Normothermic Perfusion is Superior to Cold Perfusion in Porcine Ex Situ Lung Perfusion.

BACKGROUND: Cold ex situ lung perfusion (ESLP) has demonstrated improved preservation in small animal ESLP compared to normothermic ESLP and cold static preservation. We hypothesized that cold negative pressure ventilation (NPV)-ESLP would improve graft function in a porcine transplantation model. METHODS: Four perfusate temperatures were examined with 12 hours NPV-ESLP in a large animal transplantation model. Pig lungs were allotted to four groups: (1) Normothermia (38°C, n = 6); (2) profound hypothermia (10°C, n = 6); (3) moderate hypothermia (20°C, n = 3); (4) subnormothermia (32°C, n = 3). A fifth group subnormothermic low-flow (SNLF) perfusion was examined to assess the effect of reduced cardiac output with cold perfusion (32°C, 10% cardiac output, n = 6). RESULTS: Only Normothermic and SNLF groups demonstrated acceptable oxygenation after 12 hours NPV-ESLP and were transplanted. All other groups failed prematurely. After 12 hours of ESLP, Normothermic lungs demonstrated significantly greater dynamic compliance compared to SNLF lungs (P = .03). Edema formation post-ESLP was significantly worse in the SNLF group (P = .01). There was no significant difference in pulmonary artery pressures after ESLP (P = .10); however, pulmonary vascular resistance was significantly greater in the SNLF (P = .04). Isolated left lung oxygenation 4-hours post-transplant and left lung edema formation was not significantly different between Normothermic and SNLF post-transplant (P = .09). Proinflammatory cytokines were significantly greater during SNLF-ESLP (tumor necrosis factor alpha, P < .05). CONCLUSIONS: Prolonged normothermic (38°C) NPV-ESLP is superior to 10, 20, and 32°C perfusion. Normothermic ESLP of porcine lungs results in superior graft function and reduced inflammation versus SNLF-ESLP.

Left Lung Orthotopic Transplantation in a Juvenile Porcine Model for ESLP.

Lung transplantation is the gold-standard treatment for end-stage lung disease, with over 4,600 lung transplantations performed worldwide annually. However, lung transplantation is limited by a shortage of available donor organs. As such, there is high waitlist mortality. Ex situ lung perfusion (ESLP) has increased donor lung utilization rates in some centers by 15%-20%. ESLP has been applied as a method to assess and recondition marginal donor lungs and has demonstrated acceptable short- and long-term outcomes following transplantation of extended criteria donor (ECD) lungs. Large animal (in vivo) transplantation models are required to validate ongoing in vitro research findings. Anatomic and physiologic differences between humans and pigs pose significant technical and anesthetic challenges. An easily reproducible transplant model would permit the in vivo validation of current ESLP strategies and the preclinical evaluation of various interventions designed to improve donor lung function. This protocol describes a porcine model of orthotopic left lung allotransplantation. This includes anesthetic and surgical techniques, a customized surgical checklist, troubleshooting, modifications, and the benefits and limitations of the approach.

Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: Evaluation of Lung Function and Metabolism.

Lung transplantation (LTx) remains the standard of care for end-stage lung disease. A shortage of suitable donor organs and concerns over donor organ quality exacerbated by excessive geographic transportation distance and stringent donor organ acceptance criteria pose limitations to current LTx efforts. Ex situ lung perfusion (ESLP) is an innovative technology that has shown promise in attenuating these limitations. The physiologic ventilation and perfusion of the lungs outside of the inflammatory milieu of the donor body affords ESLP several advantages over traditional cold static preservation (CSP). There is evidence that negative pressure ventilation (NPV) ESLP is superior to positive pressure ventilation (PPV) ESLP, with PPV inducing more significant ventilator-induced lung injury, pro-inflammatory cytokine production, pulmonary edema, and bullae formation. The NPV advantage is perhaps due to the homogenous distribution of intrathoracic pressure across the entire lung surface. The clinical safety and feasibility of a custom NPV-ESLP device have been demonstrated in a recent clinical trial involving extender criteria donor (ECD) human lungs. Herein, the use of this custom device is described in a juvenile porcine model of normothermic NPV-ESLP over a 12 h duration, paying particular attention to management techniques. Pre-surgical preparation, including ESLP software initialization, priming, and de-airing of the ESLP circuit, and the addition of anti-thrombotic, anti-microbial, and anti-inflammatory agents, is specified. The intraoperative techniques of central line insertion, lung biopsy, exsanguination, blood collection, cardiectomy, and pneumonectomy are described. Furthermore, particular focus is paid to anesthetic considerations, with anesthesia induction, maintenance, and dynamic modifications outlined. The protocol also specifies the custom device's initialization, maintenance, and termination of perfusion and ventilation. Dynamic organ management techniques, including alterations in ventilation and metabolic parameters to optimize organ function, are thoroughly described. Finally, the physiological and metabolic assessment of lung function is characterized and depicted in the representative results.

