The final publication is available at http://dx.doi.org/10.1016/j.jtcvs.2013.11.054. Motoaki Ohnaka, Akira Marui, Kenichi Yamahara, Kenji Minakata, Kazuhiro Yamazaki, Motoyuki Kumagai, Hidetoshi Masumoto, Shiro Tanaka, Tadashi Ikeda, Ryuzo Sakata, Effect of microRNA-145 to prevent vein graft disease in rabbits by regulation of smooth muscle cell phenotype, The Journal of Thoracic and Cardiovascular Surgery, Volume 148, Issue 2, August 2014, Pages 676-682.e2, ISSN 0022-5223. / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18544号 / 医博第3937号 / 新制||医||1006(附属図書館) / 31444 / 京都大学大学院医学研究科医学専攻 / (主査)教授 木村 剛, 教授 野田 亮, 教授 瀬原 淳子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Experiments were carried out with the objectives of developing propagation protocols for Uapaca kirkiana and Pappea capensis tree species of southern Africa, and evaluating the graft compatibility within U. Kirkiana tree clones, provenances and species. Reverse phase high performance liquid chromatography (RP-HPLC), Folin-Ciocalteau reagent, fluorescence microscopy and callus fusion methodologies were used to diagnose graft compatibility. Results indicated that U. Kirkiana culture asepsis was achieved with 0.1% w/v mercuric chloride HgCl2) and using pre-conditioned grafted trees. Sodium hypochlorite (NaOCl) improved P. Capensis seed asepsis and germination, and discarding floating seeds improved germination. Murashige and Skoog (MS) medium with 2.0 mg l-1 benzylaminopurine (BAP) and 0.3 mg l-1 casein hydrolysate (CH) was superior in shoot multiplication and 0.5 mg l-1 indole-3-butyric acid (IBA) for rooting of P. Capensis microshoots. For somatic embryogenesis, three quarter strength MS medium with 0.05 mg l-1 thidiazuron (TDZ) and 0.3 mg l-1 CH, or 0.2 mg l-1 BAP with 0.3 mg l-1 CH, were effective in germination of P. Capensis somatic embryos. For U. Kirkiana lateral shoot explants, shoot multiplication was superior on three quarter strength MS medium with 0.1 mg l-1 BAP and 0.3 mg l-1 CH. Rooting of micro-cuttings (36%) was achieved on ½ MS with 2.5 mg l-1 IBA. RP-HPLC, fluorescence microscopy and callus fusion studies showed that phenolic compounds play a major role in U. Kirkiana graft incompatibility. Less graft compatible combinations showed an increase in phenol deposits above the union and graft incompatibility was more pronounced above the union than below the union. Proliferation of parenchymatous tissues was better below the union than above the union. Fluorescence microscopy showed presence of flavonoids and polymers above the union of less graft compatible combinations. The chromatograms showed that ferulic acid was abundant and responsible for wood discolouration. The chromatograms also isolated ρara-coumaric acids which were predominant above the union of the less compatible combinations. Therefore, ρara-coumaric acids, flavonoids and polymers were implicated in graft incompatibility of U. kirkiana trees. Copyright / Thesis (PhD)--University of Pretoria, 2008. / Plant Production and Soil Science / unrestricted

Conventionally, adherent cells are cultured in vitro using flat 2D cell culture trays. However the 2D cell culture method is tedious, unreliable and does not replicate the complexity of the 3D dynamic environment of native tissue. Nowadays 3D scaffolds can be used to culture cells. However a number of challenges still exist, including the need for destructive enzymes to release confluent cells. Poly(Nisopropylacrylamide) (PNIPAAm), a temperature responsive polymer, has revolutionised the cell culture fraternity by providing a non-invasive means of harvesting adherent cells, whereby confluent cells can be spontaneously released by simply cooling the cell culture medium and without requiring enzymes. While PNIPAAm monolayer cell culturing is a promising tool for engineering cell sheets, the current technology is largely limited to the use of flat 2D substrates, which lacks structural and organisational cues for cells. The aim of this project was to develop a 3D PNIPAAm scaffold which could be used efficiently for non-invasive 3D culture of adherent cells. This project was divided into three phases: Phase 1 (preliminary phase) involved development and characterisation of cross-linked PNIPAAm hydrogels; Phase 2 involved development and characterisation of PNIPAAm grafted 3D non-woven scaffolds, while Phase 3 focused on showing proof of concept for non-invasive temperature-induced cell culture from the 3D