No description available.

No description available.

<p>PPAR agonists have been suggested as novel therapeutics for the treatment of inflammatory lung disease, such as allergic asthma. Treatment with PPAR agonists have been shown to inhibit peripheral eosinophilia in murine models of allergic asthma, which can occur through several mechanisms including decreased cytokine/chemoattractant (IL-5/eotaxin) release, decreased eosinophil migration and/or decreased eosinophil differentiation. This is the first study to show that PPARγ is expressed at the protein level in human airway eosinophils sampled from induced sputum, and confirms PPARγ protein expression in human peripheral blood eosinophils. We demonstrated the novel observation that peripheral blood eosinophil PPARγ protein expression, as measured by flow cytometry, is not different in eosinophils purified from asthmatic subjects compared to healthy controls and these observations suggest that the level of PPARγ expressed in human eosinophils is not related to asthmatic status. Our study also confirms, by real time PCR, the detection of mRNA for PPARγ in airway-derived leukocytes, collected from bronchial washings, increases 24hrs after whole lung allergen challenge. This increase is regulated by Symbicort® and Pulmicort® treatment in most subjects. This is the first study to show increased chemokinesis (random stimulated movement) of eosinophils <em>in vitro</em> at low concentrations of a PPARγ agonist. We have generated data to suggest this is through an effect on calcium signalling. We also observed that higher concentrations of PPAR agonists directly inhibit eotaxin-stimulated eosinophil directional migration. Finally, using methycellulose cultures of non-adherent mononuclear cells and CD34+ progenitor cells, we demonstrate that PPAR activation can inhibit the differentiation of eosinophils <em>in vitro</em>. Collectively, these data demonstrate that PPARγ is expressed consitutively on eosinophils in peripheral blood and airways, and suggest signaling through this receptor with nanomolar concentrations of agonist regulates eosinophilia through inhibition of both eosinophil migration and eosinophil differentiation.</p> / Doctor of Philosophy (PhD)

Adult hippocampal neurogenesis, or the process of creating new neurons in the dentate gyrus (DG) of the hippocampus, underlies learning and memory capacity. This cognitive ability is essential for humans to operate in their everyday lives, but cognitive disruption can occur in response to traumatic insult such as brain injury. Previous findings in rodent models have characterized the effect of moderate traumatic brain injury (TBI) on neurogenesis and found learning and memory shortfalls correlated with limited neurogenic capacity. While there are no substantial changes after one mild TBI, research has yet to determine if neurogenesis contributes to the worsened cognitive outcomes of repetitive mild TBI. Here, we examined the effect of neurogenesis on cognitive decline following repetitive mild TBI by utilizing AraC to limit the neurogenic capacity of the DG. Utilizing a BrdU fate-labeling strategy, we found a significant increase in the number of immature neurons that correlate learning and memory impairment. These changes were attenuated in AraC-treated animals. We further identified endothelial cell (EC)-specific EphA4 receptor as a key mediator of aberrant neurogenesis. Taken together, we conclude that increased aberrant neurogenesis contributes to learning and memory deficits after repetitive mild TBI. / Doctor of Philosophy / In the United States, millions of people experience mild traumatic brain injuries, or concussions, every year. Patients often have a lower ability to learn and recall new information, and those who go on to receive more concussions are at an increased risk of developing long-term memory-associated disorders such as dementia and chronic traumatic encephalopathy. Despite the high number of athletes and military personnel at risk for these disorders, the underlying cause of long-term learning and memory shortfalls associated with multiple concussions remains ill defined. In the brain, the hippocampus play an important role in learning and memory and is one of only two regions in the brain where new neurons are created from neural stem cells through the process of neurogenesis. Our study seeks to address the role of neurogenesis in learning and memory deficits in mice. These findings provide the foundation for future, long-term mechanistic experiments that uncover the aberrant or uncontrolled processes that derail neurogenesis after multiple concussions. In short, we found an increase in the number of newborn immature neurons that we classify as aberrant neurogenesis. Suppressing this process rescued the learning and memory problems in a rodent model of repeated concussion. These findings improve our understanding of the processes that contribute to the pathophysiology of TBI.

