Oncologic Outcomes of Interventions to Decrease Allograft Ischemia-Reperfusion Injury within Patients Undergoing Liver Transplantation for Hepatocellular Carcinoma: A Systematic Review.

Ischemia-reperfusion injury (IRI) during liver transplantation has been implicated in the recurrence of hepatocellular carcinoma (HCC). This systematic review aimed to evaluate interventions to reduce IRI during liver transplantation for HCC and their impact on oncologic outcomes. A comprehensive literature search retrieved four retrospective studies involving 938 HCC patients, utilising interventions such as post-operative prostaglandin administration, hypothermic machine perfusion, and normothermic machine perfusion. Overall, treated patients exhibited reduced post-operative hepatocellular injury and inflammation and significantly enhanced recurrence-free survival. Despite these promising results, the impact of these interventions on overall survival remains unclear. This underscores the imperative for further prospective research to comprehensively understand the efficacy of these interventions in HCC patients undergoing transplantation. The findings highlight the potential benefits of these strategies while emphasising the need for continued investigation into their overall impact.

Resection of colorectal carcinoma liver metastases: A population-based study in outcomes and factors associated with recurrent disease.

OBJECTIVE: To assess the hepatic disease-free survival (HDFS) and overall survival (OS) of patients who underwent resection of colorectal cancer liver metastases (CRCLM) in our population, and evaluate what factors are associated with these outcomes. METHODS: Patients with resected non-mucinous CRCLM between January 2013-February 2020 were retrospectively identified. Dates of diagnosis, surgery, and, if applicable, death were recorded. HDFS and OS were calculated using a census date of 24 September 2022. Separate Cox multivariate regression analyses were performed to evaluate for association between HDFS and OS and the following factors: pre-operative imaging interval (<4 weeks vs. ≥4 weeks); pre-operative imaging modality (CT only vs. MRI+CT); extrahepatic disease at time of hepatectomy (yes vs. no); tumor burden score (TBS, where TBS2 = (largest axial dimension of CRCLM)2 + (number of CRCLM)2); pT and pN; and neoadjuvant chemotherapy. RESULTS: 137 subjects (mean age, 61 ± 11 years, 86 males) were included. Associations with recurrent hepatic disease were found with chemotherapy (HR 2.11[95 % CI = 1.13-3.92]), TBS (HR 1.30[95 % CI = 1.17-1.45]), MRI+CT (HR 2.12[95 % CI = 1.29-3.48]), and extrahepatic disease at hepatectomy (HR 2.16[95 % CI = 1.08-4.35]). For mortality, associations were found with TBS (HR 1.22[95 % CI = 1.09-1.37]), pT (HR 1.45[95 % CI = 1.05-2.00]), and extrahepatic disease at hepatectomy (HR 2.10[95 % CI = 1.31-3.36]). CONCLUSION: In our population, non-imaging related factors TBS, neoadjuvant chemotherapy, pT and presence of extrahepatic disease at time of hepatectomy were associated with HDFS and/or OS. The preoperative imaging interval and use of preoperative MRI were not associated with improved patient outcomes.

Mitochondrial DNA levels in perfusate and bile during ex vivo normothermic machine correspond with donor liver quality.

Ex vivo normothermic machine perfusion (NMP) preserves donor organs and permits real-time assessment of allograft health, but the most effective indicators of graft viability are uncertain. Mitochondrial DNA (mtDNA), released consequent to traumatic cell injury and death, including the ischemia-reperfusion injury inherent in transplantation, may meet the need for a biomarker in this context. We describe a real time PCR-based approach to assess cell-free mtDNA during NMP as a universal biomarker of allograft quality. Measured in the perfusate fluid of 29 livers, the quantity of mtDNA correlated with metrics of donor liver health including International Normalized Ratio (INR), lactate, and warm ischemia time, and inversely correlated with inferior vena cava (IVC) flow during perfusion. Our findings endorse mtDNA as a simple and rapidly measured feature that can inform donor liver health, opening the possibility to better assess livers acquired from extended criteria donors to improve organ supply.

Changing liver utilization and discard rates in clinical transplantation in the ex-vivo machine preservation era.

