Normothermic ex vivo perfusion of the limb allograft depletes donor leukocytes prior to transplantation.

INTRODUCTION: The donor immune compartment plays a central role in graft rejection of the vascularised composite allograft (VCA) by contributing to 'direct presentation'. Using our limb ex vivo normothermic machine perfusion (EVNP) protocol designed for prolonged allograft preservation, this study aimed to assess whether donor leukocytes responsible for allograft rejection are mobilised from the donor compartment. METHODS: Five genetically different pig forelimbs underwent perfusion via the brachial and radial collateral artery for 6 h after 2 h of cold storage. Oxygenated haemodilute leucocyte-deplete blood was recirculated at normothermia using an extracorporeal perfusion system. Tissue perfusion was evaluated clinically and biochemically via blood perfusate. The temporal kinetics of donor leucocyte extravasation, cytokine secretion and cell-free DNA was characterised in the circulating perfusate. RESULTS: Flow cytometry revealed increasing populations of viable leukocytes over time, reaching 49 billion leukocytes by 6 h. T (3.0 × 109 cells) and B cells (3.1 × 108 cells) lymphocytes, monocytes (2.7 × 109 cells), granulocytes (8.1 × 109 cells), NK (6.3 × 108) and γδ (8.1 × 108) cells were all identified. Regulatory T cells comprised a minor population (1.6 × 107 cells). There was a cumulative increase in pro-inflammatory cytokines suggesting that the donor limb has the capacity to elicit significant inflammatory responses that could contribute to leucocyte activation and diapedesis. CONCLUSION: EVNP not only acts as a preservation tool, but could also be utilized to immunodeplete the VCA allograft prior to transplantation. This has clinical implications to mitigate acute rejection and prevent graft dysfunction and supports the future application of machine perfusion in graft preservation and immune modulation.

Randomized preclinical study of machine perfusion in vascularized composite allografts.

BACKGROUND: Attempts to improve limb preservation for transplantation using ex vivo perfusion have yielded promising results. However, metabolic acidosis, aberrant perfusate biochemistry and significant perfusion-induced oedema are reported universally. Optimizing perfusion protocols is therefore essential for maintaining tissue health. METHODS: A randomized, two-stage open preclinical trial design was used to determine the optimal temperature and mean arterial pressure for machine perfusion. Conditions compared were: normothermic machine perfusion at 70 mmHg (NMP-70); subnormothermic perfusion (28°C) at 70 mmHg; subnormothermic (28°C) perfusion at 50 mmHg; and hypothermic perfusion (10°C) at 30 mmHg. Following this, a head-to-head experiment was undertaken comparing the optimal machine perfusion with static cold storage. Paired bilateral limbs (10 in total) were randomized to either 8 h of static cold storage, or 2 h of static cold storage and 6 h of optimal machine perfusion. Both groups of limbs were then reperfused on a circuit primed with matched blood from unrelated donors for 4 h without immunosuppression. RESULTS: NMP-70 resulted in less tissue injury and stable perfusion biochemistry. Assessing reperfusion outcomes, static cold storage resulted in acidosis with increased lactate and a worsening electrolyte profile, necessitating bolus infusions of bicarbonate to prevent graft loss. Conversely, NMP-70 was associated with haemodynamic and biochemical stability. Histologically, on reperfusion with allogeneic whole blood, limbs subjected to static cold storage exhibited multifocal ischaemic injury and increased inflammation, which was absent with NMP-70. Static cold storage also resulted in significant oedema compared with NMP-70. CONCLUSION: Normothermic perfusion resulted in superior graft preservation and less reperfusion injury compared with the current static cold storage protocol.

The use of kinase inhibitors in solid organ transplantation.

INTRODUCTION: Despite the efficacy of current immunosuppression regimes used in solid organ transplantation, graft dysfunction, graft lost and antibody-mediated rejection continue to be problematic. As a result, clear attraction in exploiting key potential targets controlled by kinase phosphorylation has led to a number of studies exploring the use of these drugs in transplantation. Aim In this review, we consider the role of tyrosine kinase as a target in transplantation and summarize the relevant studies on kinase inhibitors that have been reported to date. METHODS: Narrative review of literature from inception to December 2016, using OVID interface searching EMBASE and MEDLINE databases. All studies related to kinase based immunosuppression were examined for clinical relevance with no exclusion criteria. Key ideas were extracted and referenced. CONCLUSION: The higher incidence of infections when using kinase inhibitors is an important consideration, however the number and range inhibitors and their clinical indications are likely to expand, but their exact role in transplantation is yet to be determined.

