Enhanced Mitochondrial DNA Repair Resuscitates Transplantable Lungs Donated After Circulatory Death.

BACKGROUND: Transplantation of lungs procured after donation after circulatory death (DCD) is challenging because postmortem metabolic degradation may engender susceptibility to ischemia-reperfusion (IR) injury. Because oxidative mitochondrial DNA (mtDNA) damage has been linked to endothelial barrier disruption in other models of IR injury, here we used a fusion protein construct targeting the DNA repair 8-oxoguanine DNA glycosylase-1 (OGG1) to mitochondria (mtOGG1) to determine if enhanced repair of mtDNA damage attenuates endothelial barrier dysfunction after IR injury in a rat model of lung procurement after DCD. MATERIALS AND METHODS: Lungs excised from donor rats 1 h after cardiac death were cold stored for 2 h after which they were perfused ex vivo in the absence and presence of mt-OGG1 or an inactive mt-OGG1 mutant. Lung endothelial barrier function and mtDNA integrity were determined during and at the end of perfusion, respectively. RESULTS AND CONCLUSIONS: Mitochondria-targeted OGG1 attenuated indices of lung endothelial dysfunction incurred after a 1h post-mortem period. Oxidative lung tissue mtDNA damage as well as accumulation of proinflammatory mtDNA fragments in lung perfusate, but not nuclear DNA fragments, also were reduced by mitochondria-targeted OGG1. A repair-deficient mt-OGG1 mutant failed to protect lungs from the adverse effects of DCD procurement. CONCLUSIONS: These findings suggest that endothelial barrier dysfunction in lungs procured after DCD is driven by mtDNA damage and point to strategies to enhance mtDNA repair in concert with EVLP as a means of alleviating DCD-related lung IR injury.

Somatic mitochondrial DNA mutations in early Parkinson and incidental Lewy body disease.

Somatic mutations in mitochondrial DNA (mtDNA) are hypothesized to play a role in Parkinson disease (PD), but large increases in mtDNA mutations have not previously been found in PD, potentially because neurons with high mutation levels degenerate and thus are absent in late stage tissue. To address this issue, we studied early stage PD cases and cases of incidental Lewy body disease (ILBD), which is thought to represent presymptomatic PD. We show for the first time that mtDNA mutation levels in substantia nigra neurons are significantly elevated in this group of early PD and ILBD cases.

Analysis of Plasma Products for Cellular Contaminants: Comparing Standard Preparation Methods.

BACKGROUND: Recent reports suggest that component plasma products contain significant quantities of cellular contamination. We hypothesized that leukoreduction of whole blood before preparation of derived plasma is an effective method to prevent cellular contamination of stored plasma. STUDY DESIGN: Samples of never-frozen liquid plasma prepared by standard methods (n = 25) were obtained from 3 regional blood centers that supply 3 major trauma centers. Samples were analyzed for leukocyte and platelet contamination by flow cytometry. To determine if leukoreduction of whole blood before centrifugation and expression of plasma prevents cellular contamination of liquid plasma, 1 site generated 6 additional units of liquid plasma from leukoreduced whole blood, which were then compared with units of liquid plasma derived by standard processing. RESULTS: Across all centers, each unit of never-frozen liquid plasma contained a mean of 12.8 ± 3.0 million leukocytes and a mean of 4.6 ± 2 billion platelets. Introduction of whole blood leukoreduction (LR) before centrifugation and plasma extraction essentially eliminated all contaminating leukocytes (Non-LR: 12.3 ± 2.9 million vs LR: 0.05 ± 0.05 million leukocytes) and platelets (Non-LR: 4.2 ± 0.3 billion platelets vs LR: 0.00 ± 0.00 billion platelets). CONCLUSIONS: Despite widespread belief that stored plasma is functionally acellular, testing of liquid plasma from 3 regional blood banks revealed a significant amount of previously unrecognized cellular contamination. Introduction of a leukoreduction step before whole blood centrifugation essentially eliminated detectable leukocyte and platelet contaminants from plasma. Therefore, our study highlights a straightforward and cost-effective method to eliminate cellular contamination of stored plasma.

Plasma Transfusion Products and Contamination with Cellular and Associated Pro-Inflammatory Debris.

BACKGROUND: Stored plasma products are widely regarded as being functionally acellular, obviating the need for leukoreduction. We tested the hypothesis that donor plasma is contaminated by leukocytes and platelets, which, after frozen storage, would release cellular debris in quantities sufficient to elicit significant pro-inflammatory responses. STUDY DESIGN: Samples of never-frozen liquid plasma from 2 regional Level I trauma centers were analyzed for leukocyte and platelet contamination. To determine if the cellular contamination and associated debris found in liquid plasma were at levels sufficient to evoke an innate immune response, known quantities of leukocytes were subjected to a freeze-thaw cycle, added to whole blood, and the magnitude of the inflammatory response was determined by induction of interleukin-6. RESULTS: Units of never-frozen plasma from 2 regional Level I trauma centers located in Alabama and Louisiana contained significant amounts of leukocyte contamination (Louisiana, n = 22; 17.3 ± 4.5 million vs Alabama, n = 22; 11.3 ± 2.2 million) and platelet contamination (Louisiana, n = 21; 0.86 ± 0.20 billion vs Alabama, n = 22; 1.0 ± 0.3 billion). Cellular debris from as few as 1 million leukocytes induced significant increases in interleukin-6 levels (R2 = 0.74; p < 0.0001). CONCLUSIONS: Stored plasma units from trauma center blood banks were highly contaminated with leukocytes and platelets, at levels more than 15-fold higher than sufficient to elicit ex vivo inflammatory responses. In light of paradigm shifts toward the use of more empiric plasma for treatment of hypovolemia, this study suggests that new manufacturing and quality-control processes are needed to eliminate previously unrecognized cellular contamination present in stored plasma products.

Pharmacologic Protection of Mitochondrial DNA Integrity May Afford a New Strategy for Suppressing Lung Ischemia-Reperfusion Injury.

Lung ischemia-reperfusion (IR) injury contributes to post-transplant complications, including primary graft dysfunction. Decades of reports show that reactive oxygen species generated during lung IR contribute to pulmonary vascular endothelial barrier disruption and edema formation, but the specific target molecule(s) that "sense" injury-inducing oxidant stress to activate signaling pathways culminating in pathophysiologic changes have not been established. This review discusses evidence that mitochondrial DNA (mtDNA) may serve as a molecular sentinel wherein oxidative mtDNA damage functions as an upstream trigger for lung IR injury. First, the mitochondrial genome is considerably more sensitive than nuclear DNA to oxidant stress. Multiple studies suggest that oxidative mtDNA damage could be transduced to physiologic dysfunction by pathways that are either a direct consequence of mtDNA damage per se or involve formation of proinflammatory mtDNA damage-associated molecular patterns. Second, transgenic animals or cells overexpressing components of the base excision DNA repair pathway in mitochondria are resistant to oxidant stress-mediated pathophysiologic effects. Finally, published and preliminary studies show that pharmacologic enhancement of mtDNA repair or mtDNA damage-associated molecular pattern degradation suppresses reactive oxygen species-induced or IR injury in multiple organs, including preclinical models of lung procurement for transplant. Collectively, these findings point to the interesting prospect that pharmacologic enhancement of DNA repair during procurement or ex vivo lung perfusion may increase the availability of lungs for transplant and reduce the IR injury contributing to primary graft dysfunction.