Renal hemofiltration prevents metabolic acidosis and reduces inflammation during normothermic machine perfusion of the vascularized composite allograft: A preclinical study.

INTRODUCTION: Recent experimental evidence suggests normothermic machine perfusion of the vascularized composite allograft results in improved preservation compared to static cold storage, with less reperfusion injury in the immediate post-operative period. However, metabolic acidosis is a common feature of vascularized composite allograft perfusion, primarily due to the inability to process metabolic by-products. We evaluated the impact of combined limb-kidney perfusion on markers of metabolic acidosis and inflammation in a porcine model. METHODS: Ten paired pig forelimbs were used for this study, grouped as either limb-only (LO, n = 5) perfusion, or limb-kidney (LK, n = 5) perfusion. Infrared thermal imaging was used to determine homogeneity of perfusion. Lactate, bicarbonate, base, pH, and electrolytes, along with an inflammatory profile generated via the quantification of cytokines and cell-free DNA in the perfusate were recorded. RESULTS: The addition of a kidney to a limb perfusion circuit resulted in the rapid stabilization of lactate, bicarbonate, base, and pH. Conversely, the LO circuit became progressively acidotic, correlating in a significant increase in pro-inflammatory cytokines. Global perfusion across the limb was more homogenous with LK compared to LO. CONCLUSION: The addition of a kidney during limb perfusion results in significant improvements in perfusate biochemistry, with no evidence of metabolic acidosis.

Modifying dietary patterns in cardiothoracic transplant patients to reduce cardiovascular risk: The AMEND-IT Trial.

BACKGROUND: Cardiovascular disease (CVD) is common after cardiothoracic transplantation and causes substantial morbidity. AIMS: To assess feasibility and potential effectiveness of dietary interventions to reduce CVD risk. MATERIALS AND METHODS: In a pilot intervention, we recruited patients from a tertiary hospital and randomly allocated them to a Mediterranean or low-fat diet for 12 months. Feasibility was measured by patient participation, retention, and adherence. Changes in weight, body mass index (BMI), heart rate, blood pressure, glucose markers, and blood lipids were assessed using longitudinal generalized estimating equation regression models with 95% confidence intervals. RESULTS: Of 56 heart and 60 lung transplant recipients, 52 (45%) consented, 41 were randomized, and 39 (95%) completed the study with good adherence to randomized diets. After 12 months, changes in many risk factors were seen in the Mediterranean and low-fat-diet groups, respectively, including mean BMI (-0.5 vs. 0.0 kg/m2 ), systolic/diastolic blood pressure +0.5/+0.1 vs -4.4/-3.5 mmHg; fasting glucose -0.26 vs -0.27 mmol/L; total cholesterol -0.56 vs -0.40 mmol/L. Changes in BMI and systolic/diastolic blood pressure in 49 eligible patients who did not take part were +0.7 kg/m2 and +2.5/+1.8 mmHg. DISCUSSION: Dietary interventions in cardiothoracic transplant patients are feasible and potentially beneficial. CONCLUSION: A definitive nutritional intervention study in these high-risk patients is warranted.

The effect of 1.5 T cardiac magnetic resonance on human circulating leucocytes.

Aims: Investigators have proposed that cardiovascular magnetic resonance (CMR) should have restrictions similar to those of ionizing imaging techniques. We aimed to investigate the acute effect of 1.5 T CMR on leucocyte DNA integrity, cell counts, and function in vitro, and in a large cohort of patients in vivo. Methods and results: In vitro study: peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteers, and histone H2AX phosphorylation (γ-H2AX) expression, leucocyte counts, and functional parameters were quantified using flow cytometry under the following conditions: (i) immediately following PBMC isolation, (ii) after standing on the benchside as a temperature and time control, (iii) after a standard CMR scan. In vivo study: blood samples were taken from 64 consecutive consenting patients immediately before and after a standard clinical scan. Samples were analysed for γ-H2AX expression and leucocyte counts. CMR was not associated with a significant change in γ-H2AX expression in vitro or in vivo, although there were significant inter-patient variations. In vitro cell integrity and function did not change with CMR. There was a significant reduction in circulating T cells in vivo following CMR. Conclusion: 1.5 T CMR was not associated with DNA damage in vitro or in vivo. Histone H2AX phosphorylation expression varied markedly between individuals; therefore, small studies using γ-H2AX as a marker of DNA damage should be interpreted with caution. Cardiovascular magnetic resonance was not associated with loss of leucocyte viability or function in vitro. Cardiovascular magnetic resonance was associated with a statistically significant reduction in viable leucocytes in vivo.