Altered Immunogenicity of Donor Lungs via Removal of Passenger Leukocytes Using Ex Vivo Lung Perfusion.

Passenger leukocyte transfer from the donor lung to the recipient is intrinsically involved in acute rejection. Direct presentation of alloantigen expressed on donor leukocytes is recognized by recipient T cells, promoting acute cellular rejection. We utilized ex vivo lung perfusion (EVLP) to study passenger leukocyte migration from donor lungs into the recipient and to evaluate the effects of donor leukocyte depletion prior to transplantation. For this purpose, female pigs received male left lungs either following 3 h of EVLP or retrieved using standard protocols. Recipients were monitored for 24 h and sequential samples were collected. EVLP-reduced donor leukocyte transfer into the recipient and migration to recipient lymph nodes was markedly reduced. Recipient T cell infiltration of the donor lung was significantly diminished via EVLP. Donor leukocyte removal during EVLP reduces direct allorecognition and T cell priming, diminishing recipient T cell infiltration, the hallmark of acute rejection.

The pathophysiology of chronic graft failure in the cardiac transplant patient.

Following cardiac transplantation, many patients develop chronic deterioration of graft function, which may lead to a clinical syndrome similar to native chronic heart failure (CHF). This condition of chronic cardiac graft failure (CGF) may also share pathophysiological processes comparable with that of CHF. However, the unique environment following cardiac transplantation may also contribute with a variety of unique mechanisms, deserved of special attention. This review article discusses the complex pathophysiology of CGF after cardiac transplantation, an important yet neglected condition of transplant medicine.

BNP directly immunoregulates the innate immune system of cardiac transplant recipients in vitro.

BACKGROUND: The innate immune system plays an important role in cardiac allograft rejection. BNP has frequently been reported to elevate during acute cardiac rejection, yet the explanation behind this phenomenon is unclear. We hypothesized that BNP might interact with the innate immune system in cardiac transplant recipients and devised a series of in vitro culture experiments to explore this phenomena. METHODS: PBMCs were isolated from whole blood of (total n = 40) cardiac transplant recipients. Short (24h, n = 20) and long term (72h, n = 20) co-cultures of innate cells in the presence or absence of BNP were performed. BNP was added at two specific concentrations and compared to placebo control. Innate cells were immunophenotyped using flow cytometry. RESULTS: BNP dose dependently reduced the total number of monocytes, B cells and NK cells. Furthermore, BNP co-culture impaired NK cell cytotoxicity and adhesion of non-classical monocytes (via down-regulation of CD11c). DISCUSSION: BNP has an additional physiological role of moderating components of the innate immune system. Although speculative, this could be beneficial to cardiac transplant recipients as the innate immune system is involved in allograft rejection. Further investigation is required to elucidate the mechanism behind how BNP affects immune cells and whether the same effects are consistent with the adaptive immune system.

Potential immunologic effects of statins in cancer following transplantation.

3-hydroxy-3-methyglutaryl CoA reductase inhibitors (statins) are frequently used following organ transplantation and have well reported pleiotropic effects, including immunomodulation, which may be of benefit in preventing graft rejection. However, the immunomodulatory effects of statins on cell transformation and malignancy, combined with the immunologic processes and administration of immunosuppression are almost completely unknown. The administration of immunosuppression is well recognised as the main cause of cancer following transplantation, so the addition of an immunomodulatory agent should be associated with an increased incidence of cancer, as immune surveillance and response may be suppressed, allowing cellular transformation and proliferation combined with lack of recognition to occur. This hypothetical review attempts to delineate the mode of action of statins in terms of pro/anti-carcinogenic mechanisms, while considering graft rejection and the presence of immunosuppression.

CMV infection is associated with the depletion but lack of activation of peripheral blood natural killer cells in a lung transplant cohort.

