosomes were isolated from human PL by differential ultracentrifugation, and analysed by electron microscopy and Western blotting. Bone marrow stromal cells (MSC treated with three different exosome concentrations (0.6 μg, 5 μg and 50 μg showed a significant, dose-dependent increase in cell proliferation and migration compared to the control. In addition, osteogenic differentiation assays demonstrated that exosome concentration differently affected the ability of MSC to deposit mineralised matrix. Finally, the analysis of exosome protein content revealed a higher amount of basic fibroblast growth factor (bFGF, vascular endothelial growth factor (VEGF, platelet-derived growth factor (PDGF-BB and transforming growth factor beta 1 (TGF-β1 as compared to PL. In regards to RNA content, an enrichment of small RNAs in exosomes as compared to donor platelets has been found. These results suggest that exosomes consistently contribute to PL activity and could represent an advantageous nanodelivery system for cell-free regeneration therapies.

  9. Investigation of the sample preparation and curing treatment effects on mechanical properties and bioactivity of silica rich metakaolin geopolymer

    International Nuclear Information System (INIS)

    Catauro, M.; Bollino, F.; Papale, F.; Lamanna, G.

    2014-01-01

    In many biomedical applications both the biological and mechanical behaviours of implants are of relevant interest; in the orthopaedic field, for example, favourable bioactivity and biocompatibility capabilities are necessary, but at the same time the mechanical characteristics of the implants must be such as to allow one to support the body weight. In the present work, the authors have examined the application of geopolymers with composition H 24 AlK 7 Si 31 O 79 and ratio Si/Al = 31 to be used in biomedical field, considering two different preparation methods: one of the activators (KOH) has been added as pellets in the potassium silicate solution, in the other as a water solution with 8 M concentration. Moreover, a different water content was used and only some of the synthesized samples were heat treated. The chemical and microstructural characterizations of those materials have been carried out by Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM). Subsequently, the effects of the adopted preparation on the mechanical and biological properties have been studied: compressive strength tests have demonstrated that more fragile specimens were obtained when KOH was added as a solution. The bioactivity was successfully evaluated with the soaking of the samples in a simulated body fluid (SBF) for 3 weeks. The formation of a layer of hydroxyapatite on the surface of the materials has been shown both by SEM micrographs and EDS analyses. - Highlights: • Rich metakaolin geopolymer activated with KOH/K 2 SiO 3 and thermal treatment • Mechanical and bioactivity test to evaluate consolidation and bone bonding ability • Order of addition of reactants and thermal treatment influence mechanical properties

  10. Investigation of the sample preparation and curing treatment effects on mechanical properties and bioactivity of silica rich metakaolin geopolymer

    Energy Technology Data Exchange (ETDEWEB)

    Catauro, M., E-mail: michelina.catauro@unina2.it; Bollino, F.; Papale, F.; Lamanna, G.

    2014-03-01

    In many biomedical applications both the biological and mechanical behaviours of implants are of relevant interest; in the orthopaedic field, for example, favourable bioactivity and biocompatibility capabilities are necessary, but at the same time the mechanical characteristics of the implants must be such as to allow one to support the body weight. In the present work, the authors have examined the application of geopolymers with composition H{sub 24}AlK{sub 7}Si{sub 31}O{sub 79} and ratio Si/Al = 31 to be used in biomedical field, considering two different preparation methods: one of the activators (KOH) has been added as pellets in the potassium silicate solution, in the other as a water solution with 8 M concentration. Moreover, a different water content was used and only some of the synthesized samples were heat treated. The chemical and microstructural characterizations of those materials have been carried out by Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM). Subsequently, the effects of the adopted preparation on the mechanical and biological properties have been studied: compressive strength tests have demonstrated that more fragile specimens were obtained when KOH was added as a solution. The bioactivity was successfully evaluated with the soaking of the samples in a simulated body fluid (SBF) for 3 weeks. The formation of a layer of hydroxyapatite on the surface of the materials has been shown both by SEM micrographs and EDS analyses. - Highlights: • Rich metakaolin geopolymer activated with KOH/K{sub 2}SiO{sub 3} and thermal treatment • Mechanical and bioactivity test to evaluate consolidation and bone bonding ability • Order of addition of reactants and thermal treatment influence mechanical properties.

  11. Purification-Free, Target-Selective Immobilization of a Protein from Cell Lysates.

    Science.gov (United States)

    Cha, Jaehyun; Kwon, Inchan

    2018-02-27

    Protein immobilization has been widely used for laboratory experiments and industrial processes. Preparation of a recombinant protein for immobilization usually requires laborious and expensive purification steps. Here, a novel purification-free, target-selective immobilization technique of a protein from cell lysates is reported. Purification steps are skipped by immobilizing a target protein containing a clickable non-natural amino acid (p-azidophenylalanine) in cell lysates onto alkyne-functionalized solid supports via bioorthogonal azide-alkyne cycloaddition. In order to achieve a target protein-selective immobilization, p-azidophenylalanine was introduced into an exogenous target protein, but not into endogenous non-target proteins using host cells with amber codon-free genomic DNAs. Immobilization of superfolder fluorescent protein (sfGFP) from cell lysates is as efficient as that of the purified sfGFP. Using two fluorescent proteins (sfGFP and mCherry), the authors also demonstrated that the target proteins are immobilized with a minimal immobilization of non-target proteins (target-selective immobilization). © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Platelet lysate obtained via plateletpheresis performed in standing and awake equine donors.

    Science.gov (United States)

    Sumner, Scarlett M; Naskou, Maria C; Thoresen, Merrilee; Copland, Ian; Peroni, John F

    2017-07-01

    Platelet preparations containing growth factors, attachment factors, and enzymes are appealing to enhance healing of injured tissues and as an alternative to xenogenic serum in cell culture media. Plateletpheresis is commonly used to collect platelets in human medicine but has not been validated in horses. Plateletpheresis to collect platelet concentrate was performed on six female, mixed breed, chemically restrained horses using commercially available apheresis equipment. Before and immediately after plateletpheresis, we performed physical examinations and collected blood for chemistry and coagulation panels and then again at 8, 16, 24, and 48 hours after the procedure. To produce platelet lysate, the platelet concentrate underwent two freeze-thaw cycles followed by centrifugation and filtration processing. The platelet lysate was then analyzed for cellular debris, fibrinogen, and growth factors. The collected platelet concentration contained a mean platelet yield of 390 × 10 3 /μL. Donor platelet count decreased from a mean of 193 × 10 3 /μL to 138 × 10 3 /μL after plateletpheresis, but no individual was at risk for hemorrhage. Pooled platelet lysate had minimal cellular residue and contained growth factor concentrations at 6.1 ng/mL for transforming growth factor-β1, at 3.5 ng/mL for platelet-derived growth factor-BB, and at 13.8 ng/mL for vascular endothelial growth factor-A. Plateletpheresis using commercially available apheresis equipment is a feasible option for collecting platelet concentrate from equine donors. The lysate generated from the apheresis product contains growth factors and has potential to be used as a fetal bovine serum substitute for cell culture. © 2017 AABB.

  13. Stressing effects on the charge trapping of silicon oxynitride prepared by thermal oxidation of LPCVD Si-rich silicon nitride

    International Nuclear Information System (INIS)

    Choi, H.Y.; Wong, H.; Filip, V.; Sen, B.; Kok, C.W.; Chan, M.; Poon, M.C.

    2006-01-01

    It was recently found that the silicon oxynitride prepared by oxidation of silicon-rich silicon nitride (SRN) has several important features. The high nitrogen and extremely low hydrogen content of this material allows it to have a high dielectric constant and a low trap density. The present work investigates in further detail the electrical reliability of this kind of gate dielectric films by studying the charge trapping and interface state generation induced by constant current stressing. Capacitance-voltage (C-V) measurements indicate that for oxidation temperatures of 850 and 950 deg. C, the interface trap generation is minimal because of the high nitrogen content at the interface. At a higher oxidation temperature of 1050 deg. C, a large flatband shift is found for constant current stressing. This observation can be explained by the significant reduction of the nitrogen content and the phase separation effect at this temperature as found by X-ray photoelectron spectroscopy study. In addition to the high nitrogen content, the Si atoms at the interface exist in the form of random bonding to oxygen and nitrogen atoms for samples oxidized at 850 and 950 deg. C. This structure reduces the interface bonding constraint and results in the low interface trap density. For heavily oxidized samples the trace amount of interface nitrogen atoms exist in the form of a highly constraint SiN 4 phase and the interface oxynitride layer is a random mixture of SiO 4 and SiN 4 phases, which consequently reduces the reliability against high energy electron stressing

  14. Theoretical prediction and validation of cell recovery rates in preparing platelet-rich plasma through a centrifugation.

    Science.gov (United States)

    Piao, Linfeng; Park, Hyungmin; Jo, Chris Hyunchul

    2017-01-01

    In the present study, we propose a theoretical framework to predict the recovery rates of platelets and white blood cells in the process of centrifugal separation of whole blood contained in a tube for the preparation of platelet-rich plasma. Compared to previous efforts to optimize or standardize the protocols of centrifugation, we try to further the physical background (i.e., based on the multiphase flow phenomena) of analysis to develop a universal approach that can be applied to widely different conditions. That is, one-dimensional quasi-linear partial differential equation to describe the centrifugal sedimentation of dispersed phase (red and white blood cells) in continuous phase (plasma) is derived based on the kinematic-wave theory. With the information of whole blood volume and tube geometry considered, it is possible to determine the positions of interfaces between supernatant/suspension and suspension/sediment, i.e., the particle concentration gradient in a tube, for a wide range of centrifugation parameters (time and acceleration). While establishing a theory to predict the recovery rates of the platelet and white blood cell from the pre-determined interface positions, we also propose a new correlation model between the recovery rates of plasma and platelets, which is found to be a function of the whole blood volume, centrifugal time and acceleration, and tube geometry. The present predictions for optimal condition show good agreements with available human clinical data, obtained from different conditions, indicating the universal applicability of our method. Furthermore, the dependence of recovery rates on centrifugal conditions reveals that there exist a different critical acceleration and time for the maximum recovery rate of platelets and white blood cells, respectively. The other parameters such as hematocrit, whole blood volume and tube geometry are also found to strongly affect the maximum recovery rates of blood cells, and finally, as a strategy

  15. A Comparison of Platelet Count and Enrichment Percentages in the Platelet Rich Plasma (PRP) Obtained Following Preparation by Three Different Methods.

    Science.gov (United States)

    Sabarish, Ram; Lavu, Vamsi; Rao, Suresh Ranga

    2015-02-01

    Platelet rich plasma (PRP) represents an easily accessible and rich source of autologous growth factors. Different manual methods for the preparation of PRP have been suggested. Lacuna in knowledge exists about the efficacy of PRP preparation by these different manual methods. This study was performed to determine the effects of centrifugation rate revolutions per minute (RPM) and time on the platelet count and enrichment percentages in the concentrates obtained following the three different manual methods of PRP preparation. In vitro experimental study. This was an experimental study in which platelet concentration was assessed in the PRP prepared by three different protocols as suggested by Marx R (method 1), Okuda K (method 2) and Landesberg R (method 3). A total of 60 peripheral blood samples, (n=20 per method) were obtained from healthy volunteers. Baseline platelet count was assessed for all the subjects following which PRP was prepared. The platelet count in the PRP was determined using coulter counter (Sysmex XT 2000i). The mean of the platelet count obtained and their enrichment percentage were calculated and intergroup comparison was done (Tukey's HSD test). The number of platelets and enrichment percentage in PRP prepared by method 1 was higher compared to method 2 and method 3; this difference in platelet concentrates was found to be statistically significant (p < 0.05). The centrifugation rate and time appear to be important parameters, which influence the platelet yield. Method 1 which had lower centrifugation rate and time yielded a greater platelet count and enrichment percentage.

  16. Ligamentization of tendon grafts treated with an endogenous preparation rich in growth factors: gross morphology and histology.

    Science.gov (United States)

    Sánchez, Mikel; Anitua, Eduardo; Azofra, Juan; Prado, Roberto; Muruzabal, Francisco; Andia, Isabel

    2010-04-01

    To investigate whether the application of a particular platelet-rich plasma preparation rich in growth factors (PRGF) during anterior cruciate ligament (ACL) surgery gives a potential advantage for better tendon graft ligamentization. This study included 37 volunteers who underwent either conventional (control group, n = 15) or PRGF-assisted (n = 22) ACL reconstruction with an autogenous hamstring and required second-look arthroscopy to remove hardware or loose bodies, treat meniscal tears or plica syndrome, or resect cyclops lesions at 6 to 24 months after ACL surgery. The gross morphologies of the grafts were evaluated on second-look arthroscopy by use of the full arthroscopic score (0 to 4 points) to evaluate graft thickness and apparent tension (0 to 2 points) plus synovial coverage (0 to 2 points). At the same time, biopsy specimens were harvested uniformly from the grafted tendons. In these specimens the histologic transformation of the tendon graft to ACL-like tissue was evaluated by use of the Ligament Tissue Maturity Index, and a score to assess the progression of new connective tissue enveloping the graft was created by use of 3 criteria previously used to characterize changes during ligament healing: cellularity, vascularity, and collagen properties. The overall arthroscopic evaluation of PRGF-treated grafts showed an excellent rating in 57.1% of the knees (score of 4) and a fair rating in 42.9% (score of 2 or 3). In contrast, evaluation of untreated grafts showed an excellent rating in 33.3% of the knees, a fair rating in 46.7%, and a poor rating in 20% (score of 0 or 1). Overall, arthroscopic evaluations were not statistically different between PRGF and control groups (P = .051). PRGF treatment influenced the histologic characteristics of the tendon graft, resulting in tissue that was more mature than in controls (P = .024). Histologically evident newly formed connective tissue enveloping the graft was present in 77.3% of PRGF-treated grafts and 40% of

  17. Particulate systems based on pectin/chitosan association for the delivery of manuka honey components and platelet lysate in chronic skin ulcers.

    Science.gov (United States)

    Tenci, Marika; Rossi, Silvia; Bonferoni, Maria Cristina; Sandri, Giuseppina; Boselli, Cinzia; Di Lorenzo, Arianna; Daglia, Maria; Icaro Cornaglia, Antonia; Gioglio, Luciana; Perotti, Cesare; Caramella, Carla; Ferrari, Franca

    2016-07-25

    The aim of the present work was the development of a powder formulation for the delivery of manuka honey (MH) bioactive components and platelet lysate (PL) in chronic skin ulcers. In particular pectin (PEC)/chitosan (CS) particles were prepared by ionotropic gelation in the presence of calcium chloride and subsequently characterized for particle size, hydration properties and mechanical resistance. Different experimental conditions (calcium chloride and CS concentrations; rest time in the cationic solution) were considered in order to obtain particles characterized by optimal size, hydration properties and mechanical resistance. Two different fractions of MH were examined: one (Fr1), rich in methylglyoxal and the other (Fr2), rich in polyphenols. Particles were loaded with Fr1, fraction able to enhance in vitro proliferation of human fibroblasts, and with PL. The presence of CS in Fr1-loaded particles produced an improvement in cell proliferation. Moreover, PL loading into particles did not affect the biological activity of the hemoderivative. In vivo efficacy of PL- and Fr1-loaded particles was evaluated on a rat wound model. Both treatments markedly increased wound healing to the same extent. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. The role of titanium-prepared platelet-rich fibrin in palatal mucosal wound healing and histoconduction.

    Science.gov (United States)

    Ustaoğlu, Gülbahar; Ercan, Esra; Tunali, Mustafa

    2016-10-01

    We aimed to determine the clinical effects of titanium-prepared, platelet-rich fibrin (T-PRF) on human palatal mucosal wound healing (PMWH) and to identify its effect on time-dependent changes in palatal soft-tissue thickness (PSTT) in terms of histoconduction, which is a new concept. Free gingival graft (FGG) donor sites were treated with T-PRF and compared with an untreated control group. The results of colour match and H 2 O 2 -bubbling tests for complete wound epithelization (CWE) were recorded on days 3, 7, 14 and 21. Pain level, number of analgesics used and bleeding status were recorded for the first 7 days. PSTT was measured at baseline and after 1 and 6 months. Colour match scores of the test group were significantly higher than those of the control group at 7 and 14 days. CWE was observed at a higher frequency in the test group than in the control group on day 14. Post-operative bleeding prevalence was lower in the test group than in the control group for the first 2 days. A time-dependent decrease in PSTT was observed at 1 and 6 months in the control group compared with baseline (baseline, 4.23 ± 0.62 mm; 1 month, 4.01 ± 0.68 mm; and 6 months, 3.93 ± 0.69 mm). However, no significant difference was found in the test group (baseline, 4.29 ± 0.64 mm; 1 month, 4.61 ± 0.51 mm; and 6 months, 4.51 ± 0.58 mm). The T-PRF membrane exhibited positive effects on PMWH. T-PRF, which is a promising autogenous matrix for histoconduction, may also be preferred as an autogenous alternative to connective tissue grafts in the treatment of gingival recessions and peri-implant mucosal recessions.

  19. Enhanced target-specific signal detection using an Escherichia coli lysate in multiplex microbead immunoassays with E. coli-derived recombinant antigens.

    Science.gov (United States)

    Crestani, Sandra; Leitolis, Amanda; Lima, Lucianna Freitas Oliveira; Krieger, Marco A; Foti, Leonardo

    2016-08-01

    Diverse techniques have been developed to analyze antibody-mediated responses to infections. However, the most common tests, i.e., enzyme-linked immunosorbent assays, require separate reactions for each antigen and consequently necessitate large sample volumes. Luminex technology allows the detection of multiple antibodies in a single experiment, but nonspecific binding can impair the results. Therefore, we examined the use of Escherichia coli lysates to reduce nonspecific binding and improve the results of liquid microarrays based on Luminex technology. Anti-bacteria antibodies were detected in human serum samples, as evidenced by high median fluorescence intensity (MFI) in assays performed with paramagnetic microspheres coupled with E. coli lysates. Moreover, the addition of an E. coli lysate as a blocker reduced the nonspecific binding of antigens produced by E. coli in a concentration-dependent manner. Tris-HCl reduced MFI values in negative samples, but did not affect MFI for positive samples. For microspheres coupled with different antigens, an E. coli lysate blocker significantly improved the fluorescence signals from positive samples. The addition of Tris-HCl and the E. coli lysate induced antigen-specific differences in MFI. This combination of the E. coli lysate blocker and Tris-HCl yielded a statistically significant improvement in MFI in the assays for Chagas disease and hepatitis C virus samples. However, for the Treponema pallidum p47 antigen improvement in MFI was only observed for the preparation with the E. coli blocker at a concentration of 3%. In conclusion, the addition of an E. coli lysate and Tris-HCl to the microarray assay reduced the nonspecific binding of human anti-bacteria antibodies and, therefore, increased the specific MFI. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Evaluation of intra-articular injection of autologous platelet lysate (PL) in horses with osteoarthritis of the distal interphalangeal joint.

    Science.gov (United States)

    Tyrnenopoulou, Panagiota; Diakakis, Nikolaos; Karayannopoulou, Maria; Savvas, Ioannis; Koliakos, Georgios

    2016-06-01

    Regenerative medicine has become one of the most promising therapies of equine osteoarthritis. Platelet lysate (PL) is rich in bioactive proteins and growth factors that play a crucial role in tissue healing. To evaluate the efficacy of intra-articularly injected autologous PL in equine athletes with naturally occurring osteoarthritis. Fifteen warmblood geldings aged 8-19 years with osteoarthritis of the distal interphalangeal joint were included in this study. They were randomly divided into two groups; 10 horses received intra-articular injections of PL and 5 of normal saline (controls). Before treatment, platelet-derived growth factor (PDGF) levels in basal plasma and prepared PL were estimated. Each joint was injected twice within a three-week period. Lameness was evaluated using the American Association of Equine Practitioners grading system, before treatment and 10 days after each intra-articular injection. Horses were examined fortnightly for one year. Radiographic examination was performed six months post-treatment. The generalized estimating equation test was used for statistical analysis. Acceptable levels of PDGF were detected in PLs (mean ± SD: 258.0 ± 52.3 pg/ml). The majority of horses (9/10) responded positively to PL treatment presenting lower lameness grades (p < 0.0005) compared to controls 10 days after the second injection, and returned to normal athletic activity. Radiographs revealed no changes in osteoarthritis lesions six months after treatment. One year post-injections, however, all horses relapsed to their initial degree of lameness. Intra-articularly injected autologous PL is an efficient method for temporarily managing osteoarthritis of the distal interphalangeal joint in athletic horses.

  1. Platelet lysate: a replacement for fetal bovine serum in animal cell culture?

    OpenAIRE

    Johansson, Liselott; Klinth, Jeanna; Holmqvist, Olov; Ohlson, Sten

    2003-01-01

    A new cell culture supplement, platelet lysate, was evaluated with reference to fetal bovine serum (FBS), an established industrial medium for animal cell culture. Chemical and bacteriological profiles were conducted including the presence of platelet-derived growth factor (PDGF). PDGF was detected in the platelet lysate but it was not present in FBS. The platelet lysate medium demonstrated lack of microorganisms, mycoplasma and endotoxins. The platelet lysate was investigated in culture stud...

  2. Preparation and functional evaluation of collagen oligopeptide-rich hydrolysate from fish skin with the serine collagenolytic protease from Pseudoalteromonas sp. SM9913.

    Science.gov (United States)

    Chen, Xiu-Lan; Peng, Ming; Li, Jing; Tang, Bai-Lu; Shao, Xuan; Zhao, Fang; Liu, Chang; Zhang, Xi-Ying; Li, Ping-Yi; Shi, Mei; Zhang, Yu-Zhong; Song, Xiao-Yan

    2017-11-16

    Although several serine collagenolytic proteases from bacteria were reported, none has been used to prepare bioactive collagen peptides. MCP-01 is the most abundant extracellular protease of deep-sea Pseudoalteromonas sp. SM9913 and is a serine collagenolytic protease with high efficiency on fish collagen hydrolysis. Here, we set up a pilot scale process to ferment SM9913 for extracellular protease production. With SM9913 extracellular protease as a tool, a process to prepare collagen oligopeptide-rich hydrolysate from codfish skin was set up, which was further scaled up to pilot (100 L) and plant (2000 L) levels with yields >66%. The hydrolysates from laboratory-, pilot- and plant-scales had quite similar quality, containing ~95% peptides with molecular weights lower than 3000 Da and approximately 60% lower than 1000 Da, in which collagen oilgopeptides account for approximately 95%. Bioactivity analyses showed that the hydrolysate had moisture-retention ability, antioxidant activity, and promoting effect on cell viability of human dermal fibroblasts. Safety evaluation showed that the hydrolysate was nontoxic and nonirritating to skin. Therefore, SM9913 extracellular protease is a good enzyme to prepare bioactive oligopeptides from fish skin. The results also suggest that the collagen oligopeptides-rich hydrolysate may have potentials in biomedical, functional food, pharmaceutical and cosmetic industries.

  3. Association of Alpha Tocopherol and Ag Sulfadiazine Chitosan Oleate Nanocarriers in Bioactive Dressings Supporting Platelet Lysate Application to Skin Wounds.

    Science.gov (United States)

    Bonferoni, Maria Cristina; Sandri, Giuseppina; Rossi, Silvia; Dellera, Eleonora; Invernizzi, Alessandro; Boselli, Cinzia; Cornaglia, Antonia Icaro; Del Fante, Claudia; Perotti, Cesare; Vigani, Barbara; Riva, Federica; Caramella, Carla; Ferrari, Franca

    2018-02-09

    Chitosan oleate was previously proposed to encapsulate in nanocarriers some poorly soluble molecules aimed to wound therapy, such as the anti-infective silver sulfadiazine, and the antioxidant α tocopherol. Because nanocarriers need a suitable formulation to be administered to wounds, in the present paper, these previously developed nanocarriers were loaded into freeze dried dressings based on chitosan glutamate. These were proposed as bioactive dressings aimed to support the application to wounds of platelet lysate, a hemoderivative rich in growth factors. The dressings were characterized for hydration capacity, morphological aspect, and rheological and mechanical behavior. Although chitosan oleate nanocarriers clearly decreased the mechanical properties of dressings, these remained compatible with handling and application to wounds. Preliminary studies in vitro on fibroblast cell cultures demonstrated good compatibility of platelet lysate with nanocarriers and bioactive dressings. An in vivo study on a murine wound model showed an accelerating wound healing effect for the bioactive dressing and its suitability as support of the platelet lysate application to wounds.

  4. Association of Alpha Tocopherol and Ag Sulfadiazine Chitosan Oleate Nanocarriers in Bioactive Dressings Supporting Platelet Lysate Application to Skin Wounds

    Directory of Open Access Journals (Sweden)

    Maria Cristina Bonferoni

    2018-02-01

    Full Text Available Chitosan oleate was previously proposed to encapsulate in nanocarriers some poorly soluble molecules aimed to wound therapy, such as the anti-infective silver sulfadiazine, and the antioxidant α tocopherol. Because nanocarriers need a suitable formulation to be administered to wounds, in the present paper, these previously developed nanocarriers were loaded into freeze dried dressings based on chitosan glutamate. These were proposed as bioactive dressings aimed to support the application to wounds of platelet lysate, a hemoderivative rich in growth factors. The dressings were characterized for hydration capacity, morphological aspect, and rheological and mechanical behavior. Although chitosan oleate nanocarriers clearly decreased the mechanical properties of dressings, these remained compatible with handling and application to wounds. Preliminary studies in vitro on fibroblast cell cultures demonstrated good compatibility of platelet lysate with nanocarriers and bioactive dressings. An in vivo study on a murine wound model showed an accelerating wound healing effect for the bioactive dressing and its suitability as support of the platelet lysate application to wounds.

  5. Platelet lysate gel and endothelial progenitors stimulate microvascular network formation in vitro: tissue engineering implications.

    Science.gov (United States)

    Fortunato, Tiago M; Beltrami, Cristina; Emanueli, Costanza; De Bank, Paul A; Pula, Giordano

    2016-05-04

    Revascularisation is a key step for tissue regeneration and complete organ engineering. We describe the generation of human platelet lysate gel (hPLG), an extracellular matrix preparation from human platelets able to support the proliferation of endothelial colony forming cells (ECFCs) in 2D cultures and the formation of a complete microvascular network in vitro in 3D cultures. Existing extracellular matrix preparations require addition of high concentrations of recombinant growth factors and allow only limited formation of capillary-like structures. Additional advantages of our approach over existing extracellular matrices are the absence of any animal product in the composition hPLG and the possibility of obtaining hPLG from patients to generate homologous scaffolds for re-implantation. This discovery has the potential to accelerate the development of regenerative medicine applications based on implantation of microvascular networks expanded ex vivo or the generation of fully vascularised organs.

  6. Effects of anti-aggregant, anti-inflammatory and anti-coagulant drug consumption on the preparation and therapeutic potential of plasma rich in growth factors (PRGF).

    Science.gov (United States)

    Anitua, Eduardo; Troya, María; Zalduendo, Mar; Orive, Gorka

    2015-02-01

    The prevalence and incidence of trauma-related injuries, coronary heart disease and other chronic diseases increase dramatically with age. This population sector is therefore a regular consumer of different types of drugs that may affect platelet aggregation and the coagulation cascade. We have evaluated whether the consumption of acetylsalicylic acid, acenocoumarol, glucosamine sulfate and chondroitin sulfate, and therefore their presence in blood, could interfere with the preparation and biological outcomes of plasma rich in growth factors (PRGF). Clotting time, clot retraction and platelet activation of PRGF was evaluated. PRGF growth factor content and the release of different biomolecules by tendon fibroblasts were also quantified, as well as cell proliferation and cell migration. The preparation and biological potential of PRGF is not affected by the intake of the evaluated drugs, and solely its angiogenic potential and its capacity to induce HA and fibronectin synthesis, is reduced in patients taking anti-coagulants.

  7. Platelet activation during preparation of platelet concentrates: a comparison of the platelet-rich plasma and the buffy coat methods

    NARCIS (Netherlands)

    Fijnheer, R.; Pietersz, R. N.; de Korte, D.; Gouwerok, C. W.; Dekker, W. J.; Reesink, H. W.; Roos, D.

    1990-01-01

    The activation of platelets during the preparation of platelet concentrates (PCs) by two methods was compared. To eliminate interdonor differences, 2 units of whole blood were pooled and subsequently divided into two batches. From one batch, the platelets were harvested as pelleted platelets from

  8. White blood cell fragments in platelet concentrates prepared by the platelet-rich plasma or buffy-coat methods

    NARCIS (Netherlands)

    Dijkstra-Tiekstra, M. J.; van der Schoot, C. E.; Pietersz, R. N. I.; Reesink, H. W.

    2005-01-01

    BACKGROUND AND OBJECTIVES: White blood cell (WBC) fragments in platelet concentrates (PCs) may induce allo-immunization in the recipient. MATERIALS AND METHODS: As the level of WBC fragments can differ between PCs produced using different methods, we compared PCs prepared by using the buffy-coat

  9. An Evaluation of the Accuracy of the Subtraction Method Used for Determining Platelet Counts in Advanced Platelet-Rich Fibrin and Concentrated Growth Factor Preparations

    Directory of Open Access Journals (Sweden)

    Taisuke Watanabe

    2017-01-01

    Full Text Available Platelet concentrates should be quality-assured of purity and identity prior to clinical use. Unlike for the liquid form of platelet-rich plasma, platelet counts cannot be directly determined in solid fibrin clots and are instead calculated by subtracting the counts in other liquid or semi-clotted fractions from those in whole blood samples. Having long suspected the validity of this method, we herein examined the possible loss of platelets in the preparation process. Blood samples collected from healthy male donors were immediately centrifuged for advanced platelet-rich fibrin (A-PRF and concentrated growth factors (CGF according to recommended centrifugal protocols. Blood cells in liquid and semi-clotted fractions were directly counted. Platelets aggregated on clot surfaces were observed by scanning electron microscopy. A higher centrifugal force increased the numbers of platelets and platelet aggregates in the liquid red blood cell fraction and the semi-clotted red thrombus in the presence and absence of the anticoagulant, respectively. Nevertheless, the calculated platelet counts in A-PRF/CGF preparations were much higher than expected, rendering the currently accepted subtraction method inaccurate for determining platelet counts in fibrin clots. To ensure the quality of solid types of platelet concentrates chairside in a timely manner, a simple and accurate platelet-counting method should be developed immediately.

  10. Differences in levels of platelet-derived microparticles in platelet components prepared using the platelet rich plasma, buffy coat, and apheresis procedures.

    Science.gov (United States)

    Noulsri, Egarit; Udomwinijsilp, Prapaporn; Lerdwana, Surada; Chongkolwatana, Viroje; Permpikul, Parichart

    2017-04-01

    There has been an increased interest in platelet-derived microparticles (PMPs) in transfusion medicine. Little is known about PMP status during the preparation of platelet concentrates for transfusion. The aim of this study is to compare the PMP levels in platelet components prepared using the buffy coat (BC), platelet-rich plasma platelet concentrate (PRP-PC), and apheresis (AP) processes. Platelet components were prepared using the PRP-PC and BC processes. Apheresis platelets were prepared using the Trima Accel and Amicus instruments. The samples were incubated with annexin A5-FITC, CD41-PE, and CD62P-APC. At day 1 after processing, the PMPs and activated platelets were determined using flow cytometry. Both the percentage and number of PMPs were higher in platelet components prepared using the Amicus instrument (2.6±1.8, 32802±19036 particles/μL) than in platelet components prepared using the Trima Accel instrument (0.5±0.4, 7568±5298 particles/μL), BC (1.2±0.6, 12,920±6426 particles/μL), and PRP-PC (0.9±0.6, 10731±5514 particles/μL). Both the percentage and number of activated platelets were higher in platelet components prepared using the Amicus instrument (33.2±13.9, 427553±196965 cells/μL) than in platelet components prepared using the Trima Accel instrument (16.2±6.1, 211209±87706 cells/μL), BC (12.9±3.2, 140624±41003 cells/μL), and PRP-PC (21.1±6.3, 265210±86257 cells/μL). The study suggests high variability of PMPs and activated platelets in platelet components prepared using different processes. This result may be important in validating the instruments involved in platelet blood collection and processing. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Lysate of engineered Escherichia coli supports high-level conversion of glucose to 2,3-butanediol.

    Science.gov (United States)

    Kay, Jennifer E; Jewett, Michael C

    2015-11-01

    Cell-free metabolic engineering (CFME) is emerging as a powerful approach for the production of target molecules and pathway debugging. Unfortunately, high cofactor costs, limited cofactor and energy regeneration, and low volumetric productivities hamper the widespread use and practical implementation of CFME technology. To address these challenges, we have developed a cell-free system that harnesses ensembles of catalytic proteins prepared from crude lysates, or extracts, of cells to fuel highly active heterologous metabolic conversions. As a model pathway, we selected conversion of glucose to 2,3-butanediol (2,3-BD), a medium level commodity chemical with many industrial applications. Specifically, we engineered a single strain of Escherichia coli to express three pathway enzymes necessary to make meso-2,3-BD (m2,3-BD). We then demonstrated that lysates from this strain, with addition of glucose and catalytic amounts of cofactors NAD+ and ATP, can produce m2,3-BD. Endogenous glycolytic enzymes convert glucose to pyruvate, the starting intermediate for m2,3-BD synthesis. Strikingly, with no strain optimization, we observed a maximal synthesis rate of m2,3-BD of 11.3 ± 0.1 g/L/h with a theoretical yield of 71% (0.36 g m2,3-BD/g glucose) in batch reactions. Titers reached 82 ± 8 g/L m2,3-BD in a 30 h fed-batch reaction. Our results highlight the ability for high-level co-factor regeneration in cell-free lysates. Further, they suggest exciting opportunities to use lysate-based systems to rapidly prototype metabolic pathways and carry out molecular transformations when bioconversion yields (g product/L), productivities (g product/L/h), or cellular toxicity limit commercial feasibility of whole-cell fermentation. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  12. Comparison of the platelet-rich plasma and buffy coat protocols for preparation of canine platelet concentrates.

    Science.gov (United States)

    Hoareau, Guillaume L; Jandrey, Karl E; Burges, Julie; Bremer, Daphne; Tablin, Fern

    2014-12-01

    Platelet (PLT) concentrates (PC) can be produced via the buffy coat (BC) or platelet-rich plasma (PRP) protocols. The 2 methods have not been compared with canine blood. The aims of the study were to compare the PLT, WBC, and RBC concentrations, in vitro PLT function, and markers of platelet storage lesion (PSL) in canine PC generated by 2 different protocols, and determine microbial growth throughout storage. PC from 8 healthy donor dogs were produced using 2 standard protocols, PRP and BC. PLT, WBC, and RBC counts, optical aggregometry assays, and PSL markers (pH, pCO2 , HCO3 , lactate and glucose concentrations, and LDH activity) were determined on storage days 0, 1, 3, 5, and 7. Aerobic and anaerobic bacterial cultures were also performed. Mean PLT counts were comparable between protocols and remained stable throughout storage up to day 7, while median WBC and RBC counts on day 0 were significantly higher in the BC-PC group (17,800 WBCs/μL; 195,000 RBCs/μL) than in the PRP-PC group (200 WBCs/μL; 10,000 RBCs/μL) (P = .012). In PRP-PC aggregometry, the median slope and amplitude in response to γ-thrombin and convulxin (+ ADP) were significantly decreased, and virtually absent in BC-PC during storage. PSL markers (lactate, LDH activity) were higher in BC-PC. Aerobic bacterial growth was observed in 2 PRP-PC and 1 BC-PC. This in vitro study suggests that PRP-PC had lesser WBC and RBC contamination and superior PLT function compared with BC-PC. In vivo studies are required to address safety and efficacy of PRP-PC. © 2014 American Society for Veterinary Clinical Pathology.

  13. Platelet lysate as a serum replacement for skin cell culture on biomimetic PCL nanofibers.

    Science.gov (United States)

    Sovkova, Vera; Vocetkova, Karolina; Rampichova, Michala; Mickova, Andrea; Buzgo, Matej; Lukasova, Vera; Dankova, Jana; Filova, Eva; Necas, Alois; Amler, Evzen

    2018-06-01

    Platelets are a popular source of native growth factors for tissue engineering applications. The aim of the study was to verify the use of platelet lysate as a fetal bovine serum (FBS) replacement for skin cell culture. The cytokine content of the platelet lysate was characterized using the Bio-Plex system. The cells (fibroblasts, melanocytes, and keratinocytes) were cultured on PCL nanofibrous scaffolds to mimic their natural microenvironment. The cytokine content of the platelet lysate was determined, and to the cells, a medium containing platelet lysate or platelet lysate in combination with FBS was added. The results showed that 7% (v/v) platelet lysate was sufficient to supplement 10% (v/v) FBS in the culture of fibroblasts and keratinocytes. The combination of platelet lysate and FBS had a rather inhibitory effect on fibroblasts, in contrary to keratinocytes, where the effect was synergic. Platelet lysate did not sufficiently promote proliferation in melanocytes; however, the combination of FBS and platelet lysate yielded a better outcome and resulted in bipolar morphology of the cultured melanocytes. The data indicated that platelet lysate improved cell proliferation and metabolic activity and may be used as an additive to the cell culture media.

  14. Structural Analyses of Phase Stability in Amorphous and Partially Crystallized Ge-Rich GeTe Films Prepared by Atomic Layer Deposition.

    Science.gov (United States)

    Gwon, Taehong; Mohamed, Ahmed Yousef; Yoo, Chanyoung; Park, Eui-Sang; Kim, Sanggyun; Yoo, Sijung; Lee, Han-Koo; Cho, Deok-Yong; Hwang, Cheol Seong

    2017-11-29

    The local bonding structures of Ge x Te 1-x (x = 0.5, 0.6, and 0.7) films prepared through atomic layer deposition (ALD) with Ge(N(Si(CH 3 ) 3 ) 2 ) 2 and ((CH 3 ) 3 Si) 2 Te precursors were investigated using Ge K-edge X-ray absorption spectroscopy (XAS). The results of the X-ray absorption fine structure analyses show that for all of the compositions, the as-grown films were amorphous with a tetrahedral Ge coordination of a mixture of Ge-Te and Ge-Ge bonds but without any signature of Ge-GeTe decomposition. The compositional evolution in the valence band electronic structures probed through X-ray photoelectron spectroscopy suggests a substantial chemical influence of additional Ge on the nonstoichiometric GeTe. This implies that the ALD process can stabilize Ge-abundant bonding networks like -Te-Ge-Ge-Te- in amorphous GeTe. Meanwhile, the XAS results on the Ge-rich films that had undergone post-deposition annealing at 350 °C show that the parts of the crystalline Ge-rich GeTe became separated into Ge crystallites and rhombohedral GeTe in accordance with the bulk phase diagram, whereas the disordered GeTe domains still remained, consistent with the observations of transmission electron microscopy and Raman spectroscopy. Therefore, amorphousness in GeTe may be essential for the nonsegregated Ge-rich phases and the low growth temperature of the ALD enables the achievement of the structurally metastable phases.

  15. Polymer Nanocomposite Film with Metal Rich Surface Prepared by In Situ Single-Step Formation of Palladium Nanoparticles: An Interesting Way to Combine Specific Functional Properties

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    David Thompson

    2016-10-01

    Full Text Available This paper presents a continuous single-step route that permits preparation of a thermostable polymer/metal nanocomposite film and to combine different functional properties in a unique material. More precisely, palladium nanoparticles are in situ generated in a polyimide matrix thanks to a designed curing cycle which is applied to a polyamic acid/metal precursor solution cast on a glass plate. A metal-rich surface layer which is strongly bonded to the bulk film is formed in addition to homogeneously dispersed metal nanoparticles. This specific morphology leads to obtaining an optically reflective film. The metal nanoparticles act as gas diffusion barriers for helium, oxygen, and carbon dioxide; they induce a tortuosity effect which allows dividing the gas permeation coefficients by a factor near to 2 with respect to the neat polyimide matrix. Moreover, the ability of the in situ synthesized palladium nanoparticles to entrap hydrogen is evidenced. The nanocomposite film properties can be modulated as a function of the location of the film metal-rich surface with respect to the hydrogen feed. The synthesized nanocomposite could represent a major interest for a wide variety of applications, from specific coatings for aerospace or automotive industry, to catalysis applications or sensors.

  16. The heat-compression technique for the conversion of platelet-rich fibrin preparation to a barrier membrane with a reduced rate of biodegradation.

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    Kawase, Tomoyuki; Kamiya, Mana; Kobayashi, Mito; Tanaka, Takaaki; Okuda, Kazuhiro; Wolff, Larry F; Yoshie, Hiromasa

    2015-05-01

    Platelet-rich fibrin (PRF) was developed as an advanced form of platelet-rich plasma to eliminate xenofactors, such as bovine thrombin, and it is mainly used as a source of growth factor for tissue regeneration. Furthermore, although a minor application, PRF in a compressed membrane-like form has also been used as a substitute for commercially available barrier membranes in guided-tissue regeneration (GTR) treatment. However, the PRF membrane is resorbed within 2 weeks or less at implantation sites; therefore, it can barely maintain sufficient space for bone regeneration. In this study, we developed and optimized a heat-compression technique and tested the feasibility of the resulting PRF membrane. Freshly prepared human PRF was first compressed with dry gauze and subsequently with a hot iron. Biodegradability was microscopically examined in vitro by treatment with plasmin at 37°C or in vivo by subcutaneous implantation in nude mice. Compared with the control gauze-compressed PRF, the heat-compressed PRF appeared plasmin-resistant and remained stable for longer than 10 days in vitro. Additionally, in animal implantation studies, the heat-compressed PRF was observed at least for 3 weeks postimplantation in vivo whereas the control PRF was completely resorbed within 2 weeks. Therefore, these findings suggest that the heat-compression technique reduces the rate of biodegradation of the PRF membrane without sacrificing its biocompatibility and that the heat-compressed PRF membrane easily could be prepared at chair-side and applied as a barrier membrane in the GTR treatment. © 2014 Wiley Periodicals, Inc.

  17. Topical gel formulation and stability assessment of platelet lysate based on turbidimetric method

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    Soliman Mohammadi Samani

    2015-06-01

    Full Text Available Platelet-rich growth factors have attracted attentions of scientists and clinical practitioners who are involved in wound healing and regenerative medicine extensively, according to their unprecedented potential of promoting and catalyzing healing process. Platelet-rich growth factors are cost-benefit, available and more stable than recombinant human growth factors. These appealing characteristics have converted PRGF to one of the popular candidates for treatment of variety of wounds. According to these valuable properties, we decided to formulate and assess the effect of different excipients on the stability of such valuable protein based formulations. Different excipients have been chosen according to their effective ness on the stability of proteins and their application in other similar formulations. The stabilizing effect of excipients was evaluated by measuring heat-induced aggregation of growth factors by turbidimetric assay. Glycerol, glycine and dextrose were chosen as stabilizing excipients for these formulations. The results show that dextrose has more stabilizing effect on prevention of heat induced aggregation of the platelet lysate growth factors than glycerol and glycine. All of the formulations also contained antioxidant, chelating agents, preservative and carbopol934 in order to form appropriate gel.

  18. Wound Healing Activity of a New Formulation from Platelet Lysate

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    Akram Jamshidzadeh

    2016-03-01

    Full Text Available Platelet-rich plasma (PRP is an attractive preparation in regenerative medicine due to its potential role in the healing process in different experimental models. This study was designed to investigate the wound healing activity of a new formulation of PRP. Different gel-based formulations of PRP were prepared. Open excision wounds were made on the back of male Sprague-Dawley rats, and PRP gel was administered topically once daily until the wounds healed completely (12 days. The results revealed that the tested PRP formulation significantly accelerated the wound healing process by increasing the wound contraction, tissue granulization, vascularization, and collagen regeneration. Interestingly, this study showed that there were no significant differences between the PRP and its gel-based formulation in all the above mentioned parameters. Although this investigation showed that PRP formulation had significant wound healing effects, the PRP gel-based formulation also had significant wound healing properties. This might indicate the wound healing properties of the PRP gel ingredients in the current investigation.

  19. In vitro study of the role of thrombin in platelet rich plasma (PRP) preparation: utility for gel formation and impact in growth factors release.

    Science.gov (United States)

    Huber, Stephany Cares; Cunha Júnior, José Luiz Rosenberis; Montalvão, Silmara; da Silva, Letícia Queiroz; Paffaro, Aline Urban; da Silva, Francesca Aparecida Ramos; Rodrigues, Bruno Lima; Lana, José Fabio Santos Duarte; Annichino-Bizzacchi, Joyce Maria

    2016-01-01

    The use of PRP has been studied for different fields, with promising results in regenerative medicine. Until now, there is no study in the literature evaluating thrombin levels in serum, used as autologous thrombin preparation. Therefore, in the present study we evaluated the role played by different thrombin concentrations in PRP and the impact in the release of growth factors. Also, different activators for PRP gel formation were evaluated. Thrombin levels were measured in different autologous preparations: serum, L-PRP (PRP rich in leukocytes) and T-PRP (thrombin produced through PRP added calcium gluconate). L-PRP was prepared according to the literature, with platelets and leukocytes being quantified. The effect of autologous thrombin associated or not with calcium in PRP gel was determined by measuring the time of gel formation. The relationship between thrombin concentration and release of growth factors was determined by growth factors (PDGF-AA, VEGF and EGF) multiplex analysis. A similar concentration of thrombin was observed in serum, L-PRP and T-PRP (8.13 nM, 8.63 nM and 7.56 nM, respectively) with a high variation between individuals (CV%: 35.07, 43 and 58.42, respectively). T-PRP and serum with calcium chloride showed similar results in time to promote gel formation. The increase of thrombin concentrations (2.66, 8 and 24 nM) did not promote an increase in growth factor release. The technique of using serum as a thrombin source proved to be the most efficient and reproducible for promoting PRP gel formation, with some advantages when compared to other activation methods, as this technique is easier and quicker with no need of consuming part of PRP. Noteworthy, PRP activation using different thrombin concentrations did not promote a higher release of growth factors, appearing not to be necessary when PRP is used as a suspension.

  20. Platelet Lysate: The Better Choice for Jaw Periosteal Cell Mineralization

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    Yvonne Wanner

    2017-01-01

    Full Text Available Previously, we demonstrated a high quality of minerals formed by serum-free cultured jaw periosteal cells (JPCs by Raman spectroscopy but the mineralization extent was not satisfactory. In the present study, we analyzed the proliferation and mineralization potential of human platelet lysate- (hPL- cultured JPCs in comparison to that of FCS-cultured JPCs. By cell impedance measurements, we detected significantly higher population doubling times of PL-cultured JPCs in comparison to FCS-cultured JPCs. However, this result was not based on lower proliferation activities but on diminished cell sizes which JPCs develop under PL cultivation. The measurements of the metabolic activities clearly showed significantly higher cell proliferation rates under PL culturing. Equivalent levels of the mesenchymal cell markers CD29, CD45, CD73, CD90, and CD105 were detected, but there were significantly increased MSCA-1 levels under PL cultivation. While JPCs only occasionally mineralize under FCS culture conditions, the mineralization potential was significantly stronger under PL cultivation. Moreover, in 4 of 5 analyzed patient cells, the addition of dexamethasone was proved no longer necessary for strong mineralization of PL-cultured JPCs. We conclude that in vitro cultivation of JPCs with platelet lysate is a suitable alternative to FCS culture conditions and a powerful tool for the development of high-quality TE constructs using jaw periosteal cells.

  1. Platelet Lysate: The Better Choice for Jaw Periosteal Cell Mineralization.

    Science.gov (United States)

    Wanner, Yvonne; Umrath, Felix; Waidmann, Marc; Reinert, Siegmar; Alexander, Dorothea

    2017-01-01

    Previously, we demonstrated a high quality of minerals formed by serum-free cultured jaw periosteal cells (JPCs) by Raman spectroscopy but the mineralization extent was not satisfactory. In the present study, we analyzed the proliferation and mineralization potential of human platelet lysate- (hPL-) cultured JPCs in comparison to that of FCS-cultured JPCs. By cell impedance measurements, we detected significantly higher population doubling times of PL-cultured JPCs in comparison to FCS-cultured JPCs. However, this result was not based on lower proliferation activities but on diminished cell sizes which JPCs develop under PL cultivation. The measurements of the metabolic activities clearly showed significantly higher cell proliferation rates under PL culturing. Equivalent levels of the mesenchymal cell markers CD29, CD45, CD73, CD90, and CD105 were detected, but there were significantly increased MSCA-1 levels under PL cultivation. While JPCs only occasionally mineralize under FCS culture conditions, the mineralization potential was significantly stronger under PL cultivation. Moreover, in 4 of 5 analyzed patient cells, the addition of dexamethasone was proved no longer necessary for strong mineralization of PL-cultured JPCs. We conclude that in vitro cultivation of JPCs with platelet lysate is a suitable alternative to FCS culture conditions and a powerful tool for the development of high-quality TE constructs using jaw periosteal cells.

  2. USE OF BACTERIAL LYSATES IN COMPLEX TREATMENT OF RHINOSINUSITIDIS

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    E.A. Vishneva

    2011-01-01

    Full Text Available Rhinosinusitis is a common term that means inflammation of the mucosa of paranasal and nasal cavities. In the last 10 years there was a three-fold increase in rhinosinusitis’ morbidity rates and annual increase of hospital admissions on the average of 1,5–2%. The prevalence of various forms of rhinosinusitidis among adult population is 5 to 15% and 5% among children. Such ubiquitous spreading is one of the leading factors that stipulate socio-economic burden of the disease. According to various studies, rhinosinusitidis dramatically decrease quality of life: these patients show more inferior results in tests upon pain sensitivity and social activity in comparison with patients with coronary heart disease and COPD; in approximately 26% of patients rhinosinusitidis are followed by development or progression of depression. Rhinosinusitis treatment is one of the hottest issues in modern otorhinolaryngology, that demand more financial support with every year. Achievments in immunology have become grounds for immunocorrection as part of complex treatment of such patients. Bacterial lysates are used as preventive medications.Key words: rhinosinusitidis, immunomodulators, bacterial lysates. (Voprosy sovremennoi pediatrii — Current Pediatrics. — 2011; 10 (6: 157–161

  3. Human Platelet Lysate as a Replacement for Fetal Bovine Serum in Limbal Stem Cell Therapy.

    Science.gov (United States)

    Suri, Kunal; Gong, Hwee K; Yuan, Ching; Kaufman, Stephen C

    2016-10-01

    To evaluate the use of human platelet lysate (HPL) as an alternative supplement for limbal explant culture. Culture media were prepared using either 10% pooled HPL (PHPL), single donor HPL, or fetal bovine serum (FBS). Limbal tissues, obtained from the Minnesota Lions Eye Bank, were cultured in each medium on plastic plates or on denuded amniotic membrane (AM). Immunofluorescence staining was performed for ABCG2, tumor protein p63α, and cytokeratin 3 (K3). Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was used to evaluate the expression of ABCG2 and p63. Limbal explants grown in each medium were labeled with bromodeoxyuridine (BrdU) to assess the proliferative capacity in each medium. Concentration of growth factors including epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), and platelet derived growth factor (PDGF) in HPL and PHPL was compared to that in human serum (HS). Immunofluorescence staining on AM showed prominent expression of ABCG2, p63α but sparse expression of K3 in HPL and PHPL supplemented medium. Real time-PCR showed 1.7 fold higher expression of ABCG2 in PHPL supplemented medium (p = 0.03), and similar expression of p63 in HPL and PHPL supplemented medium compared to FBS medium. The proliferation assay showed that LSCs retained their proliferative potential in HPL supplemented medium. Higher concentration of growth factors were found in HPL, compared to HS. Human platelet lysate has higher concentration of grown factors and is effective in maintaining growth and stem cell phenotype of corneal limbal explant cultures.

  4. Electrospun Gelatin–Chondroitin Sulfate Scaffolds Loaded with Platelet Lysate Promote Immature Cardiomyocyte Proliferation

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    Francesca Saporito

    2018-02-01

    Full Text Available The aim of the present work was the development of heart patches based on gelatin (G and chondroitin sulfate (CS to be used as implants to improve heart recovery after corrective surgery for critical congenital heart defects (CHD. Patches were prepared by means of electrospinning to obtain nanofibrous scaffolds and they were loaded with platelet lysate (PL as a source of growth factors to further enhance the repair process. Scaffolds were characterized for morphology and mechanical properties and for the capability to support in vitro adhesion and proliferation of dermal fibroblasts in order to assess the system’s general biocompatibility. Adhesion and proliferation of endothelial cells and cardiac cells (cardiomyocytes and cardiac fibroblasts from rat fetuses onto PL-loaded patches was evaluated. Patches presented good elasticity and high stiffness suitable for in vivo adaptation to heart contraction. CS improved adhesion and proliferation of dermal fibroblasts, as proof of their biocompatibility. Moreover, they enhanced the adhesion and proliferation of endothelial cells, a crucial mediator of cardiac repair. Cell adhesion and proliferation could be related to elastic properties, which could favor cell motility. The presence of platelet lysate and CS was crucial for the adhesion and proliferation of cardiac cells and, in particular, of cardiomyocytes: G/CS scaffold embedded with PL appeared to selectively promote proliferation in cardiomyocytes but not cardiac fibroblasts. In conclusion, G/CS scaffold seems to be a promising system to assist myocardial-repair processes in young patient, preserving cardiomyocyte viability and preventing cardiac fibroblast proliferation, likely reducing subsequent uncontrolled collagen deposition by fibroblasts following repair.

  5. Releasing growth factors from activated human platelets after chitosan stimulation: a possible bio-material for platelet-rich plasma preparation.

    Science.gov (United States)

    Shen, E-Chin; Chou, Tz-Chong; Gau, Ching-Hwa; Tu, Hsiao-Pei; Chen, Yen-Teen; Fu, Earl

    2006-10-01

    Thrombin is commonly used for activating the platelets and releasing the growth factors on the application of platelet-rich plasma (PRP). We have previously reported that chitosan can enhance rabbit platelet aggregation. In this study, the effects of chitosan on the subsequent growth factors release after human platelets activation were examined to evaluate the possibility of chitosan being used as a substitute for thrombin during PRP preparation. Human platelet activation was determined by aggregation, adhesion and alpha-granule membrane glycoprotein expression. Platelet aggregation was measured by the turbidimetric method, the adhesion was directly examined on chitosan-coated glass plates under light microscope and scanning electron microscope (SEM), and the alpha-granule membrane glycoprotein was detected by fluorescent isothiocyanate (FITC)-conjugated anti-CD61 antibody through flow cytometry. The subsequent epidermal growth factor (EGF), platelet-derived growth factor (PDGF)-AB and transforming growth factor (TGF)-beta1 release from platelets were assayed by ELISA after mixing with chitosan. The enhancing effects on the platelet adhesion and the aggregation from chitosan were observed. Under both microscopes, the adhesive platelets on the chitosan-coated plates were not only greater in number but also earlier in activation than those on the control plates. With flow cytometry, increased glycoprotein IIIa expression in platelets was detected after chitosan treatment. Greater concentrations of growth factors were measured from PRP after chitosan treatment than after the solvent treatment. Because of the observations of growth factors releasing from activated human platelets after chitosan stimulation, we suggest that chitosan may be an appropriate substitute for thrombin in PRP preparation.

  6. Assessment of preparation methods for organic phosphorus analysis in phosphorus-polluted Fe/Al-rich Haihe river sediments using solution 31P-NMR.

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    Wenqiang Zhang

    Full Text Available Fe/Al-rich river sediments that were highly polluted with phosphorus (P were used in tests to determine the optimum preparation techniques for measuring organic P (Po using solution (31P nuclear magnetic resonance spectroscopy ((31P-NMR. The optimum pre-treatment, extraction time, sediment to solution ratio and sodium hydroxide-ethylenediaminetetraacetic acid (NaOH-EDTA extractant solution composition were determined. The total P and Po recovery rates were higher from freeze- and air-dried samples than from fresh samples. An extraction time of 16 h was adequate for extracting Po, and a shorter or longer extraction time led to lower recoveries of total P and Po, or led to the degradation of Po. An ideal P recovery rate and good-quality NMR spectra were obtained at a sediment:solution ratio of 1:10, showing that this ratio is ideal for extracting Po. An extractant solution of 0.25 M NaOH and 50 mM EDTA was found to be more appropriate than either NaOH on its own, or a more concentrated NaOH-EDTA mixture for (31P-NMR analysis, as this combination minimized interference from paramagnetic ions and was appropriate for the detected range of Po concentrations. The most appropriate preparation method for Po analysis, therefore, was to extract the freeze-dried and ground sediment sample with a 0.25 M NaOH and 50 mM EDTA solution at a sediment:solution ratio of 1:10, for 16 h, by shaking. As lyophilization of the NaOH-EDTA extracts proved to be an optimal pre-concentration method for Po analysis in the river sediment, the extract was lyophilized as soon as possible, and analyzed by (31P-NMR.

  7. Preparation of a new composite combining strengthened β-tricalcium phosphate with platelet-rich plasma as a potential scaffold for the repair of bone defects

    Science.gov (United States)

    WANG, CHENGGONG; ZHONG, DA; ZHOU, XING; YIN, KE; LIAO, QIANDE; KONG, LINGYU; LIU, ANSONG

    2014-01-01

    β-tricalcium phosphate (β-TCP) and platelet-rich plasma (PRP) are commonly used in bone tissue engineering. In the present study, a new composite combining strengthened β-TCP and PRP was prepared and its morphological and mechanical properties were investigated by scanning electron microscopy (SEM) and material testing. The biocompatibility was evaluated by measuring the adhesion rate and cytotoxicity of bone marrow stromal cells (BMSCs). The strengthened β-TCP/PRP composite had an appearance like the fungus Boletus kermesinus with the PRP gel distributed on the surface of the micropores. The maximum load and load intensity were 945.6±86.4 N and 13.1±0.5 MPa, which were significantly higher than those of β-TCP (110.1±14.3 N and 1.6±0.2 MPa; P96% after 24 h, with a cell cytotoxicity value of zero. SEM micrographs revealed that following seeding of BMSCs onto the composite in high-glucose Dulbecco’s modified Eagle’s medium culture for two weeks, the cells grew well and exhibited fusiform, spherical and polygonal morphologies, as well as pseudopodial connections. The strengthened β-TCP/PRP composite has the potential to be used as a scaffold in bone tissue engineering due to its effective biocompatibility and mechanical properties. PMID:25187800

  8. Quality assessment of platelet concentrates prepared by platelet rich plasma-platelet concentrate, buffy coat poor-platelet concentrate (BC-PC and apheresis-PC methods

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    Singh Ravindra

    2009-01-01

    Full Text Available Background: Platelet rich plasma-platelet concentrate (PRP-PC, buffy coat poor-platelet concentrate (BC-PC, and apheresis-PC were prepared and their quality parameters were assessed. Study Design: In this study, the following platelet products were prepared: from random donor platelets (i platelet rich plasma - platelet concentrate (PRP-PC, and (ii buffy coat poor- platelet concentrate (BC-PC and (iii single donor platelets (apheresis-PC by different methods. Their quality was assessed using the following parameters: swirling, volume of the platelet concentrate, platelet count, WBC count and pH. Results: A total of 146 platelet concentrates (64 of PRP-PC, 62 of BC-PC and 20 of apheresis-PC were enrolled in this study. The mean volume of PRP-PC, BC-PC and apheresis-PC was 62.30±22.68 ml, 68.81±22.95 ml and 214.05±9.91 ml and ranged from 22-135 ml, 32-133 ml and 200-251 ml respectively. The mean platelet count of PRP-PC, BC-PC and apheresis-PC was 7.6±2.97 x 1010/unit, 7.3±2.98 x 1010/unit and 4.13±1.32 x 1011/unit and ranged from 3.2-16.2 x 1010/unit, 0.6-16.4 x 1010/unit and 1.22-8.9 x 1011/unit respectively. The mean WBC count in PRP-PC (n = 10, BC-PC (n = 10 and apheresis-PC (n = 6 units was 4.05±0.48 x 107/unit, 2.08±0.39 x 107/unit and 4.8±0.8 x 106/unit and ranged from 3.4 -4.77 x 107/unit, 1.6-2.7 x 107/unit and 3.2 - 5.2 x 106/unit respectively. A total of 26 units were analyzed for pH changes. Out of these units, 10 each were PRP-PC and BC-PC and 6 units were apheresis-PC. Their mean pH was 6.7±0.26 (mean±SD and ranged from 6.5 - 7.0 and no difference was observed among all three types of platelet concentrate. Conclusion: PRP-PC and BC-PC units were comparable in terms of swirling, platelet count per unit and pH. As expected, we found WBC contamination to be less in BC-PC than PRP-PC units. Variation in volume was more in BC-PC than PRP-PC units and this suggests that further standardization is required for preparation of BC

  9. Platelet lysate as a substitute for animal serum for the ex-vivo expansion of mesenchymal stem/stromal cells: present and future.

    Science.gov (United States)

    Astori, Giuseppe; Amati, Eliana; Bambi, Franco; Bernardi, Martina; Chieregato, Katia; Schäfer, Richard; Sella, Sabrina; Rodeghiero, Francesco

    2016-07-13

    The use of fetal bovine serum (FBS) as a cell culture supplement is discouraged by regulatory authorities to limit the risk of zoonoses and xenogeneic immune reactions in the transplanted host. Additionally, FBS production came under scrutiny due to animal welfare concerns. Platelet derivatives have been proposed as FBS substitutes for the ex-vivo expansion of mesenchymal stem/stromal cells (MSCs) since platelet-derived growth factors can promote MSC ex-vivo expansion. Platelet-derived growth factors are present in platelet lysate (PL) obtained after repeated freezing-thawing cycles of the platelet-rich plasma or by applying physiological stimuli such as thrombin or CaCl2.PL-expanded MSCs have been used already in the clinic, taking advantage of their faster proliferation compared with FBS-expanded preparations. Should PL be applied to other biopharmaceutical products, its demand is likely to increase dramatically. The use of fresh platelet units for the production of PL raises concerns due to limited availability of platelet donors. Expired units might represent an alternative, but further data are needed to define safety, including pathogen reduction, and functionality of the obtained PL. In addition, relevant questions concerning the definition of PL release criteria, including concentration ranges of specific growth factors in PL batches for various clinical indications, also need to be addressed. We are still far from a common definition of PL and standardized PL manufacture due to our limited knowledge of the mechanisms that mediate PL-promoting cell growth. Here, we concisely discuss aspects of PL as MSC culture supplement as a preliminary step towards an agreed definition of the required characteristics of PL for the requirements of manufacturers and users.

  10. Mechanism of SOS PR-domain autoinhibition revealed by single-molecule assays on native protein from lysate.

    Science.gov (United States)

    Lee, Young Kwang; Low-Nam, Shalini T; Chung, Jean K; Hansen, Scott D; Lam, Hiu Yue Monatrice; Alvarez, Steven; Groves, Jay T

    2017-04-28

    The guanine nucleotide exchange factor (GEF) Son of Sevenless (SOS) plays a critical role in signal transduction by activating Ras. Here we introduce a single-molecule assay in which individual SOS molecules are captured from raw cell lysate using Ras-functionalized supported membrane microarrays. This enables characterization of the full-length SOS protein, which has not previously been studied in reconstitution due to difficulties in purification. Our measurements on the full-length protein reveal a distinct role of the C-terminal proline-rich (PR) domain to obstruct the engagement of allosteric Ras independently of the well-known N-terminal domain autoinhibition. This inhibitory role of the PR domain limits Grb2-independent recruitment of SOS to the membrane through binding of Ras·GTP in the SOS allosteric binding site. More generally, this assay strategy enables characterization of the functional behaviour of GEFs with single-molecule precision but without the need for purification.

  11. Tobacco BY-2 cell-free lysate: an alternative and highly-productive plant-based in vitro translation system.

    Science.gov (United States)

    Buntru, Matthias; Vogel, Simon; Spiegel, Holger; Schillberg, Stefan

    2014-05-03

    Cell-free protein synthesis is a rapid and efficient method for the production of recombinant proteins. Usage of prokaryotic cell-free extracts often leads to non-functional proteins. Eukaryotic counterparts such as wheat germ extract (WGE) and rabbit reticulocyte lysate (RLL) may improve solubility and promote the correct folding of eukaryotic multi-domain proteins that are difficult to express in bacteria. However, the preparation of WGEs is complex and time-consuming, whereas RLLs suffer from low yields. Here we report the development of a novel cell-free system based on tobacco Bright Yellow 2 (BY-2) cells harvested in the exponential growth phase. The highly-productive BY-2 lysate (BYL) can be prepared quickly within 4-5 h, compared to 4-5 d for WGE. The efficiency of the BYL was tested using three model proteins: enhanced yellow fluorescent protein (eYFP) and two versions of luciferase. The added mRNA was optimized by testing different 5' and 3' untranslated regions (UTRs). The protein yield in batch and dialysis reactions using BYL was much higher than that of a commercial Promega WGE preparation, achieving a maximum yield of 80 μg/mL of eYFP and 100 μg/mL of luciferase, compared to only 45 μg/mL of eYFP and 35 μg/mL of luciferase in WGEs. In dialysis reactions, the BYL yielded about 400 μg/mL eYFP, representing up to 50% more of the target protein than the Promega WGE, and equivalent to the amount using 5Prime WGE system. Due to the high yield and the short preparation time the BYL represents a remarkable improvement over current eukaryotic cell-free systems.

  12. Isolation of RNA for dot hybridization by heparin-DNase I treatment of whole cell lysate.

    Science.gov (United States)

    Krawczyk, Z; Wu, C

    1987-08-15

    We have developed a new procedure for the rapid preparation of undegraded total RNA from cultured cells for specific quantitation by dot blotting analysis. Pelleted cells are resuspended in hypotonic solution containing a ribonuclease inhibitor and heparin and disrupted by freeze-thaw. Heparin is employed as an agent for nuclear lysis, dissociation of chromosomal protein, and release of mRNA from rough endoplasmic reticulum. We eliminate chromosomal DNA by digestion with DNase I and denature the RNA in the lysate with formaldehyde. After centrifugation to remove debris, the supernatant is used directly for dot blotting. All manipulations are performed in the same microfuge tube and recovery of RNA is quantitative. The procedure is especially useful for processing large numbers of samples. We illustrate its versatility by analysis of specific RNAs in Drosophila, rat, and human cell lines. In reconstruction experiments, less than 80 molecules per cell of a small RNA (beta-globin) can be detected under highly stringent hybridization conditions, using only moderately labeled double-stranded plasmid DNA probes and short film exposures.

  13. GMP-grade platelet lysate enhances proliferation and migration of tenon fibroblasts.

    Science.gov (United States)

    Carducci, Augusto; Scafetta, Gaia; Siciliano, Camilla; Carnevale, Roberto; Rosa, Paolo; Coccia, Andrea; Mangino, Giorgio; Bordin, Antonella; Vingolo, Enzo Maria; Pierelli, Luca; Lendaro, Eugenio; Ragona, Giuseppe; Frati, Giacomo; De Falco, Elena

    2016-01-01

    Tenon's fibroblasts (TFs), widely employed as in vitro model for many ophthalmological studies, are routinely cultured with FBS. Platelet Lysate (PL), a hemoderivate enriched with growth factors and cytokines has been largely tested in several clinical applications and as substitute of FBS in culture. Here, we investigate whether PL can exert biological effects on TF populations similarly to other cell types. Results show that PL significantly enhances cell proliferation and migration vs. FBS, without influencing cell size/granularity. Upregulation of EGF, VEGF, KDR, MMP2-9, FAK mRNA levels also occurs and phosphorylation of AKT but not of ERK1/2 is significantly enhanced. The inhibition of the PI3kinase/AKT pathway with the specific inhibitor wortmannin, decreases PL-induced cell migration but not proliferation. Condition supernatants containing PL show increased bioavailability of Nitric Oxide and reduced levels of 8-Iso-PGF2-alpha, correlating with cell proliferation and migration. Pro-angiogenic/inflammatory soluble factors (GRO, Angiogenin, EGF, I-309, PARC) are exclusively or greater expressed in media containing PL than FBS. GMP-grade PL preparations positively influence in vitro biological effects of TFs representing a suitable and safer alternative to FBS.

  14. Detection of circulating tumor lysate-reactive CD4+ T cells in melanoma patients

    DEFF Research Database (Denmark)

    Ladekarl, Morten; Agger, Ralf; Fleischer, Charlotte C

    2004-01-01

    PURPOSE: We wanted to study whether an allogeneic melanoma lysate would be a feasible stimulatory antigen source for detection of a peripheral CD4+ T-cell immune response in patients with medically untreated malignant melanoma. The lysate was produced from a melanoma cell line (FM3.29) which...... was found in 1 of 4 patients radically operated for localized disease, whereas no responders were seen among 7 healthy donors. The fraction of circulating lysate-activated T cells ranged from 0.0037% to 0.080% of the CD4+ population. A negative result of the assay was found occasionally, especially...... expresses high amounts of melanoma antigens. METHODS: Fresh peripheral blood was incubated with and without lysate for 6 h in the presence of anti-CD28/anti-CD49d MoAb (for costimulation). After flow cytometric estimation of the frequency of CD69+/IFN-gamma+ cells in the CD4+ population, the response...

