200 research outputs found

    Transforming growth factor-β/Smad - signalling pathway and conjunctival remodelling in vernal keratoconjunctivitis

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    Vernal keratoconjunctivitis (VKC) is a chronic ocular allergic inflammation characterized by corneal complications and the formation of giant papillae. Sma- and Mad-related proteins (Smad) modulate extracellular matrix gene expression during wound healing, inflammation and tissue remodelling.Objective To investigate the relationship between allergic inflammation and TGF-β/Smad signalling pathway, expression in VKC patients and in primary cultured conjunctival fibroblasts exposed to mediators found previously over-expressed in VKC.Methods Smad-2, -3, -7, phospho-(p)Smads, TGF-β1 and -β2 were evaluated in the conjunctiva of normal subjects (CT) and VKC patients by immunohistochemistry. The expression of Smads, pro-collagen I (PIP), TGF-β1, -β2, mitogen-activated protein kinase (p38/MAPK), c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK1/2) were also determined in conjunctival fibroblast cultures exposed to histamine, IL-4, -13, TGF-β1, IFN-γ and TNF-α using immunostaining or RT-PCR.Results Immunostaining for Smad-2, -3, pSmad-2, -3, TGF-β1, -β2 and PIP was significantly increased in VKC stroma compared with CT. In conjunctival fibroblast cultures, Smad-3 and PIP were stimulated by histamine, IL-4, -13 and TGF-β1 exposure, while PIP was reduced by IFN-γ, and TNF-α mRNA expression of Smad-3 was increased by histamine, while Smad-7 was reduced by IL-4. In addition, histamine, IL-4 and TNF-α increased JNK and ERK1/2 expression.Conclusion and Clinical Relevance The TGF-β/Smad signalling pathway is over-expressed in VKC tissues and modulated in conjunctival fibroblasts by histamine, IL-4, TGF-β1 and TNF-α. These mechanisms may be involved in fibrillar collagen production, giant papillae formation and tissue remodelling typical of VKC and might provide new therapeutic targets for its treatment. © 2010 Blackwell Publishing Ltd

    Effects of Th2 cytokines on expression of collagen, MMP-1, and TIMP-1 in conjunctival fibroblasts.

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    PURPOSE. To determine whether cytokines involved in chronic allergic conjunctival disorders may affect formation of giant papillae and tissue remodeling. METHODS. Conjunctival fibroblast cultures were challenged with different concentrations of human recombinant interleukin (IL)-4, IL-13, interferon (IFN)- and tumor necrosis factor (TNF)-. Procollagens I (PIP) and III (PIIIP), matrix metalloproteinase (MMP)-1 and -9, and tissue inhibitor of metalloproteinase (TIMP)-1 were measured in supernatants, and their respective mRNAs were evaluated by RT-PCR. RESULTS. IL-4 and -13 (10 ng/mL) significantly increased production and expression of PIP compared with nonstimulated cells, whereas IFN- elicited the opposite effect, at both the protein and mRNA levels. Both IL-4 and -13 significantly decreased production of MMP-1 and increased that of TIMP-1, whereas TNF- increased production of MMP-1 and -9. Expression of MMP-1 was reduced by IL-4 and increased by the other tested cytokines, whereas expression of TIMP-1 was increased by all tested cytokines. CONCLUSIONS. IL-4 and -13 increased production of collagen and modified the equilibrium between MMP-1 and its inhibitor, TIMP-1. These effects were partially opposed by IFN- and TNF- .( Invest Ophthalmol Vis Sci. 2003;44:183‐189) DOI

    An alternative construction of B-M and B-T unitals in Desarguesian planes

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    We present a new construction of non-classical unitals from a classical unital UU in PG(2,q2)PG(2,q^2). The resulting non-classical unitals are B-M unitals. The idea is to find a non-standard model Π\Pi of PG(2,q2)PG(2,q^2) with the following three properties: 1. points of Π\Pi are those of PG(2,q2)PG(2,q^2); 2. lines of Π\Pi are certain lines and conics of PG(2,q2)PG(2,q^2); 3. the points in UU form a non-classical B-M unital in Π\Pi. Our construction also works for the B-T unital, provided that conics are replaced by certain algebraic curves of higher degree.Comment: Keywords: unital, desarguesian plane 11 pages; ISSN: 0012-365

    Urokinase Plasminogen Activator, uPa Receptor, and Its Inhibitor in Vernal Keratoconjunctivitis

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    PURPOSE. Plasminogen activators play a role, not only in fibrinolysis but also in events such as chemotaxis, collagen degradation, and cell spreading. The serine protease urokinase (uPA) is a potent chemoattractant for leukocytes that may be involved in the pathogenesis of severe forms of allergic conjunctivitis such as vernal keratoconjunctivitis (VKC). METHODS. Tear and peripheral blood samples were obtained from 20 patients with active VKC and from 19 normal subjects who formed the control group. Levels of plasminogen activity, uPA, tissue plasminogen activator (tPA), and their inhibitor, plasminogen activator inhibitor type-1 (PAI-1) were measured in tears and plasma of patients with VKC. The presence of tPA, uPA, and urokinase receptor (uPAR) in conjunctival tissues were evaluated by immunohistochemistry. uPA, uPAR, and PAI-1 expression and production were measured in conjunctival epithelial cell and fibroblast cultures treated with cytokines. RESULTS. Tear levels of uPA and tPA and tear plasminogen activity levels were significantly greater in patients with VKC than in control subjects. Increased staining for uPA and uPAR was found in VKC tissues compared with normal conjunctiva. Both conjunctival epithelial cells and fibroblasts demonstrated an increased expression of uPAR after exposure to IL-4 or -13, whereas uPA was highly expressed by epithelial cells exposed to IL-4. PAI-1 levels in culture medium were increased in IL-4-exposed epithelial cells compared to nonstimulated cells and were decreased in fibroblast culture. CONCLUSIONS. Increased expression of fibrinolytic system components and imbalance between plasminogen activators and PAI may be involved in the pathogenesis of severe allergic conjunctivitis, thus contributing to inflammatory cell migration and tissue remodeling. (Invest Ophthalmol Vis Sci. 2005;46: 1364‐1370) DOI:10.1167/iovs.04-119

    In vitro and in vivo biological performance of modified gellan gum-based hydrogels for nucleus pulposus tissue engineering

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    Ionic- (iGG-MA) and photo-crosslinked (phGG-MA) methacrylated gellan gum hydrogels have been proposed as biomaterials for supporting nucleus pulposus (NP) regeneration and/or repair. In this study, the mechanical stability and biocompatibility of these hydrogels have been evaluated in vitro. Human intervertebral disc cells obtained from herniated patients were cultured within both hydrogels, for 1–21 days. Dynamic mechanical analysis and biological characterization (Live/ Dead assay, ATP and DNA quantification, PCR and immunocytochemistry) were performed after specific times of culturing. The in vitro study showed that both cell loading and culturing time do not affect the mechanical properties of hydrogels. In addition, the iGG-MA and phGG-MA hydrogels showed to be effective on supporting cells encapsulation and viability up to 21 days of culturing. In vivo biocompatibility screening was also performed, by subcutaneous implantation of both hydrogels in Lewis rats for the period of 10 and 18 days. Haematoxylin & eosin staining revealed that the hydrogels do not elicit necrosis, calcification or acute inflammatory reaction. The present study demonstrates that the iGG-MA and phGG-MA hydrogels support cells encapsulation and viability, and are well-tolerated, stable and non-cytotoxic in vitro and in vivo, thus possessing promising features for finding application as viable NP substitutes

    Mechanical performance and biocompatibility study of methacrylated Gellan gum hydrogels with potential for nucleus pulposus regeneration

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    Methacrylated gellan gum hydrogels, obtained either by ionic- (iGGMA) and photo-crosslinking (phGG-MA), have been investigated as potential biomaterials for supporting nucleus pulposus (NP) regeneration and/or repair [1,2]. In previous work, some advantages were attributed to GG-MA hydrogels, such as: (i) the possibility to control endothelial cells infiltration and blood vessel ingrowth’s, (ii) tunable and improved mechanical properties, and (iii) in situ gelation, within seconds to few minutes. In this study, the mechanical and biological performance of these hydrogels was firstly evaluated in vitro. Human intervertebral disc (hIVD) cells obtained from herniated patients were cultured within both hydrogels, for 1 up to 21 days. Dynamic mechanical analysis and biological characterization (calcein-AM staining, ATP and DNA quantification and PCR) were performed after specific times of culturing. A biocompatibility study was also performed in vivo, by subcutaneous implantation of acellular iGG-MA and phGG-MA hydrogels in Lewis rats for the period of 10 and 18 days. Tissue response to the hydrogels implantation was determined by histological analysis (haematoxylin-eosin staining). The in vitro study showed that both cell loading and culturing time do not have an effect on the mechanical properties of the hydrogels. Regarding their biological performance, the iGG-MA and phGG-MA hydrogels showed to be effective on supporting hIVD cells encapsulation and viability up to 21 days of culturing. Human IVD cells were homogeneously distributed within the hydrogels and maintained its round-shape morphology during culturing time. The in vivo biocompatibility study showed that iGG-MA and phGG-MA hydrogels do not elicit any deleterious effect, as denoted by the absence of necrosis and calcification, or acute inflammatory reaction. A thin fibrous capsule was observed around the implanted hydrogels. The results presented in this study indicate that the iGG-MA and phGG-MA hydrogels are stable in vitro and in vivo, support hIVD cells encapsulation and viability, and were found to be well-tolerated and non-cytotoxic in vivo, thus being potential candidates for NP regeneration

