Normothermic Preservation of Liver - What Does the Future Hold?

Recent years have demonstrated a surging interest in normothermic ex situ liver perfusion, with iterative experimental and clinical studies establishing this technology as providing obvious advantages over static cold storage. In particular, the safe prolongation of liver graft preservation even up to 1 week opens up possibilities of 'on circuit' interventions, which may radically change the logistics and scope of liver transplant practice. Such approaches are rife with potential, and have yet to be fully explored. Possibilities may include, but are not limited to mitochondrial enhancing strategies, steatotic graft defatting, on circuit addition of anti-aging compounds, altering graft immunogenic potential and gene silencing with siRNA, stem cell and nanoparticle therapies as well as ischemia free liver preservation. Ex situ machine perfusion technology as a platform for advanced graft modification strategies opens up the possibility of very specific, personalized transplant medicine, as well as the possibility of a future where organ grafts are re-used and repaired, providing utility to numerous successive surgical recipients, indefinitely.

Pan-caspase inhibitor F573 mitigates liver ischemia reperfusion injury in a murine model.

BACKGROUND: Liver ischemia reperfusion injury (IRI) remains a challenge in liver transplantation. A number of compounds have previously demonstrated efficacy in mitigating IRI. Herein, we applied three specific additive strategies to a mouse IRI screening model to determine their relative potencies in reducing such injury, with a view to future testing in a large animal and clinical ex situ normothermic perfusion setting: 1) F573, a pan-caspase inhibitor, 2) anti-inflammatory anakinra and etanrecept and 3) BMX-001, a mimetic of superoxide dismutase. METHODS: A non-lethal liver ischemia model in mice was used. Additives in the treatment groups were given at fixed time points before induction of injury, compared to a vehicle group that received no therapeutic treatment. Mice were recovered for 6 hours following the ischemic insult, at which point blood and tissue samples were obtained. Plasma was processed for transaminase levels. Whole liver tissue samples were processed for histology, markers of apoptosis, oxidative stress, and cytokine levels. RESULTS: In an in vivo murine IRI model, the F573 treatment group demonstrated statistically lower alanine aminotransferase (ALT) levels (p = 0.01), less evidence of apoptosis (p = 0.03), and lower cytokine levels compared to vehicle. The etanercept with anakinra treatment group demonstrated significantly lower cytokine levels. The BMX-001 group demonstrated significantly decreased apoptosis (p = 0.01) evident on TUNEL staining. CONCLUSIONS: The administration of pan-caspase inhibitor F573 in a murine in vivo model likely mitigates liver IRI based on decreased markers of cellular injury, decreased evidence of apoptosis, and improved cytokine profiles. Anakinra with etanercept, and BMX-001 did not demonstrate convincing efficacy at reducing IRI in this model, and likely need further optimization. The positive findings set rational groundwork for future translational studies of applying F573 during normothermic ex situ liver perfusion, with the aim of improving the quality of marginal grafts.

A Back-to-Base Experience of Human Normothermic Ex Situ Liver Perfusion: Does the Chill Kill?

Normothermic machine perfusion (NMP) has been shown to protect livers from injury between procurement and transplantation in a randomized controlled trial, where the machine was transported to and from the donor center. The aim of this study was to determine whether an alternative, more practical back-to-base approach after initial static cold storage would compromise beneficial outcomes. Between February 2015 and June 2018, a nonrandomized pilot study was performed at a single site. Outcomes of back-to-base livers (n = 26) were compared with those of grafts procured locally that underwent immediate NMP (n = 17). The primary outcome measure (safety) was defined as 30-day patient and graft survival. A total of 46 liver grafts were perfused with NMP, of which 3 were discarded based on poor ex situ perfusion function. The 30-day patient and graft survival in the back-to-base and local NMP groups were both 100% (primary outcome: safety). Despite significantly prolonged mean cold ischemia time (6 versus 3.2 hours; P = 0.001), the back-to-base livers demonstrated no difference in graft function, incidence of complications, or graft and patient survival. In conclusion, the back-to-base approach was safe, did not compromise the overall benefit of NMP, and offers a practical alternative to portable normothermic ex situ machine transport.

Clearance of transaminases during normothermic ex situ liver perfusion.