PNIPAAm grafted scaffolds. In Phase 1, PNIPAAm was cross-linked with N,N’-methylene-bis-acrylamide (MBA) using solution free-radical polymerisation to form P(PNIPAAm-co-MBA) hydrogels. A broad cross-link density (i.e. 1.1 - 9.1 Mol% MBA) was investigated, and the effect of using mixed solvents as the co-polymerisation medium. The P(PNIPAAm-co-MBA) gels proved unsuitable as a robust cell culture matrix, due to poor porosity, slow swelling/deswelling and poor mechanical properties. Subsequently, in Phase 2, polypropylene (PP), polyethylene terephthalate (PET), and nylon fibers were processed into highly porous non-woven fabric (NWF) scaffolds using a needle-punching technology. The NWF scaffolds were grafted with PNIPAAm using oxyfluorination-assisted graft polymerisation (OAGP). The OAGP method involved a 2 step process whereby the NWF was first fluorinated (direct fluorination or oxyfluorination) to introduce new functional groups on the fibre surface. The functionalised NWF scaffolds were then graft-polymerised with NIPAAm in an aqueous medium using ammonium persulphate as the initiator. Following oxyfluorination, new functional groups were detected on the surface of the NWF scaffolds, which included C-OH; C=O; CH2-CHF, and CHF-CHF. PP and nylon were both easily modified by oxyfluorination, while PET displayed very little changes to its surface groups. Improved wetting and swelling in water was observed for the oxyfluorinated polymers compared to pure NWF scaffolds. PP NWF showed the highest graft yield followed by nylon and then PET. PNIPAAm graft yield on the PP NWF was ~24 ±6 μg/cm2 on grafted pre-oxyfluorinated NWF when APS was used; which was found to be significantly higher compared to when pre-oxyfluorinated NWF was used without initiator (9 ±6 μg/cm2, p= 1.7x10-7); or when grafting was on pure PP with APS (2 ±0.3 μg/cm2, p = 8.4x10-12). This corresponded to an average PNIPAAm layer thickness of ~220 ±54 nm; 92 ± 60 nm; and 19 ± 3 nm respectively. Scanning electron microscopy (SEM) revealed a rough surface morphology and confinement of the PNIPAAm graft layer to the surface of the fibers when oxyfluorinated NWF scaffolds were used, however when pure NWF scaffolds were used during grafting, homopolymerisation was observed as a loosely bound layer on the NWF surface. The OAGP method did not affect the crystalline phase of bulk PP as was determined by X-ray diffraction (XRD), however, twin-melting thermal peaks were detected from DSC for the oxyfluorinated PP and PP-g-PNIPAAm NWF which possibly indicated crystal defects. Contact angle studies and microcalorimetric DSC showed that the PP-g-PNIPAAm NWF scaffolds exhibited thermoresponsive behaviour. Using the 2,2-Diphenyl-1-1-picrylhydrazyl (DPPH) radical method and electron-spin resonance (ESR), peroxides, as well as trapped long-lived peroxy radicals were identified on the surface of the oxyfluorinated PP NWF, which are believed to be instrumental in initiating graft polymerisation from the NWF. A free radical mechanism which is diffusion controlled was proposed for the OAGP method with initiation via peroxy radicals (RO•), as well as SO4•- and OH• radicals, whereby the latter result from decomposition of APS. In Phase 3 of this study, proof-of-concept is demonstrated for use of the PNIPAAm grafted NWF scaffolds in non-invasive culture of hepatocytes. Studies demonstrated that hepatocyte cells attached onto the 3D PNIPAAm scaffolds and remained viable in culture over long periods. The cells were released spontaneously and non-destructively as 3D multi-cellular constructs by simply cooling the cell culture medium from 37°C to 20°C, without requiring destructive enzymes. The PP-g- PNIPAAm NWF scaffolds performed the best in 3D cell culture. Additionally the CSIR is developing a thermo responsive 3D (T3D) cell culturing device, whereby the 3D thermo responsive NWF scaffolds are used in the bioreactor for cell culture. Temperature-induced cell release was also verified from the 3D Thermo responsive scaffolds in the bioreactor. This technology could lead to significant advances in improving the reliability of the in vitro cell culture model. Please cite as follows: Chetty, AS 2012, Thermoresponsive 3D scaffolds for non-invasive cell culture, PhD thesis, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-06112013-151344/ > D13/4/713/ag / Thesis (PhD)--University of Pretoria, 2012. / Chemical Engineering / unrestricted

No description available.