Caspase inhibitor IDN6556 facilitates marginal mass islet engraftment in a porcine islet autotransplant model.

BACKGROUND: Large numbers of islets are lost in the early phase after clinical islet transplantation, through apoptosis, necrosis, or innate inflammatory injury. We previously demonstrated the efficacy of a series of caspase inhibitors in mouse models on islet engraftment through reduction in early posttransplant apoptosis. We studied IDN6556, a caspase inhibitor with a first-pass effect, in a large animal (pig) intraportal marginal mass islet autotransplant model. METHODS: Total pancreatectomy and marginal mass islet autotransplantation were carried out in Yucatan miniature swine to explore the effects of IDN6556 on islet engraftment. Pigs were treated with IDN6556 at a dose of 20 mg/kg orally twice daily (n=7) or phosphate-buffered saline control (n=6) orally for 7 days, and blood glucose was monitored for 1 month. Glucose tolerance and acute insulin release were determined at 1 month. RESULTS: There were no differences in islet procurement, isolation, or islet functional parameters between the two groups. Pigs receiving IDN6556 had lower fasting blood glucose level after transplantation and a higher percentage (100% vs. 33.3%) showed fasting blood glucose levels less than 11 mM. This translated into an enhanced metabolic reserve and acute insulin release for pigs in the treatment group. CONCLUSIONS: IDN6556 led to enhanced islet engraftment in this large animal islet transplant model. Although this study has limitations including a short interval of study (1 month) and the use of unpurified islets, the results justify early clinical trials of IDN6556 in islet transplantation.

AEB071 (sotrastaurin) does not exhibit toxic effects on human islets in vitro, nor after transplantation into immunodeficient mice.

AEB071 (AEB, sotrastaurin), a specific inhibitor of protein kinase C, reduces T-lymphocyte activation and cytokine release. AEB delays islet allograft rejection in rats and prevents rejection when combined with cyclosporine. Since many immunosuppressive agents have toxic effects on the function of transplanted islets, we investigated whether this was also the case with AEB. Human islets were transplanted into Rag-knockout mice randomly assigned to vehicle control, AEB or sirolimus treatment groups. Non-fasting blood glucose levels, body weight and glucose tolerance was measured in recipients. In a separate experiment, human islets were cultured in the presence of AEB and assayed for glucose dependent insulin secretion and level of ß-cell apoptosis. Eighty-six percent of the AEB-treated recipients achieved normoglycemia following transplant (compared with none in sirolimus-treated group, p < 0.05). AEB-treated recipients exhibited similar glucose homeostasis as vehicle-treated controls, which was better than in sirolimus-treated recipients. Human islets cultured with AEB showed similar rates of ß-cell apoptosis (p = 0.98 by one-way ANOVA) and glucose stimulated insulin secretion (p = 0.15) as those cultured with vehicle. These results suggest that AEB is not associated with toxic effects on islet engraftment or function. AEB appears to be an appropriate immunosuppressive candidate for clinical trials in islet transplantation.

Sirolimus drug-eluting, hydrogel-impregnated polypropylene mesh reduces intra-abdominal adhesion formation in a mouse model.

