1,721,099 research outputs found
Ex vivo Lung Perfusion: A Platform for Lung Evaluation and Repair
Full text linkLung 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.Ph
The Role of Neutrophil Extracellular Traps in Lung Transplantation
No full textNeutrophil extracellular traps (NETs) are a product of the innate immune response. This thesis hypothesized that NETs are produced in response to various forms of lung transplant-related injury, and that the concentration of NETs in the lung predicts lung function. NETs were measured in the lungs and in EVLP perfusate in various models of porcine lung transplant-related injury and ex vivo lung perfusion (EVLP), as well as in clinical EVLP and recipient bronchial wash samples.
NETs were detected in porcine models of lung injury. NET concentration in porcine EVLP perfusate increased over time and varied according to lung injury. In clinical perfusate, NETs were significantly correlated with recipient days on the ventilator and length of stay in the ICU. This thesis demonstrates that NETs are present in lungs at various stages throughout the transplantation process, and that NETs in donor lungs could be used for predicting recipient outcomes.M.Sc
Strategies to Improve and Stabilize Extended Ex Vivo Lung Perfusion
No full textDespite the success of lung transplantation to treat end-stage lung diseases, organ shortage remains to be a major limitation. The Toronto Ex vivo lung perfusion (EVLP) protocol has improved assessment of donor lungs and provides an unique platform to repair lungs, with the goal of expanding the organ pool and improving long-term outcomes. However, the use of the technique is limited to ~12h, which limits advances in therapeutic strategies. We sought to determine whether a continuous replacement strategy or a modified feed could be used to operate EVLP to preserve lung integrity for extended perfusion time(24h). During EVLP, pig lungs were subjected to Toronto protocol, continuous perfusate replacement, or modified feed. By using a clinically relevant swine model, here we report the use of continuous perfusate replacement during prolonged EVLP preserved lung integrity for 24h. The application of this operation in EVLP will allow for advanced therapeutic interventions currently not feasible.M.H.Sc
Alpha 1 Antitrypsin Treatment of Donor Lungs: A Translational Pathway to Clinical Application
No full textLung transplantation is a life-saving therapy for patients suffering from end stage lung diseases. Despite the many advances in the field, there is a clear shortage of donors primarily explained by the low utilization rate of the donor lungs offered for transplant. This underutilization is mainly a consequence of the surgeons being cautious about using more marginal lungs, because of the concern that patients will develop primary graft dysfunction (PGD) after transplant. PGD accounts for 30% of the mortality seen during the first month after transplant and it is also related to worse long term survival. Ischemia reperfusion injury (IRI) is a well-known contributing factor to the development of PGD. There is currently no therapy to prevent or treat PGD. In order to examine potential therapies to decrease IRI and reduce the incidence of PGD, there is a need for a relevant large animal lung transplant survival model.
A1AT is a serine protease inhibitor, its main function is to act as an antiprotease for neutrophil elastase. It acts like an acute phase protein increasing its concentration during inflammation. A1AT as a drug was purified from human plasma for A1AT deficiency patients. Subsequent studies have shown that A1AT has anti-inflammatory effects in different settings. We have been studying the effect of A1AT in ischemia reperfusion injury in a systematic translational research approach testing its effects in progressively more complex models. Our hypothesis was that A1AT administered before transplantation in an attempt to reduce ischemia reperfusion injury would not have any detrimental side effects, and that A1AT dispensed to EVLP perfusate would improve human donor lungs that were previously rejected for transplantation.
The first part of the thesis is centered in developing a relevant pig lung transplant survival model to later on be able to test potential therapeutics to prevent PGD.
The second part of this work details the studies done to test alpha-1 antitrypsin (A1AT) in the pig single lung transplant survival model. Using the previously developed pig lung transplant survival model we were able to prove that A1AT is safe, well tolerated and if given to lung transplant recipient animals prior reperfusion, has beneficial effects including faster recovery and improved lung function after transplant.
For the third part of this thesis we used ex vivo lung perfusion (EVLP) as a platform to treat human lungs with A1AT. We explored the effect of A1AT treatment given during EVLP to injured human lungs that were not suitable for transplantation. For each case we divided the double lung block and treated one of the lungs in a randomized and blinded fashion. Using this approach we were able to assess the treatment effect of A1AT in the treated lung compared with the untreated one (both from the same donor), as well as compare all cases together (treatment vs control). A1AT was shown to have a beneficial effect on lung function measured by oxygenation function (pO2) and compliance. Biologically, A1AT treatment reduced lung endothelial injury (protected tight junctions) and decreased important pro-inflammatory cytokines that are relevant to endothelial dysfunction.
A1AT has been proven to have beneficial anti-inflammatory and immunomodulatory effects. A1AT is an already approved drug that has shown to be well tolerated and has the potential to improve human donor lungs that were previously rejected for transplantation.
Based on our findings, we are confident to move forward with a randomized prospective clinical trial to use A1AT to prevent PGD in human lung transplantation.Ph.D
Beyond Chronic Rejection: Tissue Remodelling in Obliterative Bronchiolitis after Lung Transplantation
Full text linkThe 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.Ph
Alpha 1 Antitrypsin Treatment of Donor Lungs: A Translational Pathway to Clinical Application
Full text linkLung transplantation is a life-saving therapy for patients suffering from end stage lung diseases. Despite the many advances in the field, there is a clear shortage of donors primarily explained by the low utilization rate of the donor lungs offered for transplant. This underutilization is mainly a consequence of the surgeons being cautious about using more marginal lungs, because of the concern that patients will develop primary graft dysfunction (PGD) after transplant. PGD accounts for 30% of the mortality seen during the first month after transplant and it is also related to worse long term survival. Ischemia reperfusion injury (IRI) is a well-known contributing factor to the development of PGD. There is currently no therapy to prevent or treat PGD. In order to examine potential therapies to decrease IRI and reduce the incidence of PGD, there is a need for a relevant large animal lung transplant survival model.
