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. 2012 Mar 27;20(6):1204–1211. doi: 10.1038/mt.2012.57

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

Jonathan C Yeung 1, Dirk Wagnetz 1, Marcelo Cypel 1, Matthew Rubacha 1, Terumoto Koike 1, Yi-Min Chun 1, Jim Hu 2, Thomas K Waddell 1, David M Hwang 1, Mingyao Liu 1, Shaf Keshavjee 1,*
PMCID: PMC3369301  PMID: 22453765

Abstract

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.

Introduction

The use of replication-deficient adenoviral gene therapy as a strategy to improve the function of donor lungs during post-transplant reperfusion has been investigated for over a decade.1,2 Gene therapy in a transplant setting is attractive because it circumvents difficulties with finite transgene expression times and low cellular transduction rates that have impeded gene replacement type applications in other settings.3,4,5 For example, we have shown that interleukin-10 (IL-10) transgene expression solely at the time of reperfusion can act in a paracrine manner to reduce ischemia–reperfusion injury in both small and large animal models.6,7 This could safely increase the clinical utilization of offered donor lungs currently turned down due to preexisting lung injury.8 However, despite these promising results, no clinical trials have yet occurred. This is partly due to logistic challenges in delivering vector to the donor lungs, which are often in a distant hospital. To attain sufficient transgene expression at the time of reperfusion, gene vector delivery must occur while the organs are still in the donor and 6–12 hours are required for effective therapeutic transgene expression.9 The logistics of transporting and delivering the viral vector to organ donors spread across the continent, concern about delays in organ retrieval while awaiting transgene expression, and potential challenges with vector-associated inflammation all limit easy clinical adoption.

Recently, we have demonstrated that acellular normothermic ex vivo lung perfusion (EVLP) can safely preserve donor lungs for up to 12 hours.10 During perfusion, metabolic activity and gas exchange function of the lungs are preserved, allowing for further real-time evaluation and therapeutic intervention to occur during the storage period. A phase 1–2 clinical trial has recently been completed by the Toronto Lung Transplant Program, which utilized EVLP to evaluate and treat questionable donor organs and demonstrated equivalent outcomes to contemporary lung transplantation.11 In addition, we have also shown that vector delivery during EVLP results in detectable transgene expression by 9 hours.12 EVLP thus appears to be an ideal platform for the clinical delivery of therapeutic adenoviral vector.

Vector-associated inflammation is an impediment to the use of adenoviral vectors for gene therapy.13 Delivery of adenoviral vectors into the lung elicits a prompt innate immune response in the form of a characteristic pattern of inflammatory cytokine and chemokine expression by alveolar macrophages.14,15 These signals recruit circulating neutrophils to the lung, which in turn propagate the inflammatory response and ultimately cause lung injury. Moreover, in the transplant setting, this preexisting inflammation could potentiate subsequent ischemia–reperfusion injury.16 Given that acellular EVLP preservation isolates the organ from the circulation of the host, we hypothesized that intratracheal delivery of adenoviral vector during EVLP would provide superior gene transduction with less inflammation as compared with in vivo delivery because circulating responder immune cells are largely absent in the lung perfusate during EVLP.

Herein, we compared ex vivo with in vivo intratracheal delivery of an E1-, E3-deleted adenoviral vector using a porcine single lung transplant model. Ex vivo delivery resulted in effective gene expression with less vector-associated inflammation and superior lung function pre- and post-transplantation.

Results

Intratracheal EVLP delivery of adenoviral vectors results in effective transgene expression

Saline vehicle control, 1 × 1010 plaque-forming units of adenovirus vector encoding GFP (AdGFP), or 1 × 1010 plaque-forming units of adenovirus vector encoding human IL-10 (AdhIL-10) were delivered intratracheally to lungs both in vivo and ex vivo via EVLP (n = 5/group). The transgene protein GFP acted as a reporter for histologic localization and the transgene protein hIL-10 acted as a quantifiable reporter using enzyme-linked immunosorbent assay. Twelve hours after the intratracheal delivery of AdGFP, GFP expression could be identified in bronchiolar and alveolar epithelium after both in vivo and ex vivo delivery (Figure 1a,b). Distribution of the virus was variable in intensity throughout the lungs; however, GFP expression could be identified in all tissue blocks taken from AdGFP-transduced lungs. Human IL-10 levels in perfusate and plasma of ex vivo and in vivo transduced lungs, respectively, indicate that transgene expression begins ~8 hours after delivery (Figure 1c). A trend toward higher hIL-10 levels was observed with ex vivo delivery (P = 0.063). No hIL-10 could be detected in AdGFP-transduced lungs.

