Organ donation and ethics - Nature

Immunology and Cell Biology (2014) 92, A1–A29 & 2014 Australasian Society for Immunology Inc. All rights reserved 0818-9641/14 www.nature.com/icb

ABSTRACTS

Organ donation and ethics Immunology and Cell Biology (2014) 92, A1–A29; doi:10.1038/icb.2014.64

COMPARATIVE SURVIVAL BENEFITS AND COSTS OF AN EPLET-BASED AND BROAD-ANTIGEN MATCHING SYSTEM IN DECEASED DONOR KIDNEY ALLOCATION AMONG INDIGENOUS AUSTRALIANS Do Nguyen Hung1, Wong Germaine2,3, Fidler Samantha4,5, Chapman Jeremy R3, Craig Jonathan C2,3, Ferrari Paolo1,6, D’Orsogna Lloyd4,5, Irish Ashley1,7, Lim Wai1,8

time with improved health benefits without disadvantaging nonindigenous recipients.

Table 1 Comparison of the outcomes of the simulated Eplet-based allocation and cost-effectiveness analyses. Data are in number (and %) or mean (and standard deviation)

1School

of Medicine & Pharmacology, University of Western Australia, Perth, 2School of Public Health, University of New South Wales, Sydney, 3Centre for Kidney Research, The Children’s Hospital at Westmead, Sydney, 4School of Pathology and Laboratory Medicine, University of Western Australia, Perth, 5Department of Clinical Immunology, Royal Perth Hospital, 6Department of Renal Medicine, Fremantle Hospital, WA, 7Department of Renal Medicine, Royal Perth Hospital, 8Department of Renal Medicine, Sir Charles Gairdner Hospital, Perth

Indigenous recipients with

Current

0-2 eplet

o10 eplet

allocation

cut-off

cut-off

—

6 (18%)

23 (68%)

reduction in waiting time (n) Waiting time (months) Actual

65 (25)

52 (24)

53 (26)

Simulation

—

32 (22)

32 (25)

Reduction

—

19 (22)

-22 (17)

—

9

39

Reallocated recipients included (n) Waiting time (months)

Background: The overall waiting time for transplantation among indigenous Australians is at least six times longer than the non-indigenous Australians with end-stage kidney disease. Such disparities are largely attributed to the HLA-antigen mismatch between donors and potential indigenous transplant candidates. Structural matching at the epitope level known as eplets, particularly at the HLA-DR locus may provide a more accurate assessment of immunological risk compared to HLA matching at the broad antigen level. Aims: To compare the benefits and costs of incorporating an Epletbased matching algorithm with the current allocation algorithm for deceased donor kidney allocation in indigenous Australians. Methods: Using deterministic decision analytical and simulation modelling, we compared the average waiting time for transplantation, the overall survival gains (in life years saved [LYS] and quality-adjusted life years [QALYs] gained) and the costs of integrating an Eplet-based allocation for deceased donor kidneys compared with the current allocation algorithm. The Eplet-based model simulated allocation of kidneys to indigenous recipients focusing only on HLA-DR eplet mismatches using predefined cut-offs of r2 and o10 eplet mismatches. Results: The average waiting time for transplantation reduced by an average of 23 (SD¼22) and 22 (SD¼17) months using the eplet-based allocation algorithm with r2 and o10 HLA-DR eplet cut-offs respectively. The average gain in QALY using the eplet-based algorithm varied between 0.01 and 0.05 QALYs, with average savings of $644 to $6,624 depending on the specified threshold. There was a small consequential loss of up to 0.01 QALYs and extra costs of $368 for non-indigenous Australians. Conclusions: Alternative allocation for indigenous kidney transplant recipients are associated with a reduction in transplant waiting

Actual

41 (32)

47 (27)

Simulation

56 (29)

54 (31)

56 (25)

Additional

13 (13)

9 (8)