Purpose of Review: This article reviews the latest data on unprotected left main (ULM) percutaneous coronary intervention (PCI) versus coronary artery bypass graft (CABG) surgery, with a focus on the NOBLE and EXCEL trials. Recent Findings: In EXCEL trial, the primary endpoint at 3 years was 15.4% in the PCI group and 14.7% in the CABG group (p = 0.02 for non-inferiority of PCI versus CABG). In NOBLE, the primary endpoint at 5 years was 28% and 18% for PCI and CABG, respectively (HR 1.51, CI 1.13–2.0, which did not meet the criteria for non-inferiority of PCI to CABG; p for superiority of CABG was 0.0044). Higher repeat revascularization and non-procedural myocardial infarction were noted in PCI group but there was no difference in all-cause or cardiac mortality between the two groups. Summary: A heart team approach with appropriate patient selection, careful assessment of LM lesions, and meticulous procedural technique makes PCI a valid alternative to CABG for ULM stenosis.

Bioactive agents are proteins that regulate cellular activities including cell migration, proliferation, differentiation and matrix synthesis. Over the last decades there has been a focused effort to understand how these agents influence repair or regeneration of bony defects. Platelet derived growth factor (PDGF) has potent chemotactic and angiogenic properties. Bone morphogenetic protein (BMP) is a known factor for osteoblasts. This study evaluated the impact of recombinant human PDGF and BMP-2 on resolution of critical bony defects (2 mm) using mouse calvarial bone cultures. Calvaria from 5-7 day neonatal CD-1 mice were dissected and cultured in Dulbecco’s Modified Eagle’s Medium under sterile conditions. In the first experiment, two different delivery systems to deliver PDGF - freeze-dried bone allograft and beta- tricalcium phosphate were compared. The second experiment analyzed bone formation in response to BMP-2 in the presence or absence of freeze-dried bone allograft. The media was changed every 2 days and the spent media were analysed for calcium release. At the end of three weeks the calvaria were processed for histological observation, biochemical analyses and neutral red staining. The results show higher bone formation in response to BMP-2 than PDGF. The presence of allograft inhibits this response. We found B-TCP to be a better delivery agent for PDGF compared to freeze-dried bone allograft. The histologic assessment showed development of new bone through intramembranous pathway that replicates native bone development in presence of BMP-2. In conclusion our study proves that incorporation of two bioactive agents- PDGF and BMP-2 in an osteoconductive scaffold can induce repair and new bone formation in mouse calvarial bone cultures. / 2020-07-30T00:00:00Z

La peau est un « avant poste » fascinant du système immunitaire. Elle forme une barrière entre l'environnement extérieur et l’organisme. Elle est aussi le point d'entrée pour les agents pathogènes, contre lesquels le système immunitaire organise des réponses adaptatives. Les acteurs de l'immunité innée de la peau contrôlent l'invasion des pathogènes et perçoivent également des changements environnementaux physiques et chimiques directs. Plusieurs composants du système immunitaire, tels que des cellules dendritiques (DCs), les macrophages (MΦ) et les mastocytes (MCs), participent à l'éradication des pathogènes et à l'initiation des réponses mémoires adaptatives. Ce qui permet une mobilisation rapide des cellules T effectrices ainsi que la sécrétion des anticorps par les cellules B à la suite d’une seconde exposition aux agents pathogènes. Les MCs qui sont des cellules résidentes du derme, jouent un rôle déterminant dans la libération de signaux d’alertes et sont classiquement considérés comme des cellules effectrices de la réaction allergique cutanée liée à l'IgE. Plusieurs observations récentes indiquent que les MCs seraient aussi impliqués dans les processus immunorégulateurs lors de l'initiation des réponses immunitaires adaptatives, dans le maintien de la tolérance périphérique aux composants de la peau et dans la régénération de la peau au cours des processus de cicatrisation. Cependant, les interactions entre les MCs et d'autres cellules immunitaires innées et adaptatives recrutées dans des conditions inflammatoires cutanées n'ont pas été élucidées en détail. Dans ce travail, nous décrivons l'utilisation d'une nouvelle souris possédant des MCs fluorescents (RMB), dans laquelle nous avons marqué les MCs FcεRI+ avec un marqueur fluorescent rouge tomato (TdT) et avec un système d'ablation conditionnelle basé sur l'expression concurrente du récepteur de la toxine diphtérique (DTR). Avec ces souris RMB, nous avons visualisé la dynamique des MCs et nous avons suivi les interactions entre les MCs et les lymphocytes T régulateurs (Tregs) après l'activation des MCs par l'IgE, dans une réaction inflammatoire typique de l'anaphylaxie cutanée passive (PCA). Dans un second volet d’étude, nous avons évalué le rôle des MCs lors d'un modèle expérimental de la greffe de peau de l'oreille, afin de révéler leur influence dans la cinétique de rejet ou prise de greffe du transplant. Nous avons constaté que 1) l'activation et la dégranulation des MCs induites par le pontage du récepteur FcεRI via des IgE couplées à un antigène multivalent sont les seules responsables de la réaction de PCA, et induisent le recrutement de Tregs ayant une grande motilité sur le site de l'inflammation. Nous avons constaté dans ces conditions, que les MCs restent immobiles, et que les Tregs établissent des contacts dynamiques avec les MCs dans le derme. 