Figure 1.

Figure 1

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Transgene expression at 12 hours after intratracheal AdGFP and AdhIL-10 vector delivery. (a) GFP expression in bronchiolar and (b) alveolar epithelium after ex vivo delivery. (green: GFP, anti-GFP; blue: nucleus, DAPI). Bar = a, 50 = µm; b = 150 µm. (c) Levels of hIL-10 present in the perfusate and plasma after ex vivo and in vivo AdhIL-10 delivery, respectively. Ex vivo and in vivo AdGFP groups included as controls show no IL-10 expression (P < 0.0001 versus respective AdGFP control, mean ± SEM, n = 5). AdGFP, adenovirus encoding green fluorescent protein; AdhIL-10, adenovirus encoding human interleukin-10; DAPI, 4′,6-diamidino-2-phenylindole; s, interlobular septum.

Delivery of AdGFP in vivo results in reduced lung function as compared with ex vivo delivery

Twelve hours after the in vivo delivery of AdGFP, lung oxygenation was significantly reduced as compared with the in vivo control group (P < 0.001; Figure 2). In contrast, ex vivo delivery of AdGFP demonstrated no significant fall in lung oxygenation when compared with the ex vivo control group. Note that oxygenation generally tends to be higher in the ex vivo lungs than the in vivo lungs. This is a physiologic feature of the ex vivo perfusion and ventilation system that provides superior recruitment of the lungs outside of the chest and more optimal ventilation perfusion matching. Compliance, airway pressure, and pulmonary vascular resistance were stable in all three ex vivo groups, further suggesting no vector-associated injury (Supplementary Figure S1).

Figure 2.

Figure 2

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Lung function as measured by P/F ratio after vector delivery. *P < 0.05 at that time point versus control, ***P < 0.001 at that time point versus respective control. Error bars represent mean ± SEM, n = 5. AdGFP, adenovirus encoding green fluorescent protein; AdhIL-10, adenovirus encoding human interleukin-10; PaO2, partial pressure of oxygen in arterial blood; P/F = PaO2/FiO2.

Histologic sections of biopsies taken from lungs 12 hours after vector delivery were blindly scored by a pathologist (D.M.H.) for inflammation similar to Piedra et al.17 In vivo AdGFP and AdhIL-10 groups had higher inflammation scores than the in vivo control group and the corresponding ex vivo groups (Figure 3). In sections from the in vivo AdGFP group, some areas of the lung were more inflamed and had more inflammatory cellular infiltrates than others. We stained these sections for GFP to see whether there was a correlation between viral transduction and inflammation. Indeed, lobules of the lung with increased GFP expression had more significant inflammation at 12 hours after AdGFP delivery (Supplementary Figure S2).

Figure 3.

Figure 3

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Histologic sections (hematoxylin and eosin) of lungs at 12 hours after vector delivery. Representative sections of (a) in vivo control, (b) ex vivo control, (c) in vivo AdhIL-10, (d) ex vivo AdhIL-10, (e) in vivo AdGFP, (f) ex vivo AdGFP are shown. Bar = 500 µm. (g) Inflammation score. *P < 0.05 as compared with corresponding ex vivo group, mean ± SEM, n = 5. AdGFP, adenovirus encoding green fluorescent protein; AdhIL-10, adenovirus encoding human interleukin-10.

We subsequently measured cytokine and chemokine expression in lung tissue biopsies obtained at the end of preservation. Ex vivo and in vivo AdGFP groups expressed significantly higher levels of IL-1β than their respective controls and IL-1β levels were higher in the in vivo groups as compared with their ex vivo correlates (Figure 4). Interestingly, IL-8 levels were higher in ex vivo transduced groups than in vivo groups. No significant differences were seen in IL-6 and TNF-α levels.