INTRODUCTION: Following lung transplantation, cytomegalovirus (CMV) has both direct and indirect adverse effects on the allograft. Natural killer cells mediate immune responses to CMV. This can be both dependent and independent of MHC class I expression. However, their role during CMV infection following lung transplantation is unknown. In this study, the immunophenotypic characteristics of NK cells were correlated with CMV infection following lung transplantation. METHODS: Seventy lung transplant recipients were included in the study. NK cells were characterised via flow cytometric analysis of CD3, CD16, CD56, CD107a, CD107b, and CD161. CMV infection was determined using an established quantitative PCR technique on peripheral blood. RESULTS: The number of peripheral blood NK cells with CD16, CD56 and CD161 phenotypes decreased in patients with CMV infection. However, there were no correlations between CMV infection and NK cell activation determined via LAMP expression. CONCLUSIONS: This study reports comparative differences in the peripheral blood NK cell repertoire in lung transplant recipients with CMV infection versus those without. However, NK cell activity did not alter with CMV infection, suggesting that CMV infection alone does not induce an NK cell response.

HMG-CoA reductase inhibitors deplete circulating classical and non-classical monocytes following human heart transplantation.

BACKGROUND: Monocytes mediate immune responses following solid organ transplantation via cytokine secretion and differentiation to macrophage/dendritic cell lineages. To date, the pleiotropic immunomodulatory effect of statins on human monocytes following human heart transplantation has yet to be elucidated. This study was designed to assess the effects of statin administration on the monocyte repertoire. METHODS: 108 patients were recruited into the study. Clinical data were collected from patients' notes. Peripheral blood immunophenotype was determined via flow cytometry (using CD11c, CD14, CD16, CD49d, CD64, CD80 and CD195). RESULTS: There were fewer circulating classical (p=0.0001) and non-classical (p=0.0013) monocytes in patients treated with a statin. CD64 expression was down-regulated (p=0.011 and p=0.049) whereas CD49d expression was up-regulated (p=0.004 and p=0.022) on classical and non-classical monocytes in this group. Patients receiving Atorvastatin had fewer circulating classical monocytes (p=0.001) compared to patients administered Pravastatin. Patients receiving Pravastatin had fewer circulating non-classical monocytes (p=0.029) compared to patients administered Atorvastatin. DISCUSSION: Statin administration alters the circulating monocyte repertoire following heart transplantation, including population size, FcgammaRI and VLA-4 adhesion molecule expression. Furthermore, different statin treatments are associated with a selective depletion of macrophage or DC (re)generating monocytes.

Natural killer cells and lung transplantation, roles in rejection, infection, and tolerance.

Despite improvements in surgical technique, organ preservation, immunosuppression, and management of infection, the long term survival following lung transplantation remains low, mainly due to immune mediated complications such as acute and chronic rejection. Almost all immunosuppressive agents used in the prophylaxis and treatment of rejection following lung transplantation are targets of T cell maturation, function or proliferation, which in theory should cause sufficient disruption of the adaptive immune system to prevent graft rejection. However the five year survival rate of only 50% suggests this is not the case. More recent evidence suggests that NK cells may play a significant role in immune processes following lung transplantation. This article reviews the literature on the potential function of NK cells in rejection, infection, malignancy and tolerance following lung transplantation.

Donor CCR5 Delta32 polymorphism and outcome following cardiac transplantation.

BACKGROUND: Chemokines regulate the recruitment and trafficking of leukocytes during an immune response. Animal models have shown correlations between chemokine production and leukocyte infiltration during allograft rejection. Also, antagonism of chemokine receptors in transplant models has produced prolonged graft survival. Individuals homozygous for a 32 base pair deletion in the CC chemokine receptor 5 (CCR5) gene have an inactive receptor. Renal transplant recipients homozygous for the deletion have been shown to survive significantly longer than those heterozygous or homozygous for the wild type allele. CCR5 ligands are upregulated during allograft rejection aiding infiltration of leukocytes. We investigated the influence of CCR5Delta32 polymorphism on outcome following human cardiac transplantation. METHODS: Recipients and corresponding donors were genotyped for CCR5Delta32 polymorphism using polymerase chain reactions. RESULTS: We found no correlation between recipient genotype and outcome following transplantation. However, there was a significant correlation between donor genotype and mortality in patients transplanted for a nonischemic condition (DD = n/a, ID = 4%, II = 25%, P = .0014). CONCLUSIONS: The induction of CCR5 expression in endomyocardial biopsy tissue is known to correlate with leukocyte graft infiltration. We suggest that donor CCR5 may be more important for leukocyte trafficking during rejection than recipient CCR5 expression. The CCR5 gene is highly conserved, and due to the small population available for this study, more work is required from other centers.

