University Of Wisconsin Solution Research Articles

Recipient T cell TIM-3 and hepatocyte galectin-9 signalling protects mouse liver transplants against ischemia-reperfusion injury

By binding to T cell immunoglobulin mucin-3 (TIM-3) on activated Th1 cells, galectin-9 (Gal-9) negatively regulates Th1-type alloimmunity. Although T cells contribute to hepatic ischemia-reperfusion injury (IRI), it is unknown whether negative T cell-dependent TIM-3 co-stimulation may rescue IR-stressed orthotopic liver transplants from innate immunity-driven inflammation. We used wild type (WT) and TIM-3 transgenic (Tg) mice (C57BL/6) as liver donors and recipients in a clinically-relevant model of hepatic cold storage (20 h at 4°C in UW solution) and syngeneic orthotopic liver transplantation (OLT). Orthotopic liver transplants in WT or TIM-3Tg→TIM-3Tg groups were resistant against IR-stress, evidenced by preserved hepatocellular function (serum ALT levels) and liver architecture (Suzuki's score). In contrast, orthotopic liver transplants in WT or TIM-3Tg→WT groups were susceptible to IRI. TIM-3 induction in circulating CD4+ T cells of the recipient: (1) depressed T-bet/IFN-γ, while amplifying GATA3 and IL-4/IL-10 expression in orthotopic liver transplants; (2) promoted T cell exhaustion (PD-1, LAG-3) phenotype; and (3) depressed neutrophil and macrophage infiltration/function in orthotopic liver transplants. In parallel studies, we documented for the first time that Gal-9, a natural TIM-3 ligand, was produced primarily by and released from IR-stressed hepatocytes, both in vivo and in vitro. Moreover, exogenous recombinant Gal-9 (rGal-9) potentiated liver resistance against IRI by depressing T cell activation and promoting apoptosis of CD4+ T cells. Harnessing TIM-3/Gal-9 signalling at the T cell-hepatocyte interface facilitates homeostasis in IR-stressed orthotopic liver transplants. Enhancing anti-oxidant hepatocyte Gal-9 potentiates liver IR-resistance. Negative regulation by recipient TIM-3+CD4+ cells provides evidence for cytoprotective functions of a discrete T cell subset, which should be spared when applying T cell-targeted immunosuppression in transplant recipients.

Serum-stabilized Naked Caspase-3 siRNA Protects Autotransplant Kidneys in a Porcine Model

The naked small interfering RNA (siRNA) of caspase-3, a key player in ischemia reperfusion injury, was effective in cold preserved and hemoreperfused kidneys, but not autotransplanted kidneys in our porcine models. Here, chemically modified serum stabilized caspase-3 siRNAs were further evaluated. The left kidney was retrieved and infused by University of Wisconsin solution with/without 0.3 mg caspase-3 or negative siRNA into the renal artery for 24-hour cold storage (CS). After an intravenous injection of 0.9 mg siRNA and right-uninephrectomy, the left kidney was autotransplanted for 2 weeks. The effectiveness of caspase-3 siRNA was confirmed by caspase-3 knockdown in the post-CS and/or post-transplant kidneys with reduced apoptosis and inflammation, while the functional caspase-3 siRNA in vivo was proved by detected caspase-3 mRNA degradation intermediates. HMGB1 protein was also decreased in the post-transplanted kidneys; correlated positively with renal IL-1β mRNA, but negatively with serum IL-10 or IL-4. The minimal off-target effects of caspase-3 siRNA were seen with favorable systemic responses. More importantly, renal function, associated with active caspase-3, HMGB1, apoptosis, inflammation, and tubulointerstitial damage, was improved by caspase-3 siRNA. Taken together, the 2-week autotransplanted kidneys were protected when caspase-3 siRNA administrated locally and systemically, which provides important evidence for future clinical trials.