  15. Comparison between mixed lysate antigen and α-actinin antigen in ELISA for serodiagnosis of trichomoniasis.

    Science.gov (United States)

    Kim, Seung-Ryong; Kim, Jung-Hyun; Park, Soon-Jung; Lee, Hye-Yeon; Kim, Yong-Suk; Kim, Yu-Mi; Hong, Yeon-Chul; Ryu, Jae-Sook

    2015-10-01

    The aim of this study was to identify an antigen suitable for ELISA for serodiagnosis of Trichomonas vaginalis (T. vaginalis) infection. Mixed lysate antigen (Ag) from eight strains of T. vaginalis and recombinant α-actinin protein was compared. The sera of three groups were examined by ELISA: 73 women infected with trichomoniasis served as a positive control, 31 male volunteers as a negative control, and 424 women attending an outpatient health screening at Hanyang University Guri Hospital. Based on the cutoff optical density for each Ag obtained with a negative control, the serosensitivity of the mixed lysate Ag (79.5%) was significantly higher than that of the α-actinin (52.1%) in the 73 patients with trichomoniasis. The specificity using lysate Ag and α-actinin was 100% and 96.8%, respectively. On the other hand, the positivity rate in 424 outpatients was 39.2% and 11.8% with mixed lysate and α-actinin Ag, respectively. Taken together, mixed lysate Ag showed higher sensitivity and specificity than α-actinin. Therefore, mixed lysate may be a better Ag than α-actinin for ELISA for the diagnosis of trichomoniasis. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  16. Development and validation of a production process of platelet lysate for autologous use.

    Science.gov (United States)

    Plöderl, Karin; Strasser, Cornelia; Hennerbichler, Simone; Peterbauer-Scherb, Anja; Gabriel, Christian

    2011-01-01

    Growth factors (GF) contained in platelets are a potential source to improve wound healing by the stimulation and acceleration of soft tissue and bone healing. This resulted in the idea that autologous platelet-rich plasma or platelet lysate (PL) containing high levels of GF might improve healing processes. Today platelet products are already applied in bone and maxillofacial surgery. In recent years, cosmetic surgery and facial rejuvenation procedures are growing steadily. New methods including platelet products aiming to induce non-surgical reduction of wrinkles upon topical injection and to minimize surgical risks in general are developed. Several point-of-care devices are already available on the market. However, the amount of PL obtained by these kits is far too high for certain applications in cosmetic surgery and they offer no possibility of storing the remaining material in a sterile manner. Therefore we developed a procedure for the sterile production of smaller amounts of PL in a closed system that can also be split into several products for repeated administration. The closed system was determined to be a bag system designed for an autologous blood donation of 100 ml whole blood. We set a special focus on the validation of the production procedure, mainly regarding sterility and platelet recovery. For validation 22 healthy volunteers were asked for a blood donation, which was centrifuged twice to obtain concentrated platelets (CP). A freeze-thaw cycle caused lysis of the CP to get approximately 8.48 ± 1.36 ml PL. We yielded satisfying results of 100% sterility and a platelet recovery of 36.92% ± 18.71%. We therefore conclude that the PL obtained is ready for studies comparing it with traditional treatments.

  17. Evaluation of human platelet lysate versus fetal bovine serum for culture of mesenchymal stromal cells.

    Science.gov (United States)

    Hemeda, Hatim; Giebel, Bernd; Wagner, Wolfgang

    2014-02-01

    Culture media for therapeutic cell preparations-such as mesenchymal stromal cells (MSCs)-usually comprise serum additives. Traditionally, fetal bovine serum is supplemented in basic research and in most clinical trials. Within the past years, many laboratories adapted their culture conditions to human platelet lysate (hPL), which further stimulates proliferation and expansion of MSCs. Particularly with regard to clinical application, human alternatives for fetal bovine serum are clearly to be preferred. hPL is generated from human platelet units by disruption of the platelet membrane, which is commonly performed by repeated freeze and thaw cycles. Such culture supplements are notoriously ill-defined, and many parameters contribute to batch-to-batch variation in hPL such as different amounts of plasma, a broad range of growth factors and donor-specific effects. The plasma components of hPL necessitate addition of anticoagulants such as heparins to prevent gelatinization of hPL medium, and their concentration must be standardized. Labels for description of hPL-such as "xenogen-free," "animal-free" and "serum free"-are not used consistently in the literature and may be misleading if not critically assessed. Further analysis of the precise composition of relevant growth factors, attachment factors, microRNAs and exosomes will pave the way for optimized and defined culture conditions. The use of hPL has several advantages and disadvantages: they must be taken into account because the choice of cell culture additive has major impact on cell preparations. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  18. Serum-converted platelet lysate can substitute for fetal bovine serum in human mesenchymal stromal cell cultures.

    Science.gov (United States)

    Mojica-Henshaw, Mariluz P; Jacobson, Pam; Morris, Julie; Kelley, Linda; Pierce, Jan; Boyer, Michael; Reems, Jo-Anna

    2013-12-01

    Fetal bovine serum (FBS) is commonly used as a serum supplement for culturing human mesenchymal stromal cells (hMSCs). However, human cells grown in FBS, especially for extended periods, risk potential exposure to bovine immunogenic proteins and infectious agents. To address this issue, we investigated the ability of a novel human platelet serum supplement to substitute for FBS in hMSC cultures. Platelet lysate-serum (PL-serum) was converted from platelet lysate-plasma (PL-plasma) that was manufactured from pooled platelet-rich plasma (PRP) apheresis units. Growth factor levels and the number of residual intact platelets in PL-serum and PL-plasma were compared with enzyme-linked immunosorbent assays and flow cytometry, respectively. Proliferation responses of hMSCs cultured in PL-serum, PL-plasma, or FBS were assessed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, the immunophenotype of harvested hMSCs was evaluated by flow cytometry and tri-lineage differentiation potential was evaluated by assessing adipogenic, osteogenic and chondrogenic development. Selected growth factor levels in PL-serum were not significantly different from PL-plasma (P > 0.05). hMSC cultures supplemented with PL-serum had comparable growth kinetics to PL-plasma, and hMSC yields were consistently greater than with FBS. hMSCs harvested from cultures supplemented with PL-serum, PL-plasma or FBS had similar cell surface phenotypes and maintained tri-lineage differentiation potential. PL-serum, similar to PL-plasma, can substitute for FBS in hMSC cultures. Use of PL-serum, in contrast to PL-plasma, has an added advantage of not requiring addition of a xenogeneic source of heparin, providing a completely xeno-free culture medium. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  19. Virally inactivated human platelet concentrate lysate induces regulatory T cells and immunosuppressive effect in a murine asthma model.

    Science.gov (United States)

    Lee, Yueh-Lun; Lee, Lin-Wen; Su, Chen-Yao; Hsiao, George; Yang, Yi-Yuan; Leu, Sy-Jye; Shieh, Ying-Hua; Burnouf, Thierry

    2013-09-01

    Platelet concentrate lysates (PCLs) are increasingly used in regenerative medicine. We have developed a solvent/detergent (S/D)-treated PCL. The functional properties of this preparation should be unveiled. We hypothesized that, due to transforming growth factor-β1 (TGF-β1) content, PCLs may exert immunosuppressive and anti-inflammatory functions. PCL was prepared by S/D treatment, oil extraction, and hydrophobic interaction chromatography. The content of TGF-β in PCL was determined by enzyme-linked immunosorbent assay. Cultured CD4+ T cells were used to investigate the effects of PCL on expression of transcription factor forkhead box P3 (Foxp3), the inhibition of T-cell proliferation, and cytokine production. The regulatory function of PCL-converted CD4+ T cells was analyzed by suppressive assay. The BALB/c mice were given PCL-converted CD4+ T cells before ovalbumin (OVA) sensitization and challenge using an asthma model. Inflammatory parameters, such as the level of immunoglobulin E (IgE), airway hyperresponsiveness (AHR), bronchial lavage fluid eosinophils, and cytokines were assayed. Recombinant human (rHu) TGF-β1 was used as control. PCL significantly enhanced the development of CD4+Foxp3+-induced regulatory T cells (iTregs). Converted iTregs produced neither Th1 nor Th2 cytokines and inhibited normal T-cell proliferation. PCL- and rHuTGF-β-converted CD4+ T cells prevented OVA-induced asthma. PCL- and rHuTGF-β-modified T cells both significantly reduced expression levels of OVA-specific IgE and significantly inhibited the development of AHR, airway eosinophilia, and Th2 responses in mice. S/D-treated PCL promotes Foxp3+ iTregs and exerts immunosuppressive and anti-inflammatory properties. This finding may help to understand the clinical properties of platelet lysates. © 2013 American Association of Blood Banks.

  20. Autologous Dendritic Cells Pulsed with Allogeneic Tumor Cell Lysate in Mesothelioma: From Mouse to Human.

    Science.gov (United States)

    Aerts, Joachim G J V; de Goeje, Pauline L; Cornelissen, Robin; Kaijen-Lambers, Margaretha E H; Bezemer, Koen; van der Leest, Cor H; Mahaweni, Niken M; Kunert, André; Eskens, Ferry A L M; Waasdorp, Cynthia; Braakman, Eric; van der Holt, Bronno; Vulto, Arnold G; Hendriks, Rudi W; Hegmans, Joost P J J; Hoogsteden, Henk C

    2018-02-15

    Purpose: Mesothelioma has been regarded as a nonimmunogenic tumor, which is also shown by the low response rates to treatments targeting the PD-1/PD-L1 axis. Previously, we demonstrated that autologous tumor lysate-pulsed dendritic cell (DC) immunotherapy increased T-cell response toward malignant mesothelioma. However, the use of autologous tumor material hampers implementation in large clinical trials, which might be overcome by using allogeneic tumor cell lines as tumor antigen source. The purpose of this study was to investigate whether allogeneic lysate-pulsed DC immunotherapy is effective in mice and safe in humans. Experimental Design: First, in two murine mesothelioma models, mice were treated with autologous DCs pulsed with either autologous or allogeneic tumor lysate or injected with PBS (negative control). Survival and tumor-directed T-cell responses of these mice were monitored. Results were taken forward in a first-in-human clinical trial, in which 9 patients were treated with 10, 25, or 50 million DCs per vaccination. DC vaccination consisted of autologous monocyte-derived DCs pulsed with tumor lysate from five mesothelioma cell lines. Results: In mice, allogeneic lysate-pulsed DC immunotherapy induced tumor-specific T cells and led to an increased survival, to a similar extent as DC immunotherapy with autologous tumor lysate. In the first-in-human clinical trial, no dose-limiting toxicities were established and radiographic responses were observed. Median PFS was 8.8 months [95% confidence interval (CI), 4.1-20.3] and median OS not reached (median follow-up = 22.8 months). Conclusions: DC immunotherapy with allogeneic tumor lysate is effective in mice and safe and feasible in humans. Clin Cancer Res; 24(4); 766-76. ©2017 AACR . ©2017 American Association for Cancer Research.

  1. Identification by genotyping of a commercial antigen preparation as the source of a laboratory contamination with Coxiella burnetii and as an unexpected rich source of control DNA

    NARCIS (Netherlands)

    Tilburg, Jeroen J. H. C.; Horrevorts, Alphons M.; Peeters, Marcel F.; Klaassen, Corne H. W.; Rossen, John W. A.

    By performing genotyping, a laboratory contamination involving Q fever was traced back to the antigen preparation used in a commercially available complement fixation test. It was established that such antigen preparations contain relatively high loads of DNA/RNA, making them potential sources of

  2. The effect of lysate of spleen cells after low dose radiation (LDR) on NK activity

    International Nuclear Information System (INIS)

    Lu Duicai; Su Liaoyuan

    2003-01-01

    To find effect of lysate of spleen cells after LDR on NK activity of CD 57 cells or non-CD 57 cells, lysate of spleen cells after LDR were extracted. McAb (anti CD 57 cells) was used to separate CD 57 cells from human peripheral blood by Panning direct method. The CD 57 cells and non-CD 57 cells were used as effective cells. K 562 cells labelled by 3 H-TdR were used as target cells. The ratio of effect cells to target cells was 10:1. NK activity of CD 57 cells or non-CD 5 -7 cells with the lysate of spleen cells after LDR was reflected by the efficiency of anti tumor cells. The 3 H-TdR incorporation in K 562 cells cultured with non-CD 57 cells was significantly lower than that with CD 57 cells. After use of the lysate of spleen cells after LDR, NK activities of CD 57 cells and non-CD 57 cells were 1.24 and 1.58 respectively. They were both increased obviously compared with control groups. The effect of anti K 562 cells of non-CD 57 cells is even greater than that of CD 57 cells. The lysate of spleen cells after LDR can enhance the effect of both non-CD 57 cells and CD 57 cells

  3. Optimizing parameters for clinical-scale production of high IL-12 secreting dendritic cells pulsed with oxidized whole tumor cell lysate

    Directory of Open Access Journals (Sweden)

    Chiang Cheryl L-L

    2011-11-01

    Full Text Available Abstract Background Dendritic cells (DCs are the most potent antigen-presenting cell population for activating tumor-specific T cells. Due to the wide range of methods for generating DCs, there is no common protocol or defined set of criteria to validate the immunogenicity and function of DC vaccines. Methods Monocyte-derived DCs were generated during 4 days of culture with recombinant granulocyte-macrophage colony stimulating factor and interleukin-4, and pulsed with tumor lysate produced by hypochlorous acid oxidation of tumor cells. Different culture parameters for clinical-scale DC preparation were investigated, including: 1 culture media; 2 culture surface; 3 duration of activating DCs with lipopolysaccharide (LPS and interferon (IFN-gamma; 4 method of DC harvest; and 5 cryomedia and final DC product formulation. Results DCs cultured in CellGenix DC media containing 2% human AB serum expressed higher levels of maturation markers following lysate-loading and maturation compared to culturing with serum-free CellGenix DC media or AIM-V media, or 2% AB serum supplemented AIM-V media. Nunclon™Δ surface, but not Corning® tissue-culture treated surface and Corning® ultra-low attachment surface, were suitable for generating an optimal DC phenotype. Recombinant trypsin resulted in reduced major histocompatibility complex (MHC Class I and II expression on mature lysate-loaded DCs, however presentation of MHC Class I peptides by DCs was not impaired and cell viability was higher compared to cell scraping. Preservation of DCs with an infusible cryomedia containing Plasma-Lyte A, dextrose, sodium chloride injection, human serum albumin, and DMSO yielded higher cell viability compared to using human AB serum containing 10% DMSO. Finally, activating DCs for 16 hours with LPS and IFN-γ stimulated robust mixed leukocyte reactions (MLRs, and high IL-12p70 production in vitro that continued for 24 hours after the cryopreserved DCs were thawed and

  4. Poly(hydroxyethyl methacrylate) based magnetic nanoparticles for plasmid DNA purification from Escherichia coli lysate

    Energy Technology Data Exchange (ETDEWEB)

    Percin, Is Latin-Small-Letter-Dotless-I k [Department of Biology, Hacettepe University, Ankara (Turkey); Karakoc, Veyis [Department of Chemistry, Biochemistry Division, Hacettepe University, Ankara (Turkey); Akgoel, Sinan [Department of Biochemistry, Ege University, Izmir (Turkey); Aksoez, Erol [Department of Biology, Hacettepe University, Ankara (Turkey); Denizli, Adil, E-mail: denizli@hacettepe.edu.tr [Department of Chemistry, Biochemistry Division, Hacettepe University, Ankara (Turkey)

    2012-07-01

    The aim of this study is to prepare poly(hydroxyethyl methacrylate-N-methacryloyl-(L)-histidine) [PHEMAH] magnetic nanoparticles for plasmid DNA (pDNA) purification from Escherichia coli (E. coli) cell lysate. Magnetic nanoparticles were produced by surfactant free emulsion polymerization. mPHEMAH nanoparticles were characterized by elemental analysis, Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), vibrating sample magnetometer (VSM), electron spin resonance (ESR), thermogravimetric analyses (TGA) and transmission electron microscopy (TEM). Surface area, average particle size and size distribution were also performed. Specific surface area of the mPHEMAH nanoparticles was found to be 1180 m{sup 2}/g. Elemental analysis of MAH for nitrogen was estimated as 0.18 mmol/g polymer. The amount of pDNA adsorbed onto the mPHEMAH nanoparticles first increased and then reached a saturation value at around 1.0 mg/mL of pDNA concentration. Compared with the mPHEMA nanoparticles (50 {mu}g/g polymer), the pDNA adsorption capacity of the mPHEMAH nanoparticles (154 mg/g polymer) was improved significantly due to the MAH incorporation into the polymeric matrix. The maximum pDNA adsorption was achieved at 25 Degree-Sign C. The overall recovery of pDNA was calculated as 92%. The mPHEMAH nanoparticles could be used six times without decreasing the pDNA adsorption capacity significantly. The results indicate that the PHEMAH nanoparticles promise high selectivity for pDNA. - Highlights: Black-Right-Pointing-Pointer Magnetic nanoparticles have several advantages over conventional adsorbents. Black-Right-Pointing-Pointer MAH acted as the pseudospecific ligand, ligand immobilization step was eliminated. Black-Right-Pointing-Pointer pDNA adsorption amount was 154 mg/g. Black-Right-Pointing-Pointer Fifty-fold capacity increase was obtained when compared to conventional matrices.

  5. Poly(hydroxyethyl methacrylate) based magnetic nanoparticles for plasmid DNA purification from Escherichia coli lysate

    International Nuclear Information System (INIS)

    Perçin, Işık; Karakoç, Veyis; Akgöl, Sinan; Aksöz, Erol; Denizli, Adil

    2012-01-01

    The aim of this study is to prepare poly(hydroxyethyl methacrylate-N-methacryloyl-(L)-histidine) [PHEMAH] magnetic nanoparticles for plasmid DNA (pDNA) purification from Escherichia coli (E. coli) cell lysate. Magnetic nanoparticles were produced by surfactant free emulsion polymerization. mPHEMAH nanoparticles were characterized by elemental analysis, Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), vibrating sample magnetometer (VSM), electron spin resonance (ESR), thermogravimetric analyses (TGA) and transmission electron microscopy (TEM). Surface area, average particle size and size distribution were also performed. Specific surface area of the mPHEMAH nanoparticles was found to be 1180 m 2 /g. Elemental analysis of MAH for nitrogen was estimated as 0.18 mmol/g polymer. The amount of pDNA adsorbed onto the mPHEMAH nanoparticles first increased and then reached a saturation value at around 1.0 mg/mL of pDNA concentration. Compared with the mPHEMA nanoparticles (50 μg/g polymer), the pDNA adsorption capacity of the mPHEMAH nanoparticles (154 mg/g polymer) was improved significantly due to the MAH incorporation into the polymeric matrix. The maximum pDNA adsorption was achieved at 25 °C. The overall recovery of pDNA was calculated as 92%. The mPHEMAH nanoparticles could be used six times without decreasing the pDNA adsorption capacity significantly. The results indicate that the PHEMAH nanoparticles promise high selectivity for pDNA. - Highlights: ► Magnetic nanoparticles have several advantages over conventional adsorbents. ► MAH acted as the pseudospecific ligand, ligand immobilization step was eliminated. ► pDNA adsorption amount was 154 mg/g. ► Fifty-fold capacity increase was obtained when compared to conventional matrices.

  6. Use of platelet lysate for bone regeneration - are we ready for clinical translation?

    Science.gov (United States)

    Altaie, Ala; Owston, Heather; Jones, Elena

    2016-02-26

    Current techniques to improve bone regeneration following trauma or tumour resection involve the use of autograft bone or its substitutes supplemented with osteoinductive growth factors and/or osteogenic cells such as mesenchymal stem cells (MSCs). Although MSCs are most commonly grown in media containing fetal calf serum, human platelet lysate (PL) offers an effective alternative. Bone marrow - derived MSCs grown in PL-containing media display faster proliferation whilst maintaining good osteogenic differentiation capacity. Limited pre-clinical investigations using PL-expanded MSCs seeded onto osteoconductive scaffolds indicate good potential of such constructs to repair bone in vivo. In an alternative approach, nude PL-coated scaffolds without seeded MSCs have been proposed as novel regenerative medicine devices. Even though methods to coat scaffolds with PL vary, in vitro studies suggest that PL allows for MSC adhesion, migration and differentiation inside these scaffolds. Increased new bone formation and vascularisation in comparison to uncoated scaffolds have also been observed in vivo. This review outlines the state-of-the-art research in the field of PL for ex vivo MSC expansion and in vivo bone regeneration. To minimise inconsistency between the studies, further work is required towards standardisation of PL preparation in terms of the starting material, platelet concentration, leukocyte depletion, and the method of platelet lysis. PL quality control procedures and its "potency" assessment are urgently needed, which could include measurements of key growth and attachment factors important for MSC maintenance and differentiation. Furthermore, different PL formulations could be tailor-made for specific bone repair indications. Such measures would undoubtedly speed up clinical translation of PL-based treatments for bone regeneration.

  7. Human platelet lysate as a promising growth-stimulating additive for culturing of stem cells and other cell types.

    Science.gov (United States)

    Shanskii, Ya D; Sergeeva, N S; Sviridova, I K; Kirakozov, M S; Kirsanova, V A; Akhmedova, S A; Antokhin, A I; Chissov, V I

    2013-11-01

    We compared the composition and biological activity of fetal calf serum and platelet lysate from donor platelet concentrate. In platelet lysate, the concentrations of alkaline phosphatase, lactate dehydrogenase, creatinine, and mineral metabolism parameters were lower, while parameters of lipid and protein metabolism were higher than in fetal calf serum. The concentrations of growth factors (platelet-derived (AA, AB, BB), vascular endothelial, insulin-like, and transforming growth factor β) in platelet lysate 1.7-148.7-fold surpassed the corresponding parameters in fetal calf serum. After replacement of fetal calf serum with platelet lysate in the culture medium (0, 25, 50, 75, and 100%), the count of multipotent mesenchymal stromal cells on day 7 (in comparison with day 1) increased by 154.8, 206.6, 228.2, 367.7, and 396.5%, respectively. Thus, platelet lysate can be an adequate non-xenogenic alternative for fetal calf serum.

  8. Immune Response Generated With the Administration of Autologous Dendritic Cells Pulsed With an Allogenic Tumoral Cell-Lines Lysate in Patients With Newly Diagnosed Diffuse Intrinsic Pontine Glioma

    Directory of Open Access Journals (Sweden)

    Daniel Benitez-Ribas

    2018-04-01

    Full Text Available Background and objectiveDiffuse intrinsic pontine glioma (DIPG is a lethal brainstem tumor in children. Dendritic cells (DCs have T-cell stimulatory capacity and, therefore, potential antitumor activity for disease control. DCs vaccines have been shown to reactivate tumor-specific T cells in both clinical and preclinical settings. We designed a phase Ib immunotherapy (IT clinical trial with the use of autologous dendritic cells (ADCs pulsed with an allogeneic tumors cell-lines lysate in patients with newly diagnosed DIPG after irradiation (radiation therapy.MethodsNine patients with newly diagnosed DIPG met enrollment criteria. Autologous dendritic cell vaccines (ADCV were prepared from monocytes obtained by leukapheresis. Five ADCV doses were administered intradermally during induction phase. In the absence of tumor progression, patients received three boosts of tumor lysate every 3 months during the maintenance phase.ResultsVaccine fabrication was feasible in all patients included in the study. Non-specific KLH (9/9 patients and specific (8/9 patients antitumor response was identified by immunologic studies in peripheral blood mononuclear cells (PBMC. Immunological responses were also confirmed in the T lymphocytes isolated from the cerebrospinal fluid (CSF of two patients. Vaccine administration resulted safe in all patients treated with this schema.ConclusionThese preliminary results demonstrate that ADCV preparation is feasible, safe, and generate a DIPG-specific immune response detected in PBMC and CSF. This strategy shows a promising backbone for future schemas of combination IT.

  9. Quantification of platelets and platelet derived growth factors from platelet-rich-plasma (PRP) prepared at different centrifugal force (g) and time.

    Science.gov (United States)

    Arora, Satyam; Doda, Veena; Kotwal, Urvershi; Dogra, Mitu

    2016-02-01

    Platelet derived biomaterials represent a key source of cytokines and growth factors extensively used for tissue regeneration; wound healing and tissue repair. Our study was to quantify platelets and growth factors released by PRP when prepared at different centrifugal force (g) and time. Our study was approved by the institutional ethical committee. One hundred millilitres of whole blood (WB) was collected in bag with CPDA as the anticoagulant(AC); (14 mL for 100 mL WB ratio). Nine aliquots of 10 mL each were made from the bag and set of three aliquots were made a group. PRP was prepared at varying centrifugal force (group A: -110 g, group B: -208 g & group C: -440 g) & time (1: -5 min, 2: -10 min & 3: -20 min). Contents of each PRP prepared were analysed. Commercial sandwich ELISA kits were used to quantify the concentrations of CD62P (Diaclone SAS; France), Platelet derived growth factors-AB (Qayee-Bio; China), transforming growth factor-β1 (DRG; Germany) and vascular endothelial growth factor (Boster Immuno Leader; USA) released in each PRP prepared. Eight volunteers were enrolled in the study (24-30 years). The baseline blood counts of all the volunteers were comparable (p ≥ 0.05). Mean ± SD of platelet yield of all nine groups ranged from 17.2 ± 4.2% to 78.7 ± 5.7%. Each PRP was activated with calcified thromboplastin to quantify the growth factors released by them. Significantly higher (p < 0.05) transforming growth factor-β1 and vascular endothelial growth factor were released compared to the baseline. Our study highlights the variation in both force (g) and time results in changes at cellular level and growth factor concentrations. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Lactobacillus rhamnosus GG Lysate Increases Re-Epithelialization of Keratinocyte Scratch Assays by Promoting Migration.

    Science.gov (United States)

    Mohammedsaeed, Walaa; Cruickshank, Sheena; McBain, Andrew J; O'Neill, Catherine A

    2015-11-05

    A limited number of studies have investigated the potential of probiotics to promote wound healing in the digestive tract. The aim of the current investigation was to determine whether probiotic bacteria or their extracts could be beneficial in cutaneous wound healing. A keratinocyte monolayer scratch assay was used to assess re-epithelialization; which comprises keratinocyte proliferation and migration. Primary human keratinocyte monolayers were scratched then exposed to lysates of Lactobacillus (L) rhamnosus GG, L. reuteri, L. plantarum or L. fermentum. Re-epithelialization of treated monolayers was compared to that of untreated controls. Lysates of L. rhamnosus GG and L. reuteri significantly increased the rate of re-epithelialization, with L. rhamnosus GG being the most efficacious. L. reuteri increased keratinocyte proliferation while L. rhamnosus GG lysate significantly increased proliferation and migration. Microarray analysis of L. rhamnosus GG treated scratches showed increased expression of multiple genes including the chemokine CXCL2 and its receptor CXCR2. These are involved in normal wound healing where they stimulate keratinocyte proliferation and/or migration. Increased protein expression of both CXCL2 and CXCR2 were confirmed by ELISA and immunoblotting. These data demonstrate that L. rhamnosus GG lysate accelerates re-epithelialization of keratinocyte scratch assays, potentially via chemokine receptor pairs that induce keratinocyte migration.

  11. Platelet lysate as an autologous alternative for fetal bovine serum in cardiovascular tissue engineering

    NARCIS (Netherlands)

    Riem Vis, P.W.; Bouten, C.V.C.; Sluijter, J.P.G.; Herwerden, van L.A.; Kluin, J.

    2010-01-01

    There is an ongoing search for alternative tissue culture sera to engineer autologous tissues, since use of fetal bovine serum (FBS) is limited under Good Tissue Practice (GTP) guidelines. We compared FBS with human Platelet-lysate (PL) in media for in vitro cell culture. A threefold increase in

  12. Platelet lysate and chondroitin sulfate loaded contact lenses to heal corneal lesions.

    Science.gov (United States)

    Sandri, Giuseppina; Bonferoni, Maria Cristina; Rossi, Silvia; Delfino, Alessio; Riva, Federica; Icaro Cornaglia, Antonia; Marrubini, Giorgio; Musitelli, Giorgio; Del Fante, Claudia; Perotti, Cesare; Caramella, Carla; Ferrari, Franca

    2016-07-25

    Hemoderivative tear substitutes contain various ephiteliotrophic factors, such as growth factors (GF), involved in ocular surface homeostasis without immunogenic properties. The aim of the present work was the loading of platelet lysate into contact lenses to improve the precorneal permanence of platelet lysate growth factors on the ocular surface to enhance the treatment of corneal lesions. To this purpose, chondroitin sulfate, a sulfated glycosaminoglycan, which is normally present in the extracellular matrix, was associated with platelet lysate. In fact, chondroitin sulfate is capable of electrostatic interaction with positively charged growth factors, in particular, with bFGF, IGF, VEGF, PDGF and TGF-β, resulting in their stabilization and reduced degradation in solution. In the present work, various types of commercially available contact lenses have been loaded with chondroitin sulfate or chondroitin sulfate in association with platelet lysate to achieve a release of growth factors directly onto the corneal surface lesions. One type of contact lenses (PureVision(®)) showed in vitro good proliferation properties towards corneal cells and were able to enhance cut closure in cornea constructs. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Calcium alginate particles for the combined delivery of platelet lysate and vancomycin hydrochloride in chronic skin ulcers.

    Science.gov (United States)

    Mori, Michela; Rossi, Silvia; Bonferoni, Maria Cristina; Ferrari, Franca; Sandri, Giuseppina; Riva, Federica; Del Fante, Claudia; Perotti, Cesare; Caramella, Carla

    2014-01-30

    The aim of the present work was the development of a powder formulation for the combined delivery of platelet lysate and of a model antibiotic drug, vancomycin hydrochloride (VCM), in chronic skin ulcers. Calcium alginate particles were prepared by freeze-drying beads obtained by ionic gelation method. The experimental conditions adopted permitted the complete loading of VCM and of PDGF AB, the growth factor chosen as representative of those contained in PL. Such particles where able to absorb PBS (mimicking wound exudate), to form a gel and to modulate the release of VCM and of PDGF AB. They are characterized by enhancement properties of human fibroblast proliferation due to PL presence. In particular, PL, when loaded in alginate particles, was able not only to increase the number of viable cells, but also the number of cells in proliferative phase. Such properties were comparable to those of fresh PL indicating the capability of calcium alginate particles to load PL bioactive substances without altering their activity. The formulation developed is characterized by an easier and a less painful administration with respect to traditional gauzes and semisolid preparations and permits the loading in the same dosage form of active substances of different nature avoiding eventual incompatibility problems. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. Two-step gasification of cattle manure for hydrogen-rich gas production: Effect of biochar preparation temperature and gasification temperature.

    Science.gov (United States)

    Xin, Ya; Cao, Hongliang; Yuan, Qiaoxia; Wang, Dianlong

    2017-10-01

    Two-step gasification process was proposed to dispose cattle manure for hydrogen rich gas production. The effect of temperature on product distribution and biochar properties were first studied in the pyrolysis-carbonization process. The steam gasification of biochar derived from different pyrolysis-carbonization temperatures was then performed at 750°C and 850°C. The biochar from the pyrolysis-carbonization temperatures of 500°C had high carbon content and low volatiles content. According to the results of gasification stage, the pyrolysis-carbonization temperature of 500°C and the gasification temperature of 850°C were identified as the suitable conditions for hydrogen production. We obtained 1.61m 3 /kg of syngas production, 0.93m 3 /kg of hydrogen yield and 57.58% of hydrogen concentration. This study shows that two-step gasification is an efficient waste-to-hydrogen energy process. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Preparation of triacylglycerols rich in omega-3 fatty acids from sardine oil using a Rhizomucor miehei lipase: focus in the EPA/DHA ratio.

    Science.gov (United States)

    Bispo, Paulo; Batista, Irineu; Bernardino, Raul J; Bandarra, Narcisa Maria

    2014-02-01

    The increasing evidence on the differential biochemical effects of eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) raises the need of n-3 highly unsaturated fatty acid concentrates with different amounts of these fatty acids. In the present work, physicochemical and enzymatic techniques were combined to obtain acylglycerols, mainly triacylglycerols (TAG), rich in n-3 fatty acids. Sardine oil was obtained by washing sardine (Sardina pilchardus) mince with a NaHCO3 solution, hydrolyzed in a KOH-ethanol solution, and concentrated with urea. The esterification reaction was performed in the stoichiometric proportion of substrates for re-esterification to TAG, with 10 % level of Rhizomucor miehei lipase based on the weight of substrates, without any solvent, during 48 h. This procedure led to approximately 88 % of acylglycerols, where more than 66 % were TAG and the concentration of n-3 fatty acids was higher than 60 %, the EPA and DHA ratio (EPA/DHA) was 4:1. The content of DHA in the unesterifed fraction (free fatty acids) increased from 20 to 54 %, while the EPA level in the same fraction decreased from 33 to 12.5 % (EPA/DHA ratio ≈1:4). Computational methods (density functional theory calculations) have been carried out at the B3LYP/6-31G(d,p) level to explain some of the experimental results.

  16. Actualización bibliográfica sobre el uso de preparaciones ricas en plaquetas en la cicatrización de heridas Updated review of the use of platelet rich preparations in wound healing

    Directory of Open Access Journals (Sweden)

    D.A. Mateo de Acosta Andino

    2010-09-01

    Full Text Available Las plaquetas poseen más de 20 factores de crecimiento en sus gránulos. Su secreción promueve la reparación de heridas. La cicatrización es un intrincado proceso en el cual intervienen no solo los factores plaquetarios proangiogénicos sino también factores antiangiogénicos. La preservación por congelación de las plaquetas empleada para su preservación, desestabiliza y activa la membrana plaquetaria alterando sus propiedades. En el campo del tratamiento de heridas recalcitrantes en pacientes debilitados o con enfermedades crónicas, el uso de preparaciones plaquetarias en forma de plasma fresco congelado, las preparaciones plaquetarias sonicadas, las preparaciones congeladas y deshidratadas y el uso de fragmentos de dermis alogénica, confiere efectos beneficiosos, logrando mejores tiempos de cierre en las heridas y menor alteración de la morfología de la cicatriz final. En el presente artículo, describiremos los estudios que han demostrado estos efectos, así como las encrucijadas actuales y los estudios que debemos esperar en un futuro no lejano.Platelets store more than 20 growth factors in their granules. Their secretion promotes wound healing and repair. The intricate wound healing process involves not only the angiogenic platelet factors but also de anti-angiogenic ones. The process of platelet preservation, such as freezing, disrupt the platelet membrane structure and their angiogenic properties causing platelet activation. In the field of wound healing and the management of recalcitrant wounds in debilitated and chronically ill patients, there have been several studies. These studies have focused on the use of the different platelet preparations such as fresh frozen plasma, frozen-dried platelet rich plasma, sonicated platelet rich plasma and the use of alogenic acelular dermis. Platelet preparations posses positive effects on wound healing with little alterations of the final scar tissue. In this article we will describe

  17. Preparation of a calcium-substituted copper-rich yttrium barium copper oxide superconductor from a spray-dried nitrate precursor

    International Nuclear Information System (INIS)

    Gyurov, G.; Khristova, I.; Peshev, P.; Abrashev, M.V.

    1993-01-01

    A calcium-substituted YBa 2 Cu 4 O 8 (1-2-4) high-temperature superconductor is synthesized from a precursor obtained by spray-drying of a nitrate solution containing the corresponding metals in a stoichiometric ratio. The synthesis takes place during one-stage heat-treatment of the precursor at 800 C in an oxygen flow under a pressure of 1 atm within a relatively short period of time, additives as well as intermediate grinding and pressing of the products being not needed. Measurements of the a.c. susceptibility have revealed a very sharp superconducting transition which is comparable with that of samples prepared under a high pressure. The transition in Ca-substituted YBa 2 Cu 4 O 8 occurs at a temperature by about 8 K higher than T c of the Ca-free phase. Raman spectra suggest that during the substitution calcium does not occupy barium positions in the YBa 2 Cu 4 O 8 lattice

  18. Overcoming the bottleneck of platelet lysate supply in large-scale clinical expansion of adipose-derived stem cells: A comparison of fresh versus three types of platelet lysates from outdated buffy coat-derived platelet concentrates.

    Science.gov (United States)

    Glovinski, Peter V; Herly, Mikkel; Mathiasen, Anders B; Svalgaard, Jesper D; Borup, Rehannah; Talman, Maj-Lis M; Elberg, Jens J; Kølle, Stig-Frederik T; Drzewiecki, Krzysztof T; Fischer-Nielsen, Anne

    2017-02-01

    Platelet lysates (PL) represent a promising replacement for xenogenic growth supplement for adipose-derived stem cell (ASC) expansions. However, fresh platelets from human blood donors are not clinically feasible for large-scale cell expansion based on their limited supply. Therefore, we tested PLs prepared via three methods from outdated buffy coat-derived platelet concentrates (PCs) to establish an efficient and feasible expansion of ASCs for clinical use. PLs were prepared by the freeze-thaw method from freshly drawn platelets or from outdated buffy coat-derived PCs stored in the platelet additive solution, InterSol. Three types of PLs were prepared from outdated PCs with platelets suspended in either (1) InterSol (not manipulated), (2) InterSol + supplemented with plasma or (3) plasma alone (InterSol removed). Using these PLs, we compared ASC population doubling time, cell yield, differentiation potential and cell surface markers. Gene expression profiles were analyzed using microarray assays, and growth factor concentrations in the cell culture medium were measured using enzyme-linked immunosorbent assay (ELISA). Of the three PL compositions produced from outdated PCs, removal of Intersol and resuspension in plasma prior to the first freezing process was overall the best. This specific outdated PL induced ASC growth kinetics, surface markers, plastic adherence and differentiation potentials comparable with PL from fresh platelets. ASCs expanded in PL from fresh versus outdated PCs exhibited different expressions of 17 overlapping genes, of which 10 were involved in cellular proliferation, although not significantly reflected by cell growth. Only minor differences in growth factor turnover were observed. PLs from outdated platelets may be an efficient and reliable source of human growth supplement allowing for large-scale ASC expansion for clinical use. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights

  19. Combinations of SPR and MS for Characterizations of Native and Recombinant Proteins in Cell Lysates

    DEFF Research Database (Denmark)

    Borch, Jonas; Roepstorff, Peter

    2006-01-01

    Surface plasmon resonance and mass spectrometry (SPR-MS) has been combined for quality check of recombinant 6xHis-tagged 14-3-3 proteins expressed in Escherichia coli. Lysates were injected over an SPR sensorchip with immobilized Ni2+ for SPR analysis of the specific Ni2+ binding response...... and stability. To validate the identity, intactness and homogeneity of the captured proteins were eluted for mass spectrometric analysis of intact molecular weight and peptide mass mapping. Additionally, the captured recombinant proteins were investigated for specific binding to known phosphorylated ligands...... of 14-3-3 proteins in order to test their activity. Specific binding of recombinant and native 14-3-3 proteins in complex mixtures to immobilized phosphopeptides and subsequent elution was also tested by SPR-MS. Ammonium sulfate precipitate fractions from lysates of E. coli expressing 14-3-3 protein...

  20. Highly Efficient In Vitro Reparative Behaviour of Dental Pulp Stem Cells Cultured with Standardised Platelet Lysate Supplementation

    Directory of Open Access Journals (Sweden)

    Pasquale Marrazzo

    2016-01-01

    Full Text Available Dental pulp is an accessible source of multipotent mesenchymal stromal cells (MSCs. The perspective role of dental pulp stem cells (DPSCs in regenerative medicine demands an in vitro expansion and in vivo delivery which must deal with the safety issues about animal serum, usually required in cell culture practice. Human platelet lysate (PL contains autologous growth factors and has been considered as valuable alternative to fetal bovine serum (FBS in cell cultures. The optimum concentration to be added of such supplement is highly dependent on its preparation whose variability limits comparability of results. By in vitro experiments, we aimed to evaluate a standardised formulation of pooled PL. A low selected concentration of PL (1% was able to support the growth and maintain the viability of the DPSCs. The use of PL in cell cultures did not impair cell surface signature typically expressed by MSCs and even upregulated the transcription of Sox2. Interestingly, DPSCs cultured in presence of PL exhibited a higher healing rate after injury and are less susceptible to toxicity mediated by exogenous H2O2 than those cultured with FBS. Moreover, PL addition was shown as a suitable option for protocols promoting osteogenic and chondrogenic differentiation of DPSCs. Taken together, our results indicated that PL is a valid substitute of FBS to culture and differentiate DPSCs for clinical-grade use.

  1. Highly Efficient In Vitro Reparative Behaviour of Dental Pulp Stem Cells Cultured with Standardised Platelet Lysate Supplementation.

    Science.gov (United States)

    Marrazzo, Pasquale; Paduano, Francesco; Palmieri, Francesca; Marrelli, Massimo; Tatullo, Marco

    2016-01-01

    Dental pulp is an accessible source of multipotent mesenchymal stromal cells (MSCs). The perspective role of dental pulp stem cells (DPSCs) in regenerative medicine demands an in vitro expansion and in vivo delivery which must deal with the safety issues about animal serum, usually required in cell culture practice. Human platelet lysate (PL) contains autologous growth factors and has been considered as valuable alternative to fetal bovine serum (FBS) in cell cultures. The optimum concentration to be added of such supplement is highly dependent on its preparation whose variability limits comparability of results. By in vitro experiments, we aimed to evaluate a standardised formulation of pooled PL. A low selected concentration of PL (1%) was able to support the growth and maintain the viability of the DPSCs. The use of PL in cell cultures did not impair cell surface signature typically expressed by MSCs and even upregulated the transcription of Sox2. Interestingly, DPSCs cultured in presence of PL exhibited a higher healing rate after injury and are less susceptible to toxicity mediated by exogenous H 2 O 2 than those cultured with FBS. Moreover, PL addition was shown as a suitable option for protocols promoting osteogenic and chondrogenic differentiation of DPSCs. Taken together, our results indicated that PL is a valid substitute of FBS to culture and differentiate DPSCs for clinical-grade use.

  2. FRET microscopy autologous tumor lysate processing in mature dendritic cell vaccine therapy

    Directory of Open Access Journals (Sweden)

    Ridolfi Ruggero

    2010-06-01

    Full Text Available Abstract Background Antigen processing by dendritic cells (DC exposed to specific stimuli has been well characterized in biological studies. Nonetheless, the question of whether autologous whole tumor lysates (as used in clinical trials are similarly processed by these cells has not yet been resolved. Methods In this study, we examined the transfer of peptides from whole tumor lysates to major histocompatibility complex class II molecules (MHC II in mature dendritic cells (mDC from a patient with advanced melanoma. Tumor antigenic peptides-MHC II proximity was revealed by Förster Resonance Energy Transfer (FRET measurements, which effectively extends the application of fluorescence microscopy to the molecular level ( Results We detected significant energy transfer between donor and acceptor-labelled antibodies against HLA-DR at the membrane surface of mDC. FRET data indicated that fluorescent peptide-loaded MHC II molecules start to accumulate on mDC membranes at 16 hr from the maturation stimulus, steeply increasing at 22 hr with sustained higher FRET detected up to 46 hr. Conclusions The results obtained imply that the patient mDC correctly processed the tumor specific antigens and their display on the mDC surface may be effective for several days. These observations support the rationale for immunogenic efficacy of autologous tumor lysates.

  3. Vaccination with OK-432 followed by TC-1 tumor lysate leads to significant antitumor effects.

    Science.gov (United States)

    Chen, I-Ju; Yen, Chih-Feng; Lin, Kun-Ju; Lee, Chyi-Long; Soong, Yung-Kuei; Lai, Chyong-Huey; Lin, Cheng-Tao

    2011-07-01

    Human papillomavirus (HPV) infects large numbers of women worldwide and is present in more than 99% of all cervical cancer. TC-1 cell is a cell line with high expression of E7 antigen of HPV type 16 and its cell lysate has been demonstrated as an ideal inducer of E7-specific, antitumor immunity. OK-432 (Picibanil), a penicillin-killed Streptococcus pyogenes, has been reported with potent immunomodulation properties in cancer treatment by stimulating the maturation of dendritic cells (DCs) and secretion of Th-1 type cytokines. The current study demonstrated that a protocol to immunize the C57BL/6 mice with OK-432 followed by treatment with TC-1 lysate can generate markedly increased immune responses of E7-specific CD4(+) T cells and a moderate increase of natural killer (NK) cell, as well as a satisfactorily protective and therapeutic antitumor effect by triggering the DCs to prime T cells. Depletion of lymphocyte subset in vivo suggested that the antitumor effects could be dominantly executed by CD8+ T cells and followed by NK cells, and both of these reactions were induced by the generation of robust E7-specific CD4(+) T helper cell response. These findings warrant OK-432 combination with tumor-lysate as an effective and safe vaccine in future clinical application of cervical cancer.

  4. PREPARATION OF NOVOLACS USING PHENOLIC RICH ...

    African Journals Online (AJOL)

    Preferred Customer

    Institute of Chemical Industry of Forest Products, CAF; National Engineering Laboratory for. Biomass Chemical Utilization; Key and Open Laboratory on Forest Chemical Engineering,. SFA, Nanjing 210042 ... INTRODUCTION ..... chemical composition of the bio-oil tends to change toward thermodynamic equilibrium and.

  5. Effect of Different Adjuvants on Protection and Side-Effects Induced by Helicobacter suis Whole-Cell Lysate Vaccination.

    Directory of Open Access Journals (Sweden)

    Iris Bosschem

    Full Text Available Helicobacter suis (H. suis is a widespread porcine gastric pathogen, which is also of zoonotic importance. The first goal of this study was to investigate the efficacy of several vaccine adjuvants (CpG-DNA, Curdlan, Freund's Complete and Incomplete, Cholera toxin, administered either subcutaneously or intranasally along with H. suis whole-cell lysate, to protect against subsequent H. suis challenge in a BALB/c infection model. Subcutaneous immunization with Freund's complete (FC/lysate and intranasal immunization with Cholera toxin (CT/lysate were shown to be the best options for vaccination against H. suis, as determined by the amount of colonizing H. suis bacteria in the stomach, although adverse effects such as post-immunization gastritis/pseudo-pyloric metaplasia and increased mortality were observed, respectively. Therefore, we decided to test alternative strategies, including sublingual vaccine administration, to reduce the unwanted side-effects. A CCR4 antagonist that transiently inhibits the migration of regulatory T cells was also included as a new adjuvant in this second study. Results confirmed that immunization with CT (intranasally or sublingually is among the most effective vaccination protocols, but increased mortality was still observed. In the groups immunized subcutaneously with FC/lysate and CCR4 antagonist/lysate, a significant protection was observed. Compared to the FC/lysate immunized group, gastric pseudo-pyloric metaplasia was less severe or even absent in the CCR4 antagonist/lysate immunized group. In general, an inverse correlation was observed between IFN-γ, IL-4, IL-17, KC, MIP-2 and LIX mRNA expression and H. suis colonization density, whereas lower IL-10 expression levels were observed in partially protected animals.

  6. PEMBUATAN BAHAN BAKU SPREADS KAYA KAROTEN DARI MINYAK SAWIT MERAH MELALUI INTERESTERIFIKASI ENZIMATIK MENGGUNAKAN REAKTOR BATCH [Preparation of Red Palm Oil Based-Spreads Stock Rich in Carotene Through Enzymatic Interesterification in Batch-type Reactor

    Directory of Open Access Journals (Sweden)

    Nur Wulandari1,2

    2012-12-01

    Full Text Available Enzymatic interesterification of red palm oil (a mixture of red palm olein/RPO and red palm stearin/RPS in 1:1 weight ratio and coconut oil (CNO blends of varying proportions using a non-specific immobilized Candida antartica lipase (Novozyme 435 was studied for the preparation of spread stock. The interesterification reaction was held in a batch-type reactor. Two substrate blends were chosen for the production of spread stock i.e. 77.5:22,5 and 82.5:17.5 (RPO/RPS:CNO, by weight through enzymatic interesterification in three different reaction times (2, 4, and 6 hours. The interesterification reactions were conducted at 60°C, 200 rpm agitation speed and 10% of Novozyme 435. The interesterified products were evaluated for their physical characteristics (slip melting point or SMP and solid fat content or SFC and chemical characteristics (carotene retention, moisture content, and free fatty acid/FFA content. All of the interesterified products had lower SFC and SMP as compared to the initial blends. The SMP and SFC increased in longer reaction times. The SMP ranged from 30.8°C to 34.9°C. The carotene retention ranged from 74.80% to 81.08%, while the moisture content and FFA content increased in longer reaction times. The interesterified products had desirable physical properties for possible use as a spread stock rich in carotene.

  7. Large-scale preparation of plasmid DNA.

    Science.gov (United States)

    Heilig, J S; Elbing, K L; Brent, R

    2001-05-01

    Although the need for large quantities of plasmid DNA has diminished as techniques for manipulating small quantities of DNA have improved, occasionally large amounts of high-quality plasmid DNA are desired. This unit describes the preparation of milligram quantities of highly purified plasmid DNA. The first part of the unit describes three methods for preparing crude lysates enriched in plasmid DNA from bacterial cells grown in liquid culture: alkaline lysis, boiling, and Triton lysis. The second part describes four methods for purifying plasmid DNA in such lysates away from contaminating RNA and protein: CsCl/ethidium bromide density gradient centrifugation, polyethylene glycol (PEG) precipitation, anion-exchange chromatography, and size-exclusion chromatography.

  8. Immune response in the lungs following oral immunization with bacterial lysates of respiratory pathogens.

    OpenAIRE

    Ruedl, C; Frühwirth, M; Wick, G; Wolf, H

    1994-01-01

    We have investigated the local immune response of the BALB/c mouse respiratory tract after oral immunization with a bacterial lysate of seven common respiratory pathogens. After two immunization on five consecutive days, we examined the immunoglobulin (immunoglobulin G [IgG], IgM, and IgA) secretion rates of cells isolated from the lungs and compared them with those of spleen cells of orally immunized and nonimmunized animals by using a new test system based on time-resolved fluorescence. The...

  9. Comparative Analysis of Click Chemistry Mediated Activity-Based Protein Profiling in Cell Lysates

    Directory of Open Access Journals (Sweden)

    Yinliang Yang

    2013-10-01

    Full Text Available Activity-based protein profiling uses chemical probes that covalently attach to active enzyme targets. Probes with conventional tags have disadvantages, such as limited cell permeability or steric hindrance around the reactive group. A tandem labeling strategy with click chemistry is now widely used to study enzyme targets in situ and in vivo. Herein, the probes are reacted in live cells, whereas the ensuing detection by click chemistry takes place in cell lysates. We here make a comparison of the efficiency of the activity-based tandem labeling strategy by using Cu(I-catalyzed and strain-promoted click chemistry, different ligands and different lysis conditions.

  10. Determination of Immunogenic Relevant Antigens in the Excretory-Secretory (ES Products and the Lysates of Ascaridia galli Larvae

    Directory of Open Access Journals (Sweden)

    S Saffi

    2008-04-01

    Full Text Available Background: Ascaridia galli, the largest nematode of small intestine of birds, especially the native poultry, may give rise to serious illness, pathological defects and economical losses even in modern poultry production systems. Although various measures have been undertaken to vaccinate poultry against A.galli, no satisfactory results were obtained so far. However, there is no report on the efficacy of excretory-secretory (ES proteins of A.galli larvae in immunization of poultry. Thus, the aim of the present research project was based on the use of the ES products of the larvae, in order to find the protective anti­gens.Methods: Five hundred native poultry were autopsied and adult A.galli was removed form their intestines. The eggs were harvested form the uterus of female worms and cultured at 25 ˚C in water containing 0.1 N sulphuric acid for almost a fort­night. The larvae were then freed mechanically and kept in Earl's salt solution for a few days. The supernatant solution of alive larvae containing the ES products of the larvae, as well as the sonicated alive and dead larvae, was analyzed by SDS-PAGE.Result: Many protein fractions of 15 kDa up to 200 kDa were demonstrated in lysate of these larvae. Using the serum of a hen, infected with a high numbers of A.galli, an immunogenic antigen was identified between 55 kDa to 72 kDa by Western blotting procedure.Conclusion: Finding the protein band between 55 and 72 kDa can be promising for preparation of vaccine, though more investigations are needed to prove the protective ability of this antigen.

  11. Treatment with platelet lysate induces endothelial differentation of bone marrow mesenchymal stem cells under fluid shear stress.

    Science.gov (United States)

    Homayouni Moghadam, Farshad; Tayebi, Tahereh; Moradi, Alireza; Nadri, Hamid; Barzegar, Kazem; Eslami, Gilda

    2014-01-01

    By considering stem cell-based therapies as a new hope for the treatment of some tragic diseases, marrow stromal cells or marrow mesenchymal stem cells (MSCs) were considered as a suitable and safe multipotential cell source for this new therapeutic approach. For this purpose, many investigations have been performed on differentiation of MSCs toward specific cell lines to overcome the demand for providing the organ specific cells for cell therapy or preparation of engineered tissues. In the present study, differentiation of MSCs to endothelial cells (ECs) by mechanical and chemical stimulation was evaluated. Fluid shear stress (FSS) was used as mechanical inducer, while platelet lysate (PL) and estradiol (E) were used as chemical induction factors. MSCs were placed under FSS with different forces (2, 5 and 10dyn/cm(2)) for different periods (6, 12 and 24 hours). In some groups, PL and E were added to the culture media to evaluate their effect on expression of EC specific markers. This investigation revealed that FSS with low tension (2.5-5 dyn/cm(2)) for a long time (24 hours) or high tension (10 dyn/cm(2)) in short time (6 hours) in the presence of PL could differentiate MSCs toward ECs. The presence of PL was necessary for initiation of endothelial differentiation, and in the absence of PL, there was not any expression of CD34 and Cadherin5 (Cdh5) among cells. Adding E to the culture medium did not change the rate of endothelial differentiation under FSS. Generated endothelial progenitors could produce von Willebrand factor (vWF) after two weeks culture and also they formed tubular structures after culture on matrigel.

  12. [The lysate and recombinant antigens in ELISA-test-systems for diagnostic of herpes simplex].

    Science.gov (United States)

    Ganova, L A; Kovtoniuk, G V; Korshun, L N; Kiseleva, E K; Tereshchenko, M I; Vudmaska, M I; Moĭsa, L N; Shevchuk, V A; Spivak, N Ia

    2014-08-01

    The lysate and recombinant antigens of various production included informula of ELISA-test-systems were analyzed. The ELISA-test-systems are used for detection of IgG to Herpes simplex virus type I and II. For testing the panel of serums PTH 201 (BBI Inc.) were used. The samples of this panel contain antibodies to Herpes simplex virus type I and II in mixed titers. The 69 serums of donors were used too (17 samples had IgG to Herpes simplex virus type I, 23 samples to Herpes simplex virus type II and 29 samples had no antibodies to Herpes simplex virus). The diagnostic capacity of mixture of recombinant antigens gG1 Herpes simplex virus type I and gG2 Herpes simplex virus type II (The research-and-production complex "DiaprofMed") was comparable with mixture of lysate antigen Herpes simplex virus type I and II (Membrane) EIE Antigen ("Virion Ltd."). In the test-systems for differentiation of IgG to Herpes simplex virus type I the recombinant antigen gG1 Herpes simplex virus type I proved to be comparable with commercial analogue Herpes simplex virus-1 gG1M ("Viral Therapeutics Inc."'). At the same time, capacity to detect IgG to Herpes simplex virus type II in recombinant protein gG2 Herpes simplex virus type II is significantly higher than in its analogue Herpes simplex virus-2 gG2c ("Viral Therapeutics Inc.").

  13. The Effect of Autologous Platelet Lysate Eye Drops: An In Vivo Confocal Microscopy Study

    Directory of Open Access Journals (Sweden)

    Antonio M. Fea

    2016-01-01

    Full Text Available Purpose. To determine the effectiveness of autologous platelet lysate (APL eye drops in patients with primary Sjögren syndrome (SS dry eye, refractory to standard therapy, in comparison with patients treated with artificial tears. We focused on the effect of APL on cornea morphology with the in vivo confocal microscopy (IVCM. Methods. Patients were assigned to two groups: group A used autologous platelet lysate QID, and group B used preservative-free artificial tears QID, for 90 days. Ophthalmological assessments included ocular surface disease index (OSDI, best corrected visual acuity (BCVA, Schirmer test, fluorescein score, and breakup time (BUT. A subgroup of patients in group A underwent IVCM: corneal basal epithelium, subbasal nerves, Langerhans cells, anterior stroma activated keratocytes, and reflectivity were evaluated. Results. 60 eyes of 30 patients were enrolled; in group A (n=20 patients mean OSDI, fluorescein score, and BUT showed significant improvement compared with group B (n=10 patients. The IVCM showed a significant increase in basal epithelium cells density and subbasal nerve plexus density and number and a decrease in Langerhans cells density (p<0.05. Conclusion. APL was found effective in the treatment of SS dry eye. IVCM seems to be a useful tool to visualize cornea morphologic modifications.

  14. The Effect of Autologous Platelet Lysate Eye Drops: An In Vivo Confocal Microscopy Study.

    Science.gov (United States)

    Fea, Antonio M; Aragno, Vittoria; Testa, Valeria; Machetta, Federica; Parisi, Simone; D'Antico, Sergio; Spinetta, Roberta; Fusaro, Enrico; Grignolo, Federico M

    2016-01-01

    Purpose. To determine the effectiveness of autologous platelet lysate (APL) eye drops in patients with primary Sjögren syndrome (SS) dry eye, refractory to standard therapy, in comparison with patients treated with artificial tears. We focused on the effect of APL on cornea morphology with the in vivo confocal microscopy (IVCM). Methods. Patients were assigned to two groups: group A used autologous platelet lysate QID, and group B used preservative-free artificial tears QID, for 90 days. Ophthalmological assessments included ocular surface disease index (OSDI), best corrected visual acuity (BCVA), Schirmer test, fluorescein score, and breakup time (BUT). A subgroup of patients in group A underwent IVCM: corneal basal epithelium, subbasal nerves, Langerhans cells, anterior stroma activated keratocytes, and reflectivity were evaluated. Results. 60 eyes of 30 patients were enrolled; in group A (n = 20 patients) mean OSDI, fluorescein score, and BUT showed significant improvement compared with group B (n = 10 patients). The IVCM showed a significant increase in basal epithelium cells density and subbasal nerve plexus density and number and a decrease in Langerhans cells density (p < 0.05). Conclusion. APL was found effective in the treatment of SS dry eye. IVCM seems to be a useful tool to visualize cornea morphologic modifications.

  15. Measuring O-GlcNAc cleavage by OGA and cell lysates on a peptide microarray.

    Science.gov (United States)

    Sharif, Suhela; Shi, Jie; Bourakba, Mostafa; Ruijtenbeek, Rob; Pieters, Roland J

    2017-09-01

    O-GlcNAcylation is a post-translational modification resulting from the addition of an N-acetylglucosamine moiety to the hydroxyl groups of serine and threonine residues of nuclear and cytoplasmic proteins. In addition, O-GlcNAcylated proteins can be phosphorylated, which suggests the possibility for crosstalk between O-GlcNAcylation and phosphorylation. Dysregulation of O-GlcNAcylation affects cell signaling, transcriptional regulation, cell cycle control and can e.g. lead to tumorigenesis and tumor metastasis. There is a strong demand for efficient analytical techniques to better detect and investigate this abundant modification and its role in cancer. Herein we demonstrated the utility of an O-GlcNAcylated peptide array to examine O-GlcNAcase (OGA) activity and substrate specificity of both purified protein as well cell lysates of different cancer cell lines. Using this microarray, we clearly observed OGA activity and also inhibition thereof by OGA inhibitor thiamet G. Interestingly, different levels of OGA activity were observed of lysates derived from different cancer cell lines. This suggests that the tool may be useful in cancer research and biomarker development. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Streamlined Membrane Proteome Preparation for Shotgun Proteomics Analysis with Triton X-100 Cloud Point Extraction and Nanodiamond Solid Phase Extraction

    Directory of Open Access Journals (Sweden)

    Minh D. Pham

    2016-05-01

    Full Text Available While mass spectrometry (MS plays a key role in proteomics research, characterization of membrane proteins (MP by MS has been a challenging task because of the presence of a host of interfering chemicals in the hydrophobic protein extraction process, and the low protease digestion efficiency. We report a sample preparation protocol, two-phase separation with Triton X-100, induced by NaCl, with coomassie blue added for visualizing the detergent-rich phase, which streamlines MP preparation for SDS-PAGE analysis of intact MP and shot-gun proteomic analyses. MP solubilized in the detergent-rich milieu were then sequentially extracted and fractionated by surface-oxidized nanodiamond (ND at three pHs. The high MP affinity of ND enabled extensive washes for removal of salts, detergents, lipids, and other impurities to ensure uncompromised ensuing purposes, notably enhanced proteolytic digestion and down-stream mass spectrometric (MS analyses. Starting with a typical membranous cellular lysate fraction harvested with centrifugation/ultracentrifugation, MP purities of 70%, based on number (not weight of proteins identified by MS, was achieved; the weight-based purity can be expected to be much higher.

  17. Validated hemoglobin-depletion approach for red blood cell lysate proteome analysis by means of 2D PAGE and Orbitrap MS.

    Science.gov (United States)

    Walpurgis, Katja; Kohler, Maxie; Thomas, Andreas; Wenzel, Folker; Geyer, Hans; Schänzer, Wilhelm; Thevis, Mario

    2012-08-01

    The analysis of the cytosolic red blood cell (RBC) proteome is negatively affected by the high intracellular amount of hemoglobin complicating the detection of low-abundant cytosolic proteins. In this study, an alternative approach for the preparation of hemoglobin-depleted RBC lysates is presented, which was established in combination with downstream 2D PAGE analysis and Orbitrap MS. Hemoglobin removal was accomplished by using HemoVoid(TM) depletion reagent, which enabled a very efficient enrichment of low-abundant proteins by simultaneously reducing the hemoglobin concentration of the sample. After defining selected sample preparation protocol characteristics including specificity/selectivity, precision and linearity, a 2D reference map (pH 4-7) of the cytosolic RBC proteome was generated and a total of 189 different proteins were identified. Thus, the presented approach proved to be highly suitable to prepare reproducible high-resolution 2D protein maps of the RBC cytosol and provides a helpful tool for future studies investigating disease- or storage-induced changes of the cytosolic RBC proteome. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Preparation of recombinant coat protein of Prunus necrotic ringspot virus.

    Science.gov (United States)

    Petrzik, K; Mráz, I; Kubelková, D

    2001-02-01

    The coat protein (CP) gene of Prunus necrotic ringspot virus (PNRSV) was cloned into pET 16b vector and expressed in Escherichia coli. CP-enriched fractions were prepared from whole cell lysate by differential centrifugation. The fraction sedimenting at 20,000 x g for 30 mins was used for preparation of a rabbit antiserum to CP. This antiserum had a titer of 1:2048 and reacted in a double-antibody sandwich ELISA (DAS-ELISA).

  19. Heat inactivation of a norovirus surrogate in cell culture lysate, abalone meat, and abalone viscera.

    Science.gov (United States)

    Park, Shin Young; Bae, San-Cheong; Ha, Sang-Do

    2015-03-01

    The current study examined the effects of temperature and heat treatment duration on murine norovirus-1 (MNV-1) from both viral cell culture lysate (7-8 log10 PFU) and experimentally contaminated abalone meat and viscera (5-6 log10 PFU) as a model of human norovirus (NoV). MNV-1 titers in cell culture lysate, abalone meat, and abalone viscera were gradually reduced to 1.93-4.55, 1.79-3.00, and 2.26-3.26 log10 PFU/ml, respectively, after treatment at 70 °C for 1-10 min. Treatment at 85 °C for 1-5 min gradually reduced MNV-1 titers in abalone meat to 2.71-4.15 log10 PFU/ml. MNV-1 titers in abalone viscera were gradually reduced to 2.91-3.46 log10 PFU/ml after treatment at 85 °C for 1-3 min. No significant difference (P > 0.05) was found in MNV-1 titers in the abalone meat and viscera among treatment groups (70 °C for 5 min, 70 °C for 3 min, and 85 °C for 1 min). Complete inactivation of MNV-1 in cell culture lysate was determined at 85 °C for ≥1 min and 100 °C for ≥0.5 min. Complete inactivation of MNV-1 in abalone was determined at 100 °C for ≥0.5 min for meat, and 85 °C for 5 min and 100 °C for ≥0.5 min for viscera. At treatments at 70 °C, the Td-values (3 log reduction time) were significantly lower (P abalone meat (6.07) and viscera (10.73). Td = 3 values were not significantly different (P > 0.05) between abalone meat (1.78) and abalone viscera (1.33) at treatments at 85 °C. This study suggests that 100 °C for ≥0.5 min could potentially be used to inactivate NoV in molluscan shellfishes, including viscera.

  20. BACTERIAL LYSATES FOR TOPICAL APPLICATION IN PREVENTION AND TREATMENT OF CHRONIC TONSILLITIS AMONG CHILDREN

    Directory of Open Access Journals (Sweden)

    T.I. Garashchenko

    2006-01-01

    Full Text Available The authors have carried on the research of the influence Imudon exerts (during topical application on the run of chronic tonsillitis among 48 children aged between 5 and 10 years old, being dispensary registrants. The sublingual application of a medicine 6 pills daily within 20 days demonstrated the frequency reduction of chronic tonsillitis acerbations by 2.9 times, as well as the reduction of total need in systemic antibacterial treatment by 10 times. Apart from that, the frequency of S. pyogenes group a exposure reduced by 3 times. The researchers noticed the tendency to normalization of pharynx biocenosis. Thus, Imudon may be recommended for the daily courses of treatment to the people, suffering from chronic tonsillitis, palatine tonsil auxesis and recurrent tonsillo-pharyngites.Key words: chronic tonsillitis, children, prevention, bacterial lysates.

  1. Increased efficiency of exogenous messenger RNA translation in a Krebs ascites cell lysate.

    Science.gov (United States)

    Metafora, S; Terada, M; Dow, L W; Marks, P A; Bank, A

    1972-05-01

    Addition of a 0.5 M KCl wash fraction from rabbit reticulocyte ribosomes causes a 3- to 10-fold increase in the extent of translation of natural mRNAs by Krebs-cell lysates. In the presence of the wash fraction, 1 pmol of rabbit or mouse 10S RNA directs the incorporation of 80 pmol of leucine into rabbit globin. The addition of human 10S RNA results in the synthesis of equal amounts of human alpha and beta chains, identified by column chromatography. The stimulation by the wash fraction is almost completely dependent on added mammalian tRNA. In contrast to the wash fraction from rabbit reticulocytes, the wash fraction isolated from Krebs-cell ribosomes is inhibitory to both endogenous and exogenous mRNA translation. The stimulation by the wash fraction from rabbit ribosomes is not specific for globin mRNAs, but also increases endogenous, phage Qbeta, and viral RNA-directed protein synthesis.

  2. Autologous platelet lysate local injections for the treatment of refractory lateral epicondylitis.

    Science.gov (United States)

    Tan, Xun-xiang; Ju, Hai-yang; Yan, Wei; Jiang, Hong-jiang; Su, Jin-ping; Dong, Hua-jun; Wang, Ling-shuang; Zou, De-bao

    2016-01-25

    The purpose of this study was to investigate the efficacy and safety of autologous platelet lysate (APL) local injections in reducing pain and improving function in patients with refractory lateral epicondylitis. A total of 56 patients with refractory lateral epicondylitis were enrolled in this study. All the patients received three injections in one course of treatment. Subjective assessments include visual analog scale (VAS) pain score and Mayo elbow score before injection (baseline) and at 1, 6, and 12 months after injection. Significant differences were observed in VAS and Mayo scores at baseline and at 1, 6, and 12 months after injection. Overall, the injections of APL improved local symptoms and all the patients recovered to normal elbow function with 12 months follow-up. Local injections of APL resulted in favorable clinical outcomes for the treatment of lateral epicondylitis. APL could be clinically effective in the treatment of lateral epicondylitis.

  3. Tailor-made purified human platelet lysate concentrated in neurotrophins for treatment of Parkinson's disease.

    Science.gov (United States)

    Chou, Ming-Li; Wu, Joe-Wei; Gouel, Flore; Jonneaux, Aurélie; Timmerman, Kelly; Renn, Ting-Yi; Laloux, Charlotte; Chang, Hung-Ming; Lin, Liang-Tzung; Devedjian, Jean-Christophe; Devos, David; Burnouf, Thierry

    2017-10-01

    Human platelet lysates (PLs), which contain multiple neurotrophins, have been proposed for treating neurodegenerative disorders, including Parkinson's disease (PD). However, current PLs suspended in plasma have high protein content and contain fibrinogen/fibrin and, following activation, also proteolytic and thrombogenic enzymes. Upon brain administration, such PLs may saturate the cerebrospinal fluid and exert neurotoxicity. We assessed whether purified PLs, concentrated in neurotrophins, protected dopaminergic neurons in PD models. Platelet concentrates were collected by apheresis and centrifuged to eliminate plasma and recover the platelets. Platelets were lysed by freeze-thaw cycles, and the 10-fold concentrated platelet pellet lysates (PPLs) were heat-treated (at 56 °C for 30 min). The heat-treated PPLs were low in total proteins, depleted in both plasma and platelet fibrinogen, and devoid of thrombogenic and proteolytic activities. They exerted very high neuroprotective activity when non-oncogenic, Lund human mesencephalic (LUHMES) cells that had differentiated into dopaminergic neurons were exposed to the MPP + neurotoxin. Heat treatment improved the neuroprotection and inactivated the neurotoxic blood-borne hepatitis C virus. PPL did not induce inflammation in BV2 microglial cells and inhibited COX-2 expression upon lipopolysaccharide exposure. Intranasal administration in mice revealed (a) diffusion of neurotrophins in the striatum and cortex, and (b) MPTP intoxication neuroprotection in the substantia nigra and striatum and the absence of neuroinflammation. These dedicated heat-treated PPLs can be a safe and valuable candidate for a therapeutic strategy for PD. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Adjuvant treatment with the bacterial lysate (OM-85 improves management of atopic dermatitis: A randomized study.