Figure 4.

Figure 4

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Proinflammatory cytokine expression in tissue at 12 hours after vector delivery. *P < 0.05; **P < 0.01 as compared with its respective control, mean ± SEM, n = 5, values expressed as pg cytokine/ mg total protein. AdGFP, adenovirus encoding green fluorescent protein; AdhIL-10, adenovirus encoding human interleukin-10; TNF-α, tumor necrosis factor.

IL-10 reduces vector-associated inflammation

IL-10 has previously been shown to have a beneficial effect on vector-associated inflammation in small animal models.16,18 Indeed, in this study, in vivo delivery of AdhIL-10 resulted in better lung function 12 hours after delivery when compared with the AdGFP group, further supporting the evidence that IL-10 can reduce vector-associated inflammation (Figure 2). Importantly, the incremental improvement provided by the ex vivo gene delivery strategy was supported by the fact that ex vivo AdhIL-10 delivery did not demonstrate any lung injury in this large animal model.

Absence of vector-associated injury is preserved post-transplantation

To assess whether vector-associated injury becomes evident upon introduction of the transfected lung to an in vivo recipient host environment, we transplanted the left lung of the AdGFP-transduced groups into another pig. Neither steroids nor immunosuppressive agents were given to avoid dampening the inflammatory response. Lung function in the ex vivo AdGFP group remained excellent. In comparison, in vivo AdGFP-transduced lungs continued to demonstrate poor lung function post-transplantation (Figure 5). Histopathologic lung inflammation scoring in post-transplant sections from tissue biopsies obtained 4 hours after transplantation demonstrates significantly higher inflammation in the in vivo AdGFP lungs (Figure 6). Proinflammatory cytokines and chemokines all became further elevated post-transplant as compared with pretransplant. In vivo AdGFP lungs had significantly higher IL-1β levels than ex vivo AdGFP lungs (P < 0.01). In vivo AdGFP lungs had significantly higher levels of IL-1β and IL-6 levels post-transplant than ex vivo AdIL-10 lungs (P < 0.01 and P < 0.001, respectively; Figure 7).

Figure 5.

Figure 5

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Lung function as measured by PaO2/FiO2 (P/F) ratio after transplantation. ***P < 0.001 as compared with ex vivo AdGFP group, post-transplant data only. AdGFP, adenovirus encoding green fluorescent protein; AdhIL-10, adenovirus encoding human interleukin-10; PaO2, partial pressure of oxygen in arterial blood; P/F = PaO2/FiO2.

Figure 6.

Figure 6

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Histologic sections (hematoxylin and eosin) of lungs at 4 hours after transplantation. Representative sections of (a) ex vivo AdGFP, (b) ex vivo AdhIL-10, (c) in vivo AdGFP are shown. Bar = 150 µm. (d) Inflammation score. *P < 0.05 as compared with corresponding ex vivo AdGFP, mean ± SEM, n = 5. AdGFP, adenovirus encoding green fluorescent protein; AdhIL-10, adenovirus encoding human interleukin-10.

Figure 7.

Figure 7

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Tissue cytokine levels at 4 hours after transplantation. *P < 0.05; **P < 0.01, ***P < 0.001; mean ± SEM, n = 5, values expressed as pg cytokine/mg total protein. AdGFP, adenovirus encoding green fluorescent protein; AdhIL-10, adenovirus encoding human interleukin-10; TNF-α, tumor necrosis factor.

Transgene expression is preserved in the early reperfusion period

In order for adenoviral gene therapy to be useful in a transplant setting, transgene expression ideally should persist into the postreperfusion period. GFP expression persisted in lungs post-transplantation in both ex vivo and in vivo transduced groups at 4 hours post-transplant (Figures 8a,b). To assess the kinetics of transgene expression in ex vivo transduced lungs, we also transplanted the ex vivo AdhIL-10 group to assess hIL-10 expression post-transplantation. As expected, lung function was excellent in the ex vivo AdhIL-10 group and significantly reduced levels of IL-6 were found in the plasma. Human IL-10 was produced at high enough levels to be detectable in the recipient plasma and increased in a linear fashion over the 4 hours of observation post-transplantation (Figure 8c).