Liver transplantation is a well-established treatment for many with end-stage liver disease. Unfortunately, the increasing organ demand has surpassed the donor supply, and approximately 30% of patients die while waiting for a suitable liver. Clinicians are often forced to consider livers of inferior quality to increase organ donation rates, but ultimately, many of those organs end up being discarded. Extensive testing in experimental animals and humans has shown that ex-vivo machine preservation allows for a more objective characterization of the graft outside the body, with particular benefit for suboptimal organs. This review focuses on the history of the implementation of ex-vivo liver machine preservation and how its enactment may modify our current concept of organ acceptability. We provide a brief overview of the major drivers of organ discard (age, ischemia time, steatosis, etc.) and how this technology may ultimately revert such a trend. We also discuss future directions for this technology, including the identification of new markers of injury and repair and the opportunity for other ex-vivo regenerative therapies. Finally, we discuss the value of this technology, considering current and future donor characteristics in the North American population that may result in a significant organ discard.

Predicting Early Graft Dysfunction and Mortality After Liver Transplant Using the De Ritis Ratio.

BACKGROUND: Predicting complications after liver transplantation (LT) remains challenging. We propose incorporating the De Ritis ratio (DRR), a widely known parameter of liver dysfunction, into current or future scoring models to predict early allograft dysfunction (EAD) and mortality after LT. METHODS: A retrospective chart review was conducted on 132 adults receiving a deceased donor LT from April 2015 to March 2020 and their matching donors. Donor variables, postoperative liver function, and DRR were correlated with the occurrence of EAD, post-transplant complications expressed by the Clavien-Dindo score, and 30-day mortality as outcome variables. RESULTS: Early allograft dysfunction was observed in 26.5% of patients and 7.6% of patients who died within 30 days after transplant. Recipients were more likely to experience EAD when receiving grafts from donation after circulatory death (P = .04), donor risk index (DRI) >2 (P = .006), ischemic injury at time-zero biopsy (P = .02), longer secondary warm ischemia time (P < .05), or higher Clavien-Dindo scores (IIIb-V; P < .001). The DRI, total bilirubin, and DRR on postoperative day 5 yielded significant associations with the primary outcomes and were used to develop the Gala-Lopez score using a weighted scoring model. This accurately predicted EAD, high Clavien-Dindo, and 30-day mortality in 75%, 81%, and 64% of patients. CONCLUSION: Including recipient and donor variables in predictive models, and for the first time DRR, as a constituent, should be regarded to predict EAD, severe complications, and 30-day mortality post-LT. Further studies will be required to validate the present findings and their applicability when using normothermic regional and machine perfusion technologies.

Graft survival after kidney transplantation with standard versus prolonged kidney procurement time.

BACKGROUND: During kidney procurement, after ice removal, kidneys located in the retroperitoneum are at risk for rewarming owing to the time taken to retrieve other abdominal and thoracic organs, which may lead to poorer outcomes. The purpose of this study was to evaluate the impact of prolonged kidney procurement time (PKP) on outcomes of kidney transplantation performed at the Queen Elizabeth II Health Sciences Centre, Halifax, Nova Scotia, Canada. METHODS: We retrospectively reviewed the cases of all adult (age ≥ 18 yr) patients who underwent kidney transplantation at the Queen Elizabeth II Health Sciences Centre between Jan. 1, 2010, and Dec. 31, 2015. We included all patients who received kidney transplants from deceased donors with a minimum follow-up period of 3 years. We defined PKP as more than 65 minutes from aortic cross-clamp to final organ extraction, and standard procurement time (SP) as 65 minutes or less. RESULTS: Among the 455 transplantation procedures performed during the study period, we reviewed the cases of 145 patients who received kidneys from Nova Scotian donors and were followed in Nova Scotia. No statistically significant differences were seen in outcomes between kidney-only (n = 46) and multiorgan (n = 99) procurement, although more organs from kidney-only donors than multiorgan donors had a Kidney Donor Profile Index score greater than 50% (32 [69.6%] v. 48 [48.5%], p < 0.01). Compared to the SP group (n = 115), the PKP group (n = 30) had a higher rate of 30-day graft loss (6.7% v. 0.0%, p < 0.01), a higher incidence of de novo formation of donor-specific antibodies (3 [10.0%] v. 1 [0.9%], p < 0.01) and a lower 5-year graft survival rate (90.0% v. 97.4%, p = 0.03). Left kidneys remained 11 minutes longer on the donor than right kidneys when multiorgan procurement was performed (p < 0.01), and their 5-year survival rate was significantly lower than that of right kidneys (p = 0.03). CONCLUSION: Procurement times longer than 65 minutes may be associated with poorer outcomes after kidney transplantation. Measures to reduce kidney exposure to rewarming during procurement may improve long-term outcomes.