2) En outre, nous avons mis en place un modèle pour identifier les paramètres moléculaires de l'interaction MC-Treg et avons constaté que le complexe de l'antigène avec l'IgE peut être présenté aux Tregs en association avec les molécules du complexe majeur d'histocompatibilité de classe II, permettant la formation des contacts stables MC-Treg. 3) En utilisant un modèle de transplantation de la peau in vivo, nous avons montré que l'ablation conditionnelle des MCs conduit à une accélération du rejet du greffon dans le cas d'une transplantation en présence d’une disparité d’antigènes d’histocompatibilité mineurs depuis une souris mâle sur une souris femelle. Nous avons également constaté un impact inattendu de l'ablation des MCs dans la greffe de peau en l’absence de disparité antigénique d'une souris femelle sur une souris femelle, conduisant à un rejet rapide. Les MCs semblent donc être essentiels pour la cicatrisation et la régénération tissulaire après greffe. (...) / The skin is a fascinating outpost of the immune system. It performs a barrier function between the outside environment and the inner body and is also a port of entry for pathogens against which the immune system mounts adapted responses. The skin innate immune defenses control pathogen invasion and perceive also direct physical and chemical environmental changes. Several component of the immune system such as dendritic cells (DC), macrophages (MΦ) and mast cells (MC) participate in initial pathogen clearance and in initiating adaptive memory responses, allowing rapid mobilization of effector T cells and secretion of B cellderived antibodies after secondary pathogen challenge. MCs residing in the dermis exert a determinant alert function through the liberation of various factors and are classically considered as effector cells in the IgE-mediated cutaneous allergic reaction. As emerging now, MC are also involved in immunoregulatory processes during the initiation of adaptive immune responses, the maintenance of peripheral tolerance to skin components and skin regeneration during wound healing. Yet, the crosstalks between MCs and other innate and adaptive immune cells recruited during cutaneous inflammatory conditions have not been elucidated in detail. Here, we report the use of a novel Mast cell fluorescent reporter mouse (RMB), in which we tagged FcεRI+ MCs, with red fluorescence marker tomato (Tdt) and with a conditional ablation system based on concurrent diphtheria toxin receptor (DTR) expression. Using these RMB mice, we visualized MC dynamics and monitored MC interactions with regulatory T lymphocytes (Tregs) after IgE-mediated activation of MCs, in a typical passive cutaneous anaphylaxis (PCA) inflammatory reaction. Using another setting, we further assessed the role of MC during experimental ear skin grafting to reveal their potential influence in skin grafting and rejection. We found that 1) the activation and degranulation of MCs induced by FcεRI crosslinking by multivalent IgE is solely responsible for the PCA reaction and induces the recruitment of highly motile regulatory T cells (Tregs) to the site of inflammation. In these conditions, we found that MC remain sessile and Tregs establish dynamic contacts with MC in the dermis. 2) Further we set up a model system to reveal the molecular requirement for MC-Treg interaction and found that antigen complexed with IgE were able to be presented to Treg in association with major histocompatibility complex class II molecules allowing the formation of stable MC-Treg contacts. 3) Using in vivo skin transplantation model, we showed that conditional ablation of MCs leads to an acceleration of skin transplant rejection in sex-mismatched model (male skin transplant to female). We also found an unexpected impact of MC conditional ablation in sex-matched skin graft (female skin transplant to female) leading to rapid rejection, implying that MCs are essential for the wound healing reaction and the regeneration of tissue continuity after grafting. The aforementioned results point out to an important immunoregulatory role of MC beyond their classically described activator functions in inflamed tissues. The fact that MC constantly interact with Treg during inflammatory processes suggest that MCs could participate in skin homeostasis by exerting tolerogenic functions. These functions remain to be elucidated at the molecular level as presented in the discussion.