BACKGROUND: One of the most promising applications of liver normothermic machine perfusion (NMP) is the potential to directly assess graft viability and injury. In most NMP studies, perfusate transaminases are utilized as markers of graft injury. Our aim was to further elucidate the metabolism of transaminases by healthy porcine livers during NMP, specifically whether such livers could clear circuit perfusate transaminases. METHODS: A highly concentrated transaminase solution was prepared from homogenized liver, with an aspartate aminotransferase (AST) level of 107,427 U/L. Three livers in the treatment group were compared to three controls, during 48 hours of NMP. In the treatment group, the circuit perfusate was injected with the transaminase solution to artificially raise the AST level to a target of 7,500 U/L. Perfusate samples were taken at two-hour intervals and analyzed for biochemistry until NMP end. Graft oxygen consumption and vascular parameters were monitored. RESULTS: Compared to controls, treated perfusions demonstrated abrupt elevations in transaminase levels (p>0.0001) and lactate dehydrogenase (LDH) (p>0.0001), which decreased over time, but never to control baseline. Liver function, as demonstrated by lactate clearance and oxygen consumption was not different between groups. The treatment group demonstrated a higher portal vein resistance (p = 0.0003), however hepatic artery resistance was similar. Treated livers had higher bile production overall (p<0.0001). CONCLUSIONS: Addition of high levels of transaminases and LDH to a healthy porcine liver during ex situ perfusion results in progressive clearance of these enzymes, suggesting preserved liver metabolism. Such tolerance tests may provide valuable indicators of prospective graft function.

HCV Eradication with Direct-Acting Antivirals Does Not Impact HCC Progression on the Waiting List or HCC Recurrence after Liver Transplantation.

Background: The introduction of direct-acting antivirals (DAA) for HCV has led to high rates of HCV eradication. Treatment of patients awaiting liver transplantation (LT) has been controversial. Recent data suggests that DAA treatment may accelerate recurrent HCC. The impact of DAA on delisting for HCC progression or recurrent HCC post-LT has not been well characterized. Methods: A retrospective review of both waitlist patients and LT recipients at a single institution was performed. Patient demographics, HCV treatment, HCC features and treatments, biopsy results, and graft and patient survival were evaluated. Patients on the LT waitlist or who were transplanted between January 2014 and December 2015 were included. Data was collected through December 2017 to have a minimum of two years of follow-up. Results: In the study period, 128 adult LT were performed. 44 patients were HCV+, and 68.2% (N=30) also had HCC. 38.6% (N=17) of HCV+ patients received DAA pre-LT, and 94.1% (N=16/17) achieved sustained virologic response (SVR) pre-LT. Among untreated HCV+ patients who underwent LT, 81.5% (N=22/27) received DAA post-LT, with 82.6% achieving SVR post-LT (N=18/22). 82.1% (N=23/28) of untreated post-LT patients underwent liver biopsy prior to therapy, and 52.2% had at least F1 METAVIR fibrosis. 87.5% (N=14/16) of active waitlist patients received DAA and achieved SVR. HCV eradication did not result in higher rates of delisting for HCC progression. Due to local HCC listing criteria of total tumor volume and AFP, 60% (N=18/30) of HCV+/HCC patients were beyond Milan criteria at the time of LT. Despite this, there was no difference in HCC recurrence rates post-LT, whether patients achieved SVR pre- or post-LT. Conclusions: These data suggest that HCV eradication pre-LT does not significantly impact waitlist time for HCV+ patients with HCC. HCV eradication does not impact rates of delisting for HCC progression or rates of HCC recurrence post-LT.

Determination of Minimal Hemoglobin Level Necessary for Normothermic Porcine Ex Situ Liver Perfusion.

BACKGROUND: In current studies of ex situ liver perfusion there exists considerable variability in perfusate composition, including the type of oxygen carrier. Herein, we aim to clarify the minimal hemoglobin level necessary during normothermic porcine ex situ liver perfusion. METHODS: Livers procured from 35 to 45 kg domestic pigs were connected to our experimental ex situ circuit (n = 10). In the treatment group, perfusate was sequentially diluted hourly to predetermined hemoglobin levels. At the end of each hemoglobin dilution, perfusate samples were analyzed for liver transaminases, lactate dehydrogenase (LD), total bilirubin, and lactate levels. Liver oxygen consumption was measured. In the control group, livers were perfused continually for a duration of 24 hours at target hemoglobin levels of 30 and 20 g/L. RESULTS: Rising liver transaminases, significantly higher lactate (P < 0.001), and LD levels (P < 0.001) were noted at lower perfusate hemoglobin levels in the treatment group. Liver oxygen utilization (P < 0.001) and hepatic artery oxygen delivery (P < 0.001) were significantly lower at lower hemoglobin levels, whereas liver vessel resistance remained relatively constant. Histology demonstrated increasing parenchymal damage at lower hemoglobin levels. In control livers, higher perfusate transaminases, higher lactate, and LD levels were noted at a perfusion hemoglobin level of 20 g/L. CONCLUSIONS: Ex situ liver function decompensated during perfusion between a mean hemoglobin level of 30 to 20 g/L, as evidenced by notably rising lactate and LD levels. This study demonstrates optimal hemoglobin concentration during normothermic ex situ liver perfusion to ensure a fully metabolically functioning graft.

Ex situ liver perfusion: Organ preservation into the future.

