The long-term success of lung transplantation has been challenged by chronic graft dysfunction, which is manifested as obliterative bronchiolitis (OB). We demonstrated that allograft airway fibrosis is a dynamic process of tissue remodelling, in which cellular and matrix components dynamically change before or after complete obliteration of the airway lumen. This dynamic process was associated with changes in expression and activity of matrix metalloproteinases (MMPs). The early inflammatory phase was associated with MMP-dependent migration of blood-borne fibrocytes, which highly express MMP-9 and MMP-12. ‘Established’ fibrosis was associated with MMP-2 and MMP-14 expressed by myofibroblasts in both human OB lesions and their animal models. In established allograft airway fibrosis, general MMP inhibition resulted in apoptosis of myofibroblasts in vivo and in vitro, while low-doses of MMP-inhibitor treatment induced upregulation of MMP-2, increased collagenolytic activity, and significantly decreased myofibroblasts and collagen. The dynamic process of tissue remodelling in established allograft airway fibrosis was supported by underlying continuous alloimmune responses, in particular, direct T-cell-myofibroblast contact. iii Modulation of tissue remodelling using a low-dose MMP inhibitor in combination with cyclosporine induced partial regression of fibrosis after its establishment. We further demonstrated the mechanism of alloimmune responses unique to the lung. Human and animal studies demonstrated that bronchioles develop de novo lymphoid tissue characterized by formation of high endothelial venules and homing of effector memory T-cells. A following study demonstrated the important role of local immunological memory maintained by the intrapulmonary lymphoid tissue in exerting effector function in allograft rejection. Collectively, the present studies support the hypothesis that tissue remodelling is an important mechanism of allograft airway fibrosis. Regulation of tissue remodelling and underlying tissue injury is important not only to arrest aberrant remodelling of allograft airways but likely to reverse aberrant remodelling and to regenerate normal tissue architecture in airways after lung transplantation.

The long-term success of lung transplantation has been challenged by chronic graft dysfunction, which is manifested as obliterative bronchiolitis (OB). We demonstrated that allograft airway fibrosis is a dynamic process of tissue remodelling, in which cellular and matrix components dynamically change before or after complete obliteration of the airway lumen. This dynamic process was associated with changes in expression and activity of matrix metalloproteinases (MMPs). The early inflammatory phase was associated with MMP-dependent migration of blood-borne fibrocytes, which highly express MMP-9 and MMP-12. ‘Established’ fibrosis was associated with MMP-2 and MMP-14 expressed by myofibroblasts in both human OB lesions and their animal models. In established allograft airway fibrosis, general MMP inhibition resulted in apoptosis of myofibroblasts in vivo and in vitro, while low-doses of MMP-inhibitor treatment induced upregulation of MMP-2, increased collagenolytic activity, and significantly decreased myofibroblasts and collagen. The dynamic process of tissue remodelling in established allograft airway fibrosis was supported by underlying continuous alloimmune responses, in particular, direct T-cell-myofibroblast contact. iii Modulation of tissue remodelling using a low-dose MMP inhibitor in combination with cyclosporine induced partial regression of fibrosis after its establishment. We further demonstrated the mechanism of alloimmune responses unique to the lung. Human and animal studies demonstrated that bronchioles develop de novo lymphoid tissue characterized by formation of high endothelial venules and homing of effector memory T-cells. A following study demonstrated the important role of local immunological memory maintained by the intrapulmonary lymphoid tissue in exerting effector function in allograft rejection. Collectively, the present studies support the hypothesis that tissue remodelling is an important mechanism of allograft airway fibrosis. Regulation of tissue remodelling and underlying tissue injury is important not only to arrest aberrant remodelling of allograft airways but likely to reverse aberrant remodelling and to regenerate normal tissue architecture in airways after lung transplantation.

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.

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.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21627号 / 医博第4433号 / 新制||医||1033(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 平井 豊博, 教授 松原 和夫, 教授 羽賀 博典 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Introduction: NKT cells are categorised as borderline between NK and T cells, sharing phenotypic and functional characteristics of both cells, demonstrating their capacity to contritube to both pro- or anti-inflammatory processes. However, the role of these cells among lung transplant recipients remains largely unknown. The aim of this study was to determine the role of NKT cells following lung transplantation. Methods: NKT cells were quantified and characterised according to markers of: activation (CD107a, CD161, NKG2D) and immunomodulation (CD200 and CD200R) in peripheral blood and BALs. NKT cell numbers and phenotypes were correlated to clinical variables: immunosuppression, acute rejection, acute infections (viral, bacterial and fungal), bronchiolitis obliterans syndrome (BOS grade), lung function, and demographic variables. Interactions between NKT cells and the transplanted lung were linked by determining the relative expression of immunomodulatory ligand CD200 in lung biopsies. In vitro models were employed to determine the role of NKT cells to acute lung injury, either alone or in combination with cells of the mononuclear phagocyte system (MPS). Results: Higher numbers of immunomodulatory NKT cells (CD200+ and CD200R+) were found as lung function decreased. Data from peripheral blood indicates that recipients whose donors or themselves had been exposed to CMV infection demonstrated increased numbers of NKT cells. Patients with active EBV infections demonstrated higher NKT cell numbers expressing CD200 and CD200R. Data from BALs, indicates that patients with active fungal infections present higher immunomodulatory (CD200R) NKT cells and lower cytotoxicity marker (CD107a). In peripheral blood, lung recipients demonstrated higher NKT cell numbers compared to healthy volunteers. However, the lower relative mean expression of functional markers in the lung transplant group suggests that cells are less active. In vitro cultures with immunosuppressants demonstrated that cell cycle inhibitors (MMF and AZA) and corticosteroids (Prednisolone) are likely to inhibit NKT cell proliferation, while calcineurin inhibitors (Cyclosporine A and Tacrolimus) decrease the relative mean expression of activation markers. Clinical observations indicate that higher doses of Azathioprine may correlate with increased NKT cell numbers and the relative expression of CD200 and CD200R. However, under these conditions the relative expression of activation marker NKG2D decreases. In vitro data from the acute injury model indicates that NKT cells are capable to migrate into the injured lung and become activated following transmigration which is facilitated by the presence of monocytes. We also observed the interaction of NKT cells with endothelial cells, monocytes and macrophages. Also, the relative mean expression of CD200 and CD200R increased at the capillary layer, regardless of injury while upregulation of activation markers (CD107a, CD161 and NKG2D) was found at the capillary layer, following injury. In contrast, the alveolar layer demonstrated a decrease in both activation and immunomodulatory markers, following acute injury. Conclusions: Despite immunosuppression, NKT cells remain present in peripheral blood and BAL following lung transplantation. NKT cell proliferation is likely to be reduced by effect of cell cycle inhibitors, while calcineurin inhibitors exert an immunomodulatory effect. Our data indicates that NKT cells can participate in inflammatory and immunomodulatory events at the alveolar bilayer. Their capacity to infiltrate the lungs was assisted by cells of the mononuclear phagocyte system (MPS), which play an important role in antigen presentation and modulation of acute injury. Further research is needed to elucidate the signals and mechanisms occurring between NKT and MPS interactions and the outcomes these populations drive in acute lung injury.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第12958号 / 論医博第2100号 / 新制||医||1011(附属図書館) / 32357 / 京都大学大学院医学研究科医学専攻 / (主査)教授 三嶋 理晃, 教授 三森 経世, 教授 浅野 雅秀 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM