Peter Urbanellis

Identification of a novel biomarker gene set with sensitivity and specificity for distinguishing between allograft rejection and tolerance

Here we examined whether the expression of a novel immunoregulatory gene set could be used to predict outcomes in murine models of rapamycin‐induced cardiac tolerance, spontaneous hepatic tolerance, and cardiac rejection. The expression of the immunoregulatory gene set was assessed with the GeXP multiplex reverse‐transcription polymerase chain reaction (RT‐PCR) analysis system, and it was correlated to the pathological and biochemical parameters of the allografts. In rejecting cardiac grafts, the increased expression of an inflammatory set of genes, which included CD45, CD4, CD25, suppressor of cytokine signaling 2, cytotoxic T lymphocyte–associated protein 4 (CTLA4), selectin lymphocyte, interferon‐γ (IFN‐γ), programmed cell death 1 (Pdcd1), and granzyme B (Gzmb), was seen 8 days after transplantation along with histological evidence of severe allograft rejection. In tolerant cardiac allografts, the expression of fibrinogen‐like protein 2 (Fgl2), Pdcd1, killer cell lectin‐like receptor G1 (Klrg1), CTLA4, and lymphocyte‐activation gene 3 was associated with tolerance. In a model of liver allograft tolerance, the increased expression of lectin galactose‐binding soluble 1, Fgl2, CD39, phosphodiesterase 3B, Klrg1, forkhead box P3 (Foxp3), and transforming growth factor β as well as the inflammatory set of genes was observed 8 to 14 days after transplantation (ie, when there was severe inflammatory injury). At a later time when the liver allografts had been fully accepted and were histologically normal, the expression of the inflammatory set of genes returned to the baseline, but the expression of the tolerogenic set of genes was still increased. Genes that were expressed in tolerant cardiac and liver allografts included Fgl2, Klrg1, and Foxp3, whereas genes associated with rejection included CD25, Gzmb, and IFN‐γ. Our data indicate that monitoring the graft expression of a novel biomarker gene set with the GeXP multiplex RT‐PCR analysis system may allow differentiation between rejection and tolerance. Liver Transpl 18:444–454, 2012.

The regulatory T cell effector molecule fibrinogen‐like protein 2 is necessary for the development of rapamycin‐induced tolerance to fully MHC‐mismatched murine cardiac allografts

Therapies that promote tolerance in solid organ transplantation will improve patient outcomes by eliminating the need for long‐term immunosuppression. To investigate mechanisms of rapamycin‐induced tolerance, C3H/HeJ mice were heterotopically transplanted with MHC‐mismatched hearts from BALB/cJ mice and were monitored for rejection after a short course of rapamycin treatment. Mice that had received rapamycin developed tolerance with indefinite graft survival, whereas untreated mice all rejected their grafts within 9 days. In vitro, splenic mononuclear cells from tolerant mice maintained primary CD4+ and CD8+ immune responses to donor antigens consistent with a mechanism that involves active suppression of immune responses. Furthermore, infection with lymphocytic choriomeningitis virus strain WE led to loss of tolerance suggesting that tolerance could be overcome by infection. Rapamycin‐induced, donor‐specific tolerance was associated with an expansion of regulatory T (Treg) cells in both the spleen and allograft and elevated plasma levels of fibrinogen‐like protein 2 (FGL2). Depletion of Treg cells with anti‐CD25 (PC61) and treatment with anti‐FGL2 antibody both prevented tolerance induction. Tolerant allografts were populated with Treg cells that co‐expressed FGL2 and FoxP3, whereas rejecting allografts and syngeneic grafts were nearly devoid of dual‐staining cells. We examined the utility of an immunoregulatory gene panel to discriminate between tolerance and rejection. We observed that Treg‐associated genes (foxp3, lag3, tgf‐β and fgl2) had increased expression and pro‐inflammatory genes (ifn‐γ and gzmb) had decreased expression in tolerant compared with rejecting allografts. Taken together, these data strongly suggest that Treg cells expressing FGL2 mediate rapamycin‐induced tolerance. Furthermore, a gene biomarker panel that includes fgl2 can distinguish between rejecting and tolerant grafts.

Targeted deletion of FGL2 leads to increased early viral replication and enhanced adaptive immunity in a murine model of acute viral hepatitis caused by LCMV WE.