Les Microvésicules (MVs) sont des marqueurs circulants de l’activation cellulaire au cours de la dysfonction du greffon et l’athérothrombose. Les MVs de la cellule bêta ou intratissulaires vasculaires, sont peu connues. Le suivi longitudinal de 19 patients transplantés d’îlots pancréatiques par les MV- PSA-NCAM+ sanguines témoigne d’une altération précoce du greffon avant l’identification des marqueurs clinique et biologique de la perte du greffon. La cinétique de libération des MVs leucocytaires, endothéliales et hépatiques suggère leur intérêt pour l’identification de la cause de la perte du greffon et pour la surveillance de l'immunosuppression. Après validation d’une méthode d’extraction douce des MVs tissulaires sur des plaques d’athérome, nous avons mesuré l’accumulation de MVs pro-sénescentes dans l’aorte de rats âgés, qui étaient réduite par l’ingestion d’EPA : DHA (6 :1), avec une baisse des propriétés pro-sénescentes identifiée sur les cellules endothéliales d’artères coronaires en culture. Le contrôle des MV nocives par cytoprotecteur du vaisseau réduirait la sénescence endothéliale. / Microvesicles (MVs) are circulating markers that reflect cellular activation during graft dysfunction and atherothrombosis. Data on graft tissue MVs or vessel are scarce. The longitudinal follow-up of 19 patients with pancreatic islets transplants showed that circulating MV-PSA-NCAM+ typifying the early graft loss of islet graft prior its detection using the clinical and biological markers of graft loss. In addition, the kinetics release of leukocyte, endothelial and hepatic MV suggest their interest in identifying the cause of graft loss and in monitoring of immunosuppression. Using a new tissular MV mild extraction process validated with arteriosclerotic plaques, we evidenced an accumulation of pro-senescent MVs in the aorta of old rats that was significantly reduced by EPA: DHA (6 :1) intake as well as their pro-senescent properties on coronary artery endothelial cell cultures. Altogether, the pharmacological control of the release of noxious MVs using vessel cytoprotectors would limit the consequences of endothelial senescence.

Graft Versus Host Disease (GVHD) is a major inflammatory complication of hematopoietic stem cell transplantation (HSCT). Such transplantations are lifesaving in treating certain conditions, such as acute myeloid leukemia, acute lymphocytic leukemia, myelodysplastic syndrome, aplastic anemia, and thalassemia. However, the subsequent presentation of GVHD can pose a lethal threat, placing the patient’s life at risk, once again. The inflammatory response of the graft’s adaptive immunity towards the host’s native cells in GVHD is said to trigger a cytokine storm. Despite its widespread use both colloquially and in the medical field, criteria for “cytokine storms” do not exist. For this reason, a meta-analysis is being conducted that examines various cytokine levels of several different disease conditions, including acute respiratory distress syndrome, chimeric antigen receptor T cells, Crohn’s disease, SARS CoV-2, rheumatoid arthritis, sepsis, and graft versus host disease. The purpose of this study is to analyze a subset of data within this larger meta-analysis, specifically interleukin-6 (IL-6) levels in GVHD. Herein, I discuss the role of IL-6 in the pathogenesis of GVHD, examine the levels of IL-6 in varying stages of GVHD, and propose future directions for using IL-6 inhibition as a treatment for GVHD.

No description available.