Lung transplantation is a life-saving therapy for patients suffering from end-stage lung disease; however, the majority of donor lungs are injured and attempts to transplant them results in a high risk of primary graft dysfunction in the recipient, a type of severe acute lung injury. Previously, a novel method of lung preservation known as ex vivo lung perfusion (EVLP) has been developed in which donor lungs are continuously perfused and ventilated at normothermia using a protective strategy. Donor lungs have been shown to tolerate at least 12 h of preservation in this manner without the accrual of injury. Hence, EVLP could act as a platform on which injured donor lungs could potentially be evaluated and repaired. To explore this concept, we utilized interleukin-10 (IL-10), an anti-inflammatory cytokine, as a prototypical drug for ex vivo delivery. Because IL-10 protein has a prolonged half-life during EVLP, we delivered recombinant IL-10 by the intravascular and intratracheal routes to clinically-rejected injured human lungs. Intratracheal delivery resulted in elevated levels of IL-10 in both tissue and perfusate whereas intravascular delivery resulted in elevated levels of IL-10 only in the perfusate over 12 h of EVLP. There was, however, no beneficial effect to either lung function or lung inflammation. This was thought to be a result of intratracheally delivered IL-10 leaking out into the perfusate where it may not be biologically active. Constant IL-10 production within the lung tissue could be achieved using a gene therapy approach. Thus, we subsequently explored the delivery of IL-10 by adenoviral gene therapy during EVLP. Ex vivo administered intratracheal adenoviral gene therapy could increase transgene protein levels within the lung. More importantly, it did so with less vector-associated inflammation when compared to in vivo delivery of adenoviral gene therapy. Having explored drug delivery, we sought to develop a large animal injury model on which to test ex vivo therapies. Given that the majority of organ donors are brain dead and therefore exposed to the injurious sequelae resulting from brain death, we developed a brain-death injury model in pig. Use of EVLP as a platform for repair necessitates an accurate recognition of both lung injury and lung improvement during EVLP. Thus, we utilized this injury model to explore the profile of physiological parameters when an injured lung is perfused during EVLP. Because of the alteration of the PO2 to oxygen content relationship of an acellular perfusate, we found that PaO2 changes are less dramatic than in the in vivo situation. However, as injured lungs begin to become edematous, the mechanical effects on the lung by the increased water content can be measured by corresponding falls in compliance and increases in airway pressure. Overall, use of EVLP demonstrates promise for reducing the organ shortage currently prevalent in clinical lung transplantation. Improved evaluation will instill confidence in transplant clinicians to transplant previously questionable organs. Lungs which prove to be injured during evaluation can potentially be repaired using IL-10 therapy as explored herein or with other therapies using the delivery methods described.

Primarily expressed by trophoblast cells, human leukocyte antigen-G (HLA-G) plays an essential role in maintaining maternal-fetal immune tolerance. Having previously been detected following heart transplantation, we sought to establish the value of HLA-G in identifying freedom from moderate or severe rejection post-heart transplant, and the capability of its expression in vitro. After assessing myocardial HLA-G expression through immunohistochemistry, we demonstrated that it was significantly more prevalent in non-rejecting than rejecting heart transplant recipients. Utilizing vascular endothelial and smooth muscle cell culture models, we also determined that while HLA-G expression remains tightly regulated, its expression in vitro can be induced following progesterone treatment in a dose-dependent manner. Hence, HLA-G may reliably identify patients with a low immunological risk of developing subsequent clinically significant rejection post-heart transplant. Furthermore, HLA-G expression can be induced in cultured endothelial and smooth muscle cells, which might represent a strategy to protect against allograft rejection and vasculopathy.