Mitochondrial damage-associated molecular patterns trigger arginase-dependent lymphocyte immunoregulation.

Tissue damage leads to loss of cellular and mitochondrial membrane integrity and release of damage-associated molecular patterns, including those of mitochondrial origin (mitoDAMPs). Here, we describe the lymphocyte response to mitoDAMPs. Using primary cells from mice and human donors, we demonstrate that natural killer (NK) cells and T cells adopt regulatory phenotypes and functions in response to mitoDAMPs. NK cell-mediated cytotoxicity, interferon gamma (IFN-γ) production, T cell proliferation, and in vivo anti-viral T cell activation are all interrupted in the presence of mitoDAMPs or mitoDAMP-rich irradiated cells in in vitro and in vivo assays. Mass spectrometry analysis of mitoDAMPs demonstrates that arginase and products of its enzymatic activity are prevalent in mitoDAMP preparations. Functional validation by arginase inhibition and/or arginine add-back shows that arginine depletion is responsible for the alteration in immunologic polarity. We conclude that lymphocyte responses to mitoDAMPs reflect a highly conserved mechanism that regulates inflammation in response to tissue injury.

The Use of Donation After Circulatory Death Organs for Simultaneous Liver-kidney Transplant: To DCD or Not to DCD?

BACKGROUND: Because of the challenges with organ scarcity, many centers performing simultaneous liver-kidney transplant (SLKT) are opting to accept donation after circulatory death (DCD) organs as a means of facilitating earlier transplant and reducing death rates on the waitlist. It has been suggested, however, that DCD organs may have inferior graft and patient survival posttransplant compared with donation after neurologic death (DND) organs. METHODS: We created a Markov model to compare the overall outcomes of accepting a DCD SLKT now versus waiting for a DND SLKT in patients waitlisted for SLKT, stratified by base Model for End-Stage Liver Disease (MELD) score (≤20, 21-30, >30). RESULTS: Waiting for DND SLKT was the preferred treatment strategy for patients with a MELD score of 30 or less (incremental value of 0.54 and 0.36 quality-adjusted life years for MELD score of 20 or less and MELD score of 21 to 30 with DND versus DCD SLKT, respectively). The option to accept a DCD SLKT became the preferred choice for those with a MELD score greater than 30 (incremental value of 0.31 quality-adjusted life years for DCD versus DND SLKT). This finding was confirmed in a probabilistic sensitivity analysis and persisted when analyzing total life years obtained for accept DCD versus do not accept DCD. CONCLUSIONS: There is a benefit to accepting DCD SLKT for patients with MELD score greater than 30. Although not accepting DCD SLKT and waiting for DND SLKT is the preferred option for patients with MELD of 30 or less, the incremental value is small.

Bioengineered human pseudoislets form efficiently from donated tissue, compare favourably with native islets in vitro and restore normoglycaemia in mice.