Hypothermic Storage of Human Hepatocytes for Transplantation

Transplantation of human hepatocytes is gaining recognition as a bridge or an alternative to orthotopic liver transplantation for patients with acute liver failure and genetic defects. Since most patients require multiple cell infusions over an extended period of time, we investigated hepatic functions in cells maintained in University of Wisconsin solution at 4°C up to 72 h. Eleven different assessments of hepatic viability and function were investigated both pre- and posthypothermic storage, including plating efficiency, caspase-3/7 activity, ammonia metabolism, and drug-metabolizing capacity of isolated hepatocytes. Long-term function, basal, and induced cytochrome P450 activities were measured after exposure to prototypical inducing agents. Cells from 47 different human liver specimens were analyzed. Viability significantly decreased in cells cold stored in UW solution, while apoptosis level and plating efficiency were not significantly different from fresh cells. Luminescent and fluorescent methods assessed phases I and II activities both pre- and post-24-72 h of cold preservation. A robust induction (up to 200-fold) of phase I enzymes was observed in cultured cells. Phase II and ammonia metabolism remained stable during hypothermic storage, although the inductive effect of culture on each metabolic activity was eventually lost. Using techniques that characterize 11 measurements of hepatic viability and function from plating efficiency, to ammonia metabolism, to phases I and II drug metabolism, it was determined that while viability decreased, the remaining viable cells in cold-stored suspensions retained critical hepatic functions for up to 48 h at levels not significantly different from those observed in freshly isolated cells.

Effects of Donor-, Pancreas-, and Isolation-Related Variables on Human Islet Isolation Outcome: A Systematic Review

Different factors have been reported to influence islet isolation outcome, but their importance varies between studies and are hampered by the small sample sizes in most studies. The purpose of this study was to perform a systematic review to assess the impact of donor-, pancreas-, and isolation-related variables on successful human islet isolation outcome. PubMed, Embase, and Web of Science were searched electronically in April 2009. All studies reporting on donor-, pancreas-, and isolation-related factors relating to prepurification and postpurification islet isolation yield and proportion of successful islet isolations were selected. Seventy-four retrospective studies had sufficient data and were included in the analyses. Higher pre- and postpurification islet yields and a higher proportion of successful islet isolations were obtained when pancreata were preserved with the two-layer method rather than University of Wisconsin solution in donors with shorter cold ischemia times (CITs) [1 h longer CIT resulted in an average decline of prepurification and postpurification yields and proportion of successful isolations of 59 islet equivalents (IEQs)/g, 54 IEQs/g, and 21%, respectively]. Higher prepurification yields and higher percentage of successful islet isolations were found in younger donors with higher body mass index. Lower yields were found in donation after brain death donors compared to donation after cardiac death donors. Higher postpurification yields were found for isolation with Serva collagenase. This review identified donor-, pancreas-, and isolation-related factors that influence islet isolation yield. Standardized reports of these factors in all future studies may improve the power and identify additional factors and thereby contribute to improving islet isolation yield.

Strategies for short-term storage of hepatocytes for repeated clinical infusions.

Hepatocyte transplantation is an upcoming treatment for patients with metabolic liver diseases. Repeated cell infusions over 1-2 days improve clinical outcome. Isolated hepatocytes are usually cold stored in preservation solutions between repeated infusions. However, during cold storage isolated hepatocytes undergo cell death. We investigated if tissue preservation and repeated isolations are better than storage of isolated hepatocytes when cold preserving human hepatocytes. Liver tissue obtained from liver surgery or organ donors was divided into two pieces. Hepatocytes were isolated by collagenase digestion. Hepatocytes were analyzed directly after isolation (fresh) or after storage for 48 h at 4°C in University of Wisconsin solution (UW cells). Liver tissue from the same donor was stored at 4°C in UW and hepatocytes were isolated after 48 h (UW tissue cells). Hepatocyte viability and function was evaluated by trypan blue exclusion, plating efficiency, ammonia metabolism, CYP 1A1/2, 2C9, 3A7, and 3A4 activities, phase II conjugation, and apoptosis evaluation by TUNEL assay and caspase-3/7 activities. Hepatocytes stored in UW showed a significantly lower viability compared to fresh cells or hepatocytes isolated from tissue stored for 48 h (54% vs. 71% vs. 79%). Plating efficiency was significantly decreased for cells stored in UW (40%) compared to fresh and UW tissue cells (63% vs. 55%). No significant differences between UW cells and UW tissue cells could be shown for CYP activities or ammonia metabolism. Hepatocytes stored in UW showed a strong increase in TUNEL-positive cells, whereas TUNEL staining in cold-stored liver tissue and hepatocytes isolated after 48 h was unchanged. This observation was confirmed by increased caspase-3/7 activities in UW cells. Although preservation of isolated hepatocytes in UW maintains function, cold storage of liver tissue and repeated hepatocyte isolations is superior to cold storage of isolated hepatocytes in preserving hepatocyte viability and function.