Angiotensin converting enzyme insertion/deletion polymorphism does not influence postcardiac transplantation hypertension onset or progression.

BACKGROUND: The angiotensin converting enzyme insertion deletion polymorphism (ACE I/D) has been associated with much cardiovascular pathology, including posttransplantation hypertension. Hypertension is a significant cause of morbidity and mortality after cardiac transplantation. We investigated the influence of the ACE I/D polymorphism on posttransplantation hypertension. METHODS: A total of 211 heart transplant recipients and 154 corresponding donors were genotyped for the ACE I/D polymorphism by polymerase chain reaction. ACE enzymatic activity was measured by spectrophotometric kinetic analysis. Sitting systolic and diastolic blood pressures were recorded at 3 consecutive visits, and the mean was calculated. Clinical data, including demographics and medication, were collected for all recipients. Results were analyzed by the chi-square test and analysis of variance, taking a p value of <0.05 to be significant. RESULTS: A total of 41.7% of the subjects were hypertensive (diastolic blood pressure >90 mm Hg) at the time of the study, with 79.6% taking at least one antihypertensive agent. We found no difference between the number of antihypertensive agents, cyclosporin dose and level, renal function, or systolic blood pressure for the different recipient or donor genotypes. We also found no significant correlation between ACE enzymatic activity and systolic or diastolic blood pressure. CONCLUSIONS: Our study of 211 recipients and 154 corresponding donors is the largest investigation of this polymorphism in a cardiac transplantation population. We found no apparent relationship between the ACE genotype (of either donor or recipient) and systemic hypertension (absolute measurements and the number or dose of antihypertensive agents used).

The effects of ACE inhibition on serum angiotensin II concentration following cardiac transplantation.

AIMS: ANGII mediates vascular neointimal formation through smooth muscle cell stimulation and enhanced production of growth factors leading to increased arterial medial layer thickness, which is a characteristic of transplant arteriosclerosis. ACE inhibition is known to be of benefit to patients with cardiovascular risk factors. We aimed to determine the effect of ACE inhibitor therapy on ACE enzymatic activity and serum ANGII levels following cardiac transplantation. METHODS: A total of 43 serum samples from eight transplant recipients were used for analysis. Samples were taken monthly from the date of transplant for the initial 6 months. ANGII was measured using sandwich ELISA. ACE enzymatic activity was measured using spectrophotometric kinetic analysis. RESULTS: There was a significant reduction in ACE enzymatic activity among individuals treated with ACE inhibitor therapy (18.0 +/- 16.6 vs 31.8 +/- 23.4, P = .008). We found significantly higher ANGII serum levels in patients receiving ACE inhibitor therapy compared to those not (2.4 +/- 2.1 vs 8.0 +/- 7.4, P = .002). There was also a significant positive correlation between ACE enzymatic activity and ANGII serum level (coefficient 0.332, P = .03). CONCLUSIONS: Our results suggest an effective ACE independent pathway for ANGII conversion. Chymase can convert ANGI with higher affinity than ACE. Also, chymase is stored in mast cells, which infiltrate the myocardium following transplantation. This data indicate that pharmacological chymase inhibition may be a possible therapeutic strategy following transplantation.

The influence of donor age on transplant coronary artery disease and survival post heart transplantation: is it safe to extend donor age?

BACKGROUND: Due to the increasing demand for suitable cardiac donor organs, acceptance criteria need to be re-evaluated. We retrospectively analyzed the effect of donor age on survival following cardiac transplantation. METHOD: Three hundred thirty-five cardiac transplant recipients and corresponding donor data were reviewed using SPSS. RESULTS: Seventy-two recipients had early posttransplant angiography or postmortem data available. The mean donor age of recipients with evidence of graft coronary artery disease (32.5 +/- 11.7 years) was significantly higher than that of recipients free of transplant coronary artery disease (TCAD) (24.8 +/- 9.4 years; P=.003). Recipient of organs from donors aged less than 50 years were less likely to develop TCAD than those of donors aged over 50 years (odds ratio 0.333; 95% CI 0.239-0.465; P=.044). TCAD also occurred much earlier posttransplantation in recipients of organ from donors over 50 years (mean time 6.5 years; median 5.0 years) than those of donors under 50 years (mean time 12.7 years; median 14.0 years). CONCLUSION: We observed no increase in mortality associated with cardiac donors over 50 years. However, increased donor age was associated with an increased incidence of TCAD.