Study Comparing Vein Integrity and Clinical Outcomes in Open Vein Harvesting and 2 Types of Endoscopic Vein Harvesting for Coronary Artery Bypass Grafting: The VICO Randomized Clinical Trial (Vein Integrity and Clinical Outcomes).

BACKGROUND: Current consensus statements maintain that endoscopic vein harvesting (EVH) should be standard care in coronary artery bypass graft surgery, but vein quality and clinical outcomes have been questioned. The VICO trial (Vein Integrity and Clinical Outcomes) was designed to assess the impact of different vein harvesting methods on vessel damage and whether this contributes to clinical outcomes after coronary artery bypass grafting. METHODS: In this single-center, randomized clinical trial, patients undergoing coronary artery bypass grafting with an internal mammary artery and with 1 to 4 vein grafts were recruited. All veins were harvested by a single experienced practitioner. We randomly allocated 300 patients into closed tunnel CO2 EVH (n=100), open tunnel CO2 EVH (n=100), and traditional open vein harvesting (n=100) groups. The primary end point was endothelial integrity and muscular damage of the harvested vein. Secondary end points included clinical outcomes (major adverse cardiac events), use of healthcare resources, and impact on health status (quality-adjusted life-years). RESULTS: The open vein harvesting group demonstrated marginally better endothelial integrity in random samples (85% versus 88% versus 93% for closed tunnel EVH, open tunnel EVH, and open vein harvesting; P<0.001). Closed tunnel EVH displayed the lowest longitudinal hypertrophy (1% versus 13.5% versus 3%; P=0.001). However, no differences in endothelial stretching were observed between groups (37% versus 37% versus 31%; P=0.62). Secondary clinical outcomes demonstrated no significant differences in composite major adverse cardiac event scores at each time point up to 48 months. The quality-adjusted life-year gain per patient was 0.11 (P<0.001) for closed tunnel EVH and 0.07 (P=0.003) for open tunnel EVH compared with open vein harvesting. The likelihood of being cost-effective, at a predefined threshold of £20 000 per quality-adjusted life-year gained, was 75% for closed tunnel EVH, 19% for open tunnel EVH, and 6% for open vein harvesting. CONCLUSIONS: Our study demonstrates that harvesting techniques affect the integrity of different vein layers, albeit only slightly. Secondary outcomes suggest that histological findings do not directly contribute to major adverse cardiac event outcomes. Gains in health status were observed, and cost-effectiveness was better with closed tunnel EVH. High-level experience with endoscopic harvesting performed by a dedicated specialist practitioner gives optimal results comparable to those of open vein harvesting. CLINICAL TRIAL REGISTRATION: URL: https://www.isrctn.com. International Standard Randomised Controlled Trial Registry Number: 91485426.

Adherence to Mediterranean and low-fat diets among heart and lung transplant recipients: a randomized feasibility study.

BACKGROUND: Heart and lung transplant recipients are at a substantially increased risk of cardiovascular disease (CVD). Since both low-fat and Mediterranean diets can reduce CVD in immunocompetent people at high risk, we assessed adherence among thoracic transplant recipients allocated to one or other of these diets for 12 months. METHODS: Forty-one transplant recipients (20 heart; 21 lung) randomized to a Mediterranean or a low-fat diet for 12 months received diet-specific education at baseline. Adherence was primarily assessed by questionnaire: 14-point Mediterranean diet (score 0-14) and 9-point low-fat diet (score 0-16) respectively, high scores indicating greater adherence. Median scores at baseline, 6 months, 12 months, and 6-weeks post-intervention were compared by dietary group. We further assessed changes in weight, body mass index (BMI) and serum triglycerides from baseline to 12 months as an additional indicator of adherence. RESULTS: In those randomized to a Mediterranean diet, median scores increased from 4 (range 1-9) at baseline, to 10 (range 6-14) at 6-months and were maintained at 12 months, and also at 6-weeks post-intervention (median 10, range 6-14). Body weight, BMI and serum triglycerides decreased over the 12-month intervention period (mean weight - 1.8 kg, BMI -0.5 kg/m2, triglycerides - 0.17 mmol/L). In the low-fat diet group, median scores were 11 (range 9-14) at baseline; slightly increased to 12 (range 9-16) at 6 months, and maintained at 12 months and 6 weeks post-intervention (median 12, range 8-15). Mean changes in weight, BMI and triglycerides were - 0.2 kg, 0.0 kg/m2 and - 0.44 mmol/L, respectively. CONCLUSIONS: Thoracic transplant recipients adhered to Mediterranean and low-fat dietary interventions. The change from baseline eating habits was notable at 6 months; and this change was maintained at 12 months and 6 weeks post-intervention in both Mediterranean diet and low-fat diet groups. Dietary interventions based on comprehensive, well-supported education sessions targeted to both patients and their family members are crucial to success. Such nutritional strategies can help in the management of their substantial CVD risk. TRIAL REGISTRATION: The IRAS trial registry ( ISRCTN63500150 ). Date of registration 27 July 2016. Retrospectively registered.