    Directory of Open Access Journals (Sweden)

    Christine Bodemer

    Full Text Available Environmental factors play a major role on atopic dermatitis (AD which shows a constant rise in prevalence in western countries over the last decades. The Hygiene Hypothesis suggesting an inverse relationship between incidence of infections and the increase in atopic diseases in these countries, is one of the working hypothesis proposed to explain this trend.This study tested the efficacy and safety of oral administration of the bacterial lysate OM-85 (Broncho-Vaxom®, Broncho-Munal®, Ommunal®, Paxoral®, Vaxoral®, in the treatment of established AD in children.Children aged 6 months to 7 years, with confirmed AD diagnosis, were randomized in a double-blind, placebo-controlled trial to receive, in addition to conventional treatment with emollients and topical corticosteroids, 3.5mg of the bacterial extract OM-85 or placebo daily for 9 months. The primary end-point was the difference between groups in the occurrence of new flares (NF during the study period, evaluated by Hazard Ratio (HR derived from conditional Cox proportional hazard regression models accounting for repeated events.Among the 179 randomized children, 170 were analysed, 88 in the OM-85 and 82 in the placebo group. As expected most children in both treatment groups experienced at least 1 NF during the study period (75 (85% patients in the OM-85 group and 72 (88% in the placebo group. Patients treated with OM-85 as adjuvant therapy had significantly fewer and delayed NFs (HR of repeated flares = 0.80; 95% confidence interval (CI: 0.67-0.96, also when potential confounding factors, as family history of atopy and corticosteroids use, were taken into account (HR = 0.82; 95% CI: 0.69-0.98. No major side effect was reported, with comparable and good tolerability for OM-85 and placebo.Results show an adjuvant therapeutic effect of a well standardized bacterial lysate OM-85 on established AD.

  5. Rapid and sensitive detection of multiple microRNAs in cell lysate by low-fouling surface plasmon resonance biosensor

    Czech Academy of Sciences Publication Activity Database

    Vaisocherová, Hana; Šípová, Hana; Víšová, Ivana; Bocková, Markéta; Špringer, Tomáš; Ermini, Maria Laura; Chadtová Song, Xue; Krejčík, Z.; Chrastinová, L.; Pastva, O.; Pimková, K.; Merkerová-Dostálová, M.; Dyr, J. E.; Homola, Jiří

    2015-01-01

    Roč. 70, August 05 (2015), s. 226-231 ISSN 0956-5663 R&D Projects: GA ČR(CZ) GBP205/12/G118 Institutional support: RVO:67985882 Keywords : Erythrocyte lysate * Polymer brushes * DNA array Subject RIV: JA - Electronics ; Optoelectronics, Electrical Engineering Impact factor: 7.476, year: 2015

  6. Platelet lysate mucohadesive formulation to treat oral mucositis in graft versus host disease patients: a new therapeutic approach.

    Science.gov (United States)

    Del Fante, Claudia; Perotti, Cesare; Bonferoni, Maria Cristina; Rossi, Silvia; Sandri, Giuseppina; Ferrari, Franca; Scudeller, Luigia; Caramella, Carla Marcella

    2011-09-01

    Optimal treatment of oral mucositis (OM) due to graft versus host disease (GvHD) is currently not available. Platelet-derived growth factors (PDGFs) have high capability for tissue healing and may play a role in repairing the mucosal barrier. The aim of the present work was to develop a mucoadhesive formulation to administer platelet lysate to oral cavity prolonging contact time of platelet lysate with oral mucosa. The mucoadhesive formulation was characterized for in vitro properties (PDGF-AB concentration, mucoadhesive properties, cytotoxicity, fibroblast proliferation, wound healing). Moreover, a preliminary clinical study on seven GvHD patients with OM refractory to other therapies was conducted, to evaluate feasibility, safety, and efficacy. GVPL (mucoadhesive gel vehicle mixed with platelet lysate)showed good mucoadhesive properties; additionally, it was characterized by good biocompatibility in vitro on fibroblasts and it was able to enhance fibroblast proliferation and wound healing, maintaining the efficacy for up to 14 days following storage at 2-8°C. In vivo, clinical response was good-to-complete in five, fair in one, none in the remaining one. The in vitro results indicate that GVPL has optimal mucoadhesive and healing enhancer properties, maintained over time (up to 14 days); preliminary clinical results suggest that oral application of platelet lysate-loaded mucoadhesive formulation is feasible, safe, well tolerated, and effective. A larger controlled randomized study is needed.

  7. Evaluation of a platelet lysate bilayered system for periodontal regeneration in a rat intrabony three-wall periodontal defect

    NARCIS (Netherlands)

    Babo, P.S.; Cai, X; Plachokova, A.S.; Reis, R.L.; Jansen, J.A.; Gomes, M.E.; Walboomers, X.F.

    2018-01-01

    With currently available therapies, full regeneration of lost periodontal tissues after periodontitis cannot be achieved. In this study, a combined compartmentalized system was tested, composed of (a) a platelet lysate (PL)-based construct, which was placed along the root aiming to regenerate the

  8. Decreased mechanical properties of heart valve tissue constructs cultured in platelet lysate as compared to fetal bovine serum

    NARCIS (Netherlands)

    Geemen, van D.; Riem Vis, P.W.; Soekhradj - Soechit, R.S.; Sluijter, J.P.G.; Liefde - van Beest, de M.; Kluin, J.; Bouten, C.V.C.

    2011-01-01

    In autologous heart valve tissue engineering, there is an ongoing search for alternatives of fetal bovine serum (FBS). Human platelet-lysate (PL) might be a promising substitute. In the present article, we aimed to examine the tissue formation, functionality, and mechanical properties of engineered

  9. Activation of NO and cytokines by low-molecular weight fractions of lysates from G+ and G bacteria

    Czech Academy of Sciences Publication Activity Database

    Kmoníčková, Eva; Dusilová, Adéla; Kostecká, Petra; Kverka, Miloslav; Zídek, Zdeněk

    2013-01-01

    Roč. 157, Suppl.1 (2013), S51-S55 ISSN 1213-8118 R&D Projects: GA ČR GAP303/12/0535 Institutional support: RVO:68378041 ; RVO:61388971 Keywords : bacteria * lysate * immunomodulation Subject RIV: FR - Pharmacology ; Medidal Chemistry; EC - Immunology (MBU-M) Impact factor: 1.661, year: 2013

  10. Development of chitosan oleate ionic micelles loaded with silver sulfadiazine to be associated with platelet lysate for application in wound healing.

    Science.gov (United States)

    Dellera, Eleonora; Bonferoni, Maria Cristina; Sandri, Giuseppina; Rossi, Silvia; Ferrari, Franca; Del Fante, Claudia; Perotti, Cesare; Grisoli, Pietro; Caramella, Carla

    2014-11-01

    In the treatment of chronic wounds, topical application of anti-infective drugs such as silver sulfadiazine (AgSD) is of primary importance to avoid infections and accelerate wound repair. AgSD is used in burns and chronic wounds for its wide antibacterial spectrum, but presents limitations due to poor solubility and cytotoxicity. In the present work polymeric micelles obtained by self-assembling of chitosan ionically modified by interaction with oleic acid were developed as carriers for AgSD to overcome the drawbacks of the drug. The AgSD loaded micelles were intended to be associated in wound healing with platelet lysate (PL), a hemoderivative rich in growth factors. Unloaded micelles demonstrated good compatibility with both fibroblasts and PL. The relevance of chitosan concentration and of the ratio between chitosan and oleic acid to the drug loading and the particle size of nanoparticles was studied. A marked increase (up to 100 times with respect to saturated solution) of AgSD concentration in micelle dispersion was obtained. Moreover, the encapsulation reduced the cytotoxic effect of the drug towards fibroblasts and the drug incompatibility with PDGF-AB (platelet derived growth factor), chosen as representative of platelet growth factors. Copyright © 2014. Published by Elsevier B.V.

  11. Assessment of bone healing ability of calcium phosphate cements loaded with platelet lysate in rat calvarial defects.

    Science.gov (United States)

    Babo, Pedro S; Carvalho, Pedro P; Santo, Vítor E; Faria, Susana; Gomes, Manuela E; Reis, Rui L

    2016-11-01

    Injectable calcium phosphate cements have been used as a valid alternative to autologous bone grafts for bone augmentation with the additional advantage of enabling minimally invasive implantation procedures and for perfectly fitting the tissue defect. Nevertheless, they have low biodegradability and lack adequate biochemical signaling to promote bone healing and remodeling. In previous in vitro studies, we observed that the incorporation of platelet lysate directly into the cement paste or loaded in hyaluronic acid microspheres allowed to modulate the cement degradation and the in vitro expression of osteogenic markers in seeded human adipose derived stem cells. The present study aimed at investigating the possible effect of this system in new bone formation when implanted in calvarial bilateral defects in rats. Different formulations were assessed, namely plain calcium phosphate cements, calcium phosphate cements loaded with human platelet lysate, hybrid injectable formulations composed of the calcium phosphate cement incorporating hyaluronin acid non-loaded microparticles (20% hyaluronin acid) or with particles loaded with platelet lysate. The degradability and new bone regrowth were evaluated in terms of mineral volume in the defect, measured by micro-computed tomography and histomorphometric analysis upon 4, 8 and 12 weeks of implantation. We observed that the incorporation of hyaluronin acid microspheres induced an overly rapid cement degradation, impairing the osteoconductive properties of the cement composites. Moreover, the incorporation of platelet lysate induced higher bone healing than the materials without platelet lysate, up to four weeks after surgery. Nevertheless, this effect was not found to be significant when compared to the one observed in the sham-treated group. © The Author(s) 2016.

  12. Intranasal Coadministration of the Cry1Ac Protoxin with Amoebal Lysates Increases Protection against Naegleria fowleri Meningoencephalitis

    Science.gov (United States)

    Rojas-Hernández, Saúl; Rodríguez-Monroy, Marco A.; López-Revilla, Rubén; Reséndiz-Albor, Aldo A.; Moreno-Fierros, Leticia

    2004-01-01

    Cry1Ac protoxin has potent mucosal and systemic adjuvant effects on antibody responses to proteins or polysaccharides. In this work, we examined whether Cry1Ac increased protective immunity against fatal Naegleria fowleri infection in mice, which resembles human primary amoebic meningoencephalitis. Higher immunoglobulin G (IgG) than IgA anti-N. fowleri responses were elicited in the serum and tracheopulmonary fluids of mice immunized by the intranasal or intraperitoneal route with N. fowleri lysates either alone or with Cry1Ac or cholera toxin. Superior protection against a lethal challenge with 5 × 104 live N. fowleri trophozoites was achieved for immunization by the intranasal route. Intranasal immunization of N. fowleri lysates coadministered with Cry1Ac increased survival to 100%; interestingly, immunization with Cry1Ac alone conferred similar protection to that achieved with amoebal lysates alone (60%). When mice intranasally immunized with Cry1Ac plus lysates were challenged with amoebae, both IgG and IgA mucosal responses were rapidly increased, but only the increased IgG response persisted until day 60 in surviving mice. The brief rise in the level of specific mucosal IgA does not exclude the role that this isotype may play in the early defense against this parasite, since higher IgA responses were detected in nasal fluids of mice intranasally immunized with lysates plus either Cry1Ac or cholera toxin, which, indeed, were the treatments that provided the major protection levels. In contrast, serum antibody responses do not seem to be related to the protection level achieved. Both acquired and innate immune systems seem to play a role in host defense against N. fowleri infection, but further studies are required to elucidate the mechanisms involved in protective effects conferred by Cry1Ac, which may be a valuable tool to improve mucosal vaccines. PMID:15271892

  13. Bone Formation with Deproteinized Bovine Bone Mineral or Biphasic Calcium Phosphate in the Presence of Autologous Platelet Lysate: Comparative Investigation in Rabbit

    Directory of Open Access Journals (Sweden)

    Carole Chakar

    2014-01-01

    Full Text Available Bone substitutes alone or supplemented with platelet-derived concentrates are widely used to promote bone regeneration but their potency remains controversial. The aim of this study was, therefore, to compare the regenerative potential of preparations containing autologous platelet lysate (APL and particles of either deproteinized bovine bone mineral (DBBM or biphasic calcium phosphate (BCP, two bone substitutes with different resorption patterns. Rabbit APL was prepared by freeze-thawing a platelet suspension. Critical-size defects in rabbit femoral condyle were filled with DBBM or DBBM+APL and BCP or BCP+APL. Rabbits were sacrificed after six weeks and newly formed bone and residual implanted material were evaluated using nondemineralized histology and histomorphometry. New bone was observed around particles of all fillers tested. In the defects filled with BCP, the newly formed bone area was greater (70%; P<0.001 while the residual material area was lower (60%; P<0.001 than that observed in those filled with DBBM. New bone and residual material area of defects filled with either APL+DBBM or APL+BCP were similar to those observed in those filled with the material alone. In summary, osteoconductivity and resorption of BCP were greater than those of DBBM, while APL associated with either DBBM or BCP did not have an additional benefit.

  14. The CAPRICE RICH detector

    Energy Technology Data Exchange (ETDEWEB)

    Basini, G. [INFN, Laboratori Nazionali di Frascati, Rome (Italy); Codino, A.; Grimani, C. [Perugia Univ. (Italy)]|[INFN, Perugia (Italy); De Pascale, M.P. [Rome Univ. `Tor Vergata` (Italy). Dip. di Fisica]|[INFN, Sezione Univ. `Tor Vergata` Rome (Italy); Cafagna, F. [Bari Univ. (Italy)]|[INFN, Bari (Italy); Golden, R.L. [New Mexico State Univ., Las Cruces, NM (United States). Particle Astrophysics Lab.; Brancaccio, F.; Bocciolini, M. [Florence Univ. (Italy)]|[INFN, Florence (Italy); Barbiellini, G.; Boezio, M. [Trieste Univ. (Italy)]|[INFN, Trieste (Italy)

    1995-09-01

    A compact RICH detector has been developed and used for particle identification in a balloon borne spectrometer to measure the flux of antimatter in the cosmic radiation. This is the first RICH detector ever used in space experiments that is capable of detecting unit charged particles, such as antiprotons. The RICH and all other detectors performed well during the 27 hours long flight.

  15. A novel platelet lysate hydrogel for endothelial cell and mesenchymal stem cell-directed neovascularization.

    Science.gov (United States)

    Robinson, Scott T; Douglas, Alison M; Chadid, Tatiana; Kuo, Katie; Rajabalan, Ajai; Li, Haiyan; Copland, Ian B; Barker, Thomas H; Galipeau, Jacques; Brewster, Luke P

    2016-05-01

    Mesenchymal stem cells (MSC) hold promise in promoting vascular regeneration of ischemic tissue in conditions like critical limb ischemia of the leg. However, this approach has been limited in part by poor cell retention and survival after delivery. New biomaterials offer an opportunity to localize cells to the desired tissue after delivery, but also to improve cell survival after delivery. Here we characterize the mechanical and microstructural properties of a novel hydrogel composed of pooled human platelet lysate (PL) and test its ability to promote MSC angiogenic activity using clinically relevant in vitro and in vivo models. This PL hydrogel had comparable storage and loss modulus and behaved as a viscoelastic solid similar to fibrin hydrogels despite having 1/4-1/10th the fibrin content of standard fibrin gels. Additionally, PL hydrogels enabled sustained release of endogenous PDGF-BB for up to 20days and were resistant to protease degradation. PL hydrogel stimulated pro-angiogenic activity by promoting human MSC growth and invasion in a 3D environment, and enhancing endothelial cell sprouting alone and in co-culture with MSCs. When delivered in vivo, the combination of PL and human MSCs improved local tissue perfusion after 8days compared to controls when assessed with laser Doppler perfusion imaging in a murine model of hind limb ischemia. These results support the use of a PL hydrogel as a scaffold for MSC delivery to promote vascular regeneration. Innovative strategies for improved retention and viability of mesenchymal stem cells (MSCs) are needed for cellular therapies. Human platelet lysate is a potent serum supplement that improves the expansion of MSCs. Here we characterize our novel PL hydrogel's desirable structural and biologic properties for human MSCs and endothelial cells. PL hydrogel can localize cells for retention in the desired tissue, improves cell viability, and augments MSCs' angiogenic activity. As a result of these unique traits, PL

  16. Electrospun silk fibroin fibers for storage and controlled release of human platelet lysate.

    Science.gov (United States)

    Pignatelli, Cataldo; Perotto, Giovanni; Nardini, Marta; Cancedda, Ranieri; Mastrogiacomo, Maddalena; Athanassiou, Athanassia

    2018-04-17

    Human platelet lysate (hPL) is a pool of growth factors and cytokines able to induce regeneration of different tissues. Despite its good potentiality as therapeutic tool for regenerative medicine applications, hPL has been only moderately exploited in this field. A more widespread adoption has been limited because of its rapid degradation at room temperature that decreases its functionality. Another limiting factor for its extensive use is the difficulty of handling the hPL gels. In this work, silk fibroin-based patches were developed to address several points: improving the handling of hPL, enabling their delivery in a controlled manner and facilitating their storage by creating a device ready to use with expanded shelf life. Patches of fibroin loaded with hPL were synthesized by electrospinning to take advantage of the fibrous morphology. The release kinetics of the material was characterized and tuned through the control of fibroin crystallinity. Cell viability assays, performed with primary human dermal fibroblasts, demonstrated that fibroin is able to preserve the hPL biological activity and prolong its shelf-life. The strategy of storing and preserving small active molecules within a naturally-derived, protein-based fibrous scaffold was successfully implemented, leading to the design of a biocompatible device, which can potentially simplify the storage and the application of the hPL on a human patient, undergoing medical procedures such as surgery and wound care. Human platelets lysate (hPL) is a mixture of growth factors and cytokines able to induce the regeneration of damaged tissues. This study aims at enclosing hPL in a silk fibroin electrospun matrix to expand its utilization. Silk fibroin showed the ability to preserve the hPL activity at temperature up to 60 °C and the manipulation of fibroin's crystallinity provided a tool to modulate the hPL release kinetic. This entails the possibility to fabricate the hPL silk fibroin patches in advance and

  17. Immune response in the lungs following oral immunization with bacterial lysates of respiratory pathogens.

    Science.gov (United States)

    Ruedl, C; Frühwirth, M; Wick, G; Wolf, H

    1994-03-01

    We have investigated the local immune response of the BALB/c mouse respiratory tract after oral immunization with a bacterial lysate of seven common respiratory pathogens. After two immunization on five consecutive days, we examined the immunoglobulin (immunoglobulin G [IgG], IgM, and IgA) secretion rates of cells isolated from the lungs and compared them with those of spleen cells of orally immunized and nonimmunized animals by using a new test system based on time-resolved fluorescence. The procedure followed the principle of the classical ELISPOT test with nitrocellulose-bottomed microtiter plates, but europium (Eu3+)-linked streptavidin rather than enzyme-conjugated streptavidin was used, with the advantage of quantifying secreted immunoglobulins instead of detecting single antibody-secreting cells. Lymphocytes isolated from the lungs of treated animals revealed significant increases in total and antigen-specific IgA synthesis compared with the rates of the controls, whereas IgG and IgM production rates showed no remarkable differences. In addition, the sera of treated mice revealed higher antigen-specific IgA titers but not increased IgM and IgG levels. We conclude that priming the gut-associated lymphoid tissue with bacterial antigens of pneumotropic microorganisms can elicit an enhanced IgA response in a distant mucosal effector site, such as the respiratory tract, according to the concept of a common mucosa-associated immune system.

  18. Immunological Characterization of Whole Tumour Lysate-Loaded Dendritic Cells for Cancer Immunotherapy

    Science.gov (United States)

    Ottobrini, Luisa; Biasin, Mara; Borelli, Manuela; Lucignani, Giovanni; Trabattoni, Daria; Clerici, Mario

    2016-01-01

    Introduction Dendritic cells play a key role as initiators of T-cell responses, and even if tumour antigen-loaded dendritic cells can induce anti-tumour responses, their efficacy has been questioned, suggesting a need to enhance immunization strategies. Matherials & Methods We focused on the characterization of bone marrow-derived dendritic cells pulsed with whole tumour lysate (TAA-DC), as a source of known and unknown antigens, in a mouse model of breast cancer (MMTV-Ras). Dendritic cells were evaluated for antigen uptake and for the expression of MHC class I/II and costimulatory molecules and markers associated with maturation. Results Results showed that antigen-loaded dendritic cells are characterized by a phenotypically semi-mature/mature profile and by the upregulation of genes involved in antigen presentation and T-cell priming. Activated dendritic cells stimulated T-cell proliferation and induced the production of high concentrations of IL-12p70 and IFN-γ but only low levels of IL-10, indicating their ability to elicit a TH1-immune response. Furthermore, administration of Antigen loaded-Dendritic Cells in MMTV-Ras mice evoked a strong anti-tumour response in vivo as demonstrated by a general activation of immunocompetent cells and the release of TH1 cytokines. Conclusion Data herein could be useful in the design of antitumoral DC-based therapies, showing a specific activation of immune system against breast cancer. PMID:26795765

  19. Human platelet lysate versus minoxidil stimulates hair growth by activating anagen promoting signaling pathways.

    Science.gov (United States)

    Dastan, Maryam; Najafzadeh, Nowruz; Abedelahi, Ali; Sarvi, Mohammadreza; Niapour, Ali

    2016-12-01

    Minoxidil and human platelet lysate (HPL) are commonly used to treat patients with hair loss. However, the roles of HPL versus minoxidil in hair follicle biology largely remain unknown. Here, we hypothesized that bulge and dermal papilla (DP) cells may express specific genes, including Kras, Erk, Akt, Shh and β-catenin after exposure to minoxidil or HPL. The mouse hair follicles were isolated on day 10 after depilation and bulge or DP regions were dissected. The bulge and DP cells were cultured for 14days in DMEM/F12 medium. Then, the cells were treated with 100μM minoxidil and 10% HPL for 10 days. Nuclear morphology was identified using DAPi staining. Reverse transcriptase and real-time polymerase chain reaction (PCR) analysis were also performed to examine the expression of Kras, Erk, Akt, Shh and β-catenin mRNA levels in the treated bulge and DP regions after organ culture. Here, we found that minoxidil influences bulge and DP cell survival (Pminoxidil treatment in both bulge and DP cells. HPL mediated Erk upregulation in both bulge and DP cells (Pminoxidil-treated bulge cells. In contrast, the expression of β-cateinin and Shh in the DP cells was not meaningfully increased after treatment with HPL. Our results suggest that minoxidil and HPL can promote hair growth by activating the main anagen inducing signaling pathways. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  20. Engineering Enriched Microenvironments with Gradients of Platelet Lysate in Hydrogel Fibers.

    Science.gov (United States)

    Santo, Vítor E; Babo, Pedro; Amador, Miguel; Correia, Cláudia; Cunha, Bárbara; Coutinho, Daniela F; Neves, Nuno M; Mano, João F; Reis, Rui L; Gomes, Manuela E

    2016-06-13

    Gradients of physical and chemical cues are characteristic of specific tissue microenvironments and contribute toward morphogenesis and tissue regeneration upon injury. Recent advances on microfluidics and hydrogel manipulation raised the possibility of generating biomimetic biomaterials enriched with bioactive factors and encapsulating cells following designs specifically tailored for a target application. The novelty of this work relies on the combination of methacrylated gellan gum (MeGG) with platelet lysate (PL), aiming to generate novel advanced 3D PL-enriched photo-cross-linkable hydrogels and overcoming the lack of adhesion sites provided by the native MeGG hydrogels. This combination takes advantage of the availability, enriched growth factor composition, and potential autologous application of PL while simultaneously preserving the ability provided by MeGG to tailor mechanical properties, protein release kinetics, and shape of the construct according to the desired goal. Incorporation of PL in the hydrogels significantly improved cellular adhesion and viability in the constructs. The use of microfluidic tools allowed the design of a fiber-like hydrogel incorporating a gradient of PL along the length of the fiber. These spatial protein gradients led to the viability and cell number gradients caused by maintenance of human umbilical vein endothelial cells (HUVECs) survival in the fibers toward the PL-enriched sections in comparison with the nonloaded MeGG sections of the fibers. Altogether, we propose a proof of concept strategy to design a PL gradient biomaterial with potential in tissue engineering approaches and analysis of cell-microenvironment interactions.

  1. Layer-by-layer assembled cell instructive nanocoatings containing platelet lysate.

    Science.gov (United States)

    Oliveira, Sara M; Santo, Vítor E; Gomes, Manuela E; Reis, Rui L; Mano, João F

    2015-04-01

    Great efforts have been made to introduce growth factors (GFs) onto 2D/3D constructs in order to control cell behavior. Platelet lysate (PL) presents itself as a cost-effective source of multiple GFs and other proteins. The instruction given by a construct-PL combination will depend on how its instructive cues are presented to the cells. The content, stability and conformation of the GFs affect their instruction. Strategies for a controlled incorporation of PL are needed. Herein, PL was incorporated into nanocoatings by layer-by-layer assembling with polysaccharides presenting different sulfation degrees (SD) and charges. Heparin and several marine polysaccharides were tested to evaluate their PL and GF incorporation capability. The consequent effects of those multilayers on human adipose derived stem cells (hASCs) were assessed in short-term cultures. Both nature of the polysaccharide and SD were important properties that influenced the adsorption of PL, vascular endothelial growth factor (VEGF), fibroblast growth factor b (FGFb) and platelet derived growth factor (PDGF). The sulfated polysaccharides-PL multilayers showed to be efficient in the promotion of morphological changes, serum-free adhesion and proliferation of high passage hASCs (P > 5). These biomimetic multilayers promise to be versatile platforms to fabricate instructive devices allowing a tunable incorporation of PL. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Absence of micronucleus formation in CHO-K1 cells cultivated in platelet lysate enriched medium.

    Science.gov (United States)

    Bernardi, Martina; Adami, Valentina; Albiero, Elena; Madeo, Domenico; Rodeghiero, Francesco; Astori, Giuseppe

    2014-03-01

    Human platelet lysate (PL) represents an effective substitute of fetal bovine serum (FBS) for mesenchymal stromal cell (MSC) cultivation. Compared to FBS, PL favors MSC proliferation significantly shortening the population doubling time and avoiding the risks related to the use of animal derivatives. Growth factors contained in the platelets are released upon platelet disruption following freezing/thawing cycles or as we have recently described by using ultrasound. We have investigated whether the increased cell proliferation achieved by using PL could induce mitotic stress and whether the potential formation of free radicals during PL production by ultrasound could cause chromosomal instability in mammalian cells. We have applied an image analysis assisted high content screening (HCS) in vitro micronucleus assay in the Chinese Hamster Ovarian K1 (CHO-K1) rodent mammalian cell line. PL was produced by sonication; for the micronucleus assay, CHO-K1 cells were exposed to increasing concentrations of PL. Cytokinesis was blocked by cytochalasin B, nuclei were stained with bisbenzimide and images were acquired and analyzed automatically using an HCS system, both with a 20× and a 10× objective. Our results suggest that growth stimulus induced by the use of PL did not significantly increase micronucleus formation in CHO-K1 cells compared to negative control. Micronucleus testing in conjunction with HCS could represent a valid tool to evaluate the safety of ancillary materials used in the production of cell-based medicinal products. Copyright © 2013 Elsevier GmbH. All rights reserved.

  3. Culture of human cell lines by a pathogen-inactivated human platelet lysate.

    Science.gov (United States)

    Fazzina, R; Iudicone, P; Mariotti, A; Fioravanti, D; Procoli, A; Cicchetti, E; Scambia, G; Bonanno, G; Pierelli, L

    2016-08-01

    Alternatives to the use of fetal bovine serum (FBS) have been investigated to ensure xeno-free growth condition. In this study we evaluated the efficacy of human platelet lysate (PL) as a substitute of FBS for the in vitro culture of some human cell lines. PL was obtained by pools of pathogen inactivated human donor platelet (PLT) concentrates. Human leukemia cell lines (KG-1, K562, JURKAT, HL-60) and epithelial tumor cell lines (HeLa and MCF-7) were cultured with either FBS or PL. Changes in cell proliferation, viability, morphology, surface markers and cell cycle were evaluated for each cell line. Functional characteristics were analysed by drug sensitivity test and cytotoxicity assay. Our results demonstrated that PL can support growth and expansion of all cell lines, although the cells cultured in presence of PL experienced a less massive proliferation compared to those grown with FBS. We found a comparable percentage of viable specific marker-expressing cells in both conditions, confirming lineage fidelity in all cultures. Functionality assays showed that cells in both FBS- and PL-supported cultures maintained their normal responsiveness to adriamycin and NK cell-mediated lysis. Our findings indicate that PL is a feasible serum substitute for supporting growth and propagation of haematopoietic and epithelial cell lines with many advantages from a perspective of process standardization, ethicality and product safety.

  4. The use of lumbar epidural injection of platelet lysate for treatment of radicular pain.

    Science.gov (United States)

    Centeno, Christopher; Markle, Jason; Dodson, Ehren; Stemper, Ian; Hyzy, Matthew; Williams, Christopher; Freeman, Michael

    2017-11-25

    Epidural steroid injections (ESI) are the most common pain management procedure performed in the US, however evidence of efficacy is limited. In addition, there is early evidence that the high dose of corticosteroids used can have systemic side effects. We describe the results of a case series evaluating the use of platelet lysate (PL) epidural injections for the treatment of lumbar radicular pain as an alternative to corticosteroids. Registry data was obtained for patients (N = 470) treated with PL epidural injections presenting with symptoms of lumbar radicular pain and MRI findings that were consistent with symptoms. Collected outcomes included numeric pain score (NPS), functional rating index (FRI), and a modified single assessment numeric evaluation (SANE) rating. Patients treated with PL epidurals reported significantly lower (p < .0001) NPS and FRI change scores at all time points compared to baseline. Post-treatment FRI change score means exceeded the minimal clinically important difference beyond 1 month. Average modified SANE ratings showed 49.7% improvement at 24 months post-treatment. Twenty-nine (6.3%) patients reported mild adverse events related to treatment. Patients treated with PL epidurals reported significant improvements in pain, exceeded the minimal clinically important difference (MCID) for FRI, and reported subjective improvement through 2-year follow-up. PL may be a promising substitute for corticosteroid.

  5. Heparin concentration is critical for cell culture with human platelet lysate.

    Science.gov (United States)

    Hemeda, Hatim; Kalz, Jana; Walenda, Gudrun; Lohmann, Michael; Wagner, Wolfgang

    2013-09-01

    Culture media for mesenchymal stromal cells (MSCs) are generally supplemented with fetal bovine serum. Human platelet lysate (hPL) has been proven to be a very effective alternative without the risk of xenogeneic infections or immune reactions. In contrast to fetal bovine serum, hPL comprises plasma, and anticoagulants-usually unfractionated heparin (UFH)-need to be added to prevent gel formation. Cultures of MSCs in hPL media with various concentrations of UFH and enoxaparin, a low-molecular-weight heparin (LMWH), were systematically compared with regard to proliferation, fibroblastoid colony-forming unit frequency, immunophenotype and in vitro differentiation. At least 0.61 IU/mL UFH or 0.024 mg/mL LMWH was necessary for reliable prevention of coagulation of hPL pools used in this study. Higher concentrations impaired cellular proliferation in a dose-dependent manner even without benzyl alcohol, which is commonly added to heparins as a bacteriostatic agent. Colony-forming unit frequency was also reduced at higher heparin concentrations, particularly with LMWH, whereas no significant effect was observed on cellular morphology or immunophenotype. High concentrations of heparins reduced the in vitro differentiation toward adipogenic and osteogenic lineages. Heparin concentration is critical for culture of MSCs in hPL media; this is of particular relevance for cellular therapy where cell culture procedures need to be optimized and standardized. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  6. Human platelet lysate supports the formation of robust human periodontal ligament cell sheets.

    Science.gov (United States)

    Tian, Bei-Min; Wu, Rui-Xin; Bi, Chun-Sheng; He, Xiao-Tao; Yin, Yuan; Chen, Fa-Ming

    2018-04-01

    The use of stem cell-derived sheets has become increasingly common in a wide variety of biomedical applications. Although substantial evidence has demonstrated that human platelet lysate (PL) can be used for therapeutic cell expansion, either as a substitute for or as a supplement to xenogeneic fetal bovine serum (FBS), its impact on cell sheet production remains largely unexplored. In this study, we manufactured periodontal ligament stem cell (PDLSC) sheets in vitro by incubating PDLSCs in sheet-induction media supplemented with various ratios of PL and FBS, i.e. 10% PL without FBS, 7.5% PL + 2.5% FBS, 5% PL + 5% FBS, 2.5% PL + 7.5% FBS or 10% FBS without PL. Cultures with the addition of all the designed supplements led to successful cell sheet production. In addition, all the resultant cellular materials exhibited similar expression profiles of matrix-related genes and proteins, such as collagen I, fibronectin and integrin β1. Interestingly, the cell components within sheets generated by media containing both PL and FBS exhibited improved osteogenic potential. Following in vivo transplantation, all sheets supported significant new bone formation. Our data suggest that robust PDLSC sheets can be produced by applying PL as either an alternative or an adjuvant to FBS. Further examination of the relevant influences of human PL that benefit cell behaviour and matrix production will pave the way towards optimized and standardized conditions for cell sheet production. Copyright © 2017 John Wiley & Sons, Ltd.

  7. Global phenotypic characterisation of human platelet lysate expanded MSCs by high-throughput flow cytometry.

    Science.gov (United States)

    Reis, Monica; McDonald, David; Nicholson, Lindsay; Godthardt, Kathrin; Knobel, Sebastian; Dickinson, Anne M; Filby, Andrew; Wang, Xiao-Nong

    2018-03-02

    Mesenchymal stromal cells (MSCs) are a promising cell source to develop cell therapy for many diseases. Human platelet lysate (PLT) is increasingly used as an alternative to foetal calf serum (FCS) for clinical-scale MSC production. To date, the global surface protein expression of PLT-expended MSCs (MSC-PLT) is not known. To investigate this, paired MSC-PLT and MSC-FCS were analysed in parallel using high-throughput flow cytometry for the expression of 356 cell surface proteins. MSC-PLT showed differential surface protein expression compared to their MSC-FCS counterpart. Higher percentage of positive cells was observed in MSC-PLT for 48 surface proteins, of which 13 were significantly enriched on MSC-PLT. This finding was validated using multiparameter flow cytometry and further confirmed by quantitative staining intensity analysis. The enriched surface proteins are relevant to increased proliferation and migration capacity, as well as enhanced chondrogenic and osteogenic differentiation properties. In silico network analysis revealed that these enriched surface proteins are involved in three distinct networks that are associated with inflammatory responses, carbohydrate metabolism and cellular motility. This is the first study reporting differential cell surface protein expression between MSC-PLT and MSC-FSC. Further studies are required to uncover the impact of those enriched proteins on biological functions of MSC-PLT.

  8. Platelet lysate as replacement for fetal bovine serum in mesenchymal stromal cell cultures.

    Science.gov (United States)

    Bieback, Karen

    2013-10-01

    Mesenchymal stromal cells (MSC) emerged as highly attractive in cell-based regenerative medicine. Initially thought to provide cells capable of differentiation towards mesenchymal cell types (osteoblasts, chondrocytes, adipocytes etc.), by and by potent immunoregulatory and pro-regenerative activities have been discovered, broadening the field of potential applications from bone and cartilage regeneration to wound healing and treatment of autoimmune diseases. Due to the limited frequency in most tissue sources, ex vivo expansion of MSC is required compliant with good manufacturing practice (GMP) guidelines to yield clinically relevant cell doses. Though, still most manufacturing protocols use fetal bovine serum (FBS) as cell culture supplement to isolate and to expand MSC. However, the high lot-to-lot variability as well as risk of contamination and immunization call for xenogenic-free culture conditions. In terms of standardization, chemically defined media appear as the ultimate achievement. Since these media need to maintain all key cellular and therapy-relevant features of MSC, the development of chemically defined media is still - albeit highly investigated - only in its beginning. The current alternatives to FBS rely on human blood-derived components: plasma, serum, umbilical cord blood serum, and platelet derivatives like platelet lysate. Focusing on quality aspects, the latter will be addressed within this review.

  9. Limulus amoebocyte lysate test via an open-microcavity optical biosensor

    Science.gov (United States)

    Scudder, Jonathan; Ye, Jing Yong

    2018-02-01

    Almost since its discovery, Limulus amoebocyte lysate (LAL) testing has been an important part of the pharmaceutical quality control toolkit. It allows for in vitro endotoxin testing, which has replaced tests using animals, such as using rabbits' thermal response to judge pyrogenicity of test samples, thus leading to a less expensive and faster test of parenteral pharmaceuticals and medical devices that contact blood or cerebrospinal fluid. However, limited by the detection mechanisms of the LAL assays currently used in industry, further improvement in their performance is challenging. To address the growing demand on optimizing LAL assays for increased test sensitivity and reduced assay time, we have developed an LAL assay approach based on a detection mechanism that is different from those being used in industry, namely, gel-clot, turbidimetric, and chromogenic detection. Using a unique open-microcavity photonic-crystal biosensor to monitor the change in the refractive index due to the reaction between LAL regents and endotoxins, we have demonstrated that this approach has improved the LAL assay sensitivity by 200 times compared with the commercial standard methods, reduced the time needed for the assay by more than half, and eliminated the necessity to incubate the test samples. This study opens up the possibility of using the significantly improved LAL assays for a wide range of applications.

  10. Platelet Lysate-Modified Porous Silicon Microparticles for Enhanced Cell Proliferation in Wound Healing Applications.

    Science.gov (United States)

    Fontana, Flavia; Mori, Michela; Riva, Federica; Mäkilä, Ermei; Liu, Dongfei; Salonen, Jarno; Nicoletti, Giovanni; Hirvonen, Jouni; Caramella, Carla; Santos, Hélder A

    2016-01-13

    The new frontier in the treatment of chronic nonhealing wounds is the use of micro- and nanoparticles to deliver drugs or growth factors into the wound. Here, we used platelet lysate (PL), a hemoderivative of platelets, consisting of a multifactorial cocktail of growth factors, to modify porous silicon (PSi) microparticles and assessed both in vitro and ex vivo the properties of the developed microsystem. PL-modified PSi was assessed for its potential to induce proliferation of fibroblasts. The wound closure-promoting properties of the microsystem were then assessed in an in vitro wound healing assay. Finally, the PL-modified PSi microparticles were evaluated in an ex vivo experiment over human skin. It was shown that PL-modified PSi microparticles were cytocompatible and enhanced the cell proliferation in different experimental settings. In addition, this microsystem promoted the closure of the gap between the fibroblast cells in the wound healing assay, in periods of time comparable with the positive control, and induced a proliferation and regeneration process onto the human skin in an ex vivo experiment. Overall, our results show that PL-modified PSi microparticles are suitable microsystems for further development toward applications in the treatment of chronic nonhealing wounds.

  11. Rapid and specific purification of Argonaute-small RNA complexes from crude cell lysates.

    Science.gov (United States)

    Flores-Jasso, C Fabián; Salomon, William E; Zamore, Phillip D

    2013-02-01

    Small interfering RNAs (siRNAs) direct Argonaute proteins, the core components of the RNA-induced silencing complex (RISC), to cleave complementary target RNAs. Here, we describe a method to purify active RISC containing a single, unique small RNA guide sequence. We begin by capturing RISC using a complementary 2'-O-methyl oligonucleotide tethered to beads. Unlike other methods that capture RISC but do not allow its recovery, our strategy purifies active, soluble RISC in good yield. The method takes advantage of the finding that RISC partially paired to a target through its siRNA guide dissociates more than 300 times faster than a fully paired siRNA in RISC. We use this strategy to purify fly Ago1- and Ago2-RISC, as well as mouse AGO2-RISC. The method can discriminate among RISCs programmed with different guide strands, making it possible to deplete and recover specific RISC populations. Endogenous microRNA:Argonaute complexes can also be purified from cell lysates. Our method scales readily and takes less than a day to complete.

  12. Clinical Benefit of Allogeneic Melanoma Cell Lysate-Pulsed Autologous Dendritic Cell Vaccine in MAGE-Positive Colorectal Cancer Patients

    DEFF Research Database (Denmark)

    Toh, Han Chong; Wang, Who-Whong; Chia, Whay Kuang

    2009-01-01

    PURPOSE: We evaluated the clinical benefit of an allogeneic melanoma cell lysate (MCL)-pulsed autologous dendritic cell (DC) vaccine in advanced colorectal cancer patients expressing at least one of six MAGE-A antigens overexpressed by the cell line source of the lysate. EXPERIMENTAL DESIGN: DCs...... were cultured from peripheral blood mononuclear cells (PBMC), pulsed with the allogeneic MCL, and matured using cytokines that achieved high CD83- and CCR7-expressing DCs. Each patient received up to 10 intradermal vaccinations (3-5 x 10(6) cells per dose) at biweekly intervals. RESULTS: Twenty......-free for >27 and >37 months, respectively. This result is particularly meaningful as all patients had progressive disease before treatment. Overall, DC vaccination was associated with a serial decline in regulatory T cells. Using an antibody array, we characterized plasma protein profiles in responding...

  13. A midgut lysate of the Riptortus pedestris has antibacterial activity against LPS O-antigen-deficient Burkholderia mutants.

    Science.gov (United States)

    Jang, Ho Am; Seo, Eun Sil; Seong, Min Young; Lee, Bok Luel

    2017-02-01

    Riptortus pedestris, a common pest in soybean fields, harbors a symbiont Burkholderia in a specialized posterior midgut region of insects. Every generation of second nymphs acquires new Burkholderia cells from the environment. We compared in vitro cultured Burkholderia with newly in vivo colonized Burkholderia in the host midgut using biochemical approaches. The bacterial cell envelope of in vitro cultured and in vivo Burkholderia differed in structure, as in vivo bacteria lacked lipopolysaccharide (LPS) O-antigen. The LPS O-antigen deficient bacteria had a reduced colonization rate in the host midgut compared with that of the wild-type Burkholderia. To determine why LPS O-antigen-deficient bacteria are less able to colonize the host midgut, we examined in vitro survival rates of three LPS O-antigen-deficient Burkholderia mutants and lysates of five different midgut regions. The LPS O-antigen-deficient mutants were highly susceptible when cultured with the lysate of a specific first midgut region (M1), indicating that the M1 lysate contains unidentified substance(s) capable of killing LPS O-antigen-deficient mutants. We identified a 17 kDa protein from the M1 lysate, which was enriched in the active fractions. The N-terminal sequence of the protein was determined to be a soybean Kunitz-type trypsin inhibitor. These data suggest that the 17 kDa protein, which was originated from a main soybean source of the R. pedestris host, has antibacterial activity against the LPS O-antigen deficient (rough-type) Burkholderia. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Feasibility and Efficiency of Human Bone Marrow Stromal Cell Culture with Allogeneic Platelet Lysate-Supplementation for Cell Therapy against Stroke

    Directory of Open Access Journals (Sweden)

    Chengbo Tan

    2016-01-01

    Full Text Available Currently, there is increasing interest in human bone marrow stromal cells (hBMSCs as regeneration therapy against cerebral stroke. The aim of the present study was to evaluate the feasibility and validity of hBMSC cultures with allogeneic platelet lysates (PLs. Platelet concentrates (PC were harvested from healthy volunteers and made into single donor-derived PL (sPL. The PL mixtures (mPL were made from three different sPL. Some growth factors and platelet cell surface antigens were detected by enzyme-linked immunosorbent assay (ELISA. The hBMSCs cultured with 10% PL were analyzed for their proliferative potential, surface markers, and karyotypes. The cells were incubated with superparamagnetic iron oxide (SPIO agents and injected into a pig brain. MRI and histological analysis were performed. Consequently, nine lots of sPL and three mPL were prepared. ELISA analysis showed that PL contained adequate growth factors and a particle of platelet surface antigens. Cell proliferation capacity of PLs was equivalent to or higher than that of fetal calf serum (FCS. No contradiction in cell surface markers and no chromosomal aberrations were found. The MRI detected the distribution of SPIO-labeled hBMSCs in the pig brain. In summary, the hBMSCs cultured with allogeneic PL are suitable for cell therapy against stroke.

  15. A novel dressing for the combined delivery of platelet lysate and vancomycin hydrochloride to chronic skin ulcers: Hyaluronic acid particles in alginate matrices.

    Science.gov (United States)

    Rossi, S; Mori, M; Vigani, B; Bonferoni, M C; Sandri, G; Riva, F; Caramella, C; Ferrari, F

    2018-06-15

    The aim of the present work was to develop a medication allowing for the combined delivery of platelet lysate (PL) and an anti-infective model drug, vancomycin hydrochloride (VCM), to chronic skin ulcers. A simple method was set up for the preparation of hyaluronic acid (HA) core-shell particles, loaded with PL and coated with calcium alginate, embedded in a VCM containing alginate matrix. Two different CaCl 2 concentrations were investigated to allow for HA/PL core-shell particle formation. The resulting dressings were characterized for mechanical and hydration properties and tested in vitro (on fibroblasts) and ex-vivo (on skin biopsies) for biological activity. They were found of sufficient mechanical strength to withstand packaging and handling stress and able to absorb a high amount of wound exudate and to form a protective gel on the lesion area. The CaCl 2 concentration used for shell formation did not affect VCM release from the alginate matrix, but strongly modified the release of PGFAB (chosen as representative of growth factors present in PL) from HA particles. In vitro and ex vivo tests provided sufficient proof of concept of the ability of dressings to improve skin ulcers healing. Copyright © 2018 Elsevier B.V. All rights reserved.

  16. Sponge-Like Dressings Based on the Association of Chitosan and Sericin for the Treatment of Chronic Skin Ulcers. II. Loading of the Hemoderivative Platelet Lysate.

    Science.gov (United States)

    Mori, Michela; Rossi, Silvia; Ferrari, Franca; Bonferoni, Maria C; Sandri, Giuseppina; Riva, Federica; Tenci, Marika; Del Fante, Claudia; Nicoletti, Giovanni; Caramella, Carla

    2016-03-01

    Platelet lysate (PL) was loaded into dressings based on chitosan glutamate (CSG) low and high molecular weight, sericin (Ser), and glycine (Gly). A synergic effect of Ser and PL on fibroblast proliferation was proved in vitro. Two different PL loading approaches were considered: the first provided to prepare dressings by freeze-drying a mixture of PL and CSG/Gly/Ser solution, the second approach consisted in the extemporarily loading of PL in the CSG/Gly/Ser freeze-dried dressings. As for the first approach, PL loading did not produce any variation in dressing mechanical properties. Such dressings absorbed a high amount (about 8-fold of dry weight) of phosphate-buffered saline (fluid mimicking wound exudate), forming a gel with pseudoplastic and elastic properties. Platelet-derived growth factor AB assay indicated that neither freeze-drying nor the excipients alter PL growth factor content. As for the second approach, mechanical and rheological properties of the gel formed upon PL absorption enabled to choose a PL loading of about 90 μL/cm(2). Upon contact with fibroblasts, all PL loaded formulations increased the number not only of viable cells but also of those in the proliferative phase. Histological studies effected on human skin strips pointed out the positive effect of PL loaded dressings on dermal matrix reconstruction. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  17. Composition of growth factors and cytokines in lysates obtained from fresh versus stored pathogen-inactivated platelet units.

    Science.gov (United States)

    Sellberg, Felix; Berglund, Erik; Ronaghi, Martin; Strandberg, Gabriel; Löf, Helena; Sommar, Pehr; Lubenow, Norbert; Knutson, Folke; Berglund, David

    2016-12-01

    Platelet lysate is a readily available source of growth factors, and other mediators, which has been used in a variety of clinical applications. However, the product remains poorly standardized and the present investigation evaluates the composition of platelet lysate obtained from either fresh or stored pathogen-inactivated platelet units. Platelet pooled units (n = 10) were obtained from healthy blood donors and tested according to standard procedures. All units were pathogen inactivated using amotosalen hydrochloride and UVA exposure. Platelet lysate was subsequently produced at two separate time-points, either from fresh platelet units or after 5 days of storage, by repeated freeze-thaw cycles. The following mediators were determined at each time-point: EGF, FGF-2, VEGF, IGF-1, PDGF-AB/BB, BMP-2, PF4, TGF-β isoform 1, IL-1β, IL-2, IL-6, IL-10, IL-12p70, 1L-17A, TNF-α, and IFN-γ. The concentration of growth factors and cytokines was affected by time in storage. Notably, TGF-β, PDGF-AB/BB, and PF4 showed an increase of 27.2% (p product, which potentially may influence the clinical effects. Copyright © 2016. Published by Elsevier Ltd.

  18. Research: Rags to Rags? Riches to Riches?

    Science.gov (United States)

    Bracey, Gerald W.

    2004-01-01

    Everyone has read about what might be called the "gold gap"--how the rich in this country are getting richer and controlling an ever-larger share of the nation's wealth. The Century Foundation has started publishing "Reality Check", a series of guides to campaign issues that sometimes finds gaps in these types of cherished delusions. The guides…

  19. Kings Today, Rich Tomorrow

    DEFF Research Database (Denmark)

    Fattoum, Asma

    2013-01-01

    This study investigates the King vs. Rich dilemma that founder-CEOs face at IPO. When undertaking IPO, founders face two options. They can either get rich, but then run the risk of losing the control over their firms; or they can remain kings by introducing defensive mechanisms, but this is likel...

  20. Developments on RICH detectors

    International Nuclear Information System (INIS)

    Besson, P.; Bourgeois, P.

    1996-01-01

    The RICH (ring imaging Cherenkov) detector which is dedicated to Cherenkov radiation detection is described. An improvement made by replacing photo sensible vapor with solid photocathode is studied. A RICH detector prototype with a CsI photocathode has been built in Saclay and used with Saturne. The first results are presented. (A.C.)

  1. Purification of two high molecular weight proteases from rabbit reticulocyte lysate

    International Nuclear Information System (INIS)

    Hough, R.; Pratt, G.; Rechsteiner, M.

    1987-01-01

    The authors have purified two large proteases from rabbit reticulocyte lysate. The enzymes are so similar in their chromatographic behavior that each is the only significant contaminant of the other during the final stages of purification. At pH 7.8, both hydrolyze 125 I-α-casein and 4-methylcoumaryl-7-amide (MCA) derivatives with tyrosine, phenylalanine or arginine at the P 1 position. The larger, ATP-dependent enzyme degrades ubiquitin-lysozyme conjugates, but it does not degrade unmodified lysozyme. Hydrolysis of Suc-Leu-Leu-Val-Tyr-MCA by this enzyme is also stimulated two-fold in the presence of ATP. The protease has a molecular weight of 950,000 based on sedimentation, gel filtration and non-denaturing PAGE. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the protease is composed of a number of subunits with molecular masses between 32 and 110 kDa. Densitometric analysis showed equivalent amounts of the two larger chains, and the presence of one copy of each in the native enzyme would be consistent with an M/sub r/ of 950,000. The smaller protease has a molecular weight of 700,000 and is composed of 8 to 10 subunits ranging from 21,000 to 32,000. It cleaves ubiquitin-lysozyme conjugates only slightly, and hydrolysis of conjugates or fluorogenic peptide substrates is not stimulated by ATP. This protease appears similar, if not identical, to the multicatalytic protease complex first purified by Wilk and Orlowski

  2. Human platelet lysate supplementation of mesenchymal stromal cell delivery: issues of xenogenicity and species variability.

    Science.gov (United States)

    Allen, Ashley B; Butts, Emily B; Copland, Ian B; Stevens, Hazel Y; Guldberg, Robert E

    2017-10-01

    Immunogenicity of fetal bovine serum (FBS) poses a problem for its use in the propagation of autologous mesenchymal stromal cells (MSCs) for cell therapy. Human platelet lysate (hPL), an enriched growth factor solution containing mitogenic and angiogenic cues, has potential utility in replacing FBS for human MSC (hMSC) delivery strategies. Despite its potentiation of hMSC number in vitro, little is known concerning its capacity to supplement implanted hMSC-seeded constructs and promote tissue regeneration in vivo. In this study, we tested the effects of incorporating hPL in cell-seeded constructs implanted subcutaneously into immunocompromised rats, investigated in vitro interactions between hPL and rat MSCs (rMSCs) and determined interspecies variability in the PL product [hPL vs rat PL (rPL)] and its effect on cultured MSCs (hPL/hMSCs vs rPL/rMSCs). The overarching aim was to determine the utility of hPL to foster MSC survival in preclinical rodent models. Exposure to hPL-supplemented media resulted in rMSC death, by a process attributable to heat-labile proteins, but not membrane attack complex formation. In the in vitro syngeneic model, the rodent product proved fundamentally distinct from the human product, with rPL having substantially lower growth factor content than hPL. Moreover, contrary to the positive effects of hPL on hMSC expansion, rPL did not reduce rMSC doubling time for the serum concentrations examined. When tested in vivo, hPL did not improve cell survival within hydrogel constructs through 2 weeks postimplantation. In summary, this study highlights the many facets of xenogenicity and interspecies variability that must be considered in the preclinical evaluation of hPL. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  3. Donor age of human platelet lysate affects proliferation and differentiation of mesenchymal stem cells.

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    Michael Lohmann

    Full Text Available The regenerative potential declines upon aging. This might be due to cell-intrinsic changes in stem and progenitor cells or to influences by the microenvironment. Mesenchymal stem cells (MSC raise high hopes in regenerative medicine. They are usually culture expanded in media with fetal calf serum (FCS or other serum supplements such as human platelet lysate (HPL. In this study, we have analyzed the impact of HPL-donor age on culture expansion. 31 single donor derived HPLs (25 to 57 years old were simultaneously compared for culture of MSC. Proliferation of MSC did not reveal a clear association with platelet counts of HPL donors or growth factors concentrations (PDGF-AB, TGF-β1, bFGF, or IGF-1, but it was significantly higher with HPLs from younger donors (45 years. Furthermore, HPLs from older donors increased activity of senescence-associated beta-galactosidase (SA-βgal. HPL-donor age did not affect the fibroblastoid colony-forming unit (CFU-f frequency, immunophenotype or induction of adipogenic differentiation, whereas osteogenic differentiation was significantly lower with HPLs from older donors. Concentrations of various growth factors (PDGF-AB, TGF-β1, bFGF, IGF-1 or hormones (estradiol, parathormone, leptin, 1,25 vitamin D3 were not associated with HPL-donor age or MSC growth. Taken together, our data support the notion that aging is associated with systemic feedback mechanisms acting on stem and progenitor cells, and this is also relevant for serum supplements in cell culture: HPLs derived from younger donors facilitate enhanced expansion and more pronounced osteogenic differentiation.

  4. The effects of platelet lysate patches on the activity of tendon-derived cells.

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    Costa-Almeida, Raquel; Franco, Albina R; Pesqueira, Tamagno; Oliveira, Mariana B; Babo, Pedro S; Leonor, Isabel B; Mano, João F; Reis, Rui L; Gomes, Manuela E

    2018-03-01

    Platelet-derived biomaterials are widely explored as cost-effective sources of therapeutic factors, holding a strong potential for endogenous regenerative medicine. Particularly for tendon repair, treatment approaches that shift the injury environment are explored to accelerate tendon regeneration. Herein, genipin-crosslinked platelet lysate (PL) patches are proposed for the delivery of human-derived therapeutic factors in patch augmentation strategies aiming at tendon repair. Developed PL patches exhibited a controlled release profile of PL proteins, including bFGF and PDGF-BB. Additionally, PL patches exhibited an antibacterial effect by preventing the adhesion, proliferation and biofilm formation by S. aureus, a common pathogen in orthopaedic surgical site infections. Furthermore, these patches supported the activity of human tendon-derived cells (hTDCs). Cells were able to proliferate over time and an up-regulation of tenogenic genes (SCX, COL1A1 and TNC) was observed, suggesting that PL patches may modify the behavior of hTDCs. Accordingly, hTDCs deposited tendon-related extracellular matrix proteins, namely collagen type I and tenascin C. In summary, PL patches can act as a reservoir of biomolecules derived from PL and support the activity of native tendon cells, being proposed as bioinstructive patches for tendon regeneration. Platelet-derived biomaterials hold great interest for the delivery of therapeutic factors for applications in endogenous regenerative medicine. In the particular case of tendon repair, patch augmentation strategies aiming at shifting the injury environment are explored to improve tendon regeneration. In this study, PL patches were developed with remarkable features, including the controlled release of growth factors and antibacterial efficacy. Remarkably, PL patches supported the activity of native tendon cells by up-regulating tenogenic genes and enabling the deposition of ECM proteins. This patch holds great potential towards

  5. Platelet lysate induces chondrogenic differentiation of umbilical cord-derived mesenchymal stem cells.

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    Hassan, Ghmkin; Bahjat, Mohammad; Kasem, Issam; Soukkarieh, Chadi; Aljamali, Majd

    2018-01-01

    Articular cartilage has a poor capacity for self-repair, and thus still presents a major challenge in orthopedics. Mesenchymal stem cells (MSCs) are multipotent stem cells with the potential to differentiate into chondrocytes in the presence of transforming growth factor beta (TGF-β). Platelet lysate (PL) contains a relatively large number of growth factors, including TGF-β, and has been shown to ameliorate cartilage repair. Here, we investigated the ability of PL to direct chondrogenic differentiation of MSCs along with other standard differentiation components in a pellet culture system. We isolated and expanded MSCs from human umbilical cords using a PL-supplemented medium and characterized the cells based on immunophenotype and potential for differentiation to adipocytes and osteocytes. We further cultured MSCs as pellets in a chondrogenic-differentiation medium supplemented with PL. After 21 days, the pellets were processed for histological analysis and stained with alician blue and acridine orange. The expression of SOX9 was investigated using RT-PCR. MSCs maintained their stemness characteristics in the PL-supplemented medium. However, the distribution of cells in the pellets cultured in the PL-supplemented chondrogenic differentiation medium had a greater similarity to cartilage tissue-derived chondrocytes than to the negative control. The intense alician blue staining indicated an increased production of mucopolysaccharides in the differentiated pellets, which also showed elevated expression of SOX9 . Our data suggest that MSCs could be differentiated to chondrocytes in the presence of PL and absence of exogenous TGF-β. Further research needs to be conducted to understand the exact role and potential of PL in chondrogenic differentiation and chondrocyte regeneration.

  6. Platelet lysate enhances synovial fluid multipotential stromal cells functions: Implications for therapeutic use.

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    Altaie, Ala; Baboolal, Thomas G; Wall, Owen; Jones, Elena; McGonagle, Dennis

    2018-03-01

    Although intra-articular injection of platelet products is increasingly used for joint regenerative approaches, there are few data on their biological effects on joint-resident multipotential stromal cells (MSCs), which are directly exposed to the effects of these therapeutic strategies. Therefore, this study investigated the effect of platelet lysate (PL) on synovial fluid-derived MSCs (SF-MSCs), which in vivo have direct access to sites of cartilage injury. SF-MSCs were obtained during knee arthroscopic procedures (N = 7). Colony forming unit-fibroblast (CFU-F), flow-cytometric phenotyping, carboxyfluorescein succinimidyl ester-based immunomodulation for T-cell and trilineage differentiation assays were performed using PL and compared with standard conditions. PL-enhanced SF-MSC (PL-MSC) proliferation as CFU-F colonies was 1.4-fold larger, and growing cultures had shorter population-doubling times. PL-MSCs and fetal calf serum (FCS)-MSCs had the same immunophenotype and similar immunomodulation activities. In chondrogenic and osteogenic differentiation assays, PL-MSCs produced 10% more sulfated-glycosaminoglycan (sGAG) and 45% less Ca ++ compared with FCS-MSCs, respectively. Replacing chondrogenic medium transforming growth factor-β3 with 20% or 50% PL further increased sGAG production of PL-MSCs by 69% and 95%, respectively, compared with complete chondrogenic medium. Also, Dulbecco's Modified Eagle's Medium high glucose (HG-DMEM) plus 50% PL induced more chondrogenesis compared with HG-DMEM plus 10% FCS and was comparable to complete chondrogenic medium. This is the first study to assess SF-MSC responses to PL and provides biological support to the hypothesis that PL may be capable of modulating multiple functional aspects of joint resident MSCs with direct access to injured cartilage. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  7. Umbilical Cord Blood Platelet Lysate as Serum Substitute in Expansion of Human Mesenchymal Stem Cells.

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    Shirzad, Negin; Bordbar, Sima; Goodarzi, Alireza; Mohammad, Monire; Khosravani, Pardis; Sayahpour, Froughazam; Baghaban Eslaminejad, Mohamadreza; Ebrahimi, Marzieh

    2017-10-01

    The diverse clinical applications for human mesenchymal stem cells (hMSCs) in cellular therapy and regenerative medicine warrant increased focus on developing adequate culture supplements devoid of animal-derived products. In the present study, we have investigated the feasibility of umbilical cord blood-platelet lysate (UCB-PL) as a standard substitute for fetal bovine serum (FBS) and human peripheral blood-PL (PB-PL). In this experimental study, platelet concentrates (PC) from UCB and human PB donors were frozen, melted, and sterilized to obtain PL. Quality control included platelet cell counts, sterility testing (viral and microbial), total protein concentrations, growth factor levels, and PL stability. The effects of UCB-PL and PB-PL on hMSCs proliferation and differentiation into osteocytes, chondrocytes, and adipocytes were studied and the results compared with FBS. UCB-PL contained high levels of protein content, platelet-derived growth factor- AB (PDGF-AB), and transforming growth factor (TGF) compared to PB-PL. All growth factors were stable for at least nine months post-storage at -70˚C. hMSCs proliferation enhanced following treatment with UCB-PL. With all three supplements, hMSCs could differentiate into all three lineages. PB-PL and UCB-PL both were potent in hMSCs proliferation. However, PB promoted osteoblastic differentiation and UCB-PL induced chondrogenic differentiation. Because of availability, ease of use and feasible standardization of UCB-PL, we have suggested that UCB-PL be used as an alternative to FBS and PB-PL for the cultivation and expansion of hMSCs in cellular therapy. Copyright© by Royan Institute. All rights reserved.

  8. Potency testing of mesenchymal stromal cell growth expanded in human platelet lysate from different human tissues.

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    Fazzina, R; Iudicone, P; Fioravanti, D; Bonanno, G; Totta, P; Zizzari, I G; Pierelli, L

    2016-08-25

    Mesenchymal stromal cells (MSCs) have been largely investigated, in the past decade, as potential therapeutic strategies for various acute and chronic pathological conditions. MSCs isolated from different sources, such as bone marrow (BM), umbilical cord tissue (UCT) and adipose tissue (AT), share many biological features, although they may show some differences on cumulative yield, proliferative ability and differentiation potential. The standardization of MSCs growth and their functional amplification is a mandatory objective of cell therapies. The aim of this study was to evaluate the cumulative yield and the ex vivo amplification potential of MSCs obtained from various sources and different subjects, using defined culture conditions with a standardized platelet lysate (PL) as growth stimulus. MSCs isolated from BM, UCT and AT and expanded in human PL were compared in terms of cumulative yield and growth potential per gram of starting tissue. MSCs morphology, phenotype, differentiation potential, and immunomodulatory properties were also investigated to evaluate their biological characteristics. The use of standardized PL-based culture conditions resulted in a very low variability of MSC growth. Our data showed that AT has the greater capacity to generate MSC per gram of initial tissue, compared to BM and UCT. However, UCT-MSCs replicated faster than AT-MSCs and BM-MSCs, revealing a greater proliferation capacity of this source irrespective of its lower MSC yield. All MSCs exhibited the typical MSC phenotype and the ability to differentiate into all mesodermal lineages, while BM-MSCs showed the most prominent immunosuppressive effect in vitro. The adoption of standardized culture conditions may help researchers and clinicians to reveal particular characteristics and inter-individual variability of MSCs sourced from different tissues. These data will be beneficial to set the standards for tissue collection and MSCs clinical-scale expansion both for cell banking

  9. Platelet lysate supports the in vitro expansion of human periodontal ligament stem cells for cytotherapeutic use.

    Science.gov (United States)

    Wu, Rui-Xin; Yu, Yang; Yin, Yuan; Zhang, Xi-Yu; Gao, Li-Na; Chen, Fa-Ming

    2017-08-01

    Human platelet lysate (PL) produced under optimal conditions of standardization and safety has been increasingly suggested as the future 'gold standard' supplement to replace fetal bovine serum (FBS) for the ex vivo propagation of mesenchymal stem cells for translational medicine and cell therapy applications. However, the multifaceted effects of PL on tissue-specific stem cells remain largely unexplored. In the present study, we investigated the stem cell behaviours of human periodontal ligament stem cells (PDLSCs) in media with or without PL. Our data indicate that human PL, either as an adjuvant for culture media or as a substitute for FBS, supports the proliferation and expansion of human PDLSCs derived from either 'young' or 'old' donors to the same extent as FBS, without interfering with their immunomodulatory capacities. Although PL appears to inhibit the in vitro differentiation of 'young' or 'old' PDLSCs, their decreased osteogenic potential may be restored to similar or higher levels compared with FBS-expanded cells. PL- and FBS-expanded PDLSCs exhibited a similar potential to form mineralized nodules and expressed similar levels of osteogenic genes. Our data indicate that large clinically relevant quantities of PDLSCs may be yielded by the use of human PL; however, further analysis of its precise composition and function will pave the way for determining optimized, defined culture conditions. In addition to the potential increase in patient safety, our findings highlight the need for further research to develop the potential of PL-expanded PDLSCs for clinical use. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  10. Donor Age of Human Platelet Lysate Affects Proliferation and Differentiation of Mesenchymal Stem Cells

    Science.gov (United States)

    Lohmann, Michael; Walenda, Gudrun; Hemeda, Hatim; Joussen, Sylvia; Drescher, Wolf; Jockenhoevel, Stefan; Hutschenreuter, Gabriele; Zenke, Martin; Wagner, Wolfgang

    2012-01-01

    The regenerative potential declines upon aging. This might be due to cell-intrinsic changes in stem and progenitor cells or to influences by the microenvironment. Mesenchymal stem cells (MSC) raise high hopes in regenerative medicine. They are usually culture expanded in media with fetal calf serum (FCS) or other serum supplements such as human platelet lysate (HPL). In this study, we have analyzed the impact of HPL-donor age on culture expansion. 31 single donor derived HPLs (25 to 57 years old) were simultaneously compared for culture of MSC. Proliferation of MSC did not reveal a clear association with platelet counts of HPL donors or growth factors concentrations (PDGF-AB, TGF-β1, bFGF, or IGF-1), but it was significantly higher with HPLs from younger donors (45 years). Furthermore, HPLs from older donors increased activity of senescence-associated beta-galactosidase (SA-βgal). HPL-donor age did not affect the fibroblastoid colony-forming unit (CFU-f) frequency, immunophenotype or induction of adipogenic differentiation, whereas osteogenic differentiation was significantly lower with HPLs from older donors. Concentrations of various growth factors (PDGF-AB, TGF-β1, bFGF, IGF-1) or hormones (estradiol, parathormone, leptin, 1,25 vitamin D3) were not associated with HPL-donor age or MSC growth. Taken together, our data support the notion that aging is associated with systemic feedback mechanisms acting on stem and progenitor cells, and this is also relevant for serum supplements in cell culture: HPLs derived from younger donors facilitate enhanced expansion and more pronounced osteogenic differentiation. PMID:22662236

  11. Purification of CD47-streptavidin fusion protein from bacterial lysate using biotin-agarose affinity chromatography.

    Science.gov (United States)

    Salehi, Nasrin; Peng, Ching-An

    2016-07-08

    CD47 is a widely expressed transmembrane glycoprotein that modulates the activity of a plethora of immune cells via its extracellular domain. Therefore, CD47 plays important roles in the regulation of immune responses and may serve as targets for the development of immunotherapeutic agents. To make sure CD47 functionality is intact under the process of protein conjugation, CD47-streptavidin fusion protein was expressed and purified because it can easily bind to biotin-tagged materials via the unique biotin-streptavidin affinity. In this study, gene sequences of CD47 extracellular domain (CD47ECD) and core streptavidin (coreSA) with a total 834 bp were inserted into pET20b plasmid to construct recombinant plasmid encoding CD47-SA fusion gene. After bacteria transformation, the CD47-SA fusion protein was expressed by isopropyl-β-d-thiogalactopyranoside (IPTG) induction. The collected bacteria lysate was loaded on biotinylated agarose to proceed the purification of CD47-SA fusion protein. Due to the unexpected high affinity between biotin and coreSA, standard washing and elution approaches (e.g., varying pH, using biotin, and applying guanidine hydrochloride) reported for biotin-streptavidin affinity chromatography were not able to separate the target fusion protein. Instead, using low concentration of the non-ionic detergent Triton X-100 followed with alkaline buffer could efficiently weaken the binding between biotin and coreSA, thereby eluting out CD47-SA fusion protein from the biotin agarose column. The purified CD47-SA fusion protein was further characterized by molecular biology methods and its antiphagocytic functionality was confirmed by the phagocytosis assay. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:949-958, 2016. © 2016 American Institute of Chemical Engineers.

  12. Ligament Tissue Engineering Using a Novel Porous Polycaprolactone Fumarate Scaffold and Adipose Tissue-Derived Mesenchymal Stem Cells Grown in Platelet Lysate

    Science.gov (United States)

    Wagner, Eric R.; Bravo, Dalibel; Dadsetan, Mahrokh; Riester, Scott M.; Chase, Steven; Westendorf, Jennifer J.; Dietz, Allan B.; van Wijnen, Andre J.; Yaszemski, Michael J.