Lung transplantation is a life-saving therapy for patients suffering from end-stage lung disease; however, the majority of donor lungs are injured and attempts to transplant them results in a high risk of primary graft dysfunction in the recipient, a type of severe acute lung injury. Previously, a novel method of lung preservation known as ex vivo lung perfusion (EVLP) has been developed in which donor lungs are continuously perfused and ventilated at normothermia using a protective strategy. Donor lungs have been shown to tolerate at least 12 h of preservation in this manner without the accrual of injury. Hence, EVLP could act as a platform on which injured donor lungs could potentially be evaluated and repaired. To explore this concept, we utilized interleukin-10 (IL-10), an anti-inflammatory cytokine, as a prototypical drug for ex vivo delivery. Because IL-10 protein has a prolonged half-life during EVLP, we delivered recombinant IL-10 by the intravascular and intratracheal routes to clinically-rejected injured human lungs. Intratracheal delivery resulted in elevated levels of IL-10 in both tissue and perfusate whereas intravascular delivery resulted in elevated levels of IL-10 only in the perfusate over 12 h of EVLP. There was, however, no beneficial effect to either lung function or lung inflammation. This was thought to be a result of intratracheally delivered IL-10 leaking out into the perfusate where it may not be biologically active. Constant IL-10 production within the lung tissue could be achieved using a gene therapy approach. Thus, we subsequently explored the delivery of IL-10 by adenoviral gene therapy during EVLP. Ex vivo administered intratracheal adenoviral gene therapy could increase transgene protein levels within the lung. More importantly, it did so with less vector-associated inflammation when compared to in vivo delivery of adenoviral gene therapy. Having explored drug delivery, we sought to develop a large animal injury model on which to test ex vivo therapies. Given that the majority of organ donors are brain dead and therefore exposed to the injurious sequelae resulting from brain death, we developed a brain-death injury model in pig. Use of EVLP as a platform for repair necessitates an accurate recognition of both lung injury and lung improvement during EVLP. Thus, we utilized this injury model to explore the profile of physiological parameters when an injured lung is perfused during EVLP. Because of the alteration of the PO2 to oxygen content relationship of an acellular perfusate, we found that PaO2 changes are less dramatic than in the in vivo situation. However, as injured lungs begin to become edematous, the mechanical effects on the lung by the increased water content can be measured by corresponding falls in compliance and increases in airway pressure. Overall, use of EVLP demonstrates promise for reducing the organ shortage currently prevalent in clinical lung transplantation. Improved evaluation will instill confidence in transplant clinicians to transplant previously questionable organs. Lungs which prove to be injured during evaluation can potentially be repaired using IL-10 therapy as explored herein or with other therapies using the delivery methods described.

Zugl.: Halle (Saale), Univ., Diss., 2008

Thesis (M.Res.(Med.))--University of Hong Kong, 2009. / Includes bibliographical references (p. 105-124).

The pulp organ is retained in the pulp chamber of teeth and maintains the biological and physiological vitality of the surrounding dentin. It works as a biosensor and generates secondary dentine and tertiary dentine to resist tooth abrasion and pathogenic stimuli (Zhang and Yelick, 2010). However, dental pulp is vulnerable to injury (Smulson and Sieraski, 1989). Most people experience some irreversible pulpal diseases during their lifetime. Hence, pulp regeneration is one of the research tasks in dentistry that attracts much attention. Stem cell transplantation is a plausible strategy for the regeneration of dental pulp organ. Dental pulp stem cells (DPSCs)derived from heavy or inflamed dental pulps have the natural advantage in pulp regeneration due to its dentinogenic potentiality (Huang et al., 2009). DPSCs are delivered into prepared root canal, which then differentiate into odontoblasts, fibroblasts, and other kinds of cells. It was shown that these transplanted DPSCs were able to produce dentin and formed a dentin-pulp like tissue both in vitro and in vivo(Huang, 2009).However, low survival rate of the transplanted cells is a common problem in pulp regeneration. Overexpression of Bcl-2 could enhance cell anti-apoptotic ability. Studies of many kinds of cell transplantation showed that a large number of cells died upon grafting and a large proportion of cell death seemed to have occurred due to apoptosis (Liu et al., 2013; Zhang et al., 2001).The aim of this study was to improve cell survival through making DPSCs overexpress lymphoma 2 (Bcl-2) protein.Bcl-2 is a proto-oncogene which playsa significant role in (anti) apoptosis. Former studies in the literature have provided evidences that overexpressing Bcl-2 could reduce cell apoptosis. However, this strategy has not been studied in the modification of DPSCs. In this study, DPSCs were isolated from discarded third molars of adults and manipulated to overexpressing Bcl-2. Proliferation of modified DPSCs was analyzed by static batch culture, CCK-8 test and BrdU based proliferation test. Apoptosis of modified DPSCs was analyzed by measuring DNA fragments in the cells. Modified DPSCs generated a higher maximum cell population during static batch culture and showed higher viability (the ratio of live cells to total cells). CCK-8 test showed that the population of modified DPSCs increased faster than control group cells and wild type cells. Modified DPSCs were not better than the other cells in proliferative ability, but had lower apoptosis level when culturing in serum free medium. Hence, overexpressing Bcl-2 could increase cell population, the mechanism is to help DPSCs survival rather than promote the proliferative ability of cells. / published_or_final_version / Dentistry / Master / Master of Philosophy

published_or_final_version / Medicine / Master / Doctor of Medicine

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

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