Characterizing the early inflammatory contribution of the donor kidney following reperfusion.

BACKGROUND: Donor kidneys contain a large reservoir of passenger leucocytes that contribute to acute rejection via direct alloantigen presentation and pro-inflammatory cytokine secretion. However, the early contribution of these cells following revascularization has not previously been described. We performed a secondary, high-volume preservation flush following cold storage to characterize the inflammatory contribution of the donor kidney upon reperfusion. METHODS: Porcine kidneys were retrieved using a protocol analogous to current UK clinical practice. Following 2 h of cold static preservation, kidneys underwent a secondary flush with Ringer's solution. The venous effluent was collected and leucocytes phenotyped via flow cytometry. Inflammatory mediators, including cytokines and cell-free DNA, were then assessed to determine the inflammatory contribution of the donor kidney. RESULTS: Upon reperfusion, a significant population of donor-derived CD45 + leucocytes mobilized from the renal vasculature via the renal vein [mean 4.738 × 10 8 (SD 1.348 × 10 8 )]. Within this population, T cells were dominant, representing >60% of the leucocyte repertoire. Granulocytes, monocytes and natural killer cells were also identified, but in comparatively lower numbers. Significant concentrations of cytokines and cell-free DNA were also eluted upon reperfusion. CONCLUSIONS: The donor kidney contains a significant immune load that rapidly mobilizes following reperfusion. Performing a secondary preservation flush prior to implantation may reduce this inflammatory burden via diversion of donor leucocytes and inflammatory mediators from entry into the recipient circulation. This may modulate direct presentation and reduce the inflammatory contribution of the donor kidney following transplantation.

Strategies to Reduce Ischemia Reperfusion Injury in Vascularized Composite Allotransplantation of the Limb.

An important and often underinvestigated contributor to solid organ transplant rejection is ischemia reperfusion injury. This pathophysiological response releases damaging reactive oxygen species and cell stress signals that initiate inflammation, which has a critical role in priming the immune system for allorecognition. In time, this renders graft dysfunction and how this response is mediated in composite tissues remains unknown. Current protocols are drawn from solid organ transplantation with little scientific basis as to how this informs current hand transplantation practices. In addition to preservation flush and allograft cooling, machine perfusion is placing itself experimentally as a concept that could act to promote viability and increase the critical ischemic window, which is especially beneficial at a time of limited donors. With the increasing prevalence worldwide of hand transplantation, we review the potential contribution of ischemia reperfusion injury to hand allograft rejection including both current and experimental strategies.

Mucopolysaccharide diseases: a complex interplay between neuroinflammation, microglial activation and adaptive immunity.

Mucopolysaccharide (MPS) diseases are lysosomal storage disorders (LSDs) caused by deficiencies in enzymes required for glycosaminoglycan (GAG) catabolism. Mucopolysaccharidosis I (MPS I), MPS IIIA, MPS IIIB and MPS VII are deficient in the enzymes α-L-Iduronidase, Heparan-N-Sulphatase, N-Acetylglucosaminidase and Beta-Glucuronidase, respectively. Enzyme deficiency leads to the progressive multi-systemic build-up of heparan sulphate (HS) and dermatan sulphate (DS) within cellular lysosomes, followed by cell, tissue and organ damage and in particular neurodegeneration. Clinical manifestations of MPS are well established; however as lysosomes represent vital components of immune cells, it follows that lysosomal accumulation of GAGs could affect diverse immune functions and therefore influence disease pathogenesis. Theoretically, MPS neurodegeneration and GAGs could be substantiating a threat of danger and damage to alert the immune system for cellular clearance, which due to the progressive nature of MPS storage would propagate disease pathogenesis. Innate immunity appears to have a key role in MPS; however the extent of adaptive immune involvement remains to be elucidated. The current literature suggests a complex interplay between neuroinflammation, microglial activation and adaptive immunity in MPS disease.