    2015-01-01

    Purpose: Surgical reconstruction of intra-articular ligament injuries is hampered by the poor regenerative potential of the tissue. We hypothesized that a novel composite polymer “neoligament” seeded with progenitor cells and growth factors would be effective in regenerating native ligamentous tissue. Methods: We synthesized a fumarate-derivative of polycaprolactone fumarate (PCLF) to create macro-porous scaffolds to allow cell–cell communication and nutrient flow. Clinical grade human adipose tissue-derived human mesenchymal stem cells (AMSCs) were cultured in 5% human platelet lysate (PL) and seeded on scaffolds using a dynamic bioreactor. Cell growth, viability, and differentiation were examined using metabolic assays and immunostaining for ligament-related markers (e.g., glycosaminoglycans [GAGs], alkaline phosphatase [ALP], collagens, and tenascin-C). Results: AMSCs seeded on three-dimensional (3D) PCLF scaffolds remain viable for at least 2 weeks with proliferating cells filling the pores. AMSC proliferation rates increased in PL compared to fetal bovine serum (FBS) (p < 0.05). Cells had a low baseline expression of ALP and GAG, but increased expression of total collagen when induced by the ligament and tenogenic growth factor fibroblast growth factor 2 (FGF-2), especially when cultured in the presence of PL (p < 0.01) instead of FBS (p < 0.05). FGF-2 and PL also significantly increased immunostaining of tenascin-C and collagen at 2 and 4 weeks compared with human fibroblasts. Summary: Our results demonstrate that AMSCs proliferate and eventually produce a collagen-rich extracellular matrix on porous PCLF scaffolds. This novel scaffold has potential in stem cell engineering and ligament regeneration. PMID:26413793

  13. Equine platelet lysate as an alternative to fetal bovine serum in equine mesenchymal stromal cell culture - too much of a good thing?

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    Russell, K A; Koch, T G

    2016-03-01

    Multipotent mesenchymal stromal cells (MSC) are often culture-expanded in vitro. Presently, expansion medium (EM) for MSC is supplemented with fetal bovine serum (FBS). However, increasing cost, variable composition and potential risks associated with bovine antigens call for alternatives. Platelet lysate (PL) has shown promise as an alternative supplement. To determine how equine umbilical cord blood (CB) MSC proliferate in EM enriched with PL or FBS at various concentrations. Randomised dose escalation study. Platelet concentrate was generated from 5 equine whole blood samples through a double centrifugation method and standardised to 1 × 10(12) platelets/l prior to a freeze/thaw cycle to produce PL. Pooled PL or pooled FBS was added to EM at concentrations of 5% to 60%. Proliferation of 4 equine CB-MSC cultures was determined after 4 days using a resazurin semiquantitative assay. Cord blood-MSC proliferated with a dose-dependent response with no significant difference found between PL and FBS up to a 30% concentration. Beyond 30%, proliferation fell in the PL-cultured cells, while continued dose-dependent proliferation was noted in the FBS-cultured cells. Despite reduced cell numbers in high PL concentrations, live/dead staining revealed that adherent cells remained viable. Expansion medium enriched with PL can support short-term equine CB-MSC proliferation at conventional culture concentrations. Based on the unexpected suppression of CB-MSC at higher PL concentrations, an in vivo dose study is indicated to investigate if combinational therapies of CB-MSC and platelet-rich plasma are associated with synergistic or antagonistic effect on CB-MSC function. © 2015 EVJ Ltd.

  14. Human Platelet Lysate as a Xeno Free Alternative of Fetal Bovine Serum for the In Vitro Expansion of Human Mesenchymal Stromal Cells.

    Science.gov (United States)

    Mohammadi, Saeed; Nikbakht, Mohsen; Malek Mohammadi, Ashraf; Zahed Panah, Mahdi; Ostadali, Mohammad Reza; Nasiri, Hajar; Ghavamzadeh, Ardeshir

    2016-07-01

    Mesenchymal stromal cells (MSCs) are employed in various different clinical settings in order to modulate immune response. Human autologous and allogeneic supplements including platelet derivatives such as platelet lysate (PL), platelet-released factors (PRF) and serum are assessed in clinical studies to replace fetal bovine serum (FBS). The immunosuppressive activity and multi-potential characteristic of MSCs appear to be maintained when the cells are expanded in platelet derivatives. Platelet-rich plasma was collected from umbrical cord blood (UCB). Platelet-derived growth factors obtained by freeze and thaw methods. CD62P expression was determined by flow cytometry. The concentration of PDGF-BB and PDGF-AB was detemined by ELISA. We tested the ability of a different concentration of PL-supplemented medium to support the ex vivo expansion of Wharton's jelly derived MSCs. We also investigated the biological/functional properties of expanded MSCs in presence of different concentration of PL. The conventional karyotyping was performed in order to study the chromosomal stability. The gene expression of Collagen I and II aggrecan and SOX-9 in the presence of different concentrations of PL was evaluated by Real-time PCR. We observed 5% and 10% PL, causing greater effects on proliferation of MSCs .These cells exhibited typical morphology, immunophenotype and differentiation capacity. The genetic stability of these derivative cells from Wharton's jelly was demonstrated by a normal karyotype. Furthermore, the results of Real-time PCR analysis showed that the expression of chondrocyte specific genes was higher in MSCs in the presence of 5% and 10% PL, compared with FBS supplement. We demonstrated that PL could be used as an alternative safe source of growth factors for expansion of MSCs and also maintained similar growing potential and phenotype without any effect on chromosomal stability.

  15. Canine Platelet Lysate Is Inferior to Fetal Bovine Serum for the Isolation and Propagation of Canine Adipose Tissue- and Bone Marrow-Derived Mesenchymal Stromal Cells

    Science.gov (United States)

    Russell, Keith A.; Gibson, Thomas W. G.; Chong, Andrew; Co, Carmon; Koch, Thomas G.

    2015-01-01

    Background Mesenchymal stromal cells (MSC) are increasingly investigated for their clinical utility in dogs. Fetal bovine serum (FBS) is a common culture supplement used for canine MSC expansion. However, FBS content is variable, its clinical use carries risk of an immune response, and its cost is increasing due to global demand. Platelet lysate (PL) has proven to be a suitable alternative to FBS for expansion of human MSC. Hypothesis and Objectives We hypothesized that canine adipose tissue (AT) and bone marrow (BM) MSC could be isolated and expanded equally in PL and FBS at conventionally-used concentrations with differentiation of these MSC unaffected by choice of supplement. Our objectives were to evaluate the use of canine PL in comparison with FBS at four stages: 1) isolation, 2) proliferation, 3) spontaneous differentiation, and 4) directed differentiation. Results 1) Medium with 10% PL was unable to isolate MSC. 2) MSC, initially isolated in FBS-supplemented media, followed a dose-dependent response with no significant difference between PL and FBS cultures at up to 20% (AT) or 30% (BM) enrichment. Beyond these respective peaks, proliferation fell in PL cultures only, while a continued dose-dependent proliferation response was noted in FBS cultures. 3) Further investigation indicated PL expansion culture was inducing spontaneous adipogenesis in concentrations as low as 10% and as early as 4 days in culture. 4) MSC isolated in FBS, but expanded in either FBS or PL, maintained ability to undergo directed adipogenesis and osteogenesis, but not chondrogenesis. Conclusions/Significance Canine PL did not support establishment of MSC colonies from AT and BM, nor expansion of MSC, which appear to undergo spontaneous adipogenesis in response to PL exposure. In vivo studies are warranted to determine if concurrent use of MSC with any platelet-derived products such as platelet-rich plasma are associated with synergistic, neutral or antagonistic effects. PMID:26353112

  16. Canine Platelet Lysate Is Inferior to Fetal Bovine Serum for the Isolation and Propagation of Canine Adipose Tissue- and Bone Marrow-Derived Mesenchymal Stromal Cells.

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    Keith A Russell

    Full Text Available Mesenchymal stromal cells (MSC are increasingly investigated for their clinical utility in dogs. Fetal bovine serum (FBS is a common culture supplement used for canine MSC expansion. However, FBS content is variable, its clinical use carries risk of an immune response, and its cost is increasing due to global demand. Platelet lysate (PL has proven to be a suitable alternative to FBS for expansion of human MSC.We hypothesized that canine adipose tissue (AT and bone marrow (BM MSC could be isolated and expanded equally in PL and FBS at conventionally-used concentrations with differentiation of these MSC unaffected by choice of supplement. Our objectives were to evaluate the use of canine PL in comparison with FBS at four stages: 1 isolation, 2 proliferation, 3 spontaneous differentiation, and 4 directed differentiation.1 Medium with 10% PL was unable to isolate MSC. 2 MSC, initially isolated in FBS-supplemented media, followed a dose-dependent response with no significant difference between PL and FBS cultures at up to 20% (AT or 30% (BM enrichment. Beyond these respective peaks, proliferation fell in PL cultures only, while a continued dose-dependent proliferation response was noted in FBS cultures. 3 Further investigation indicated PL expansion culture was inducing spontaneous adipogenesis in concentrations as low as 10% and as early as 4 days in culture. 4 MSC isolated in FBS, but expanded in either FBS or PL, maintained ability to undergo directed adipogenesis and osteogenesis, but not chondrogenesis.Canine PL did not support establishment of MSC colonies from AT and BM, nor expansion of MSC, which appear to undergo spontaneous adipogenesis in response to PL exposure. In vivo studies are warranted to determine if concurrent use of MSC with any platelet-derived products such as platelet-rich plasma are associated with synergistic, neutral or antagonistic effects.

  17. Innate immune responses of equine monocytes cultured in equine platelet lysate.

    Science.gov (United States)

    Naskou, Maria C; Norton, Natalie A; Copland, Ian B; Galipeau, Jacques; Peroni, John F

    2018-01-01

    Platelet lysate (PL) has been extensively used for the laboratory expansion of human mesenchymal stem cells (MSC) in order to avoid fetal bovine serum (FBS) which has been associated with immune-mediated host reactions and transmission of bovine-origin microbial contaminants. Before suggesting the routine use of PL for MSC culture, we wanted to further investigate whether PL alone might trigger inflammatory responses when exposed to reactive white blood cells such as monocytes. Our objectives were to evaluate the inflammatory profile of equine monocytes cultured with equine PL (ePL) and to determine if ePL can modulate the expression of inflammatory cytokines in lipopolysaccharide (LPS)-stimulated monocytes. In a first experiment, equine monocytes were isolated and incubated with donor horse serum (DHS), FBS, six individual donors ePL or pooled ePL from all horses. In a second experiment, monocytes were stimulated with E. coli LPS in the presence of 1, 5 or 10% DHS and/or pooled ePL. After 6h of incubation, cell culture supernatants were assayed via ELISA for production of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and Interleukin 1β (IL-1β) as well as for the anti-inflammatory Interleukin 10 (IL-10). Equine monocytes incubated with pooled ePL produced significantly less TNF-α and significantly more IL-10 than monocytes incubated in FBS. A statistically significant difference was not identified for the production of IL-1β. The second experiment showed that pooled ePL added to LPS-stimulated equine monocytes resulted in a significant reduction in TNF-α and IL-1β production. IL-10 production was not significantly upregulated by the addition of ePL to LPS-stimulated monocytes. Finally, the addition of ePL to LPS-stimulated monocytes in the presence of various concentrations of DHS resulted to statistically significant decrease of TNF-α and IL-1β compared to the control groups. This is the first study to demonstrate that ePL suppresses

  18. Dendritic Cells Loaded with Pancreatic Cancer Stem Cells (CSCs) Lysates Induce Antitumor Immune Killing Effect In Vitro

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    Yin, Tao; Shi, Pengfei; Gou, Shanmiao; Shen, Qiang; Wang, Chunyou

    2014-01-01

    According to the cancer stem cells (CSCs) theory, malignant tumors may be heterogeneous in which a small population of CSCs drive the progression of cancer. Because of their intrinsic abilities, CSCs may survive a variety of treatments and then lead to therapeutic resistance and cancer recurrence. Pancreatic CSCs have been reported to be responsible for the malignant behaviors of pancreatic cancer, including suppression of immune protection. Thus, development of immune strategies to eradicate pancreatic CSCs may be of great value for the treatment of pancreatic cancer. In this study, we enriched pancreatic CSCs by culturing Panc-1 cells under sphere-forming conditions. Panc-1 CSCs expressed low levels of HLA-ABC and CD86, as measured by flow cytometry analysis. We further found that the Panc-1 CSCs modulate immunity by inhibiting lymphocyte proliferation which is promoted by phytohemagglutinin (PHA) and anti-CD3 monoclonal antibodies. The monocyte derived dendritic cells (DCs) were charged with total lysates generated from Panc-1 CSCs obtained from tumor sphere culturing. After co-culturing with lymphocytes at different ratios, the Panc-1 CSCs lysates modified DC effectively promoted lymphocyte proliferation. The activating efficiency reached 72.4% and 74.7% at the ratios of 1∶10 and 1∶20 with lymphocytes. The activated lymphocytes secreted high levels of INF-γ and IL-2, which are strong antitumor cytokines. Moreover, Panc-1 CSCs lysates modified DC induced significant cytotoxic effects of lymphocytes on Panc-1 CSCs and parental Panc-1 cells, respectively, as shown by lactate dehydrogenase (LDH) assay. Our study demonstrates that the development of CSCs-based vaccine is a promising strategy for treating pancreatic cancer. PMID:25521461

  19. Dendritic cells loaded with pancreatic Cancer Stem Cells (CSCs lysates induce antitumor immune killing effect in vitro.

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    Tao Yin

    Full Text Available According to the cancer stem cells (CSCs theory, malignant tumors may be heterogeneous in which a small population of CSCs drive the progression of cancer. Because of their intrinsic abilities, CSCs may survive a variety of treatments and then lead to therapeutic resistance and cancer recurrence. Pancreatic CSCs have been reported to be responsible for the malignant behaviors of pancreatic cancer, including suppression of immune protection. Thus, development of immune strategies to eradicate pancreatic CSCs may be of great value for the treatment of pancreatic cancer. In this study, we enriched pancreatic CSCs by culturing Panc-1 cells under sphere-forming conditions. Panc-1 CSCs expressed low levels of HLA-ABC and CD86, as measured by flow cytometry analysis. We further found that the Panc-1 CSCs modulate immunity by inhibiting lymphocyte proliferation which is promoted by phytohemagglutinin (PHA and anti-CD3 monoclonal antibodies. The monocyte derived dendritic cells (DCs were charged with total lysates generated from Panc-1 CSCs obtained from tumor sphere culturing. After co-culturing with lymphocytes at different ratios, the Panc-1 CSCs lysates modified DC effectively promoted lymphocyte proliferation. The activating efficiency reached 72.4% and 74.7% at the ratios of 1∶10 and 1∶20 with lymphocytes. The activated lymphocytes secreted high levels of INF-γ and IL-2, which are strong antitumor cytokines. Moreover, Panc-1 CSCs lysates modified DC induced significant cytotoxic effects of lymphocytes on Panc-1 CSCs and parental Panc-1 cells, respectively, as shown by lactate dehydrogenase (LDH assay. Our study demonstrates that the development of CSCs-based vaccine is a promising strategy for treating pancreatic cancer.

  20. Platelet lysate 3D scaffold supports mesenchymal stem cell chondrogenesis: an improved approach in cartilage tissue engineering.

    Science.gov (United States)

    Moroz, Andrei; Bittencourt, Renata Aparecida Camargo; Almeida, Renan Padron; Felisbino, Sérgio Luis; Deffune, Elenice

    2013-01-01

    Articular lesions are still a major challenge in orthopedics because of cartilage's poor healing properties. A major improvement in therapeutics was the development of autologous chondrocytes implantation (ACI), a biotechnology-derived technique that delivers healthy autologous chondrocytes after in vitro expansion. To obtain cartilage-like tissue, 3D scaffolds are essential to maintain chondrocyte differentiated status. Currently, bioactive 3D scaffolds are promising as they can deliver growth factors, cytokines, and hormones to the cells, giving them a boost to attach, proliferate, induce protein synthesis, and differentiate. Using mesenchymal stem cells (MSCs) differentiated into chondrocytes, one can avoid cartilage harvesting. Thus, we investigated the potential use of a platelet-lysate-based 3D bioactive scaffold to support chondrogenic differentiation and maintenance of MSCs. The MSCs from adult rabbit bone marrow (n = 5) were cultivated and characterized using three antibodies by flow cytometry. MSCs (1 × 10(5)) were than encapsulated inside 60 µl of a rabbit platelet-lysate clot scaffold and maintained in Dulbecco's Modified Eagle Medium Nutrient Mixture F-12 supplemented with chondrogenic inductors. After 21 days, the MSCs-seeded scaffolds were processed for histological analysis and stained with toluidine blue. This scaffold was able to maintain round-shaped cells, typical chondrocyte metachromatic extracellular matrix deposition, and isogenous group formation. Cells accumulated inside lacunae and cytoplasm lipid droplets were other observed typical chondrocyte features. In conclusion, the usage of a platelet-lysate bioactive scaffold, associated with a suitable chondrogenic culture medium, supports MSCs chondrogenesis. As such, it offers an alternative tool for cartilage engineering research and ACI.

  1. Comparison of clinical grade human platelet lysates for cultivation of mesenchymal stromal cells from bone marrow and adipose tissue

    DEFF Research Database (Denmark)

    Juhl, Morten; Tratwal, Josefine; Follin, Bjarke

    2016-01-01

    be devoid of any animal derived components. We have evaluated whether human Platelet Lysate (hPL) could be an attractive alternative to animal supplements. METHODS: MSCs from bone marrow (BMSCs) and adipose tissue-derived stromal cells (ASCs) obtained from three donors were culture expanded in three...... culture conditions with 10% fetal bovine serum (FBS). Cell morphology, proliferation, phenotype, genomic stability, and differentiation potential were analyzed. RESULTS: Regardless of manufacturer, BMSCs and ASCs cultured in hPL media showed a significant increase in proliferation capacity compared to FBS...

  2. Data from SILAC-based quantitative analysis of lysates from mouse microglial cells treated with Withaferin A (WA).

    Science.gov (United States)

    Narayan, Malathi; Seeley, Kent W; Jinwal, Umesh K

    2016-06-01

    Mass spectrometry data collected in a study analyzing the effect of withaferin A (WA) on a mouse microglial (N9) cell line is presented in this article. Data was collected from SILAC-based quantitative analysis of lysates from mouse microglial cells treated with either WA or DMSO vehicle control. This article reports all the proteins that were identified in this analysis. The data presented here is related to the published research article on the effect of WA on the differential regulation of proteins in mouse microglial cells [1]. Mass spectrometry data has also been deposited in the ProteomeXchange with the identifier PXD003032.

  3. Tumour cell lysate-loaded dendritic cell vaccine induces biochemical and memory immune response in castration-resistant prostate cancer patients.

    Science.gov (United States)

    Reyes, D; Salazar, L; Espinoza, E; Pereda, C; Castellón, E; Valdevenito, R; Huidobro, C; Inés Becker, M; Lladser, A; López, M N; Salazar-Onfray, F

    2013-09-17

    Recently, we produced a tumour antigen-presenting cells (TAPCells) vaccine using a melanoma cell lysate, called TRIMEL, as an antigen source and an activation factor. Tumour antigen-presenting cells induced immunological responses and increased melanoma patient survival. Herein, we investigated the effect of TAPCells loaded with prostate cancer cell lysates (PCCL) as an antigen source, and TRIMEL as a dendritic cell (DC) activation factor; which were co-injected with the Concholepas concholepas haemocyanin (CCH) as an adjuvant on castration-resistant prostate cancer (CRPC) patients. The lysate mix capacity, for inducing T-cell activation, was analysed by flow cytometry and Elispot. Delayed-type hypersensitivity (DTH) reaction against PCCL, frequency of CD8(+) memory T cells (Tm) in blood and prostate-specific antigen (PSA) levels in serum were measured in treated patients. The lysate mix induced functional mature DCs that were capable of activating PCCL-specific T cells. No relevant adverse reactions were observed. Six out of 14 patients showed a significant decrease in levels of PSA. DTH(+) patients showed a prolonged PSA doubling-time after treatment. Expansion of functional central and effector CD8(+) Tm were detected. Treatment of CRPC patients with lysate-loaded TAPCells and CCH as an adjuvant is safe: generating biochemical and memory immune responses. However, the limited number of cases requires confirmation in a phase II clinical trial.

  4. Cultivation and irradiation of human fibroblasts in a medium enriched with platelet lysate for obtaining feeder layer in epidermal cell culture

    International Nuclear Information System (INIS)

    Yoshito, Daniele

    2011-01-01

    For over 30 years, the use of culture medium, enriched with bovine serum, and murines fibroblasts, with the rate of proliferation controlled by irradiation or by share anticarcinogenic drugs, has been playing successfully its role in assisting in the development of keratinocytes in culture, for clinical purposes. However, currently there is a growing concern about the possibility of transmitting prions and animals viruses to transplanted patients. Taking into account this concern, the present work aims to cultivate human fibroblasts in a medium enriched with human platelets lysate and determine the irradiation dose of these cells, for obtaining feeder layer in epidermal cell culture. For carrying out the proposed objective, platelets lysis has standardized, this lysate was used for human fibroblasts cultivation and the irradiation dose enough to inhibit its duplication was evaluated. Human keratinocytes were cultivated in these feeder layers, in culture medium enriched with the lysate. With these results we conclude that the 10% platelets lysate promoted a better adhesion and proliferation of human fibroblasts and in all dose levels tested (60 to 300 Gy), these had their mitotic activity inactivated by ionizing irradiation, being that the feeder layers obtained with doses from 70 to 150 Gy were those that provided the best development of keratinocytes in medium containing 2.5% of human platelet lysate. Therefore, it was possible to standardize both the cultivation of human fibroblasts as its inactivation for use as feeder layer in culture of keratinocytes, so as to eliminate xenobiotics components. (author)

  5. Rapid and simple purification of elastin-like polypeptides directly from whole cells and cell lysates by organic solvent extraction.

    Science.gov (United States)

    VerHeul, Ross; Sweet, Craig; Thompson, David H

    2018-03-26

    Elastin-like polypeptides (ELP) are a well-known class of proteins that are being increasingly utilized in a variety of biomedical applications, due to their beneficial physicochemical properties. A unifying feature of ELP is their demonstration of a sequence tunable inverse transition temperature (Tt) that enables purification using a simple, straightforward process called inverse transition cycling (ITC). Despite the utility of ITC, the process is inherently limited to ELP with an experimentally accessible Tt. Since the underlying basis for the ELP Tt is related to its high overall hydrophobicity, we anticipated that ELP would be excellent candidates for purification by organic extraction. We report the first method for rapidly purifying ELP directly from whole E. coli cells or clarified lysates using pure organic solvents and solvent mixtures, followed by aqueous back extraction. Our results show that small ELP and a large ELP-fusion protein can be isolated in high yield from whole cells or cell lysates with greater than 95% purity in less than 30 min and with very low levels of LPS and DNA contamination.

  6. High DNA stability in white blood cells and buffy coat lysates stored at ambient temperature under anoxic and anhydrous atmosphere.

    Directory of Open Access Journals (Sweden)

    Anne-Lise Fabre

    Full Text Available Conventional storage of blood-derived fractions relies on cold. However, lately, ambient temperature preservation has been evaluated by several independent institutions that see economic and logistic advantages in getting rid of the cold chain. Here we validated a novel procedure for ambient temperature preservation of DNA in white blood cell and buffy coat lysates based on the confinement of the desiccated biospecimens under anoxic and anhydrous atmosphere in original hermetic minicapsules. For this validation we stored encapsulated samples either at ambient temperature or at several elevated temperatures to accelerate aging. We found that DNA extracted from stored samples was of good quality with a yield of extraction as expected. Degradation rates were estimated from the average fragment size of denatured DNA run on agarose gels and from qPCR reactions. At ambient temperature, these rates were too low to be measured but the degradation rate dependence on temperature followed Arrhenius' law, making it possible to extrapolate degradation rates at 25°C. According to these values, the DNA stored in the encapsulated blood products would remain larger than 20 kb after one century at ambient temperature. At last, qPCR experiments demonstrated the compatibility of extracted DNA with routine DNA downstream analyses. Altogether, these results showed that this novel storage method provides an adequate environment for ambient temperature long term storage of high molecular weight DNA in dehydrated lysates of white blood cells and buffy coats.

  7. High DNA stability in white blood cells and buffy coat lysates stored at ambient temperature under anoxic and anhydrous atmosphere

    Science.gov (United States)

    Luis, Aurélie; Colotte, Marthe; Tuffet, Sophie; Bonnet, Jacques

    2017-01-01

    Conventional storage of blood-derived fractions relies on cold. However, lately, ambient temperature preservation has been evaluated by several independent institutions that see economic and logistic advantages in getting rid of the cold chain. Here we validated a novel procedure for ambient temperature preservation of DNA in white blood cell and buffy coat lysates based on the confinement of the desiccated biospecimens under anoxic and anhydrous atmosphere in original hermetic minicapsules. For this validation we stored encapsulated samples either at ambient temperature or at several elevated temperatures to accelerate aging. We found that DNA extracted from stored samples was of good quality with a yield of extraction as expected. Degradation rates were estimated from the average fragment size of denatured DNA run on agarose gels and from qPCR reactions. At ambient temperature, these rates were too low to be measured but the degradation rate dependence on temperature followed Arrhenius’ law, making it possible to extrapolate degradation rates at 25°C. According to these values, the DNA stored in the encapsulated blood products would remain larger than 20 kb after one century at ambient temperature. At last, qPCR experiments demonstrated the compatibility of extracted DNA with routine DNA downstream analyses. Altogether, these results showed that this novel storage method provides an adequate environment for ambient temperature long term storage of high molecular weight DNA in dehydrated lysates of white blood cells and buffy coats. PMID:29190767

  8. The virion N protein of infectious bronchitis virus is more phosphorylated than the N protein from infected cell lysates

    International Nuclear Information System (INIS)

    Jayaram, Jyothi; Youn, Soonjeon; Collisson, Ellen W.

    2005-01-01

    Because phosphorylation of the infectious bronchitis virus (IBV) nucleocapsid protein (N) may regulate its multiple roles in viral replication, the dynamics of N phosphorylation were examined. 32 P-orthophosphate labeling and Western blot analyses confirmed that N was the only viral protein that was phosphorylated. Pulse labeling with 32 P-orthophosphate indicated that the IBV N protein was phosphorylated in the virion, as well as at all times during infection in either chicken embryo kidney cells or Vero cells. Pulse-chase analyses followed by immunoprecipitation of IBV N proteins using rabbit anti-IBV N polyclonal antibody demonstrated that the phosphate on the N protein was stable for at least 1 h. Simultaneous labeling with 32 P-orthophosphate and 3 H-leucine identified a 3.5-fold increase in the 32 P: 3 H counts per minute (cpm) ratio of N in the virion as compared to the 32 P: 3 H cpm ratio of N in the cell lysates from chicken embryo kidney cells, whereas in Vero cells the 32 P: 3 H cpm ratio of N from the virion was 10.5-fold greater than the 32 P: 3 H cpm ratio of N from the cell lysates. These studies are consistent with the phosphorylation of the IBV N playing a role in assembly or maturation of the viral particle

  9. Vaccination with melanoma lysate-pulsed dendritic cells, of patients with advanced colorectal carcinoma: report from a phase I study

    DEFF Research Database (Denmark)

    Burgdorf, S K; Fischer, A; Claesson, M H

    2006-01-01

    Immune therapy have shown new and exciting perspectives for cancer treatment. Aim of our study was to evaluate toxicity and possible adverse effects from vaccination of patients with advanced colorectal cancer with autologous dendritic cells (DC) pulsed with lysate from a newly developed melanoma...... contained 3-5 x 10(6) DCs. Five of the six patients received all five vaccines. The treatment was well tolerated in all patients without any observed vaccine-correlated adverse effects. Treatment with this DC-based cancer vaccine proved safe and non-toxic.......Immune therapy have shown new and exciting perspectives for cancer treatment. Aim of our study was to evaluate toxicity and possible adverse effects from vaccination of patients with advanced colorectal cancer with autologous dendritic cells (DC) pulsed with lysate from a newly developed melanoma...... and selected melanoma cell line enriched in expression of MAGE-A antigens and deficient in expression of melanoma differentiation antigens: tyrosinase, MART-1 and gp100. Vaccinations were administered intradermally on the proximal thigh with a total of five given vaccines at 2 weeks intervals. Each vaccine...

  10. Strain-dependent augmentation of tight-junction barrier function in human primary epidermal keratinocytes by Lactobacillus and Bifidobacterium lysates.

    Science.gov (United States)

    Sultana, Reshma; McBain, Andrew J; O'Neill, Catherine A

    2013-08-01

    In this study, we investigated whether probiotic lysates can modify the tight-junction function of human primary keratinocytes. The keratinocytes were grown on cell culture inserts and treated with lysates from Bifidobacterium longum, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus fermentum, or Lactobacillus rhamnosus GG. With the exception of L. fermentum (which decreased cell viability), all strains markedly enhanced tight-junction barrier function within 24 h, as assessed by measurements of transepithelial electrical resistance (TEER). However, B. longum and L. rhamnosus GG were the most efficacious, producing dose-dependent increases in resistance that were maintained for 4 days. These increases in TEER correlated with elevated expression of tight-junction protein components. Neutralization of Toll-like receptor 2 abolished both the increase in TEER and expression of tight-junction proteins induced by B. longum, but not L. rhamnosus GG. These data suggest that some bacterial strains increase tight-junction function via modulation of protein components but the different pathways involved may vary depending on the bacterial strain.

  11. The effects of platelet lysate on maturation, fertilization and embryo development of NMRI mouse oocytes at germinal vesicle stage.

    Science.gov (United States)

    Pazoki, Hassan; Eimani, Hussein; Farokhi, Farah; Shahverdi, Abdol-Hossein; Tahaei, Leila Sadat

    2016-04-01

    Improving in vitro maturation could increase the rate of pregnancy from oocytes matured in vitro. Consequently, patients will be prevented from using gonadotropin with its related side effects. In this study, the maturation medium was enriched by platelet lysate (PL), then maturation and subsequent developments were monitored. Oocytes at germinal vesicle stage with cumulus cells (cumulus-oocyte complex) and without cumulus cells (denuded oocytes) were obtained from mature female mice. The maturation medium was enriched by 5 and 10 % PL and 5 % PL + 5 % fetal bovine serum (FBS) as experimental groups; the control groups' media consisted of 5 and 10 % FBS. After 18 h, the matured oocytes were collected and, after fertilization, subsequent development was monitored. The rates of maturation, fertilization and 2-cell embryo development for the denuded oocyte groups in experimental media 5 % PL and 5 % PL + 5 % FBS were significantly higher than those of the control groups ( P platelet lysate could improve the maturation rate in the absence of granulosa cells compared to media with FBS. This extract also had positive effects on fertilization and embryo development.

  12. Evaluation of human platelet lysate and dimethyl sulfoxide as cryoprotectants for the cryopreservation of human adipose-derived stem cells.

    Science.gov (United States)

    Wang, Chuan; Xiao, Ran; Cao, Yi-Lin; Yin, Hong-Yu

    2017-09-09

    Cryopreservation provides an effective technique to maintain the functional properties of human adipose-derived stem cells (ASCs). Dimethylsulfoxide (DMSO) and fetal bovine serum (FBS) are frequently used as cryoprotectants for this purpose. However, the use of DMSO can result in adverse effects and toxic reactions and FBS can introduce risks of viral, prion, zoonose contaminations and evoke immune responses after injection. It is therefore crucial to reduce DMSO concentrations and use serum-free solution in the cryopreservation process. Human platelet lysate (PL) is a promising candidate for use as an alternative to DMSO and FBS. Therefore, in this study, with an aim to identify a cryoprotective agent for ASC cryopreservation, we determined the viability, proliferation potential, phenotype, and differentiation potential of fresh ASCs and ASCs cryopreserved using different combinations of three cryoprotective agents: fetal bovine serum (FBS), dimethylsulfoxide (DMSO), and human platelet lysate (PL). The viability of the ASCs cryopreserved with 90% FBS and 10% DMSO, 95% FBS and 5% DMSO, and 97% PL and 3% DMSO was >80%, and the proliferation potentials, cell phenotypes, and differentiation potentials of these groups were similar to those of fresh ASCs. Together, our findings suggest that a combination of 97% PL and 3% DMSO is an ideal cryoprotective agent for the efficient cryopreservation of human ASCs. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. The CBM RICH project

    Energy Technology Data Exchange (ETDEWEB)

    Adamczewski-Musch, J. [GSI Darmstadt (Germany); Becker, K.-H. [University Wuppertal (Germany); Belogurov, S. [ITEP Moscow (Russian Federation); Boldyreva, N. [PNPI Gatchina (Russian Federation); Chernogorov, A. [ITEP Moscow (Russian Federation); Deveaux, C. [University Gießen (Germany); Dobyrn, V. [PNPI Gatchina (Russian Federation); Dürr, M. [University Gießen (Germany); Eom, J. [Pusan National University (Korea, Republic of); Eschke, J. [GSI Darmstadt (Germany); Höhne, C. [University Gießen (Germany); Kampert, K.-H. [University Wuppertal (Germany); Kleipa, V. [GSI Darmstadt (Germany); Kochenda, L. [PNPI Gatchina (Russian Federation); Kolb, B. [GSI Darmstadt (Germany); Kopfer, J. [University Wuppertal (Germany); Kravtsov, P. [PNPI Gatchina (Russian Federation); Lebedev, S.; Lebedeva, E. [University Gießen (Germany); Leonova, E. [PNPI Gatchina (Russian Federation); and others

    2014-12-01

    The Compressed Baryonic Matter (CBM) experiment will study the properties of super dense nuclear matter by means of heavy ion collisions at the future FAIR facility. An integral detector component is a large Ring Imaging Cherenkov detector with CO{sub 2} gas radiator, which will mainly serve for electron identification and pion suppression necessary to access rare dileptonic probes like e{sup +}e{sup −} decays of light vector mesons or J/Ψ. We describe the design of this future RICH detector and focus on results obtained by building a CBM RICH detector prototype tested at CERN-PS.

  14. Neutron rich nuclei

    International Nuclear Information System (INIS)

    Foucher, R.

    1979-01-01

    If some β - emitters are particularly interesting to study in light, medium, and heavy nuclei, another (and also) difficult problem is to know systematically the properties of these neutron rich nuclei far from the stability line. A review of some of their characteristics is presented. How far is it possible to be objective in the interpretation of data is questioned and implications are discussed

  15. Tritium-labelled leukotriene B4 binding to the guinea-pig spleen membrane preparation: a rich tissue source for a high-affinity leukotriene B4 receptor site

    International Nuclear Information System (INIS)

    Cheng, J.B.; Cheng, E.I.; Kohi, F.; Townley, R.G.

    1986-01-01

    Intact human granulocytes contain a leukotriene (LT) B4 receptor binding site, but the limited supply of these cells could adversely affect further progress of the study of this receptor. To select a tissue homogenate rich for this site, we have characterized the binding of highly specific [ 3 H]LTB4 to guinea-pig spleen membranes and we have determined the ability of LTB4 to compete with [ 3 H]LTB4 for binding sites in the membranes of 10 nonspleen tissues. In the spleen membrane, MgCl2 and CaCl2 enhanced [ 3 H]LTB4 binding, but NaCl and KCl decreased it. Spleen [ 3 H] LTB4 binding was a function of protein concentration and was rapid, reversible, stereoselective and saturable. Kinetic analyses showed that the rate constant for association and dissociation at 25 0 C was 0.47 nM-1 min-1 and 0.099 min-1, respectively. A Scatchard plot of the data of the equilibrium experiment resulted a straight line with a dissociation constant of 1.8 nM and a density of 274 fmol/mg of protein. Moreover, the LTB4/[ 3 H]LTB4 competition study performed at 4 or 25 0 C revealed the inhibitory constant (Ki) of LTB4 to be in the nanomolar range. The rank order of agents competing for spleen [ 3 H]LTB4 binding was: LTB4 (Ki = 2.8 nM) greater than 20-hydroxy-LTB4 (23 nM) greater than LTA4 (48 nM) greater than LTA4 methyl ester (0.13 microM) greater than 20-carboxy-LTB4 (greater than 6.6 microM) greater than or equal to arachidonic acid (0.15mM) = FPL-55,712 and FPL-57,231 (0.1-0.2 mM). Competition studies further indicated that felodipine, a 1,4-dihyropyridine Ca++ channel blocker, exhibited micromolar inhibition of spleen [ 3 H]LTB4 binding

  16. The CBM RICH project

    Energy Technology Data Exchange (ETDEWEB)

    Adamczewski-Musch, J. [GSI Helmholtzzentrum für Schwerionenforschung GmbH, D-64291 Darmstadt (Germany); Akishin, P. [Laboratory of Information Technologies, Joint Institute for Nuclear research (JINR-LIT), Dubna (Russian Federation); Becker, K.-H. [Department of Physics, University of Wuppertal, D-42097 Wuppertal (Germany); Belogurov, S. [SSC RF ITEP, 117218 Moscow (Russian Federation); Bendarouach, J. [Institute of Physics II and Institute of Applied Physics, Justus Liebig University Giessen, D-35392 Giessen (Germany); Boldyreva, N. [National Research Centre “Kurchatov Institute” B.P. Konstantinov Petersburg Nuclear Physics Institute, 188300 Gatchina (Russian Federation); Chernogorov, A. [SSC RF ITEP, 117218 Moscow (Russian Federation); Deveaux, C. [Institute of Physics II and Institute of Applied Physics, Justus Liebig University Giessen, D-35392 Giessen (Germany); Dobyrn, V. [National Research Centre “Kurchatov Institute” B.P. Konstantinov Petersburg Nuclear Physics Institute, 188300 Gatchina (Russian Federation); Dürr, M. [Institute of Physics II and Institute of Applied Physics, Justus Liebig University Giessen, D-35392 Giessen (Germany); Eschke, J. [GSI Helmholtzzentrum für Schwerionenforschung GmbH, D-64291 Darmstadt (Germany); Förtsch, J. [Department of Physics, University of Wuppertal, D-42097 Wuppertal (Germany); Heep, J.; Höhne, C. [Institute of Physics II and Institute of Applied Physics, Justus Liebig University Giessen, D-35392 Giessen (Germany); Kampert, K.-H. [Department of Physics, University of Wuppertal, D-42097 Wuppertal (Germany); and others

    2017-02-11

    The CBM RICH detector is an integral component of the future CBM experiment at FAIR, providing efficient electron identification and pion suppression necessary for the measurement of rare dileptonic probes in heavy ion collisions. The RICH design is based on CO{sub 2} gas as radiator, a segmented spherical glass focussing mirror with Al+MgF{sub 2} reflective coating, and Multianode Photomultipliers for efficient Cherenkov photon detection. Hamamatsu H12700 MAPMTs have recently been selected as photon sensors, following an extensive sensor evaluation, including irradiation tests to ensure sufficient radiation hardness of the MAPMTs. A brief overview of the detector design and concept is given, results on the radiation hardness of the photon sensors are shown, and the development of a FPGA-TDC based readout chain is discussed.

  17. The CLEO RICH detector

    International Nuclear Information System (INIS)

    Artuso, M.; Ayad, R.; Bukin, K.; Efimov, A.; Boulahouache, C.; Dambasuren, E.; Kopp, S.; Li, Ji; Majumder, G.; Menaa, N.; Mountain, R.; Schuh, S.; Skwarnicki, T.; Stone, S.; Viehhauser, G.; Wang, J.C.; Coan, T.E.; Fadeyev, V.; Maravin, Y.; Volobouev, I.; Ye, J.; Anderson, S.; Kubota, Y.; Smith, A.

    2005-01-01

    We describe the design, construction and performance of a Ring Imaging Cherenkov Detector (RICH) constructed to identify charged particles in the CLEO experiment. Cherenkov radiation occurs in LiF crystals, both planar and ones with a novel 'sawtooth'-shaped exit surface. Photons in the wavelength interval 135-165nm are detected using multi-wire chambers filled with a mixture of methane gas and triethylamine vapor. Excellent π/K separation is demonstrated

  18. Gut-targeted immunonutrition boosting natural killer cell activity using Saccharomyces boulardii lysates in immuno-compromised healthy elderly subjects.

    Science.gov (United States)

    Naito, Yasuhiro; Marotta, Francesco; Kantah, Makoto K; Zerbinati, Nicola; Kushugulova, Almagul; Zhumadilov, Zhaxybay; Illuzzi, Nicola; Sapienza, Chiara; Takadanohara, Hiroshi; Kobayashi, Riyichi; Catanzaro, Roberto

    2014-04-01

    The aim of this study was to assess the immunomodulatory effect of KC-1317 (a symbiotic mixture containing Saccharomyces boulardii lysate in a cranberry, colostrum-derived lactoferrin, fragaria, and lactose mixture) supplementation in immune-compromised but otherwise healthy elderly subjects. A liquid formulation of KC-1317 was administered in a randomized controlled trial (RCT) fashion to healthy volunteers (65-79 years) previously selected for low natural killer (NK) cell activity, and this parameter was checked at the completion of the study. A significant improvement in NK cell activity of KC-1317 consumers was observed as compared to placebo at the end of 2 months. Although preliminary, these beneficial immune-modulatory effects of KC-1317 in aged individuals might indicate its employment within a wider age-management strategy.

  19. Data from SILAC-based quantitative analysis of lysates from mouse microglial cells treated with Withaferin A (WA

    Directory of Open Access Journals (Sweden)

    Malathi Narayan

    2016-06-01

    Full Text Available Mass spectrometry data collected in a study analyzing the effect of withaferin A (WA on a mouse microglial (N9 cell line is presented in this article. Data was collected from SILAC-based quantitative analysis of lysates from mouse microglial cells treated with either WA or DMSO vehicle control. This article reports all the proteins that were identified in this analysis. The data presented here is related to the published research article on the effect of WA on the differential regulation of proteins in mouse microglial cells [1]. Mass spectrometry data has also been deposited in the ProteomeXchange with the identifier http://www.ebi.ac.uk/pride/archive/projects/PXD003032.

  20. Human Platelet Lysate versus Fetal Calf Serum: These Supplements Do Not Select for Different Mesenchymal Stromal Cells.

    Science.gov (United States)

    Fernandez-Rebollo, Eduardo; Mentrup, Birgit; Ebert, Regina; Franzen, Julia; Abagnale, Giulio; Sieben, Torsten; Ostrowska, Alina; Hoffmann, Per; Roux, Pierre-François; Rath, Björn; Goodhardt, Michele; Lemaitre, Jean-Marc; Bischof, Oliver; Jakob, Franz; Wagner, Wolfgang

    2017-07-11

    Culture medium of mesenchymal stromal cells (MSCs) is usually supplemented with either human platelet lysate (HPL) or fetal calf serum (FCS). Many studies have demonstrated that proliferation and cellular morphology are affected by these supplements - it is therefore important to determine if they favor outgrowth of different subpopulations and thereby impact on the heterogeneous composition of MSCs. We have isolated and expanded human bone marrow-derived MSCs in parallel with HPL or FCS and demonstrated that HPL significantly increases proliferation and leads to dramatic differences in cellular morphology. Remarkably, global DNA-methylation profiles did not reveal any significant differences. Even at the transcriptomic level, there were only moderate changes in pairwise comparison. Furthermore, the effects on proliferation, cytoskeletal organization, and focal adhesions were reversible by interchanging to opposite culture conditions. These results indicate that cultivation of MSCs with HPL or FCS has no systematic bias for specific cell types.

  1. CBM RICH geometry optimization

    Energy Technology Data Exchange (ETDEWEB)

    Mahmoud, Tariq; Hoehne, Claudia [II. Physikalisches Institut, Giessen Univ. (Germany); Collaboration: CBM-Collaboration

    2016-07-01

    The Compressed Baryonic Matter (CBM) experiment at the future FAIR complex will investigate the phase diagram of strongly interacting matter at high baryon density and moderate temperatures in A+A collisions from 2-11 AGeV (SIS100) beam energy. The main electron identification detector in the CBM experiment will be a RICH detector with a CO{sub 2} gaseous-radiator, focusing spherical glass mirrors, and MAPMT photo-detectors being placed on a PMT-plane. The RICH detector is located directly behind the CBM dipole magnet. As the final magnet geometry is now available, some changes in the RICH geometry become necessary. In order to guarantee a magnetic field of 1 mT at maximum in the PMT plane for effective operation of the MAPMTs, two measures have to be taken: The PMT plane is moved outwards of the stray field by tilting the mirrors by 10 degrees and shielding boxes have been designed. In this contribution the results of the geometry optimization procedure are presented.

  2. Purification of Proteins From Cell-Culture Medium or Cell-Lysate by High-Speed Counter-Current Chromatography Using Cross-Axis Coil Planet Centrifuge

    Science.gov (United States)

    Shibusawa, Yoichi; Ito, Yoichiro

    2014-01-01

    This review describes protein purifications from cell culture medium or cell-lysate by high speed counter-current chromatography using the cross-axis coil planet centrifuge. Purifications were performed using aqueous two phase systems composed of polyethylene glycols and dextrans. PMID:25360182

  3. Long-term cytokine and growth factor release from equine platelet-rich fibrin clots obtained with two different centrifugation protocols.

    Science.gov (United States)

    Jiménez-Aristizabal, Román F; López, Catalina; Álvarez, María E; Giraldo, Carlos; Prades, Marta; Carmona, Jorge U

    2017-09-01

    To compare the temporal release (over three weeks) of tumor necrosis factor alpha (TNF-α), interleukin 4 (IL-4), IL-1 receptor antagonist (IL-1ra), platelet-derived growth factor BB (PDGF-BB) and transforming growth factor beta-1 (TGF-β 1 ) from two platelet-rich fibrin (PRF) preparations from equine blood obtained at either 240g/8min or 416g/10min. Whole blood from 10 horses was used to obtain PRF clots by two different centrifugation protocols. After 1h of rest, PRF clots were deposited in wells with culture medium, which was changed at 6h, 24h and then every 48h to 21days. Cytokines and GFs were measured by ELISA at 1h (serum supernatants from PRF clots) and all time points of culture medium change. A negative control (plasma) and a positive control (blood lysate) were also included. There were no relevant differences between the two protocols for the temporal release of proteins. However, a significant (p=0.01) effect of time was noted. All cytokines were detected after 6h of PRF clot culture until day 21. GF were detected at 1h until day 21. The concentrations for these proteins diminished gradually over time. A highly significant (p=0.01) correlation was noticed between all the proteins evaluated. Leukocytes enmeshed in PRF clots were able to produce cytokines, TGF-β 1 and PDGF-BB. These findings demonstrate a paramount role of leukocytes in wound healing induced or modified by PRF clots in mammals. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Human serum and platelet lysate are appropriate xeno-free alternatives for clinical-grade production of human MuStem cell batches.

    Science.gov (United States)

    Saury, Charlotte; Lardenois, Aurélie; Schleder, Cindy; Leroux, Isabelle; Lieubeau, Blandine; David, Laurent; Charrier, Marine; Guével, Laëtitia; Viau, Sabrina; Delorme, Bruno; Rouger, Karl

    2018-05-02

    Canine MuStem cells have demonstrated regenerative efficacy in a dog model of muscular dystrophy, and the recent characterization of human counterparts (hMuStem) has highlighted the therapeutic potential of this muscle-derived stem cell population. To date, these cells have only been generated in research-grade conditions. However, evaluation of the clinical efficacy of any such therapy will require the production of hMuStem cells in compliance with good manufacturing practices (GMPs). Because the current use of fetal bovine serum (FBS) to isolate and expand hMuStem cells raises several ethical, safety, and supply concerns, we assessed the use of two alternative xeno-free blood derivatives: human serum (HS) and a human platelet lysate (hPL). hMuStem cells were isolated and expanded in vitro in either HS-supplemented or hPL-supplemented media and the proliferation rate, clonogenicity, myogenic commitment potential, and oligopotency compared with that observed in FBS-supplemented medium. Flow cytometry and high-throughput 3'-digital gene expression RNA sequencing were used to characterize the phenotype and global gene expression pattern of hMuStem cells cultured with HS or hPL. HS-supplemented and hPL-supplemented media both supported the isolation and long-term proliferation of hMuStem cells. Compared with FBS-based medium, both supplements enhanced clonogenicity and allowed for a reduction in growth factor supplementation. Neither supplement altered the cell lineage pattern of hMuStem cells. In vitro differentiation assays revealed a decrease in myogenic commitment and in the fusion ability of hMuStem cells when cultured with hPL. In return, this reduction of myogenic potential in hPL-supplemented cultures was rapidly reversed by substitution of hPL with HS or fibrinogen-depleted hPL. Moreover, culture of hMuStem cells in hPL hydrogel and fibrinogen-depleted hPL demonstrated that myogenic differentiation potential is maintained in heparin-free hPL derivatives. Our

  5. Platelet-rich fibrin matrix for facial plastic surgery.

    Science.gov (United States)

    Sclafani, Anthony P; Saman, Masoud

    2012-05-01

    Platelets are known primarily for their role in hemostasis, but there is increasing interest in the effect of platelets on wound healing. Platelet isolates such as platelet-rich plasma have been advocated to enhance and accelerate wound healing. This article describes the use of a novel preparation, platelet-rich fibrin matrix (PRFM), for facial plastic surgery applications such as volume augmentation, fat transfer supplementation, and as an adjunct to open surgical procedures. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. Platelet-rich fibrin or platelet-rich plasma – which one is better? an opinion

    Directory of Open Access Journals (Sweden)

    Shweta Bansal

    2017-01-01

    Full Text Available The healing of hard and soft tissue in mediated by a wide range of intracellular and extracellular events that are regulated by signaling proteins. Platelets can play a crucial role in periodontal regeneration as they are the reservoirs of growth factors and cytokines which are the key factors for regeneration of bone and maturation of soft tissue. Platelet-rich plasma (PRP is first generation platelet concentrate. However, the short duration of cytokine release and its poor mechanical properties have resulted in search of new material. Platelet-rich fibrin (PRF is a natural fibrin-based biomaterial prepared from an anticoagulant-free blood harvest without any artificial biochemical modification (no bovine thrombin is required that allows obtaining fibrin membranes enriched with platelets and growth factors. The slow polymerization during centrifugation, fibrin-based structure, ease of preparation, minimal expense makes PRF somewhat superior in some aspect to PRP.

  7. Lithium-Rich Nanoscale Li 1.2 Mn 0.54 Ni 0.13 Co 0.13 O 2 Cathode Material Prepared by Co-Precipitation Combined Freeze Drying (CP-FD) for Lithium-Ion Batteries

    Energy Technology Data Exchange (ETDEWEB)

    Bai, Ying; Li, Yu; Wu, Chuan; Lu, Jun; Li, Hui; Liu, Zhaolin; Zhong, Yunxia; Chen, Shi; Zhang, Cunzhong; Amine, Khalil; Wu, Feng

    2015-07-14

    Nanoscale Li-rich Li1.2Mn0.54Ni0.13Co0.13O2 material is synthesized by a co-precipitation combined freeze drying (CP-FD) method, and compared with a conventional co-precipitation method combined vacuum drying (CP-VD). With the combination of X-ray diffraction (XRD) and scanning electron microscopy (SEM), it is found that the sample from CP-FD method consists of a pure phase with good crystallinity and small, homogenous particles (100-300 nm) with uniform particle size distribution. Inductively coupled plasma spectroscopy (ICP) shows that the sample has a stoichiometric ratio of n((Li)): n((Mn)): n((Ni)): n((Co))=9: 4: 1: 1; and its Brunauer-Emmett-Teller (BET) specific surface area is 5.749 m(2)g(-1). This sample achieves excellent electrochemical properties: its initial discharge capacities are 298.9 mAhg(-1) at 0.1C (20 mAg(-1)), 246.1 mAhg(-1) at 0.5C, 215.8 mAhg(-1) at 1C, and 154.2 mAhg(-1) at 5C (5C charge and 5C discharge), as well as good cycling performance. In addition, the Li+ chemical diffusion coefficient of Li1.2Mn0.54Ni0.13Co0.13O2 material prepared by the CP-FD method is 4.59 x 10(-11) cm(2) s(-1), which is higher than that of the Li1.2Mn0.54Ni0.13Co0.13O2 material prepared by CP-VD. This phenomenon illustrates the potential for Li1.2Mn0.54Ni0.13Co0.13O2 with good rate performance synthesized by CP-FD method.

  8. Microarray R-based analysis of complex lysate experiments with MIRACLE.

    Science.gov (United States)

    List, Markus; Block, Ines; Pedersen, Marlene Lemvig; Christiansen, Helle; Schmidt, Steffen; Thomassen, Mads; Tan, Qihua; Baumbach, Jan; Mollenhauer, Jan

    2014-09-01

    Reverse-phase protein arrays (RPPAs) allow sensitive quantification of relative protein abundance in thousands of samples in parallel. Typical challenges involved in this technology are antibody selection, sample preparation and optimization of staining conditions. The issue of combining effective sample management and data analysis, however, has been widely neglected. This motivated us to develop MIRACLE, a comprehensive and user-friendly web application bridging the gap between spotting and array analysis by conveniently keeping track of sample information. Data processing includes correction of staining bias, estimation of protein concentration from response curves, normalization for total protein amount per sample and statistical evaluation. Established analysis methods have been integrated with MIRACLE, offering experimental scientists an end-to-end solution for sample management and for carrying out data analysis. In addition, experienced users have the possibility to export data to R for more complex analyses. MIRACLE thus has the potential to further spread utilization of RPPAs as an emerging technology for high-throughput protein analysis. Project URL: http://www.nanocan.org/miracle/. Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press.

  9. A comparative study of Mono Mac 6 cells, isolated mononuclear cells and Limulus amoebocyte lysate assay in pyrogen testing

    DEFF Research Database (Denmark)

    Moesby, Lise; Jensen, S; Hansen, E W

    1999-01-01

    ) and Staphylococcus aureus was comparable to that of MNC. Aspergillus niger and Candida albicans induced IL-6 in isolated MNC, but not in MM6. The detection limit for Salmonella typhimurium in the MM6 assay was comparable to that of the LAL assay. As expected, S. aureus and C. albicans did not show any LAL activity......Pyrogen induced secretion of interleukin 6 (IL-6) in Mono Mac 6 (MM6) cells was measured. The ability of the MM6 cell culture to detect pyrogens was compared to the Limulus amoebocyte lysate (LAL) test and isolated mononuclear cells (MNC). The detection limit of MM6 for lipopolysaccharide (LPS....... A. niger and Influenza virus showed some activity in the LAL test, but could not be detected by MM6 cells. In conclusion, the MM6 assay is a good supplement to the current pyrogen assays for detection of LPS, S. aureus and S. typhimurium, but the MM6 assay could not detect A. niger, C. albicans...

  10. Disposable Amperometric Immunosensor for the Determination of Human P53 Protein in Cell Lysates Using Magnetic Micro-Carriers

    Directory of Open Access Journals (Sweden)

    María Pedrero

    2016-11-01

    Full Text Available An amperometric magnetoimmunosensor for the determination of human p53 protein is described in this work using a sandwich configuration involving the covalent immobilization of a specific capture antibody onto activated carboxylic-modified magnetic beads (HOOC-MBs and incubation of the modified MBs with a mixture of the target protein and horseradish peroxidase-labeled antibody (HRP-anti-p53. The resulting modified MBs are captured by a magnet placed under the surface of a disposable carbon screen-printed electrode (SPCE and the amperometric responses are measured at −0.20 V (vs. an Ag pseudo-reference electrode, upon addition of hydroquinone (HQ as a redox mediator and H2O2 as the enzyme substrate. The magnetoimmunosensing platform was successfully applied for the detection of p53 protein in different cell lysates without any matrix effect after a simple sample dilution. The results correlated accurately with those provided by a commercial ELISA kit, thus confirming the immunosensor as an attractive alternative for rapid and simple determination of this protein using portable and affordable instrumentation.

  11. A versatile coupled cell-free transcription-translation system based on tobacco BY-2 cell lysates.

    Science.gov (United States)

    Buntru, Matthias; Vogel, Simon; Stoff, Katrin; Spiegel, Holger; Schillberg, Stefan

    2015-05-01

    Cell-free protein synthesis is a powerful method for the high-throughput production of recombinant proteins, especially proteins that are difficult to express in living cells. Here we describe a coupled cell-free transcription-translation system based on tobacco BY-2 cell lysates (BYLs). Using a combination of fractional factorial designs and response surface models, we developed a cap-independent system that produces more than 250 μg/mL of functional enhanced yellow fluorescent protein (eYFP) and about 270 μg/mL of firefly luciferase using plasmid templates, and up to 180 μg/mL eYFP using linear templates (PCR products) in 18 h batch reactions. The BYL contains actively-translocating microsomal vesicles derived from the endoplasmic reticulum, promoting the formation of disulfide bonds, glycosylation and the cotranslational integration of membrane proteins. This was demonstrated by expressing a functional full-size antibody (∼ 150 μg/mL), the model enzyme glucose oxidase (GOx) (∼ 7.3 U/mL), and a transmembrane growth factor (∼ 25 μg/mL). Subsequent in vitro treatment of GOx with peptide-N-glycosidase F confirmed the presence of N-glycans. Our results show that the BYL can be used as a high-throughput expression and screening platform that is particularly suitable for complex and cytotoxic proteins. © 2014 Wiley Periodicals, Inc.

  12. Implementation of a Multiplex and Quantitative Proteomics Platform for Assessing Protein Lysates Using DNA-Barcoded Antibodies.

    Science.gov (United States)

    Lee, Jinho; Geiss, Gary K; Demirkan, Gokhan; Vellano, Christopher P; Filanoski, Brian; Lu, Yiling; Ju, Zhenlin; Yu, Shuangxing; Guo, Huifang; Bogatzki, Lisa Y; Carter, Warren; Meredith, Rhonda K; Krishnamurthy, Savitri; Ding, Zhiyong; Beechem, Joseph M; Mills, Gordon B

    2018-06-01

    Molecular analysis of tumors forms the basis for personalized cancer medicine and increasingly guides patient selection for targeted therapy. Future opportunities for personalized medicine are highlighted by the measurement of protein expression levels via immunohistochemistry, protein arrays, and other approaches; however, sample type, sample quantity, batch effects, and "time to result" are limiting factors for clinical application. Here, we present a development pipeline for a novel multiplexed DNA-labeled antibody platform which digitally quantifies protein expression from lysate samples. We implemented a rigorous validation process for each antibody and show that the platform is amenable to multiple protocols covering nitrocellulose and plate-based methods. Results are highly reproducible across technical and biological replicates, and there are no observed "batch effects" which are common for most multiplex molecular assays. Tests from basal and perturbed cancer cell lines indicate that this platform is comparable to orthogonal proteomic assays such as Reverse-Phase Protein Array, and applicable to measuring the pharmacodynamic effects of clinically-relevant cancer therapeutics. Furthermore, we demonstrate the potential clinical utility of the platform with protein profiling from breast cancer patient samples to identify molecular subtypes. Together, these findings highlight the potential of this platform for enhancing our understanding of cancer biology in a clinical translation setting. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Evaluation of a platelet lysate bilayered system for periodontal regeneration in a rat intrabony three-wall periodontal defect.

    Science.gov (United States)

    Babo, Pedro S; Cai, Xinjie; Plachokova, Adelina S; Reis, Rui L; Jansen, John; Gomes, Manuela E; Walboomers, X Frank

    2018-02-01

    With currently available therapies, full regeneration of lost periodontal tissues after periodontitis cannot be achieved. In this study, a combined compartmentalized system was tested, composed of (a) a platelet lysate (PL)-based construct, which was placed along the root aiming to regenerate the root cementum and periodontal ligament, and (b) a calcium phosphate cement composite incorporated with hyaluronic acid microspheres loaded with PL, aiming to promote the regeneration of alveolar bone. This bilayered system was assessed in a 3-wall periodontal defect in Wistar rats. The periodontal healing and the inflammatory response of the materials were scored for a period up to 6 weeks after implantation. Furthermore, histomorphometrical measurements were performed to assess the epithelial downgrowth, the formation of alveolar bone, and the formation of new connective tissue attachment. Our data showed that the stabilization of platelet-origin proteins on the root surface increased the overall periodontal healing score and restricted the formation of long epithelial junctions. Nevertheless, the faster degradation of the cement component with incorporated hyaluronic acid microspheres compromised the stability of the system, which hampered the periodontal regeneration. Overall, in this work, we proved the positive therapeutic effect of the immobilization of a PL-based construct over the root surface in a combined compartmentalized system to assist predictable healing of functional periodontium. Therefore, after optimization of the hard tissue analogue, the system should be further elaborated in (pre)clinical validation studies. Copyright © 2017 John Wiley & Sons, Ltd.

  14. Development of an Injectable Calcium Phosphate/Hyaluronic Acid Microparticles System for Platelet Lysate Sustained Delivery Aiming Bone Regeneration.

    Science.gov (United States)

    Babo, Pedro S; Santo, Vítor E; Gomes, Manuela E; Reis, Rui L

    2016-11-01

    Despite the biocompatibility and osteoinductive properties of calcium phosphate (CaP) cements their low biodegradability hampers full bone regeneration. Herein the incorporation of CaP cement with hyaluronic acid (HAc) microparticles loaded with platelet lysate (PL) to improve the degradability and biological performance of the cements is proposed. Cement formulations incorporating increasing weight ratios of either empty HAc microparticles or microparticles loaded with PL (10 and 20 wt%) are developed as well as cements directly incorporating PL. The direct incorporation of PL improves the mechanical properties of the plain cement, reaching values similar to native bone. Morphological analysis shows homogeneous particle distribution and high interconnectivity between the HAc microparticles. The cements incorporating PL (with or without the HAc microparticles) present a sustained release of PL proteins for up to 8 d. The sustained release of PL modulates the expression of osteogenic markers in seeded human adipose tissue derived stem cells, thus suggesting the stimulatory role of this hybrid system toward osteogenic commitment and bone regeneration applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Differentiation of Rat bone marrow Mesenchymal stem cells into Adipocytes and Cardiomyocytes after treatment with platelet lysate.

    Science.gov (United States)

    Homayouni Moghadam, Farshad; Tayebi, Tahereh; Barzegar, Kazem

    2016-01-01

    Mesenchymal stem cells (MSCs) are multipotential cells and their therapeutic potency is under intense investigation. Studying the effect of different induction factors on MSCs could increase our knowledge about the differentiation potency of these cells. One of the most important sources of these factors in mammalian body is platelet. Platelet lysate (PL) contains many growth factors and therefore, it can be used as a differentiation inducer. In the present study, the effect of PL on differentiation of rat bone marrow MSCs into cardiomyocytes was studied. To study the differentiation-inducing effect of PL, MSCs were treated with 2.5, 5 and 10% PL. Early results of this study showed that PL in high concentrations (10%) induces adipogenic differentiation of MSCs. Therefore, to evaluate differentiation to cardiomyocytes, MSCs were cultured in media containing lower levels of PL (2.5% and 5%) and then cardiomyogenic differentiation was induced by treatment with 5-azacytidine. Differentiation of MSCs was evaluated using direct observation of beating cells, immunostaining and real-time PCR techniques. The results of qPCR showed that treatment with PL alone increased the expression of cardiac alpha actinin (CAA) being predictable by earlier observation of beating cells in PL-treated groups. The results of staining assays against cardiac alpha actinin also showed that there were stained cells in PL-treated groups. The results of the present study showed that PL is a powerful induction factor for differentiation of MSCs into different cell lines such as cardiomyocytes and adipocytes.

  16. HEMOXCell, a New Oxygen Carrier Usable as an Additive for Mesenchymal Stem Cell Culture in Platelet Lysate-Supplemented Media.

    Science.gov (United States)

    Le Pape, Fiona; Cosnuau-Kemmat, Lucie; Richard, Gaëlle; Dubrana, Frédéric; Férec, Claude; Zal, Franck; Leize, Elisabeth; Delépine, Pascal

    2017-04-01

    Human mesenchymal stem cells (MSCs) are promising candidates for therapeutic applications such as tissue engineering. However, one of the main challenges is to improve oxygen supply to hypoxic areas to reduce oxygen gradient formation while preserving MSC differentiation potential and viability. For this purpose, a marine hemoglobin, HEMOXCell, was evaluated as an oxygen carrier for culturing human bone marrow MSCs in vitro for future three-dimensional culture applications. Impact of HEMOXCell on cell growth and viability was assessed in human platelet lysate (hPL)-supplemented media. Maintenance of MSC features, such as multipotency and expression of MSC specific markers, was further investigated by biochemical assays and flow cytometry analysis. Our experimental results highlight its oxygenator potential and indicate that an optimal concentration of 0.025 g/L HEMOXCell induces a 25%-increase of the cell growth rate, preserves MSC phenotype, and maintains MSC differentiation properties; a two-fold higher concentration induces cell detachment without altering cell viability. Our data suggest the potential interest of HEMOXCell as a natural oxygen carrier for tissue engineering applications to oxygenate hypoxic areas and to maintain cell viability, functions and "stemness." These features will be further tested within three-dimensional scaffolds. © 2017 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

  17. The effect of Platelet Lysate on osteoblast proliferation associated with a transient increase of the inflammatory response in bone regeneration.

    Science.gov (United States)

    Ruggiu, Alessandra; Ulivi, Valentina; Sanguineti, Francesca; Cancedda, Ranieri; Descalzi, Fiorella

    2013-12-01

    Platelet Lysate (PL) contains a cocktail of growth factors and cytokines, which actively participates in tissue repair and its clinical application has been broadly described. The aim of this study was to assess the regenerative potential of PL for bone repair. We demonstrated that PL stimulation induces a transient increase of the inflammatory response in quiescent human osteoblasts, via NF-kB activation, COX-2 induction, PGE2 production and secretion of pro-inflammatory cytokines. Furthermore, we showed that long-term PL stimulation enhances proliferation of actively replicating osteoblasts, without affecting their differentiation potential, along with changes of cell morphology, resulting in increased cell density at confluence. In confluent resting osteoblasts, PL treatment induced resumption of proliferation, change in cell morphology and increase of cell density at confluence. A burst of PL treatment (24-h) was sufficient to trigger such processes in both conditions. These results correlated with up-regulation of the proliferative and survival pathways ERKs and Akt and with cell cycle re-activation via induction of CyclinD1 and phosphorylation of Rb, following PL stimulation. Our findings demonstrate that PL treatment results in activation and expansion of resting osteoblasts, without affecting their differentiation potential. Therefore PL represents a good therapeutic candidate in regenerative medicine for bone repair. Copyright © 2013 Elsevier Ltd. All rights reserved.

  18. Long-term safety and efficacy of autologous platelet lysate drops for treatment of ocular GvHD.

    Science.gov (United States)

    Pezzotta, S; Del Fante, C; Scudeller, L; Rossi, G C; Perotti, C; Bianchi, P E; Antoniazzi, E

    2017-01-01

    Current ocular GvHD (oGvHD) treatments are suboptimal. We investigated the safety and efficacy of long-term continuous treatment with autologous platelet lysate (PL) drops in patients with oGvHD Dry Eye Syndrome (DES) score 2-3 refractory to topical conventional therapy. Ophthalmic evaluation was performed at 6 month intervals. Symptoms were assessed using the Glaucoma Symptom Scale (GSS). Patients were defined 'responders' when showing a reduction at least one grade on National Institutes of Health Eye Score from baseline at the 6 month visit. Thirty-one patients were included, and 16 (51%) completed 36 months of follow-up (range 6.5-72.7). At 6 months all patients were classified as responders: median GSS symptom score decreased from 70 to 41 (33 at 36 months), median GSS function score reduced from 68 to 46 (33 at 36 months) (all P<0.001). Median Tear Break Up Time improved from 3 to 6 s after 6 months and was maintained over time. All signs improved at 6 and 36 months (clinical and statistical significance). No severe adverse events occurred. Long-term treatment with PL drops is secure and effective for oGvHD and can be an efficient therapy option from initial stages of oGvHD to prevent permanent ocular impairment and improving quality of life.

  19. Capillary zone electrophoresis-multiple reaction monitoring from 100 pg of RAW 264.7 cell lysate digest.

    Science.gov (United States)

    Sun, Liangliang; Li, Yihan; Champion, Matthew M; Zhu, Guijie; Wojcik, Roza; Dovichi, Norman J

    2013-06-07

    Capillary zone electrophoresis-multiple/single reaction monitoring (CZE-MRM/SRM), which employed an electrokinetically driven sheath-flow electrospray interface, was used for the rapid and highly sensitive detection of protein analytes in complex tryptic digests. MRM channels were developed against a commercial exponential mixture of bovine proteins. Five proteins spanning four orders of magnitude concentration range were confidently detected from only 2.5 ng of the digest mixture; the mass detection limits (S/N = 3) of two detected proteins, alpha-casein and glutamate dehydrogenase were about 600 zmol and 30 amol, respectively. This technique was then applied to a RAW 264.7 cell lysate digest. Three proteins were confidently and reproducibly detected from 100 pg of this digest. The sample amount corresponds to the approximate protein content from a single cell, which suggests that CZE-MRM may be a useful analytical tool in chemical cytometry. In addition to providing highly sensitive detection of proteins in complex mixtures, this system is highly rapid; migration time of the protein digests was less than 10 min.