Mounting effective innate and adaptive immune responses are critical for viral clearance and the generation of long lasting immunity. It is known that production of inhibitory factors may result in the inability of the host to clear viruses, resulting in chronic viral persistence. Fibrinogen-like protein 2 (FGL2) has been identified as a novel effector molecule of CD4+CD25+ Foxp3+ regulatory T (Treg) cells that inhibits immune activity by binding to FCγRIIB expressed primarily on antigen presenting cells (APC). In this study, we show that infection of mice with Lymphocytic Choriomeningitis Virus WE (LCMV WE) leads to increased plasma levels of FGL2, which were detected as early as 2 days post-infection (pi) and persisted until day 50 pi. Mice deficient in FGL2 (fgl2−/−) had increased viral titers of LCMV WE in the liver early p.i but cleared the virus by day 12 similar to wild type mice. Dendritic cells (DC) isolated from the spleens of LCMV WE infected fgl2−/− had increased expression of the DC maturation markers CD80 and MHC Class II compared to wild type (fgl2+/+). Frequencies of CD8+ and CD4+ T cells producing IFNγ in response to ex vivo peptide re-stimulation isolated from the spleen and lymph nodes were also increased in LCMV WE infected fgl2 −/− mice. Increased frequencies of CD8+ T cells specific for LCMV tetramers GP33 and NP396 were detected within the liver of fgl2−/− mice. Plasma from fgl2−/− mice contained higher titers of total and neutralizing anti-LCMV antibody. Enhanced anti-viral immunity in fgl2−/− mice was associated with increased levels of serum alanine transaminase (ALT), hepatic necrosis and inflammation following LCMV WE infection. These data demonstrate that targeting FGL2 leads to early increased viral replication but enhanced anti-viral adaptive T & B cell responses. Targeting FGL2 may enhance the efficacy of current anti-viral therapies for hepatotropic viruses.

PPAR-gamma activation is associated with reduced liver ischemia-reperfusion injury and altered tissue-resident macrophages polarization in a mouse model

Background PPAR-gamma (γ) is highly expressed in macrophages and its activation affects their polarization. The effect of PPAR-γ activation on Kupffer cells (KCs) and liver ischemia-reperfusion injury (IRI) has not yet been evaluated. We investigated the effect of PPAR-γ activation on KC-polarization and IRI. Materials and methods Seventy percent (70%) liver ischemia was induced for 60mins. PPAR-γ-agonist or vehicle was administrated before reperfusion. PPAR-γ-antagonist was used to block PPAR-γ activation. Liver injury, necrosis, and apoptosis were assessed post-reperfusion. Flow-cytometry determined KC-phenotypes (pro-inflammatory Nitric Oxide +, anti-inflammatory CD206+ and anti-inflammatory IL-10+). Results Liver injury assessed by serum AST was significantly decreased in PPAR-γ-agonist versus control group at all time points post reperfusion (1hr: 3092±105 vs 4469±551; p = 0.042; 6hr: 7041±1160 vs 12193±1143; p = 0.015; 12hr: 5746±328 vs 8608±1259; p = 0.049). Furthermore, liver apoptosis measured by TUNEL-staining was significantly reduced in PPAR-γ-agonist versus control group post reperfusion (1hr:2.46±0.49 vs 6.90±0.85%;p = 0.001; 6hr:26.40±2.93 vs 50.13±8.29%; p = 0.048). H&E staining demonstrated less necrosis in PPAR-γ-agonist versus control group (24hr:26.66±4.78 vs 45.62±4.57%; p = 0.032). The percentage of pro-inflammatory NO+ KCs was significantly lower at all post reperfusion time points in the PPAR-γ-agonist versus control group (1hr:28.49±4.99 vs 53.54±9.15%; p = 0.040; 6hr:5.51±0.54 vs 31.12±9.58%; p = 0.009; 24hr:4.15±1.50 vs 17.10±4.77%; p = 0.043). In contrast, percentage of anti-inflammatory CD206+ KCs was significantly higher in PPAR-γ-agonist versus control group prior to IRI (8.62±0.96 vs 4.88 ±0.50%; p = 0.04). Administration of PPAR-γ-antagonist reversed the beneficial effects on AST, apoptosis, and pro-inflammatory NO+ KCs. Conclusion PPAR-γ activation reduces IRI and decreases the pro-inflammatory NO+ Kupffer cells. PPAR-γ activation can become an important tool to improve outcomes in liver surgery through decreasing the pro-inflammatory phenotype of KCs and IRI.