The contribution of the donor vascularised hand and face allograft in transplant rejection: An immunological perspective.

Overcoming immunological rejection remains a barrier to the safe adoption of Vascularised Composite Allotransplantation (VCA). To mitigate this risk, clinical protocols have been derived from solid organ transplantation, targeting recipient immunomodulation, yet VCA is unique. Face and hand composite allografts are composed of multiple different tissues, each with their own immunological properties. Experimental work suggests that allografts carry variable numbers and populations of donor leukocytes in an organ specific manner. Ordinarily, these passenger leukocytes are transferred from the donor graft into the recipient circulation after transplantation. Whether alloantigen presentation manifests as acute allograft rejection or transplant tolerance is unknown. This review aims to characterise the immunological properties of the constituent parts of the donor face and hand, the potential fate of donor leukocytes and to consider theoretical graft specific interventions to mitigate early rejection.

Developing an Injury-Free 15 Hour Preservation Protocol of Donor Porcine Kidneys Using Normothermic Machine Perfusion.

Normothermic machine perfusion (NMP) offers a superior alternative to hypothermic preservation but is currently time limited. Extending this time could electivise transplantation and enable physiologic assessments of functionality. Porcine kidneys were retrieved, stored on ice for 3.5 hours before being placed onto a NMP circuit for 12 hours. Hemodynamics, biochemistry, and urine output were assessed. After 12 hours, kidneys were scored using the clinical assessment score. Biopsies were collected for histological assessment. Kidneys demonstrated continual improvements in hemodynamics. Perfusate sodium concentrations remained within physiologic parameters. Sodium bicarbonate increased over-time with corresponding decreases in lactate, demonstrating active renal gluconeogenesis and Cori cycle processes. Urine production began immediately and was sustained, indicating renal functionality. Under the clinical perfusion assessment score, all kidneys received a score of 1 and would be considered suitable for transplantation. Histological assessment revealed kidneys were injury free. Our NMP protocol safely preserves kidneys for over 15 hours. Successful perfusion was achieved with stable hemodynamics and biochemistry, with maintained urination. Importantly, kidneys remained in optimal health, with no evidence of injury. This may enable electivisation of transplantation, while reducing hypothermic injury.

Characterisation of the T cell and dendritic cell repertoire in a murine model of mucopolysaccharidosis I (MPS I).

BACKGROUND: Mucopolysaccharidosis I (MPS I) is a metabolic disorder caused by α-L-Iduronidase (IDUA) deficiency, resulting in lysosomal accumulation of heparan (HS) and dermatan sulphate (DS). This has been reported in microglia, yet currently the effect of IDUA deficiency on T cells and dendritic cells (DC) and their functionality in disease pathogenesis remains unclear. METHODS: Peripheral blood was collected from 3 month old C57BL/6 MPS I (n = 11) and wildtype (WT) (n = 6) mice. T cell and DC phenotype and functional characteristics were identified by flow cytometry. RESULTS: MPS I mice exhibited a reduction in DC (p = <0.001) along with CD8+ cytotoxic (p = 0.01) and CD4+ T helper (p = 0.032) cells, compared to WT controls. MPS I DC displayed a significant decrease in cell surface CD123 (p = 0.02) and CD86 (p = 0.006) expression. Furthermore, CD45RB expression was significantly reduced on T helper cells in the MPS I population (p = 0.019). CONCLUSION: We report a reduction in circulating DC and T cells in the MPS I mouse; indicative of adaptive immune dysfunction. DC reduction may occur in response to down-regulation of the IL-3 receptor (CD123), necessary for DC survival. We also report down-regulation of cell surface CD86, a molecule required for T cell co-stimulation. T helper cell down-regulation of CD45RB is redolent of an anti-inflammatory phenotype with poor proliferative capacity. The definitive causes of our findings and the consequences and role that these findings play in the pathogenesis of MPS are unclear, but may be in response to lysosomal storage of unmetabolized HS and DS.