BACKGROUND: Prosthetic mesh is used frequently in abdominal wall hernia reconstruction but is prone to postoperative adhesion formation. Complications resulting from intra-abdominal adhesions represent a considerable clinical and cost burden. We, herein, investigate the antiproliferative and antiadhesiogenic properties of sirolimus and hydrogel-impregnated, drug-eluting mesh to decrease such complications in a mouse model of abdominal wall hernia repair. METHODS: A 1 × 1cm(2) polypropylene mesh from 1 of 3 groups (group 1, plain control; group 2, hydrogel [2% agarose]; and group 3, hydrogel + 10 mcg sirolimus) was implanted operatively into the peritoneal cavity of BALB/c mice and followed for up to 4 weeks. Adhesions were scored by percent surface area of mesh (range, 0-100%), severity (range, 0-3), and tenacity (range, 0-4). Representative samples were assessed by scanning electron microscopy. RESULTS: Mesh impregnated with the combination of hydrogel and sirolimus led to a significant decrease in adhesion formation. The percent surface area of adhesional attachment to mesh was decreased from 100.0 ± 0% in the plain mesh control group versus 18 ± 8% (P < .001) in the combined impregnated mesh group. Similarly, adhesion severity scores were decreased from a score of 2.9 ± 0.1 (plain mesh) versus 1.4 ± 0.1 (sirolimus/hydrogel-impregnated mesh) (P < .001). Scores for tenacity were also decreased markedly from 3.5 ± 0.2 (plain mesh) versus 1.5 ± 0.1 (sirolimus/hydrogel-impregnated mesh (P < .001). CONCLUSION: Creation of a sirolimus drug-eluting and hydrogel-impregnated polypropylene mesh resulted in marked decrease of adhesion formation in this mouse model, was well tolerated without side effects, and has potential for clinical application.

Histopaque provides optimal mouse islet purification kinetics: comparison study with Ficoll, iodixanol and dextran.

Islet transplantation has become a very promising treatment for type 1 diabetes. To facilitate further clinical improvements in this exciting field, rodent islets are used to evaluate new strategies and modifications. One method to purify islets is on a density gradient, although the optimal gradient component can be debated. N=6 separate mouse islet isolations were used and the resulting islets were separated and purified on either a Ficoll, Histopaque, Dextran or Iodixanol gradient. Islets were assessed for recovery, viability, purity and in vitro functionality. Aliquots were transplanted into diabetic mice to assess in vivo functionality and survival. There was no difference in the number of islets recovered across groups nor in the size of recovered islets. Use of a Ficoll or Histopaque gradient led to the most pure and viable islets in comparison to Dextran and Iodixanol. Functionally, islets isolated on a Ficoll gradient had the highest glucose-stimulated insulin release in vitro while performing equally to Histopaque and Dextran gradients in vivo. Using a Ficoll gradient, however, comes at a higher monetary cost. We recommend using a Histopaque gradient, which led to the isolation of viable and functional islets with a reduced cost as compared to a Ficoll gradient.

The caspase inhibitor IDN-6556 (PF3491390) improves marginal mass engraftment after islet transplantation in mice.

BACKGROUND: Islet transplantation has become a viable option for selected type 1 diabetic patients; however, a significant portion need to return to exogenous insulin. The predominant factors include impaired islet engraftment and early islet loss. Caspase inhibition is a potent way to improve islet engraftment, but all tested compounds so far have not been clinically relevant. IDN-6556 (PF3491390) has already been used clinically and can be delivered orally with high portal vein concentrations. METHODS: Mice were given a marginal mass islet graft of either mouse or human islets and treated with either IDN-6556 (10 or 20 mg/kg ip bid) or vehicle and followed for diabetes reversal. At 1 month post-transplant, mice were subjected to a glucose tolerance test and an assessment of graft mass. In separate experiments, human islets were cultured with IDN-6556 or vehicle to assess for islet survival and viability. RESULTS: In both syngeneic mouse islets and human islets transplanted into immunodeficient mice, IDN-6556 (20 mg/kg) given for 7 days post-transplant led to a significantly enhanced rate of diabetes reversal as compared to vehicle. In addition, mice receiving caspase inhibitor displayed improved glucose tolerance and graft survival at the 1-month point. We also found protective effects in vitro for islet viability and marked reduction in apoptosis in vivo. CONCLUSION: Taken together, these results demonstrate the effectiveness of caspase inhibition with IDN-6556 on islet transplantation and in particular islet engraftment and survival.