Figure 8.

Figure 8

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Transgene expression following transplantation. (a) In vivo AdGFP, (b) ex vivo AdGFP (green: GFP, anti-GFP; blue: nucleus, DAPI). Bar = 150 µm. (c) Human IL-10 levels in plasma of transplanted pig following ex vivo AdhIL-10 delivery, mean ± SEM, n = 5. AdGFP, adenovirus encoding green fluorescent protein; AdhIL-10, adenovirus encoding human interleukin-10; DAPI, 4′,6-diamidino-2-phenylindole; s, interlobular septum.

Discussion

All donor organs undergoing transplantation must undergo the process of ischemia/reperfusion and the attendant risks and complications thereof. In clinical lung transplantation, ischemia–reperfusion injury is the leading cause of primary graft dysfunction and the leading cause of death in the early post-transplant period.19 It is also becoming increasingly evident that early or primary graft dysfunction is linked to increased rejection and late graft dysfunction.20,21 Concern for this condition leads to a highly conservative selection of offered donor lungs by transplant clinicians and thus results in a utilization of only 20%. This translates into high wait-list mortality for potential lung transplant recipients. Fortunately, the predictability of timing and limited duration of ischemia and reperfusion renders ischemia–reperfusion injury particularly amenable to gene therapy strategies. The duration of transgene expression need not be lifelong and the cellular transduction rate need not be complete.

In the past, we have shown that intratracheal IL-10 delivery by adenoviral gene therapy to the donor can reduce ischemia–reperfusion injury in small and large animal lung transplant models.6,7 Despite success in preclinical models, an obstacle to starting gene therapy clinical trials in transplantation is the difficulty with viral vector delivery to organ donors. Preclinical studies examining donor vector delivery suggest that a minimum of 6–12 hours is needed for transgene expression before organ retrieval can commence.9 Further lengthening of this time and the addition of corticosteroids can help reduce the impact of vector-associated inflammation.16 Therefore, safe clinical translation of this strategy might often require the delay of donor organ retrieval for at least 12 hours. During this time, brain-death or intensive care–related complications could further injure donor organs, including those not receiving gene therapy.22 When combined with the already complicated logistics of multiorgan donor procurement for multiple transplant centers, widespread clinical implementation of this strategy remains a challenge.

Ideally, vector delivery would occur after organ retrieval and transport to the recipient hospital; however, the current standard of lung preservation, cold static preservation, reduces lung metabolism to the point that essentially no transgene expression will have occurred by the time it is needed at reperfusion.9 In this study, we described the use of EVLP as a platform for ex vivo donor lung gene therapy and compared it with the conventional in vivo gene therapy approach. Transgene expression after EVLP gene therapy with AdGFP demonstrated localization to epithelial cells both pre- and post-transplantation. Although we recognize that not all cells were transduced with our current vector delivery technique, applications where the transgene product acts in a paracrine manner (such as IL-10) can still be effective. To that end, we have previously shown the ability to effectively transduce human lungs ex vivo with AdIL-10 and have demonstrated improved function and an effective decrease in inflammation related to brain death.23

EVLP gene therapy isolates the donor organ from the immune system of the host, i.e., circulating cells and other organ systems during transduction. This greatly aids the targeting of gene therapy vectors to the lung, particularly for strategies that might need intravascular vector delivery such as endothelial NO synthase gene therapy described by Suda et al.24 More importantly, a major benefit of isolating the donor organ during vector delivery is the absence of circulating leukocytes. Although the ex vivo lung continues to harbor leukocytes in its alveolar and interstitial compartment in the form of alveolar and interstitial macrophages, interstitial neutrophils, and bronchus-associated lymphocytes, among others, attraction of circulating leukocytes into the lung remains a major part of generating a strong inflammatory response.25 Adenoviral vector–associated inflammation is well characterized in the lung and remains a challenge for adenoviral gene therapy.14,26 Following intratracheal delivery, capsid proteins elicit an almost immediate innate immune response in the form of cytokine release. These cytokines recruit inflammatory cells from the circulation into the lung which further propagate inflammation. Absence of these circulating inflammatory cells appears to arrest this process as ex vivo AdGFP delivery resulted in improved measures of lung function and histology as compared with in vivo AdGFP delivery. In addition, the IL-1β response observed was higher in the in vivo group pre- and post-transplantation. Surprisingly, IL-8 levels were higher in ex vivo transduced groups pretransplantation. As a strong neutrophil chemokine, we would have expected IL-8 to be elevated in both in vivo and ex vivo groups.27 Perhaps a lack of neutrophil responders ex vivo resulted in an early increased production of IL-8; however, further study would be needed to confirm this speculation. The in vivo AdhIL-10 group in this large animal study once again had better lung function as compared with the AdGFP group, supporting the benefit of IL-10 against vector-associated inflammation first observed in the rat.16