In recent years, remarkable progress has occurred in the development of technologies to support ex situ liver perfusion. Building upon extensive preclinical studies in large animal models, pilot and randomized clinical trials have been initiated, and preliminary outcomes suggest more optimal protection of both standard and extended criteria liver grafts. There currently exists an incredible opportunity and need to further refine this technology, determine appropriate viability measures to predict usable liver grafts, and to explore potent protective additive strategies to further optimize the quality of extended criteria organs. These findings will have major bearing in expanding the limited liver donor pool, and may save lives where up to a quarter of listed patients die on wait-lists. Herein we offer a brief overview of the history and current status of ex situ liver perfusion, and discuss future directions that will likely have major impact on the practice of clinical liver transplantation.

Engraftment Site and Effectiveness of the Pan-Caspase Inhibitor F573 to Improve Engraftment in Mouse and Human Islet Transplantation in Mice.

BACKGROUND: Islet transplantation is an effective therapy in type 1 diabetes and recalcitrant hypoglycemia. However, there is an ongoing need to circumvent islet loss posttransplant. We explore herein the potential of the pan-caspase inhibitor F573 to mitigate early apoptosis-mediated islet death within portal and extrahepatic portal sites in mice. METHODS: Mouse or human islets were cultured in standard media ±100 µM F573 and subsequently assessed for viability and apoptosis via terminal deoxynucleotidyl transferase dUTP nick end labeling staining and caspase-3 activation. Diabetic mice were transplanted with syngeneic islets placed under the kidney capsule (KC) or into the subcutaneous deviceless (DL) site at a marginal islet dose (150 islets), or into the portal vein (PV) at a full dose (500 islets). Human islets were transplanted under the KC of diabetic immunodeficient mice at a marginal dose (500 islet equivalents). Islets were cultured in the presence of F573, and F573 was administered subcutaneously on days 0 to 5 posttransplant. Control mice were transplanted with nontreated islets and were injected with saline. Graft function was measured by nonfasting blood glucose and glucose tolerance testing. RESULTS: F573 markedly reduced human and mouse islet apoptosis after in vitro culture (P < 0.05 and P < 0.05, respectively). Furthermore, F573 improved human islet function when transplanted under the KC (P < 0.05); whereas F573 did not enhance murine islet marginal KC transplants. Conversely, F573 significantly improved mouse islet engraftment in the PV and DL site (P < 0.05 and P < 0.05, respectively). CONCLUSIONS: The pan-caspase inhibitor F573 markedly reduces human and mouse islet apoptosis and improves engraftment most effectively in the portal and DL subcutaneous sites.

Reparixin, a CXCR1/2 inhibitor in islet allotransplantation.

Quality of life in Type 1 diabetic patients may be improved with islet transplantation, but lifelong immunosuppression is required to prevent rejection. Allo-immune response is a key player in graft dysfunction and although the adaptive immune response is well characterized, the effect of the innate immune reaction after transplantation is only recently becoming appreciated. In this study, we address how the innate response affects long-term outcomes in a murine islet allotransplant model. CTLA-4 Ig treatment is known to significantly prolong kidney subcapsular islet allograft survival and enhance glucose tolerance. The combination of CTLA-4 Ig with reparixin, which blocks against inflammatory neutrophil infiltration, yielded no long-term graft survival in an intrahepatic allotransplant model but had similar long-term graft survival in the kidney subcapsular model. Seven days after transplant, serum blood IFN-γ levels were significantly lower in the CTLA-4 Ig with reparixin treatment group compared to controls. IL-12p70 cytokine levels were increased with combination treatment, a positive modulation of the inflammatory response to the allograft. Furthermore, KC GRO, also known as CXCL1, was decreased in serum 7 d after transplant. Histologically, we found that immune cell infiltrate, CD4+ and CD8+ T cell populations along with both CXCR1+ and CXCR2+ cell populations were decreased within the CTLA-4 Ig and reparixin islet transplant graft. Overall these data provide insight into the down regulation of T-cell recruitment by CTLA-4 Ig and decreased neutrophil activation and recruitment with reparixin after long-term islet graft survival.

Harnessing the Foreign Body Reaction in Marginal Mass Device-less Subcutaneous Islet Transplantation in Mice.