  20. Evaluation of Peripheral Blood and Cord Blood Platelet Lysates in Isolation and Expansion of Multipotent Mesenchymal Stromal Cells

    Directory of Open Access Journals (Sweden)

    Ioanna Christou

    2018-02-01

    Full Text Available Background: Multipotent Mesenchymal Stromal Cells (MSCs are used in tissue engineering and regenerative medicine. The in vitro isolation and expansion of MSCs involve the use of foetal bovine serum (FBS. However, many concerns have been raised regarding the safety of this product. In this study, alternative additives derived either from peripheral or cord blood were tested as an FBS replacement. Methods: Platelet lysates (PL from peripheral and cord blood were used for the expansion of MSCs. The levels of growth factors in peripheral blood (PB and cord blood (CB PLs were determined using the Multiple Reaction Monitoring (MRM. Finally, the cell doubling time (CDT, tri-lineage differentiation and phenotypic characterization of the MSCs expanded with FBS and PLs were determined. Results: MSCs treated with culture media containing FBS and PB-PL, were successfully isolated and expanded, whereas MSCs treated with CB-PL could not be maintained in culture. Furthermore, the MRM analysis yielded differences in growth factor levels between PB-PL and CB-PL. In addition, the MSCs were successfully expanded with FBS and PB-PL and exhibited tri-lineage differentiation and stable phenotypic characteristics. Conclusion: PB-PL could be used as an alternative additive for the production of MSCs culture medium applied to xenogeneic-free expansion and maintenance of MSCs in large scale clinical studies.

  1. Clinical and Immunological Effects in Patients with Advanced Non-Small Cell Lung-Cancer after Vaccination with Dendritic Cells Exposed to an Allogeneic Tumor Cell Lysate*

    DEFF Research Database (Denmark)

    Engell-Noerregaard, Lotte; Kvistborg, Pia; Zocca, Mai-Britt

    2013-01-01

    Background: We evaluated the clinical and immunological effects of dendritic cell (DC) vaccination of patients with NSCLC. Autologous DCs were pulsed with a MAGE containing allogeneic melanoma cell lysate (MelCancerVac®, Dandrit Biotech, Copenhagen, Denmark). Imiquimod cream, proleukin and celeco......Background: We evaluated the clinical and immunological effects of dendritic cell (DC) vaccination of patients with NSCLC. Autologous DCs were pulsed with a MAGE containing allogeneic melanoma cell lysate (MelCancerVac®, Dandrit Biotech, Copenhagen, Denmark). Imiquimod cream, proleukin...... and celecoxib were used as adjuvants to the vaccines. The objective of the study was to evaluate specific T cell response in vitro by IFN EliSpot. Secondary objec- tives were overall survival, response and quality of life (QoL). Results: Twenty-two patients initiated the vaccination program consisting of ten...

  2. LHCB RICH gas system proposal

    CERN Document Server

    Bosteels, Michel; Haider, S

    2001-01-01

    Both LHCb RICH will be operated with fluorocarbon as gas radiator. RICH 1 will be filled with 4m^3 of C4F10 and RICH 2 with 100m^3 of CF4. The gas systems will run as a closed loop circulation and a gas recovery system within the closed loop is planned for RICH 1, where the recovery of the CF4 will only be realised during filling and emptying of the detector. Inline gas purification is foreseen for the gas systems in order to limit water and oxygen impurities.

  3. Analyzing Protein Changes in Guinea Pig Tissue Lysates Using Non-guinea Pig Specific Antibodies: Procedures for Western Blotting and Examples Using 16 Individual Antibodies for Common CNS Proteins

    National Research Council Canada - National Science Library

    Johnson, Erik A; Daugherty, Kelly S

    2006-01-01

    ... behavioral and protein changes due to the absence of guinea pig-specific antibodies. We have developed a procedure to determine the specificity of commercially available, non-guinea pig-specific antibodies in guinea pig lysates...

  4. Distribution of AAV8 particles in cell lysates and culture media changes with time and is dependent on the recombinant vector

    Directory of Open Access Journals (Sweden)

    Bryan A Piras

    2016-01-01

    Full Text Available With clinical trials ongoing, efficient clinical production of adeno-associated virus (AAV to treat large numbers of patients remains a challenge. We compared distribution of AAV8 packaged with Factor VIII (FVIII in cell culture media and lysates on days 3, 5, 6, and 7 post-transfection and found increasing viral production through day 6, with the proportion of viral particles in the media increasing from 76% at day 3 to 94% by day 7. Compared to FVIII, AAV8 packaged with Factor IX and Protective Protein/Cathepsin A vectors demonstrated a greater shift from lysate towards media from day 3 to 6, implying that particle distribution is dependent on recombinant vector. Larger-scale productions showed that the ratio of full-to-empty AAV particles is similar in media and lysate, and that AAV harvested on day 6 post-transfection provides equivalent function in mice compared to AAV harvested on day 3. This demonstrates that AAV8 production can be optimized by prolonging the duration of culture post-transfection, and simplified by allowing harvest of media only, with disposal of cells that contain 10% or less of total vector yield. Additionally, the difference in particle distribution with different expression cassettes implies a recombinant vector-dependent processing mechanism which should be taken into account during process development.

  5. Information rich display design

    International Nuclear Information System (INIS)

    Welch, Robin; Braseth, Alf Ove; Veland, Oeystein

    2004-01-01

    This paper presents the concept Information Rich Displays. The purpose of Information Rich Displays (IRDs) is to condensate prevailing information in process displays in such a way that each display format (picture) contains more relevant information for the user. Compared to traditional process control displays, this new concept allows the operator to attain key information at a glance and at the same time allows for improved monitoring of larger portions of the process. This again allows for reduced navigation between both process and trend displays and ease the cognitive demand on the operator. This concept has been created while working on designing display prototypes for the offshore petroleum production facilities of tomorrow. Offshore installations basically consist of wells, separation trains (where oil, gas and water are separated from each other), an oil tax measurement system (where oil quality is measured and the pressure increased to allow for export), gas compression (compression of gas for export) and utility systems (water treatment, chemical systems etc.). This means that an offshore control room operator has to deal with a complex process that comprises several functionally different systems. The need for a new approach to offshore display format design is in particular based on shortcomings in today's designs related to the keyhole effect, where the display format only reveals a fraction of the whole process. Furthermore, the upcoming introduction of larger off- and on-shore operation centres will increase the size and complexity of the operators' work domain. In the light of the increased demands on the operator, the proposed IRDs aim to counter the negative effects this may have on the workload. In this work we have attempted to classify the wide range of different roles an operator can have in different situations. The information content and amount being presented to the operator in a display should be viewed in context of the roles the

  6. FUNCTIONALIZATION OF 3D FIBROUS SCAFFOLDS PREPARED USING CENTRIFUGAL SPINNING WITH LIPOSOMES AS A SIMPLE DRUG DELIVERY SYSTEM

    Directory of Open Access Journals (Sweden)

    Michala Rampichová

    2017-06-01

    Full Text Available 3D materials supporting cell adhesion, infiltration and proliferation are crucial for bone tissue engineering. In the current study we combined PCL fibers prepared using centrifugal spinning with adhered liposomes filled with platelet lysate as a natural source of growth factors. The scaffold was seeded with MG-63 cells and tested in vitro as a potential drug delivery system for bone tissue engineering.

  7. Human platelet lysate improves human cord blood derived ECFC survival and vasculogenesis in three dimensional (3D) collagen matrices.

    Science.gov (United States)

    Kim, Hyojin; Prasain, Nutan; Vemula, Sasidhar; Ferkowicz, Michael J; Yoshimoto, Momoko; Voytik-Harbin, Sherry L; Yoder, Mervin C

    2015-09-01

    Human cord blood (CB) is enriched in circulating endothelial colony forming cells (ECFCs) that display high proliferative potential and in vivo vessel forming ability. Since diminished ECFC survival is known to dampen the vasculogenic response in vivo, we tested how long implanted ECFC survive and generate vessels in three-dimensional (3D) type I collagen matrices in vitro and in vivo. We hypothesized that human platelet lysate (HPL) would promote cell survival and enhance vasculogenesis in the 3D collagen matrices. We report that the percentage of ECFC co-cultured with HPL that were alive was significantly enhanced on days 1 and 3 post-matrix formation, compared to ECFC alone containing matrices. Also, co-culture of ECFC with HPL displayed significantly more vasculogenic activity compared to ECFC alone and expressed significantly more pro-survival molecules (pAkt, p-Bad and Bcl-xL) in the 3D collagen matrices in vitro. Treatment with Akt1 inhibitor (A-674563), Akt2 inhibitor (CCT128930) and Bcl-xL inhibitor (ABT-263/Navitoclax) significantly decreased the cell survival and vasculogenesis of ECFC co-cultured with or without HPL and implicated activation of the Akt1 pathway as the critical mediator of the HPL effect on ECFC in vitro. A significantly greater average vessel number and total vascular area of human CD31(+) vessels were present in implants containing ECFC and HPL, compared to the ECFC alone implants in vivo. We conclude that implantation of ECFC with HPL in vivo promotes vasculogenesis and augments blood vessel formation via diminishing apoptosis of the implanted ECFC. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Mesenchymal Stromal Cells Implantation in Combination with Platelet Lysate Product Is Safe for Reconstruction of Human Long Bone Nonunion.

    Science.gov (United States)

    Labibzadeh, Narges; Emadedin, Mohsen; Fazeli, Roghayeh; Mohseni, Fatemeh; Hosseini, Seyedeh Esmat; Moghadasali, Reza; Mardpour, Soura; Azimian, Vajiheh; Ghorbani Liastani, Maede; Mirazimi Bafghi, Ali; Baghaban Eslaminejad, Mohamadreza; Aghdami, Nasser

    2016-01-01

    Nonunion is defined as a minimum of 9 months since injury without any visible progressive signs of healing for 3 months. Recent literature has shown that the application of mesenchymal stromal cells is safe, in vitro and in vivo, for treating long bone nonunion. The present study was performed to investigate the safety of mesenchymal stromal cell (MSC) implantation in combination with platelet lysate (PL) product for treating human long bone nonunion. In this case series clinical trial, orthopedic surgeons visited eighteen patients with long bone nonunion, of whom 7 complied with the eligibility criteria. These patients received mesenchymal stromal cells (20 million cells implanted once into the nonunion site using a fluoroscopic guide) in combination with PL product. For evaluation of the effects of this intervention all the patients were followed up by taking anterior-posterior and lateral X-rays of the affected limb before and 1, 3, 6, and 12 months after the implantation. All side effects (local or systemic, serious or non-serious, related or unrelated) were observed during this time period. From a safety perspective the MSC implantation in combination with PL was very well tolerated during the 12 months of the trial. Four patients were healed; based on the control Xray evidence, bony union had occurred. Results from the present study suggest that the implantation of bone marrow-derived MSCs in combination with PL is safe for the treatment of nonunion. A double blind, controlled clinical trial is required to assess the efficacy of this treatment (Registration Number: NCT01206179).

  9. Senescence and quiescence in adipose-derived stromal cells: Effects of human platelet lysate, fetal bovine serum and hypoxia.

    Science.gov (United States)

    Søndergaard, Rebekka Harary; Follin, Bjarke; Lund, Lisbeth Drozd; Juhl, Morten; Ekblond, Annette; Kastrup, Jens; Haack-Sørensen, Mandana

    2017-01-01

    Adipose-derived stromal cells (ASCs) are attractive sources for cell-based therapies. The hypoxic niche of ASCs in vivo implies that cells will benefit from hypoxia during in vitro expansion. Human platelet lysate (hPL) enhances ASC proliferation rates, compared with fetal bovine serum (FBS) at normoxia. However, the low proliferation rates of FBS-expanded ASCs could be signs of senescence or quiescence. We aimed to determine the effects of hypoxia and hPL on the expansion of ASCs and whether FBS-expanded ASCs are senescent or quiescent. ASCs expanded in FBS or hPL at normoxia or hypoxia until passage 7 (P7), or in FBS until P5 followed by culture in hPL until P7, were evaluated by proliferation rates, cell cycle analyses, gene expression and β-galactosidase activity. hPL at normoxia and hypoxia enhanced proliferation rates and expression of cyclins, and decreased G0/G1 fractions and expression of p21 and p27, compared with FBS. The shift from FBS to hPL enhanced cyclin levels, decreased p21 and p27 levels and tended to decrease G0/G1 fractions. Hypoxia does not add to the effect of hPL during ASC expansion with regard to proliferation, cell cycle regulation and expression of cyclins, p21 and p27. hPL rejuvenates FBS-expanded ASCs with regard to cell cycle regulation and expression of cyclins, p21 and p27. This indicates a reversible arrest. Therefore, we conclude that ASCs expanded until P7 are not senescent regardless of culture conditions. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  10. Human platelet lysate is a successful alternative serum supplement for propagation of monocyte-derived dendritic cells.

    Science.gov (United States)

    Švajger, Urban

    2017-04-01

    Clinical protocols for dendritic cell (DC) generation from monocytes require the use of animal serum-free supplements. Serum-free media can also require up to 1% of serum supplementation. In addition, recommendations based on the 3Rs (Refinement, Reduction, Replacement) principle also recommend the use of non-animal sera in in vitro studies. The aim of this study was to explore the potential use of platelet lysate (PL) for generation of optimally differentiated DCs from monocytes. Cells were isolated from buffy coats from healthy volunteers using immunomagnetic selection. DCs were differentiated in RPMI1640 supplemented with either 10% fetal bovine serum (FBS), 10% AB serum or 10% PL with the addition of granulocyte monocyte colony stimulating factor and interleukin-4. Generated DCs were assessed for their morphology, viability, endocytotic capacity, surface phenotype (immature, mature and tolerogenic DCs) and activation of important signaling pathways. DC function was evaluated on the basis of their allostimulatory capacity, cytokine profile and ability to induce different T-helper subsets. DCs generated with PL displayed normal viability, morphology and endocytotic capacity. Their differentiation and maturation phenotype was comparable to FBS-cultured DCs. They showed functional plasticity and up-regulated tolerogenic markers in response to their environment. PL-cultured mature DCs displayed unhindered allostimulatory potential and the capacity to induce Th1 responses. The use of PL allowed for activation of crucial signaling proteins associated with DC differentiation and maturation. This study demonstrates for the first time that human PL represents a successful alternative to FBS in differentiation of DCs from monocytes. DCs display the major phenotypic and functional characteristics compared with existing culture protocols. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  11. Impact of human platelet lysate on the expansion and chondrogenic capacity of cultured human chondrocytes for cartilage cell therapy.

    Science.gov (United States)

    Sykes, J G; Kuiper, J H; Richardson, J B; Roberts, S; Wright, K T; Kuiper, N J

    2018-05-01

    High hopes have been pinned on regenerative medicine strategies in order to prevent the progression of cartilage damage to osteoarthritis, particularly by autologous chondrocyte implantation (ACI). The loss of chondrocyte phenotype during in vitro monolayer expansion, a necessary step to obtain sufficient cell numbers, may be a key limitation in ACI. In this study, it was determined whether a shorter monolayer expansion approach could improve chondrogenic differentiation. The effects of two supplement types, foetal bovine serum (FBS) and Stemulate™ (a commercial source of human platelet lysate), on the expansion and re-differentiation potential of human chondrocytes, isolated from five individuals, were compared. Chondrocytes were expanded with 10 % FBS or 10 % Stemulate™. Pellets were cultured for 28 d in chondrogenic differentiation medium and assessed for the presence of cartilage matrix molecules and genes associated with chondrogenicity. Stemulate™ significantly enhanced the proliferation rate [average population doubling times: FBS, 25.07 ± 6.98 d (standard error of the mean, SEM) vs. Stemulate™, 13.10 ± 2.57 d (SEM)]. Sulphated glycosaminoglycans (sGAG), total collagen and qRT-PCR analyses of cartilage genes showed that FBS-expanded chondrocytes demonstrated significantly better chondrogenic capacity than Stemulate™-expanded chondrocytes. Histologically, FBS-expanded chondrocyte pellets appeared to be more stable, with a more intense staining for toluidine blue, indicating a greater chondrogenic capacity. Although Stemulate™ positively influenced chondrocyte proliferation, it had a negative effect on chondrogenic differentiation potential. This suggested that, in the treatment of cartilage defects, Stemulate™ might not be the ideal supplement for expanding chondrocytes (which maintained a chondrocyte phenotype) and, hence, for cell therapies (including ACI).

  12. Impact of human platelet lysate on the expansion and chondrogenic capacity of cultured human chondrocytes for cartilage cell therapy

    Directory of Open Access Journals (Sweden)

    JG Sykes

    2018-05-01

    Full Text Available High hopes have been pinned on regenerative medicine strategies in order to prevent the progression of cartilage damage to osteoarthritis, particularly by autologous chondrocyte implantation (ACI. The loss of chondrocyte phenotype during in vitro monolayer expansion, a necessary step to obtain sufficient cell numbers, may be a key limitation in ACI. In this study, it was determined whether a shorter monolayer expansion approach could improve chondrogenic differentiation. The effects of two supplement types, foetal bovine serum (FBS and Stemulate™ (a commercial source of human platelet lysate, on the expansion and re-differentiation potential of human chondrocytes, isolated from five individuals, were compared. Chondrocytes were expanded with 10 % FBS or 10 % Stemulate™. Pellets were cultured for 28 d in chondrogenic differentiation medium and assessed for the presence of cartilage matrix molecules and genes associated with chondrogenicity. Stemulate™ significantly enhanced the proliferation rate [average population doubling times: FBS, 25.07 ± 6.98 d (standard error of the mean, SEM vs. Stemulate™, 13.10 ± 2.57 d (SEM]. Sulphated glycosaminoglycans (sGAG, total collagen and qRT-PCR analyses of cartilage genes showed that FBS-expanded chondrocytes demonstrated significantly better chondrogenic capacity than Stemulate™-expanded chondrocytes. Histologically, FBS-expanded chondrocyte pellets appeared to be more stable, with a more intense staining for toluidine blue, indicating a greater chondrogenic capacity. Although Stemulate™ positively influenced chondrocyte proliferation, it had a negative effect on chondrogenic differentiation potential. This suggested that, in the treatment of cartilage defects, Stemulate™ might not be the ideal supplement for expanding chondrocytes (which maintained a chondrocyte phenotype and, hence, for cell therapies (including ACI.

  13. Platelet lysate activates quiescent cell proliferation and reprogramming in human articular cartilage: Involvement of hypoxia inducible factor 1.

    Science.gov (United States)

    Nguyen, Van Thi; Cancedda, Ranieri; Descalzi, Fiorella

    2018-03-01

    The idea of rescuing the body self-repair capability lost during evolution is progressively gaining ground in regenerative medicine. In particular, growth factors and bioactive molecules derived from activated platelets emerged as promising therapeutic agents acting as trigger for repair of tissue lesions and restoration of tissue functions. Aim of this study was to assess the potential of a platelet lysate (PL) for human articular cartilage repair considering its activity on progenitor cells and differentiated chondrocytes. PL induced the re-entry in the cell cycle of confluent, growth-arrested dedifferentiated/progenitor cartilage cells. In a cartilage permissive culture environment, differentiated cells also resumed proliferation after exposure to PL. These findings correlated with an up-regulation of the proliferation/survival pathways ERKs and Akt and with an induction of cyclin D1. In short- and long-term cultures of articular cartilage explants, we observed a release of proliferating chondroprogenitors able to differentiate and form an "in vitro" tissue with properties of healthy articular cartilage. Moreover, in cultured cartilage cells, PL induced a hypoxia-inducible factor (HIF-1) alpha increase, its nuclear relocation and the binding to HIF-1 responsive elements. These events were possibly related to the cell proliferation because the HIF-1 inhibitor acriflavine inhibited HIF-1 binding to HIF-1 responsive elements and cell proliferation. Our study demonstrates that PL induces quiescent cartilage cell activation and proliferation leading to new cartilage formation, identifies PL activated pathways playing a role in these processes, and provides a rationale to the application of PL for therapeutic treatment of damaged articular cartilage. Copyright © 2017 John Wiley & Sons, Ltd.

  14. Human platelet lysate in mesenchymal stromal cell expansion according to a GMP grade protocol: a cell factory experience.

    Science.gov (United States)

    Becherucci, Valentina; Piccini, Luisa; Casamassima, Serena; Bisin, Silvia; Gori, Valentina; Gentile, Francesca; Ceccantini, Riccardo; De Rienzo, Elena; Bindi, Barbara; Pavan, Paola; Cunial, Vanessa; Allegro, Elisa; Ermini, Stefano; Brugnolo, Francesca; Astori, Giuseppe; Bambi, Franco

    2018-05-02

    The use of platelet lysate (PL) for the ex-vivo expansion of mesenchymal stromal/stem cells (MSCs) was initially proposed by Doucet et al. in 2005, as an alternative to animal serum. Moreover, regulatory authorities discourage the use of fetal bovine serum (FBS) or other animal derivatives, to avoid risk of zoonoses and xenogeneic immune reactions. Even if many studies investigated PL composition, there still are some open issues related to its use in ex-vivo MSC expansion, especially according to good manufacturing practice (GMP) grade protocols. As an authorized cell factory, we report our experience using standardized PL produced by Azienda Ospedaliero Universitaria Meyer Transfusion Service for MSC expansion according to a GMP grade clinical protocol. As suggested by other authors, we performed an in-vitro test on MSCs versus MSCs cultured with FBS that still represents the best way to test PL batches. We compared 12 MSC batches cultured with DMEM 5% PL with similar batches cultured with DMEM 10% FBS, focusing on the MSC proliferation rate, MSC surface marker expression, MSC immunomodulatory and differentiation potential, and finally MSC relative telomere length. Results confirmed the literature data as PL increases cell proliferation without affecting the MSC immunophenotype, immunomodulatory potential, differentiation potential and relative telomere length. PL can be considered a safe alternative to FBS for ex-vivo expansion of MSC according to a GMP grade protocol. Our experience confirms the literature data: a large number of MSCs for clinical applications can be obtained by expansion with PL, without affecting the MSC main features. Our experience underlines the benefits of a close collaboration between the PL producers (transfusion service) and the end users (cell factory) in a synergy of skills and experiences that can lead to standardized PL production.

  15. Human platelet lysate stimulates high-passage and senescent human multipotent mesenchymal stromal cell growth and rejuvenation in vitro.

    Science.gov (United States)

    Griffiths, Sarah; Baraniak, Priya R; Copland, Ian B; Nerem, Robert M; McDevitt, Todd C

    2013-12-01

    Multipotent mesenchymal stromal cells (MSCs) are clinically useful because of their immunomodulatory and regenerative properties, but MSC therapies are limited by the loss of self-renewal and cell plasticity associated with ex vivo expansion culture and, on transplantation, increased immunogenicity from xenogen exposure during culture. Recently, pooled human platelet lysate (hPL) has been used as a culture supplement to promote MSC growth; however, the effects of hPL on MSCs after fetal bovine serum (FBS) exposure remain unknown. MSCs were cultured in medium containing FBS or hPL for up to 16 passages, and cell size, doubling time and immunophenotype were determined. MSC senescence was assessed by means of a fluorometric assay for endogenous β-galactosidase expression. MSCs cultured with FBS for different numbers of passages were switched to hPL conditions to evaluate the ability of hPL to "rescue" the proliferative capacity of MSCs. hPL culture resulted in more rapid cell proliferation at earlier passages (passage 5 or earlier) than remove FBS; by day 4, hPL (5%) yielded an MSC doubling time of 1.28 days compared with 1.52 days in 16% FBS. MSCs cultured first in FBS and switched to hPL proliferated more and demonstrated less β-galactosidase production and smaller cell sizes than remove MSCs continuously propagated in FBS. hPL enables rapid expansion of MSCs without adversely affecting immunophenotype. hPL culture of aged and senescent MSCs demonstrated cellular rejuvenation, reflected by decreased doubling time and smaller cell size. These results suggest that expansion of MSCs in hPL after FBS exposure can enhance cell phenotype and proliferative capacity. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  16. Comparison of human platelet lysate alternatives using expired and freshly isolated platelet concentrates for adipose-derived stromal cell expansion.

    Science.gov (United States)

    Dessels, Carla; Durandt, Chrisna; Pepper, Michael S

    2018-03-19

    Pooled human platelet lysate (pHPL) has been used to expand adipose-derived stromal cells (ASCs) and can be formulated using fresh or expired buffy coats (BCs) which are then resuspended in either plasma or an additive solution. Not much is known about the effects that expired products and additive solutions have on ASC expansion, and the need for quality control and release criteria has been expressed. This pilot study compared proliferation, cell size, morphology and immunophenotype of ASCs expanded in the different pHPL alternatives versus foetal bovine serum (FBS). Quality control criteria were assessed prior to and during the manufacture of the pHPL alternatives. ASCs were then expanded in 1%, 2.5%, 5% or 10% of the different pHPL alternatives or in 10% FBS. Cell size, morphology, cell number and immunophenotype were measured using microscopy and flow cytometry. The majority of the pHPL alternatives were within the recommended ranges for the quality control criteria. ASCs expanded in the pHPL alternatives were smaller in size, displayed a tighter spindle-shaped morphology, increased cell growth and had a similar immunophenotype (with the exception of CD34 and CD36) when compared to ASCs expanded in FBS. Here we report on the effects that expired BC products and additive solutions have on ASC expansion. When taken together, our findings indicate that all of the pHPL alternatives can be considered to be suitable replacements for FBS for ASC expansion, and that expired BC products can be used as an alternative to fresh BC products.

  17. A novel whole-cell lysate kinase assay identifies substrates of the p38 MAPK in differentiating myoblasts

    Directory of Open Access Journals (Sweden)

    Knight James DR

    2012-03-01

    Full Text Available Abstract Background The p38α mitogen-activated protein kinase (MAPK is a critical mediator of myoblast differentiation, and does so in part through the phosphorylation and regulation of several transcription factors and chromatin remodelling proteins. However, whether p38α is involved in processes other than gene regulation during myogenesis is currently unknown, and why other p38 isoforms cannot compensate for its loss is unclear. Methods To further characterise the involvement of p38α during myoblast differentiation, we developed and applied a simple technique for identifying relevant in vivo kinase substrates and their phosphorylation sites. In addition to identifying substrates for one kinase, the technique can be used in vitro to compare multiple kinases in the same experiment, and we made use of this to study the substrate specificities of the p38α and β isoforms. Results Applying the technique to p38α resulted in the identification of seven in vivo phosphorylation sites on six proteins, four of which are cytoplasmic, in lysate derived from differentiating myoblasts. An in vitro comparison with p38β revealed that substrate specificity does not discriminate these two isoforms, but rather that their distinguishing characteristic appears to be cellular localisation. Conclusion Our results suggest p38α has a novel cytoplasmic role during myogenesis and that its unique cellular localisation may be why p38β and other isoforms cannot compensate for its absence. The substrate-finding approach presented here also provides a necessary tool for studying the hundreds of protein kinases that exist and for uncovering the deeper mechanisms of phosphorylation-dependent cell signalling.

  18. Manufacture of endothelial colony-forming progenitor cells from steady-state peripheral blood leukapheresis using pooled human platelet lysate.

    Science.gov (United States)

    Siegel, Georg; Fleck, Erika; Elser, Stefanie; Hermanutz-Klein, Ursula; Waidmann, Marc; Northoff, Hinnak; Seifried, Erhard; Schäfer, Richard

    2018-05-01

    Endothelial colony-forming progenitor cells (ECFCs) are promising candidates for cell therapies. However, ECFC translation to the clinic requires optimized isolation and manufacture technologies according to good manufacturing practice (GMP). ECFCs were manufactured from steady-state peripheral blood (PB) leukapheresis (11 donors), using GMP-compliant technologies including pooled human platelet (PLT) lysate, and compared to human umbilical cord endothelial cells, human aortic endothelial cells, and human cerebral microvascular endothelial cells. Specific variables assessed were growth kinetics, phenotype, trophic factors production, stimulation of tube formation, and Dil-AcLDL uptake. ECFCs could be isolated from PB leukapheresis units with mean processed volume of 5411 mL and mean white blood cell (WBC) concentration factor of 8.74. The mean frequency was 1.44 × 10 -8 ECFCs per WBC, corresponding to a mean of 177.8 ECFCs per apheresis unit. Expandable for up to 12 cumulative population doublings, calculated projection showed that approximately 730 × 10 3 ECFCs could be manufactured from 1 apheresis unit. ECFCs produced epidermal growth factor, hepatocyte growth factor, vascular endothelial growth factor (VEGF)-A, PLT-derived growth factor-B, interleukin-8, and monocyte chemoattractant protein-1, featured high potential for capillary-like tubes formation, and showed no telomerase activity. They were characterized by CD29, CD31, CD44, CD105, CD117, CD133, CD144, CD146, and VEGF-R2 expression, with the most common subpopulation CD34+CD117-CD133-. Compared to controls, ECFCs featured greater Dil-AcLDL uptake and higher expression of CD29, CD31, CD34, CD44, CD144, and VEGF-R2. Here we show that isolation of ECFCs with proangiogenic profile from steady-state PB leukapheresis is feasible, marking a first step toward ECFC product manufacture according to GMP. © 2018 AABB.

  19. Integrated Automation of High-Throughput Screening and Reverse Phase Protein Array Sample Preparation

    DEFF Research Database (Denmark)

    Pedersen, Marlene Lemvig; Block, Ines; List, Markus

    into automated robotic high-throughput screens, which allows subsequent protein quantification. In this integrated solution, samples are directly forwarded to automated cell lysate preparation and preparation of dilution series, including reformatting to a protein spotter-compatible format after the high......-throughput screening. Tracking of huge sample numbers and data analysis from a high-content screen to RPPAs is accomplished via MIRACLE, a custom made software suite developed by us. To this end, we demonstrate that the RPPAs generated in this manner deliver reliable protein readouts and that GAPDH and TFR levels can...

  20. An Improved Cluster Richness Estimator

    Energy Technology Data Exchange (ETDEWEB)

    Rozo, Eduardo; /Ohio State U.; Rykoff, Eli S.; /UC, Santa Barbara; Koester, Benjamin P.; /Chicago U. /KICP, Chicago; McKay, Timothy; /Michigan U.; Hao, Jiangang; /Michigan U.; Evrard, August; /Michigan U.; Wechsler, Risa H.; /SLAC; Hansen, Sarah; /Chicago U. /KICP, Chicago; Sheldon, Erin; /New York U.; Johnston, David; /Houston U.; Becker, Matthew R.; /Chicago U. /KICP, Chicago; Annis, James T.; /Fermilab; Bleem, Lindsey; /Chicago U.; Scranton, Ryan; /Pittsburgh U.

    2009-08-03

    Minimizing the scatter between cluster mass and accessible observables is an important goal for cluster cosmology. In this work, we introduce a new matched filter richness estimator, and test its performance using the maxBCG cluster catalog. Our new estimator significantly reduces the variance in the L{sub X}-richness relation, from {sigma}{sub lnL{sub X}}{sup 2} = (0.86 {+-} 0.02){sup 2} to {sigma}{sub lnL{sub X}}{sup 2} = (0.69 {+-} 0.02){sup 2}. Relative to the maxBCG richness estimate, it also removes the strong redshift dependence of the richness scaling relations, and is significantly more robust to photometric and redshift errors. These improvements are largely due to our more sophisticated treatment of galaxy color data. We also demonstrate the scatter in the L{sub X}-richness relation depends on the aperture used to estimate cluster richness, and introduce a novel approach for optimizing said aperture which can be easily generalized to other mass tracers.

  1. Mitochondrial ceramide-rich macrodomains functionalize Bax upon irradiation.

    Directory of Open Access Journals (Sweden)

    Hyunmi Lee

    Full Text Available Evidence indicates that Bax functions as a "lipidic" pore to regulate mitochondrial outer membrane permeabilization (MOMP, the apoptosis commitment step, through unknown membrane elements. Here we show mitochondrial ceramide elevation facilitates MOMP-mediated cytochrome c release in HeLa cells by generating a previously-unrecognized mitochondrial ceramide-rich macrodomain (MCRM, which we visualize and isolate, into which Bax integrates.MCRMs, virtually non-existent in resting cells, form upon irradiation coupled to ceramide synthase-mediated ceramide elevation, optimizing Bax insertion/oligomerization and MOMP. MCRMs are detected by confocal microscopy in intact HeLa cells and isolated biophysically as a light membrane fraction from HeLa cell lysates. Inhibiting ceramide generation using a well-defined natural ceramide synthase inhibitor, Fumonisin B1, prevented radiation-induced Bax insertion, oligomerization and MOMP. MCRM deconstruction using purified mouse hepatic mitochondria revealed ceramide alone is non-apoptogenic. Rather Bax integrates into MCRMs, oligomerizing therein, conferring 1-2 log enhanced cytochrome c release. Consistent with this mechanism, MCRM Bax isolates as high molecular weight "pore-forming" oligomers, while non-MCRM membrane contains exclusively MOMP-incompatible monomeric Bax.Our recent studies in the C. elegans germline indicate that mitochondrial ceramide generation is obligate for radiation-induced apoptosis, although a mechanism for ceramide action was not delineated. Here we demonstrate that ceramide, generated in the mitochondrial outer membrane of mammalian cells upon irradiation, forms a platform into which Bax inserts, oligomerizes and functionalizes as a pore. We posit conceptualization of ceramide as a membrane-based stress calibrator, driving membrane macrodomain organization, which in mitochondria regulates intensity of Bax-induced MOMP, and is pharmacologically tractable in vitro and in vivo.

  2. Scalability and process transfer of mesenchymal stromal cell production from monolayer to microcarrier culture using human platelet lysate.

    Science.gov (United States)

    Heathman, Thomas R J; Stolzing, Alexandra; Fabian, Claire; Rafiq, Qasim A; Coopman, Karen; Nienow, Alvin W; Kara, Bo; Hewitt, Christopher J

    2016-04-01

    The selection of medium and associated reagents for human mesenchymal stromal cell (hMSC) culture forms an integral part of manufacturing process development and must be suitable for multiple process scales and expansion technologies. In this work, we have expanded BM-hMSCs in fetal bovine serum (FBS)- and human platelet lysate (HPL)-containing media in both a monolayer and a suspension-based microcarrier process. The introduction of HPL into the monolayer process increased the BM-hMSC growth rate at the first experimental passage by 0.049 day and 0.127/day for the two BM-hMSC donors compared with the FBS-based monolayer process. This increase in growth rate in HPL-containing medium was associated with an increase in the inter-donor consistency, with an inter-donor range of 0.406 cumulative population doublings after 18 days compared with 2.013 in FBS-containing medium. Identity and quality characteristics of the BM-hMSCs are also comparable between conditions in terms of colony-forming potential, osteogenic potential and expression of key genes during monolayer and post-harvest from microcarrier expansion. BM-hMSCs cultured on microcarriers in HPL-containing medium demonstrated a reduction in the initial lag phase for both BM-hMSC donors and an increased BM-hMSC yield after 6 days of culture to 1.20 ± 0.17 × 10(5) and 1.02 ± 0.005 × 10(5) cells/mL compared with 0.79 ± 0.05 × 10(5) and 0.36 ± 0.04 × 10(5) cells/mL in FBS-containing medium. This study has demonstrated that HPL, compared with FBS-containing medium, delivers increased growth and comparability across two BM-hMSC donors between monolayer and microcarrier culture, which will have key implications for process transfer during scale-up. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  3. Removal of endotoxins from bacteriophage preparations by extraction with organic solvents.

    Directory of Open Access Journals (Sweden)

    Bożena Szermer-Olearnik

    Full Text Available Lipopolysaccharide (LPS, endotoxin, pyrogen constitutes a very troubling contaminant of crude phage lysates produced in Gram-negative bacteria. Toxicity of LPS depends on the strong innate immunity response including the cytokines. Therefore, its removal is important for bacteriophage applications. In this paper, we present a procedure for extractive removal of endotoxin from bacteriophage preparations with water immiscible solvents (1-octanol or 1-butanol. During extraction most of the phage lytic activity is retained in the aqueous phase, while endotoxin accumulates in the organic solvent. The levels of endotoxin (expressed as endotoxin units, EU in the aqueous bacteriophage-containing fraction determined by limulus amebocyte lysate or EndoLISA assay were exceptionally low. While the initial endotoxin levels in the crude phage lysates ranged between 10(3 and 10(5 EU/ml the average level after organic extraction remaining in the aqueous fraction was 5.3 EU/ml. These values when related to phage titers decreased from 10(3-10(5 EU/10(9 PFU (plaque forming units down to an average of 2.8 EU/10(9 PFU. The purification procedure is scalable, efficient and applicable to all the bacteriophages tested: T4, HAP1 (E. coli and F8 (P. aeruginosa.

  4. Gene expression profiling of chicken cecal tonsils and ileum following oral exposure to soluble and PLGA-encapsulated CpG ODN, and lysate of Campylobacter jejuni.

    Science.gov (United States)

    Taha-Abdelaziz, Khaled; Alkie, Tamiru Negash; Hodgins, Douglas C; Yitbarek, Alexander; Shojadoost, Bahram; Sharif, Shayan

    2017-12-01

    Campylobacter jejuni (C. jejuni) is a leading bacterial cause of food-borne illness in humans. Contaminated chicken meat is an important source of infection for humans. Chickens are not clinically affected by colonization, and immune responses following natural infection have limited effects on bacterial load in the gut. Induction of intestinal immune responses may possibly lead to a breakdown of the commensal relationship of chickens with Campylobacter. We have recently shown that soluble and poly D, L-lactic-co-glycolic acid (PLGA)-encapsulated CpG oligodeoxynucleotide (ODN) as well as C. jejuni lysate, are effective in reducing the intestinal burden of C. jejuni in chickens; however, the mechanisms behind this protection have yet to be determined. The present study was undertaken to investigate the mechanisms of host responses conferred by these treatments. Chickens were treated orally with soluble CpG ODN, or PLGA-encapsulated CpG ODN, or C. jejuni lysate, and expression of cytokines and antimicrobial peptides was evaluated in cecal tonsils and ileum using quantitative RT-PCR. Oral administration of soluble CpG ODN upregulated the expression of interferon (IFN)-γ, interleukin (IL)-1β, CXCLi2, transforming growth factor (TGF)-β4/1, IL-10 and IL-13, while treatment with PLGA-encapsulated CpG ODN upregulated the expression of IL-1β, CXCLi2, TGF-β4/1, IL-13, avian β-defensin (AvBD) 1, AvBD2 and cathelicidin 3 (CATHL-3). C. jejuni lysate upregulated the expression of IFN-γ, IL-1β, TGF-β4/1, IL-13, AvBD1, and CATHL-3. In conclusion, induction of cytokine and antimicrobial peptides expression in intestinal microenvironments may provide a means of reducing C. jejuni colonization in broiler chickens, a key step in reducing the incidence of campylobacteriosis in humans. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Platelet-rich fibrin: Evolution of a second-generation platelet concentrate

    Directory of Open Access Journals (Sweden)

    Sunitha Raja V

    2008-01-01

    Full Text Available Platelet-rich plasma (PRP is a platelet concentrate that has been used widely to accelerate soft-tissue and hard-tissue healing. The preparation of PRP has been described by several authors. Platelet-rich fibrin (PRF was first described by Choukroun et al. in France. It has been referred to as a second-generation platelet concentrate, which has been shown to have several advantages over traditionally prepared PRP. Its chief advantages include ease of preparation and lack of biochemical handling of blood, which makes this preparation strictly autologous. This article describes the evolution of this novel platelet concentrate, referred to as PRF.

  6. Overcoming the bottleneck of platelet lysate supply in large-scale clinical expansion of adipose-derived stem cells

    DEFF Research Database (Denmark)

    Glovinski, Peter Viktor; Herly, Mikkel; Mathiasen, Anders B

    2017-01-01

    , we tested PLs prepared via three methods from outdated buffy coat-derived platelet concentrates (PCs) to establish an efficient and feasible expansion of ASCs for clinical use. METHODS: PLs were prepared by the freeze-thaw method from freshly drawn platelets or from outdated buffy coat-derived PCs...

  7. Adaptive Reactive Rich Internet Applications

    Science.gov (United States)

    Schmidt, Kay-Uwe; Stühmer, Roland; Dörflinger, Jörg; Rahmani, Tirdad; Thomas, Susan; Stojanovic, Ljiljana

    Rich Internet Applications significantly raise the user experience compared with legacy page-based Web applications because of their highly responsive user interfaces. Although this is a tremendous advance, it does not solve the problem of the one-size-fits-all approach1 of current Web applications. So although Rich Internet Applications put the user in a position to interact seamlessly with the Web application, they do not adapt to the context in which the user is currently working. In this paper we address the on-the-fly personalization of Rich Internet Applications. We introduce the concept of ARRIAs: Adaptive Reactive Rich Internet Applications and elaborate on how they are able to adapt to the current working context the user is engaged in. An architecture for the ad hoc adaptation of Rich Internet Applications is presented as well as a holistic framework and tools for the realization of our on-the-fly personalization approach. We divided both the architecture and the framework into two levels: offline/design-time and online/run-time. For design-time we explain how to use ontologies in order to annotate Rich Internet Applications and how to use these annotations for conceptual Web usage mining. Furthermore, we describe how to create client-side executable rules from the semantic data mining results. We present our declarative lightweight rule language tailored to the needs of being executed directly on the client. Because of the event-driven nature of the user interfaces of Rich Internet Applications, we designed a lightweight rule language based on the event-condition-action paradigm.2 At run-time the interactions of a user are tracked directly on the client and in real-time a user model is built up. The user model then acts as input to and is evaluated by our client-side complex event processing and rule engine.

  8. Platelet-rich plasma, plasma rich in growth factors and simvastatin in the regeneration and repair of alveolar bone.

    Science.gov (United States)

    Rivera, César; Monsalve, Francisco; Salas, Juan; Morán, Andrea; Suazo, Iván

    2013-12-01

    Platelet preparations promote bone regeneration by inducing cell migration, proliferation and differentiation in the area of the injury, which are essential processes for regeneration. In addition, several studies have indicated that simvastatin (SIMV), widely used for the treatment of hypercholesterolemia, stimulates osteogenesis. The objective of this study was to evaluate the effects of treatment with either platelet-rich plasma (PRP) or plasma rich in growth factors (PRGF) in combination with SIMV in the regeneration and repair of alveolar bone. The jaws of Sprague Dawley rats (n=18) were subjected to rotary instrument-induced bone damage (BD). Animals were divided into six groups: BD/H 2 O (n=3), distilled water without the drug and alveolar bone damage; BD/H 2 O/PRP (n=3), BD and PRP; BD/H 2 O/PRGF (n=3), BD and PRGF; BD/SIMV (n=3), BD and water with SIMV; BD/SIMV/PRP (n=3), BD, PRP and SIMV; and BD/SIMV/PRGF (n=3), BD, PRGF and SIMV. Conventional histological analysis (hematoxylin and eosin staining) revealed that the BD/SIMV group showed indicators for mature bone tissue, while the BD/SIMV/PRP and BD/SIMV/PRGF groups showed the coexistence of indicators for mature and immature bone tissue, with no statistical differences between the platelet preparations. Simvastatin did not improve the effect of platelet-rich plasma and plasma rich in growth factors. It was not possible to determine which platelet preparation produced superior effects.

  9. Effects of sodium citrate and acid citrate dextrose solutions on cell counts and growth factor release from equine pure-platelet rich plasma and pure-platelet rich gel.

    Science.gov (United States)

    Giraldo, Carlos E; Álvarez, María E; Carmona, Jorge U

    2015-03-14

    There is a lack information on the effects of the most commonly used anticoagulants for equine platelet rich plasmas (PRPs) elaboration on cell counts and growth factor release from platelet rich gels (PRGs). The aims of this study were 1) to compare the effects of the anticoagulants sodium citrate (SC), acid citrate dextrose solution A (ACD-A) and ACD-B on platelet (PLT), leukocyte (WBC) and on some parameters associated to platelet activation including mean platelet volume (MPV) and platelet distribution width (PDW) between whole blood, pure PRP (P-PRP) and platelet-poor plasma (PPP); 2) to compare transforming growth factor beta 1 (TGF-β(1)) and platelet-derived growth factor isoform BB (PDGF-BB) concentrations in supernatants from pure PRG (P-PRG), platelet-poor gel (PPG), P-PRP lysate (positive control) and plasma (negative control); 3) to establish the possible correlations between all the studied cellular and molecular parameters. In all cases the three anticoagulants produced P-PRPs with significantly higher PLT counts compared with whole blood and PPP. The concentrations of WBCs were similar between P-PRP and whole blood, but significantly lower in PPP. The type of anticoagulant did not significantly affect the cell counts for each blood component. The anticoagulants also did not affect the MPV and PDW parameters. Independently of the anticoagulant used, all blood components presented significantly different concentrations of PDGF-BB and TGF-β(1). The highest growth factor (GF) concentrations were observed from P-PRP lysates, followed by PRG supernatants, PPP lysates, PPG supernatants and plasma. Significant correlations were observed between PLT and WBC counts (ρ = 0.80), PLT count and TGF-β(1) concentration (ρ = 0.85), PLT count and PDGF-BB concentration (ρ = 0.80) and PDGF-BB and TGF-β(1) concentrations (ρ = 0.75). The type of anticoagulant was not correlated with any of the variables evaluated. The anticoagulants did not

  10. Is buffer a good proxy for a crowded cell-like environment? A comparative NMR study of calmodulin side-chain dynamics in buffer and E. coli lysate.

    Directory of Open Access Journals (Sweden)

    Michael P Latham

    Full Text Available Biophysical studies of protein structure and dynamics are typically performed in a highly controlled manner involving only the protein(s of interest. Comparatively fewer such studies have been carried out in the context of a cellular environment that typically involves many biomolecules, ions and metabolites. Recently, solution NMR spectroscopy, focusing primarily on backbone amide groups as reporters, has emerged as a powerful technique for investigating protein structure and dynamics in vivo and in crowded "cell-like" environments. Here we extend these studies through a comparative analysis of Ile, Leu, Val and Met methyl side-chain motions in apo, Ca(2+-bound and Ca(2+, peptide-bound calmodulin dissolved in aqueous buffer or in E. coli lysate. Deuterium spin relaxation experiments, sensitive to pico- to nano-second time-scale processes and Carr-Purcell-Meiboom-Gill relaxation dispersion experiments, reporting on millisecond dynamics, have been recorded. Both similarities and differences in motional properties are noted for calmodulin dissolved in buffer or in lysate. These results emphasize that while significant insights can be obtained through detailed "test-tube" studies, experiments performed under conditions that are "cell-like" are critical for obtaining a comprehensive understanding of protein motion in vivo and therefore for elucidating the relation between motion and function.

  11. Comparison of osteo/odontogenic differentiation of human adult dental pulp stem cells and stem cells from apical papilla in the presence of platelet lysate.

    Science.gov (United States)

    Abuarqoub, Duaa; Awidi, Abdalla; Abuharfeil, Nizar

    2015-10-01

    Human dental pulp cells (DPSCs) and stem cells from apical papilla have been used for the repair of damaged tooth tissues. Human platelet lysate (PL) has been suggested as a substitute for fetal bovine serum (FBS) for large scale expansion of dental stem cells. However, biological effects and optimal concentrations of PL for proliferation and differentiation of human dental stem cells remain to be elucidated. DPSCs and SCAP cells were isolated from impacted third molars of young healthy donors, at the stage of root development and identified by markers using flow cytometry. For comparison the cells were cultured in media containing PL (1%, 5% and 10%) and FBS, with subsequent induction for osteogenic/odontogenic differentiation. The cultures were analyzed for; morphology, growth characteristics, mineralization potential (Alizarin Red method) and differentiation markers using ELISA and real time -polymerase chain reaction (qPCR). The proliferation rates of DPSCs and SCAP significantly increased when cells were treated with 5% PL (7X doubling time) as compared to FBS. 5% PL also enhanced mineralized differentiation of DPSCs and SCAP, as indicated by the measurement of alkaline phosphatase activity, osteocalcin and osteopontin, calcium deposition and q-PCR. Our findings suggest that using 5% platelet lysate, proliferation and osteo/odontogenesis of DPSCs and SCAP for a short period of time (15 days), was significantly improved. This may imply its use as an optimum concentration for expansion of dental stem cells in bone regeneration. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Personnel Preparation.

    Science.gov (United States)

    Fair, George, Ed.; Stodden, Robert, Ed.

    1981-01-01

    Three articles comprise a section on personnel preparation in vocational education. Articles deal with two inservice programs in career/vocational education for the handicapped and a project to train paraprofessionals to assist special educators in vocational education. (CL)

  13. Solution preparation

    International Nuclear Information System (INIS)

    Seitz, M.G.

    1982-01-01

    Reviewed in this statement are methods of preparing solutions to be used in laboratory experiments to examine technical issues related to the safe disposal of nuclear waste from power generation. Each approach currently used to prepare solutions has advantages and any one approach may be preferred over the others in particular situations, depending upon the goals of the experimental program. These advantages are highlighted herein for three approaches to solution preparation that are currently used most in studies of nuclear waste disposal. Discussion of the disadvantages of each approach is presented to help a user select a preparation method for his particular studies. Also presented in this statement are general observations regarding solution preparation. These observations are used as examples of the types of concerns that need to be addressed regarding solution preparation. As shown by these examples, prior to experimentation or chemical analyses, laboratory techniques based on scientific knowledge of solutions can be applied to solutions, often resulting in great improvement in the usefulness of results

  14. Platelet adhesiveness: the effect of centrifugation on the measurement of adhesiveness in platelet-rich plasma

    Science.gov (United States)

    McBride, J. A.

    1968-01-01

    Platelet adhesiveness has been measured in citrated whole blood and in platelet-rich plasma obtained from normal subjects, splenectomized patients, and from patients in whom the diagnosis of recurrent venous thrombosis had been made. The duration of centrifugation used in the preparation of platelet-rich plasma was found to have a profound effect on the measurement of platelet adhesiveness because the figure for platelet adhesiveness measured in platelet-rich plasma obtained by centrifugation was considerably lower than that found in citrated whole blood. This effect was particularly marked when platelet-rich plasma was obtained from subjects in whom platelet adhesiveness measured in whole blood was increased. PMID:5699080

  15. The STAR-RICH Detector

    CERN Document Server

    Lasiuk, B; Braem, André; Cozza, D; Davenport, M; De Cataldo, G; Dell'Olio, L; Di Bari, D; Di Mauro, A; Dunlop, J C; Finch, E; Fraissard, Daniel; Franco, A; Gans, J; Ghidini, B; Harris, J W; Horsley, M; Kunde, G J; Lasiuk, B; Lesenechal, Y; Majka, R D; Martinengo, P; Morsch, Andreas; Nappi, E; Paic, G; Piuz, François; Posa, F; Raynaud, J; Salur, S; Sandweiss, J; Santiard, Jean-Claude; Satinover, J; Schyns, E M; Smirnov, N; Van Beelen, J; Williams, T D; Xu, Z

    2002-01-01

    The STAR-RICH detector extends the particle idenfication capabilities of the STAR spectrometer for charged hadrons at mid-rapidity. It allows identification of pions and kaons up to ~3 GeV/c and protons up to ~5 GeV/c. The characteristics and performance of the device in the inaugural RHIC run are described.

  16. SOFTWARE SUPPORT FOR RICH PICTURES

    DEFF Research Database (Denmark)

    Valente, Andrea; Marchetti, Emanuela

    2010-01-01

    Rich pictures (RP) are common in object-oriented analysis and design courses, but students seem to have problems in integrating them in their projects' workflow. A new software tool is being developed, specific for RP authoring. To better understand students' issues and working practice with RP...

  17. PREPARATIVE SKIN PREPARATION AND SURGICAL WOUND INFECTION

    Directory of Open Access Journals (Sweden)

    Anjanappa

    2015-01-01

    Full Text Available BACKGROUND AND OBJECTIVE: It is an established fact now that the normal skin of healthy human beings harbours a rich bacterial fl ora. Normally considered non - pathogenic , these organisms way be a potential source of infection of the surgical wound. Approximately 20% of the resident flora is beyond the reach of surgical scrubs and antiseptics. The goal of surgical preparation of the skin with antiseptics is to remove transient and pathogenic microorganisms on the skin surface and to reduce the resident flora to a low level. Povidone iodine (I odophors and chlorhexidine are most often used antiseptics for pre - operative skin preparation. OBJECTIVES : To evaluate the efficacy of povidone iodine alone and in combination with antiseptic agent containing alcoholic chlorhexidine in preoperative skin p reparation by taking swab culture. (2 To compare the rate of postoperative wound infection in both the groups. METHODS: One hundred patients (fifty in each group undergoing clean elective surgery with no focus of infection on the body were included in th e study. The pre - operative skin preparation in each group is done with the respective antiseptic regimen. In both the groups after application of antiseptics , sterile saline swab culture was taken immediately from site of incision. In cases which showed gr owth of organisms , the bacteria isolated were identified by their morphological and cultural characteristics. Grams staining , coagulase test and antibiotic sensitivity test were done wherever necessary and difference in colonization rates was determined as a measure of efficacy of antiseptic regimen. RESULTS: The results of the study showed that when compared to povidone iodine alone , using a combination of povidone iodine and alcoholic solution of chlorhexidine , the colonization rates of the site of incisi on were reduced significantly. As for the rate of post - operative wound infection , it is also proven that wound infections are also

  18. Effects of Plasma Rich in Growth Factors and Platelet-Rich Fibrin on Proliferation and Viability of Human Gingival Fibroblasts

    Science.gov (United States)

    Vahabi, Surena; Vaziri, Shahram; Torshabi, Maryam

    2015-01-01

    Objectives: Platelet preparations are commonly used to enhance bone and soft tissue regeneration. Considering the existing controversies on the efficacy of platelet products for tissue regeneration, more in vitro studies are required. The aim of the present study was to compare the in vitro effects of plasma rich in growth factors (PRGF) and platelet-rich fibrin (PRF) on proliferation and viability of human gingival fibroblasts (HGFs). Materials and Methods: Anitua’s PRGF and Choukran’s PRF were prepared according to the standard protocols. After culture periods of 24, 48 and 72 hours, proliferation of HGFs was evaluated by the methyl thiazol tetrazolium assay. Statistical analysis was performed using one-way ANOVA followed by Tukey-Kramer’s multiple comparisons and P-valuesPRGF treatment induced statistically significant (PPRGF than in PRF group (PPRGF had a strong stimulatory effect on HGF cell viability and proliferation compared to PRF. PMID:26877740

  19. Production of conjugated linoleic acid-rich potato chips.

    Science.gov (United States)

    Jain, Vishal P; Proctor, Andrew

    2007-01-01

    Conjugated linoleic acid (CLA) is found primarily in diary and beef products, but the health benefits of CLA can only be realized if they are consumed at much greater levels than a normal healthy dietary intake. We have recently shown that a CLA-rich soy oil can be produced by simple isomerization of linoleic acid in soy oil by photoirradiation. This oil may allow greatly increased dietary CLA without significantly elevating fat intake. The objective of this study was to prepare CLA-rich potato chips by frying in CLA-rich soy oil. Soy oil was photoisomerized in the presence of iodine catalyst with UV/visible light. The irradiated oil was clay processed to remove the residual iodine and this oil was then used to fry potato chips. Oil was extracted from fried chips and analyzed for its CLA content with gas chromatography. A 1-oz serving of CLA-rich potato chips contained approximately 2.4 g CLA as compared to 0.1 g CLA in 3-oz serving of steak fillet and 0.06 g CLA in 8-oz serving of whole milk. The peroxide value of the oil extracted from potato chips was found to be 1 meq/1000 g sample, which was within the acceptable commercial standards. This study may lead to the commercialization of CLA-rich food products.

  20. Sample preparation

    International Nuclear Information System (INIS)

    Anon.

    1992-01-01

    Sample preparation prior to HPLC analysis is certainly one of the most important steps to consider in trace or ultratrace analysis. For many years scientists have tried to simplify the sample preparation process. It is rarely possible to inject a neat liquid sample or a sample where preparation may not be any more complex than dissolution of the sample in a given solvent. The last process alone can remove insoluble materials, which is especially helpful with the samples in complex matrices if other interactions do not affect extraction. Here, it is very likely a large number of components will not dissolve and are, therefore, eliminated by a simple filtration process. In most cases, the process of sample preparation is not as simple as dissolution of the component interest. At times, enrichment is necessary, that is, the component of interest is present in very large volume or mass of material. It needs to be concentrated in some manner so a small volume of the concentrated or enriched sample can be injected into HPLC. 88 refs

  1. Generation of mesenchymal stromal cells in the presence of platelet lysate: a phenotypic and functional comparison of umbilical cord blood- and bone marrow-derived progenitors

    Science.gov (United States)

    Avanzini, Maria Antonietta; Bernardo, Maria Ester; Cometa, Angela Maria; Perotti, Cesare; Zaffaroni, Nadia; Novara, Francesca; Visai, Livia; Moretta, Antonia; Del Fante, Claudia; Villa, Raffaella; Ball, Lynne M.; Fibbe, Willem E.; Maccario, Rita; Locatelli, Franco

    2009-01-01

    Background Mesenchymal stromal cells are employed in various different clinical settings in order to modulate immune response. However, relatively little is known about the mechanisms responsible for their immunomodulatory effects, which could be influenced by both the cell source and culture conditions. Design and Methods We tested the ability of a 5% platelet lysate-supplemented medium to support isolation and ex vivo expansion of mesenchymal stromal cells from full-term umbilical-cord blood. We also investigated the biological/functional properties of umbilical cord blood mesenchymal stromal cells, in comparison with platelet lysate-expanded bone marrow mesenchymal stromal cells. Results The success rate of isolation of mesenchymal stromal cells from umbilical cord blood was in the order of 20%. These cells exhibited typical morphology, immunophenotype and differentiation capacity. Although they have a low clonogenic efficiency, umbilical cord blood mesenchymal stromal cells may possess high proliferative potential. The genetic stability of these cells from umbilical cord blood was demonstrated by a normal molecular karyotype; in addition, these cells do not express hTERT and telomerase activity, do express p16ink4a protein and do not show anchorage-independent cell growth. Concerning alloantigen-specific immune responses, umbilical cord blood mesenchymal stromal cells were able to: (i) suppress T- and NK-lymphocyte proliferation, (ii) decrease cytotoxic activity and (iii) only slightly increase interleukin-10, while decreasing interferon-γ secretion, in mixed lymphocyte culture supernatants. While an indoleamine 2,3-dioxygenase-specific inhibitor did not reverse mesenchymal stromal cell-induced suppressive effects, a prostaglandin E2-specific inhibitor hampered the suppressive effect of both umbilical cord blood- and bone marrow-mesenchymal stromal cells on alloantigen-induced cytotoxic activity. Mesenchymal stromal cells from both sources expressed HLA

  2. Integrated culture platform based on a human platelet lysate supplement for the isolation and scalable manufacturing of umbilical cord matrix-derived mesenchymal stem/stromal cells.

    Science.gov (United States)

    de Soure, António M; Fernandes-Platzgummer, Ana; Moreira, Francisco; Lilaia, Carla; Liu, Shi-Hwei; Ku, Chen-Peng; Huang, Yi-Feng; Milligan, William; Cabral, Joaquim M S; da Silva, Cláudia L

    2017-05-01

    Umbilical cord matrix (UCM)-derived mesenchymal stem/stromal cells (MSCs) are promising therapeutic candidates for regenerative medicine settings. UCM MSCs have advantages over adult cells as these can be obtained through a non-invasive harvesting procedure and display a higher proliferative capacity. However, the high cell doses required in the clinical setting make large-scale manufacturing of UCM MSCs mandatory. A commercially available human platelet lysate-based culture supplement (UltraGRO TM , AventaCell BioMedical) (5%(v/v)) was tested to effectively isolate UCM MSCs and to expand these cells under (1) static conditions, using planar culture systems and (2) stirred culture using plastic microcarriers in a spinner flask. The MSC-like cells were isolated from UCM explant cultures after 11 ± 2 days. After five passages in static culture, UCM MSCs retained their immunophenotype and multilineage differentiation potential. The UCM MSCs cultured under static conditions using UltraGRO TM -supplemented medium expanded more rapidly compared with UCM MSCs expanded using a previously established protocol. Importantly, UCM MSCs were successfully expanded under dynamic conditions on plastic microcarriers using UltraGRO TM -supplemented medium in spinner flasks. Upon an initial 54% cell adhesion to the beads, UCM MSCs expanded by >13-fold after 5-6 days, maintaining their immunophenotype and multilineage differentiation ability. The present paper reports the establishment of an easily scalable integrated culture platform based on a human platelet lysate supplement for the effective isolation and expansion of UCM MSCs in a xenogeneic-free microcarrier-based system. This platform represents an important advance in obtaining safer and clinically meaningful MSC numbers for clinical translation. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  3. Stimulation with lysates of Aspergillus terreus, Candida krusei and Rhizopus oryzae maximizes cross-reactivity of anti-fungal T cells.

    Science.gov (United States)

    Deo, Shivashni S; Virassamy, Balaji; Halliday, Catriona; Clancy, Leighton; Chen, Sharon; Meyer, Wieland; Sorrell, Tania C; Gottlieb, David J

    2016-01-01

    Invasive fungal diseases caused by filamentous fungi and yeasts are significant causes of morbidity and mortality in immunosuppressed hematology patients. We previously published a method to expand Aspergillus fumigatus-specific T cells for clinical cell therapy. In the present study, we investigated expansion of T cells specific for other fungal pathogens and creation of a broadly reactive panfungal T-cell product. Fungal strains selected were those frequently observed in the clinical hematology setting and included Aspergillus, Candida, Fusarium, Rhizopus and Lomentospora/Scedosporium. Four T-cell cultures specific to each fungus were established. We selected lysates of Aspergillus terreus, Candida krusei and Rhizopus oryzae to expand panfungal T cells. Allelic restriction of anti-fungal activity was determined through the use of specific major histocompatibility complex class II-blocking antibodies. Individual T-cell cultures specific to each fungus could be expanded in vitro, generating predominantly CD4(+) T cells of which 8% to 20% were fungus-specific. We successfully expanded panfungal T cells from the peripheral blood (n = 8) and granulocyte-colony-stimulating factor-primed stem cell products (n = 3) of normal donors by using a combination of lysates from Aspergillus terreus, Candida krusei and Rhizopus oryzae. Anti-fungal activity was mediated through human leukocyte antigen (HLA)-DR alleles and was maintained when antigen-presenting cells from partially HLA-DRB1-matched donors were used to stimulate T cells. We demonstrate a method to manufacture panfungal T-cell products with specificity against a range of clinical fungal pathogens by use of the blood and stem cells of healthy donors as the starting material. The safety and efficacy of these products will need to be tested clinically. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  4. Analysis of glutathione in supernatants and lysates of a human proximal tubular cell line from perfusion culture upon intoxication with cadmium chloride by HPLC and LC-ESI-MS

    NARCIS (Netherlands)

    Hahn, Hans; Huck, Christian W; Rainer, Matthias; Najam-ul-Haq, Muhammad; Bakry, Rania; Abberger, Thomas; Jennings, Paul; Pfaller, Walter; Bonn, Günther K

    A simple and highly effective reversed-phase (RP) high-performance liquid chromatography (HPLC) method is described for analysing glutathione (GSH) and glutathione disulfide (GSSG) in out-flowing supernatants and lysates of perfusion cell cultures of human kidney cells (HK-2 cells) continuously

  5. Analyzing Protein Changes in Guinea Pig Tissue Lysates Using Non-guinea Pig Specific Antibodies: Procedures for Western Blotting and Examples Using 16 Individual Antibodies for Common CNS Proteins

    Science.gov (United States)

    2006-05-01

    guinea pig model does present a significant problem...trying to correlate behavioral and protein changes due to the absence of guinea pig -specific antibodies. We...have developed a procedure to determine the specificity of commercially available, non- guinea pig -specific antibodies in guinea pig lysates.

  6. Preparing hydraulic cement from oil-shale residue

    Energy Technology Data Exchange (ETDEWEB)

    1921-08-28

    A process for preparation of hydraulic cement from oil-shale residue is characterized in that, as flux is used, rich-in-lime poor-in-sulfur portland-cement clinker, by which the usual gypsum addition, is avoided.

  7. Valoración de endotoxinas bacterianas en el inyectable ácido zoledrónico mediante la prueba de lisado del amebocito de Limulus Assessment of bacterial endotoxins in Zoledronic acid injectable drug by using Limulus amebocyte lysate test

    Directory of Open Access Journals (Sweden)

    Nancy Burguet Lago

    2012-09-01

    Full Text Available Objetivo: valorar las endotoxinas bacterianas por la técnica del lisado del amebocito de Limulus para el producto inyectable ácido zoledrónico, por el método de gelificación. Métodos: el ensayo se realizó mediante dos pruebas: 1 confirmación de la sensibilidad del lisado etiquetado, para lo cual se preparó una curva estándar con diluciones seriadas dobles de endotoxina por cuadruplicado; y 2 el ensayo del producto de inhibición, en el que se prepararon diluciones seriadas dobles de endotoxina con agua apirogénica y con las muestras de los lotes a ensayar sin sobrepasar la máxima dilución válida. Se determinó el punto final y se calculó la media geométrica. Se definió la dilución de trabajo, la cual se validó por cuadruplicado en tres lotes consecutivos. Resultados: la sensibilidad del lisado resultó 0,03125 UE/mL. La máxima dilución válida fue de 112 UE/mL y la dilución de trabajo 1/100. La cantidad de endotoxinas bacterianas presentes en tres lotes del producto inyectable no sobrepasó el límite establecido, por lo que cumplió con las especificaciones de calidad establecidas para el ensayo. Conclusión: la estandarización de las condiciones del método por gelificación, hace que este resulte eficaz, confiable, rápido y de fácil ejecución, por lo que puede emplearse como ensayo de rutina en el control de la calidad del inyectable analizado.Objective: To asses the presence of bacterial endotoxins in Zoledronic Acid injectable drug by using the Limulus amebocyte lysate test, particularly by the gelling procedure. Methods: The assay was performed in two tests: the first was the confirmation of labeled lysate sensitivity by preparing a standard curve with serial double dilutions of endotoxins four times, and the second was the inhibition product test in which serial double dilutions of endotoxins were prepared with apyrogenic water and with samples from the batches to be tested, without exceeding the maximum valid

  8. HTML5 rich media foundation

    CERN Document Server

    David, Matthew

    2010-01-01

    Learn about the new ways in which video and audio can be easily embedded into your HTML5 Web pages. Discover how you can create new Web media content and how JavaScript, CSS, and SVG can be integrated to create a compelling, rich media foundation for your work. HTML 5, is the first major update to the core language of the Web in over a decade The focus of this book is on innovations that most directly effect Web site design and multimedia integration The companion Web site features working demonstrations and tutorial media for hands-on pract

  9. Conservation and Biodiversity of Rich Fens

    DEFF Research Database (Denmark)

    Andersen, Dagmar Kappel

    2014-01-01

    Rich fen is a habitat type dependent on a constant supply of nutrient poor, calcium rich groundwater. A high, stable groundwater table, relatively high pH combined with nutrient poor conditions support a special and very species rich vegetation including many rare and threatened plant species. In...

  10. Radioprotective preparation

    International Nuclear Information System (INIS)

    Stefanova, D.; Frattadochi, A.; Gattavecchia, E.; Ferri, E.; Tonnelli, D.

    1988-01-01

    The invention is intended for radiation injuries prophylaxis in mammals. It has an well expressed radioprotective effect against acute gamma irradiation on cellular level as well as a prolonged action when applied up to 48 hours before the acute irradiation. The preparation is a coprecipitate of the natural tripeptide glutathione (reduced form) and polyvinyl pyrrolidone (pvp) in ratio 30-60/70-40. It is obtained by incubation method with subsequent lyophilization from water solution of the initial components. The molecular mass of the pvp is 20 till 360.10 3 . 2 claims

  11. Target preparation

    International Nuclear Information System (INIS)

    Hinn, G.M.

    1984-01-01

    A few of the more interesting of the 210 targets prepared in the Laboratory last year are listed. In addition the author continues to use powdered silver mixed with /sup 9,10/BeO to produce sources for accelerator radio dating of Alaskan and South Polar snow. Currently, he is trying to increase production by multiple sample processing. Also the author routinely makes 3 μg/cm 2 cracked slacked carbon stripper foils and is continuing research with some degree of success in making enriched 28 Si targets starting with the oxide

  12. Pathogen reduction through additive-free short-wave UV light irradiation retains the optimal efficacy of human platelet lysate for the expansion of human bone marrow mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    Sabrina Viau

    Full Text Available We recently developed and characterized a standardized and clinical grade human Platelet Lysate (hPL that constitutes an advantageous substitute for fetal bovine serum (FBS for human mesenchymal stem cell (hMSC expansion required in cell therapy procedures, avoiding xenogenic risks (virological and immunological and ethical issues. Because of the progressive use of pathogen-reduced (PR labile blood components, and the requirement of ensuring the viral safety of raw materials for cell therapy products, we evaluated the impact of the novel procedure known as THERAFLEX UV-Platelets for pathogen reduction on hPL quality (growth factors content and efficacy (as a medium supplement for hMSC expansion. This technology is based on short-wave ultraviolet light (UV-C that induces non-reversible damages in DNA and RNA of pathogens while preserving protein structures and functions, and has the main advantage of not needing the addition of any photosensitizing additives (that might secondarily interfere with hMSCs.We applied the THERAFLEX UV-Platelets procedure on fresh platelet concentrates (PCs suspended in platelet additive solution and prepared hPL from these treated PCs. We compared the quality and efficacy of PR-hPL with the corresponding non-PR ones. We found no impact on the content of five cytokines tested (EGF, bFGF, PDGF-AB, VEGF and IGF-1 but a significant decrease in TGF-ß1 (-21%, n = 11, p<0.01. We performed large-scale culture of hMSCs from bone marrow (BM during three passages and showed that hPL or PR-hPL at 8% triggered comparable BM-hMSC proliferation as FBS at 10% plus bFGF. Moreover, after proliferation of hMSCs in an hPL- or PR-hPL-containing medium, their profile of membrane marker expression, their clonogenic potential and immunosuppressive properties were maintained, in comparison with BM-hMSCs cultured under FBS conditions. The potential to differentiate towards the adipogenic and osteogenic lineages of hMSCs cultured in parallel

  13. Pathogen reduction through additive-free short-wave UV light irradiation retains the optimal efficacy of human platelet lysate for the expansion of human bone marrow mesenchymal stem cells.