P11: Targeted modulation of hepatocellular stress pathways during biopreservation affects storage outcome

Primary hepatocytes are central to a number of in vitro applications including hepatotoxicity testing for drug development, potential uses in cell therapy, and as research tools for improving organ transplantation. Further, hepatocyte derivatives such as the human hepatoma cell line C3A serve important roles in other technologies such as bioartificial liver support systems. While it is well characterized that molecular stress pathways are activated in response to biopreservation associated stresses thereby resulting in cell death and system failure, it remains unclear whether the specific molecular pathways activated are universal or cell specific. We hypothesized that given the vastly different characteristics of hepatocyte cell systems, cell type specific stress pathways may be activated under different storage regimes. To this end, we compared the responses of two hepatic cell types following both hypothermic and hypoxic normothermic storage and examined storage related stress signaling. Normal hepatocytes (NHEP) and C3A were stored under either hypothermic (4 °C) conditions for 18hrs to 3d or hypoxic normothermic (37 °C) conditions for 1–14 d. Respective growth media, HBSS (Balanced Salt Solution), and ViaSpan (UW solution) were utilized as storage media with and without the addition of salubrinal or resveratrol to examine targeted molecular pathway modulation. Viability was assessed at 0, 24 and 48 h post-storage. Apoptotic and necrotic populations were assessed via flow cytometry and fluorescent microscopy post-storage. Protein immunoblotting was performed to evaluate alterations in ER stress pathway proteins and apoptotic signaling proteins. Viability studies demonstrated a significant difference in modulation study outcomes for the two cell types. This was noted as salubrinal addition to HBSS stored C3A yielded significant improvement in viability (∼60%) while having no significant impact on NHEPs. Conversely, resveratrol addition to HBSS resulted in a significant decrease in C3A viability (∼80%) whereas it conferred a marked improvement on NHEP survival (∼25%). Interestingly, when the modulators were utilized under a hypoxic normothermic regime a differential response was observed as salubrinal negatively impacted C3A viability (∼50% decrease) while yielding improvement to NHEP survival (∼30% increase) and resveratrol yielded decreased viability in both cell types. Investigations into cell death populations demonstrated marked changes in the number of cells undergoing apoptosis and necrosis that was influenced by the use of these molecular modulators. Further, immunoblot analyses revealed alterations to protein levels associated with apoptotic activation (pro-caspase cleavage) as well as implications of ER stress induced cell death signaling (calnexin cleavage). These data demonstrate a differential response in hepatic cell systems that is dependent upon storage temperature, basal media and modulator addition. This is important for the processing and storage of biologics as it demonstrates that universal protocols may not be effective for all cell types and conditions and a more specific and directed approach may be necessary for optimal efficacy.

A08 Session 2: Biopreservation of biologics: Translation from stem cells to cancer : Differential activation of stress pathways in human mesenchymal stem cells following biopreservation.

Research on human mesenchymal stem cells (hMSC) has expanded considerably in recent years as continued investigations have demonstrated their potential for reestablishment of in vivo functions. Further, as hMSC studies have increased our understanding of their utility, this has in turn driven the development of hMSC-based technologies. These technologies range from the exploitation of innate in vivo functions to target and repair damaged tissue, to removal and purification of hMSC for use in numerous other settings such as tissue engineering, cell therapies, etc. Essential to these developments is the ability to process and maintain these unique biologics without the loss of viability and functionality. As such, bioprocessing has become a limiting factor in the downstream use of hMSC as non-uniform samples and variation in function impairs efficacy, outcome and application of these advancements. In an effort to improve hMSC bioprocessing protocols, this study focused on the analysis and modulation of hMSC stress response following biopreservation. Two biopreservation stress regimes (hypothermic storage and hypoxic normothermic storage) were utilized to examine cell stress pathway activation. Specifically the unfolded protein response (UPR) was examined as a mediator of post-storage stress response given its recent link to preservation failure in other cell systems. Additionally, the incorporation of two chemical agents (i.e. salubrinal and resveratrol) during storage was conducted to examine the effect of targeted molecular modulation. The results of these studies demonstrated a differential response of hMSC to targeted modulation dependent upon storage temperature and carrier medium. The addition of resveratrol to MSCGM (complete growth media), HBSS (balanced salt solution) or ViaSpan (UW solution) yielded marked improvement to immediate post-storage viability (increases of 53%, 50%, and 10%, respectively) and regrowth of hMSC following hypothermic storage. Interestingly, salubrinal addition yielded modest improvement to MSCGM and HBSS stored hMSC survival (16% and 8% increases) while its addition to ViaSpan resulted in decreased viability and impaired ability to repopulate. In contrast, use of these modulators during hypoxic normothermic storage in either MSCGM or HBSS yielded negative impacts as growth inhibition or cell loss was observed in the samples. Further in-depth analyses revealed changes to both apoptotic and necrotic populations post-storage through the use of these modulators. Western blot analysis confirmed changes in the apoptotic signaling pathways as well as implicated the involvement of the UPR pathway signaling following storage. These findings illustrate the potential for storage specific cell stress pathway modulation to improve hMSC biopreservation. Further, the differential response of pathway modulation demonstrates that a more in-depth understanding of hMSC response is needed to improve each step throughout the biopreservation process (pre-storage processing, preservation and post-preservation recovery) to enable the delivery of optimal hMSC samples.