7.59

7.60

7.64

—

0.01

0.05

$521,632

$520,042

$515,008

—

$1,590

$6,624

Cost-effectiveness analysis Indigenous recipient Total benefit (QALY gained) Incremental benefit (QALY gained) Total cost ($) Incremental cost ($) Non-indigenous recipients Total benefit (QALY gained) Incremental benefit (QALY gained) Total cost ($) Incremental cost ($)

9.27

9.27

9.26

—

o 0.01

0.01

$311,547

$311,901

$311,915

—

$354

$368

DCD PANCREAS TRANSPLANTATION – AN UNDERUTILISED RESOURCE IN AUSTRALIA Jameson Carolyn1,2, Rajkumar T3, Kable K3, Yuen L1, Ryan B1, Lam V1,2, Hawthorne W1,2,4, Allen R1,2, Pleass H1,2 of Surgery, Westmead Hospital, Sydney, 2University of Pancreas Transplant Unit, Westmead Hospital, Sydney, 4Westmead Millennium Institute, Westmead Hospital, Sydney 1Department

Sydney, 3Renal &

Aims: To review patient and allograft outcomes following simultaneous pancreas/kidney (SPK) transplantation from DCD donors. Methods: Our prospective transplant database (1987-current) was used to identify SPK transplant recipients from DCD donors and outcomes of both recipients and allografts were examined.

Abstracts A2

Results: Three individual DCD donor SPK transplant recipients were identified. Transplants were performed in 2007, 2012 and 2014. All recipients had Type I diabetes and diabetic nephropathy. Median donor age was 17 years (range 12–18 years) and median recipient age was 34 years (range 27–44 years). Average duration of dialysis prior to transplantation was 16.3 months (range 8–29 months). HLA mismatch for each patient was; 0/6, 5/6, and 4/6. Cold ischaemic time was 8 hours 32 mins (range 6 hrs 53 mins to 9 hrs 24 mins) for kidney and 11 hours 13 mins (range 9 hrs 17 mins - 12 hrs 54 mins) for pancreas. Warm ischaemic time was 18.7 mins (range 14–28 mins). There was no delayed pancreatic allograft function. However 2 of 3 recipients required renal dialysis. To date, all patients are alive, 3 of 3 pancreatic allografts are functioning and 2 of 3 renal allografts. Conclusions: Our results are in keeping with those in the published DCD literature. We have a history of successfully transplanting DCD renal allografts since 1995 and have now successfully transplanted simultanous pancreas and kidney allografts from 3 DCD donors. We believe this is a potentially underutilised resource in Australia.

Results: We identified five major themes: justifying donor sacrifice (ensuring reasonable graft survival, warranting adherence, reciprocal donor benefits, perceived urgency); championing recipient outcomes (responsibility for patient advocacy, maximizing survival, earning trust and confidence); cognizance of boundaries (avoiding coercion, guarding against external pressure, minimizing conflict of interest, emphasizing patient accountability, maintaining center performance); entrenched inequalities (self-perpetuating disadvantage, exclusivity of suitable donors, selective recommendations); contending with infrastructural inadequacy (limited transparency, geographical disadvantage, frustrating inefficiency, visibility of transplantation). Conclusions: There is substantial variability in the attitudes and preferences of nephrologists regarding recipient eligibility, which may explain some of the differences in living donor kidney transplantation rates. To target inequalities in access to living donor transplantation an explicit pathway for patient education and referral to transplant services and comprehensive guidelines, which include recommendations on complex medical and psychosocial considerations, might promote transparent decision-making.