    Fractional-order operators: Boundary problems, heat equations

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    The first half of this work gives a survey of the fractional Laplacian (and related operators), its restricted Dirichlet realization on a bounded domain, and its nonhomogeneous local boundary conditions, as treated by pseudodifferential methods. The second half takes up the associated heat equation with homogeneous Dirichlet condition. Here we recall recently shown sharp results on interior regularity and on LpL_p-estimates up to the boundary, as well as recent H\"older estimates. This is supplied with new higher regularity estimates in L2L_2-spaces using a technique of Lions and Magenes, and higher LpL_p-regularity estimates (with arbitrarily high H\"older estimates in the time-parameter) based on a general result of Amann. Moreover, it is shown that an improvement to spatial CC^\infty -regularity at the boundary is not in general possible.Comment: 29 pages, updated version, to appear in a Springer Proceedings in Mathematics and Statistics: "New Perspectives in Mathematical Analysis - Plenary Lectures, ISAAC 2017, Vaxjo Sweden

    Disinfection of ocular cells and tissues by atmospheric-pressure cold plasma

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    Low temperature plasmas have been proposed in medicine as agents for tissue disinfection and have received increasing attention due to the frequency of bacterial resistance to antibiotics. Our previous studies (1) demonstrated that atmospheric-pressure cold plasma (APCP) generated by a new portable device that ionizes a flow of helium gas inactivated ocular pathogens in vitro. This study explored whether APCP inactivates ocular pathogens without causing significant tissue damage. We tested the APCP effects on cultured Pseudomonas aeruginosa, Staphylococcus aureus, A. fumigatus, ocular cells (conjunctival fibroblasts and keratocytes) and ex-vivo cornea. Exposure to APCP for 0.5–5 min significantly reduced microbial viability (colony-forming units) but not human cell viability (MTT assay and Tunel analysis). Since our previous study indicated that exposure to plasma increases intracellular reactive oxygen species (ROS) production, ROS levels in APCP exposed microorganisms and keratocytes were analyzed by 2’,7’-dichlorofluorescein diacetate (HDCF-DA) fluorescence. The potential genotoxic effects of plasma on cells and tissues were evaluated by analyses of thymine dimers (TD), genes and proteins involved in DNA damage and repair (OGG1, GPX, NRF2) at set time intervals. High levels of intracellular reactive oxygen species (ROS) were found in exposed microorganisms and cells. Immunoassay confirmed no induction of thymine dimers in corneal tissues. Conversely, a transient expression of genes and proteins recruited following oxidative stress was determined in ocular cells and corneas by qRT-PCR and Western blotting. In conclusion, a short application of APCP appears to be an efficient and rapid ocular disinfectant with ROS production likely causing pathogen killing and no substantial effects on ocular cells and tissues. The same APCP treatment to conjuntival fibroblasts and keratocytes caused a time-restricted formation of ROS and a change in some stress-response genes

    In vitro concurrent endothelial and osteogenic commitment of adipose-derived stem cells and their genomical analyses through CGH array: novel strategies to increase the succesfull engraftement of a tissue engineered bone grafts

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    In the field of tissue engineering, adult stem cells are increasingly recognized as an important tool for in vitro reconstructed tissue-engineered grafts. In the world of cell therapies, mesenchymal stem cells from bone marrow or adipose tissue are undoubtedly the most promising progenitors for tissue engineering applications. In this setting, adipose-derived stem cells (ASC) are generally similar to those derived from bone marrow and are most conveniently extracted from tissue removed in elective cosmetic liposuction procedures; they also show a great potential for endothelization. The aim of the present work was to investigate how the co-commitment into a vascular and bone phenotype of ASC could be a usefull tools for improving the in vitro and in vivo reconstruction of a vascularized bone graft. Human ASC obtained from abdominoplasty procedures were loaded in a hydroxyapatite clinical-grade scaffold, co-differentiated and tested for proliferation, cell distribution, and osteogenic and vasculogenic gene expression. The chromosomal stability of the cultures was investigated using the CGH array for 3D cultures. ASC adhesion, distribution, proliferation and gene expression not only demonstrated a full osteogenic and vasculogenic commitment in vitro and in vivo, but also showed that endothelization strongly improves their osteogenic commitment. In the end, genetic analyses confirmed that no genomical alteration in long-term in vitro culture of ASC in 3D scaffolds occurs
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