Typescript. / Thesis (Ph. D.)--University of Hawaii, 1968. / Bibliography: leaves 93-98. / xii, 98 l graphs

Typescript. / Thesis (Ph. D.)--University of Hawaii, 1964. / Bibliography: leaves [80]-86. / vi, 86 l mounted illus

Zugl.: Giessen, Univ., Diss., 2008 / Text engl.

Zugl.: Giessen, Univ., Diss., 2008

Objective: To assess the safety and efficacy of lyophilized milled human amnion as a wound dressing of experimentally created equine distal limb wounds. Animals: Four clinically normal adult horses (3 Thoroughbred and 1 Paint, median age 11 years) obtained via donation. Procedures: One forelimb of each horse was randomly assigned to the treatment group, and the contralateral limb was assigned as the control. Full-thickness skin wounds were created on each metacarpus. Treatment limb wounds were dressed with lyophilized, milled, human-derived amnion material delivered under triple antibiotic ointment. Control wounds were dressed with triple antibiotic ointment. All wounds were covered in non-adherent dressings and distal limb bandages were applied. Digital photographs were taken of the wounds at each bandage change, performed every 2-4 days throughout a 98-day study period. Biopsies were collected at days 7, 21, 35, and 84. Results: One horse developed unilateral cellulitis that resolved with additional treatment. All treatment limbs exhibited an inflammatory response characterized by focal edema and discharge from the wounds. Wounds were completely epithelialized in control limbs sooner than treatment limbs in all horses, although there was no statistical difference between control (mean 46.8 days) and treatment (mean 51.8 days) wounds. Histologic scores were better in control wounds than in amnion-treated wounds at all time points. Conclusions and Clinical Relevance: Because wounds treated with amnion material in this study exhibited an inappropriate inflammatory response that resulted in delayed time to wound closure, human lyophilized milled amnion is not recommended for use in equine wound management. / Master of Science

Pendant la grossesse, les aquaporines (AQPs) exprimées au sein des membranes fœtales sont essentielles pour assurer l’homéostasie du volume de liquide amniotique (LA), mais leur régulation par l’insuline n’a jamais été explorée chez les femmes diabétiques.Le but de notre étude était de préciser le rôle des AQPs 1, 3, 8 et 9 au sein des membranes fœtales chez des parturientes diabétiques et d’étudier la régulation de leur expression par l’insuline.A partir des 129 membranes fœtales, réparties selon 4 populations (36 témoins, 35 diabètes de type 1 (DT1), 17 diabètes de type 2 (DT2) et 41 diabètes gestationnels (DG)), nous avons établi un profil d’expression qualitatif et quantitatif des gènes des AQPs. Dans un second temps, nous avons étudié la régulation par l’insuline de l’expression des AQPs 3 et 9 au sein d’explants d’amnion et de chorion. L’expression ARN et protéique des AQPs au sein de nos différents fragments de membranes fœtales a été étudiée par RT-PCR quantitative et ELISA. Des membranes fœtales issues de grossesses non pathologiques, séparées en ses 2 feuillets (amnion et chorion), ont été utilisées pour étudier la régulation de l’expression des gènes des AQPs 3 et 9 par l’insuline ainsi que la voie de signalisation de l’insuline au sein de l’amnion. Un test au glycérol tritié a permis l’étude fonctionnelle de l’insuline sur les AQPs. Un inhibiteur de la phosphatidyl-inositol 3-kinase (PI3K) est utilisé pour analyser le signal intracellulaire de l’insuline.L’expression du gène de l’AQP 3 est significativement plus faible dans les groupes DT2 et DG. Au sein d’explants de membranes fœtales non diabétiques, il a été observé au sein de l’amnion (mais pas du chorion), une répression significative par l’insuline de l’expression ARN et protéique des gènes AQPs 3 et 9 qui est bloquée par l’inhibiteur de PI3K. Au sein des membranes fœtales, la répression de l’AQP 3 observée in vivo, est permise par l’hyperinsulinisme connu des patientes atteintes de DT2 ou de DG. / During pregnancy, aquaporins (AQPs) expressed in fetal membranes are essential for controlling the homeostasis of the amniotic volume, but their regulation by insulin was never explored in diabetic women.The aim of our study was to investigate the involvement of AQP 1, 3, 8, and 9 expressed in fetal membranes in diabetic parturient women, and the control of their expression by insulin.From 129 fetal membranes in 4 populations, (controls, type 1 (T1D), type 2 (T2D) and gestational diabetes (GD)), we established an expression AQPs profile. In a 2nd step, the amnion was used to study control of the expression and functions of AQPs 3 and 9 by insulin.The expression of transcripts and proteins of AQPs was studied by qRT-PCR and ELISA. We analysed the regulation by insulin of the expression of AQPs 3 and 9 in the amnion. A tritiated glycerol test enabled us to measure the impact of insulin on the functional characteristics. Using an inhibitor of phosphatidylinositol 3-kinase (PI3K) we analysed the insulin intracellular signaling pathway.Expression of AQP3 protein was significantly weaker in groups T2D and GD. In non-diabetic fetal membranes, we showed for the amnion (not for the chorion) a significant repression by insulin of the ARN expression of AQPs 3 and 9, which was blocked by PI3K inhibitor.In fetal membranes, the repression of AQP3 protein expression and functions observed in vivo is allowed by the hyper-insulinism described in pregnant women with T2D or GD.

Zugl.: Giessen, Univ., Diss., 2009

Freie Universiẗat, Diss., 2006--Berlin. / Dateiformat: zip, Dateien im PDF-Format.