AIMS/HYPOTHESIS: Islet transplantation is a treatment option that can help individuals with type 1 diabetes become insulin independent, but inefficient oxygen and nutrient delivery can hamper islet survival and engraftment due to the size of the islets and loss of the native microvasculature. We hypothesised that size-controlled pseudoislets engineered via centrifugal-forced-aggregation (CFA-PI) in a platform we previously developed would compare favourably with native islets, even after taking into account cell loss during the process. METHODS: Human islets were dissociated and reaggregated into uniform, size-controlled CFA-PI in our microwell system. Their performance was assessed in vitro and in vivo over a range of sizes, and compared with that of unmodified native islets, as well as islet cell clusters formed by a conventional spontaneous aggregation approach (in which dissociated islet cells are cultured on ultra-low-attachment plates). In vitro studies included assays for membrane integrity, apoptosis, glucose-stimulated insulin secretion assay and total DNA content. In vivo efficacy was determined by transplantation under the kidney capsule of streptozotocin-treated Rag1-/- mice, with non-fasting blood glucose monitoring three times per week and IPGTT at day 60 for glucose response. A recovery nephrectomy, removing the graft, was conducted to confirm efficacy after completing the IPGTT. Architecture and composition were analysed by histological assessment via insulin, glucagon, pancreatic polypeptide, somatostatin, CD31 and von Willebrand factor staining. RESULTS: CFA-PI exhibit markedly increased uniformity over native islets, as well as substantially improved glucose-stimulated insulin secretion (8.8-fold to 11.1-fold, even after taking cell loss into account) and hypoxia tolerance. In vivo, CFA-PI function similarly to (and potentially better than) native islets in reversing hyperglycaemia (55.6% for CFA-PI vs 20.0% for native islets at 500 islet equivalents [IEQ], and 77.8% for CFA-PI vs 55.6% for native islets at 1000 IEQ), and significantly better than spontaneously aggregated control cells (55.6% for CFA-PI vs 0% for spontaneous aggregation at 500 IEQ, and 77.8% CFA-PI vs 33.4% for spontaneous aggregation at 1000 IEQ; p < 0.05). Glucose clearance in the CFA-PI groups was improved over that in the native islet groups (CFA-PI 18.1 mmol/l vs native islets 29.7 mmol/l at 60 min; p < 0.05) to the point where they were comparable with the non-transplanted naive normoglycaemic control mice at a low IEQ of 500 IEQ (17.2 mmol/l at 60 min). CONCLUSIONS/INTERPRETATION: The ability to efficiently reformat dissociated islet cells into engineered pseudoislets with improved properties has high potential for both research and therapeutic applications.

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.

Ferroptosis-inducing agents compromise in vitro human islet viability and function.

Human islet transplantation has been hampered by donor cell death associated with the islet preparation procedure before transplantation. Regulated necrosis pathways are biochemically and morphologically distinct from apoptosis. Recently, ferroptosis was identified as a non-apoptotic form of iron-dependent regulated necrosis implicated in various pathological conditions. Mediators of islet oxidative stress, including glutathione peroxidase-4 (GPX4), have been identified as inhibitors of ferroptosis, and mechanisms that affect GPX4 function can impact islet function and viability. Ferroptosis has not been investigated directly in human islets, and its relevance in islet transplantation remains unknown. Herein, we sought to determine whether in vitro human islet viability and function is compromised in the presence of two distinct ferroptosis-inducing agents (FIA), erastin or RSL3, and whether these effects could be rescued with ferroptosis inhibitors, ferrostatin-1 (Fer-1), or desferrioxamine (DFO). Viability, as assessed by lactate dehydrogenase (LDH) release, revealed significant death in erastin- and RSL3-treated islets, 20.3% ± 3.8 and 24.4% ± 2.5, 24 h post culture, respectively. These effects were ameliorated in islets pre-treated with Fer-1 or the iron chelator, desferrioxamine (DFO). Stimulation index, a marker of islet function revealed a significant reduction in function in erastin-treated islets (control 1.97 ± 0.13 vs. 50 µM erastin 1.32 ± 0.1) (p < 0.05). Fer-1 and DFO pre-treatment alone did not augment islet viability or function. Pre-treatment of islets with erastin or Fer-1 did not impact in vivo engraftment in an immunodeficient mouse transplant model. Our data reveal that islets are indeed susceptible to ferroptosis in vitro, and induction of this novel cell death modality leads to compromised islet function, which can be recoverable in the presence of the ferroptosis inhibitors. The in vivo impact of this pathway in islet transplantation remains elusive given the constraints of our study, but warrants continued investigation.

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.

BMX-001, a novel redox-active metalloporphyrin, improves islet function and engraftment in a murine transplant model.