The feasibility of therapeutic gene therapy for ischemia–reperfusion injury depends on effective transgene expression during the early reperfusion period. This study demonstrated excellent GFP expression following reperfusion in both ex vivo and in vivo transduced lungs. Furthermore, to rule out the possibility that the GFP detected was made pretransplant, we transplanted lungs from the EVLP AdhIL-10 group and measured hIL-10 in the plasma post-transplantation. Human IL-10 transgene product was detectable in the plasma of the recipient and increased in a linear fashion, suggesting a constant rate of production from the transplanted donor lung immediately following reperfusion.

This study is limited in that post-transplant outcomes were followed only for the early reperfusion period. Further study with survival models of lung transplantation would allow for a prolonged safety assessment and better characterization of transgene expression kinetics.

In summary, we have demonstrated an effective, clinically applicable strategy for ex vivo gene therapy to treat donor lungs before transplantation. EVLP-based gene therapy appears to be a safe method of adenoviral vector delivery with an E1-, E3-deleted adenoviral vector. Our results demonstrate that ex vivo application of gene therapy represents a unique application of gene therapy using current vector technology that is superior to conventional in vivo application of adenoviral gene therapy. We demonstrated effective therapeutic gene transduction of the donor lung with a decreased inflammatory response and improved post-transplant lung function. Future directions could include combining ex vivo delivery with helper-dependent adenoviral vectors or other improved vector systems for further efficacy or immunologic benefit. Investigation of ex vivo vector delivery strategies in the perfusate for endothelial targeting would also be an avenue for future research. Despite the relatively recent introduction of EVLP, there has been significant interest and adoption by the lung transplantation community and the promising results of a recent clinical trial will further encourage its development and adoption.11 We hope that novel applications using gene therapy during EVLP will transform clinical lung transplantation improving both transplant activity and outcomes.

Materials and Methods

Animals. Male Yorkshire pigs weighing 25–35 kg were utilized for studies. All animals received humane care in compliance with the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research and the “Guide for the Care of Laboratory Animals” published by the National Institutes of Health. The Animal Care Committee of the Toronto General Research Institute approved all studies.

Gene vector. A first generation (E1-, E3-deleted) serotype 5 adenoviral vector under the control of a cytomegalovirus promoter and containing the GFP or hIL-10 complementary DNA was obtained from the Gene Transfer Vector Core of the University of Iowa College of Medicine (Iowa City, IA).

Anesthesia. Pigs were sedated with ketamine (40 mg/kg intramuscularly), anesthetized with inhaled isoflurane (5%) and maintained with propofol (5–8 mg/kg/hour intravenously), and fentanyl citrate (2–20 mg/kg/hour intravenously) for the duration of all surgeries. The airway was secured by tracheostomy and intubation with a size 8.5 French endotracheal tube.

In vivo viral delivery. Following induction of anesthesia, 1 × 1010 plaque-forming units of AdGFP or 1 × 1010 plaque-forming unit of AdhIL-10 were diluted with normal saline to a final volume of 10 ml. Control groups received 10 ml of saline only. Polyethylene tubing (4.7 Fr PE160; Becton Dickenson, Sparks, MD) was then inserted into the port of a bronchoscope (Olympus Canada, Markham, Ontario, Canada). Approximately 1.0 ml of the diluted virus mixture was then injected intratracheally via the tube into each of the segmental bronchi of the left lung. After delivery, an inspiratory hold was performed to a peak airway pressure of 25 cmH2O and the lungs ventilated at a higher volume and rate (10 ml/kg at 12 breaths/minute) for a period of 10 minutes. At 12 hours after viral delivery, the lungs were retrieved from the pigs as detailed by Pierre et al.28 and placed on ice. Preparation of the recipient for transplantation followed immediately afterward and the left lung transplanted as previously described.28