Functional evaluation of human donation after cardiac death donor hearts using a continuous isolated myocardial perfusion technique: Potential for expansion of the cardiac donor population

To investigate the resuscitation potential and contractile function in adult human donation after cardiac death (DCD) hearts by ex vivo perfusion. With institutional review board approval and under the DCD protocol at the University of Wisconsin (UW) Organ Procurement Organization, 5 brain dead (BD) and 5 DCD donor hearts were evaluated. All BD hearts were declined for clinical transplantation because of coronary artery disease, advanced age, or social history. All hearts were preserved by flushing and cold storage with UW solution. By using our ex vivo perfusion system, the left ventricular end systolic pressure-volume relationship (LV-ESPVR) was assessed for 2 hours of oxygenated blood reperfusion. All BD (n = 5) and 4 DCD hearts were successfully resuscitated. One DCD heart was unable to be resuscitated due to prolonged warm ischemic time (WIT; 174 minutes). Mean WIT for resuscitated DCD hearts (from extubation to flushing with cold UW solution) was 34 ± 3 minutes (range, 26 to 40 minutes); mean cold ischemic time for BD donors was 211 ± 31 minutes compared with 177 ± 64 minutes for DCD donors. The calculated LV-ESPVRs for BD hearts after 1 and 2 hours of reperfusion were 6.9 ± 0.7 and 5.7 ± 1.0 mm Hg/mL, respectively; LV-ESPVRs for DCD hearts after 1 and 2 hours of reperfusion were 5.6 ± 1.5 (P = .45) and 3.0 ± 0.7 mm Hg/mL (P = .07), respectively. We successfully resuscitated and measured ex vivo cardiac function in human DCD and BD donor hearts. Resuscitation potential in DCD hearts was achieved when the WIT was less than 40 minutes. Contractile performance in DCD hearts tended to be lower compared with BD hearts. Further investigation with longer reperfusion periods seems warranted.

Use of IGL–1 Preservation Solution in Liver Transplantation

University of Wisconsin (UW) solution has been known as the standard solution for liver graft preservation. Alternative preservation solutions have been used in liver transplantation, such as histidine-tryptophan-ketoglutarate (HTK) and Celsior solution. Institut Georges Lopez-1 (IGL--1) is a new preservation solution with lower potassium and lower viscosity than UW solution that has recently been used in liver transplant. Data from 178 patients who received transplants from August 2008 to June 2013 at Hospital Santa Isabel, Blumenau, Brazil, were analyzed. All patients received grafts from brain death donors. In November 2011 we started to use IGL--1 as an alternate preservation solution. Therefore, 53 patients using IGL--1 preserved grafts were compared to 125 using HTK solution. The donor age in the HTK group ranged from 11–77 years, with a mean of 43.4 ± 4.8. In the IGL--1 group donor age ranged from 9–62 years, with a mean of 35.8 ± 4.5. Cold ischemia time in the HTK group ranged from 85–1145 minutes, mean 443.5 ± 183.5 minutes. In the IGL--1 group, cold ischemia time ranged from 85–670 minutes, mean 329.3 ± 134.8 minutes. The overall operative mortality rate was 14% (25 patients); in the HTK group, 14.4% (18 patients); and in the IGL--1 group, 13.4% (7 patients). One graft in the HTK group presented with primary non-function (PNF), 0.7%; there were none in the IGL--1 group. In our study, IGL--1 has been shown to be safe to use as a preservation solution for liver transplantation. Early post-transplant graft function was comparable to that observed with HTK solution, although a tendency for lower alanine aminotransferase levels was noticed. IGL--1 has been shown to be safe, cost efficient, and an effective preservation solution.