Islet transplantation has become a well-established therapy for select patients with type 1 diabetes. Viability and engraftment can be compromised by the generation of oxidative stress encountered during isolation and culture. We evaluated whether the administration of BMX-001 (MnTnBuOE-2-PyP5+ [Mn(III) meso-tetrakis-(N-b-butoxyethylpyridinium-2-yl)porphyrin]) and its earlier derivative, BMX-010 (MnTE-2-PyP [Mn(III) meso-tetrakis-(N-methylpyridinium-2-yl)porphyrin]) could improve islet function and engraftment outcomes. Long-term culture of human islets with BMX-001, but not BMX-010, exhibited preserved in vitro viability. Murine islets isolated and cultured for 24 hours with 34 µmol/L BMX-001 exhibited improved insulin secretion (n = 3 isolations, P < .05) in response to glucose relative to control islets. In addition, 34 µmol/L BMX-001-supplemented murine islets exhibited significantly reduced apoptosis as indicated by terminal deoxynucleotidyl transferase dUTP nick end labeling, compared with nontreated control islets (P < .05). Murine syngeneic islets transplanted under the kidney capsule at a marginal dose of 150 islets revealed 58% of 34 µmol/L BMX-001-treated islet recipients became euglycemic (n = 11 of 19) compared with 19% of nontreated control islet recipients (n = 3 of 19, P < .05). Of murine recipients receiving a marginal dose of human islets cultured with 34 µmol/L BMX-001, 92% (n = 12 of 13) achieved euglycemia compared with 57% of control recipients (n = 8 of 14, P = .11). These results demonstrate that the administration of BMX-001 enhances in vitro viability and augments murine marginal islet mass engraftment.

Beta Cell Death by Cell-free DNA and Outcome After Clinical Islet Transplantation.

BACKGROUND: Optimizing engraftment and early survival after clinical islet transplantation is critical to long-term function, but there are no reliable, quantifiable measures to assess beta cell death. Circulating cell-free DNA (cfDNA) derived from beta cells has been identified as a novel biomarker to detect cell loss and was recently validated in new-onset type 1 diabetes and in islet transplant patients. METHODS: Herein we report beta cell cfDNA measurements after allotransplantation in 37 subjects and the correlation with clinical outcomes. RESULTS: A distinctive peak of cfDNA was observed 1 hour after transplantation in 31 (83.8%) of 37 subjects. The presence and magnitude of this signal did not correlate with transplant outcome. The 1-hour signal represents dead beta cells carried over into the recipient after islet isolation and culture, combined with acute cell death post infusion. Beta cell cfDNA was also detected 24 hours posttransplant (8/37 subjects, 21.6%). This signal was associated with higher 1-month insulin requirements (P = 0.04), lower 1-month stimulated C-peptide levels (P = 0.01), and overall worse 3-month engraftment, by insulin independence (receiver operating characteristic-area under the curve = 0.70, P = 0.03) and beta 2 score (receiver operating characteristic-area under the curve = 0.77, P = 0.006). CONCLUSIONS: cfDNA-based estimation of beta cell death 24 hours after islet allotransplantation correlates with clinical outcome and could predict early engraftment.

Low energy X-ray (grenz ray) treatment of purified islets prior to allotransplant markedly decreases passenger leukocyte populations.

Grenz rays, or minimally penetrating X-rays, are known to be an effective treatment of certain recalcitrant immune-mediated skin diseases, but their use in modulating allograft rejection has not been tested. We examined the capacity of grenz ray treatment to minimize islet immunogenicity and extend allograft survival in a mouse model. In a preliminary experiment, 1 of 3 immunologically intact animals demonstrated long-term acceptance of their grenz ray treated islet allograft. Further experiments revealed that 28.6% (2 of 7) grenz ray treated islet allografts survived >60 d. A low dose of 20Gy, was important; a 4-fold increase in radiation resulted in rapid graft failure, and transplanting a higher islet mass did not alter this outcome. To determine whether increased islet allograft survival after grenz treatment would be masked by immunosuppression, we treated the recipients with CTLA-4 Ig, and found an additive effect, whereby 17.5% more animals accepted the graft long-term versus those with CTLA-4 Ig alone. Cell viability assays verified that islet integrity was maintained after treatment with 20Gy. As well, through splenocyte infiltration analysis, donor CD4+ T cell populations 24-hours after transplant were decreased by more than16-fold in recipients receiving irradiated islets compared with control. Donor CD8+ T cell populations, although less prevalent, decreased in all treatment groups compared with control. Our results suggest that brief treatment of isolated islets with low energy grenz rays before allotransplantation can significantly reduce passenger leukocytes and promote graft survival, possibly by inducing donor dendritic cells to differentiate toward a tolerogenic phenotype.