    Science.gov (United States)

    Viau, Sabrina; Chabrand, Lucie; Eap, Sandy; Lorant, Judith; Rouger, Karl; Goudaliez, Francis; Sumian, Chryslain; Delorme, Bruno

    2017-01-01

    We recently developed and characterized a standardized and clinical grade human Platelet Lysate (hPL) that constitutes an advantageous substitute for fetal bovine serum (FBS) for human mesenchymal stem cell (hMSC) expansion required in cell therapy procedures, avoiding xenogenic risks (virological and immunological) and ethical issues. Because of the progressive use of pathogen-reduced (PR) labile blood components, and the requirement of ensuring the viral safety of raw materials for cell therapy products, we evaluated the impact of the novel procedure known as THERAFLEX UV-Platelets for pathogen reduction on hPL quality (growth factors content) and efficacy (as a medium supplement for hMSC expansion). This technology is based on short-wave ultraviolet light (UV-C) that induces non-reversible damages in DNA and RNA of pathogens while preserving protein structures and functions, and has the main advantage of not needing the addition of any photosensitizing additives (that might secondarily interfere with hMSCs). We applied the THERAFLEX UV-Platelets procedure on fresh platelet concentrates (PCs) suspended in platelet additive solution and prepared hPL from these treated PCs. We compared the quality and efficacy of PR-hPL with the corresponding non-PR ones. We found no impact on the content of five cytokines tested (EGF, bFGF, PDGF-AB, VEGF and IGF-1) but a significant decrease in TGF-ß1 (-21%, n = 11, p<0.01). We performed large-scale culture of hMSCs from bone marrow (BM) during three passages and showed that hPL or PR-hPL at 8% triggered comparable BM-hMSC proliferation as FBS at 10% plus bFGF. Moreover, after proliferation of hMSCs in an hPL- or PR-hPL-containing medium, their profile of membrane marker expression, their clonogenic potential and immunosuppressive properties were maintained, in comparison with BM-hMSCs cultured under FBS conditions. The potential to differentiate towards the adipogenic and osteogenic lineages of hMSCs cultured in parallel in the

  14. Haemolytic and Antimicrobial Activites of Saponin-Rich Extracts from Guar Meal

    Science.gov (United States)

    Saponin-rich GM extract was prepared by refluxing 25 g of GM with 250 ml of EtOH/H2O (1:1, v/v) for 3 h then filtering and distilling EtOH at 50oC. The refluxed extract was partitioned with equal volume of BuOH obtaining crude saponin-rich GM extract with 4.8 ± 0.6% DM of GM that was purified by RP...

  15. The leucine-rich repeat structure.

    Science.gov (United States)

    Bella, J; Hindle, K L; McEwan, P A; Lovell, S C

    2008-08-01

    The leucine-rich repeat is a widespread structural motif of 20-30 amino acids with a characteristic repetitive sequence pattern rich in leucines. Leucine-rich repeat domains are built from tandems of two or more repeats and form curved solenoid structures that are particularly suitable for protein-protein interactions. Thousands of protein sequences containing leucine-rich repeats have been identified by automatic annotation methods. Three-dimensional structures of leucine-rich repeat domains determined to date reveal a degree of structural variability that translates into the considerable functional versatility of this protein superfamily. As the essential structural principles become well established, the leucine-rich repeat architecture is emerging as an attractive framework for structural prediction and protein engineering. This review presents an update of the current understanding of leucine-rich repeat structure at the primary, secondary, tertiary and quaternary levels and discusses specific examples from recently determined three-dimensional structures.

  16. Evaluating Platelet-Rich Therapy for Facial Aesthetics and Alopecia: A Critical Review of the Literature.

    Science.gov (United States)

    Motosk O, Catherine C; Khouri, Kimberly S; Poudrier, Grace; Sinno, Sammy; Hazen, Alexes

    2018-05-01

    Despite the growing popularity of platelet-rich plasma, existing evidence supporting its efficacy remains controversial due to the lack of large-scale studies and standardized protocols for preparation and application. This article reviews its use in facial rejuvenation, fat grafting, acne scarring, and androgenic alopecia. Emphasis is placed on comparing methods of platelet-rich plasma preparation and application across studies. A systematic review was performed for articles published between 2006 and 2015. All clinical studies and case reports that addressed platelet-rich plasma alone and/or in combination with fat grafting for facial rejuvenation, acne scarring, or androgenic alopecia were included. Of the 22 articles included in the analysis, seven studies used platelet-rich plasma alone for facial rejuvenation, seven in combination with fat grafting, two for treatment of acne scarring, and six for treatment of androgenic alopecia. Individual study procedures, means of evaluation, and significant results are summarized. Although the majority of studies in this review report positive results, significant variation exists in preparation protocols and in the number and frequency of clinical treatments. The majority of studies report positive results for all indications evaluated in this review, but the procedure is limited by the lack of a standardized method for preparation and application of platelet-rich plasma. The extent to which significant variability in platelet-rich plasma preparation and/or application methods may affect clinical outcomes is not completely clear. In the interim, we present a consolidation of platelet-rich plasma treatment techniques and outcomes currently in use to help guide physicians in their clinical practice.

  17. Platelet-rich fibrin in the treatment of periodontal bone defects.

    Science.gov (United States)

    Ranganathan, Aravindhan T; Chandran, Chitraa R

    2014-05-01

    Periodontitis is characterized by the formation of true pockets, bone loss and attachment loss. Various techniques have been attempted in the past to truly regenerate the lost periodontal structures, albeit with variable outcome. In this evolution, the technique being tried out widely is the use of platelet rich concentrates, namely platelet-rich fibrin (PRF). In this report, we present a case of surgical treatment of osseous bone defects namely two walled crater and dehiscence treated in posterior teeth with autologously prepared platelet rich fibrin mixed with hydroxy apatite bone graft and PRF in the form of a membrane. Our results showed clinical improvements in all the clinical parameters postoperatively namely the pocket depth reduction and gain in attachment level and hence, PRF can be used alone or in combination with the bone graft to yield successful clinical results in treating periodontal osseous defects. Platelet-rich fibrin is an effective alternative to platelet-rich plasma (PRP) in reconstructing bone defects.

  18. Rich Language Analysis for Counterterrorism

    Science.gov (United States)

    Guidère, Mathieu; Howard, Newton; Argamon, Shlomo

    Accurate and relevant intelligence is critical for effective counterterrorism. Too much irrelevant information is as bad or worse than not enough information. Modern computational tools promise to provide better search and summarization capabilities to help analysts filter and select relevant and key information. However, to do this task effectively, such tools must have access to levels of meaning beyond the literal. Terrorists operating in context-rich cultures like fundamentalist Islam use messages with multiple levels of interpretation, which are easily misunderstood by non-insiders. This chapter discusses several kinds of such “encryption” used by terrorists and insurgents in the Arabic language, and how knowledge of such methods can be used to enhance computational text analysis techniques for use in counterterrorism.

  19. Platelet Rich Plasma- mechanism of action and clinical applications

    Directory of Open Access Journals (Sweden)

    Cristina N. Cozma

    2016-11-01

    Full Text Available Platelet-rich plasma (PRP is a blood-derived fraction containing high level of platelets, a high concentration of leukocytes and growth factors. PRP therapy has been growing as a viable treatment alternative for a number of clinical applications and has a potential benefit for use in wound healing. Nowadays platelet rich plasma is used in stimulating wound healing in skin and soft tissue ulcerations, accelerating wound healing in diabetic patients and facilitating bone proliferation in orthopedic and trauma surgery. It has also applications in maxillofacial surgery, spinal surgery, plastic and esthetic surgery, heart surgery and burns. This review of the literature shows a limited number of studies realized on humans that sustain PRP applications in orthopedic and plastic surgery. As the use of PRP increases, more properly structured clinical studies are necessary to confirm the results and to establish clearly the techniques of preparing, the conditions and the clinical indications of applying this therapy.

  20. Platelet-rich-fibrin: A novel root coverage approach

    Directory of Open Access Journals (Sweden)

    Anilkumar K

    2009-01-01

    Full Text Available Treatment of gingival recession has become an important therapeutic issue due to increasing cosmetic demand. Multiple surgical procedures have been developed to obtain predictable esthetic root coverage. More specifically, after periodontal regenerative surgery, the aim is to achieve complete wound healing and regeneration of the periodontal unit. A recent innovation in dentistry is the preparation and use of platelet-rich plasma (PRP, a concentrated suspension of the growth factors, found in platelets. These growth factors are involved in wound healing and postulated as promoters of tissue regeneration. This paper reports the use of PRF membrane for root coverage on the labial surfaces of the mandibular anterior teeth. This was accomplished using laterally displaced flap technique with platelet rich fibrin (PRF membrane at the recipient site.

  1. Long-Term Expansion in Platelet Lysate Increases Growth of Peripheral Blood-Derived Endothelial-Colony Forming Cells and Their Growth Factor-Induced Sprouting Capacity.

    Science.gov (United States)

    Tasev, Dimitar; van Wijhe, Michiel H; Weijers, Ester M; van Hinsbergh, Victor W M; Koolwijk, Pieter

    2015-01-01

    Efficient implementation of peripheral blood-derived endothelial-colony cells (PB-ECFCs) as a therapeutical tool requires isolation and generation of a sufficient number of cells in ex vivo conditions devoid of animal-derived products. At present, little is known how the isolation and expansion procedure in xenogeneic-free conditions affects the therapeutical capacity of PB-ECFCs. The findings presented in this study indicate that human platelet lysate (PL) as a serum substitute yields twice more colonies per mL blood compared to the conventional isolation with fetal bovine serum (FBS). Isolated ECFCs displayed a higher proliferative ability in PL supplemented medium than cells in FBS medium during 30 days expansion. The cells at 18 cumulative population doubling levels (CPDL) retained their proliferative capacity, showed higher sprouting ability in fibrin matrices upon stimulation with FGF-2 and VEGF-A than the cells at 6 CPDL, and displayed low β-galactosidase activity. The increased sprouting of PB-ECFCs at 18 CPDL was accompanied by an intrinsic activation of the uPA/uPAR fibrinolytic system. Induced deficiency of uPA (urokinase-type plasminogen activator) or uPAR (uPA receptor) by siRNA technology completely abolished the angiogenic ability of PB-ECFCs in fibrin matrices. During the serial expansion, the gene induction of the markers associated with inflammatory activation such as VCAM-1 and ICAM-1 did not occur or only to limited extent. While further propagation up to 31 CPDL proceeded at a comparable rate, a marked upregulation of inflammatory markers occurred in all donors accompanied by a further increase of uPA/uPAR gene induction. The observed induction of inflammatory genes at later stages of long-term propagation of PB-ECFCs underpins the necessity to determine the right time-point for harvesting of sufficient number of cells with preserved therapeutical potential. The presented isolation method and subsequent cell expansion in platelet lysate

  2. Platelet lysate as a novel serum-free media supplement for the culture of equine bone marrow-derived mesenchymal stem cells.

    Science.gov (United States)

    Naskou, Maria C; Sumner, Scarlett M; Chocallo, Anna; Kemelmakher, Hannah; Thoresen, Merrilee; Copland, Ian; Galipeau, Jacques; Peroni, John F

    2018-03-22

    Mesenchymal stem cells (MSCs) produced for clinical purposes rely on culture media containing fetal bovine serum (FBS) which is xenogeneic and has the potential to significantly alter the MSC phenotype, rendering these cells immunogenic. As a result of bovine-derived exogenous proteins expressed on the cell surface, MSCs may be recognized by the host immune system as non-self and be rejected. Platelet lysate (PL) may obviate some of these concerns and shows promising results in human medicine as a possible alternative to FBS. Our goal was to evaluate the use of equine platelet lysate (ePL) pooled from donor horses in place of FBS to culture equine MSCs. We hypothesized that ePL, produced following apheresis, will function as the sole media supplement to accelerate the expansion of equine bone marrow-derived MSCs without altering their phenotype and their immunomodulatory capacity. Platelet concentrate was obtained via plateletpheresis and ePL were produced via freeze-thaw and centrifugation cycles. Population doublings (PD) and doubling time (DT) of bone marrow-derived MSCs (n = 3) cultured with FBS or ePL media were calculated. Cell viability, immunophenotypic analysis, and trilineage differentiation capacity of MSCs were assessed accordingly. To assess the ability of MSCs to modulate inflammatory responses, E. coli lipopolysaccharide (LPS)-stimulated monocytes were cocultured with MSCs cultured in the two different media formulations, and cell culture supernatants were assayed for the production of tumor necrosis factor (TNF)-α. Our results showed that MSCs cultured in ePL media exhibited similar proliferation rates (PD and DT) compared with those cultured in FBS at individual time points. MSCs cultured in ePL showed a statistically significant increased viability following a single washing step, expressed similar levels of MSC markers compared to FBS, and were able to differentiate towards the three lineages. Finally, MSCs cultured in ePL efficiently suppressed

  3. Optimized human platelet lysate as novel basis for a serum-, xeno-, and additive-free corneal endothelial cell and tissue culture.

    Science.gov (United States)

    Thieme, Daniel; Reuland, Lynn; Lindl, Toni; Kruse, Friedrich; Fuchsluger, Thomas

    2018-02-01

    The expansion of donor-derived corneal endothelial cells (ECs) is a promising approach for regenerative therapies in corneal diseases. To achieve the best Good Manufacturing Practice standard the entire cultivation process should be devoid of nonhuman components. However, so far, there is no suitable xeno-free protocol for clinical applications. We therefore introduce a processed variant of a platelet lysate for the use in corneal cell and tissue culture based on a Good Manufacturing Practice-grade thrombocyte concentrate. This processed human platelet lysate (phPL), free of any animal components and of anticoagulants such as heparin with a physiological ionic composition, was used to cultivate corneal ECs in vitro and ex vivo in comparison to standard cultivation with fetal calf serum (FCS). Human donor corneas were cut in quarters while 2 quarters of each cornea were incubated with the respective medium supplement. Three fields of view per quarter were taken into account for the analysis. Evaluation of phPL as a medium supplement in cell culture of immortalized EC showed a superior viability compared with FCS control with reduced cell proliferation. Furthermore, the viability during the expansion of primary cells is significantly (3-fold ±0.5) increased with phPL compared with FCS standard medium. Quartering donor corneas was traumatic for the endothelium and therefore resulted in increased EC loss. Interestingly, however, cultivation of the quartered pieces for 2 weeks in 0.1-mg/ml pHPL in Biochrome I showed a 21 (±10) % EC loss compared with 67 (±12) % EC loss when cultivated in 2% FCS in Biochrome I. The cell culture protocol with pHPL as FCS replacement seems to be superior to the standard FCS protocols with respect to EC survival. It offers a xeno-free and physiological environment for corneal endothelial cells. This alternative cultivation protocol could facilitate the use of EC for human corneal cell therapy. Copyright © 2017 John Wiley & Sons, Ltd.

  4. Long-Term Expansion in Platelet Lysate Increases Growth of Peripheral Blood-Derived Endothelial-Colony Forming Cells and Their Growth Factor-Induced Sprouting Capacity.

    Directory of Open Access Journals (Sweden)

    Dimitar Tasev

    Full Text Available Efficient implementation of peripheral blood-derived endothelial-colony cells (PB-ECFCs as a therapeutical tool requires isolation and generation of a sufficient number of cells in ex vivo conditions devoid of animal-derived products. At present, little is known how the isolation and expansion procedure in xenogeneic-free conditions affects the therapeutical capacity of PB-ECFCs.The findings presented in this study indicate that human platelet lysate (PL as a serum substitute yields twice more colonies per mL blood compared to the conventional isolation with fetal bovine serum (FBS. Isolated ECFCs displayed a higher proliferative ability in PL supplemented medium than cells in FBS medium during 30 days expansion. The cells at 18 cumulative population doubling levels (CPDL retained their proliferative capacity, showed higher sprouting ability in fibrin matrices upon stimulation with FGF-2 and VEGF-A than the cells at 6 CPDL, and displayed low β-galactosidase activity. The increased sprouting of PB-ECFCs at 18 CPDL was accompanied by an intrinsic activation of the uPA/uPAR fibrinolytic system. Induced deficiency of uPA (urokinase-type plasminogen activator or uPAR (uPA receptor by siRNA technology completely abolished the angiogenic ability of PB-ECFCs in fibrin matrices. During the serial expansion, the gene induction of the markers associated with inflammatory activation such as VCAM-1 and ICAM-1 did not occur or only to limited extent. While further propagation up to 31 CPDL proceeded at a comparable rate, a marked upregulation of inflammatory markers occurred in all donors accompanied by a further increase of uPA/uPAR gene induction. The observed induction of inflammatory genes at later stages of long-term propagation of PB-ECFCs underpins the necessity to determine the right time-point for harvesting of sufficient number of cells with preserved therapeutical potential.The presented isolation method and subsequent cell expansion in platelet

  5. Robust Optical Richness Estimation with Reduced Scatter

    Energy Technology Data Exchange (ETDEWEB)

    Rykoff, E.S.; /LBL, Berkeley; Koester, B.P.; /Chicago U. /Chicago U., KICP; Rozo, E.; /Chicago U. /Chicago U., KICP; Annis, J.; /Fermilab; Evrard, A.E.; /Michigan U. /Michigan U., MCTP; Hansen, S.M.; /Lick Observ.; Hao, J.; /Fermilab; Johnston, D.E.; /Fermilab; McKay, T.A.; /Michigan U. /Michigan U., MCTP; Wechsler, R.H.; /KIPAC, Menlo Park /SLAC

    2012-06-07

    Reducing the scatter between cluster mass and optical richness is a key goal for cluster cosmology from photometric catalogs. We consider various modifications to the red-sequence matched filter richness estimator of Rozo et al. (2009b), and evaluate their impact on the scatter in X-ray luminosity at fixed richness. Most significantly, we find that deeper luminosity cuts can reduce the recovered scatter, finding that {sigma}{sub ln L{sub X}|{lambda}} = 0.63 {+-} 0.02 for clusters with M{sub 500c} {approx}> 1.6 x 10{sup 14} h{sub 70}{sup -1} M{sub {circle_dot}}. The corresponding scatter in mass at fixed richness is {sigma}{sub ln M|{lambda}} {approx} 0.2-0.3 depending on the richness, comparable to that for total X-ray luminosity. We find that including blue galaxies in the richness estimate increases the scatter, as does weighting galaxies by their optical luminosity. We further demonstrate that our richness estimator is very robust. Specifically, the filter employed when estimating richness can be calibrated directly from the data, without requiring a-priori calibrations of the red-sequence. We also demonstrate that the recovered richness is robust to up to 50% uncertainties in the galaxy background, as well as to the choice of photometric filter employed, so long as the filters span the 4000 {angstrom} break of red-sequence galaxies. Consequently, our richness estimator can be used to compare richness estimates of different clusters, even if they do not share the same photometric data. Appendix A includes 'easy-bake' instructions for implementing our optimal richness estimator, and we are releasing an implementation of the code that works with SDSS data, as well as an augmented maxBCG catalog with the {lambda} richness measured for each cluster.

  6. Floristic richness and endemism in the Flora of Ethiopia and Eritrea

    DEFF Research Database (Denmark)

    Friis, Ib

    2009-01-01

    An analysis of the distribution of species richness and endemism on the floristic regions that have been used for the preparation of the Flora of Ethiopia and Eritrea; the article is based on a previously published and more comprehensive study of the flora of the entire Horn of Africa.......An analysis of the distribution of species richness and endemism on the floristic regions that have been used for the preparation of the Flora of Ethiopia and Eritrea; the article is based on a previously published and more comprehensive study of the flora of the entire Horn of Africa....

  7. A high-throughput 2D-analytical technique to obtain single protein parameters from complex cell lysates for in silico process development of ion exchange chromatography.

    Science.gov (United States)

    Kröner, Frieder; Elsäßer, Dennis; Hubbuch, Jürgen

    2013-11-29

    The accelerating growth of the market for biopharmaceutical proteins, the market entry of biosimilars and the growing interest in new, more complex molecules constantly pose new challenges for bioseparation process development. In the presented work we demonstrate the application of a multidimensional, analytical separation approach to obtain the relevant physicochemical parameters of single proteins in a complex mixture for in silico chromatographic process development. A complete cell lysate containing a low titre target protein was first fractionated by multiple linear salt gradient anion exchange chromatography (AEC) with varying gradient length. The collected fractions were subsequently analysed by high-throughput capillary gel electrophoresis (HT-CGE) after being desalted and concentrated. From the obtained data of the 2D-separation the retention-volumes and the concentration of the single proteins were determined. The retention-volumes of the single proteins were used to calculate the related steric-mass action model parameters. In a final evaluation experiment the received parameters were successfully applied to predict the retention behaviour of the single proteins in salt gradient AEC. Copyright © 2013 Elsevier B.V. All rights reserved.

  8. Ligament Tissue Engineering Using a Novel Porous Polycaprolactone Fumarate Scaffold and Adipose Tissue-Derived Mesenchymal Stem Cells Grown in Platelet Lysate.

    Science.gov (United States)

    Wagner, Eric R; Bravo, Dalibel; Dadsetan, Mahrokh; Riester, Scott M; Chase, Steven; Westendorf, Jennifer J; Dietz, Allan B; van Wijnen, Andre J; Yaszemski, Michael J; Kakar, Sanjeev

    2015-11-01

    Surgical reconstruction of intra-articular ligament injuries is hampered by the poor regenerative potential of the tissue. We hypothesized that a novel composite polymer "neoligament" seeded with progenitor cells and growth factors would be effective in regenerating native ligamentous tissue. We synthesized a fumarate-derivative of polycaprolactone fumarate (PCLF) to create macro-porous scaffolds to allow cell-cell communication and nutrient flow. Clinical grade human adipose tissue-derived human mesenchymal stem cells (AMSCs) were cultured in 5% human platelet lysate (PL) and seeded on scaffolds using a dynamic bioreactor. Cell growth, viability, and differentiation were examined using metabolic assays and immunostaining for ligament-related markers (e.g., glycosaminoglycans [GAGs], alkaline phosphatase [ALP], collagens, and tenascin-C). AMSCs seeded on three-dimensional (3D) PCLF scaffolds remain viable for at least 2 weeks with proliferating cells filling the pores. AMSC proliferation rates increased in PL compared to fetal bovine serum (FBS) (p ligament and tenogenic growth factor fibroblast growth factor 2 (FGF-2), especially when cultured in the presence of PL (p engineering and ligament regeneration.

  9. Donor T cells primed on leukemia lysate-pulsed recipient APCs mediate strong graft-versus-leukemia effects across MHC barriers in full chimeras.

    Science.gov (United States)

    Ghosh, Arnab; Koestner, Wolfgang; Hapke, Martin; Schlaphoff, Verena; Länger, Florian; Baumann, Rolf; Koenecke, Christian; Cornberg, Markus; Welte, Karl; Blazar, Bruce R; Sauer, Martin G

    2009-04-30

    Antigen-presenting cells (APCs) of host origin drive graft-versus-leukemia (GVL) effects but can also trigger life-threatening graft-versus-host disease (GVHD) after hematopoietic cell transplantation (HCT) across major histocompatibility complex (MHC) barriers. We show that in vitro priming of donor lymphocytes can circumvent the need of recipient-derived APCs in vivo for mediating robust GVL effects and significantly diminishes the risk of severe GVHD. In vitro, generated and expanded T cells (ETCs) mediate anti-leukemia effects only when primed on recipient-derived APCs. Loading of APCs in vitro with leukemia cell lysate, chimerism status of the recipient, and timing of adoptive transfer after HCT are important factors determining the outcome. Delayed transfer of ETCs resulted in strong GVL effects in leukemia-bearing full chimera (FC) and mixed chimera (MC) recipients, which were comparable with the GVL/GVHD rates observed after the transfer of naive donor lymphocyte infusion (DLI). Upon early transfer, GVL effects were more pronounced with ETCs but at the expense of significant GVHD. The degree of GVHD was most severe in MCs after transfer of ETCs that had been in vitro primed either on nonpulsed recipient-derived APCs or with donor-derived APCs.

  10. Back to basics: the untreated rabbit reticulocyte lysate as a competitive system to recapitulate cap/poly(A) synergy and the selective advantage of IRES-driven translation.

    Science.gov (United States)

    Soto Rifo, Ricardo; Ricci, Emiliano P; Décimo, Didier; Moncorgé, Olivier; Ohlmann, Théophile

    2007-01-01

    Translation of most eukaryotic mRNAs involves the synergistic action between the 5' cap structure and the 3' poly(A) tail at the initiation step. The poly(A) tail has also been shown to stimulate translation of picornavirus internal ribosome entry sites (IRES)-directed translation. These effects have been attributed principally to interactions between eIF4G and poly(A)-binding protein (PABP) but also to the participation of PABP in other steps during translation initiation. As the rabbit reticulocyte lysate (RRL) does not recapitulate this cap/poly(A) synergy, several systems based on cellular cell-free extracts have been developed to study the effects of poly(A) tail in vitro but they generally exhibit low translational efficiency. Here, we describe that the non-nuclease-treated RRL (untreated RRL) is able to recapitulate the effects of poly(A) tail on translation in vitro. In this system, translation of a capped/polyadenylated RNA was specifically inhibited by either Paip2 or poly(rA), whereas translation directed by HCV IRES remained unaffected. Moreover, cleavage of eIF4G by FMDV L protease strongly stimulated translation directed by the EMCV IRES, thus recapitulating the competitive advantage that the proteolytic processing of eIF4G confers to IRES-driven RNAs.

  11. Freeze-thaw lysates of Plasmodium falciparum-infected red blood cells induce differentiation of functionally competent regulatory T cells from memory T cells.

    Science.gov (United States)

    Finney, Olivia C; Lawrence, Emma; Gray, Alice P; Njie, Madi; Riley, Eleanor M; Walther, Michael

    2012-07-01

    In addition to naturally occurring regulatory T (nTreg) cells derived from the thymus, functionally competent Treg cells can be induced in vitro from peripheral blood lymphocytes in response to TCR stimulation with cytokine costimulation. Using these artificial stimulation conditions, both naïve as well as memory CD4(+) T cells can be converted into induced Treg (iTreg) cells, but the cellular origin of such iTreg cells in vivo or in response to more physiologic stimulation with pathogen-derived antigens is less clear. Here, we demonstrate that a freeze/thaw lysate of Plasmodium falciparum schizont extract (PfSE) can induce functionally competent Treg cells from peripheral lymphocytes in a time- and dose-dependent manner without the addition of exogenous costimulatory factors. The PfSE-mediated induction of Treg cells required the presence of nTreg cells in the starting culture. Further experiments mixing either memory or naïve T cells with antigen presenting cells and CFSE-labeled Treg cells identified CD4(+) CD45RO(+) CD25(-) memory T cells rather than Treg cells as the primary source of PfSE-induced Treg cells. Taken together, these data suggest that in the presence of nTreg cells, PfSE induces memory T cells to convert into iTreg cells that subsequently expand alongside PfSE-induced effector T cells. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Comparison of allogeneic platelet lysate and fetal bovine serum for in vitro expansion of equine bone marrow-derived mesenchymal stem cells.

    Science.gov (United States)

    Seo, Jong-pil; Tsuzuki, Nao; Haneda, Shingo; Yamada, Kazutaka; Furuoka, Hidefumi; Tabata, Yasuhiko; Sasaki, Naoki

    2013-10-01

    Mesenchymal stem cells (MSCs) are promising candidates for cell-based therapy and tissue engineering approaches. Fetal bovine serum (FBS) is commonly used for in vitro MSC expansion; however, the use of FBS may be associated with ethical, scientific, and safety issues. This study aimed to compare the ability of allogeneic platelet lysate (PL) and FBS to cause equine bone marrow-derived MSC expansion. MSCs were isolated from bone marrow aspirate in media supplemented with either PL or FBS, and cell proliferation properties and characteristics were examined. There were no significant differences in MSC yield, colony-forming unit-fibroblast (CFU-F) assay, and population doubling time between PL and FBS cultures. In addition, both PL-MSCs and FBS-MSCs showed similar results in term of ALP staining, osteogenic differentiation, and RT-PCR, although there were subtle differences in morphology, growth pattern, and adhesive properties. These results suggest that PL is a suitable alternative to FBS for use in equine MSC expansion, without the problems related to FBS use. Published by Elsevier India Pvt Ltd.

  13. The Potential of GMP-Compliant Platelet Lysate to Induce a Permissive State for Cardiovascular Transdifferentiation in Human Mediastinal Adipose Tissue-Derived Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Camilla Siciliano

    2015-01-01

    Full Text Available Human adipose tissue-derived mesenchymal stem cells (ADMSCs are considered eligible candidates for cardiovascular stem cell therapy applications due to their cardiac transdifferentiation potential and immunotolerance. Over the years, the in vitro culture of ADMSCs by platelet lysate (PL, a hemoderivate containing numerous growth factors and cytokines derived from platelet pools, has allowed achieving a safe and reproducible methodology to obtain high cell yield prior to clinical administration. Nevertheless, the biological properties of PL are still to be fully elucidated. In this brief report we show the potential ability of PL to induce a permissive state of cardiac-like transdifferentiation and to cause epigenetic modifications. RTPCR results indicate an upregulation of Cx43, SMA, c-kit, and Thy-1 confirmed by immunofluorescence staining, compared to standard cultures with foetal bovine serum. Moreover, PL-cultured ADMSCs exhibit a remarkable increase of both acetylated histones 3 and 4, with a patient-dependent time trend, and methylation at lysine 9 on histone 3 preceding the acetylation. Expression levels of p300 and SIRT-1, two major regulators of histone 3, are also upregulated after treatment with PL. In conclusion, PL could unravel novel biological properties beyond its routine employment in noncardiac applications, providing new insights into the plasticity of human ADMSCs.

  14. The Potential of GMP-Compliant Platelet Lysate to Induce a Permissive State for Cardiovascular Transdifferentiation in Human Mediastinal Adipose Tissue-Derived Mesenchymal Stem Cells

    Science.gov (United States)

    Bordin, Antonella; Ponti, Donatella; Iudicone, Paola; Rendina, Erino Angelo; Calogero, Antonella; Pierelli, Luca; Ibrahim, Mohsen; De Falco, Elena

    2015-01-01

    Human adipose tissue-derived mesenchymal stem cells (ADMSCs) are considered eligible candidates for cardiovascular stem cell therapy applications due to their cardiac transdifferentiation potential and immunotolerance. Over the years, the in vitro culture of ADMSCs by platelet lysate (PL), a hemoderivate containing numerous growth factors and cytokines derived from platelet pools, has allowed achieving a safe and reproducible methodology to obtain high cell yield prior to clinical administration. Nevertheless, the biological properties of PL are still to be fully elucidated. In this brief report we show the potential ability of PL to induce a permissive state of cardiac-like transdifferentiation and to cause epigenetic modifications. RTPCR results indicate an upregulation of Cx43, SMA, c-kit, and Thy-1 confirmed by immunofluorescence staining, compared to standard cultures with foetal bovine serum. Moreover, PL-cultured ADMSCs exhibit a remarkable increase of both acetylated histones 3 and 4, with a patient-dependent time trend, and methylation at lysine 9 on histone 3 preceding the acetylation. Expression levels of p300 and SIRT-1, two major regulators of histone 3, are also upregulated after treatment with PL. In conclusion, PL could unravel novel biological properties beyond its routine employment in noncardiac applications, providing new insights into the plasticity of human ADMSCs. PMID:26495284

  15. Phenotypical and functional characteristics of mesenchymal stem cells from bone marrow: comparison of culture using different media supplemented with human platelet lysate or fetal bovine serum

    Science.gov (United States)

    2012-01-01

    Introduction Mesenchymal stem cells (MSCs) are multipotent cells able to differentiate into several mesenchymal lineages, classically derived from bone marrow (BM) but potentially from umbilical cord blood (UCB). Although they are becoming a good tool for regenerative medicine, they usually need to be expanded in fetal bovine serum (FBS)-supplemented media. Human platelet lysate (HPL) has recently been proposed as substitute for safety reasons, but it is not yet clear how this supplement influences the properties of expanded MSCs. Methods In the present study, we compared the effect of various media combining autologous HPL with or without FBS on phenotypic, proliferative and functional (differentiation, cytokine secretion profile) characteristics of human BM-derived MSCs. Results Despite less expression of adipogenic and osteogenic markers, MSCs cultured in HPL-supplemented media fully differentiated along osteoblastic, adipogenic, chondrogenic and vascular smooth muscle lineages. The analyses of particular specific proteins expressed during osteogenic differentiation (calcium-sensing receptor (CaSR) and parathormone receptor (PTHR)) showed their decrease at D0 before any induction for MSC cultured with HPL mostly at high percentage (10%HPL). The cytokine dosage showed a clear increase of proliferation capacity and interleukin (IL)-6 and IL-8 secretion. Conclusions This study shows that MSCs can be expanded in media supplemented with HPL that can totally replace FBS. HPL-supplemented media not only preserves their phenotype as well as their differentiation capacity, but also shortens culture time by increasing their growth rate. PMID:22333342

  16. Investigation of the bacteriophage community in induced lysates of undefined mesophilic mixed-strain DL-cultures using classical and metagenomic approaches.

    Science.gov (United States)

    Muhammed, Musemma K; Olsen, Mette L; Kot, Witold; Neve, Horst; Castro-Mejía, Josué L; Janzen, Thomas; Hansen, Lars H; Nielsen, Dennis S; Sørensen, Søren J; Heller, Knut J; Vogensen, Finn K

    2018-05-02

    To investigate the notion that starter cultures can be a reservoir of bacteriophages (phages) in the dairy environment, strains of three DL-starters (undefined mesophilic mixed-strain starters containing Lactococcus lactis subsp. lactis biovar. diacetylactis and Leuconostoc species) were selected and induced by mitomycin C, and the whole starters were induced spontaneously as well as by mitomycin C. Frequency of induction of 17%, 26% and 12% was estimated among the isolates of the three starters, with majority of the induced phages mostly showing morphological similarity to known P335 phages, and with a fraction of them showing atypical features. Sequences of P335 quasi-species phages were found to be the most frequent entities in almost all metaviromes derived from the induced lysates. However, sequences of Sk1virus phages (previously 936 phages) were emerged as the predominant entities following spontaneous induction of one of the starters, suggesting a phage-carrier state. Sequences of other phages such as 949, 1706, C2virus (previously c2 phages) and Leuconostoc species could also be observed but with a lower relative frequency. Taken together, the majority of the P335 quasi-species phages could represent the induced viral community of the starters and the remaining phage groups mainly represent the background ambient viral community. Copyright © 2018 Elsevier B.V. All rights reserved.

  17. Cell-Free Systems Based on CHO Cell Lysates: Optimization Strategies, Synthesis of "Difficult-to-Express" Proteins and Future Perspectives.

    Directory of Open Access Journals (Sweden)

    Lena Thoring

    Full Text Available Nowadays, biotechnological processes play a pivotal role in target protein production. In this context, Chinese Hamster Ovary (CHO cells are one of the most prominent cell lines for the expression of recombinant proteins and revealed as a safe host for nearly 40 years. Nevertheless, the major bottleneck of common in vivo protein expression platforms becomes obvious when looking at the production of so called "difficult-to-express" proteins. This class of proteins comprises in particular several ion channels and multipass membrane proteins as well as cytotoxic proteins. To enhance the production of "difficult-to-express" proteins, alternative technologies were developed, mainly based on translationally active cell lysates. These so called "cell-free" protein synthesis systems enable an efficient production of different classes of proteins. Eukaryotic cell-free systems harboring endogenous microsomal structures for the synthesis of functional membrane proteins and posttranslationally modified proteins are of particular interest for future applications. Therefore, we present current developments in cell-free protein synthesis based on translationally active CHO cell extracts, underlining the high potential of this platform. We present novel results highlighting the optimization of protein yields, the synthesis of various "difficult-to-express" proteins and the cotranslational incorporation of non-standard amino acids, which was exemplarily demonstrated by residue specific labeling of the glycoprotein Erythropoietin and the multimeric membrane protein KCSA.

  18. Proton-rich nuclear statistical equilibrium

    International Nuclear Information System (INIS)

    Seitenzahl, I.R.; Timmes, F.X.; Marin-Lafleche, A.; Brown, E.; Magkotsios, G.; Truran, J.

    2008-01-01

    Proton-rich material in a state of nuclear statistical equilibrium (NSE) is one of the least studied regimes of nucleosynthesis. One reason for this is that after hydrogen burning, stellar evolution proceeds at conditions of an equal number of neutrons and protons or at a slight degree of neutron-richness. Proton-rich nucleosynthesis in stars tends to occur only when hydrogen-rich material that accretes onto a white dwarf or a neutron star explodes, or when neutrino interactions in the winds from a nascent proto-neutron star or collapsar disk drive the matter proton-rich prior to or during the nucleosynthesis. In this Letter we solve the NSE equations for a range of proton-rich thermodynamic conditions. We show that cold proton-rich NSE is qualitatively different from neutron-rich NSE. Instead of being dominated by the Fe-peak nuclei with the largest binding energy per nucleon that have a proton-to-nucleon ratio close to the prescribed electron fraction, NSE for proton-rich material near freezeout temperature is mainly composed of 56Ni and free protons. Previous results of nuclear reaction network calculations rely on this nonintuitive high-proton abundance, which this Letter explains. We show how the differences and especially the large fraction of free protons arises from the minimization of the free energy as a result of a delicate competition between the entropy and nuclear binding energy.

  19. Proliferation-promoting effect of platelet-rich plasma on human adipose-derived stem cells and human dermal fibroblasts.

    Science.gov (United States)

    Kakudo, Natsuko; Minakata, Tatsuya; Mitsui, Toshihito; Kushida, Satoshi; Notodihardjo, Frederik Zefanya; Kusumoto, Kenji

    2008-11-01

    This study evaluated changes in platelet-derived growth factor (PDGF)-AB and transforming growth factor (TGF)-beta1 release from platelets by platelet-rich plasma activation, and the proliferation potential of activated platelet-rich plasma and platelet-poor plasma on human adipose-derived stem cells and human dermal fibroblasts. Platelet-rich plasma was prepared using a double-spin method, with the number of platelets counted in each preparation stage. Platelet-rich and platelet-poor plasma were activated with autologous thrombin and calcium chloride, and levels of platelet-released PDGF-AB and TGF-beta1 were determined by enzyme-linked immunosorbent assay. Cells were cultured for 1, 4, or 7 days in serum-free Dulbecco's Modified Eagle Medium supplemented with 5% whole blood plasma, nonactivated platelet-rich plasma, nonactivated platelet-poor plasma, activated platelet-rich plasma, or activated platelet-poor plasma. In parallel, these cells were cultured for 1, 4, or 7 days in serum-free Dulbecco's Modified Eagle Medium supplemented with 1%, 5%, 10%, or 20% activated platelet-rich plasma. The cultured human adipose-derived stem cells and human dermal fibroblasts were assayed for proliferation. Platelet-rich plasma contained approximately 7.9 times as many platelets as whole blood, and its activation was associated with the release of large amounts of PDGF-AB and TGF-beta1. Adding activated platelet-rich or platelet-poor plasma significantly promoted the proliferation of human adipose-derived stem cells and human dermal fibroblasts. Adding 5% activated platelet-rich plasma to the medium maximally promoted cell proliferation, but activated platelet-rich plasma at 20% did not promote it. Platelet-rich plasma can enhance the proliferation of human adipose-derived stem cells and human dermal fibroblasts. These results support clinical platelet-rich plasma application for cell-based, soft-tissue engineering and wound healing.

  20. Pooled human platelet lysate versus fetal bovine serum—investigating the proliferation rate, chromosome stability and angiogenic potential of human adipose tissue-derived stem cells intended for clinical use

    DEFF Research Database (Denmark)

    Trojahn Kølle, Stig-Frederik; Oliveri, Roberto S; Glovinski, Peter V

    2013-01-01

    Because of an increasing focus on the use of adipose-derived stem cells (ASCs) in clinical trials, the culture conditions for these cells are being optimized. We compared the proliferation rates and chromosomal stability of ASCs that had been cultured in Dulbecco's modified Eagle's Medium (DMEM) ......) supplemented with either pooled human platelet lysate (pHPL) or clinical-grade fetal bovine serum (FBS) (DMEM(pHPL) versus DMEM(FBS))....

  1. LHCb RICH1 Engineering Design Review Report

    CERN Document Server

    Brook, N; Metlica, F; Muir, A; Phillips, A; Buckley, A; Gibson, V; Harrison, K; Jones, C R; Katvars, S G; Lazzeroni, C; Storey, J; Ward, CP; Wotton, S; Alemi, M; Arnabaldi, C; Bellunato, T F; Calvi, M; Matteuzzi, C; Musy, M; Negri, P; Perego, D L; Pessina, G; Chamonal, R; Eisenhardt, S; Lawrence, J; McCarron, J; Muheim, F; Playfer, S; Walker, A; Cuneo, S; Fontanelli, F; Gracco, Valerio; Mini, G; Musico, P; Petrolini, A; Sannino, M; Bates, A; MacGregor, A; O'Shea, V; Parkes, C; Paterson, S; Petrie, D; Pickford, A; Rahman, M; Soler, F; Allebone, L; Barber, J H; Cameron, W; Clark, D; Dornan, Peter John; Duane, A; Egede, U; Hallam, R; Howard, A; Plackett, R; Price, D; Savidge, T; Vidal-Sitjes, G; Websdale, D M; Adinolfi, M; Bibby, J H; Cioffi, C; Gligorov, Vladimir V; Harnew, N; Harris, F; McArthur, I A; Newby, C; Ottewell, B; Rademacker, J; Senanayake, R; Somerville, L P; Soroko, A; Smale, N J; Topp-Jørgensen, S; Wilkinson, G; Yang, S; Benayoun, M; Khmelnikov, V A; Obraztsov, V F; Densham, C J; Easo, S; Franek, B; Kuznetsov, G; Loveridge, P W; Morrow, D; Morris, JV; Papanestis, A; Patrick, G N; Woodward, M L; Aglieri-Rinella, G; Albrecht, A; Braem, André; Campbell, M; D'Ambrosio, C; Forty, R W; Frei, C; Gys, Thierry; Jamet, O; Kanaya, N; Losasso, M; Moritz, M; Patel, M; Piedigrossi, D; Snoeys, W; Ullaland, O; Van Lysebetten, A; Wyllie, K

    2005-01-01

    This document describes the concepts of the engineering design to be adopted for the upstream Ring Imaging Cherenkov detector (RICH1) of the reoptimized LHCb detector. Our aim is to ensure that coherent solutions for the engineering design and integration for all components of RICH1 are available, before proceeding with the detailed design of these components.

  2. Island Species Richness Increases with Habitat Diversity

    NARCIS (Netherlands)

    Hortal, J.; Triantis, K.A.; Meiri, S.; Thebault, E.M.C.; Sfenthourakis, S.

    2009-01-01

    Species richness is commonly thought to increase with habitat diversity. However, a recent theoretical model aiming to unify niche and island biogeography theories predicted a hump-shaped relationship between richness and habitat diversity. Given the contradiction between model results and previous

  3. Platelet-rich plasma in otolaryngology.

    Science.gov (United States)

    Stavrakas, M; Karkos, P D; Markou, K; Grigoriadis, N

    2016-12-01

    Platelet-rich plasma is a novel material that is being used more frequently in many surgical specialties. A literature review on the current and potential uses of platelet-rich plasma in otolaryngology was performed. There is limited evidence on the use of platelet-rich plasma in otolaryngology compared with other specialties: only 11 studies on various subspecialties (otology, rhinology and laryngology) were included in the final review. Based on the limited number of studies, we cannot draw safe conclusions about the value of platelet-rich plasma in otolaryngology. Nevertheless, the available literature suggests that platelet-rich plasma holds promise for future research and may have a number of clinical applications.

  4. Fostering Technology-Rich Service-Learning Experiences between School Librarians and Teacher Education Programs

    Science.gov (United States)

    Shepherd, Craig E.; Dousay, Tonia; Kvenild, Cassandra; Meredith, Tamara

    2015-01-01

    School libraries are untapped resources for fieldwork by preservice teachers. Many school librarians have expertise in pedagogy and standards-based curriculum development, both for information literacy and for technology integration. By forging partnerships with teacher-preparation programs, school librarians can provide fieldwork sites rich in…

  5. The biology of platelet-rich plasma and its application in oral surgery: literature review.

    NARCIS (Netherlands)

    Nikolidakis, D.; Jansen, J.A.

    2008-01-01

    Platelet-rich plasma (PRP) is a new approach in tissue regeneration and a developing area for clinicians and researchers. It is used in various surgical fields, including oral and maxillofacial surgery. PRP is prepared from the patient's own blood and contains growth factors that influence wound

  6. The LHCb RICH Upgrade: Development of the DCS and DAQ system.

    CERN Multimedia

    Cavallero, Giovanni

    2018-01-01

    The LHCb experiment is preparing for an upgrade during the second LHC long shutdown in 2019-2020. In order to fully exploit the LHC flavour physics potential with a five-fold increase in the instantaneous luminosity, a trigger-less readout will be implemented. The RICH detectors will require new photon detectors and a brand new front-end electronics. The status of the integration of the RICH photon detector modules with the MiniDAQ, the prototype of the upgraded LHCb readout architecture, has been reported. The development of the prototype of the RICH Upgrade Experiment Control System, integrating the DCS and DAQ partitions in a single FSM, has been described. The status of the development of the RICH Upgrade Inventory, Bookkeeping and Connectivity database has been reported as well.

  7. Influence of Silane modified nano silica on the corrosion protection of zinc rich coating

    International Nuclear Information System (INIS)

    Nguyen Thuy Duong; To Thi Xuan Hang; Trinh Anh Truc; Pham Gia Vu; Bui Van Truoc; Thai Hoang

    2015-01-01

    Zinc rich coatings are the best effective primers for corrosion protection of carbon steel in aggressive conditions. For traditional zinc rich primer the zinc content is very high, more than 90 wt.%. The coating adhesion is decreased with the increase of zinc content, so that it is necessary to decrease the zinc content by using additives. In this study the nano silica modified by N-(2-Aminoethyl)-3-aminopropyltrimethoxysilane was prepared and incorporated in zinc rich epoxy coatings containing 85 wt.% zinc powder. The corrosion protection performance of coatings was evaluated by electrochemical impedance spectroscopy. The results obtained show that the presence of nano silica improved corrosion protection of zinc rich epoxy coating and the best protection was obtained with 3 wt.% nano silica. (author)

  8. Removal process of prion and parvovirus from human platelet lysates used as clinical-grade supplement for ex vivo cell expansion.

    Science.gov (United States)

    Kao, Yu-Chun; Bailey, Andy; Samminger, Bernhard; Tanimoto, Junji; Burnouf, Thierry

    2016-07-01

    Pooled human platelet lysate (HPL) is becoming the new gold standard as supplement for ex vivo cell culture for clinical protocols. However, the risk of pathogen contamination of HPL increases with the platelet pool size. We hypothesized that hollow fiber anion exchange membrane chromatography using QyuSpeed D (QSD) could remove resistant and untested bloodborne pathogens, such as parvoviruses and prions, from HPL-supplemented growth media without substantially affecting their capacity to support ex vivo cell expansion. Frozen or thawed platelet concentrates were serum-converted and centrifuged for obtaining HPL that was added to various growth media (ca. 100 mL), filtered through a 0.6-mL QSD membrane and characterized for proteins, growth factors and chemical composition. Capacity to expand Chinese hamster ovary, periodontal ligament, gingival fibroblast cells and Wharton's jelly mesenchymal stromal cells was studied. Removal of porcine parvovirus (PPV) and of the 263K prion strain of hamster-adapted scrapie was studied by spiking experiments following international guidelines. QSD had minimal impact on HPL-supplemented medium composition in proteins, growth factors and chemical content, nor capacity to expand and differentiate cells. In addition, QSD could remove ≥5.58 log10 [TCID50/mL] and ≥3.72 log10 of PPV and the 263K prion, respectively. QSD hollow fiber chromatography can be used to improve the virus and prion safety of HPL-supplemented media to safely expand cells for clinical protocols. These data bring new perspectives for increasingly safer use of pooled HPL in cell therapy and regenerative medicine applications. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  9. Evaluation of the limulus amoebocyte lysate test in conjunction with a gram negative bacterial plate count for detecting irradiation of chicken

    Science.gov (United States)

    Scotter, Susan L.; Wood, Roger; McWeeny, David J.

    A study to evaluate the potential of the Limulus amoebocyte lysate (LAL) test in conjuction with a Gram negative bacteria (GNB) plate count for detecting the irradiation of chicken is described. Preliminary studies demonstrated that chickens irradiated at an absorbed dose of 2.5 kGy could be differentiated from unirradiated birds by measuring levels of endotoxin and of numbers of GNB on chicken skin. Irradiated birds were found to have endotoxin levels similar to those found in unirradiated birds but significantly lower numbers of GNB. In a limited study the test was found to be applicable to birds from different processors. The effect of temperature abuse on the microbiological profile, and thus the efficacy of the test, was also investigated. After temperature abuse, the irradiated birds were identifiable at worst up to 3 days after irradiation treatment at the 2.5 kGy level and at best some 13 days after irradiation. Temperature abuse at 15°C resulted in rapid recovery of surviving micro-organisms which made differentiation of irradiated and unirradiated birds using this test unreliable. The microbiological quality of the bird prior to irradiation treatment also affected the test as large numbers of GNB present on the bird prior to irradiation treatment resulted in larger numbers of survivors. In addition, monitoring the developing flora after irradiation treatment and during subsequent chilled storage also aided differentiation of irradiated and unirradiated birds. Large numbers of yeasts and Gram positive cocci were isolated from irradiated carcasses whereas Gram negative oxidative rods were the predominant spoilage flora on unirradiated birds.

  10. Evaluation of the Limulus amoebocyte lysate test in conjunction with a gram negative bacterial plate count for detecting irradiation of chicken

    International Nuclear Information System (INIS)

    Scotter, S.L.; Wood, R.; McWeeny, D.J.

    1990-01-01

    A study to evaluate the potential of the Limulus amoebocyte lysate (LAL) test in conjunction with a Gram negative bacterial (GNB) plate count for detecting the irradiation of chicken is described. Preliminary studies demonstrated that chickens irradiated at an absorbed dose of 2.5 kGy could be differentiated from unirradiated birds by measuring levels of endotoxin and of numbers of GNB on chicken skin. Irradiated birds were found to have endotoxin levels similar to those found in unirradiated birds but significantly lower numbers of GNB. In a limited study the test was found to be applicable to birds from different processors. The effect of temperature abuse on the microbiological profile, and thus the efficacy of the test, was also investigated. After temperature abuse, the irradiated birds were identifiable at worst up to 3 days after irradiation treatment at the 2.5 kGy level and at best some 13 days after irradiation. Temperature abuse at 15 0 C resulted in rapid recovery of surviving micro-organisms which made differentiation of irradiated and unirradiated birds using this test unreliable. The microbiological quality of the bird prior to irradiation treatment also affected the test as large numbers of GNB present on the bird prior to irradiation treatment resulted in larger numbers of survivors. In addition, monitoring the developing flora after irradiation treatment amd during subsequent chilled storage also aided differentiation of irradiated and unirradiated birds. Large numbers of yeast and Gram positive cocci were isolated from irradiated carcasses whereas Gram negative oxidative rods were the predominant spoilage flora on unirradiated birds. (author)

  11. Phase 1/2 study of immunotherapy with dendritic cells pulsed with autologous tumor lysate in patients with refractory bone and soft tissue sarcoma.

    Science.gov (United States)

    Miwa, Shinji; Nishida, Hideji; Tanzawa, Yoshikazu; Takeuchi, Akihiko; Hayashi, Katsuhiro; Yamamoto, Norio; Mizukoshi, Eishiro; Nakamoto, Yasunari; Kaneko, Shuichi; Tsuchiya, Hiroyuki

    2017-05-01

    There are limited options for the curative treatment of refractory bone and soft tissue sarcomas. The purpose of this phase 1/2 study was to assess the immunological and clinical effects of dendritic cells (DCs) pulsed with autologous tumor lysate (TL) in patients with advanced bone and soft tissue sarcomas. Thirty-seven patients with metastatic or recurrent sarcomas were enrolled in this study. Peripheral blood mononuclear cells obtained from the patients were suspended in media containing interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor. Subsequently, these cells were treated with TL, tumor necrosis factor α, and OK-432. The DCs were injected into the inguinal or axillary region. One treatment course comprised 6 weekly DC injections. The toxicity, clinical response (tumor volume, serum interferon-γ [IFN-γ], and serum IL-12), and oncological outcomes were observed. In total, 47 courses of DC therapy were performed in 37 patients. No severe adverse events or deaths associated with the DC injections were observed in the study patients. Increased serum IFN-γ and IL-12 levels were observed 1 month after the DC injection. Among the 37 patients, 35 patients were assessed for clinical responses: 28 patients showed tumor progression, 6 patients had stable disease, and 1 patient showed a partial response 8 weeks after the DC injection. The 3-year overall and progression-free survival rates of the patients were 42.3% and 2.9%, respectively. Although DC therapy appears safe and resulted in an immunological response in patients with refractory sarcoma, it resulted in an improvement of the clinical outcome in only a small number of patients. Cancer 2017;123:1576-1584. © 2017 American Cancer Society. © 2017 American Cancer Society.

  12. Evaluation of the Limulus amoebocyte lysate test in conjunction with a gram negative bacterial plate count for detecting irradiation of chicken

    Energy Technology Data Exchange (ETDEWEB)

    Scotter, S L; Wood, R; McWeeny, D J [Ministry of Agriculture, Fisheries and Food, Norwich (UK). Food Science Lab.

    1990-01-01

    A study to evaluate the potential of the Limulus amoebocyte lysate (LAL) test in conjunction with a Gram negative bacterial (GNB) plate count for detecting the irradiation of chicken is described. Preliminary studies demonstrated that chickens irradiated at an absorbed dose of 2.5 kGy could be differentiated from unirradiated birds by measuring levels of endotoxin and of numbers of GNB on chicken skin. Irradiated birds were found to have endotoxin levels similar to those found in unirradiated birds but significantly lower numbers of GNB. In a limited study the test was found to be applicable to birds from different processors. The effect of temperature abuse on the microbiological profile, and thus the efficacy of the test, was also investigated. After temperature abuse, the irradiated birds were identifiable at worst up to 3 days after irradiation treatment at the 2.5 kGy level and at best some 13 days after irradiation. Temperature abuse at 15{sup 0}C resulted in rapid recovery of surviving micro-organisms which made differentiation of irradiated and unirradiated birds using this test unreliable. The microbiological quality of the bird prior to irradiation treatment also affected the test as large numbers of GNB present on the bird prior to irradiation treatment resulted in larger numbers of survivors. In addition, monitoring the developing flora after irradiation treatment amd during subsequent chilled storage also aided differentiation of irradiated and unirradiated birds. Large numbers of yeast and Gram positive cocci were isolated from irradiated carcasses whereas Gram negative oxidative rods were the predominant spoilage flora on unirradiated birds. (author).

  13. Human platelet lysate successfully promotes proliferation and subsequent chondrogenic differentiation of adipose-derived stem cells: a comparison with articular chondrocytes.

    Science.gov (United States)

    Hildner, F; Eder, M J; Hofer, K; Aberl, J; Redl, H; van Griensven, M; Gabriel, C; Peterbauer-Scherb, A

    2015-07-01

    Fetal calf serum (FCS) bears a potential risk for carrying diseases and eliciting immune reactions. Nevertheless, it still represents the gold standard as medium supplement in cell culture. In the present study, human platelet lysate (PL) was tested as an alternative to FCS for the expansion and subsequent chondrogenic differentiation of human adipose-derived stem cells (ASCs). ASCs were expanded with 10% FCS (group F) or 5% PL (group P). Subsequently, three-dimensional (3D) micromass pellets were created and cultured for 5 weeks in chondrogenic differentiation medium. Additionally, the de- and redifferentiation potential of human articular chondrocytes (HACs) was evaluated and compared to ASCs. Both HACs and ASCs cultured with PL showed strongly enhanced proliferation rates. Redifferentiation of HACs was possible for cells expanded up to 3.3 population doublings (PD). At this stage, PL-expanded HACs demonstrated better redifferentiation potential than FCS-expanded cells. ASCs could also be differentiated following extended passaging. Glycosaminoglycan (GAG) quantification and qRT-PCR of 10 cartilage related markers demonstrated a tendency for increased chondrogenic differentiation of PL-expanded ASCs compared to cells expanded with FCS. Histologically, collagen type II but also collagen type X was mainly present in group P. The present study demonstrates that PL strongly induces proliferation of ASCs, while the chondrogenic differentiation potential is retained. HACs also showed enhanced proliferation and even better redifferentiation when previously expanded with PL. This suggests that PL is superior to FCS as a supplement for the expansion of ASCs and HACs, particularly with regard to chondrogenic (re)differentiation. Copyright © 2013 John Wiley & Sons, Ltd.

  14. Production of human platelet lysate by use of ultrasound for ex vivo expansion of human bone marrow-derived mesenchymal stromal cells.

    Science.gov (United States)

    Bernardi, Martina; Albiero, Elena; Alghisi, Alberta; Chieregato, Katia; Lievore, Chiara; Madeo, Domenico; Rodeghiero, Francesco; Astori, Giuseppe

    2013-08-01

    A medium supplemented with fetal bovine serum (FBS) is of common use for the expansion of human mesenchymal stromal cells (MSCs). However, its use is discouraged by regulatory authorities because of the risk of zoonoses and immune reactions. Human platelet lysate (PL) obtained by freezing/thawing disruption of platelets has been proposed as a possible substitute of FBS. The process is time-consuming and not well standardized. A new method for obtaining PL that is based on the use of ultrasound is proposed. Platelet sonication was performed by submerging platelet-containing plastic bags in an ultrasonic bath. To evaluate platelet lysis we measured platelet-derived growth factor-AB release. PL efficiency was tested by expanding bone marrow (BM)-MSCs, measuring population doubling time, differentiation capacity and immunogenic properties. Safety was evaluated by karyotyping expanded cells. After 30 minutes of sonication, 74% of platelet derived growth factor-AB was released. PL enhanced BM-MSC proliferation rate compared with FBS. The mean cumulative population doubling (cPD) of cells growth in PL at 10%, 7.5% and 5% was better compared with cPD obtained with 10% FBS. PD time (hours) of MSCs with PL obtained by sonication was shorter than for cPD with PL obtained by freezing/thawing (18.9 versus 17.4, P < 0.01). BM mononucleated cells expressed MSC markers and were able to differentiate into adipogenic, osteogenic and chondrogenic lineages. When BM-MSCs and T cells were co-cultured in close contact, immunosuppressive activity of BM-MSCs was maintained. Cell karyotype showed no genetic alterations. The proposed method for the production of PL by sonication could be a safe, efficient and fast substitute of FBS, without the potential risks of FBS. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  15. High-resolution molecular validation of self-renewal and spontaneous differentiation in adipose-tissue derived human mesenchymal stem cells cultured in human platelet lysate

    Science.gov (United States)

    Dudakovic, Amel Dudakovic; Camilleri, Emily; Riester, Scott M.; Lewallen, Eric A.; Kvasha, Sergiy; Chen, Xiaoyue; Radel, Darcie J.; Anderson, Jarett M.; Nair, Asha A.; Evans, Jared M.; Krych, Aaron J.; Smith, Jay; Deyle, David R.; Stein, Janet L.; Stein, Gary S.; Im, Hee-Jeong; Cool, Simon M.; Westendorf, Jennifer J.; Kakar, Sanjeev; Dietz, Allan B.; van Wijnen, Andre J.

    2014-01-01

    Improving the effectiveness of adipose-tissue derived human mesenchymal stromal/stem cells (AMSCs) for skeletal therapies requires a detailed characterization of mechanisms supporting cell proliferation and multi-potency. We investigated the molecular phenotype of AMSCs that were either actively proliferating in platelet lysate or in a basal non-proliferative state. Flow cytometry combined with high-throughput RNA sequencing (RNASeq) and RT-qPCR analyses validate that AMSCs express classic mesenchymal cell surface markers (e.g., CD44, CD73/NT5E, CD90/THY1 and CD105/ENG). Expression of CD90 is selectively elevated at confluence. Self-renewing AMSCs express a standard cell cycle program that successively mediates DNA replication, chromatin packaging, cyto-architectural enlargement and mitotic division. Confluent AMSCs preferentially express genes involved in extracellular matrix (ECM) formation and cellular communication. For example, cell cycle-related biomarkers (e.g., cyclins E2 and B2, transcription factor E2F1) and histone-related genes (e.g., H4, HINFP, NPAT) are elevated in proliferating AMSCs, while ECM genes are strongly upregulated (>10 fold) in quiescent AMSCs. AMSCs also express pluripotency genes (e.g., POU5F1, NANOG, KLF4) and early mesenchymal markers (e.g., NES, ACTA2) consistent with their multipotent phenotype. Strikingly, AMSCs modulate expression of WNT signaling components and switch production of WNT ligands (from WNT5A/WNT5B/WNT7B to WNT2/WNT2B), while up-regulating WNT-related genes (WISP2, SFRP2 and SFRP4). Furthermore, post-proliferative AMSCs spontaneously express fibroblastic, osteogenic, chondrogenic and adipogenic biomarkers when maintained in confluent cultures. Our findings validate the biological properties of self-renewing and multi-potent AMSCs by providing high-resolution quality control data that support their clinical versatility. PMID:24905804

  16. Development of large-scale manufacturing of adipose-derived stromal cells for clinical applications using bioreactors and human platelet lysate.

    Science.gov (United States)

    Haack-Sørensen, Mandana; Juhl, Morten; Follin, Bjarke; Harary Søndergaard, Rebekka; Kirchhoff, Maria; Kastrup, Jens; Ekblond, Annette

    2018-04-17

    In vitro expanded adipose-derived stromal cells (ASCs) are a useful resource for tissue regeneration. Translation of small-scale autologous cell production into a large-scale, allogeneic production process for clinical applications necessitates well-chosen raw materials and cell culture platform. We compare the use of clinical-grade human platelet lysate (hPL) and fetal bovine serum (FBS) as growth supplements for ASC expansion in the automated, closed hollow fibre quantum cell expansion system (bioreactor). Stromal vascular fractions were isolated from human subcutaneous abdominal fat. In average, 95 × 10 6 cells were suspended in 10% FBS or 5% hPL medium, and loaded into a bioreactor coated with cryoprecipitate. ASCs (P0) were harvested, and 30 × 10 6 ASCs were reloaded for continued expansion (P1). Feeding rate and time of harvest was guided by metabolic monitoring. Viability, sterility, purity, differentiation capacity, and genomic stability of ASCs P1 were determined. Cultivation of SVF in hPL medium for in average nine days, yielded 546 × 10 6 ASCs compared to 111 × 10 6 ASCs, after 17 days in FBS medium. ASCs P1 yields were in average 605 × 10 6 ASCs (PD [population doublings]: 4.65) after six days in hPL medium, compared to 119 × 10 6 ASCs (PD: 2.45) in FBS medium, after 21 days. ASCs fulfilled ISCT criteria and demonstrated genomic stability and sterility. The use of hPL as a growth supplement for ASCs expansion in the quantum cell expansion system provides an efficient expansion process compared to the use of FBS, while maintaining cell quality appropriate for clinical use. The described process is an obvious choice for manufacturing of large-scale allogeneic ASC products.

  17. Human platelet lysate is a feasible candidate to replace fetal calf serum as medium supplement for blood vascular and lymphatic endothelial cells.

    Science.gov (United States)

    Hofbauer, Pablo; Riedl, Sabrina; Witzeneder, Karin; Hildner, Florian; Wolbank, Susanne; Groeger, Marion; Gabriel, Christian; Redl, Heinz; Holnthoner, Wolfgang

    2014-09-01

    As angiogenic and lymphangiogenic key players, endothelial cells (ECs) are promising candidates for vascular regenerative therapies. To culture ECs in vitro, fetal calf serum (FCS) is most often used. However, some critical aspects of FCS usage, such as possible internalization of xenogeneic proteins and prions, must be considered. Therefore, the aim of this project was to determine if human platelet lysate (hPL) is a suitable alternative to FCS as medium supplement for the culture of blood vascular and lymphatic endothelial cells. The usability of hPL was tested by analysis of endothelial surface marker expression, metabolic activity and vasculogenic potential of outgrowth ECs (OECs), human umbilical vein ECs (HUVECs), and lymphatic ECs (LECs). Expression of EC markers CD31, VEGFR2, VE-cadherin and CD146 did not differ significantly between the EC types cultured in FCS or hPL. In addition, OECs, HUVECs and LECs formed tube-like structures on Matrigel when cultured in hPL and FCS. With the use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid assays, we found that the metabolic activity of OECs and LECs was slightly decreased when hPL was used. However, HUVECs and LECs did not show a significant decrease in metabolic activity, and HUVECs showed a slightly higher activity at low seeding densities. The use of hPL on different EC types did not reveal any substantial negative effects on EC behavior. Thus, hPL appears to be a favorable candidate to replace FCS as a medium supplement in the culture of ECs. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  18. Inactivated human platelet lysate with psoralen: a new perspective for mesenchymal stromal cell production in Good Manufacturing Practice conditions.

    Science.gov (United States)

    Castiglia, Sara; Mareschi, Katia; Labanca, Luciana; Lucania, Graziella; Leone, Marco; Sanavio, Fiorella; Castello, Laura; Rustichelli, Deborah; Signorino, Elena; Gunetti, Monica; Bergallo, Massimiliano; Bordiga, Anna Maria; Ferrero, Ivana; Fagioli, Franca

    2014-06-01

    Mesenchymal stromal cells (MSC) are ideal candidates for regenerative and immunomodulatory therapies. The use of xenogeneic protein-free Good Manufacturing Practice-compliant growth media is a prerequisite for clinical MSC isolation and expansion. Human platelet lysate (HPL) has been efficiently implemented into MSC clinical manufacturing as a substitute for fetal bovine serum (FBS). Because the use of human-derived blood materials alleviates immunologic risks but not the transmission of blood-borne viruses, the aim of our study was to test an even safer alternative than HPL to FBS: HPL subjected to pathogen inactivation by psoralen (iHPL). Bone marrow samples were plated and expanded in α-minimum essential medium with 10% of three culture supplements: HPL, iHPL and FBS, at the same time. MSC morphology, growth and immunophenotype were analyzed at each passage. Karyotype, tumorigenicity and sterility were analyzed at the third passage. Statistical analyses were performed. The MSCs cultivated in the three different culture conditions showed no significant differences in terms of fibroblast colony-forming unit number, immunophenotype or in their multipotent capacity. Conversely, the HPL/iHPL-MSCs were smaller, more numerous, had a higher proliferative potential and showed a higher Oct-3/4 and NANOG protein expression than did FBS-MSCs. Although HPL/iHPL-MSCs exhibit characteristics that may be attributable to a higher primitive stemness than FBS-MSCs, no tumorigenic mutations or karyotype modifications were observed. We demonstrated that iHPL is safer than HPL and represents a good, Good Manufacturing Practice-compliant alternative to FBS for MSC clinical production that is even more advantageous in terms of cellular growth and stemness. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  19. Neutron rich nuclei around 132Sn

    International Nuclear Information System (INIS)

    Bhattacharya, Sarmishtha

    2016-01-01

    The neutron rich nuclei with few particles or holes in 132 Sn have various experimental and theoretical interest to understand the evolution of nuclear structure around the doubly magic shell closure Z=50 and N=82. Some of the exotic neutron rich nuclei in this mass region are situated near waiting points in the r-process path and are of special astrophysical interest. Neutron rich nuclei near 132 Sn have been studied using fission fragment spectroscopy. The lifetime of low lying isomeric states have been precisely measured and the beta decay from the ground and isomeric states have been characterized using gamma-ray spectroscopy

  20. Firm size diversity, functional richness, and resilience

    Science.gov (United States)

    Garmestani, A.S.; Allen, Craig R.; Mittelstaedt, J.D.; Stow, C.A.; Ward, W.A.

    2006-01-01

    This paper applies recent advances in ecology to our understanding of firm development, sustainability, and economic development. The ecological literature indicates that the greater the functional richness of species in a system, the greater its resilience - that is, its ability to persist in the face of substantial changes in the environment. This paper focuses on the effects of functional richness across firm size on the ability of industries to survive in the face of economic change. Our results indicate that industries with a richness of industrial functions are more resilient to employment volatility. ?? 2006 Cambridge University Press.

  1. Growth factor and pro-inflammatory cytokine contents in platelet-rich plasma (PRP), plasma rich in growth factors (PRGF), advanced platelet-rich fibrin (A-PRF), and concentrated growth factors (CGF).