Cold Storage of Rat Hepatocyte Spheroids

Cell-based therapies for liver disease rely on a high-quality supply of hepatocytes and a means for storage during transportation from site of isolation to site of usage. Unfortunately, frozen cryopreservation is associated with unacceptable loss of hepatocyte viability after thawing. The purpose of this study was to optimize conditions for cold storage of rat hepatocyte spheroids without freezing. Rat hepatocytes were isolated by a two-step perfusion method; hepatocyte spheroids were formed during 48 h of rocked culture in serum-free medium (SFM). Spheroids were then maintained in rocked culture at 37 °C (control condition) or cold stored at 4 °C for 24 or 48 h in six different cold storage solutions: SFM alone; SFM + 1 mM deferoxamine (Def); SFM + 1 μM cyclosporin A (CsA); SFM + 1 mM Def + 1 μM CsA, University of Wisconsin (UW) solution alone, UW + 1 mM Def. Performance metrics after cold storage included viability, gene expression, albumin production, and functional activity of cytochrome P450 enzymes and urea cycle proteins. We observed that cold-induced injury was reduced significantly by the addition of the iron chelator (Def) to both SFM and UW solution. Performance metrics (ammonia detoxification, albumin production) of rat hepatocyte spheroids stored in SFM + Def for 24 h were significantly increased from SFM alone and approached those in control conditions, while performance metrics after cold storage in SFM alone or cold storage for 48 h were both significantly reduced. A serum-free medium supplemented with Def allowed hepatocyte spheroids to tolerate 24 h of cold storage with less than 10% loss in viability and functionality. Further research is warranted to optimize a solution for extended cold storage of hepatocyte spheroids.

Absence of Allograft TLR4 Receptor Signaling Attenuates the Increased Early Inflammation in Allografts Subjected to Prolonged Cold Ischemic Storage.

Endogenous memory CD8 T cells infiltrate MHC-mismatched cardiac allografts and produce IFN-γ in response to donor class I MHC within 24 hrs after graft reperfusion in mice. Our recent studies have indicated that prolonged cold ischemic storage provokes intense inflammation within hours after allograft reperfusion and promotes endogenous memory CD8 T cell rejection of the allograft. This study tested the role of graft TLR4 signaling in the early post-transplant inflammation and downstream alloimmune response to allografts subjected to prolonged cold ischemic storage. Wild type (WT) BALBc (H-2d) or BALB/c.TLR4-/-hearts were subjected to 8 hours of cold ischemic storage in University of Wisconsin solution and then transplanted to C57BL/6 (H-2b) mice. CD4 and CD8 T cell, macrophage, and neutrophil infiltration into allografts on day 2 post-transplant was assessed by flow cytometry and immunohistochemistry. Levels of mRNA encoding inflammatory cytokines were measured by qPCR. Marked decreases in neutrophil, but not CD4 and CD8 T cell and macrophage, infiltration were observed in TLR4-/- vs. WT BALB/c allografts on day 2 post-transplant. This decrease was associated with marked decreases in the intragraft mRNA expression of the neutrophil chemoattractants CXCL1 and CXCL2 on day 2. The absence of allograft TLR4 signaling did not affect intragraft mRNA expression of CXCL10 and the acute phase cytokines, TNF-α and IL-1β. On day 7, TLR4-deficient allografts had virtually no infiltrating CD4 T cells, macrophages or neutrophils and decreased CD8 T cell infiltration that was accompanied by decreases in mRNA expression in perforin, granzyme B and FasL. These data indicate that the absence of TLR4 signaling in allografts subjected to prolonged cold ischemic storage inhibits the early post-reperfusion inflammation and inflammation at times post-transplant when wild-type allografts are undergoing acute rejection. The results suggest that therapeutic targeting of the TLR4 will reduce the negative impact of early inflammatory events on allograft outcome.