Comparison of Continuous Normothermic Ex Vivo Kidney Perfusion to Dynamic and Static Hypothermic Preservation Techniques in Porcine Kidneys Donated after Cardiac Death

Background Strategies to decrease preservation-related injury in renal grafts donated after circulatory death (DCD) urgently needed. This highlights the importance of direct comparison of dynamic preservation technologies for kidneys. Our prior work has suggested superior graft function of continuous normothermic ex vivo kidney perfusion (NEVKP) over static cold storage. Here we investigated whether NEVKP could promote functional recovery compared to hypothermic machine perfusion (HMP). Methods 15 porcine kidneys were exposed to 30min of warm ischemia, then subjected to either 16hrs of SCS, 16hrs of HMP (LifePort® 1.0) or 16hrs of NEVKP prior to autotransplantation (n=5 each group). Animals were followed for 8 days. Graft function and histology were assessed. Results Grafts preserved by NEVKP demonstrated improved graft function, and more rapid graft recovery, with the mean peak serum creatinine of 3.66 ± 1.33mg/dl, occurring on day 1, compared with 8.82 ± 3.17mg/dl in the HMP group, occurring on day 2, and 10.5 ± 5.38mg/dl in the SCS group, occurring on day 3. Differences between daily serum creatinine levels reached significance between NEVKP and HMP on day 1 (p=0.002), day 2 (p=0.004) and day 3 (p=0.024), and between HMP and SCS on day 3 (p=0.016) and day 4 (p=0.046). Injury scores [scaled from 0 to 3] were lower in both perfused groups (NEVKP, HMP): score: 1 (1-1.5) compared to SCS group: score: 1.5(1-3) in wedge biopsies taken on postoperative day 8, however this did not reach statistical significance (p>0.05). Similarly, inflammation scores were not statistically different between any groups. Figure. No caption available. Conclusion In this DCD model of renal autotransplantation, we demonstrated that NEVKP significantly improved initial graft function compared to either HMP or SCS. Thus, normothermic perfusion may provide superior preservation options for DCD renal grafts than conventional hypothermic methods. The biologic mechanisms underlying normothermic preservation present new opportunities to advance these findings.

Normothermic Ex-vivo Kidney Perfusion Improves Function of Marginal Renal Grafts that were Subjected to Prolonged Ischemia Prior to Preservation

Background Normothermic ex-vivo kidney perfusion (NEVKP) is an emerging technique for renal graft preservation. We investigated whether NEVKP could promote improved marginal graft function compared to cold storage in a model of donation after cardiac death. Methods Kidneys from 30kg Yorkshire pigs were removed following 30, 60, 90, or 120 minutes of warm ischemia (WI). These grafts were then preserved in either cold histidine-tryptophan-ketoglutarate solution (CS) or subjected to pressure-controlled NEVKP for 8 hours prior to heterotopic autotransplantation. Results Prolonging WI time prior to kidney retrieval and subsequent storage in CS resulted in grafts that demonstrated incremental posttransplant increases in serum creatinine with grafts subjected to 120min of WI having persistent elevation (POD7: 13.45±3.50mg/dl vs baseline: 1.1±0.33mg/dl p<0.01, n=4). During NEVKP perfusion, 120min WI grafts cleared lactate from perfusion solution (0hr: 10.48±0.93mmol/L vs 7hr: 1.48±0.85mmol/L, p<0.01, n=5), had decreasing intra-renal resistance (0hr: 2.26±0.9mmHg/mL/min vs 7hr: 0.37±0.6mmHg/mL/min, p<0.01), and continuous urine production. Posttransplantation, 120min WI grafts with NEVKP, compared to CS, demonstrated significantly decreased serum creatinine peak values (POD4: 12.62±2.34mg/dl vs POD5: 18.95±1.11mg/dL, p=0.001) and higher creatinine clearance (POD4: 6.61±4.03mL/min vs 0.35±0.30mL/min, p=0.02 and POD7: 26.31±11.54mL/min vs 9.78±4.6mL/min, p=0.03). On POD7, serum creatinine returned to baseline values in the NEVKP group (POD7: 4.88±5.57mg/dL vs baseline: 1.02±0.16mg/dL, p=0.16) but not the CS group (POD7: 13.45±3.50mg/dl vs baseline: 1.1±0.33mg/dl p<0.01, n=4). Histology from 120min WI NEVKP grafts at POD7 demonstrated decreased tubular injury scores compared to cold CS grafts (1.8+/-0.8 vs. 3.0+/-0.0, p=0.03) as assessed by a blinded pathologist. Conclusion Kidney grafts subjected to 120min of WI before retrieval showed significant improvement in function following 8hrs of continuous pressure-controlled NEVKP compared to CS. This suggests NEVKP could be utilized to expand the donor pool through the consideration of extreme marginal grafts for transplantation.