Ex vivo viral delivery. Double lung blocks were retrieved from the pigs as detailed by Pierre et al.28 and EVLP started as detailed by Cypel et al.10 for 12 hours. In brief, following retrieval of the double lung block and excision of the heart, cannulae (Vitrolife, Kungsbacka, Sweden) were connected to the pulmonary artery and left atrium and the trachea intubated. A custom XVIVO pack was utilized (GISH, Pack #11512), which consists of a circuit containing a pediatric reservoir, a centrifugal pump head, an oxygenator, a heat exchanger, and a leukocyte filter. This circuit was mounted onto a centrifugal pump setup and then primed with 1.5 l of Steen solution (Vitrolife), 10,000 U of heparin, 500 mg of Solu-Medrol, and 1 g of Cefazolin. The lungs were then placed within an XVIVO dome and connected to the circuit. A gradual increase in anterograde perfusion flow was delivered over the next hour to a maximum of 40% of the estimated cardiac output. Warming of the lung was gradual over the initial 30 minutes to a maximum temperature of 37 °C. Ventilation of the lungs at a rate of 7 beats/minute, 7 ml/kg using a volume control mode of ventilation and an FiO2 of 21% was started when the lungs reached 34 °C. Lung physiologic parameters including pulmonary artery pressure, pulmonary venous pressure, perfusate flow rate, dynamic compliance, peak airway pressure, partial pressure of oxygen in arterial blood, and partial pressure of oxygen in the prelung perfusate (inflow) were measured hourly. Following the initial 1-hour period, vector delivery to the lung was performed as described above. At 12 hours after delivery, the lungs were cooled on the circuit and placed on ice. Preparation of the recipient for transplantation followed immediately afterward and the left lung was transplanted as detailed by Pierre et al.28

Biopsies. Biopsy of the lung was performed by ligation of a superficial portion of lung tissue with 0 silk ties. One portion of the biopsy was immediately snap frozen. The other portion was inflated with 10% buffered formalin using a 25 gauge needle and the stored in 10% buffered formalin. After 24 hours of formalin storage, the storage solution was changed to 70% ethanol and embedding in paraffin shortly thereafter. Biopsies, perfusate, and plasma samples were taken 12 hours after vector delivery. Plasma samples were taken hourly post-transplant and final biopsies were taken 4 hours after transplantation.

Histopathologic assessment. Paraffin-embedded tissues were sectioned at 5-µm thickness, stained with by hematoxylin and eosin, and examined for pathological changes under light microscopy. A pulmonary pathologist (D.M.H.) and an investigator (J.C.Y.) evaluated mid-sagittal slices of lung sections in a randomized and blinded fashion to assess histopathological grading of inflammation using the following parameters similar to the method of Piedra et al.: parenchymal inflammation, peribronchial inflammation, and perivascular inflammation.17 The severity of each finding was graded on a four-point scale as follows: 0, absent; 1, mild; 2, moderate; and 3, severe and then summed to make a final composite injury score.

GFP staining. For GFP immunohistochemistry, formalin-fixed, paraffin-embedded tissue sections (4-µm thick) were mounted on positively charged microscope slides. Antigen retrieval was performed with microwave heating in citrate buffer (10 mmol/l sodium citrate, 0.05% Tween 20, pH 6.0). Endogenous peroxidase and biotin activities were blocked, respectively, using 3% hydrogen peroxide and avidin/biotin blocking kit (Lab Vision, Fremont, CA). After blocking for 15 minutes with 10% normal horse serum diluted in casein solution (Dako, Carpinteria, CA), polyclonal rabbit anti-GFP primary antibody (Ab290; Abcam, Cambridge, MA) was applied at 1:1,000 dilution and incubated at room temperature for 30 minutes. The detection procedure was performed using a biotinylated goat anti-rabbit secondary antibody (Vector Laboratories, Burlingame, CA) for 30 minutes and horseradish peroxidase–conjugated ultrastreptavidin labeling reagent (ID Labs, London, Ontario, Canada) for 30 minutes. Color development was performed with freshly prepared diaminobenzidine solution (Vector Laboratories). Finally, sections were counterstained lightly with Mayer's hematoxylin to better display nuclei. These slides were then imaged on a Nikon 80i microscope (Nikon Instruments, Melville, NY) following Köhler alignment and images were recorded using an attached charge-coupled device camera using ACT-4U software.