Graft rejection - endogenous or allogeneic?

The presence and persistence of alloantigen is necessary for graft-specific T-cell-mediated immunity. However, specificity comprises only a single facet of an extremely complex process. Evidence is accruing to suggest that immunogenicity could be manipulated by endogenous ligands released during tissue injury. Stress molecules are significantly up-regulated following transplantation and stimulate conserved receptors on a range of leucocytes, including dendritic cells (DCs). The DCs are essential for co-stimulation and the induction of adaptive immunity. Stress signals can act as an adjuvant leading to DC maturation and activation. DCs stimulated by endogens exhibit enhanced alloantigen presentation, co-stimulation and production of pro-inflammatory cytokines, such as interleukin-1ß (IL-1ß) and IL-18. Inflammasomes have a major role in IL-1ß/IL-18 production and secretion, and can be stimulated by endogens. Importantly, the polarization toward inflammatory T helper type 17 cells as opposed to regulatory T cells is dependent upon, among other factors, IL-1ß. This highlights an important differentiation pathway that may be influenced by endogenous signals. Minimizing graft damage and stress expression should hypothetically be advantageous, and we feel that this area warrants further research, and may provide novel treatment modalities with potential clinical benefit.

Bionic Prostheses: The Emerging Alternative to Vascularised Composite Allotransplantation of the Limb.

Twenty years have surpassed since the first vascularised composite allotransplantation (VCA) of the upper limb. This is an opportunity to reflect on the position of VCA as the gold standard in limb reconstruction. The paucity of recipients, tentative clinical outcomes, and insufficient scientific progress question whether VCA will remain a viable treatment option for the growing numbers of amputees. Bionic technology is advancing at a rapid pace. The prospect of widely available, affordable, safely applied prostheses with long-standing functional benefit is appealing. Progress in the field stems from the contributions made by engineering, electronic, computing and material science research groups. This review will address the ongoing reservations surrounding VCA whilst acknowledging the future impact of bionic technology as a realistic alternative for limb reconstruction.

The Use of Hemoglobin-Based Oxygen Carriers in Ex Vivo Machine Perfusion of Donor Organs for Transplantation.

There has been significant progress in the development of ex vivo machine perfusion for the nonischemic preservation of donor organs. However, several complications remain, including the logistics of using human blood for graft oxygenation and hemolysis occurring as a result of mechanical technology. Recently, hemoglobin-based oxygen carriers, originally developed for use as blood substitutes, have been studied as an alternative to red blood cell-based perfusates. Although research in this field is somewhat limited, the findings are promising. We offer a brief review of the use of hemoglobin-based oxygen carriers in ex vivo machine perfusion and discuss future directions that will likely have a major impact in progressing oxygen carrier use in clinical practice.

Understanding immunological response to desensitisation strategies in highly sensitised potential kidney transplant patients.

Sensitisation to human leukocyte antigen (HLA) represents a significant barrier to kidney transplantation. Antibody removal and immune modulation strategies, known as 'desensitisation', aim to reduce levels of circulating HLA antibodies and increase transplant opportunities for highly sensitised patients (HSPs). However, the effects of desensitisation are generally transient and maintaining low or absent HLA antibody levels remains a substantial challenge. Furthermore, several studies report variation in patient response, with a proportion of desensitised patients able to replenish or maintain levels of circulating HLA specific antibodies despite receiving treatment to remove antibodies, antibody-producing plasma cells and their precursor B-cells. Various factors that influence the response to desensitisation have been proposed. However, the immune system is central, with differences in cytokine and leukocyte repertoire (i.e. the persistence of HLA antibody producing long-lived plasma cells (LLPCs) residing in the bone marrow) critical to desensitisation. Various cytokines are involved in commitment of B-cells to the LLPC fate, including interleukin (IL)-6, IL-21, B-cell activation factor (BAFF), a proliferation-inducing ligand (APRIL) and C-X-C motif chemokine 12 (CXCL12). Several studies have investigated variation in patient response to desensitisation with various immunological factors proposed as predictive biomarkers. However, this review reveals a need for larger studies to validate existing findings and a need for better understanding of the complex effects of desensitisation on immune profiles.