Transplantation of Human Pancreatic Endoderm Cells Reverses Diabetes Post Transplantation in a Prevascularized Subcutaneous Site.

Beta-cell replacement therapy is an effective means to restore glucose homeostasis in select humans with autoimmune diabetes. The scarcity of "healthy" human donor pancreata restricts the broader application of this effective curative therapy. "ß-Like" cells derived from human embryonic stem cells (hESC), with the capacity to secrete insulin in a glucose-regulated manner, have been developed in vitro, with limitless capacity for expansion. Here we report long-term diabetes correction in mice transplanted with hESC-derived pancreatic endoderm cells (PECs) in a prevascularized subcutaneous site. This advancement mitigates chronic foreign-body response, utilizes a device- and growth factor-free approach, facilitates in vivo differentiation of PECs into glucose-responsive insulin-producing cells, and reliably restores glycemic control. Basal and stimulated human C-peptide secretion was detected throughout the study, which was abolished upon graft removal. Recipient mice demonstrated physiological clearance of glucose in response to metabolic challenge and safely retrieved grafts contained viable glucose regulatory cells.

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.

Long-term function and optimization of mouse and human islet transplantation in the subcutaneous device-less site.

Clinical islet transplantation has routinely been demonstrated to be an efficacious means of restoring glycemic control in select patients with autoimmune diabetes. Notwithstanding marked progress and improvements, the broad-spectrum application of this treatment option is restricted by the complications associated with intrahepatic portal cellular infusion and the scarcity of human donor pancreata. Recent progress in stem cell biology has demonstrated that the potential to expand new ß cells for clinical transplantation is now a reality. As such, research focus is being directed toward optimizing safe extrahepatic transplant sites to house future alternative ß cell sources for clinical use. The present study expands on our previous development of a prevascularized subcutaneous device-less (DL) technique for cellular transplantation, by demonstrating long-term (>365 d) durable syngeneic murine islet graft function. Furthermore, histological analysis of tissue specimens collected immediately post-DL site creation and acutely post-human islet transplantation demonstrates that this technique results in close apposition of the neovascularized collagen to the transplanted cells without dead space, thereby avoiding hypoxic luminal dead-space. Murine islets transplanted into the DL site created by a larger luminal diameter (6-Fr.) (n = 11), reversed diabetes to the similar capacity as our standard DL method (5-Fr.)(n = 9). Furthermore, glucose tolerance testing did not differ between these 2 transplant groups (p > 0 .05). Taken together, this further refinement of the DL transplant approach facilitates a simplistic means of islet infusion, increases the transplant volume capacity and may provide an effective microenvironment to house future alternative ß cell sources.

Photoacoustic imaging of angiogenesis in a subcutaneous islet transplant site in a murine model.

Islet transplantation (IT) is an established clinical therapy for select patients with type-1 diabetes. Clinically, the hepatic portal vein serves as the site for IT. Despite numerous advances in clinical IT, limitations remain, including early islet cell loss posttransplant, procedural complications, and the inability to effectively monitor islet grafts. Hence, alternative sites for IT are currently being explored, with the subcutaneous space as one potential option. When left unmodified, the subcutaneous space routinely fails to promote successful islet engraftment. However, when employing the previously developed subcutaneous "deviceless" technique, a favorable microenvironment for islet survival and function is established. In this technique, an angiocatheter was temporarily implanted subcutaneously, which facilitated angiogenesis to promote subsequent islet engraftment. This technique has been employed in preclinical animal models, providing a sufficient means to develop techniques to monitor functional aspects of the graft such as angiogenesis. Here, we utilize photoacoustic imaging to track angiogenesis during the priming of the subcutaneous site by the implanted catheter at 1 to 4 weeks postcatheter. Quantitative analysis on vessel densities shows gradual growth of vasculature in the implant position. These results demonstrate the ability to track angiogenesis, thus facilitating a means to optimize and assess the pretransplant microenvironment.

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.