Tendon injuries are a significant cause of morbidity in performance horses with superficial digital flexor tendon injury reported to represent up to 43% of overall Thoroughbred racehorse injuries. Natural repair is slow and results in inferior structural organization and biomechanical properties and, therefore, reinjury is common. The inability of tendon to regenerate after injury, or to heal with mechanical properties comparable to the original tissue, is likely attributable to low vascularity and cellularity of the tissue, low number of resident progenitor cells, and healing under weight-bearing conditions. Strategies to improve tendon healing have focused on enhancing the metabolic response of tenocytes, modulating the organization of the newly synthesized extracellular matrix, or administering progenitor cells to enhance repair. Significant research effort has been directed at the use of adult mesenchymal stem cells as a source of progenitor cells for equine tendon repair and recent clinical applications have utilized adult autologous stem cells derived either from adipose tissue or bone marrow aspirates. Isolation of a homogenous population of stem cells from bone marrow is time-consuming, and there is much variation in cell numbers, cell viability and growth rates among samples. Recently, a population of progenitor cells has been isolated from equine flexor tendons, thus providing an alternative source of progenitor cells from the target tissue for therapeutic intervention. The interaction between cells and the extracellular matrix (ECM) is an important factor in regulation of cell function. Proliferation, migration, differentiation and gene expression of many cell types are altered by adhesion to and interaction with matrix proteins and the extracellular environment. Tendon progenitor cells reside within a niche that comprises primarily parallel collagen fibers, and this niche plays an important role in regulating their function and differentiation. Culture conditions replicating this environment could be beneficial for both cell growth and matrix gene expression. The objectives of the study were to compare cell growth kinetics and biosynthetic capabilities of bone marrow mesenchymal stem cells (BMMSCs) and tendon derived progenitor cells (TPCs) cultured on commercially available bovine, highly purified bovine, porcine, and rattus collagen sources and standard tissue culture surfaces. We hypothesized that collagen type I matrix would preferentially support TPC proliferation and up regulate gene expression for collagens and organizational components of tendon and therefore provide a culture system and progenitor cell type with advantages over the current practice of BMMSC expansion on standard cell culture plastic surfaces. Cells were isolated from 6 young adult horses, expanded, and cultured on collagen-coated tissue culture plates, and no collagen control for 7 days. Samples were analyzed for cell number on days 4 and 7, and for mRNA expression of collagen type I, collagen type III, cartilage oligomeric matrix protein (COMP), and decorin on day 7. Glycosaminoglycan (GAG) synthesis was analyzed on day 7. Differences of cell number between collagen groups and cell type, and in gene expression and GAG synthesis between collagen groups and cell types, were evaluated by use of mixed-model repeated measures ANOVA. Pair-wise comparisons were made on significant differences identified with ANOVA using Tukey's post hoc test. Statistical significance was set at P<0.05. A statistical significant (P=0.05) increase in cell number for TPCs grown on rattus collagen versus control on day 4 was observed. No difference in GAG synthesis or expression of collagen type I, collagen type III, COMP or decorin mRNA was observed between collagen groups and non-collagen controls for either cell type on day 7. TPCs cultured on all collagen types yielded more cells than similarly cultured BMMSCs on day 4, but only porcine collagen was superior on day 7. TPCs synthesized more GAG than BMMSCs when cultured on control surfaces only. BMMSCs expressed more collagen type I mRNA when cultured on control, porcine and highly-purified collagen, and more collagen type III when cultured on control, porcine, highly-purified collagen, and rattus collagen, than TPCs. Tendon-progenitor cells expressed significantly more COMP when cultured on control and all collagen types, and decorin when cultured on porcine, highly purified bovine and bovine collagen when compared to BMMSCs. The results of this study revealed an advantage to culturing TPCs on randomly organized rattus collagen during the early growth phase. The beneficial effects of collagen-coated surfaces on cell proliferation is likely related to increased surface area for attachment and expansion provided by the random collagen matrix, and/or collagen-cell interactions. Tendon progenitor cells showed superior growth kinetics and expression of the matrix organizational components, COMP and decorin, than similarly cultured BMMSCs that expressed more collagen types III and I. TPCs synthesize more GAG compared to BMMSCs when cultured on plastic surfaces and there was no induction by collagen. Tendon progenitor cells should be considered as an alternative source of progenitor cells for injured equine tendons. Further in vitro studies characterizing factors that influence gene expression of both cell types is warranted. / Master of Science