A1AT is a serine protease inhibitor, its main function is to act as an antiprotease for neutrophil elastase. It acts like an acute phase protein increasing its concentration during inflammation. A1AT as a drug was purified from human plasma for A1AT deficiency patients. Subsequent studies have shown that A1AT has anti-inflammatory effects in different settings. We have been studying the effect of A1AT in ischemia reperfusion injury in a systematic translational research approach testing its effects in progressively more complex models. Our hypothesis was that A1AT administered before transplantation in an attempt to reduce ischemia reperfusion injury would not have any detrimental side effects, and that A1AT dispensed to EVLP perfusate would improve human donor lungs that were previously rejected for transplantation.
The first part of the thesis is centered in developing a relevant pig lung transplant survival model to later on be able to test potential therapeutics to prevent PGD.
The second part of this work details the studies done to test alpha-1 antitrypsin (A1AT) in the pig single lung transplant survival model. Using the previously developed pig lung transplant survival model we were able to prove that A1AT is safe, well tolerated and if given to lung transplant recipient animals prior reperfusion, has beneficial effects including faster recovery and improved lung function after transplant.
For the third part of this thesis we used ex vivo lung perfusion (EVLP) as a platform to treat human lungs with A1AT. We explored the effect of A1AT treatment given during EVLP to injured human lungs that were not suitable for transplantation. For each case we divided the double lung block and treated one of the lungs in a randomized and blinded fashion. Using this approach we were able to assess the treatment effect of A1AT in the treated lung compared with the untreated one (both from the same donor), as well as compare all cases together (treatment vs control). A1AT was shown to have a beneficial effect on lung function measured by oxygenation function (pO2) and compliance. Biologically, A1AT treatment reduced lung endothelial injury (protected tight junctions) and decreased important pro-inflammatory cytokines that are relevant to endothelial dysfunction.
A1AT has been proven to have beneficial anti-inflammatory and immunomodulatory effects. A1AT is an already approved drug that has shown to be well tolerated and has the potential to improve human donor lungs that were previously rejected for transplantation.
Based on our findings, we are confident to move forward with a randomized prospective clinical trial to use A1AT to prevent PGD in human lung transplantation.Ph.D
Beyond Chronic Rejection: Tissue Remodelling in Obliterative Bronchiolitis after Lung Transplantation
No full textThe 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.Ph
RNA interference in organ transplantation: next-generation medicine?
No full textIschemia-reperfusion injury (IRI) is a major factor in graft quality and organ function after transplantation. Gene silencing through RNA interference (RNAi) may be used to suppress genes related to IRI and modify a graft before transplantation. RNAi is a natural process of posttranscriptional gene regulation that gained a lot of attention after winning the Nobel Prize in Medicine in 2006. Hence, several strategies have been used to deliver siRNA, and pretransplant machine perfusion presents a unique opportunity to deliver siRNA to the target organ during ex situ preservation. This book chapter discusses RNAi in the field of organ transplantation and considers whether this technique may be used as next-generation medicine. It gives a brief overview of the discovery of RNAi and its mechanism of action. In addition, studies using RNAi to target genes related to IRI in liver, kidney, lung, and heart transplantation are discussed.</p
Enhanced Coagulatory and Inflammatory Response Associated with Complement-1 Inhibitor (C1-INH) Administration in a Pig Lung Transplant Survival Model
No full textComplement inhibition has been shown to be beneficial in the treatment of ischemia-reperfusion injury. The effects of complement-1 inhibitor (C1-INH) were evaluated in a pig lung transplant model. Left single lung transplantation was performed in animals randomized to either a control group or treated with 200 U/kg of C1-INH. Allograft functions were assessed at 1 hour and 72 hours after reperfusion. All control animals survived to sacrifice on postoperative day 3. Two out of 4 animals in the treatment arm, however, developed graft failure and experiments were terminated early. Autopsy showed major pulmonary artery thrombus in one and bronchial casts in both failed treatment animals. The C1-INH group exhibited enhanced coagulation activation as well as more severe acute lung injury and inflammation. Early complement activation was weaker in the control group. The safety of C1-INH needs to be reconsidered as a potential therapy for ischemia-reperfusion induced injury in lung transplantation.M.Sc
The Role of Neutrophil Extracellular Traps in Lung Transplantation
Full text linkNeutrophil extracellular traps (NETs) are a product of the innate immune response. This thesis hypothesized that NETs are produced in response to various forms of lung transplant-related injury, and that the concentration of NETs in the lung predicts lung function. NETs were measured in the lungs and in EVLP perfusate in various models of porcine lung transplant-related injury and ex vivo lung perfusion (EVLP), as well as in clinical EVLP and recipient bronchial wash samples.
NETs were detected in porcine models of lung injury. NET concentration in porcine EVLP perfusate increased over time and varied according to lung injury. In clinical perfusate, NETs were significantly correlated with recipient days on the ventilator and length of stay in the ICU. This thesis demonstrates that NETs are present in lungs at various stages throughout the transplantation process, and that NETs in donor lungs could be used for predicting recipient outcomes.M.Sc
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