Cancer procoagulant (EC 3.4.22.26) is an enzyme that is derived from tumour and foetal tissue, but not normal tissue. It is a direct activator of factor X and has been isolated from amnion-chorion membranes as well as from extracts and cells from human melanoma. The presence of cancer procoagulant has been associated with the malignant phenotype, as well as having a particularly high activity in metastatic cells. Cancer procoagulant activity is elevated in the serum of early stage breast cancer patients and decreased to normal in the advanced stages of the disease. In this study, cancer procoagulant was successfully isolated from amnion-chorion membranes and purified to homogeneity. The molecular weight of cancer procoagulant was determined using SDS-PAGE and was found to be 68 kDa. Cancer procoagulant was delipidated and it was shown that its activity was increased by the presence of lipids in a dose-dependent manner. Recovery of cancer procoagulant after delipidation is poor, consequently, a larger mass of sample is required to obtain sufficient amounts of delipidated material for N-terminal amino acid analysis. The optimum pH of cancer procoagulant was determined to be pH 8 and its optimal temperature was found to be 50°C. Novel synthetic substrates were designed to assay for cancer procoagulant activity. Currently, 2 potential candidates have been identified, namely, PQVR-AMC and AVSQSKP-AMC. Cancer procoagulant-induced expression of cytokines is differently modulated in the less aggressive MCF-7 cell line as compared to the metastatic and more aggressive MDA-MB-231 cell line. There are marked similarities in the inflammatory response produced by cancer procoagulant in hTERT-HDLEC and MDA-MB-231 cells, which are both associated with migratory capacity. Furthermore, cancer procoagulant-induced PDGF-β expression in hTERT-HDLEC and MDA-MB-231 cells could point to involvement of cancer procoagulant in wound healing and metastatic spread, respectively. Cancer procoagulant induced the motility of MDA-MB-231, MCF-7 and hTERT- cells in vitro in a time- and dose-dependent manner. Together, these results suggest that cancer procoagulant plays a role in the migration of breast cancer cells as well as the migration of endothelial cells.

BACKGROUND: Cells, growth factors and scaffold are the 3 fundamental factors currently proposed necessary for tissue regeneration. The use of these components has to be orchestrated precisely for ideal functional tissue formation. Growth factors enhance cellular activities that may lead to angiogenesis, cell proliferation and extracellular matrix biosynthesis. Due to the complexity of biochemical reactions a single growth factor may have limited effect. In order to explore a mixed profile of growth factors, a new biomaterial containing multiple growth factors derived from human Amniotic Membrane was chosen to compare with a known single growth factor (rhPDGF-BB). AIM: To compare the potential for enhanced bone formation by a morselized amniotic membrane suspension (AmnioSpark) with a known single cytokine PDGF-BB (GEM21,Lynch) under ex-vivo calvaria culture conditions. MATERIALS AND METHODS: 45 Calvaria from 7-9 day neonatal CD-1 mice were surgically harvested under sterile conditions. The calvaria were split through the mid sagittal suture to create 90 test specimens. A 2mm diameter critical size defect was created by biopsy punch thru the center of each calvarial specimen. This defect was bridged with a non-crosslinked type I collagen membrane of the same diameter to act as a scaffold. To compare AmnioSpark (AGF) potential for tissue regeneration against a known single cytokine PDGF-BB, the calvarial specimen were divided into six experimental groups: 1) Defect only, 2) Defect + scaffold, 3) Defect + scaffold + a single dose of (rhPDGF-BB ) a known bone stimulant, 4) Defect + Scaffold + 4 doses (day 0,3,5,7) of ( rhPDGF-BB), 5) Defect + scaffold + a single dose of (AGF) and 6) Defect + scaffold + 4 doses (day 0,3,5,7) of (AGF). Each test group had (N=5). A unique static tissue culture method was used with DMEM medium supplemented with ascorbic acid (150 ug/ml) and bovine serum albumin (5 mg/ml) without fetal calf serum to enhance bone formation for up to 7 weeks. Culture medium was changed every 2 days after day 3 and the harvested media was used for the following analyses: A) Alkaline phosphatase (ALP) as an osteoblastic activity indicator, and B) Tartrate Resistant Acid phosphatase (TRAP) as an osteoclastic bone remodeling activity indicator1. Macro photography and Scanning electron microscope (SEM) image analysis at different magnifications was performed to evaluate surface conditions. Histological analysis was performed with light microscope images on standard 4 um sections using H&E, Tri chrome, Picrosirius red and a fluorescence stain for RUNX2 as an osteoblast marker. RESULTS: With a single dose of test material ALP activity in the AGF group was significantly higher at 5 and 7 days. In addition ALP activity was significantly higher compared to all groups for up to 3 weeks post-application in the multiple dose AGF group (P<0.05). In contrast there was a dramatic decline in ALP in all other groups within the first week. TRAP activity was not detectable in any group. SEM images showed that osteoblast like cells accumulated and new tissue formation occurred over the surface of the scaffold obliterating the defect/membrane interface at 21 days with the AGF stimulus while in the PDGF-BB group the scaffold was still distinguishable from surrounding bone with no new tissue formation or cell migration . Histologic images confirmed an organized distribution of cells along the surface of the scaffold and new bone formation around the periphery of the defect in the AGF group (FIG42), while no bone formation or cell migration occurred in PDGF-BB group (FIG 35-38). Further diagnostic stains confirmed the presence of active osteoblasts (RUNX2)and the production of collagens I and II ( Masson Tri Chrome and PSR). CONCLUSION: Our results indicate that growth factors from amniotic extract (AGF) have the potential to enhance calvarial bone regeneration under an ex-vivo culture condition. These findings suggest that AGF could be a candidate for use as a new type of therapeutic material for regenerative medicine.