M. Rubacha

Technique for Prolonged Normothermic Ex Vivo Lung Perfusion

BACKGROUND The inhibition of cellular metabolism induced by hypothermia obviates the possibility of substantial reparative processes occurring during organ preservation. The aim of this study was to develop a technique of extended (12-hour) ex vivo lung perfusion (EVLP) at normothermia for assessment and protective maintenance of the donor lung. METHODS Six double-lung blocks from 35-kg pigs and 5 single human lungs were subjected to 12 hours of normothermic EVLP using acellular Steen Solution. In the animal studies, the left lung was transplanted into recipients at the end of EVLP and reperfused for 4 hours to evaluate the impact of prolonged EVLP on post-transplant lung function. A protective mode of mechanical ventilation with controlled perfusion flows and pressures in the pulmonary vasculature were employed during EVLP. Lung oxygenation capacity (DeltaPo(2)), pulmonary vascular resistance and airway pressures were evaluated in the system. Red blood cells were added to the perfusate to a hematocrit of 20% at the end of human lung EVLP to study lung functional assessment with and without cells. RESULTS Lung function was stable during 12 hours of EVLP. This stability during prolonged normothermic EVLP translated into excellent post-transplant lung function (Pao(2)/Fio(2): 527 +/- 22 mm Hg), low edema formation (wet/dry ratio: 5.24 +/- 0.38) and preserved lung histology after transplantation. The acellular perfusion assessment of lung function accurately correlated with post-transplant graft function. CONCLUSIONS Twelve hours of EVLP at physiologic temperatures using an acellular perfusate is achievable and maintains the donor lungs without inflicting significant added injury. This system can be used to assess, maintain and treat injured donor lungs.

Normothermic Ex Vivo Perfusion Prevents Lung Injury Compared to Extended Cold Preservation for Transplantation

Treatment of injured donor lungs ex vivo to accelerate organ recovery and ameliorate reperfusion injury could have a major impact in lung transplantation. We have recently demonstrated a feasible technique for prolonged (12 h) normothermic ex vivo lung perfusion (EVLP). This study was performed to examine the impact of prolonged EVLP on ischemic injury. Pig donor lungs were cold preserved in Perfadex® for 12 h and subsequently divided into two groups: cold static preservation (CSP) or EVLP at 37°C with Steen™ solution for a further 12 h (total 24 h preservation). Lungs were then transplanted and reperfused for 4 h. EVLP preservation resulted in significantly better lung oxygenation (PaO2 531 ± 43 vs. 244 ± 49 mmHg, p < 0.01) and lower edema formation rates after transplantation. Alveolar epithelial cell tight junction integrity, evaluated by zona occludens‐1 protein staining, was disrupted in the cell membranes after prolonged CSP but not after EVLP. The maintenance of integrity of barrier function during EVLP translates into significant attenuation of reperfusion injury and improved graft performance after transplantation. Integrity of functional metabolic pathways during normothermic perfusion was confirmed by effective gene transfer and GFP protein synthesis by lung alveolar cells. In conclusion, EVLP prevents ongoing injury associated with prolonged ischemia and accelerates lung recovery.

Ex Vivo Adenoviral Vector Gene Delivery Results in Decreased Vector-associated Inflammation Pre- and Post–lung Transplantation in the Pig

Acellular normothermic ex vivo lung perfusion (EVLP) is a novel method of donor lung preservation for transplantation. As cellular metabolism is preserved during perfusion, it represents a potential platform for effective gene transduction in donor lungs. We hypothesized that vector-associated inflammation would be reduced during ex vivo delivery due to isolation from the host immune system response. We compared ex vivo with in vivo intratracheal delivery of an E1-, E3-deleted adenoviral vector encoding either green fluorescent protein (GFP) or interleukin-10 (IL-10) to porcine lungs. Twelve hours after delivery, the lung was transplanted and the post-transplant function assessed. We identified significant transgene expression by 12 hours in both in vivo and ex vivo delivered groups. Lung function remained excellent in all ex vivo groups after viral vector delivery; however, as expected, lung function decreased in the in vivo delivered adenovirus vector encoding GFP (AdGFP) group with corresponding increases in IL-1β levels. Transplanted lung function was excellent in the ex vivo transduced lungs and inferior lung function was seen in the in vivo group after transplantation. In summary, ex vivo delivery of adenoviral gene therapy to the donor lung is superior to in vivo delivery in that it leads to less vector-associated inflammation and provides superior post-transplant lung function.

Long pentraxin PTX3 deficiency worsens LPS-induced acute lung injury

ObjectiveLong pentraxin PTX3 is an inflammatory mediator and a component of the humoral arm of innate immunity. PTX3 expression is increased in animals with acute lung injury (ALI) and in patients with sepsis or acute respiratory distress syndrome and is considered to be a potential biomarker for these diseases. However, the role of PTX3 in the pathogenesis of ALI is not fully understood. We hypothesized that PTX3, as an important immune modulator, may determine the severity of ALI.MethodsLipopolysaccharide (LPS) was intra-tracheally administrated to PTX3 knock-out (PTX3-KO) and wild-type (WT) mice. Lung injury, neutrophil infiltration, cell death, fibrin deposition, and tissue factor expression in the lung were determined. Local and systemic inflammatory responses were assessed by measuring cytokines in the lung and plasma.ResultsLPS instillation induced ALI in both PTX3-KO and WT mice. Interestingly, PTX3 deficiency significantly increased the magnitude/extent of lung injury compared to that in WT mice. The severe lung injury was accompanied by elevated neutrophil infiltration, cell death, and fibrin deposition in the lung. PTX3 deficiency also enhanced LPS-induced tissue factor expression/activation in the lung and increased tumor necrosis factor-alpha and monocyte chemoattractant protein-1 levels in the plasma.ConclusionOur data suggest that the endogenously expressed PTX3 plays a protective role in the pathogenesis of ALI and that a lack of PTX3 may enhance neutrophil recruitment, cell death, activation of coagulation cascades, and inflammatory responses in the lung.