Soluble FGL2 Regulated Innate Immunity and Promoted Renal Self-Recovery After Ischemia Reperfusion Injury in a Porcine Kidney Auto-Transplantation Model.

Background Regulatory T cells (Treg) protect kidney against ischemia reperfusion (IR) injury via suppressing innate immunity, but the mechanism has not been fully clarified. Soluble fibrinogen-like protein 2 (sFGL2), a novel effector of Treg, may affect apoptosis and inflammation. This study investigated the role of sFGL2 in renal IR injury in a porcine kidney auto-transplantation model. Materials and methods The left kidney was retrieved from mini pigs and infused by University of Wisconsin solution into the renal artery with the renal artery and vein clamped for 24-h cold storage. After the right nephrectomy, the left kidney was auto-transplanted into the right for 2 weeks. 3 pigs were sacrificed at day 2, 5, 7, 10 and 14 post-transplantation respectively. Collected renal tissues and daily blood samples were stored for further analyses. Results Both serum creatinine and blood urea nitrogen were maximized during day 2 to 5 and followed by a gradual recovery over 2 weeks. The similar change trends were showed in histological damage, apoptosis and myeloperoxidase+ cells in the kidney, as well as circulating TNF-α and IFN-γ. Serum sFGL2 presented a fluctuating increase and reached a peak at day 10. The expression of sFGL2 and its receptor FcγRIIB in the kidney was notably increased from day 5 to 10. Conclusion The raised sFGL2 together with FcγRIIB during renal recovery after IR injury regulated innate immunity in terms of apoptosis and inflammation. Therefore, sFGL2 might be a potential renoprotective mediator that promoted renal self-repairing and remodeling in this 2-week porcine auto-transplantation model.Figure: No Caption available.

A Novel Hyperbranched Polyglycerol-Based Solution for Donor Organ Preservation: A Comparison With University of Wisconsin Solution in Hypothermic Preservation of Mouse Donor Hearts.

A Novel Hyperbranched Polyglycerol-Based Solution for Donor Organ Preservation: A Comparison With University of Wisconsin Solution in Hypothermic Preservation of Mouse Donor Hearts.

The of Institute George Lopez (IGL-1) Solution in Liver Graft Preservation - Experience With 100 Liver Transpant in a Single Center.

University of Wisconsin solution has been known as the standard solution for liver graft preservation. Alternative preservation solutions have been used in liver transplantation, such as histidine-tryptophan-ketoglutarate (HTK, CustodiolTM) and Celsior solution. Institut Georges Lopez-1 (IGL-1) is a new preservation solution with lower potassium and lower viscosity than UW solution that has recently been used in liver transplant. Data was collected from 100 consecutive liver transplants performed from January 2012 through December 2013 at Hospital Santa Isabel, Blumenau, Brazil. Liver grafts were preserved with IGL-1 solution. All patients were adults and received whole grafts from brain death donors. The mean donor age was 38.0±16.3 years. Cold ischemia time was 361±151 minutes. The mean recipient age was 52.3±11.6 years. The MELD score was 19.19±8.87. First day post-transplant liver enzymes, ALT and AST, levels were 593.5±66 U/L and 665.5±64.2 U/L. None patient presented primary non-function (PNF). In our study, IGL-1 has been shown to be a safe alternative as preservation solution for liver transplantation.

Alpha-1-Antitrypsin Treatment Prevents Hepatic Ischemia-Reperfusion Injury in Mice.