    Science.gov (United States)

    Masuki, Hideo; Okudera, Toshimitsu; Watanebe, Taisuke; Suzuki, Masashi; Nishiyama, Kazuhiko; Okudera, Hajime; Nakata, Koh; Uematsu, Kohya; Su, Chen-Yao; Kawase, Tomoyuki

    2016-12-01

    The development of platelet-rich fibrin (PRF) drastically simplified the preparation procedure of platelet-concentrated biomaterials, such as platelet-rich plasma (PRP), and facilitated their clinical application. PRF's clinical effectiveness has often been demonstrated in pre-clinical and clinical studies; however, it is still controversial whether growth factors are significantly concentrated in PRF preparations to facilitate wound healing and tissue regeneration. To address this matter, we performed a comparative study of growth factor contents in PRP and its derivatives, such as advanced PRF (A-PRF) and concentrated growth factors (CGF). PRP and its derivatives were prepared from the same peripheral blood samples collected from healthy donors. A-PRF and CGF preparations were homogenized and centrifuged to produce extracts. Platelet and white blood cell counts in A-PRF and CGF preparations were determined by subtracting those counts in red blood cell fractions, supernatant acellular serum fractions, and A-PRF/CGF exudate fractions from those counts of whole blood samples. Concentrations of growth factors (TGF-β1, PDGF-BB, VEGF) and pro-inflammatory cytokines (IL-1β, IL-6) were determined using ELISA kits. Compared to PRP preparations, both A-PRF and CGF extracts contained compatible or higher levels of platelets and platelet-derived growth factors. In a cell proliferation assay, both A-PRF and CGF extracts significantly stimulated the proliferation of human periosteal cells without significant reduction at higher doses. These data clearly demonstrate that both A-PRF and CGF preparations contain significant amounts of growth factors capable of stimulating periosteal cell proliferation, suggesting that A-PRF and CGF preparations function not only as a scaffolding material but also as a reservoir to deliver certain growth factors at the site of application.

  2. Thermodynamics of neutron-rich nuclear matter

    Energy Technology Data Exchange (ETDEWEB)

    López, Jorge A., E-mail: jorgelopez@utep.edu [Department of Physics, University of Texas at El Paso, El Paso, Texas 79968, U.S.A (United States); Porras, Sergio Terrazas, E-mail: sterraza@uacj.mx; Gutiérrez, Araceli Rodríguez, E-mail: al104010@alumnos.uacj.mx [Universidad Autónoma de Ciudad Juárez, Ciudad Juárez, Chihuahua, México (Mexico)

    2016-07-07

    This manuscript presents methods to obtain properties of neutron-rich nuclear matter from classical molecular dynamics. Some of these are bulk properties of infinite nuclear matter, phase information, the Maxwell construction, spinodal lines and symmetry energy.

  3. Preparing for Surgery

    Science.gov (United States)

    ... Events Advocacy For Patients About ACOG Preparing for Surgery Home For Patients Search FAQs Preparing for Surgery ... Surgery FAQ080, August 2011 PDF Format Preparing for Surgery Gynecologic Problems What is the difference between outpatient ...

  4. Leveraging data rich environments using marketing analytics

    OpenAIRE

    Holtrop, Niels

    2017-01-01

    With the onset of what is popularly known as “big data”, increased attention is being paid to creating value from these data rich environments. Within the field of marketing, the analysis of customer and market data supported by models is known as marketing analytics. The goal of these analyses is to enhance managerial decision making regarding marketing problems. However, before these data rich environments can be used to guide managerial decision making, firms need to grasp the process of d...

  5. The Role of a Platelet Lysate-Based Compartmentalized System as a Carrier of Cells and Platelet-Origin Cytokines for Periodontal Tissue Regeneration.

    Science.gov (United States)

    Babo, Pedro S; Cai, Xinjie; Plachokova, Adelina S; Reis, Rui L; Jansen, John A; Gomes, Manuela E; Walboomers, X Frank

    2016-10-01

    Currently available clinical therapies are not capable to regenerate tissues that are lost by periodontitis. Tissue engineering can be applied as a strategy to regenerate reliably the tissues and function of damaged periodontium. A prerequisite for this regeneration is the colonization of the defect with the adequate cell populations. In this study, we proposed a bilayered system composed of (1) a platelet lysate (PL)-based construct produced by crosslinking of PL proteins with genipin (gPL) for the delivery of rat periodontal ligament cells (rat-PDLCs); combined with (2) an injectable composite consisting of calcium phosphate cement incorporated with PL-loaded poly(d, l-lactic-co-glycolic acid) microspheres. This system was expected to promote periodontal regeneration by the delivery of adequate progenitor cells and providing a stable system enriched with adequate cytokines and growth factors for the orchestration of tissue regrowth in periodontal defects. The bilayered system was tested in a three-wall intrabony defect in rats and the healing of periodontal tissue was assessed 6 weeks after surgery. Results showed that the bilayered system was able to promote the regrowth of functional periodontal tissues, both with (cells + gPL) and without the loading of PDLCs (gPL). Significant connective tissue attachment (45.0 ± 15.0% and 64.0 ± 15.0% for gPL and cells + gPL group, respectively) and new bone area (33.8 ± 21% and 21.3 ± 3% for gPL and cells + gPL group, respectively) were observed. Nevertheless, rat PDLCs delivered with gPL construct in the defect area were hardly visible 6 weeks after surgery and did not contribute for the regeneration of new periodontal tissue. Overall, our findings show that the bilayered system promotes the stabilization of PL proteins on the root surface and has a positive effect in the repair of periodontal tissues both in quality and in quantity.

  6. LITHIUM-RICH GIANTS IN GLOBULAR CLUSTERS

    Energy Technology Data Exchange (ETDEWEB)

    Kirby, Evan N.; Cohen, Judith G. [California Institute of Technology, 1200 E. California Boulevard, MC 249-17, Pasadena, CA 91125 (United States); Guhathakurta, Puragra [UCO/Lick Observatory and Department of Astronomy and Astrophysics, University of California, 1156 High Street, Santa Cruz, CA 95064 (United States); Zhang, Andrew J. [The Harker School, 500 Saratoga Avenue, San Jose, CA 95129 (United States); Hong, Jerry [Palo Alto High School, 50 Embarcadero Road, Palo Alto, CA, 94301 (United States); Guo, Michelle [Stanford University, 450 Serra Mall, Stanford, CA 94305 (United States); Guo, Rachel [Irvington High School, 41800 Blacow Road, Fremont, CA 94538 (United States); Cunha, Katia [Observatório Nacional, São Cristóvão Rio de Janeiro (Brazil)

    2016-03-10

    Although red giants deplete lithium on their surfaces, some giants are Li-rich. Intermediate-mass asymptotic giant branch (AGB) stars can generate Li through the Cameron–Fowler conveyor, but the existence of Li-rich, low-mass red giant branch (RGB) stars is puzzling. Globular clusters are the best sites to examine this phenomenon because it is straightforward to determine membership in the cluster and to identify the evolutionary state of each star. In 72 hours of Keck/DEIMOS exposures in 25 clusters, we found four Li-rich RGB and two Li-rich AGB stars. There were 1696 RGB and 125 AGB stars with measurements or upper limits consistent with normal abundances of Li. Hence, the frequency of Li-richness in globular clusters is (0.2 ± 0.1)% for the RGB, (1.6 ± 1.1)% for the AGB, and (0.3 ± 0.1)% for all giants. Because the Li-rich RGB stars are on the lower RGB, Li self-generation mechanisms proposed to occur at the luminosity function bump or He core flash cannot explain these four lower RGB stars. We propose the following origin for Li enrichment: (1) All luminous giants experience a brief phase of Li enrichment at the He core flash. (2) All post-RGB stars with binary companions on the lower RGB will engage in mass transfer. This scenario predicts that 0.1% of lower RGB stars will appear Li-rich due to mass transfer from a recently Li-enhanced companion. This frequency is at the lower end of our confidence interval.

  7. The choice of the conditions to receive the electrolytic zinc powders for metal-rich compositions

    Directory of Open Access Journals (Sweden)

    A. V. Patrushev

    2015-03-01

    Full Text Available In the work a method of obtaining highly dispersed zinc powders by electrolysis and comparison of the properties of zinc-rich compositions prepared using as a pigment zinc powders obtained by different methods is provided. Conducted measurements have shown that the electrical conductivity of zinc-rich coatings comprising electrolytic zinc powder does not inferior to the conductivity of the film with PZHD-0 powder obtained by the  evaporation-condensation method, despite the significant difference in the amount of zinc pigment. On the basis of the received data one can conclude that the use of electrolytic zinc powder as a pigment will significantly save zinc.

  8. Basic characteristics of plasma rich in growth factors (PRGF): blood cell components and biological effects.

    Science.gov (United States)

    Nishiyama, Kazuhiko; Okudera, Toshimitsu; Watanabe, Taisuke; Isobe, Kazushige; Suzuki, Masashi; Masuki, Hideo; Okudera, Hajime; Uematsu, Kohya; Nakata, Koh; Kawase, Tomoyuki

    2016-11-01

    Platelet-rich plasma (PRP) is widely used in regenerative medicine because of its high concentrations of various growth factors and platelets. However, the distribution of blood cell components has not been investigated in either PRP or other PRP derivatives. In this study, we focused on plasma rich in growth factors (PRGF), a PRP derivative, and analyzed the distributions of platelets and white blood cells (WBCs). Peripheral blood samples were collected from healthy volunteers ( N  = 14) and centrifuged to prepare PRGF and PRP. Blood cells were counted using an automated hematology analyzer. The effects of PRP and PRGF preparations on cell proliferation were determined using human periosteal cells. In the PRGF preparations, both red blood cells and WBCs were almost completely eliminated, and platelets were concentrated by 2.84-fold, whereas in the PRP preparations, both platelets and WBCs were similarly concentrated by 8.79- and 5.51-fold, respectively. Platelet counts in the PRGF preparations were positively correlated with platelet counts in the whole blood samples, while the platelet concentration rate was negatively correlated with red blood cell counts in the whole blood samples. In contrast, platelet counts and concentration rates in the PRP preparations were significantly influenced by WBC counts in whole blood samples. The PRP preparations, but not the PRGF preparations, significantly suppressed cell growth at higher doses in vitro. Therefore, these results suggest that PRGF preparations can clearly be distinguished from PRP preparations by both inclusion of WBCs and dose-dependent stimulation of periosteal cell proliferation in vitro.

  9. Electrocatalysts Prepared by Galvanic Replacement

    Directory of Open Access Journals (Sweden)

    Athanasios Papaderakis

    2017-03-01

    Full Text Available Galvanic replacement is the spontaneous replacement of surface layers of a metal, M, by a more noble metal, Mnoble, when the former is treated with a solution containing the latter in ionic form, according to the general replacement reaction: nM + mMnoblen+ → nMm+ + mMnoble. The reaction is driven by the difference in the equilibrium potential of the two metal/metal ion redox couples and, to avoid parasitic cathodic processes such as oxygen reduction and (in some cases hydrogen evolution too, both oxygen levels and the pH must be optimized. The resulting bimetallic material can in principle have a Mnoble-rich shell and M-rich core (denoted as Mnoble(M leading to a possible decrease in noble metal loading and the modification of its properties by the underlying metal M. This paper reviews a number of bimetallic or ternary electrocatalytic materials prepared by galvanic replacement for fuel cell, electrolysis and electrosynthesis reactions. These include oxygen reduction, methanol, formic acid and ethanol oxidation, hydrogen evolution and oxidation, oxygen evolution, borohydride oxidation, and halide reduction. Methods for depositing the precursor metal M on the support material (electrodeposition, electroless deposition, photodeposition as well as the various options for the support are also reviewed.

  10. Modeling Aquatic Macroinvertebrate Richness Using Landscape Attributes

    Directory of Open Access Journals (Sweden)

    Marcia S. Meixler

    2015-01-01

    Full Text Available We used a rapid, repeatable, and inexpensive geographic information system (GIS approach to predict aquatic macroinvertebrate family richness using the landscape attributes stream gradient, riparian forest cover, and water quality. Stream segments in the Allegheny River basin were classified into eight habitat classes using these three landscape attributes. Biological databases linking macroinvertebrate families with habitat classes were developed using life habits, feeding guilds, and water quality preferences and tolerances for each family. The biological databases provided a link between fauna and habitat enabling estimation of family composition in each habitat class and hence richness predictions for each stream segment. No difference was detected between field collected and modeled predictions of macroinvertebrate families in a paired t-test. Further, predicted stream gradient, riparian forest cover, and total phosphorus, total nitrogen, and suspended sediment classifications matched observed classifications much more often than by chance alone. High gradient streams with forested riparian zones and good water quality were predicted to have the greatest macroinvertebrate family richness and changes in water quality were predicted to have the greatest impact on richness. Our findings indicate that our model can provide meaningful landscape scale macroinvertebrate family richness predictions from widely available data for use in focusing conservation planning efforts.

  11. Species richness, area and climate correlates

    DEFF Research Database (Denmark)

    Nogues, David Bravo; Bastos Araujo, Miguel

    2006-01-01

    affects: (1) the selection of climate variables entering a species richness model; and (2) the accuracy of models in predicting species richness in unsampled grid cells. Location Western Europe. Methods Models are developed for European plant, breeding bird, mammal and herptile species richness using...... seven climate variables. Generalized additive models are used to relate species richness, climate and area. Results We found that variation in the grid cell area was large (50 × 50 km: 8-3311 km2; 220 × 220: 193-55,100 km2), but this did not affect the selection of variables in the models. Similarly...... support the assumption that variation in near-equal area cells may be of second-order importance for models explaining or predicting species richness in relation to climate, although there is a possibility that drops in accuracy might increase with grid cell size. The results are, however, contingent...

  12. Cultivation and irradiation of human fibroblasts in a medium enriched with platelet lysate for obtaining feeder layer in epidermal cell culture; Cultivo e irradiacao de fibroblastos humanos em meio enriquecido com lisado de plaquetas para obtencao de camada de sustentacao em culturas de celulas da epiderme

    Energy Technology Data Exchange (ETDEWEB)

    Yoshito, Daniele

    2011-07-01

    For over 30 years, the use of culture medium, enriched with bovine serum, and murines fibroblasts, with the rate of proliferation controlled by irradiation or by share anticarcinogenic drugs, has been playing successfully its role in assisting in the development of keratinocytes in culture, for clinical purposes. However, currently there is a growing concern about the possibility of transmitting prions and animals viruses to transplanted patients. Taking into account this concern, the present work aims to cultivate human fibroblasts in a medium enriched with human platelets lysate and determine the irradiation dose of these cells, for obtaining feeder layer in epidermal cell culture. For carrying out the proposed objective, platelets lysis has standardized, this lysate was used for human fibroblasts cultivation and the irradiation dose enough to inhibit its duplication was evaluated. Human keratinocytes were cultivated in these feeder layers, in culture medium enriched with the lysate. With these results we conclude that the 10% platelets lysate promoted a better adhesion and proliferation of human fibroblasts and in all dose levels tested (60 to 300 Gy), these had their mitotic activity inactivated by ionizing irradiation, being that the feeder layers obtained with doses from 70 to 150 Gy were those that provided the best development of keratinocytes in medium containing 2.5% of human platelet lysate. Therefore, it was possible to standardize both the cultivation of human fibroblasts as its inactivation for use as feeder layer in culture of keratinocytes, so as to eliminate xenobiotics components. (author)

  13. Aluminum-rich mesoporous MFI - type zeolite single crystals

    DEFF Research Database (Denmark)

    Kustova, Marina; Kustov, Arkadii; Christensen, Christina Hviid

    2005-01-01

    Zeolitcs are crystalline materials, which are widely used as solid acid catalysts and supports in many industrial processes. Recently, mesoporous MFI-type zeolite single crystals were synthesized by use of carbon particles as a mesopore template and sodium aluminate as the aluminum Source....... With this technique, only zeolites with relatively low Al contents were reported (Si/Al ratio about 100). In this work, the preparation of aluminum-rich mesoporous MFI-type zeolite single crystals (Si/Al similar to 16-50) using aluminum isopropoxide as the aluminum Source is reported for the first time. All samples...... are characterized by X-ray powder diffraction (XRD), scanning electron microscopy (SEM), ammonia temperature programmed desorption (NH3-TPD), and N-2 adsorption measurements. The obtained zeolites combine the high crystallinity and the characteristic micropores of zeolites with an intracrystalline mesopore system...

  14. Structure of Light Neutron-rich Nuclei

    International Nuclear Information System (INIS)

    Dlouhy, Zdenek

    2007-01-01

    In this contribution we searched for irregularities in various separation energies in the frame of mass measurement of neutron-rich nuclei at GANIL. On this basis we can summarize that the new doubly magic nuclei are 8 He, 22 O and 24 O. They are characterized by extra stability and, except 24 O, they cannot accept and bind additional neutrons. However, if we add to these nuclei a proton we obtain 9 Li and 25 F which are the core for two-neutron halo nucleus 11 Li and enables that fluorine can bound even 6 more neutrons, respectively. In that aspect the doubly magic nuclei in the neutron-rich region can form the basis either for neutron halo or very neutron-rich nuclei. (Author)

  15. Origin of the latitudinal richness gradient

    DEFF Research Database (Denmark)

    Engemann, Kristine; Sandel, Brody Steven; Enquist, Brian J.

    2015-01-01

    Spatial variation in richness patterns must be due to variation in rates of speciation, extinction, immigration and emigration. Hotspots of diversity can occur either because they are hotspots of speciation (cradles) or cold spots of extinction (museums) – two major hypotheses that make contrasting...... predictions for the phylogenetic structure of communities. We test these hypotheses by comparing centers of species richness and phylogenetic clustering for vascular plants in the New World. Range maps for 88,417 plant species were extracted from the Botanical Information and Ecology Network (BIEN) database...... and combined with the BIEN mega phylogeny of >80,000 species. We calculated the Phylogenetic Diversity Index (PDI) and Net Relatedness Index (NRI) for each cell in a 100×100 km grid using a new computationally efficient algorithm. Species richness patterns were compared to patterns of PDI and NRI. We found...

  16. Platelet-Rich Plasma Increases Pigmentation.

    Science.gov (United States)

    Uysal, Cagri A; Ertas, Nilgun Markal

    2017-11-01

    Platelet-rich plasma (PRP) is an autologous solution of plasma containing 4 to 7 times the baseline concentration of human platelets. Platelet-rich plasma has been widely popular in facial rejuvenation to attenuate wrinkles and has been practically used. The authors have been encountering various patients of increased hiperpigmentation following PRP applications that were performed to attenuate the postinflammatory hiperpigmentation especially after laser treatment. The authors have been using PRP for facial rejuvenation in selected patients and in 1 patient the authors have encountered increased pigmentation over the pigmented skin lesions that were present before the application. The authors recommend that the PRP might increase pigmentation especially in the face region and precautions might be taken before and after the application. Platelet-rich plasma should not be used for the treatment of post inflammatory hiperpigmentation.

  17. Fast Photon Detection for COMPASS RICH1

    CERN Document Server

    Abbon, P; Angerer, H; Apollonio, M; Birsa, R; Bordalo, P; Bradamante, F; Bressan, A; Busso, L; Chiosso, M; Ciliberti, P; Colantoni, M L; Costa, S; Dibiase, N; Dafni, T; Dalla Torre, S; Diaz, V; Duic, v; Delagnes, E; Deschamps, H; Eyrich, W; Faso, D; Ferrero, A; Finger, M; Finger, M Jr; Fischer, H; Gerassimov, S; Giorgi, M; Gobbo, B; Hagemann, R; Von Harrach, D; Heinsius, F H; Joosten, R; Ketzer, B; Königsmann, K; Kolosov, V N; Konorov, I; Kramer, D; Kunne, F; Levorato, S; Maggiora, A; Magnon, A; Mann, A; Martin, A; Menon, G; Mutter, A; Nähle, O; Neyret, D; Nerling, F; Pagano, P; Paul, S; Panebianco, S; Panzieri, D; Pesaro, G; Pizzolotto, C; Polak, J; Rebourgeard, P; Rocco, E; Robinet, F; Schiavon, P; Schill, C; Schoenmeier, P; Silva, L; Slunecka, M; Steiger, L; Sozzi, F; Sulc, M; Svec, M; Tessarotto, F; Teufel, A; Wollny, H

    2006-01-01

    The new photon detection system for COMPASS RICH-1 has been designed to cope with the demanding requests of operation at high beam intensity and at high trigger rates. The detection technique in the central region of RICH-1 has been changed with a system based on multianode photomultipliers coupled to individual fused silica lens telescopes and to a fast, almost dead time free readout system based on the MAD-4 amplifier-discriminator and the F1 TDC-chip. The new photon detection system design and construction are described, as well as its first response in the experiment.

  18. Hamman-Rich syndrome in a goldsmith

    International Nuclear Information System (INIS)

    Kirchner, J.; Stein, A.; Jacobi, V.; Viel, K.

    1997-01-01

    We report the case of a 54-year-old goldsmith admitted because of dyspnea on exertion, persistent cough, and weakness under the suspicion of exogenous allergic alveolitis. He rapidly developed progressive lung fibrosis with exitus letalis 7 weeks after admission. Radiological examination (chest X-ray and HRCT) first showed ground glass opacities, and later rapid development of severe interstitial pattern with architectural distraction. The findings were similar to idiopathic lung fibrosis; however, the rare Hamman-Rich syndrome was confirmed by progressive course of the disease. Correlations between Hamann-Rich syndrome and idiopathic lung fibrosis are discussed. (orig.) [de

  19. Application of platelet-rich plasma and platelet-rich fibrin in fat grafting: basic science and literature review.

    Science.gov (United States)

    Liao, Han-Tsung; Marra, Kacey G; Rubin, J Peter

    2014-08-01

    Due to the natural properties of fat, fat grafting remains a popular procedure for soft tissue volume augmentation and reconstruction. However, clinical outcome varies and is technique dependent. Platelet-rich plasma (PRP) contains α-granules, from which multiple growth factors such as platelet-derived growth factor, transforming growth factor-β, vascular endothelial growth factor, and epidermal growth factor can be released after activation. In recent years, the scope of PRP therapies has extended from bone regeneration, wound healing, and healing of musculoskeletal injuries, to enhancement of fat graft survival. In this review, we focus on the definition of PRP, the different PRP preparation and activation methods, and growth factor concentrations. In addition, we discuss possible mechanisms for the role of PRP in fat grafting by reviewing in vitro studies with adipose-derived stem cells, preadipocytes, and adipocytes, and preclinical and clinical research. We also review platelet-rich fibrin, a so-called second generation PRP, and its slow-releasing biology and effects on fat grafts compared to PRP in both animal and clinical research. Finally, we provide a general foundation on which to critically evaluate earlier studies, discuss the limitations of previous research, and direct plans for future experiments to improve the optimal effects of PRP in fat grafting.

  20. Effects of Plasma Rich in Growth Factors and Platelet-Rich Fibrin on Proliferation and Viability of Human Gingival Fibroblasts

    Directory of Open Access Journals (Sweden)

    Surena Vahabi

    2016-01-01

    Full Text Available Objectives: Platelet preparations are commonly used to enhance bone and soft tissue regeneration. Considering the existing controversies on the efficacy of platelet products for tissue regeneration, more in vitro studies are required. The aim of the present study was to compare the in vitro effects of plasma rich in growth factors (PRGF and platelet-rich fibrin (PRF on proliferation and viability of human gingival fibroblasts (HGFs.Materials and Methods: Anitua's PRGF and Choukran's PRF were prepared according to the standard protocols. After culture periods of 24, 48 and 72 hours, proliferation of HGFs was evaluated by the methyl thiazol tetrazolium assay. Statistical analysis was performed using one-way ANOVA followed by Tukey-Kramer’s multiple comparisons and P-values<0.05 were considered statistically significant.Results: PRGF treatment induced statistically significant (P<0.001 proliferation of HGF cells compared to the negative control (100% viability at 24, 48 and 72 hours in values of 123%±2.25%, 102%±2.8% and 101%±3.92%, respectively. The PRF membrane treatment of HGF cells had a statistically significant effect on cell proliferation (21%±1.73%, P<0.001 at 24 hours compared to the negative control. However, at 48 and 72 hours after treatment, PRF had a negative effect on HGF cell proliferation and caused 38% and 60% decrease in viability and proliferation compared to the negative control, respectively. The HGF cell proliferation was significantly higher in PRGF than in PRF group (P< 0.001.Conclusion: This study demonstrated that PRGF had a strong stimulatory effect on HGF cell viability and proliferation compared to PRF.

  1. Intratendon Delivery of Leukocyte-Poor Platelet-Rich Plasma Improves Healing Compared With Leukocyte-Rich Platelet-Rich Plasma in a Rabbit Achilles Tendinopathy Model.

    Science.gov (United States)

    Yan, Ruijian; Gu, Yanjia; Ran, Jisheng; Hu, Yejun; Zheng, Zefeng; Zeng, Mengfeng; Heng, Boon Chin; Chen, Xiao; Yin, Zi; Chen, Weishan; Shen, Weiliang; Ouyang, Hongwei

    2017-07-01

    Chronic tendinopathy is a commonly occurring clinical problem that affects both athletes and inactive middle-aged patients. Although some studies have shown that different platelet-rich plasma (PRP) preparations could exert various therapeutic effects in vitro, the role of leukocytes in PRP has not yet been defined under tendinopathy conditions in vivo. This study compared the effects of the intratendon delivery of leukocyte-poor PRP (Lp-PRP) versus leukocyte-rich PRP (Lr-PRP) in a rabbit chronic tendinopathy model in vivo. Controlled laboratory study. Four weeks after a local injection of collagenase in the Achilles tendon, the following treatments were randomly administered on the lesions: injections of (1) 200 μL of Lp-PRP (n = 8), (2) 200 μL of Lr-PRP (n = 8), or (3) 200 μL of saline (n = 8). Healing outcomes were assessed at 4 weeks after therapy with magnetic resonance imaging (MRI), cytokine quantification, real-time polymerase chain reaction analysis of gene expression, histology, and transmission electron microscopy (TEM). MRI revealed that the Lr-PRP and saline groups displayed higher signal intensities compared with the Lp-PRP group with T2 mapping. Histologically, the Lp-PRP group displayed significantly better general scores compared with the Lr-PRP ( P = .001) and saline ( P tendon healing and is a preferable option for the clinical treatment of tendinopathy. PRP is widely used in the clinical management of chronic tendinopathy. However, the clinical results are ambiguous. It is imperative to understand the influence of leukocytes on PRP-mediated tissue healing in vivo, which could facilitate the better clinical management of chronic tendinopathy. Further studies are needed to translate our findings to the clinical setting.

  2. Platlet Rich Plasma (PRP) Improves Fat Grafting Outcomes.

    Science.gov (United States)

    Modarressi, Ali

    2013-01-01

    Autologous fat transfer offers many qualities of a ideal soft tissue filler. Main advantages of fat grafting ensue from the fact that the lipoaspirate tissue is an abundant source of regenerative pluripotential cells. However, the reported rates of fat cell survival vary greatly in the medical literature (10-90%). Different techniques of harvesting, processing, and reinjecting the fat cells are so claimed to be responsible for these differences, without any agreement concerning the best way to process. To address this important disadvantage, we propose the addition of autologous platelet rich plasma (PRP) which is known as a natural reservoir of growth factors stimulating tissue repair and regeneration. This approach is completely autologous and immediately employed without any type of preconditioning. Platelets rich plasma (PRP) preparation included bleeding of 8 ml of blood from patient's peripheral vein in Regen Lab© tubes containing sodium citrate anticoagulant. The whole blood was centrifugated at 1500 g during 3 min. As Regen-tubes contained a special gel separator, 99 % of red blood cells were discarded from the plasma at the bottom of the gel, and >90% of platelets were harvested in 4 ml of plasma on the top of the gel, called the platelet-rich plasma (PRP). The purified fat prepared by Coleman technique was mixed with different amount of PRP for in vitro, in vivo (mice) and clinical experiments: >50% of PRP for skin rejuvenation, superficial scars correction, infraorbital region, ..., and for 20% of PRP with 80% of purified fat for deep filler indication (nasolabial folds, lips, or soft tissue defect). In vitro studies demonstrated that PRP increased fat cells survival rate and stem cells differentiation. Animal models showed that fat graft survival rate was significantly increased by addition of PRP. Several clinical cases confirmed the improvement of wound healing and fat grafting survival in facial reconstruction and aesthetic cases by association of

  3. Electrochemical study of lithium insertion into carbon-rich polymer-derived silicon carbonitride ceramics

    International Nuclear Information System (INIS)

    Kaspar, Jan; Mera, Gabriela; Nowak, Andrzej P.; Graczyk-Zajac, Magdalena; Riedel, Ralf

    2010-01-01

    This paper presents the lithium insertion into carbon-rich polymer-derived silicon carbonitride (SiCN) ceramic synthesized by the thermal treatment of poly(diphenylsilylcarbodiimide) at three temperatures, namely 1100, 1300, and 1700 o C under 0.1 MPa Ar atmosphere. At lower synthesis temperatures, the material is X-ray amorphous, while at 1700 o C, the SiCN ceramic partially crystallizes. Anode materials prepared from these carbon-rich SiCN ceramics without any fillers and conducting additives were characterized using cyclic voltammetry and chronopotentiometric charging/discharging. We found that the studied silicon carbonitride ceramics demonstrate a promising electrochemical behavior during lithium insertion/extraction in terms of capacity and cycling stability. The sample synthesized at 1300 o C exhibits a reversible capacity of 392 mAh g -1 . Our study confirms that carbon-rich SiCN phases are electrochemically active materials in terms of Li inter- and deintercalation.

  4. Platelet-rich plasma and chronic wounds: remaining fibronectin may influence matrix remodeling and regeneration success.

    Science.gov (United States)

    Moroz, Andrei; Deffune, Elenice

    2013-11-01

    Platelet-rich plasma has been largely used as a therapeutic option for the treatment of chronic wounds of different etiologies. The enhanced regeneration observed after the use of platelet-rich plasma has been systematically attributed to the growth factors that are present inside platelets' granules. We hypothesize that the remaining plasma and platelet-bound fibronectin may act as a further bioactive protein in platelet-rich plasma preparations. Recent reports were analyzed and presented as direct evidences of this hypotheses. Fibronectin may directly influence the extracellular matrix remodeling during wound repair. This effect is probably through matrix metalloproteinase expression, thus exerting an extra effect on chronic wound regeneration. Physicians should be well aware of the possible fibronectin-induced effects in their future endeavors with PRP in chronic wound treatment. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  5. Strontium-rich injectable hybrid system for bone regeneration

    Energy Technology Data Exchange (ETDEWEB)

    Neves, Nuno, E-mail: nsmneves@gmail.com [Instituto de Investigação e Inovação em Saúde, Universidade do Porto (Portugal); INEB — Instituto de Engenharia Biomédica, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto (Portugal); FMUP — Faculdade de Medicina da Universidade do Porto, Departamento de Cirurgia, Serviço de Ortopedia, Alameda Prof. Hernâni Monteiro, 4200-319 Porto (Portugal); Campos, Bruno B. [FCUP — Faculdade de Ciências da Universidade do Porto, Centro de Investigação em Química, Departamento de Química e Bioquímica, Rua do Campo Alegre 1021/1055, 4169-007 Porto (Portugal); Almeida, Isabel F.; Costa, Paulo C. [FFUP — Faculdade de Farmácia da Universidade do Porto, Laboratório de Tecnologia Farmacêutica, Departamento de Ciências do Medicamento, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto (Portugal); Cabral, Abel Trigo [FMUP — Faculdade de Medicina da Universidade do Porto, Departamento de Cirurgia, Serviço de Ortopedia, Alameda Prof. Hernâni Monteiro, 4200-319 Porto (Portugal); and others

    2016-02-01

    Current challenges in the development of scaffolds for bone regeneration include the engineering of materials that can withstand normal dynamic physiological mechanical stresses exerted on the bone and provide a matrix capable of supporting cell migration and tissue ingrowth. The objective of the present work was to develop and characterize a hybrid polymer–ceramic injectable system that consists of an alginate matrix crosslinked in situ in the presence of strontium (Sr), incorporating a ceramic reinforcement in the form of Sr-rich microspheres. The incorporation of Sr in the microspheres and in the vehicle relies on the growing evidence that Sr has beneficial effects in bone remodeling and in the treatment of osteopenic disorders and osteoporosis. Sr-rich porous hydroxyapatite microspheres with a uniform size and a mean diameter of 555 μm were prepared, and their compression strength and friability tested. A 3.5% (w/v) ultrapure sodium alginate solution was used as the vehicle and its in situ gelation was promoted by the addition of calcium (Ca) or Sr carbonate and Glucone-δ-lactone. Gelation times varied with temperature and crosslinking agent, being slower for Sr than for Ca, but adequate for injection in both cases. Injectability was evaluated using a device employed in vertebroplasty surgical procedures, coupled to a texture analyzer in compression mode. Compositions with 35% w of microspheres presented the best compromise between injectability and compression strength of the system, the force required to extrude it being lower than 100 N. Micro CT analysis revealed a homogeneous distribution of the microspheres inside the vehicle, and a mean inter-microspheres space of 220 μm. DMA results showed that elastic behavior of the hybrid is dominant over the viscous one and that the higher storage modulus was obtained for the 3.5%Alg–35%Sr-HAp-Sr formulation. - Highlights: • We developed a Sr rich viscoelastic hybrid system (alginate matrix crosslinked in

  6. Experiments with neutron-rich isomeric beams

    International Nuclear Information System (INIS)

    Rykaczewski, K.; Lewitowicz, M.; Pfuetzner, M.

    1998-01-01

    A review of experimental results obtained on microsecond-isomeric states in neutron-rich nuclei produced in fragmentation reactions and studied with SISSI-Alpha-LISE3 spectrometer system at GANIL Caen is given. The perspectives of experiments based on secondary reactions with isomeric beams are presented

  7. Development of a Rich Picture editor

    DEFF Research Database (Denmark)

    Valente, Andrea; Marchetti, Emanuela

    2010-01-01

    rich picture practice into software proved difficult, therefore, we decided to follow a user-centered approach: design and implement a prototype with basic functionalities, then run a usability test with a few students and professionals. The feedback collected in the test validated our hypothesis circa...

  8. Leveraging data rich environments using marketing analytics

    NARCIS (Netherlands)

    Holtrop, Niels

    2017-01-01

    With the onset of what is popularly known as “big data”, increased attention is being paid to creating value from these data rich environments. Within the field of marketing, the analysis of customer and market data supported by models is known as marketing analytics. The goal of these analyses is

  9. Proximity focusing RICH with TOF capabilities

    International Nuclear Information System (INIS)

    Korpar, S.; Adachi, I.; Fujita, K.; Fukushima, T.; Gorisek, A.; Hayashi, D.; Iijima, T.; Ikado, T.; Ishikawa, T.; Kawai, H.; Kozakai, Y.; Krizan, P.; Kuratani, A.; Mazuka, Y.; Nakagawa, T.; Nishida, S.; Ogawa, S.; Pestotnik, R.; Seki, T.; Sumiyoshi, T.; Tabata, M.; Unno, Y.

    2007-01-01

    A proximity focusing RICH counter with a multi-channel micro-channel plate (MCP) PMT was tested as a time-of-flight counter. Cherenkov photons emitted in the radiator medium as well as in the entrance window of the PMT were used for the time-of-flight measurement, and an excellent performance of the counter could be demonstrated

  10. Probing luminescence centers in Na rich feldspar

    DEFF Research Database (Denmark)

    Prasad, Amit Kumar; Lapp, Torben; Kook, Myung Ho

    2016-01-01

    our understanding of the luminescence mechanisms and recombination sites, in a sample of Na rich plagioclase feldspar (oligoclase). Both the UV and violet–blue emissions show resonant excitations arising from a distribution of energy levels. We propose, contrary to the general understanding...

  11. Technology-Rich Schools Up Close

    Science.gov (United States)

    Levin, Barbara B.; Schrum, Lynne

    2013-01-01

    This article observes that schools that use technology well have key commonalities, including a project-based curriculum and supportive, distributed leadership. The authors' research into tech-rich schools revealed that schools used three strategies to integrate technology successfully. They did so by establishing the vision and culture,…

  12. Platelet-rich fibrin: the benefits.

    Science.gov (United States)

    Kumar, Yuvika Raj; Mohanty, Sujata; Verma, Mahesh; Kaur, Raunaq Reet; Bhatia, Priyanka; Kumar, Varun Raj; Chaudhary, Zainab

    2016-01-01

    Current published data presents confusing results about the effects of platelet-rich fibrin on bone, and there is a need for studies that throw light on its effect. Our main objective therefore was to evaluate (by fractal analysis) osseous regeneration in extraction sockets with and without platelet-rich fibrin in a study with a substantial sample and a reliable technique to calibrate its effects on bone cells. We also assessed the soft tissue response. Thirty-four patients had their bilaterally impacted third molars (68 surgical sites) extracted in this split-mouth study, following which platelet-rich fibrin was placed in one of the sockets. Patients were followed up clinically and radiographically, and a pain score and fractal analysis were used to evaluate healing of soft tissue and bone, respectively. We conclude that platelet-rich fibrin improves healing of both soft and hard tissues. Although osseous healing did not differ significantly between the groups, healing of soft tissue as judged by the pain score was significantly better in the experimental group. Copyright © 2015 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

  13. Power Divider for Waveforms Rich in Harmonics

    Science.gov (United States)

    Sims, William Herbert, III

    2005-01-01

    A method for dividing the power of an electronic signal rich in harmonics involves the use of an improved divider topology. A divider designed with this topology could be used, for example, to propagate a square-wave signal in an amplifier designed with a push-pull configuration to enable the generation of more power than could be generated in another configuration.

  14. Stem cell secretome-rich nanoclay hydrogel: a dual action therapy for cardiovascular regeneration

    Science.gov (United States)

    Waters, Renae; Pacelli, Settimio; Maloney, Ryan; Medhi, Indrani; Ahmed, Rafeeq P. H.; Paul, Arghya

    2016-03-01

    A nanocomposite hydrogel with photocrosslinkable micro-porous networks and a nanoclay component was successfully prepared to control the release of growth factor-rich stem cell secretome. The proven pro-angiogenic and cardioprotective potential of this new bioactive system provides a valuable therapeutic platform for cardiac tissue repair and regeneration.A nanocomposite hydrogel with photocrosslinkable micro-porous networks and a nanoclay component was successfully prepared to control the release of growth factor-rich stem cell secretome. The proven pro-angiogenic and cardioprotective potential of this new bioactive system provides a valuable therapeutic platform for cardiac tissue repair and regeneration. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr07806g

  15. The study of a Mg-rich epoxy primer for protection of AZ91D magnesium alloy

    Energy Technology Data Exchange (ETDEWEB)

    Lu Xiangyu [School of Materials Science and Engineering, Beijing University of Chemical Technology, Beijing 100029 (China); Zuo Yu, E-mail: zuoy@mail.buct.edu.c [School of Materials Science and Engineering, Beijing University of Chemical Technology, Beijing 100029 (China); Zhao Xuhui; Tang Yuming; Feng Xingguo [School of Materials Science and Engineering, Beijing University of Chemical Technology, Beijing 100029 (China)

    2011-01-15

    Research highlights: {yields} A Mg-rich epoxy primer was prepared by adding pure magnesium particles in epoxy coating. Cross scratch testing results showed that in 3% NaCl solution the Mg-rich primer showed better protection for AZ91D magnesium alloy than the same epoxy primer without Mg addition. {yields} The open circuit potential of AZ91D alloy in NaCl solution decreased after coated with Mg-rich coating, suggesting that cathodic protection effect of the Mg-rich coating on AZ91D alloy was present. {yields} EIS studies showed that during the immersion tests of AZ91D alloy with Mg-rich coating the magnesium particles in coating dissolved with the charge-transfer resistance R{sub ct} at the magnesium particle/coating interface decreased and the double-layer capacitance Q{sub dl} increased. While the coating resistance remained stable for a long time and corrosion of the AZ91D alloy substrate was obviously delayed. - Abstract: A Mg-rich epoxy primer was prepared by adding pure magnesium particles to an epoxy coating. The coating properties were studied with electrochemical impedance spectroscopy (EIS), scanning electronic microscopy (SEM) and X-ray diffraction (XRD). The Mg-rich primer showed better protection for AZ91D magnesium alloy than the same epoxy primer without Mg addition. The open circuit potential measurements showed cathodic protection effect of the Mg-rich primer on AZ91D alloy. Cross scratch testing showed that the Mg-rich primer provided better protection for the substrate than original epoxy coating. The precipitation of Mg(OH){sub 2} in the coating also provided some degree of barrier protection.

  16. The study of a Mg-rich epoxy primer for protection of AZ91D magnesium alloy

    International Nuclear Information System (INIS)

    Lu Xiangyu; Zuo Yu; Zhao Xuhui; Tang Yuming; Feng Xingguo

    2011-01-01

    Research highlights: → A Mg-rich epoxy primer was prepared by adding pure magnesium particles in epoxy coating. Cross scratch testing results showed that in 3% NaCl solution the Mg-rich primer showed better protection for AZ91D magnesium alloy than the same epoxy primer without Mg addition. → The open circuit potential of AZ91D alloy in NaCl solution decreased after coated with Mg-rich coating, suggesting that cathodic protection effect of the Mg-rich coating on AZ91D alloy was present. → EIS studies showed that during the immersion tests of AZ91D alloy with Mg-rich coating the magnesium particles in coating dissolved with the charge-transfer resistance R ct at the magnesium particle/coating interface decreased and the double-layer capacitance Q dl increased. While the coating resistance remained stable for a long time and corrosion of the AZ91D alloy substrate was obviously delayed. - Abstract: A Mg-rich epoxy primer was prepared by adding pure magnesium particles to an epoxy coating. The coating properties were studied with electrochemical impedance spectroscopy (EIS), scanning electronic microscopy (SEM) and X-ray diffraction (XRD). The Mg-rich primer showed better protection for AZ91D magnesium alloy than the same epoxy primer without Mg addition. The open circuit potential measurements showed cathodic protection effect of the Mg-rich primer on AZ91D alloy. Cross scratch testing showed that the Mg-rich primer provided better protection for the substrate than original epoxy coating. The precipitation of Mg(OH) 2 in the coating also provided some degree of barrier protection.

  17. Particle size effect of Ni-rich cathode materials on lithium ion battery performance

    International Nuclear Information System (INIS)

    Hwang, Ilkyu; Lee, Chul Wee; Kim, Jae Chang; Yoon, Songhun

    2012-01-01

    Graphical abstract: The preparation condition of Ni-rich cathode materials was investigated. When the retention time was short, a poor cathode performance was observed. For long retention time condition, cathode performance displayed a best result at pH 12. Highlights: ► Ni-rich cathode materials (LiNi 0.8 Co 0.15 Al 0.05 O 2 ) were prepared by co-precipitation method using separate addition of Al salt. ► Particle size of Ni-rich cathode materials became larger with increase of retention time and solution pH. ► Cathode performance was poor for low retention time. ► Optimal pH for co-precipitation was 12. -- Abstract: Herein, Ni-rich cathode materials (LiNi 0.8 Co 0.15 Al 0.05 O 2 ) in lithium ion batteries are prepared by a separate addition of Ni/Co salt and Al sol solution using a continuously stirred tank reactor. Retention time and solution pH were controlled in order to obtain high performance cathode material. Particle size increase was observed with a higher retention time of the reactants. Also, primary and secondary particles became smaller according to an increase of solution pH, which was probably due to a decrease of growth rate. From the cathode application, a high discharge capacity (175 mAh g −1 ), a high initial efficiency (90%) and a good cycleability were observed in the cathode material prepared under pH 12 condition, which was attributed to its well-developed layered property and the optimal particle size. However, rate capability was inversely proportional to the particle size, which was clarified by a decrease of charge-transfer resistance measured in the electrochemical impedance spectroscopy.

  18. The SAMPLE experience: The development of a rich media online mathematics learning environment

    OpenAIRE

    Chang, Jen

    2006-01-01

    This report documents the development of Sample Architecture for Mathematically Productive Learning Experiences (SAMPLE), a rich media, online, mathematics learning environment created to meet the needs of middle school educators. It explores some of the current pedagogical challenges in mathematics education, and their amplified impacts when coupled with under-prepared teachers, a decidedly wide-spread phenomenon. The SAMPLE publishing experience is discussed in terms of its instructional de...

  19. The HADES-RICH upgrade using Hamamatsu H12700 MAPMTs with DiRICH FEE + Readout

    Science.gov (United States)

    Patel, V.; Traxler, M.

    2018-03-01

    The High Acceptance Di-Electron Spectrometer (HADES) is operational since the year 2000 and uses a hadron blind RICH detector for electron identification. The RICH photon detector is currently replaced by Hamamatsu H12700 MAPMTs with a readout system based on the DiRICH front-end module. The electronic readout chain is being developed as a joint effort of the HADES-, CBM- and PANDA collaborations and will also be used in the photon detectors for the upcoming Compressed Baryonic Matter (CBM) and PANDA experiments at FAIR . This article gives a brief overview on the photomultipliers and their quality assurance test measurements, as well as first measurements of the new DiRICH front-end module in final configurations.

  20. RICH Detector for Jefferson Labs CLAS12

    Science.gov (United States)

    Trotta, Richard; Torisky, Ben; Benmokhtar, Fatiha

    2015-10-01

    Jefferson Lab (Jlab) is performing a large-scale upgrade to its Continuous Electron Beam Accelerator Facility (CEBAF) up to 12GeV beams. The Large Acceptance Spectrometer (CLAS12) in Hall B is being upgraded and a new hybrid Ring Imaging Cherenkov (RICH) detector is being developed to provide better kaon - pion separation throughout the 3 to 8 GeV/c momentum range. This detector will be used for a variety of Semi-Inclusive Deep Inelastic Scattering experiments. Cherenkov light can be accurately detected by a large array of sophisticated Multi-Anode Photomultiplier Tubes (MA-PMT) and heavier particles, like kaons, will span the inner radii. We are presenting our work on the creation of the RICH's geometry within the CLAS12 java framework. This development is crucial for future calibration, reconstructions and analysis of the detector.

  1. Complexation-Induced Phase Separation: Preparation of Metal-Rich Polymeric Membranes

    KAUST Repository

    Villalobos, Luis Francisco

    2017-01-01

    The majority of state-of-the-art polymeric membranes for industrial or medical applications are fabricated by phase inversion. Complexation induced phase separation (CIPS)—a surprising variation of this well-known process—allows direct fabrication

  2. [The study of anticoagulants selection in platelet-rich plasma preparation].

    Science.gov (United States)

    Hua, Lei; Lai, Gui; Zhenjun, Liu; Guie, Ma

    2015-07-01

    To investigate the effect of the anticoagulants on PRP quality, so as to clarify the appropriate anticoagulant used in PRP production. The microstructure change of platelets collected via heparin, citrate, acid citrate dextrose (ACD) and citrate-theophylline-adenosine-dipyridamole ( CTAD) was observed by TEM following time course. The extent of spontaneous activation of platelets in four groups was detected by measuring sP-selectin in plasma. The TGF-β1 release amount of activated PRP of four groups was measured. CTAD is superior to other anticoagulants in maintaining the integrity of platelet structures for a long time and preventing platelet spontaneous activation. ACD slightly surpassed heparin and citrate in above two aspects. ACD-PRP and CTAD-PRP released significantly more TGF-β1 compared with heparin and citrate. The PRP quality and biological effects were strongly associated with the type of Anticoagulants. ACD and CTAD are optimal anticoagulants in PRP production for they can maintain platelet viability at a high level.

  3. Fusion expression and high-level preparation of a glycine-rich ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-09-15

    Sep 15, 2009 ... SK66, a derivative of the gene cg13551 of Drosophila containing 66 amino acid peptide with N-terminal serine and C-terminal lysine, shows high antimicrobial activities. To obtain it in large amounts, the mature DNA fragment of SK66 was acquired from the pMD18-T-SK66 simple vector using PCR and then.

  4. Prevention of renal dysfunction by nutraceuticals prepared from oil rich plant foods

    Directory of Open Access Journals (Sweden)

    Sahar Y. Al-Okbi

    2014-08-01

    Conclusions: Administration of the studied nutraceuticals proved to possess protective role against cisplatin-induced nephrotoxicity, chromosomal aberration and abnormal sperms. All studied nutraceuticals showed complete safety.

  5. Improving the performance of dairy cattle with a xylanase-rich exogenous enzyme preparation.

    Science.gov (United States)

    Romero, J J; Macias, E G; Ma, Z X; Martins, R M; Staples, C R; Beauchemin, K A; Adesogan, A T

    2016-05-01

    The objective of this experiment was to examine effects of adding 2 exogenous fibrolytic enzymes (EFE) to the total mixed ration (TMR) on the performance of lactating dairy cows (experiment 1) and the kinetics of ruminal degradation of the diet (experiment 2). Twelve EFE had been screened in a series of in vitro assays that identified the most potent EFE and their optimal doses for increasing the digestibility of bermudagrass. In experiment 1, 66 Holstein cows (21±5 d in milk) were grouped by previous milk production and parity (45 multiparous and 21 primiparous) and assigned randomly to 1 of the following 3 treatments: (1) control (CON, untreated), (2) Xylanase Plus [2A, 1mL/kg of TMR dry matter (DM); Dyadic International, Jupiter, FL], and (3) a 75:25 (vol/vol) mixture of Cellulase Plus and Xylanase Plus EFE (3A, 3.4mL/kg of TMR DM; Dyadic International). The EFE were sprayed twice daily onto a TMR (10% bermudagrass silage, 35% corn silage, 5% alfalfa-orchardgrass hay mixture, and 50% concentrates; DM basis) and fed for a 14-d training and covariate period and a 70-d measurement period. Experiment 2 aimed to examine the in situ DM ruminal degradability and ruminal fermentation measurements of the diets fed in experiment 1. Three ruminally fistulated lactating Holstein cows were assigned to the diets. The experiment had a 3×3 Latin square design with 23-d periods. In experiment 1, application of 2A increased intakes (kg/d) of DM (23.5 vs. 22.6), organic matter (21.9 vs. 20.9), and crude protein (3.9 vs. 3.7) and tended to increase yields (kg/d) of fat-corrected milk (41.8 vs. 40.7) and milk fat (1.48 vs. 1.44). In particular, 2A increased milk yield (kg/d) during wk 3 (41.2 vs. 39.8, tendency), 6 (41.9 vs. 40.1), and 7 (42.1 vs. 40.4), whereas 3A increased milk yield (kg/d) during wk 6 (41.5 vs. 40.1, tendency), 8 (41.8 vs. 40.0), and 9 (40.9 vs. 39.5, tendency). In experiment 2, EFE treatment did not affect ruminal DM degradation kinetics or ruminal pH, ammonia-N, and volatile fatty acid concentration. Application of 2A to the bermudagrass-based TMR increased DM intake and milk production, implying that this EFE could be used to increase the performance of lactating dairy cows fed diets containing up to 10% bermudagrass. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  6. Three-dimensional structure and cytokine distribution of platelet-rich fibrin.

    Science.gov (United States)

    Bai, Meng-Yi; Wang, Ching-Wei; Wang, Jyun-Yi; Lin, Ming-Fang; Chan, Wing P

    2017-02-01

    Previous reports have revealed that several cytokines (including platelet-derived growth factor-BB, transforming growth factors-β1 and insulin-like growth factor-1) can enhance the rate of bone formation and synthesis of extracellular matrix in orthopaedics or periodontology. This study aimed to determine the concentration of cytokines within platelet-rich fibrin microstructures and investigate whether there are differences in the different portions of platelet-rich fibrin, which has implications for proper clinical use of platelet-rich fibrin gel. Whole blood was obtained from six New Zealand rabbits (male, 7 to 39 weeks old, weight 2.7-4 kg); it was then centrifuged for preparation of platelet-rich fibrin gels and harvest of plasma. The resultant platelet-rich fibrin gels were used for cytokine determination, histological analyses and scanning electron microscopy. All plasmas obtained were subject to the same cytokine determination assays for the purpose of comparison. Cytokines platelet-derived growth factor-BB and transforming growth factor-β1 formed concentration gradients from high at the red blood cell end of the platelet-rich fibrin gel (p=1.88×10-5) to low at the plasma end (p=0.19). Insulin-like growth factor-1 concentrations were similar at the red blood cell and plasma ends. The porosities of the platelet-rich fibrin samples taken in sequence from the red blood cell end to the plasma end were 6.5% ± 4.9%, 24.8% ± 7.5%, 30.3% ± 8.5%, 41.4% ± 12.3%, and 40.3% ± 11.7%, respectively, showing a gradual decrease in the compactness of the platelet-rich fibrin network. Cytokine concentrations are positively associated with platelet-rich fibrin microstructure and portion in a rabbit model. As platelet-rich fibrin is the main entity currently used in regenerative medicine, assessing cytokine concentration and the most valuable portion of PRF gels is essential and recommended to all physicians.

  7. Three-dimensional structure and cytokine distribution of platelet-rich fibrin

    Directory of Open Access Journals (Sweden)

    Meng-Yi Bai

    Full Text Available OBJECTIVES: Previous reports have revealed that several cytokines (including platelet-derived growth factor-BB, transforming growth factors-β1 and insulin-like growth factor-1 can enhance the rate of bone formation and synthesis of extracellular matrix in orthopaedics or periodontology. This study aimed to determine the concentration of cytokines within platelet-rich fibrin microstructures and investigate whether there are differences in the different portions of platelet-rich fibrin, which has implications for proper clinical use of platelet-rich fibrin gel. METHODS: Whole blood was obtained from six New Zealand rabbits (male, 7 to 39 weeks old, weight 2.7-4 kg; it was then centrifuged for preparation of platelet-rich fibrin gels and harvest of plasma. The resultant platelet-rich fibrin gels were used for cytokine determination, histological analyses and scanning electron microscopy. All plasmas obtained were subject to the same cytokine determination assays for the purpose of comparison. RESULTS: Cytokines platelet-derived growth factor-BB and transforming growth factor-β1 formed concentration gradients from high at the red blood cell end of the platelet-rich fibrin gel (p=1.88×10-5 to low at the plasma end (p=0.19. Insulin-like growth factor-1 concentrations were similar at the red blood cell and plasma ends. The porosities of the platelet-rich fibrin samples taken in sequence from the red blood cell end to the plasma end were 6.5% ± 4.9%, 24.8% ± 7.5%, 30.3% ± 8.5%, 41.4% ± 12.3%, and 40.3% ± 11.7%, respectively, showing a gradual decrease in the compactness of the platelet-rich fibrin network. CONCLUSION: Cytokine concentrations are positively associated with platelet-rich fibrin microstructure and portion in a rabbit model. As platelet-rich fibrin is the main entity currently used in regenerative medicine, assessing cytokine concentration and the most valuable portion of PRF gels is essential and recommended to all physicians.

  8. Ring recognition in the CBM RICH detector

    International Nuclear Information System (INIS)

    Lebedev, S.; Ososkov, G.; Hoehne, C.

    2007-01-01

    Two algorithms of ring recognition, a standalone ring finder (using only RICH information) and an algorithm based on the information from vertex tracks are described. The fake ring problem and its solution using a set of two-dimensional cuts or an artificial neural network are discussed. Results of a comparative study are given. All developed algorithms were tested on large statistics of simulated events and were then included into the CBM framework for common use

  9. Proton radioactivity from proton-rich nuclei

    International Nuclear Information System (INIS)

    Guzman, F.; Goncalves, M.; Tavares, O.A.P.; Duarte, S.B.; Garcia, F.; Rodriguez, O.

    1999-03-01

    Half-lives for proton emission from proton-rich nuclei have been calculated by using the effective liquid drop model of heavy-particle decay of nuclei. It is shown that this model is able to offer results or spontaneous proton-emission half-life-values in excellent agreement with the existing experimental data. Predictions of half-life-values for other possible proton-emission cases are present for null orbital angular momentum. (author)

  10. A facile cathode design combining Ni-rich layered oxides with Li-rich layered oxides for lithium-ion batteries

    Energy Technology Data Exchange (ETDEWEB)

    Song, Bohang; Li, Wangda; Yan, Pengfei; Oh, Seung-Min; Wang, Chong-Min; Manthiram, Arumugam

    2016-09-01

    A facile synthesis method was developed to prepare xLi2MnO3·(1-x)LiNi0.7Co0.15Mn0.15O2 (x = 0, 0.03, 0.07, 0.10, 0.20, and 0.30 as molar ratio) cathode materials, combining the advantages of high specific capacity from Ni-rich layered phase and surface chemical stability from Li-rich layered phase. X-ray diffraction (XRD), high resolution transmission electron microscope (HRTEM) and electrochemical charge/discharge performance confirm the formation of a Li-rich layered phase with C2/m symmetry. Most importantly, high-angle annular dark-field (HAADF) scanning transmission electron microscopy (STEM) reveals a spatial relationship that Li-rich nano-domain islands are integrated into a conventional Ni-rich layered matrix (R$\\bar{3}$m). This is the first time that Li-rich phase has been directly observed inside a particle at the nano-scale, when the overall composition of layered compounds (Li1+δNixMnyM1-x-y-δO2, M refers to transition metal elements) is Ni-rich (x > 0.5) rather than Mn-rich (y > 0.5). Remarkably, xLi2MnO3·(1-x)LiNi0.7Co0.15Mn0.15O2 cathode with optimized x value shows superior electrochemical performance at C/3, i.e., 170 mA h g-1 with 90.3 % of capacity retention after 400 cycles at 25 °C and 164 mA h g-1 with 81.3 % capacity retention after 200 cycles at 55 °C.

  11. Evidence that the primary effect of phosphorylation of eukaryotic initiation factor 2(alpha) in rabbit reticulocyte lysate is inhibition of the release of eukaryotic initiation factor-2.GDP from 60 S ribosomal subunits

    International Nuclear Information System (INIS)

    Gross, M.; Redman, R.; Kaplansky, D.A.

    1985-01-01

    The phosphorylation of eukaryotic initiation factor (eIF) 2 alpha that occurs when rabbit reticulocyte lysate is incubated in the absence of hemin or with poly(I.C) causes inhibition of polypeptide chain initiation by preventing a separate factor (termed RF) from promoting the exchange of GTP for GDP on eIF-2. When lysate was incubated in the presence of hemin and [ 14 C] eIF-2 or [alpha- 32 P]GTP, the authors observed binding of eIF-2 and GDP or GTP to 60 S ribosomal subunits that was slightly greater than that bound to 40 S subunits and little binding to 80 S ribosomes. When incubation was in the absence of hemin or in the presence of hemin plus 0.1 microgram/ml poly(I.C), eIF-2 and GDP binding to 60 S subunits was increased 1.5- to 2-fold, that bound to 80 S ribosomes was almost as great as that bound to 60 S subunits, and that bound to 40 S subunits was unchanged. The data indicate that about 40% of the eIF-2 that becomes bound to 60 S subunits and 80 S ribosomes in the absence of hemin or with poly(I.C) is eIF-2(alpha-P) and suggest that the eIF-2 and GDP bound is probably in the form of a binary complex. The rate of turnover of GDP (presumably eIF-2.GDP) on 60 S subunits and 80 S ribosomes in the absence of hemin is reduced to less than 10% the control rate, because the dissociation of eIF-2.GDP is inhibited. Our findings suggest that eIF-2.GTP binding to and eIF-2.GDP release from 60 S subunits may normally occur and serve to promote subunit joining

  12. Materials Preparation Center

    Data.gov (United States)

    Federal Laboratory Consortium — MPC is recognized throughout the worldwide research community for its unique capabilities in purification, preparation, and characterization of: rare earth metals,...

  13. Reconstruction and calibration strategies for the LHCb RICH detector

    CERN Multimedia

    2006-01-01

    - LHCb particle identification - LHCb ring pattern recognition algorithm requirements - RICH pattern recognition - Cherenkov angle reconstruction online - Online PID - Hough transform - Metropolis- Hastings Markov chains - PID online: physics performances - Rich PID Callibration

  14. Bar quenching in gas-rich galaxies

    Science.gov (United States)

    Khoperskov, S.; Haywood, M.; Di Matteo, P.; Lehnert, M. D.; Combes, F.

    2018-01-01

    Galaxy surveys have suggested that rapid and sustained decrease in the star-formation rate (SFR), "quenching", in massive disk galaxies is frequently related to the presence of a bar. Optical and near-IR observations reveal that nearly 60% of disk galaxies in the local universe are barred, thus it is important to understand the relationship between bars and star formation in disk galaxies. Recent observational results imply that the Milky Way quenched about 9-10 Gyr ago, at the transition between the cessation of the growth of the kinematically hot, old, metal-poor thick disk and the kinematically colder, younger, and more metal-rich thin disk. Although perhaps coincidental, the quenching episode could also be related to the formation of the bar. Indeed the transfer of energy from the large-scale shear induced by the bar to increasing turbulent energy could stabilize the gaseous disk against wide-spread star formation and quench the galaxy. To explore the relation between bar formation and star formation in gas rich galaxies quantitatively, we simulated gas-rich disk isolated galaxies. Our simulations include prescriptions for star formation, stellar feedback, and for regulating the multi-phase interstellar medium. We find that the action of stellar bar efficiently quenches star formation, reducing the star-formation rate by a factor of ten in less than 1 Gyr. Analytical and self-consistent galaxy simulations with bars suggest that the action of the stellar bar increases the gas random motions within the co-rotation radius of the bar. Indeed, we detect an increase in the gas velocity dispersion up to 20-35 km s-1 at the end of the bar formation phase. The star-formation efficiency decreases rapidly, and in all of our models, the bar quenches the star formation in the galaxy. The star-formation efficiency is much lower in simulated barred compared to unbarred galaxies and more rapid bar formation implies more rapid quenching.

  15. COMPASS mirror wall of RICH 1

    CERN Multimedia

    Patrice Loïez

    2001-01-01

    The COMPASS experiment uses ring imaging Cherenkov (RICH) counters to identify particles produced in high-energy muon collisions, to better understand the spin structure of the nucleon. Charged particles moving faster than the speed of light in the medium through which they are travelling emit a cone of Cherenkov radiation in the direction of their motion. The light in this cone is reflected from these mirrors onto a photo detector so that the size of the cone can be measured, which gives the energy of the particle.

  16. Modeling a neutron rich nuclei source

    Energy Technology Data Exchange (ETDEWEB)

    Mirea, M.; Bajeat, O.; Clapier, F.; Ibrahim, F.; Mueller, A.C.; Pauwels, N.; Proust, J. [Institut de Physique Nucleaire, IN2P3/CNRS, 91 - Orsay (France); Mirea, M. [Institute of Physics and Nuclear Engineering, Tandem Lab., Bucharest (Romania)

    2000-07-01

    The deuteron break-up process in a suitable converter gives rise to intense neutron beams. A source of neutron rich nuclei based on the neutron induced fission can be realised using these beams. A theoretical optimization of such a facility as a function of the incident deuteron energy is reported. The model used to determine the fission products takes into account the excitation energy of the target nucleus and the evaporation of prompt neutrons. Results are presented in connection with a converter-target specific geometry. (author000.

  17. HTML5 Designing Rich Internet Applications

    CERN Document Server

    David, Matthew

    2010-01-01

    Implement the powerful new multimedia and interactive capabilities offered by HTML5, including style control tools, illustration tools, video, audio, and rich media solutions. Understand how HTML5 is changing the Web development game with this full-color, project-based treatment that shows you-not just tells you-what HTML5 can do for your Web sites. Reinforce your practical understanding of the new standard with demo applications and tutorials, so that execution is one short step away. The companion website, visualizetheweb.com, is packed full of extra information, online code libraries, and

  18. How countries become rich and reduce poverty

    DEFF Research Database (Denmark)

    Whitfield, Lindsay

    2012-01-01

    For the sake of less developed countries, it is time to adjust the discussion of international development assistance on poverty reduction. This article attempts to do so by reviewing new and old literature explaining why some countries are rich and others are poor. History has repeatedly shown...... that building up capabilities in manufacturing and improving the productivity of agriculture are the keys to wealth creation and long-term sustained poverty reduction. Furthermore, industrialisation and increased agricultural productivity are interdependent processes. Discussion about ending world poverty needs...

  19. Mars: a water-rich planet

    International Nuclear Information System (INIS)

    Carr, M.H.

    1987-01-01

    Good geomorphic evidence is presented for a planet that was once water rich, and that a lower limit on the amount of water available for a given Martian watershed may be estimated by assuming that the volume of material eroded was equal to the volume of water available. This estimate, coupled with high latitude water estimates of 50 to 100 m gives a global inventory of about 500 m total water in the subsurface. It was emphasized that this is a lower limit as considerable water may be bound in weathered debris and in primary minerals

  20. Modeling a neutron rich nuclei source

    International Nuclear Information System (INIS)

    Mirea, M.; Bajeat, O.; Clapier, F.; Ibrahim, F.; Mueller, A.C.; Pauwels, N.; Proust, J.; Mirea, M.

    2000-01-01

    The deuteron break-up process in a suitable converter gives rise to intense neutron beams. A source of neutron rich nuclei based on the neutron induced fission can be realised using these beams. A theoretical optimization of such a facility as a function of the incident deuteron energy is reported. The model used to determine the fission products takes into account the excitation energy of the target nucleus and the evaporation of prompt neutrons. Results are presented in connection with a converter-target specific geometry. (authors)

  1. Structure of neutron-rich nuclei

    International Nuclear Information System (INIS)

    Nazarewicz, W.; Oak Ridge National Lab., TN; Warsaw Univ.

    1997-11-01

    One of the frontiers of today's nuclear science is the ''journey to the limits'': of atomic charge and nuclear mass, of neutron-to-proton ratio, and of angular momentum. The new data on exotic nuclei are expected to bring qualitatively new information about the fundamental properties of the nucleonic many-body system, the nature of the nuclear interaction, and nucleonic correlations at various energy-distance scales. In this talk, current developments in nuclear structure of neutron-rich nuclei are discussed from a theoretical perspective

  2. Rapidly involuting congenital hemangioma (RICH): a brief case report

    OpenAIRE

    Scalise, Robert; Bolton, Joanna; Gibbs, Neil F

    2014-01-01

    Congenital hemangiomas (CH) are benign vascular neoplasms that proliferate in utero and have completed development by birth. Two subtypes of CH are recognized: rapidly involuting congenital hemangiomas (RICH) and non-involuting congenital hemangiomas (NICH). Involution of the RICH subtype often begins in the first weeks of life. NICH does not involute, allowing the distinction between RICH and NICH. We report a case of an infant with RICH occurring on the scalp, examined at birth and followed...

  3. Mixed Media Richness and Computer-Mediated Communications

    OpenAIRE

    Atkins, Anthony B.

    2006-01-01

    Mixed richness communications occur when a participant in a conversation receives a different media or combination of media than they transmit. Mixed richness communications occur in the workplace when technical, physiological or practical limitations prevent the use of the same media on both ends of a conversation. Prior research in CMC has focused on same-richness communications, and the design guidelines that are available for same-richness communications may not be applicable to mixed-r...

  4. A high-throughput sample preparation method for cellular proteomics using 96-well filter plates.

    Science.gov (United States)

    Switzar, Linda; van Angeren, Jordy; Pinkse, Martijn; Kool, Jeroen; Niessen, Wilfried M A

    2013-10-01

    A high-throughput sample preparation protocol based on the use of 96-well molecular weight cutoff (MWCO) filter plates was developed for shotgun proteomics of cell lysates. All sample preparation steps, including cell lysis, buffer exchange, protein denaturation, reduction, alkylation and proteolytic digestion are performed in a 96-well plate format, making the platform extremely well suited for processing large numbers of samples and directly compatible with functional assays for cellular proteomics. In addition, the usage of a single plate for all sample preparation steps following cell lysis reduces potential samples losses and allows for automation. The MWCO filter also enables sample concentration, thereby increasing the overall sensitivity, and implementation of washing steps involving organic solvents, for example, to remove cell membranes constituents. The optimized protocol allowed for higher throughput with improved sensitivity in terms of the number of identified cellular proteins when compared to an established protocol employing gel-filtration columns. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Comparison of the effects of platelet-rich or growth factor-rich plasma on intestinal anastomosis healing in pigs.

    Science.gov (United States)

    Giusto, Gessica; Vercelli, Cristina; Iussich, Selina; Tursi, Massimiliano; Perona, Giovanni; Gandini, Marco

    2017-06-19

    The use of autologous platelet-rich plasma (PRP) and plasma rich in growth factors (PRGF) has been proposed for the treatment of several acute and chronic syndromes, such as corneal epithelial defects and dry eye syndrome, gum bleeding during oral surgery, and in orthopaedic surgery. We hypothesized that PRGF, rather than PRP, could be more effective because of its intrinsic characteristics in promoting the healing of intestinal anastomosis. The purpose of the present study was to evaluate and compare the effects of PRP and PRGF on various parameters of anastomotic healing in a swine model. Eight female pigs were randomly assigned to two groups and subjected to hand sewn jeujuno-jejunal appositional extramucosal anastomoses. For each animal, a total of six anastomoses were performed: two were considered controls and received no treatment, while the remaining four anastomoses were treated with PRP or PRGF of which both were prepared at a platelet concentration that was respectively 3.4-fold and 2.81-fold higher than the original platelet count. In each animal, either PRP or PRGF was used as a treatment, to avoid interference among products. Animals were euthanized after 8 days and the anastomoses were evaluated and compared for the presence of adhesions, anastomotic leakage, bursting pressure, and histological appearance. The concentration of platelets in PRP was 3.41-fold higher (range, 3.20-4.24) that the concentration in whole blood, while the concentration in PRGF was 2.81-fold higher (range, 2.89-4.88). The results obtained from the present study highlighted that there are no differences between anastomotic samples treated with either PRP or PRGF preparations, except for a significant increase in epithelization of the intestinal mucosa at the anastomotic site in the PRGF group. Both PRP and PRGF suspensions should be considered a safe strategy and represent a relatively low-cost technology that is flexible enough to be applied in several therapeutic fields. No

  6. Platelet-rich plasma regenerative medicine sports medicine, orthopedic, and recovery of musculoskeletal injuries

    CERN Document Server

    Santana, Maria; Belangero, William; Luzo, Angela

    2014-01-01

    Platelet-Rich Plasma (PRP) has gained tremendous popularity in recent years as a treatment option for specialties including Orthopedics, Dentistry, Sports Medicine, Otorhinolaryngology, Neurosurgery, Ophthalmology, Urology, Vascular, Cardiothoracic and Maxillofacial Surgery, and Veterinarian Medicine. Nowadays, PRP and Stem Cell Science have added an exciting dimension to tissue repair. This book begins by giving the reader a broad overview of current progress as well as a discussion of the technical aspects of preparation and therapeutic use of autologous PRP. It is followed by a review of platelet structure, function and major growth factors in PRP (PDGF and TGFβ).The third chapter outlines the basic principles of biochemical cellular metabolism that increases the efficacy of PRP. Analogous to the preparation of soil for a garden, restoring cellular health should be the first consideration in Regenerative Medicine. Standardization of PRP preparation to clinical use still remains a challenging prospect. In ...