BACKGROUND: Islet transplantation is a successful ß-cell replacement therapy for selected patients with type 1 diabetes mellitus. However, despite early insulin independence, long-term graft attrition gradually reverts recipients to exogenous insulin dependency. Undoubtedly, as insulin producing stem cell therapies progress, a transplant site that is retrievable is desirable. This prerequisite is currently incompatible with intrahepatic islet transplantation. Herein, we evaluate the functional capacity of a prevascularized subcutaneous site to accommodate marginal islet mass transplantation in mice. METHODS: Syngeneic mouse islets (150) were transplanted either under the kidney capsule (KC), into a prevascularized subcutaneous device-less (DL) site, or into the unmodified subcutaneous (SC) tissue. The DL site was created 4 weeks before diabetes induction and islet transplantation through the transient placement of a 5-Fr vascular catheter. Recipient mice were monitored for glycemic control and intraperitoneal glucose tolerance. RESULTS: A marginal islet mass transplanted into the DL site routinely reversed diabetes (n = 13 of 18) whereas all SC islet recipients failed to restore glycemic control (n = 0 of 10, P < 0.01, log-rank). As anticipated, nearly all islet-KC mice (n = 15 of 16) became euglycemic posttransplant. The DL recipients' glucose profiles were comparable to KC islet grafts, postintrapertioneal glucose tolerance testing, whereas SC recipients remained hyperglycemic postglucose challenge. All normoglycemic mice maintained graft function for 100 days until graft retrieval. DL and KC islet grafts stained positively for insulin, microvessels, and a collagen scaffold. CONCLUSIONS: The device-less prevascularized approach supports marginal mass islet engraftment in mice.

Antiaging Glycopeptide Protects Human Islets Against Tacrolimus-Related Injury and Facilitates Engraftment in Mice.

Clinical islet transplantation has become an established treatment modality for selected patients with type 1 diabetes. However, a large proportion of transplanted islets is lost through multiple factors, including immunosuppressant-related toxicity, often requiring more than one donor to achieve insulin independence. On the basis of the cytoprotective capabilities of antifreeze proteins (AFPs), we hypothesized that supplementation of islets with synthetic AFP analog antiaging glycopeptide (AAGP) would enhance posttransplant engraftment and function and protect against tacrolimus (Tac) toxicity. In vitro and in vivo islet Tac exposure elicited significant but reversible reduction in insulin secretion in both mouse and human islets. Supplementation with AAGP resulted in improvement of islet survival (Tac(+) vs. Tac+AAGP, 31.5% vs. 67.6%, P < 0.01) coupled with better insulin secretion (area under the curve: Tac(+) vs. Tac+AAGP, 7.3 vs. 129.2 mmol/L/60 min, P < 0.001). The addition of AAGP reduced oxidative stress, enhanced insulin exocytosis, improved apoptosis, and improved engraftment in mice by decreasing expression of interleukin (IL)-1ß, IL-6, keratinocyte chemokine, and tumor necrosis factor-α. Finally, transplant efficacy was superior in the Tac+AAGP group and was similar to islets not exposed to Tac, despite receiving continuous treatment for a limited time. Thus, supplementation with AAGP during culture improves islet potency and attenuates long-term Tac-induced graft dysfunction.

Ischaemic heart preconditioning in rats with adjuvant-induced arthritis.

BACKGROUND: Adjuvant-induced arthritis (AIA) in rats is a model of chronic systemic inflammation and a model of rheumatoidarthritis in humans. AIM: To investigate effectiveness of ischaemic preconditioning (IPC) in reducing the area of myocardial infarction in rats with AIA. METHODS: The study was performed in vivo in male SPRD/Mol/Lod rats. Animals were assigned to the experimental group (n = 15) or the control group (n = 14). In the experimental group, AIA was induced by subcutaneous administration of 1 mLof Freund's complete adjuvant, and the experiment was performed after 14 days. In control (healthy) animals, no procedures were performed prior to the proper experiment. Animals were anaesthesized (by intraperitoneal administration of ketamineand xylazine) and put on a ventilator. Then, myocardial infarction was induced, preceded by IPC in some animals. To induceinfarction, left main coronary artery (LMCA) was occluded for 30 min, followed by 60 min of reperfusion. IPC protocol consisted of LMCA occlusion for 3 min, followed by 5 min of reperfusion and a second LMCA occlusion for 7 min. Weevaluated a percentage ratio of the infarct size to the risk area (IS/RA). Necrosis area was stained with tetrazolium, and thearea supplied by LMCA was determined using Evans blue. All areas were determined by planimetry. We used nonparametric Kruskal-Wallis rank ANOVA with multiple comparisons, and the results were shown as median values and 25th and 75th percentiles. P < 0.05 was considered statistically significant. RESULTS: In the control group with IPC (n = 7), the IS/RA ratio of 25% (23-38) was significantly reduced compared to thecontrol group without IPC (n = 7) (58% [57-63], p < 0.05). In the AIA group with IPC (n = 7), the IS/RA ratio of 58% (51-65)did not differ significantly compared to the AIA group without IPC (n = 8) (65% [62-71]). CONCLUSIONS: Our findings indicate that IPC in rats with AIA does not result in a significant reduction of myocardial necrosisarea induced by 30 min of ischaemia and 60 min of reperfusion. This effect might have been related to the presence ofchronic systemic inflammation. Absent or reduced benefits of IPC may be one reason for an increased cardiovascular risk inpatients with rheumatoid arthritis.