Warm ischemia associated with non-heart beating donation negatively affects early graft function and long-term survival. Recent evidence shows that treatment with acute phase protein alpha-1-antitrypsine (A1AT) reduces cell death and inflammation in a kidney ischemia-reperfusion injury (IRI) model. For liver, protective effects of A1AT on IRI are unknown. We investigated if A1AT treatment protects against hepatic injury in vitro and in a mouse model of hepatic IRI. Methods The effect of human A1AT (Zemaira, CSL Behring, 2.5 mg/ml) on cell viability was tested on Huh7 cells (MTT assay). Both normothermic (37 °C) and hypothermic conditions (18h 4 °C medium or UW solution followed by 24h medium 37 °C) were tested. The effect of A1AT on hepatic IRI was tested in C57BL6 mice, subjected to partial hepatic warm ischemia for 75 min by clamping the left hepatic artery and vein. Mice were either sham operated without induction of hepatic IRI (n=3), or IV injected with PBS n=7) or 10 mg/kg A1AT (n=5) 10 min prior to IRI. Serum at 6 and 24h after IRI was tested for AST, ALT and LDH Results Huh7 viability was increased for A1AT treated cells (1.3x ctrl, p=0.009). Increased viability was even more pronounced after cooling and rewarming with both medium (1.8x ctrl, p=0.006) or UW (1.9x, p=0.019). In vivo, compared to sham operated mice, levels of AST, ALT and LDH were all significantly increased at 6 and 24h after warm hepatic IRI but less in A1AT mice (p≤0.05). In mice treated with A1AT the protective effects were most apparent 24h after IRI, with significant reduction in ALT (mean±SEM 4235±699 U/L vs. 1506±542.4) and LDH (9890±2476 vs. 460±378 U/L) (p<0.05). At peak of injury 6h after IRI, only ALT was significantly reduced (13583±1267 U/L PBS vs. 7620±1829 A1AT, p=0.042). Conclusion Treatment with A1AT shows protective effects in Huh7 cells and attenuates hepatic IRI in mice. This result warrants further research on using A1AT for preemptive treatment of donors during organ procurement or liver graft cold storage. (research support from CSL Behring)Figure: No Caption available.DISCLOSURES:Berger, M.: Employee, CSL Behring.

Endogenous Memory CD8 T Cells Exhibit Increased Early Proliferation in Cardiac Allografts Subjected to Prolonged Ischemia.

Introduction: Memory T cells induced to microbial infections provide protection to subsequent exposures but can have cross-reactivity with other antigens including allogeneic MHC molecules. High numbers of memory T cells with donor reactivity in human and non-human primate recipients prior to transplant are associated with poorer allograft survival and resistance to costimulatory blockade strategies given to induce long-term graft survival. Rodent transplant models have failed to replicate the detrimental impact of endogenous memory T cells on allograft survival and tolerance induction, raising questions about the sufficiency of these models to study this component of alloimmunity. Methods: A/J (H-2a) hearts were subjected to 0.5 or 8 h of cold ischemic storage in UW solution before being heterotopically transplanted to C57BL/6 (H-2b) recipients. BrdU (1 mg) was injected intraperitoneally on days 0 and +1 and graft infiltrating T cell proliferation was assessed on day 2 by flow cytometry. Results: We have recently shown a direct association between increasing duration of cold ischemic storage and numbers of early graft infiltrating memory CD8 T cells that markedly up-regulate effector functions and directly mediate costimulatory blockade resistant rejection of cardiac allografts in a mouse transplant model. Here we show that endogenous memory CD8 T cells exhibit increased early proliferation in grafts subjected to prolonged ischemia that is supported by memory CD4 T cells. Recipient depletion of CD4 T cells or treatment with MR1 (anti-CD40L) at transplant significantly decreased early memory CD8 T cell proliferation and extended survival of grafts subjected to prolonged cold ischemia. Conclusion: These studies provide novel insights into mechanisms underlying endogenous memory CD8 T cell mediated graft injury and the poor outcomes associated with cadaver donor grafts.

Graft Function and Incidence of Biliary Stenosis After Use of UW or IGL1 Preservation Solutions for Liver Transplantation.

Graft Function and Incidence of Biliary Stenosis After Use of UW or IGL1 Preservation Solutions for Liver Transplantation.

Removal of Donor HLA Class I Proteins with Papain: Translation for Possible Whole Organ Practices.

Papain is a protease with potential use in transplantation because of its capacity to remove HLA Class 1 proteins from donor organs. However, its proteolytic activity has not been studied under conditions suitable for use in preservation and perfusion of donor organs, namely under a temperature of 4°C and dissolution in Belzer UW solution. Under these two conditions, we first test whether papain is active against a test substrate (the milk protein casein) and then whether it is active against HLA-expressing peripheral blood lymphocytes (PBLs). We found that papain at a range of concentrations is nearly as active in cleaving the test substrate in Belzer UW solution as it is in distilled water. In distilled water, papain is as active in cleaving a test substrate at a temperature of 4°C as compared to its optimal temperature of 37°C, if the incubation time is extended from 10 minutes to three hours. This finding also holds true if papain is dissolved in Belzer UW solution. In PBLs, papain cleaved off HLA Class 1 proteins as effectively at 4°C as at 37°C, provided the incubation time was also extended to three hours. These findings suggest that papain has potential use in the transplant setting to eliminate donor organ HLA class 1 expression, a method to reduce their likelihood of immunological rejection.