Yes / Background: Melanin is synthesized by melanocytes in the basal layer of the epidermis. When transferred to surrounding keratinocytes it is the key UVR-protective biopolymer responsible for skin pigmentation. Most melanin is observable in the proliferative basal layer of the epidermis, and only sparsely distributed in the stratifying/differentiating epidermis. The latter has been explained, despite formal evidence, to ‘melanin degradation’ in supra-basal layers. Objectives: Our aim was to re-evaluate this currently-accepted basis for melanin distribution in the human skin epidermis, and whether this pattern is altered after a regenerative stimulus. Methods: Normal epidermis of adult human skin, at rest and after tape-stripping, was analysed by a range of (immuno)histochemical and high-resolution microscopy techniques. In vitro models of melanin granule uptake by human keratinocytes were attempted. Results: We propose a wholly different fate for melanin in the human epidermis. Our evidence indicates that the bulk of melanin is inherited only by the non-differentiating daughter cell post mitosis in progenitor keratinocytes, via asymmetric organelle inheritance. Moreover, this preferred pattern of melanin distribution can switch to a symmetric or equal daughter cell inheritance mode under conditions of stress including regeneration. Conclusions: We provide in this preliminary report a plausible and histologically-supportable explanation for how human skin pigmentation is efficiently organized in the epidermis. Steady state epidermis pigmentation may involve much less redox-sensitive melanogenesis than previously thought, and at least some pre-made melanin may be available for re-use. The epidermal-melanin unit may be an excellent example to study organelle distribution via asymmetric or symmetric inheritance in response to micro-environment and tissue demands. / Walgreens Boots Alliance

No description available.

Endometriosis affects 5-10% of women and is characterized by the growth of endometrial tissue outside of the uterus. Treatment for endometriosis primarily focuses on symptom relief, is short term with severe side effects and often leads to recurrence of the condition. Establishing new blood supply is a fundamental requirement for endometriosis lesions growth. This has led to the idea that antiangiogenic therapy may be a successful approach for inhibiting endometriosis. Recent evidence indicates that endothelial progenitor cells (EPCs) contribute to neoangiogenesis of endometriotic lesions. These EPCs are recruited to the lesion site by stromal cell-derived factor-1 (SDF-1). We hypothesize that SDF-1 is central to the neoangiogenesis and survival of endometriotic lesions and that administration of SDF-1 blocking antibody will inhibit lesion growth by inhibiting angiogenesis in a murine model of endometriosis. Immunohistochemistry for SDF-1 and CD34 was performed on human endometriosis and normal endometrial samples. Quantification of SDF-1 and EPCs was performed in the blood of endometriosis patients and controls using ELISA and flow cytometry, respectively. A new mouse model of endometriosis was developed using BALB/c-Rag2-/-/IL2rg-/- mice to investigate role of SDF-1 in neoangiogenesis. Either SDF-1 blocking antibody or an isotype control was administered on a weekly basis for four weeks. Weekly samples of peripheral blood from mice were analyzed for SDF-1, other cytokines of interest and EPCs. Mice were euthanized at seven weeks to observe lesion growth and blood vessel development. Our results indicate overabundance of SDF-1 and CD34+ progenitor cells in human endometriotic lesions compared to eutopic endometrium. In the mouse model, SDF-1 and circulating EPC levels decreased from pre-treatment levels after one week, and remained constant over the course of the treatment in both SDF-1 blocking antibody and isotype control groups. In the SDF-1 blocking group, reduced vascularity of lesions, identified by immunofluorescence staining for CD31, was revealed compared to isotype controls. These findings suggest that SDF-1 may be responsible for CD34+ progenitor cell recruitment to the neoangiogenic sites in endometriosis. Blocking of SDF-1 reduces neovascularization of human endometriotic lesions in a mouse model. Further studies on blocking SDF-1 in combination with other antiangiogenic agents are needed. / Thesis (Master, Anatomy & Cell Biology) -- Queen's University, 2011-12-21 19:34:43.054