A post-preservation vascular flush removes significant populations of donor leukocytes prior to lung transplantation.

BACKGROUND: Donor leukocytes are intrinsically involved in acute lung allograft rejection, via self-presentation of donor antigens to recipient leukocytes. Therapeutic modalities to remove donor leukocytes are currently unavailable. We evaluated if a vascular flush immediately following preservation can be used for this purpose. METHODS: A post-preservation flush was performed with STEEN solution in n = 6 porcine lungs following static cold storage. The first 500 ml effluent from the left atrium was collected and an inflammatory profile performed. RESULTS: A total of 1.17 billion (±2.8 × 108) viable leukocytes were identified within the effluent. T cells were the dominant cell population, representing 82% of the total mobilised leukocytes, of which <0.01% were regulatory T cells. IL-18 was the most abundant cytokine, with a mean concentration of 84,216 pg (±153,552 pg). In addition, there was a mean concentration of 8819 ng (±4415) cell-free mitochondrial DNA. CONCLUSION: There is an immediate transfer of donor leukocytes, cytokines and damage-associated molecular patterns following reperfusion. Such a pro-inflammatory donor load may enhance alloantigen presentation and drive recipient alloimmune responses. A post-preservation flush may therefore be an effective method for reducing the immune burden of the donor lung prior to transplantation.

Hemodynamics and Metabolic Parameters in Normothermic Kidney Preservation Are Linked With Donor Factors, Perfusate Cells, and Cytokines.

Kidney transplantation is the best renal-replacement option for most patients with end-stage renal disease. Normothermic machine preservation (NMP) of the kidney has been studied extensively during the last two decades and implemented in clinical trials. Biomarker research led to success in identifying molecules with diagnostic, predictive and therapeutic properties in chronic kidney disease. However, perfusate biomarkers and potential predictive mechanisms in NMP have not been identified yet. Twelve discarded human kidneys (n = 7 DBD, n = 5 DCD) underwent NMP for up to 24 h. Eight were perfused applying urine recirculation (URC), four with replacement of urine (UR) using Ringer's lactate. The aim of our study was to investigate biomarkers (NGAL, KIM-1, and L-FABP), cells and cytokines in the perfusate in context with donor characteristics, perfusate hemodynamics and metabolic parameters. Cold ischemia time did not correlate with any of the markers. Perfusates of DBD kidneys had a significantly lower number of leukocytes after 6 h of NMP compared to DCD. Arterial flow, pH, NGAL and L-FABP correlated with donor creatinine and eGFR. Arterial flow was higher in kidneys with lower perfusate lactate. Perfusate TNF-α was higher in kidneys with lower arterial flow. The cytokines IL-1ß and GM-CSF decreased during 6 h of NMP. Kidneys with more urine output had lower perfusate KIM-1 levels. Median and 6-h values of lactate, arterial flow, pH, NGAL, KIM-1, and L-FABP correlated with each other indicating a 6-h period being applicable for kidney viability assessment. The study results demonstrate a comparable cytokine and cell profile in perfusates with URC and UR. In conclusion, clinically available perfusate and hemodynamic parameters correlate well with donor characteristics and measured biomarkers in a discarded human NMP model.

Pro-IL-1ß Is an Early Prognostic Indicator of Severe Donor Lung Injury During Ex Vivo Lung Perfusion.

BACKGROUND: Ex vivo lung perfusion (EVLP) is used to evaluate and recondition extended criteria donor lungs for transplantation. Interleukin-1ß (IL-1ß) has been identified as a prognostic indicator of nonrecovery during EVLP. This may be an effect of inflammasome activation or cellular necrosis following donation and graft preservation. Delineating the mechanism of IL-1ß release is required. METHODS: The inactive intracellular precursor molecule, pro-IL-1ß, was characterized along with the pro-IL-1ß processing enzyme, caspase-1, in the perfusate of n = 20 human lungs that had undergone EVLP (n = 10 lungs that failed to recover and were discarded versus n = 10 lungs that reconditioned and were transplanted). In an experimental porcine model, n = 8 lungs underwent EVLP and were randomized to receive either a specific NLRP3 inflammasome inhibitor or control. RESULTS: Significant increases in pro-IL-1ß and caspase-1 were observed in the perfusate from human lungs that did not recondition during EVLP compared with those that successfully reconditioned and were used for transplantation. Within the porcine EVLP, NLRP3 inflammasome inhibition reduced IL-1ß within the perfusate compared with controls, but this had no impact on lung function, hemodynamics, or inflammation. CONCLUSIONS: Our data suggest that pro-IL-1ß is passively released following cellular necrosis of the donor lung.