Improved Islet Yield From UW Solution Preserved Pancreas With Dimethyl Fumarate Supplementation.

Background: One of the major obstacles in clinical islet transplantation is the insufficient number of viable islets derived from preserved pancreas. During pancreas preservation and islet isolation, islets suffer from hypoxia and oxidant stress. In responses to oxidative stress, Nuclear factor erythroid-derived 2 factor (Nrf2) promotes expression of genes encoding antioxidant and detoxifying enzyme. Dimethyl fumarate, a novel Nrf2 activator, is also an intermediate in the citric acid cycle used by cells to produce energy in the form of ATP. We hypothesized that supplementation of preservation solution with DMF may improve islet yield by protecting islet during pancreas preservation. Method: 8-week old male Sprague Dawley rats were used as pancreas donors. The common bile duct was cannulated and the pancreas was removed en bloc with duodenum and spleen and preserved in UW (UW group) or UW + DMF, 50uM (DMF group) at 4°C for 24 hours after flushing by same preservation solution via aorta. After 24 hour cold storage, the ADP/ATP ratio and expression of antioxidant proteins in pancreatic tissue were evaluated. Pancreas was then digested by collagenase and isolated rat islets were examined by assessing islet yield and cure rates after transplantation to diabetic nude mice. Results: The islet yield (IEQ) in DMF group was significantly higher than UW group (969.3 ± 273.7 vs 412 ± 80.8; respectively, P<0.05). The cure rate after transplantation in DMF group (57.1%) was similar with UW group (66.7%, P>0.05). Likewise the percentage of viable and 8-OHDG positive cells in the isolated islets were comparable in the two groups. After 24 hour preservation, the ADP/ATP ratio of pancreas in DMF group was 4-fold lower than in the UW group. Enhanced expressions of HO-1 and NQO1 of pancreas were detected in DMF group. Conclusion: UW supplementation with DMF significantly improved the islet yield compared with UW cold storage alone. The islet function and viability were comparable in both groups. Thus DMF may protect islets and reduce islet loss during preservation and isolation procedure by enhancing the citric acid cycle and antioxidant defense system.

Sub-normothermic preservation of donor hearts for transplantation using a novel solution, Somah: A comparative pre-clinical study

Hearts preserved ex vivo at extreme hypothermia (4°C) undergo time-dependent irreversible injury. Our studies using a novel solution, Somah, suggest that hearts are viably preserved at 21°C. In this study we evaluate the relative efficacy of Somah for preservation of hearts at 21°C when compared with the clinically used Celsior and University of Wisconsin (UWS) solutions. Porcine hearts arrested by cardioplegia at 21°C using Somah, Celsior or UWS solution were stored in the respective solutions at 21°C (n = 5) for 5 hours and then reperfused ex vivo for functional assessment. We assessed development of edema, cardiac tissue high-energy phosphate (HEP; ATP + creatine phosphate) levels and release of cardiac enzymes. Alterations in left ventricular wall thicknesses and functional parameters were examined by 2-dimensional (2D) echocardiography. Changes in myocardial oxygen consumption (MVO2) and lactate utilization were assessed at reperfusion. Heart weights were unaltered during 5-hour storage in all groups. After storage, HEP levels were 28.33 ± 5.51, 10.20 ± 2.78 and 5.92 ± 1.46 nmol/liter per milligram protein (p < 0.001) in the Somah, Celsior and UWS group hearts, respectively. Upon reanimation, 2D echocardiography showed edema in the Celsior and UWS hearts; prompt attainment of physiologic function was associated with rapid establishment of aerobic metabolism not requiring stimulatory interventions in the Somah hearts, but not in the Celsior/UWS hearts. Percent fractional area change, ejection fraction and stroke volume were significantly higher (p < 0.001) in Somah hearts than in Celsior and UWS group hearts. Increased synthesis of HEP, rapid metabolic switch and optimal function together provide evidence that hearts procured for transplantation are preserved in a superior viable condition at 21°C with Somah, but not with other commonly used clinical preservation solutions.