  7. The effect of platelet-rich fibrin on implant stability.

    Science.gov (United States)

    Öncü, Elif; Alaaddinoğlu, E Emine

    2015-01-01

    Achieving accelerated implant osseointegration could make immediate or early loading of implants more predictable. Platelet-rich fibrin (PRF) is frequently used to accelerate soft and hard tissue healing. The activated platelets in PRF release growth factors, resulting in cellular proliferation, collagen synthesis, and osteoid production. The aim of this study was to compare the stability of dental implants inserted in a one-stage surgical protocol with or without PRF application. Twenty healthy patients with adequate alveolar bone and two or more adjacent missing teeth extracted at least 6 months previously were included in this study. A minimum of two tapered implants (Ankylos, Dentsply/Friadent) were placed in each patient. After surgical preparation of the implant sockets, PRF that had been prepared preoperatively was placed randomly into one of the sockets (PRF+). The acellular plasma portion of PRF was used to wet the implant placed into the PRF-coated socket. Resonance frequency measurements were made after implant placement and at 1 week and 1 month postoperatively. Mean implant stability quotients (ISQs) of the PRF+ implants was 69.3 ± 10.5, and mean ISQs for the PRF- implants was 64.5 ± 12.2 at the end of the first week. The mean ISQs at 4 weeks postoperatively were 77.1 ± 7.1 for the PRF+ group and 70.5 ± 7.7 for the PRF- group. In this study, PRF application increased implant stability during the early healing period, as evidenced by higher ISQ values. Simple application of this material seems to provide faster osseointegration.

  8. Documents preparation and review

    International Nuclear Information System (INIS)

    1999-01-01

    Ignalina Safety Analysis Group takes active role in assisting regulatory body VATESI to prepare various regulatory documents and reviewing safety reports and other documentation presented by Ignalina NPP in the process of licensing of unit 1. The list of main documents prepared and reviewed is presented

  9. Domain walls collision in Fe-rich and Co-rich glass covered microwires

    Directory of Open Access Journals (Sweden)

    Gonzalez J.

    2013-01-01

    Full Text Available We report the results of the investigation of domain walls propagation in Fe-rich and Co-rich microwires performed using Sixtus-Tonks and magneto-optical Kerr effect techniques. It was found that under certain experimental conditions we are able to create the regime of the motion of two domain walls moving to opposite directions which terminates by the collision of the domain walls. Also the domain walls collision was visualized using magneto-optical Kerr effect microscope when the surface giant Barkhausen jump induced by circular magnetic field has been observed.

  10. Magnesium-rich Basalts on Mercury

    Science.gov (United States)

    Martel, L. M. V.

    2013-05-01

    X-ray and gamma-ray spectrometers on NASA's MESSENGER spacecraft are making key measurements regarding the composition and properties of the surface of Mercury, allowing researchers to more clearly decipher the planet's formation and geologic history. The origin of the igneous rocks in the crust of Mercury is the focus of recent research by Karen Stockstill-Cahill and Tim McCoy (National Museum of Natural History, Smithsonian Institution), along with Larry Nittler and Shoshana Weider (Carnegie Institution of Washington) and Steven Hauck II (Case Western Reserve University). Using the well-known MELTS computer code Stockstill-Cahill and coauthors worked with MESSENGER-derived and rock-analog compositions to constrain petrologic models of the lavas that erupted on the surface of Mercury. Rock analogs included a partial melt of the Indarch meteorite and a range of Mg-rich terrestrial rocks. Their work shows the lavas on Mercury are most similar to terrestrial magnesian basalt (with lowered FeO content). The implications of the modeling are that Mg-rich lavas came from high-temperature sources in Mercury's mantle and erupted at high temperature with exceptionally low viscosity into thinly bedded and laterally extensive flows, concepts open to further evaluation by laboratory experiments and by geologic mapping of Mercury's surface using MESSENGER's imaging system and laser altimeter to document flow features and dimensions.

  11. Sports medicine applications of platelet rich plasma.

    Science.gov (United States)

    Mishra, Allan; Harmon, Kimberly; Woodall, James; Vieira, Amy

    2012-06-01

    Platelet rich plasma (PRP) is a powerful new biologic tool in sports medicine. PRP is a fraction of autologous whole blood containing and increased number of platelets and a wide variety of cytokines such as platelet derived growth factor (PDGF), vascular endothelial growth factor (VEGF) and transforming growth factor beta-1 (TGF-B1), fibroblast growth factor (FGF), Insulin-like growth factor-1 (IGF-1) among many others. Worldwide interest in this biologic technology has recently risen sharply. Basic science and preclinical data support the use of PRP for a variety of sports related injuries and disorders. The published, peer reviewed, human data on PRP is limited. Although the scientific evaluation of clinical efficacy is in the early stages, elite and recreational athletes already use PRP in the treatment of sports related injuries. Many questions remain to be answered regarding the use of PRP including optimal formulation, including of leukocytes, dosage and rehabilitation protocols. In this review, a classification for platelet rich plasma is proposed and the in-vitro, preclinical and human investigations of PRP applications in sports medicine will be reviewed as well as a discussion of rehabilitation after a PRP procedure. The regulation of PRP by the World Anti-Doping Agency will also be discussed. PRP is a promising technology in sports medicine; however, it will require more vigorous study in order to better understand how to apply it most effectively.

  12. Habitability constraints on water-rich exoplanets

    Science.gov (United States)

    Noack, Lena; Höning, Dennis; Rivoldini, Attilio; Heistracher, Clemens; Zimov, Nastasia; Journaux, Baptiste; Lammer, Helmut; Van Hoolst, Tim; Hendrik Bredehöft, Jan

    2016-04-01

    This research addresses the characterization, modelling, thermal evolution and possible habitability of water-rich exoplanets. Water is necessary for the origin and survival of life as we know it. In the search for habitable worlds, water-rich planets therefore seem obvious candidates. The water layer on such planets could be hundreds of kilometers deep. Depending on the temperature profile and the pressure gradient, it is likely that at great depths a significant part of the water layer is solid high pressure ice. Whether the solid ice layer extends to the bottom of the water layer, or if a shallow lower ocean forms above the silicate mantle, depends amongst others on the thermal state of the planet. We therefore model the thermal evolution of water-rich planets with a 1D parameterized model. Depth-dependent profiles for thermodynamic properties as well as pressure and gravity are obtained by solving the Poisson equation for the gravity and the hydrostatic pressure equation for pre-defined mass and composition (in terms of iron, silicates and water) [1]. For density, equations of state are applied. For the simulation of the thermal evolution of water-rich planets, several parameters (as initial temperatures or layer thicknesses) are unknown. We therefore employ a quantitatve study with more than 20'000 simulations, where we investigated which parameters have the largest influence on the appearance of a lower ocean, i.e. the possible melting of high-pressure ice by heat flowing out of the silicate mantle [2]. We find that the surface temperature has the largest influence on the thickness of water layers, for which a lower ocean can still form between the high-pressure ice layer and the silicate mantle. For higher surface temperatures, not only entirely liquid oceans are possible for deeper water shells, also a liquid ocean can form under high-pressure ice layers of hundreds of kilometer thickness (for a 1 Earth-mass planet). Deeper down, the lower ocean can still

  13. Using ecological niche modeling to determine avian richness hotspots

    Directory of Open Access Journals (Sweden)

    R. Mirzaei

    2017-04-01

    Full Text Available Understanding distributions of wildlife species is a key step towards identifying biodiversity hotspots and designing effective conservation strategies. In this paper, the spatial pattern of diversity of birds in Golestan Province, Iran was estimated. Ecological niche modeling was used to determine distributions of 144 bird species across the province using a maximum entropy algorithm. Richness maps across all birds, and separately for rare and threatened species, were prepared as approximations to hotspots. Results showed close similarity between hotspots for all birds and those for rare birds; hotspots were concentrated in the southern and especially the southwestern parts of the province. Hotspots for threatened birds tended more to the central and especially the western parts of the province, which include coastal habitats. Based on three criteria, it is clear that the western part is the most important area of the province in terms of bird Faunas. Despite some shortcomings, hotspot analysis for birds could be applied to guide conservation efforts and provide useful tool towards efficient conservation action.

  14. Ion transport by mitochondria-rich cells in toad skin

    DEFF Research Database (Denmark)

    Larsen, Erik Hviid; Ussing, H H; Spring, K R

    1987-01-01

    The optical sectioning video imaging technique was used for measurements of the volume of mitochondria-rich (m.r.) cells of the isolated epithelium of toad skin. Under short-circuit conditions, cell volume decreased by about 14% in response to bilateral exposure to Cl-free (gluconate substitution....... Unilateral exposure to a Cl-free solution did not prevent ouabain-induced cell swelling. It is concluded that m.r. cells have an amiloride-blockable Na conductance in the apical membrane, a ouabain-sensitive Na pump in the basolateral membrane, and a passive Cl permeability in both membranes. From...... the initial rate of ouabain-induced cell volume increase the active Na current carried by a single m.r. cell was estimated to be 9.9 +/- 1.3 pA. Voltage clamping of the preparation in the physiological range of potentials (0 to -100 mV, serosa grounded) resulted in a cell volume increase with a time course...

  15. Platelet-rich plasma for bone healing and regeneration.

    Science.gov (United States)

    Oryan, Ahmad; Alidadi, Soodeh; Moshiri, Ali

    2016-01-01

    Successful healing of large bone defects (LBDs) is a complicated phenomenon because the body's natural ability often fails to effectively repair the LBDs. New modalities should be utilized to increase the quality and accelerate bone healing. Platelet concentrates in different forms can be considered an attractive option for such purpose. Platelets as a natural source of growth factors, cytokines, and other micro and macromolecules are hypothesized to improve bone healing. This review has covered important concepts regarding platelet-rich plasma (PRP) including mechanisms of action, preparation protocols and their differences, and factors affecting the PRP efficacy during bone healing. In addition, the most recent studies in different levels which evaluated the role of PRP on bone repair has been reviewed and discussed to clarify the controversies and conflicts, and to illustrate a future prospective and directions for orthopedic surgeons to overcome current limitations and difficulties. As the efficacy of PRP is dependent on various factors, the outcome of PRP therapy is variable and unpredictable in orthopedic patients. Therefore, it is still too soon to suggest PRP as the first line treatment option in complicated bone injuries such as LBDs and nonunions. However, combination of PRP with natural and synthetic biomaterials can enhance the effectiveness of PRP.

  16. Group Work Tests for Context-Rich Problems

    Science.gov (United States)

    Meyer, Chris

    2016-05-01

    The group work test is an assessment strategy that promotes higher-order thinking skills for solving context-rich problems. With this format, teachers are able to pose challenging, nuanced questions on a test, while providing the support weaker students need to get started and show their understanding. The test begins with a group discussion phase, when students are given a "number-free" version of the problem. This phase allows students to digest the story-like problem, explore solution ideas, and alleviate some test anxiety. After 10-15 minutes of discussion, students inform the instructor of their readiness for the individual part of the test. What follows next is a pedagogical phase change from lively group discussion to quiet individual work. The group work test is a natural continuation of the group work in our daily physics classes and helps reinforce the importance of collaboration. This method has met with success at York Mills Collegiate Institute, in Toronto, Ontario, where it has been used consistently for unit tests and the final exam of the grade 12 university preparation physics course.

  17. The LHCb RICH system: current detector performance and status of the upgrade program

    CERN Document Server

    Fiorini, Massimiliano

    2016-01-01

    LHCb is a precision experiment devoted to the study of CP violation and rare decays of b and c quarks, and to the search for new physics beyond the Standard Model at the Large Hadron Collider (LHC) at CERN. The Ring-Imaging Cherenkov (RICH) system is a key component of the LHCb experiment: it consists of two RICH detectors that provide charged particle identification over a wide momentum range (2-100 GeV/c) and angular acceptance (15-300 mrad). The LHCb RICH system has been performing extremely well during Run 1 and is providing the LHCb experiment also in Run 2 with a robust, reliable and precise particle identification system. Performance of the RICH detectors measured from data will be presented, with special reference to its dependence on calibration parameters and event multiplicities. The LHCb experiment is preparing for an upgrade during the second LHC long shutdown (2019-2020) in order to fully exploit the LHC flavour physics potential. A five-fold increase in instantaneous luminosity is foreseen reac...

  18. Is torrefaction of polysaccharides-rich biomass equivalent to carbonization of lignin-rich biomass?

    Science.gov (United States)

    Bilgic, E; Yaman, S; Haykiri-Acma, H; Kucukbayrak, S

    2016-01-01

    Waste biomass species such as lignin-rich hazelnut shell (HS) and polysaccharides-rich sunflower seed shell (SSS) were subjected to torrefaction at 300°C and carbonization at 600°C under nitrogen. The structural variations in torrefied and carbonized biomasses were compared. Also, the burning characteristics under dry air and pure oxygen (oxy-combustion) conditions were investigated. It was concluded that the effects of carbonization on HS are almost comparable with the effects of torrefaction on SSS in terms of devolatilization and deoxygenation potentials and the increases in carbon content and the heating value. Consequently, it can be proposed that torrefaction does not provide efficient devolatilization from the lignin-rich biomass while it is relatively more efficient for polysaccharides-rich biomass. Heat-induced variations in biomass led to significant changes in the burning characteristics under both burning conditions. That is, low temperature reactivity of biomass reduced considerably and the burning shifted to higher temperatures with very high burning rates. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Species richness of arbuscular mycorrhizal fungi: associations with grassland plant richness and biomass

    Czech Academy of Sciences Publication Activity Database

    Hiiesalu, Inga; Pärtel, M.; Davison, J.; Gerhold, P.; Metsis, M.; Moora, M.; Öpik, M.; Vasar, M.; Zobel, M.; Wilson, S. D.

    2014-01-01

    Roč. 203, č. 1 (2014), s. 233-244 ISSN 1469-8137 R&D Projects: GA MŠk EE2.3.30.0048 Institutional research plan: CEZ:AV0Z60050516 Institutional support: RVO:67985939 Keywords : belowground plant richness * diversity * productivity Subject RIV: EH - Ecology, Behaviour Impact factor: 6.545, year: 2013

  20. Release kinetics of platelet-derived and plasma-derived growth factors from autologous plasma rich in growth factors.

    Science.gov (United States)

    Anitua, Eduardo; Zalduendo, Mari Mar; Alkhraisat, Mohammad Hamdan; Orive, Gorka

    2013-10-01

    Many studies have evaluated the biological effects of platelet rich plasma reporting the final outcomes on cell and tissues. However, few studies have dealt with the kinetics of growth factor delivery by plasma rich in growth factors. Venous blood was obtained from three healthy volunteers and processed with PRGF-Endoret technology to prepare autologous plasma rich in growth factors. The gel-like fibrin scaffolds were then incubated in triplicate, in a cell culture medium to monitor the release of PDGF-AB, VEGF, HGF and IGF-I during 8 days of incubation. A leukocyte-platelet rich plasma was prepared employing the same technology and the concentrations of growth factors and interleukin-1β were determined after 24h of incubation. After each period, the medium was collected, fibrin clot was destroyed and the supernatants were stored at -80°C until analysis. The growth factor delivery is diffusion controlled with a rapid initial release by 30% of the bioactive content after 1h of incubation and a steady state release when almost 70% of the growth factor content has been delivered. Autologous fibrin matrix retained almost 30% of the amount of the growth factors after 8 days of incubation. The addition of leukocytes to the formula of platelet rich plasma did not increase the concentration of the growth factors, while it drastically increased the presence of pro-inflammatory IL-1β. Further studies employing an in vitro inflammatory model would be interesting to study the difference in growth factors and pro-inflammatory cytokines between leukocyte-free and leukocyte-rich platelet rich plasma. Copyright © 2013 Elsevier GmbH. All rights reserved.

  1. imFASP: An integrated approach combining in-situ filter-aided sample pretreatment with microwave-assisted protein digestion for fast and efficient proteome sample preparation.

    Science.gov (United States)

    Zhao, Qun; Fang, Fei; Wu, Ci; Wu, Qi; Liang, Yu; Liang, Zhen; Zhang, Lihua; Zhang, Yukui

    2016-03-17

    An integrated sample preparation method, termed "imFASP", which combined in-situ filter-aided sample pretreatment and microwave-assisted trypsin digestion, was developed for preparation of microgram and even nanogram amounts of complex protein samples with high efficiency in 1 h. For imFASP method, proteins dissolved in 8 M urea were loaded onto a filter device with molecular weight cut off (MWCO) as 10 kDa, followed by in-situ protein preconcentration, denaturation, reduction, alkylation, and microwave-assisted tryptic digestion. Compared with traditional in-solution sample preparation method, imFASP method generated more protein and peptide identifications (IDs) from preparation of 45 μg Escherichia coli protein sample due to the higher efficiency, and the sample preparation throughput was significantly improved by 14 times (1 h vs. 15 h). More importantly, when the starting amounts of E. coli cell lysate decreased to nanogram level (50-500 ng), the protein and peptide identified by imFASP method were improved at least 30% and 44%, compared with traditional in-solution preparation method, suggesting dramatically higher peptide recovery of imFASP method for trace amounts of complex proteome samples. All these results demonstrate that the imFASP method developed here is of high potential for high efficient and high throughput preparation of trace amounts of complex proteome samples. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Platelet Rich Plasma and Knee Surgery

    Directory of Open Access Journals (Sweden)

    Mikel Sánchez

    2014-01-01

    Full Text Available In orthopaedic surgery and sports medicine, the knee joint has traditionally been considered the workhorse. The reconstruction of every damaged element in this joint is crucial in achieving the surgeon’s goal to restore the knee function and prevent degeneration towards osteoarthritis. In the last fifteen years, the field of regenerative medicine is witnessing a boost of autologous blood-derived platelet rich plasma products (PRPs application to effectively mimic and accelerate the tissue healing process. The scientific rationale behind PRPs is the delivery of growth factors, cytokines, and adhesive proteins present in platelets and plasma, as well as other biologically active proteins conveyed by the plasma such as fibrinogen, prothrombin, and fibronectin; with this biological engineering approach, new perspectives in knee surgery were opened. This work describes the use of PRP to construct and repair every single anatomical structure involved in knee surgery, detailing the process conducted in ligament, meniscal, and chondral surgery.

  3. The neutrino opacity of neutron rich matter

    Energy Technology Data Exchange (ETDEWEB)

    Alcain, P.N., E-mail: pabloalcain@gmail.com [Departamento de Física, FCEyN, UBA and IFIBA, Conicet, Pabellón 1, Ciudad Universitaria, 1428 Buenos Aires (Argentina); IFIBA-CONICET (Argentina); Dorso, C.O. [Departamento de Física, FCEyN, UBA and IFIBA, Conicet, Pabellón 1, Ciudad Universitaria, 1428 Buenos Aires (Argentina); IFIBA-CONICET (Argentina)

    2017-05-15

    The study of neutron rich matter, present in neutron star, proto-neutron stars and core-collapse supernovae, can lead to further understanding of the behavior of nuclear matter in highly asymmetric nuclei. Heterogeneous structures are expected to exist in these systems, often referred to as nuclear pasta. We have carried out a systematic study of neutrino opacity for different thermodynamic conditions in order to assess the impact that the structure has on it. We studied the dynamics of the neutrino opacity of the heterogeneous matter at different thermodynamic conditions with semiclassical molecular dynamics model already used to study nuclear multifragmentation. For different densities, proton fractions and temperature, we calculate the very long range opacity and the cluster distribution. The neutrino opacity is of crucial importance for the evolution of the core-collapse supernovae and the neutrino scattering.

  4. Phosphorus Chemistry in Oxygen Rich Stars

    Science.gov (United States)

    Bernal, Jacob; Schmidt, Deborah; Anderson, Julie; Ziurys, Lucy M.

    2017-06-01

    Observations of PO and PN have been carried out at the Arizona Radio Observatory at 1, 2, and 3 mm. Multiple transitions of PO and PN have been detected towards the O-rich AGB stars TX Cam and RCas. Data obtained toward supergiant stars VY Canis Majoris and NML Cyg have also been analyzed. Abundances were obtained for these molecules in all four objects using the radiative transfer code ESCAPADE, which is suitable for symmetric and asymmetric stellar outflows. The abundances of PN and PO were found to be in the range 10^{-8} - 10^{-7} relative to H_{2}. While PN appears to be a parent molecule formed by LTE chemistry near the stellar photosphere, PO appears to be created further out from the star at r > 400 R_*.

  5. RICH High Voltages & PDF Analysis @ LHCb

    CERN Multimedia

    Fanchini, E

    2009-01-01

    In the LHCb experiment an important issue is the identification of the hadrons of the final states of the B mesons decays. Two RICH subdetectors are devoted to this task, and the Hybrid Photon Detectors (HPDs) are the photodetectors used to detect Cherenkov light. In this poster there is a description of how the very high voltage (-18 KV) supply stability used to power the HPDs is monitored. It is also presented the basics of a study which can be done with the first collision data: the analysis of the dimuons from the Drell-Yan process. This process is well known and the acceptance of the LHCb detector in terms of pseudorapidity will be very useful to improve the knowledge of the proton structure functions or, alternatively, try to estimate the luminosity from it.

  6. Plasma rich in growth factors in dentistry

    Directory of Open Access Journals (Sweden)

    Ana Glavina

    2017-06-01

    Full Text Available Background Plasma rich in growth factors (PRGF has wider use in many fields of dentistry due to its endogenous biocompatible regenerative potential i.e., their potential to stimulate and accelerate tissue healing and bone regeneration. Aims This review shows the increasing use of PRGF technology in various fields of dentistry. Methods In the last nine years PubMed has been searched in order to find out published articles upon PRGF in dentistry and 36 papers have been included. Results PRGF technology has many advantages with positive clinical and biological outcomes in tissue healing and bone regeneration. Conclusion In order to determine the most effective therapeutic value for patients, further research is required.

  7. Platelet-rich plasma: applications in dermatology.

    Science.gov (United States)

    Conde Montero, E; Fernández Santos, M E; Suárez Fernández, R

    2015-03-01

    In recent years, the use of platelet-rich plasma has increased notably in a range of diseases and settings. Uses of these products now go beyond skin rejuvenation therapy in patients with facial ageing. Good outcomes for other dermatological indications such as skin ulcers and, more recently, alopecia have been reported in case series and controlled studies. However, these indications are not currently included in the labeling given that stronger scientific evidence is required to support their real benefits. With the increased use of these products, dermatologists need to become familiar with the underlying biological principles and able to critically assess the quality and outcomes of the studies of these products in different skin diseases. Copyright © 2013 Elsevier España, S.L.U. and AEDV. All rights reserved.

  8. [Plant signaling peptides. Cysteine-rich peptides].

    Science.gov (United States)

    Ostrowski, Maciej; Kowalczyk, Stanisław

    2015-01-01

    Recent bioinformatic and genetic analyses of several model plant genomes have revealed the existence of a highly abundant group of signaling peptides that are defined as cysteine-rich peptides (CRPs). CRPs are usually in size between 50 and 90 amino acid residues, they are positively charged, and they contain 4-16 cysteine residues that are important for the correct conformational folding. Despite the structural differences among CRP classes, members from each class have striking similarities in their molecular properties and function. The present review presents the recent progress in research on signaling peptides from several families including: EPF/EPFL, SP11/SCR, PrsS, RALF, LURE, and some other peptides belonging to CRP group. There is convincing evidence indicating multiple roles for these CRPs as signaling molecules during the plant life cycle, ranging from stomata development and patterning, self-incompatibility, pollen tube growth and guidance, reproductive processes, and nodule formation.

  9. VOSGES, a long and rich geologic history

    Science.gov (United States)

    Dominique, Carteaux; Cyrille, Delangle; Sophie, Demangel

    2015-04-01

    The study of geology in scientific classes is often too theoretical and abstract for the pupils. How can teachers make the link between some samples of rocks observed in a practical class and the geologic story of the region? There's nothing better than outdoor education to establish a relationship between the rock observed in macroscopic and microscopic scale in the classroom,with the outcrop scale and the landscape scale in the field: all of them are the result of a fascinating geologic history.Our pupils are lucky enough to live at the heart of a modest mountain massif that has a very rich geologic story: the massif from Vosges situated in the east of France. During two expeditions we show the students all the following tectonic processes: Accretion at the scale of the landscape with the Rhenish Ditch (tectonic and volcanic markers) Obductionis observed due to ophiolites found in the massive of Thalhorn (peridotite, gabbro and sedimentary marine rocks of great depth). Collisionis illuminated with numerous sites like the schists of Steige, the phyllite of Villé, the gneisses of Climont. Subductionis captured bystudying the outcrops of magmatic rocks within the continental crust (andesite, diorite, granodiorite). At each of the stops we have the students, from a hand sample, to findits story in a more global context. So the theory becomes reality. A study of thin slides of rocks observed on the ground finishes these exits and so various scales of understanding are approached. The long and rich geologic history of Vosges maybe reconstituted on hundreds of million years, allowing certainly giving another aspect to the living environment of our pupils.

  10. Sulfur rich microporous polymer enables rapid and efficient removal of mercury(II) from water.

    Science.gov (United States)

    Xu, Dan; Wu, Winston Duo; Qi, Hao-Jun; Yang, Rui-Xia; Deng, Wei-Qiao

    2018-04-01

    Design and synthesis of adsorbents for efficient decontamination of hazardous contaminants Hg 2+ from wastewater, based on a facile and economical strategy, is an attractive target. Here, a novel sulfur rich microporous polymer (sulfur content of 31.4 wt %) with high surface area as well as densely populated sulfur atom with fast accessibility was reported to remove mercury (II) from water. The as prepared polymer (SMP) exhibited high binding affinity, high adsorption capacities, rapid adsorption kinetics, and good recyclability for Hg 2+ . The adsorption capacity of SMP was 595.2 mg g -1 . Furthermore, SMP could reduce trace concentrations of Hg 2+ from 200 p. p. b. to a level below drinking water standards (2 p. p. b.) within 3 min. This work allows large-scale production of sulfur rich porous materials for the practical application in water treatment. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Preparation of bromine fluoride

    International Nuclear Information System (INIS)

    Domange, Pr; Duflo, J.

    1958-05-01

    This note addresses the preparation of bromine fluoride. It indicates the implemented process for the reaction, used products (fluorine and bromine), and column characteristics. It describes the operating mode. Apparatus drawing is provided

  12. Dukovany ASSET mission preparation

    Energy Technology Data Exchange (ETDEWEB)

    Kouklik, I [NPP Dukovany (Czech Republic)

    1997-12-31

    We are in the final stages of the Dukovany ASSET mission 1996 preparation. I would like to present some of our recent experiences. Maybe they would be helpful to other plants, that host ASSET missions in future.

  13. Sperm preparation for fertilization

    NARCIS (Netherlands)

    Gadella, B.M.

    2014-01-01

    Description This book contains 19 chapters that discuss theoretical and applied andrology for domestic, zoo and wild animals. Topics include semen and its constituents; sperm production and harvest; determinants of sperm morphology; sperm preparation for fertilization; practical aspects of semen

  14. Dukovany ASSET mission preparation

    International Nuclear Information System (INIS)

    Kouklik, I.

    1996-01-01

    We are in the final stages of the Dukovany ASSET mission 1996 preparation. I would like to present some of our recent experiences. Maybe they would be helpful to other plants, that host ASSET missions in future

  15. FERMILAB: Preparing to collide

    International Nuclear Information System (INIS)

    Anon.

    1992-01-01

    Against the background of stringent Environment, Safety and Health (ES&H) regulations mandated by the US Department of Energy for all national Labs, Fermilab prepared to mount the next major Tevatron proton-antiproton collider run

  16. Calcium-rich biochar from the pyrolysis of crab shell for phosphorus removal.

    Science.gov (United States)

    Dai, Lichun; Tan, Furong; Li, Hong; Zhu, Nengmin; He, Mingxiong; Zhu, Qili; Hu, Guoquan; Wang, Li; Zhao, Jie

    2017-08-01

    Calcium-rich biochars (CRB) prepared through pyrolysis of crab shell at various temperatures were characterized for physicochemical properties and P removal potential. Elemental analysis showed that CRB was rich in calcium (22.91%-36.14%), while poor in carbon (25.21%-9.08%). FTIR, XRD and TG analyses showed that calcite-based CRB was prepared at temperature ≤600 °C, while lime-based CRB was prepared at temperature ≥700 °C. Phosphorus removal experiment showed that P removal efficiencies in 80 mg P/L phosphate solution and biogas effluent ranged from 26% to 11%, respectively, to about 100% and 63%, respectively, depending on the pyrolysis temperature of the resulting biochar. Specifically, compared to common used CaCO 3 and Ca(OH) 2 , P removal potential of calcite-based CRB was much higher than that of CaCO 3 ; while that of lime-based CRB was close to that of Ca(OH) 2 . These results suggested that CRB was competent for P removal/recovery from wastewater. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Animal models to assess the therapeutic efficacy of human serum and serum-converted platelet lysates for dry eye syndrome: Seeing is believing.

    Science.gov (United States)

    Tseng, Ching-Li; Seghatchian, Jerard; Burnouf, Thierry

    2015-08-01

    There is much interest in the clinical use of serum-converted human blood or platelet concentrates in regenerative medicine, most specifically for wound healing and tissue repair of soft and hard tissues. The scientific rationale supporting the clinical efficacy of these preparations is based on the expectation that their physiological mixture of natural growth factors can orchestrate cell expansion and differentiation in vivo. However, a lack of standardization and regulatory oversight of these blood materials maintain a perception of uncertainty in the scientific and medical community on the value of these preparations for some clinical indications. More studies are needed to understand the mechanism of action underlying their expected efficacy and standardize their use, and benefit from their biological versatility. One application of serum is as eye drop for treating dry eye syndrome (DES), a multifactorial disease of the ocular surface, which has a prevalence of 15% of more in the population. DES can lead to chronic inflammation of the ocular surface, surface impairment in the cornea and conjunctiva, and, in patients with Sjogren syndrome, result in a disruption of the ocular surface epithelium. Objective experimental assessment of safety and efficacy of serum eye drops can help establish scientific rationale in optimal product composition and use. This can be achieved, first, through cell cultures with relevant cell models, before considering, then, animal studies using DES animal models. Several models have been evaluated and are reported in this concise review. The model we have developed encompasses the use of rabbits, where their eyes are treated with 0.1% benzalkonium chloride (BAC), a common preservative in ophthalmic agents, 3 times daily for 4 weeks. This relatively mild treatment results in moderate DES pathology, with a stable shortage of tear secretion throughout a 7-week study period, which we found suitable for assessing efficacy of serum eye

  18. Platelet-rich plasma: why intra-articular? A systematic review of preclinical studies and clinical evidence on PRP for joint degeneration.

    Science.gov (United States)

    Filardo, G; Kon, E; Roffi, A; Di Matteo, B; Merli, M L; Marcacci, M

    2015-09-01

    The aim of this review was to analyze the available evidence on the clinical application of this biological approach for the injective treatment of cartilage lesions and joint degeneration, together with preclinical studies to support the rationale for the use of platelet concentrates, to shed some light and give indications on what to treat and what to expect from intra-articular injections of platelet-rich plasma (PRP). All in vitro, in vivo preclinical and clinical studies on PRP injective treatment in the English language concerning the effect of PRP on cartilage, synovial tissue, menisci, and mesenchymal stem cells were considered. A systematic review on the PubMed database was performed using the following words: (platelet-rich plasma or PRP or platelet concentrate or platelet lysate or platelet supernatant) and (cartilage or chondrocytes or synoviocytes or menisci or mesenchymal stem cells). Fifty-nine articles met the inclusion criteria: 26 were in vitro, 9 were in vivo, 2 were both in vivo and in vitro, and 22 were clinical studies. The analysis showed an increasing number of published studies over time. Preclinical evidence supports the use of PRP injections that might promote a favourable environment for joint tissues healing. Only a few high-quality clinical trials have been published, which showed a clinical improvement limited over time and mainly documented in younger patients not affected by advanced knee degeneration. Besides the limits and sometimes controversial findings, the preclinical literature shows an overall support toward this PRP application. An intra-articular injection does not just target cartilage; instead, PRP might influence the entire joint environment, leading to a short-term clinical improvement. Many biological variables might influence the clinical outcome and have to be studied to optimize PRP injective treatment of cartilage degeneration and osteoarthritis.

  19. A PUFA-rich diet improves fat oxidation following saturated fat-rich meal.

    Science.gov (United States)

    Stevenson, Jada L; Miller, Mary K; Skillman, Hannah E; Paton, Chad M; Cooper, Jamie A

    2017-08-01

    To determine substrate oxidation responses to saturated fatty acid (SFA)-rich meals before and after a 7-day polyunsaturated fatty acid (PUFA)-rich diet versus control diet. Twenty-six, normal-weight, adults were randomly assigned to either PUFA or control diet. Following a 3-day lead-in diet, participants completed the pre-diet visit where anthropometrics and resting metabolic rate (RMR) were measured, and two SFA-rich HF meals (breakfast and lunch) were consumed. Indirect calorimetry was used to determine fat oxidation (Fox) and energy expenditure (EE) for 4 h after each meal. Participants then consumed a PUFA-rich diet (50 % carbohydrate, 15 % protein, 35 % fat, of which 21 % of total energy was PUFA) or control diet (50 % carbohydrate, 15 % protein, 35 % fat, of which 7 % of total energy was PUFA) for the next 7 days. Following the 7-day diet, participants completed the post-diet visit. From pre- to post-PUFA-rich diet, there was no change in RMR (16.3 ± 0.8 vs. 16.4 ± 0.8 kcal/20 min) or in incremental area under the curve for EE (118.9 ± 20.6-126.9 ± 14.1 kcal/8h, ns). Fasting respiratory exchange ratio increased from pre- to post-PUFA-rich diet only (0.83 ± 0.1-0.86 ± 0.1, p diet (0.03 ± 0.1-0.23 ± 0.1 g/15 min for cumulative Fox; p diet initiates greater fat oxidation after eating occasional high SFA meals compared to a control diet, an effect achieved in 7 days.

  20. Benchmarking sample preparation/digestion protocols reveals tube-gel being a fast and repeatable method for quantitative proteomics.

    Science.gov (United States)

    Muller, Leslie; Fornecker, Luc; Van Dorsselaer, Alain; Cianférani, Sarah; Carapito, Christine

    2016-12-01

    Sample preparation, typically by in-solution or in-gel approaches, has a strong influence on the accuracy and robustness of quantitative proteomics workflows. The major benefit of in-gel procedures is their compatibility with detergents (such as SDS) for protein solubilization. However, SDS-PAGE is a time-consuming approach. Tube-gel (TG) preparation circumvents this drawback as it involves directly trapping the sample in a polyacrylamide gel matrix without electrophoresis. We report here the first global label-free quantitative comparison between TG, stacking gel (SG), and basic liquid digestion (LD). A series of UPS1 standard mixtures (at 0.5, 1, 2.5, 5, 10, and 25 fmol) were spiked in a complex yeast lysate background. TG preparation allowed more yeast proteins to be identified than did the SG and LD approaches, with mean numbers of 1979, 1788, and 1323 proteins identified, respectively. Furthermore, the TG method proved equivalent to SG and superior to LD in terms of the repeatability of the subsequent experiments, with mean CV for yeast protein label-free quantifications of 7, 9, and 10%. Finally, known variant UPS1 proteins were successfully detected in the TG-prepared sample within a complex background with high sensitivity. All the data from this study are accessible on ProteomeXchange (PXD003841). © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Influence of apical enlargement and complementary canal preparation with the Self-Adjusting File on endotoxin reduction in retreatment cases.

    Science.gov (United States)

    Silva, E J N L; Ferreira, V M; Silva, C C; Herrera, D R; De-Deus, G; Gomes, B P

    2017-07-01

    To compare the effectiveness of large apical preparations and complementary canal preparation with the Self-Adjusting File (SAF) in removing endotoxins from the root canal of teeth with apical periodontitis. Ten single-rooted and single-canaled teeth with post-treatment apical periodontitis were selected. Endotoxin samples were taken after removal of the root filling (S1), after chemomechanical preparation (CMP) using 2.5% NaOCl and an R25 file (S2), after CMP using 2.5% NaOCl and an R40 file (S3) and after complementary CMP using the SAF system (S4). Limulus amebocyte lysate (LAL) was used to measure endotoxin levels. The Friedman and Wilcoxon tests were used to compare endotoxin levels at each clinical intervention (P file was able to significantly reduce endotoxin levels (P file (P  0.05) following the use of the R40 instrument. Apical enlargement protocols were effective in significantly reducing endotoxin levels. Complementary preparation with the SAF system failed to eliminate residual endotoxin contents beyond those obtained with the R40 instrument. © 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd.

  2. A novel glutamine-rich putative transcriptional adaptor protein (TIG-1), preferentially expressed in placental and bone-marrow tissues.

    Science.gov (United States)

    Abraham, S; Solomon, W B

    2000-09-19

    We used a subtractive hybridization protocol to identify novel expressed sequence tags (ESTs) corresponding to mRNAs whose expression was induced upon exposure of the human leukemia cell line K562 to the phorbol ester 12-O-tetradecanolyphorbol-13-acetate (TPA). The complete open reading frame of one of the novel ESTs, named TIG-1, was obtained by screening K562 cell and placental cDNA libraries. The deduced open reading frame of the TIG-1 cDNA encodes for a glutamine repeat-rich protein with a predicted molecular weight of 63kDa. The predicted open reading frame also contains a consensus bipartite nuclear localization signal, though no specific DNA-binding domain is found. The corresponding TIG-1 mRNA is ubiquitously expressed. Placental tissue expresses the TIG-1 mRNA 200 times more than the lowest expressing tissues such as kidney and lung. There is also preferential TIG-1 mRNA expression in cells of bone-marrow lineage.In-vitro transcription/translation of the TIG-1 cDNA yielded a polypeptide with an apparent molecular weight of 97kDa. Using polyclonal antibodies obtained from a rabbit immunized with the carboxy-terminal portion of bacterially expressed TIG-1 protein, a polypeptide with molecular weight of 97kDa was identified by Western blot analyses of protein lysates obtained from K562 cells. Cotransfection assays of K562 cells, using a GAL4-TIG-1 fusion gene and GAL4 operator-CAT, indicate that the TIG-1 protein may have transcriptional regulatory activity when tethered to DNA. We hypothesize that this novel glutamine-rich protein participates in a protein complex that regulates gene transcription. It has been demonstrated by Naar et al. (Naar, A.M., Beaurang, P.A., Zhou, S., Abraham, S., Solomon, W.B., Tjian, R., 1999, Composite co-activator ARC mediates chromatin-directed transcriptional activation. Nature 398, 828-830) that the amino acid sequences of peptide fragments obtained from a polypeptide found in a complex of proteins that alters chromatin

  3. The hospital preparation of radiopharmaceuticals

    International Nuclear Information System (INIS)

    The subject is covered in sections: introduction; preparation ((general - sterilization), production areas (laboratories), working methods for injections, working methods for oral preparations and iodination procedures); analytical testing (general, standards common to injections and oral preparations, standards for injections, standards for oral preparations); reliable methods of preparing sup(99m)Tc-radiopharmaceuticals and 51 Cr-red cells; commercial radiopharmaceutical kits. (U.K.)

  4. Preparations of high density (Th,U)O2 pellets

    International Nuclear Information System (INIS)

    Akabori, Mitsuo; Ikawa, Katsuichi

    1986-07-01

    Preparations of high density and homogeneous (Th,U)O 2 pellets by a powder metallurgy method were examined. (Th,U)O 2 powders were prepared by calcining coprecipitates of ammonium uranate and thorium hydroxide derived from nitrates and mixed sols, and by calcining mixed oxalates precipitated from nitrates. (Th,U)O 2 pellets were characterized with respect to sinterability, lattice parameter, microstructure, homogeneity and stoichiometry. Sintering atmospheres had a significant effect upon all the properties of the derived pellets. The sinterability of (Th,U)O 2 was most favourable in oxidizing and reducing atmospheres for ThO 2 -rich and UO 2 -rich compositions, respectively, and can be enhanced by presence of water vapour in sintering atmospheres. In addition, highly homogeneous (Th,U)O 2 pellets with 99 % in theoretical density were derived from the sol powders. (author)

  5. Human cord blood-derived platelet lysate enhances the therapeutic activity of adipose-derived mesenchymal stromal cells isolated from Crohn's disease patients in a mouse model of colitis.

    Science.gov (United States)

    Forte, Dorian; Ciciarello, Marilena; Valerii, Maria Chiara; De Fazio, Luigia; Cavazza, Elena; Giordano, Rosaria; Parazzi, Valentina; Lazzari, Lorenza; Laureti, Silvio; Rizzello, Fernando; Cavo, Michele; Curti, Antonio; Lemoli, Roberto M; Spisni, Enzo; Catani, Lucia

    2015-09-09

    Due to their immunomodulatory properties, mesenchymal stromal cells (MSCs) have been used for auto-immune disease treatment. Crohn disease (CD) and ulcerative colitis are two major inflammatory bowel diseases (IBDs), resulting from pathological immune responses to environmental or microbial antigens. Preclinical and clinical studies have suggested that MSC-based cellular therapy hold promising potential for IBD treatment. However, open issues include the selection of the proper cell dose, the source and the optimal route of administration of MSCs for more effective results. Platelet lysate has gained clinical interest due to its efficacy in accelerating wound healing. Thus, we propose to combine the administration of MSCs with a human umbilical cord blood-derived platelet lysate (hCBPL) as a novel strategy to improve MSC-based therapy for IBD resolution. Colitis was induced in 8-week-old C57BL/6J mice by daily oral administration of dextran sulphate sodium (DSS) (1.5 % w/v in tap water) for 9 days. MSCs were isolated from adipose tissue of CD patients (adCD-MSCs), expanded in proliferation medium, resuspended in hCBPL or PBS and administrated via enema for three times (1 × 10(6) cells/mouse/time) every other day starting on day +7 from DSS induction. The colitis evolution was evaluated by daily monitoring of body weight, stool consistency and bleeding. Histopathological analysis was performed. Inflammatory cytokine plasma levels were determined. adCD-MSCs stained with lipophilic membrane dye Nile Red, were injected in DSS mice as described above. Colon section of mice sacrificed 24 hours after last cell administration, were analyzed by confocal microscopy. We found that adCD-MSCs could be easily isolated and expanded from CD patients. Upon injection, adCD-MSCs exerted a therapeutic effect on DSS-induced colitis. Moreover, hCBPL increased adCD-MSCs efficacy by significantly reducing colitis scores, extension of the colon inflamed area and plasma levels of

  6. Solid-state reaction synthesis and aqueous durability of Ce-doped zirconolite-rich ceramics

    Energy Technology Data Exchange (ETDEWEB)

    Wen, Guanjun [State Key Laboratory Cultivation Base for Nonmetal Composite and Functional Materials, Southwest University of Science and Technology, Mianyang, Sichuan 621010 (China); Zhang, Kuibao, E-mail: xiaobao320@163.com [State Key Laboratory Cultivation Base for Nonmetal Composite and Functional Materials, Southwest University of Science and Technology, Mianyang, Sichuan 621010 (China); Yin, Dan [State Key Laboratory Cultivation Base for Nonmetal Composite and Functional Materials, Southwest University of Science and Technology, Mianyang, Sichuan 621010 (China); Zhang, Haibin, E-mail: hbzhang@imr.ac.cn [Institute of Nuclear Physics and Chemistry, China Academy of Engineering Physics, Mianyang 621900 (China)

    2015-11-15

    In this study, Ce-doped zirconolite-rich ceramics were prepared by solid-state reaction process using cerium as the surrogate of tetravalence actinide nuclide. The occupancy of Ce in the waste forms was investigated. The aqueous durability of Ce-doped zirconolite-rich ceramic was examined as well. The results show that zirconolite and pseudobrookite coexisted after being sintered at 1200 °C for 6 h. Meanwhile, perovskite is inevitable generated during the process. CeO{sub 2} can be successfully incorporated into the lattice structure of the zirconolite-rich ceramics. The maximum containing capacity of CeO{sub 2} is up to 14.95 wt% or y = 0.4. The normalized elemental leaching rates of Ce and Ca are fairly constant in low values of 1.2 × 10{sup −6} and 2.3 × 10{sup −2} g m{sup −2} d{sup −1} after 28 days. The normalized leaching rate of Fe is also in a low value of 2.9 × 10{sup −4} g m{sup −2} d{sup −1} after 7 days. - Highlights: • Ce-doped zirconolite-rich ceramic was produced at 1200 °C. • Pseudobrookite-type Fe{sub 2}TiO{sub 5} was employed to incorporate Fe element. • Ce{sup 3+} and Ce{sup 4+} coexisted in the Ce-doped zirconolite-rich waste form after being sintered at 1200 °C for 6 h. • The leaching rate of Ca was relatively higher than that of borosilicate glasses.

  7. Efficacy of leukocyte- and platelet-rich fibrin in wound healing: a randomized controlled clinical trial.

    Science.gov (United States)

    Chignon-Sicard, Bérengère; Georgiou, Charalambos A; Fontas, Eric; David, Sylvain; Dumas, Pierre; Ihrai, Tarik; Lebreton, Elisabeth

    2012-12-01

    Application of platelet concentrates to wounds could speed healing. Leukocyte- and platelet-rich fibrin, a relatively recent development, stands out from the other preparations. This prospective, randomized, controlled clinical trial studied the rate of healing of postoperative hand wounds after a single application of leukocyte- and platelet-rich fibrin. Eligible patients were healthy individuals older than 18 years who had been scheduled for elective McCash (open palm) surgery for Dupuytren disease at the Plastic and Hand Surgery Department of Nice's University Hospital between August of 2007 and February of 2010. The control group received the reference care of petroleum jelly mesh (Vaselitulle), and test patients had leukocyte- and platelet-rich fibrin applied. The primary endpoint was healing delay measured in postoperative days. Secondary endpoints included pain, bleeding, and wound exudate. The trial was carried out as a single-blind trial. Among the 68 randomized patients, 33 patients in the leukocyte- and platelet-rich fibrin group and 31 in the Vaselitulle group were analyzed. Primary endpoint analysis showed a median healing delay of 24 days (interquartile range, 18 to 28 days) for the fibrin group and 29 days (interquartile range, 26 to 35 days) for the Vaselitulle group (p = 0.014, log-rank test). Postoperative pain assessment, bleeding, and exudate were always lower for the fibrin group, but not significantly so. The authors trial demonstrates that a single leukocyte- and platelet-rich fibrin application on fresh postoperative hand wounds shows a median improvement of 5 days in comparison with the standard treatment. Therapeutic, II.

  8. arXiv The Future of RICH Detectors through the Light of the LHCb RICH

    CERN Document Server

    D'Ambrosio, C.; Easo, S.; Petrolini, A.; Ullaland, O.

    2017-12-21

    The limitations in performance of the present RICH system in the LHCb experiment are given by the natural chromatic dispersion of the gaseous Cherenkov radiator, the aberrations of the optical system and the pixel size of the photon detectors. Moreover, the overall PID performance can be affected by high detector occupancy as the pattern recognition becomes more difficult with high particle multiplicities. This paper shows a way to improve performance by systematically addressing each of the previously mentioned limitations. These ideas are applied in the present and future upgrade phases of the LHCb experiment. Although applied to specific circumstances, they are used as a paradigm on what is achievable in the development and realisation of high precision RICH detectors.

  9. Preparation of aluminum nitride-silicon carbide nanocomposite powder by the nitridation of aluminum silicon carbide

    NARCIS (Netherlands)

    Itatani, K.; Tsukamoto, R.; Delsing, A.C.A.; Hintzen, H.T.J.M.; Okada, I.

    2002-01-01

    Aluminum nitride (AlN)-silicon carbide (SiC) nanocomposite powders were prepared by the nitridation of aluminum-silicon carbide (Al4SiC4) with the specific surface area of 15.5 m2·g-1. The powders nitrided at and above 1400°C for 3 h contained the 2H-phases which consisted of AlN-rich and SiC-rich

  10. Generation of Autologous Platelet-Rich Plasma by the Ultrasonic Standing Waves.

    Science.gov (United States)

    Wu, Yue; Kanna, Murugappan Suresh; Liu, Chenhui; Zhou, Yufeng; Chan, Casey K

    2016-08-01

    Platelet-rich plasma (PRP) is a volume of autologous plasma that has a higher platelet concentration above baseline. It has already been approved as a new therapeutic modality and investigated in clinics, such as bone repair and regeneration, and oral surgery, with low cost-effectiveness ratio. At present, PRP is mostly prepared using a centrifuge. However, this method has several shortcomings, such as long preparation time (30 min), complexity in operation, and contamination of red blood cells (RBCs). In this paper, a new PRP preparation approach was proposed and tested. Ultrasound waves (4.5 MHz) generated from piezoelectric ceramics can establish standing waves inside a syringe filled with the whole blood. Subsequently, RBCs would accumulate at the locations of pressure nodes in response to acoustic radiation force, and the formed clusters would have a high speed of sedimentation. It is found that the PRP prepared by the proposed device can achieve higher platelet concentration and less RBCs contamination than a commercial centrifugal device, but similar growth factor (i.e., PDGF-ββ). In addition, the sedimentation process under centrifugation and sonication was simulated using the Mason-Weaver equation and compared with each other to illustrate the differences between these two technologies and to optimize the design in the future. Altogether, ultrasound method is an effective method of PRP preparation with comparable outcomes as the commercially available centrifugal products.

  11. IMPROVEMENT OF THE RICHNESS ESTIMATES OF maxBCG CLUSTERS

    International Nuclear Information System (INIS)

    Rozo, Eduardo; Rykoff, Eli S.; Koester, Benjamin P.; Hansen, Sarah; Becker, Matthew; Bleem, Lindsey; McKay, Timothy; Hao Jiangang; Evrard, August; Wechsler, Risa H.; Sheldon, Erin; Johnston, David; Annis, James; Scranton, Ryan

    2009-01-01

    Minimizing the scatter between cluster mass and accessible observables is an important goal for cluster cosmology. In this work, we introduce a new matched filter richness estimator, and test its performance using the maxBCG cluster catalog. Our new estimator significantly reduces the variance in the L X -richness relation, from σ lnLx 2 = (0.86±0.02) 2 to σ lnLx 2 = (0.69±0.02) 2 . Relative to the maxBCG richness estimate, it also removes the strong redshift dependence of the L X -richness scaling relations, and is significantly more robust to photometric and redshift errors. These improvements are largely due to the better treatment of galaxy color data. We also demonstrate the scatter in the L X -richness relation depends on the aperture used to estimate cluster richness, and introduce a novel approach for optimizing said aperture which can easily be generalized to other mass tracers.

  12. Effect of carbo-nitride-rich and oxide-rich inclusions on the pitting susceptibility of depleted uranium

    International Nuclear Information System (INIS)

    Pu, Zhen; Chen, Xianglin; Meng, Xiandong; Wu, Yanping; Shen, Liang; Wang, Qingfu; Liu, Tianwei; Shuai, Maobing

    2017-01-01

    Highlights: •The Volta potential differences relative to the matrix are positive for both types of inclusions. •Both types of inclusions are cathodic in the “inclusion/matrix” microgalvanic couples. •The oxide-rich inclusions show a larger Volta potential value of about 115 mV than the carbo-nitride-rich inclusions. •The oxide-rich inclusions give stronger local galvanic coupling with the matrix. •The oxide-rich inclusions are more predisposed to initiate pitting corrosion. -- Abstract: The effects of carbo-nitride-rich and oxide-rich inclusions on the pitting susceptibility of depleted uranium were investigated by electrochemical corrosion measurements, optical microscopy, scanning Kelvin probe force microscopy (SKPFM), and SEM. The results of the potentiodynamic polarization tests suggest that oxide-rich inclusions are more likely to induce pitting corrosion than carbo-nitride-rich inclusions. This enhanced corrosion may be explained by the strong local galvanic coupling between the oxide-rich inclusion and the surrounding matrix, which, from the sight of SKPFM analysis, exhibits a 115 V higher Volta potential than the coupling between the carbo-nitride-rich inclusions and the matrix, respectively.

  13. Conservation and viability of refrigerated platelet-rich plasma from New Zealand rabbits

    OpenAIRE

    Lucas Vilela Perroni Silva; Duvaldo Eurides; Marina Greco Magalhães Guerra de Andrade; Marcelo Carrijo da Costa; Vinicius Candido Ferreira; Marina de Oliveira Silva; Ednaldo Carvalho Guimarães

    2016-01-01

    Fifteen male New Zealand rabbits were used in this study, with the aim of storing their platelet-rich plasma (PRP) for 30 days at 4-6 °C to investigate its conservation and viability during this period. Thirty samples of PRP were prepared and sorted into three equal groups (G1, G2, and G3), and every three days a sample was taken out for evaluationof the number of platelets, mean platelet volume (MPV), pH of the plasma, aggregation post addition of calcium thromboplastin, and for the presence...

  14. Magnetic properties of ND Rich Melt-Spun ND-FE-B alloy

    Directory of Open Access Journals (Sweden)

    Grujić Aleksandar

    2005-01-01

    Full Text Available As a part of these experimental investigations of melt-spun Nd-Fe-B alloy with Nd rich content in relation to Nd2Fe14B prepared by rapid quenching process for optimally selected cooling rate and heat treatment, the influence of the chosen chemical composition on magnetic properties was observed. The results of X-ray diffraction, Mössbauer spectroscopy phase analysis and magnetic measurement of investigated melt-spun Nd14.5Fe78.5B7 alloy are presented to bring some new information concerning the relation between their structure and magnetic properties.

  15. Platelet Preparations for Use in Facial Rejuvenation and Wound Healing: A Critical Review of Current Literature.

    Science.gov (United States)

    Sclafani, Anthony P; Azzi, James

    2015-08-01

    In facial plastic surgery, the potential for direct delivery of growth factors from platelet preparations has been of particular interest for use in facial rejuvenation, recovery after facial surgery, and wound healing. A literature search was conducted through PubMed for the terms PRP, PRFM, platelet-rich plasma, platelet-rich fibrin matrix, platelet preparations, platelet therapy, growth factors, platelet facial, platelet facial rejuvenation, platelet wound healing, platelet plastic surgery. Articles pertaining to the use of platelet preparations in facial surgery and wound healing in plastic surgery after 2001 were included. Thirteen in vitro studies showed use of platelet-rich plasma (PRP) and platelet-rich fibrin matrix (PRFM) had a significant effect on cellular activity. Twenty-four out of 28 animal studies exhibited favorable results with use of a platelet preparation, including five of six studies that showed enhanced fat graft survival with addition of a platelet preparation. Twenty-three case series and clinical trials were identified, only two of which showed no differences. Twenty-one reported favorable results with use of various platelet preparations. A total of 47 studies used PRP, four studies evaluated Leukocyte-rich PRP, and fourteen studies used PRFM. The vast majority of studies examined show a significant and measurable effect on cellular changes, wound healing, and facial esthetic outcomes with use of platelet preparations, both topical and injectable. One must also consider possible publication bias against null results that may have had an influence on the data that were available for review. However, the preponderance of studies suggests that platelet preparations might represent an as-of-yet untapped adjunct in facial plastic surgery.

  16. Vertebrate richness and biogeography in the Big Thicket of Texas

    Science.gov (United States)

    Michael H MacRoberts; Barbara R. MacRoberts; D. Craig Rudolph

    2010-01-01

    The Big Thicket of Texas has been described as rich in species and a “crossroads:” a place where organisms from many different regions meet. We examine the species richness and regional affiliations of Big Thicket vertebrates. We found that the Big Thicket is neither exceptionally rich in vertebrates nor is it a crossroads for vertebrates. Its vertebrate fauna is...

  17. Synthesis and study of neutron-rich nuclides

    International Nuclear Information System (INIS)

    Luo Yixiao

    1995-01-01

    During the past few years our understanding of the decay properties and nuclear structure has been extended in a systematic fashion for the neutron-rich nuclei. This review will first sketch the production and identification of the neutron-rich nuclei throughout the whole mass region, and will then discuss the impressive progress in the studies of the exotic decay properties and nuclear structure of neutron-rich nuclei. Their astrophysical implications will also be outlined

  18. Plasma rico en plaquetas Platelet -rich plasma

    Directory of Open Access Journals (Sweden)

    J. González Lagunas

    2006-04-01

    Full Text Available El Plasma Rico en Plaquetas es una suspensión concentrada de la sangre centrifugada que contiene elevadas concentraciones de trombocitos. Durante los últimos años, este producto ha aparecido de forma repetida en publicaciones científicas y en medios de comunicación generales como un producto que por sus características induce la curación y regeneración de los tejidos. La premisa de su uso es que las elevadas concentraciones de plaquetas en el PRP, liberan cantidades significativas de factores de crecimiento. En este artículo se van a recoger las evidencias científicas que se han presentado en la literatura médica con respecto al PRP y a la curación ósea, así como las diferentes aplicaciones clínicas que se han sugerido.Platelet-rich plasma is a by-product of centrifuged whole blood that contains high levels of thrombocytes. In the last decade, scientific and media interest has been generated by this product that apparently has the capacity of inducing and promoting tissue healing and regeneration. The premise of its use is that the large number of platelets in PRP release significant amounts of growth factors. In this paper, a critical review of the medical literature regarding PRP and bone healing will be presented. Also, the suggested clinical applications of the product will be addressed.

  19. The AQUA-RICH atmospheric neutrino experiment

    CERN Document Server

    Antonioli, P; Bellagamba, L; Chesi, Enrico Guido; Cindolo, F; De Pasquale, S; Ekelöf, T J C; Garbini, M; Giusti, P; Grossheim, A; Pesci, A; Learned, J G; Margotti, A; Pinfold, James L; Sartorelli, G; Séguinot, Jacques; Tarantino, A; Weilhammer, Peter; Ypsilantis, Thomas; Zichichi, A; Zuber, K

    1999-01-01

    We describe a 125 m diameter spherical detector containing 1 Mt of water, capable of high rate observation of atmospheric neutrino events (30000/y). The ring imaging Cherenkov (RICH) technique is used to measure velocity, momentum and direction of particles produced by neutrinos interacting in water. The detector will be sited outdoors (under a 50 m water shield) in a natural (further excavated) pit, probably in Sicily. Spherical reflecting mirrors focus Cherenkov light produced by secondaries from interacting neutrinos. Photons are detected by 5310 hybrid photodiodes (HPDs) of 1 m diameter each with 396 pads of 45*45 mm/sup 2/ on the photocathode surface, demagnified to 9*9 mm/sup 2/ on the silicon sensor. For most tracks the ring width will be dominated by multiple scattering which should allow momentum to be determined. Hadrons of momentum p

  20. A Metadata-Rich File System

    Energy Technology Data Exchange (ETDEWEB)

    Ames, S; Gokhale, M B; Maltzahn, C

    2009-01-07

    Despite continual improvements in the performance and reliability of large scale file systems, the management of file system metadata has changed little in the past decade. The mismatch between the size and complexity of large scale data stores and their ability to organize and query their metadata has led to a de facto standard in which raw data is stored in traditional file systems, while related, application-specific metadata is stored in relational databases. This separation of data and metadata requires considerable effort to maintain consistency and can result in complex, slow, and inflexible system operation. To address these problems, we have developed the Quasar File System (QFS), a metadata-rich file system in which files, metadata, and file relationships are all first class objects. In contrast to hierarchical file systems and relational databases, QFS defines a graph data model composed of files and their relationships. QFS includes Quasar, an XPATH-extended query language for searching the file system. Results from our QFS prototype show the effectiveness of this approach. Compared to the defacto standard, the QFS prototype shows superior ingest performance and comparable query performance on user metadata-intensive operations and superior performance on normal file metadata operations.

  1. Luminescence characterization of a sodium rich feldspar

    International Nuclear Information System (INIS)

    Correcher, V.; Sanchez M, L.; Garcia G, J.; Rivera, T.

    2006-01-01

    This paper reports on the radioluminescence (RL) and thermoluminescence (TL) properties of a sodium rich feldspar ((Na,K)[AlSi 3 O 8 ]) with a mean molecular composition of orthoclase (Or) and albite (Ab) of Or 1 Ab 99 . Despite the complexity of the luminescence signals of the sample, it is possible to determine six different emission bands at about 300, 380, 420, 460, 550 and 680 nm. The 300 nm emission can be associated to structural defects related to the recombination process in which the Na + ion diffusion-limited is involved. The UV-blue emission band at (i) 380 nm is characteristic of mineral phases containing SiO 4 tetrahedral and could be related to intrinsic defects in the lattice, (ii) the 420 nm band could be associated to the presence of Cu (II) ions placed next to the hole traps or the recombination on a centre formed from a hole-oxygen atom adjacent to two Al atoms (Al-O-Al) and (iii) the 460 nm waveband could be due to the presence of Ti 4+ . The green and red emissions are respectively associated to the presence of Mn 2+ and Fe 3+ ions. The ratio between the relative intensities, peaked at 290 (the more intense waveband) and 550 nm is about 10:1 in both TL and RL; this fact indicates that the efficiency of recombination centres does no changes regardless on the type of the process. (Author)

  2. Investigating equality: The poverty and riches indices

    International Nuclear Information System (INIS)

    Eliazar, Iddo

    2016-01-01

    As noise is omnipresent, real-world quantities measured by scientists and engineers are commonly obtained in the form of statistical distributions. In turn, perhaps the most compact representation of a given statistical distribution is via the mean-variance approach: the mean manifesting the distribution’s ‘typical’ value, and the variance manifesting the magnitude of the distribution’s fluctuations about its mean. The mean-variance approach is based on an underlying Euclidean-geometry perspective. So very often real-world quantities of interest are non-negative sizes, and their measurements yield statistical size distributions. In this paper, and in the context of size distributions, we present an alternative to the Euclidean-based mean-variance approach: a mean-equality approach that is based on an underlying socioeconomic perspective. We establish two equality indices that score, on a unit-interval scale, the intrinsic ‘egalitarianism’ of size distributions: (i) the poverty equality index which is particularly sensitive to the existence of very small “poor” sizes; (ii) the riches equality index which is particularly sensitive to the existence of very large “rich” sizes. These equality indices, their properties, their computation, their application, and their connections to the mean-variance approach – are explored and described comprehensively.

  3. Coal pyrolysis under hydrogen-rich gases

    Energy Technology Data Exchange (ETDEWEB)

    Liao, H.; Sun, C.; Li, B.; Liu, Z. [Chinese Academy of Sciences, Taiyuan (China). State Key Laboratory of Coal Conversion, Institute of Coal Chemistry

    1998-04-01

    To improve the economy of the pyrolysis process by reducing the hydrogen cost, it is suggested to use cheaper hydrogen-rich gases such as coke-oven gas (COG) or synthesis gas (SG) instead of pure hydrogen. The pyrolysis of Chinese Xianfeng lignite which was carried out with real COG and SG at 3-5 MPa, a final temperature of 650{degree}C and a heating rate of 5{degree}C/min in a 10g fixed-bed reactor is compared with coal pyrolysis with pure hydrogen and nitrogen under the same conditions. The results indicate that compared with hydropyrolysis at the same total pressure, the total conversion and tar yields from coal pyrolysis with COG and SG decreases while the unwanted water increases. However, at the same H{sub 2} partial pressure, the tar yields and yields of BBTX, PCX and naphthalene from the pyrolysis of coal with COG and SG are all significantly higher than those of hydropyrolysis. Therefore, it is possible to use COG and SG instead of pure hydrogen. 8 refs., 3 figs., 6 tabs.

  4. Platelet rich fibrin in jaw defects

    Science.gov (United States)

    Nica, Diana; Ianes, Emilia; Pricop, Marius

    2016-03-01

    Platelet rich fibrin (PRF) is a tissue product of autologous origin abundant in growth factors, widely used in regenerative procedures. Aim of the study: Evaluation of the regenerative effect of PRF added in the bony defects (after tooth removal or after cystectomy) Material and methods: The comparative nonrandomized study included 22 patients divided into 2 groups. The first group (the test group) included 10 patients where the bony defects were treated without any harvesting material. The second group included 12 patients where the bony defects were filled with PRF. The bony defect design was not critical, with one to two walls missing. After the surgeries, a close clinically monitoring was carried out. The selected cases were investigated using both cone beam computer tomography (CBCT) and radiographic techniques after 10 weeks postoperatively. Results: Faster bone regeneration was observed in the bony defects filled with PRF comparing with the not grafted bony defects. Conclusions: PRF added in the bony defects accelerates the bone regeneration. This simplifies the surgical procedures and decreases the economic costs.

  5. The HERMES dual-radiator RICH detector

    CERN Document Server

    Jackson, H E

    2003-01-01

    The HERMES experiment emphasizes measurements of semi-inclusive deep-inelastic scattering. Most of the hadrons produced lie between 2 and 10 GeV, a region in which it had not previously been feasible to separate pions, kaons, and protons with standard particle identification (PID) techniques. The recent development of new clear, large, homogeneous and hydrophobic silica aerogel material with a low index of refraction offered the means to apply RICH PID techniques to this difficult momentum region. The HERMES instrument uses two radiators, C sub 4 F sub 1 sub 0 , a heavy fluorocarbon gas, and a wall of silica aerogel tiles. A lightweight spherical mirror constructed using a newly perfected technique to make resin-coated carbon-fiber surfaces of optical quality provides optical focusing on a photon detector consisting of 1934 photomultiplier tubes (PMT) for each detector half. The PMT array is held in a soft steel matrix to provide shielding against the residual field of the main spectrometer magnet. Ring recon...

  6. Structure of neutron-rich nuclei

    International Nuclear Information System (INIS)

    Nazarewicz, W.

    2000-01-01

    Complete text of publication follows. The uncharted regions of the (N,Z) plane contain information that can answer many questions of fundamental importance for science: How many protons and neutrons can be clustered together by the strong interaction to form a bound nucleus? What are the proton and neutron magic numbers of the exotic nuclei? What are the properties of very short-lived exotic nuclei with extreme neutron-to-proton ratios? What is the effective nucleon-nucleon interaction in a nucleus that has a very large neutron excess? Nuclear life far from stability is different from that around the stability line; the promised access to completely new combinations of proton and neutron numbers offers prospects for new structural phenomena. The main objective of this talk is to discuss some of the challenges and opportunities of research with exotic nuclei. The covered topics will include: Theoretical challenges; Skins and halos in heavy nuclei; Shape coexistence in exotic nuclei; Beta-decays of neutron-rich nuclei. (author)

  7. Photoconduction in silicon rich oxide films

    Energy Technology Data Exchange (ETDEWEB)

    Luna-Lopez, J A; Carrillo-Lopez, J; Flores-Gracia, F J; Garcia-Salgado, G [CIDS-ICUAP, Benemerita Universidad Autonoma de Puebla. Ed. 103 D and C, col. San Manuel, Puebla, Pue. Mexico 72570 (Mexico); Aceves-Mijares, M; Morales-Sanchez, A, E-mail: jluna@buap.siu.m, E-mail: jluna@inaoep.m [INAOE, Luis Enrique Erro No. 1, Apdo. 51, Tonantzintla, Puebla, Mexico 72000 (Mexico)

    2009-05-01

    Photoconduction of silicon rich oxide (SRO) thin films were studied by current-voltage (I-V) measurements, where ultraviolet (UV) and white (Vis) light illumination were applied. SRO thin films were deposited by low pressure chemical vapour deposition (LPCVD) technique, using SiH{sub 4} (silane) and N{sub 2}O (nitrous oxide) as reactive gases at 700 {sup 0}. The gas flow ratio, Ro = [N{sub 2}O]/[SiH{sub 4}] was used to control the silicon excess. The thickness and refractive index of the SRO films were 72.0 nm, 75.5 nm, 59.1 nm, 73.4 nm and 1.7, 1.5, 1.46, 1.45, corresponding to R{sub o} = 10, 20, 30 and 50, respectively. These results were obtained by null ellipsometry. Si nanoparticles (Si-nps) and defects within SRO films permit to obtain interesting photoelectric properties as a high photocurrent and photoconduction. These effects strongly depend on the silicon excess, thickness and structure type. Two different structures (Al/SRO/Si and Al/SRO/SRO/Si metal-oxide-semiconductor (MOS)-like structures) were fabricated and used as devices. The photocurrent in these structures is dominated by the generation of carriers due to the incident photon energies ({approx}3.0-1.6 eV and 5 eV). These structures showed large photoconductive response at room temperature. Therefore, these structures have potential applications in optoelectronics devices.

  8. The source rock characters of U-rich granite

    Energy Technology Data Exchange (ETDEWEB)

    Mingyue, Feng; Debao, He [CNNC Key Laboratory of Uranium Resources Exploration and Evaluation Technology, Beijing Research Institute of Uranium Geology (China)

    2012-03-15

    This paper discusses the stratum composition, lithological association, uranium content of crust and the activation, migration, concentration of uranium at each tectonic cycle in South China. The authors point out that the source rock of U-rich granite is U-rich continental crust which is rich in Si, Al and K. The lithological association is mainly composed of terrestrial clastic rocks formation of mudstone and sandstone, mingled with intermediate-acidic, mafic pyroclastic rocks and carbonate rocks formation. During tectonic movements, the rocks had undergone regional metamorphism, migmatitization,