Normothermic Ex-Vivo Kidney Perfusion Restores the Genetic Profile of Marginal Kidney Grafts Subjected to Warm Ischemia

Background Increasing evidence demonstrates the superiority of normothermic ex-vivo kidney perfusion (NEVKP) over cold storage (CS) for grafts sensitive to ischemia-reperfusion injury, including kidneys procured following donation-after-cardiac-death (DCD). To account for this improvement, we investigated whether NEVKP-preservation returns the genetic profiles of DCD grafts to that of unmanipulated naïve kidneys. Methods Kidneys from 30kg Yorkshire pigs were removed following 30 minutes of warm ischemia in a model of DCD. These grafts were then stored in either static cold histidine-tryptophan-ketoglutarate solution (CS) or subjected to pressure-controlled NEVKP for 8 hours prior to heterotopic autotransplantation. Kidney biopsies were collected on POD3 and gene expression was compared with naïve porcine kidneys utilizing the Affymetrix GeneChip® Porcine Gene 1.0 ST Array platform examining over 23,000 transcripts. Validation was performed using quantitative real-time polymerase chain reaction. Results During NEVKP storage, DCD grafts demonstrated favourable perfusion characteristics including progressive lactate clearance (0hr: 10.29 +/-0.48 mmol/L vs 8hr: 1.67+/-0.67 mmol/L, n=5, P<0.01), decreasing intra-renal resistance (0hr:1.63+/-0.20 mmHg/mL/min vs 8hr:0.41+/-0.13 mmHg/mL/min, n=5, p<0.01), and continuous urine production. Post-transplantation graft function significantly improved with NEVKP compared to CS with decreased peak serum creatinine (POD1: 4.0+/-1.15mg/dL vs POD3: 12.0+/-0.78mg/dL, n=5, p<0.01) and higher creatinine clearance on POD3 (39.6+/-11.8mL/min vs 2.6+/-0.9ml/min, n=5, p<0.01). Genomic comparison of grafts subjected to NEVKP on POD3 showed significant differences in only 27 genes when compared to naïve porcine kidneys (Table 1, >±2-fold changes in expression over naïve, n=3, P<0.05, FDR p-value<0.20). In contrast, 668 genes were significantly differentially expressed between grafts stored with CS on POD3 and naïve kidneys (Table 2, >±2-fold changes in expression over naïve, n=3, P<0.05, FDR p-value<0.20). Table. No title available. Table. No title available. Conclusions NEVKP restored damaged kidney grafts in a model of DCD with a genomic profile closely resembling naïve kidneys. Conversely, ongoing injury was evident in DCD grafts following CS through increased expression of genes related to inflammation, apoptosis, and repair. Together, these findings support the use of NEVKP for storage of DCD grafts that are more susceptible to ischemia-reperfusion injury.

Normothermic ex vivo kidney perfusion for graft quality assessment prior to transplantation

Normothermic ex vivo kidney perfusion (NEVKP) represents a novel approach for graft preservation and functional improvement in kidney transplantation. We investigated whether NEVKP also allows graft quality assessment before transplantation. Kidneys from 30‐kg pigs were recovered in a model of heart‐beating donation (group A) after 30 minutes (group B) or 60 minutes (group C) (n = 5/group) of warm ischemia. After 8 hours of NEVKP, contralateral kidneys were resected, grafts were autotransplanted, and the pigs were followed for 3 days. After transplantation, renal function measured based on peak serum creatinine differed significantly among groups (P < .05). Throughout NEVKP, intrarenal resistance was lowest in group A and highest in group C (P < .05). intrarenal resistance at the initiation of NEVKP correlated with postoperative renal function (P < .001 at NEVKP hour 1). Markers of acid‐base homeostasis (pH, HCO3–, base excess) differed among groups (P < .05) and correlated with posttransplantation renal function (P < .001 for pH at NEVKP hour 1). Similarly, lactate and aspartate aminotransferase were lowest in noninjured grafts versus donation after circulatory death kidneys (P < .05) and correlated with posttransplantation kidney function (P < .001 for lactate at NEVKP hour 1). In conclusion, assessment of perfusion characteristics and clinically available perfusate biomarkers during NEVKP allows the prediction of posttransplantation graft function. Thus, NEVKP might allow decision‐making regarding whether grafts are suitable for transplantation.

FGL2 EXPRESSION INDUCES GRAFT SURVIVAL IN THE ABSENCE OF IMMUNOSUPPRESSION: 2066

A. Bartczak1, W. He2, I. Shalev2, M. Mendicino2, J. Zhang2, X. Ma2, P. Urbanellis3, R. Khattar2, E. Kaplovitch2, M.J. Phillips4, O.A. Adeyi4, D.R. Grant4, G.A. Levy4 1Institute Of Medical Science, University of Toronto, Toronto/ ON/CANADA, 2University Of Toronto Transplantation Institute, University Health Network, Toronto/CANADA, 3University Of Toronto Transplantation Institute, University Health Network, Toronto/Ontario/ CANADA, 4Multiorgan Transplant Program, Toronto General Hospital, Toronto/ON/CANADA