Kinetics of lactate metabolism during acellular normothermic ex vivo lung perfusion.

BACKGROUND Plasma lactate has been used as a marker of poor prognosis in clinical conditions. However, the relationship between lactate production and lung function during acellular normothermic ex vivo lung perfusion (EVLP) is unclear. We investigated the kinetics of lactate metabolism during EVLP and the correlation of this marker with outcomes after transplant. METHODS Human donor lungs in our clinical EVLP trial (CLs; n = 28) and rejected donor lungs for experimental use (Els; n = 8) were perfused ex vivo using the Toronto technique. Lactate level, lactate/pyruvate (L/P) ratio, and glucose level in the perfusate were measured. In CLs, we examined the relationship between lactate metabolism during EVLP and early post-transplant outcomes. The hypoxia-inducible factor 1 sub-unit 1α (HIF-1α) level in lung tissue was examined in ELs. RESULTS We performed double-lung EVLP in CLs and single-lung EVLP in ELs. In CLs, the lactate and L/P ratios at the end of EVLP had no correlation with early post-transplant outcomes despite lactate elevation during EVLP. Although lactate elevation was also present in all ELs, we were able to identify 2 groups based on L/P ratio at the end of EVLP. The group with the high L/P ratio had higher airway pressure during EVLP and higher HIF-1α in lung tissue at the end of EVLP. CONCLUSIONS Lactate increases seen in the EVLP perfusate most often represent physiologic lactate production by the lung in a setting with reduced lactate clearance. Thus, patients who underwent transplantation after EVLP had good outcomes despite lactate elevation during EVLP.

INTRATRACHEAL DELIVERY OF ADENOVIRAL VECTOR EX VIVO RESULTS IN REDUCED VECTOR-ASSOCIATED INFLAMMATION IN LUNG PRE- AND POST- TRANSPLANTATION: 450

Introduction The use of adenovirus mediated IL-10(AdhIL-10) gene therapy is a promising method to reduce lung inflammation and can reduce ischemia-reperfusion injury following lung transplantation. However, the transient inflammatory response to the adenoviral vector itself limits the clinical adoption of this technique. Normothermic ex vivo lung perfusion has recently been developed as a method for donor lung evaluation and repair and is currently undergoing clinical trials. We sought to test the hypothesis that adenoviral vector delivery to the donor lung during ex vivo perfusion prior to transplantation reduces vector-associated inflammation. Methods Yorkshire pigs were randomly assigned to either the ex vivo or in vivo group. Animals were then randomly and blindly assigned to receive either: vehicle control, 1E10pfu AdhIL-10, or 1E10pfu AdGFP into the left lung. Left lungs from pigs transduced with AdGFP were then transplanted into an allogenic pig and observed for 4h. Left lung transplantation of pigs transduced with AdhIL-10 ex vivo was used as control. Lung function (P/F ratio), histology, and cytokine profile were measured. Results Levels of hIL-10 and GFP were increased in their respective groups. In vivo delivery of AdGFP caused decreased lung function both preand post-transplantation. In contrast, lungs transfected ex vivo with either AdGFP or AdhIL-10 demonstrated excellent function during EVLP and post-transplantation. (Figure 1) Moreover, significantly higher cytokine levels were found in the in vivo transduced groups. (Figure 2) Conclusion Ex vivo AdGFP delivery resulted in superior lung function and decreased inflammation when compared to in vivo delivery. This suggests that ex vivo delivery could be a superior strategy for the delivery of therapeutic adenoviral vectors. While the IL-10 transgene product appeared to mitigate much of the vector associated inflammation in vivo, this effect is likely limited to the IL-10 transgene. Ex vivo gene delivery would be logistically simpler and applicable to a variety of transgenes.

The Role of Protein-protein Interactions in Mechanotransduction: Implications in Ventilator Induced Lung Injury

Critically ill patients often require mechanical ventilation to support their breathing. This is especially true for patients with Acute Respiratory Distress Syndrome, where therapeutic intervention still remains largely ineffective. A clinical study supported by the National Institutes of Health has indicated that low tidal volume ventilation is beneficial, which has established mechanical ventilation an important contributor to lung injury in these patients. Mechanical ventilation can lead to increased production of cytokines and chemokines related to inflammation and tissue damage. Further understanding of mechanotransduction may reveal targeting strategies for therapeutic intervention. It is known that cells can sense mechanical forces across the plasma membrane through a variety of mechanisms. In addition, intracellular force sensors have been proposed to play an important role in conversion of physical forces into biochemical signals through protein-protein interactions. In this chapter we reviewed this novel mechanism for mechanosensation and mechanotransduction, and proposed to inhibit Src protein tyrosine kinase activation as a potential therapy for ventilator induced lung injury.