For GFP immunofluorescence, formalin-fixed, paraffin-embedded tissue sections (4-µm thick) were mounted on positively charged microscope slides. Antigen retrieval was performed with microwave heating in EDTA buffer (1 mmol/l EDTA, 0.05% Tween 20, pH 9.0). Endogenous peroxidase and biotin activities were blocked, respectively, using 3% hydrogen peroxide and avidin/biotin blocking kit (Lab Vision). After blocking for 15 minutes with 10% normal horse serum diluted in casein solution (Dako), polyclonal rabbit anti-GFP primary antibody (Ab290; Abcam) was applied at 1:1,000 dilution and incubated at room temperature for 30 minutes. An anti-rabbit antibody conjugated with Alexa Fluor 488 was then applied at 1:200 dilution and incubated at room temperature for 60 minutes. Slides were then washed and mounted with Vectashield mounting medium with 4′,6-diamidino-2-phenylindole and immediately imaged on a Nikon 80i microscope and images recorded using an attached charge-coupled device camera using ACT-4U software.

Homogenization of lung tissue. Lung tissue homogenization and protein extraction were performed as previously described.7 Tissue frozen in liquid nitrogen was homogenized and crushed into a powder in a mortar and pestle cooled with dry ice. Lung tissue (50 mg) was then put into a microcentrifuge tube and 1 ml of lysis buffer was added. These mixtures were then sonicated for 10 seconds on ice three times and then centrifuged at 4 °C at 10,000 r.c.f. for 15 minutes. The supernatant was then aliquoted and stored at −80 °C until analysis.

Inflammatory profile in lung tissue biopsies. Supernatants of lung tissues, perfusate samples, and plasma were assayed in duplicate using specific enzyme-linked immunosorbent assay kits for human IL-10, and porcine IL-6, IL-8, tumor necrosis factor-α, and IL-1β, respectively (R&D Systems, Minneapolis, MN). The enzyme-linked immunosorbent assay kit for hIL-10 does not cross-react with porcine IL-10. The optical density of each well was read at 450 nm and corrected at a wavelength of 540 nm according to the manufacturer's instructions with an NM-600 microplate reader (Dynatech Laboratories, Chantilly, VA). The final concentration was calculated by converting the OD readings against a standard curve.

Statistics. All results were expressed as mean ± SEM. For comparisons between the two groups at all time points, two-way analysis of variance was utilized with a Bonferroni correction for multiple comparisons (Figures 2 and 5). For comparisons between three or more groups, one-way analysis of variance was utilized again with Bonferroni correction (Figures 3, 4, 7 and 8). Student's t-test was utilized for comparing two groups (Figure 1). P values <0.05 were considered significant.

SUPPLEMENTARY MATERIAL Figure S1. Physiologic parameters following ex vivo adenoviral vector delivery. Figure S2. Inflammation in AdGFP delivered in vivo follows cellular transduction.

Acknowledgments

This study was supported by grants from the Canadian Institute of Health Research (MOP-64370, MOP-13270, and MOP-42546) and by an Astellas Pharma transplantation grant. The Steen solution was provided by Vitrolife. J.C.Y. was supported by the Surgeon-Scientist Program at the University of Toronto.

Supplementary Material

Figure S1.

Physiologic parameters following ex vivo adenoviral vector delivery.

Click here for additional data file. (33KB, pdf)
Figure S2.

Inflammation in AdGFP delivered in vivo follows cellular transduction.

Click here for additional data file. (127.8KB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1.

Physiologic parameters following ex vivo adenoviral vector delivery.

Click here for additional data file. (33KB, pdf)
Figure S2.

Inflammation in AdGFP delivered in vivo follows cellular transduction.

Click here for additional data file. (127.8KB, pdf)

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