Detecting rejection after mouse Islet transplantation utilizing islet protein-stimulated ELISPOT.

Improved posttransplant monitoring and on-time detection of rejection could improve islet transplantation outcome. The present study explored the possibility of detecting harmful events after mouse islet transplantation measuring the immune responsiveness against islet extracts. Mouse islet transplantations were performed using various donor/recipient combinations, exploring autoimmune (NOD/SCID to NOD, n = 6) and alloimmune events (C57BL/6 to BALB/c, n = 20), a combination of both (C57BL/6 to NOD, n = 8), the absence of both (BALB/c to BALB/c, n = 21), or naive, nontransplanted control mice (n = 14). The immune reactivity was measured by ELISPOT, looking at the ex vivo release of IFN-γ from splenocytes stimulated by islet donor extracts (sonicated islets). The immune reactivity was not altered in the syngeneic and autoimmune models, demonstrating similar levels as nontransplanted controls (p = 0.46 and p = 0.6). Conversely, the occurrence of an allogeneic rejection alone or in combination to autoimmunity was associated to an increase in the level of immune reactivity (p = 0.023 and p = 0.003 vs. respective controls). The observed increase was transient and lost in the postrejection period or after treatment with CTLA4-Ig. Overall, allogeneic rejection was associated to a transient increase in the reactivity of splenocytes against islet proteins. Such a strategy has the potential to improve islet graft monitoring in human and should be further explored.

Vascular endothelial growth factor expression in hepatic epithelioid hemangioendothelioma: Implications for treatment and surgical management.

Epithelioid hemangioendothelioma (EHE) is a low-grade, malignant vascular tumor that most commonly presents within the liver. Patients with hepatic EHE are often candidates for liver transplantation as the disease is usually multifocal at diagnosis. Although these patients achieve excellent early outcomes post-transplant, there are very few data regarding tumor markers that can further direct chemotherapy in hepatic EHE to prevent recurrent disease. The purpose of this study was to analyze the expression of the angiogenic factor vascular endothelial growth factor (VEGF) and its receptors in hepatic EHE. Six patients with hepatic EHE were assessed for liver transplantation at our center. Pathology specimens of primary and recurrent EHE were analyzed by hematoxylin and eosin staining and by immunofluorescence for VEGF, fetal liver kinase 1 (Flk-1), and fms-related tyrosine kinase 1 (Flt-1) expression. Five patients underwent liver transplantation, and 1 patient underwent liver resection. Biopsy-proven recurrent EHE occurred in 3 patients. VEGF expression was present in 100% of the EHE specimens examined, whereas Flt-1 expression was present in only 1 sample, and Flk-1 was not observed in any of the specimens. In 1 patient with recurrent hepatic EHE post-liver transplantation, a progressive increase in the VEGF fluorescence intensity and distribution was observed. In conclusion, in this series, VEGF expression was observed in all hepatic EHE specimens analyzed. These data suggest that anti-VEGF chemotherapeutic agents will be of use in patients with hepatic EHE, particularly as a means of reducing the tumor volume prior to resection, as a means of treating unresectable or metastatic disease, or as an adjuvant therapy in the setting of liver transplantation.

Liraglutide, a long-acting human glucagon-like peptide 1 analogue, improves human islet survival in culture.

The culture of human islets is associated with approximately 10-20% islet loss, occasionally preventing transplantation. Preconditioning of the islets to improve postculture yields would be of immediate benefit, with the potential to increase both the number of transplanted patients and their metabolic reserve. In this study, the effect of liraglutide, a long-acting human glucagon-like peptide 1 analogue, on cultured human islets was examined. Culture with liraglutide (1 micromol/l) was associated with a preservation of islet mass (significantly more islets at 24 and 48 h, compared to control; P < or = 0.05 at 24 and 48 h) and with the presence of larger islets (P < or = 0.05 at 48 h). These observations were supported by reduced apoptosis rates after 24 h of treatment. We also demonstrated that human islet engraftment is improved in C57Bl/6-RAG(-/-) mice treated with liraglutide 200 microg/kg sc twice daily (P < or = 0.05), suggesting that liraglutide should be continued after transplantation. Overall, these data demonstrate the beneficial effect of liraglutide on cultured human islets, preserving islet mass. They support the design of clinical studies looking at the effect of liraglutide in clinical islet transplantation.