Substrate for the Myocardial Inflammation-Heart Failure Hypothesis Identified Using Novel USPIO Methodology.

OBJECTIVES: The purpose of this study was to identify where ultrasmall superparamagnetic particles of iron oxide (USPIO) locate to in myocardium, develop a methodology that differentiates active macrophage uptake of USPIO from passive tissue distribution; and investigate myocardial inflammation in cardiovascular diseases. BACKGROUND: Myocardial inflammation is hypothesized to be a key pathophysiological mechanism of heart failure (HF), but human evidence is limited, partly because evaluation is challenging. USPIO-magnetic resonance imaging (MRI) potentially allows specific identification of myocardial inflammation but it remains unclear what the USPIO-MRI signal represents. METHODS: Histological validation was performed using a murine acute myocardial infarction (MI) model. A multiparametric, multi-time-point MRI methodology was developed, which was applied in patients with acute MI (n = 12), chronic ischemic cardiomyopathy (n = 7), myocarditis (n = 6), dilated cardiomyopathy (n = 5), and chronic sarcoidosis (n = 5). RESULTS: USPIO were identified in myocardial macrophages and myocardial interstitium. R1 time-course reflected passive interstitial distribution whereas multi-time-point R2* was also sensitive to active macrophage uptake. R2*/R1 ratio provided a quantitative measurement of myocardial macrophage infiltration. R2* behavior and R2*/R1 ratio were higher in infarcted (p = 0.001) and remote (p = 0.033) myocardium in acute MI and in chronic ischemic cardiomyopathy (infarct: p = 0.008; remote p = 0.010), and were borderline higher in DCM (p = 0.096), in comparison to healthy controls, but were no different in myocarditis or sarcoidosis. An R2*/R1 threshold of 25 had a sensitivity and specificity of 90% and 83%, respectively, for detecting active USPIO uptake. CONCLUSIONS: USPIO are phagocytized by cardiac macrophages but are also passively present in myocardial interstitium. A multiparametric multi-time-point MRI methodology specifically identifies active myocardial macrophage infiltration. Persistent active macrophage infiltration is present in infarcted and remote myocardium in chronic ischemic cardiomyopathy, providing a substrate for HF.

Ex Vivo Lung Perfusion Improves the Inflammatory Signaling Profile of the Porcine Donor Lung Following Transplantation.

BACKGROUND: Primary graft dysfunction and allograft rejection represent major caveats to successful lung transplantation. Reducing inflammation in donor lungs before transplantation may improve outcomes. Evidence exists that ex vivo lung perfusion (EVLP) can alter the donor lung environment, although the mechanisms remain unclear. This study aimed to characterize the inflammatory signaling profile of the lung following standard and EVLP transplant and delineate the immediate impact on the recipient circulation. METHODS: Female recipient pigs (n = 12) were randomized to undergo left lung transplantation from male donors either using the gold standard protocol (static cold storage) or following 3 hours of EVLP. The relative phosphorylation of 44 phosphokinases and the relative expression of 35 apoptosis-related molecules were profiled within the donor lung 24 hours posttransplantation. RESULTS: A global profile of mitochondrial salvage and cell survival was observed in the EVLP lung tissue compared with lungs undergoing standard transplantation. This included increased phosphorylation of downstream prosignaling kinases, including ERK1/2 and FAK. In addition, there was upregulated expression of the antiapoptotic proteins Bcl-2, HSP-70, LIVIN, and PON2 with downregulation of apoptosis inducing mitochondrial associated molecules, including clusterin, cytochrome C, and HTRA2/OMI. In the early postoperative period, there were significantly lower levels of circulating mitochondrial DNA in recipients receiving EVLP lungs compared with a standard transplant (P = 0.016). Genomic DNA did not differ between groups, with donor DNA undetectable at all time points. CONCLUSIONS: EVLP alters the inflammatory signaling profile of the donor lung before transplantation, with a global cell survival and antiapoptotic signature.