Osteoarthritis (OA) ist eine degenerative Erkrankung des hyalinen Knorpels. Knorpel ist ein avaskuläres Gewebe. Wenn dieser beschädigt wird, ist es schwierig, ihn zu reparieren. Der Gelenkknorpel ist verantwortlich für die glatte, reibungs- und schmerzlose Bewegung des Kniegelenks. Schwere Verluste oder die komplette Zerstörung des Gelenkknorpels führen zu hoher Reibung und Schmerzen bei der Bewegung des Kniegelenks, wie es oft in den späten Stadien der OA der Fall ist. Der komplette Gelenkersatz bleibt die ultimative Lösung. Jedoch gibt es viele andere Möglichkeitenden Knorpel über die Implantation von Stammzellen zu reparieren oder zu regenerieren, jedoch oft mit schwerwiegenden Folgen. Die Transplantation embryonaler Stammzellen kann beispielsweise zu Teratombildung führen. Die Nutzung von induzierten pluripotenten Stammzellen ermöglicht die Virusintegration in das Genom. Alternativ entstand das Konzept der Vorläufer- oder Reparaturzellen in situ. Beispielsweise fand man in späten Stadien der Osteoarthritis im menschlichen Knorpel chondrogene Vorläuferzellen mit migratorischen Fähigkeiten (CPCs). Bei Knorpelregeneration mit diesen Zellen sind bisher keine Risokofaktoren bekannt. Sie haben eine enorme Fähigkeit für die Knorpelreparatur ohne das schwerwiegende Risikofaktoren bisher bekannt waren. Allerdings bestehen noch Fragen zum Beispiel wie man CPCs in situ induziert, um das Gewebe auf physiologische Weise zu reparieren. Zweitens haben CPCs eine begrenzte Lebensdauer, zumindest in vitro. Darüber hinaus gibt es keine verfügbare optimierte Methode, um eine vollständige chondrogene Differenzierung von Stammzellen zu erreichen. Vor kurzem wurden primäre Zilien gefunden, die hilfreich für die Stammzelldifferenzierung sein könnten. Diese Zilien arbeiten als Dual-Sensor für mechanochemische Signale. Dieser Sensor wurde auch auf CPCs gefunden, bei Chondrozyten gewonnen aus Kiefergelenken (TMJ) von Discoidin-Domänen- Rezeptor-1- Knockout- Mäusen (DDR- 1 KO). OA ist nicht nur auf die großen Gelenke beschränkt, sondern wirkt sich auch auf die kleinen Gelenke wie das Kiefergelenk aus. Es ist gut bekannt, dass Chondrozyten im Gelenkknorpel keinen direkten Zell-zu-Zell-Kontakt besitzen vielmehr beruht die Kommunikation auf Zell-Matrix - Wechselwirkungen über Zellrezeptoren z.B Integrine oder DDRs. DDR -1- KO-Mäuse zeigen typische Symptome der Arthrose des Kiefergelenkknorpels. Die aus dem Kiefergelenknorpel der DDR - 1 KO Mäuse abgeleiteten Chondrozyten behielten ihre Arthroseeigenschaften. Einerseits wiesen sie eine hohe Expression von runx2 und Kollagen Typ I als typische osteogene Signaturen auf sowie andererseits eine geringe Expression von sox9, Kollagen Typ II und Aggrecan, welche relevant für die chondrogene Differenzierung sind. Die osteoarthritischen Charakteristika könnten zu einem normalen Chondrozyten- Typ umgekehrt werden über den Knockdown von runx2 oder der Exposition dieser Zellen in einer dreidimensionalen Umgebung und in Gegenwart von extrazellulärer Matrix (ECM) -Komponenten wie Laminin und Nidogen. Die Umkehr in Richtung des chondrogenen Phänotyps ist auch für die Pathogenese der Meniskusdegeneration von großer Bedeutung. Der Meniskus ist in den meisten Fällen der Ausgangspunkt für die Entwicklung von OA des Kniegelenks. Der Meniskus ist ein Bindegewebsknorpel, der als Stoßdämpfer wirkt. Hierbei verschlimmert eine Menikusschädigung die OA Pathogenese durch verstärkten Knorpelabbau. Der innere Teil des Meniskus ist avaskulärer Natur und hat eine sehr begrenzte Eigenreparaturfähigkeit. Es gibt jedoch andere Möglichkeiten, wie die teilweise Entfernung des Meniskus, die zu einer kurzfristigen Entlastung führt. Dies verhindert jedoch nicht die langfristigen Folgen, die letztlich zur Entwicklung von OA führen. Der innere Teil des menschlichen Meniskus birgt einzigartige Vorläuferzellen (MPC) und kann zur Meniskus-Regeneration über den TGFß -Signalweg aktiviert werden. Darüber hinaus wurden Maus-Meniskus-Progenitorzellen (MMPCs) in gesundem Meniskusgewebe untersucht. Diese Zellen wurden mittels immunohistochemischen Techniken ex vivo charakterisiert und behielten ihre Stammzelleigenschaften auch in vitro. Mit der Anwendung verschiedener Stammzellen zur Therapie der Knorpelregeneration sind viele kritische Konsequenzen assoziiert. Im Fokus standen deshalb gewebespezifische Zellen auch in situ Vorläuferzellen genannt, die bereits in erkranktem Knorpel vorhanden sind. Diese Zellen können sich in chondrogener Richtung entwickeln. Hierfür benötigen sie möglicherweise nur geringe Manipulationen, um daraufhin hyalinen Knorpel zu produzieren.

No description available.