Protein kinase C inhibitor, AEB-071, acts complementarily with cyclosporine to prevent islet rejection in rats.

BACKGROUND: AEB-071 (AEB) is a specific inhibitor of protein kinase C, which prevents T-lymphocyte activation. The present study investigated the effect of AEB on rat islet allotransplantation alone or in combination with CTLA4-Ig, mycophenolate mofetil, or cyclosporine A (CsA). METHODS: A rodent allogeneic islet transplant model (Lewis to Wistar Furth) was used to investigate the efficacy of AEB as an immunosuppressive agent. Furthermore, the Lewis rat was used to screen for any AEB associated toxicities on glucose homeostasis in vivo. RESULTS: AEB alone (30 mg/kg per os [p.o.] two times per day [bid]) delayed rejection to a median survival time of 22 days (vs. 7 days in control vehicle-treated animals, P<0.05). When combined with CsA (5 mg/kg p.o. bid), AEB prolonged survival from 12 (CsA alone) to over 100 days in 80% of animals (P<0.05). No delay in allograft rejection (above that resulting from AEB alone) was observed when AEB was combined with a sub-therapeutic dose of CTLA4-Ig or mycophenolate mofetil, nor low dose of CsA. The frequency of allospecific interferon-gamma-secreting splenocytes, assessed ex vivo by enzyme-linked immunosorbent spot (ELISPOT) assay, was lower in AEB-treated recipients compared with controls (P<0.05). AEB treatment did not alter the intraperitoneal glucose tolerance, the glucose-dependent insulin release, or the insulin content of the native pancreas. CONCLUSIONS: These data suggest that AEB is an appropriate immunosuppressive agent for islet transplantation, as it can prolong islet graft survival alone or in combination with CsA, without toxicity on glucose metabolism.

Porcine marginal mass islet autografts resist metabolic failure over time and are enhanced by early treatment with liraglutide.

Although insulin independence is maintained in most islet recipients at 1 yr after transplant, extended follow-up has revealed that many patients will eventually require insulin therapy. Previous studies have shown that islet autografts are prone to chronic failure in large animals and humans, suggesting that nonimmunological events contribute to islet graft functional decay. Early intervention with therapies that promote graft stability should provide a measurable benefit over time. In this study, the efficacy of the long-acting glucagon-like peptide-1 analog liraglutide was explored in a porcine marginal mass islet autograft transplant model. Incubation with liraglutide enhanced porcine islet survival and function after prolonged culture. Most vehicle-treated (83%) and liraglutide-treated (80%) animals became insulin independent after islet autotransplantation. Although liraglutide therapy did not improve insulin independence rates or blood glucose levels after transplant, a significant increase in insulin secretion and acute-phase insulin response was observed in treated animals. Surprisingly, no evidence for deterioration of graft function was observed in any of the transplanted animals over more than 18 months of follow-up despite significant weight gain; in fact, an enhanced response to glucose developed over time even in control animals. Histological analysis showed that intraportally transplanted islets remained highly insulin positive, retained alpha-cells, and did not form amyloid deposits. This study demonstrates that marginal mass porcine islet autografts have stable long-term function, even in the presence of an increasing metabolic demand. These results are discrepant with previous large animal studies and suggest that porcine islets may be resistant to metabolic failure.

Effect of different induction strategies on effector, regulatory and memory lymphocyte sub-populations in clinical islet transplantation.

This prospective study assessed lymphocyte subsets in the peripheral blood of 42 islet allograft recipients using flow cytometry from 2 weeks and up to 2 years post-transplantation. Subjects received daclizumab (n = 16), Thymoglobulin (n = 12) or alemtuzumab (n = 14). Alemtuzumab was associated with an early (within 1 month) and transient (up to 6 months) increase in the frequency of CD3(+) CD4(+) Foxp3(+) T cells, while daclizumab induced a near complete loss of these cells (P