Random Fasting BSL for each patient 10

PATIENTS’ ATTITUDES TOWARDS LIVING KIDNEY DONATION: SYSTEMATIC REVIEW AND THEMATIC SYNTHESIS OF QUALITATIVE RESEARCH

BSL (mmol/l)

8

Hanson Camilla1,2, Chadban Steven3,4, Chapman Jeremy5, Craig Jonathan1,2, Wong Germaine1,2,5, Ralph Angelique1,2, Tong Allison1,2

6

4

of Public Health, University of Sydney, 2Centre for Kidney Research, The Children’s Hospital at Westmead, Sydney, 3Department of Renal Medicine, Royal Prince Alfred Hospital, Sydney, 4School of Medicine, University of Sydney, 5Centre for Transplant and Renal Research, Westmead Hospital, Sydney 1School

2

0

0

1

3

12

24

36

48

60

72

84

Time since Transplant (months)

‘‘EVERYONE’S GOT THEIR OWN THRESHOLD.’’ NEPHROLOGISTS’ PERSPECTIVES ON PATIENT ELIGIBILITY AND ACCESS TO LIVING DONOR KIDNEY TRANSPLANTATION Hanson Camilla1,2, Chadban Steven3,4, Chapman Jeremy5, Craig Jonathan1,2, Wong Germaine1,2,5, Tong Allison1,2 1School of Public Health, University of Sydney, 2Centre for Kidney Research, The Children’s Hospital at Westmead, Sydney, 3Department of Renal Medicine, Royal Prince Alfred Hospital, Sydney, 4School of Medicine, University of Sydney, 5Centre for Transplant and Renal Research, Westmead Hospital, Sydney

Aims: Wide variations in access to living kidney donation are apparent. We aimed to describe nephrologists’ beliefs and attitudes towards recipient eligibility and access to living donor kidney transplantation to determine if these may impact decisions regarding patient suitability for living kidney donor transplantation. Methods: Face-to-face semi-structured interviews were conducted with 41 nephrologists from 22 centers in Australian and New Zealand centres. Transcripts were analyzed thematically.

Immunology and Cell Biology

Aims: Living kidney donation offers superior outcomes over deceased organ donation, but incurs psychosocial and ethical challenges for recipients due to the risks imposed upon their living donor. We aimed to describe the beliefs, attitudes and expectations of patients with chronic kidney disease (CKD) towards living donor kidney transplantation. Methods: A comprehensive search for qualitative studies was conducted to February 2013, and identified 37 studies, involving 1769 participants. Thematic synthesis was used to analyse the findings. Results: We identified six major themes: prioritising own health (better graft survival, accepting risk, aversion to dialysis); guilt and responsibility (jeopardising donor health, anticipating donor regret, causing donor inconvenience); ambivalence and uncertainty (doubting transplant urgency, insufficient information, unfamiliarity, prognostic uncertainty); seeking decisional validation (a familial obligation, alleviating family burden, reciprocal benefits for donors, respecting donor autonomy, external reassurance, religious approval); needing social support (avoiding family conflict, unrelenting indebtedness, emotional isolation); and cautious donor recruitment (selfadvocacy, lacking self-confidence, avoiding donor coercion, emotional vulnerability, respecting religious and cultural taboos). Conclusions: Enhanced education and psychosocial support may help clarify, validate and address patients’ concerns. This may encourage informed decision-making, increase access to living kidney donation and improve psychosocial functioning after transplantation.

Abstracts A3 Cautious donor recruitment Self-advocacy Lacking self-confidence Emotional vulnerability Respecting cultural and religious taboos • Avoiding donor coercion • • • •

Prioritizing own health • Desperate aversion to dialysis • Better graft survival • Accepting risk

• • • • • •

Decisionalconflict

Seeking decisional validation A familial obligation Alleviating family burden Reciprocal benefits for donors Social considerations Respecting donor autonomy External reassurance Religious approval

Shared responsibility

Guilt and responsibility • Jeopardizing donor health • Anticipating donor regret • Causing donor inconvenience

Needing social support • Emotional isolation • Unrelenting indebtedness • Avoiding family conflict

• • • •

Key:

Ambivalence and uncertainty Doubting transplant urgency Insufficient information Confronted by unfamiliarity Prognostic uncertainty

Barriers and challenges

EXPERIENCES OF NEW ZEALAND’S LIVE LIVER DONORS AND THE ETHICAL IMPLICATIONS FOR INCREASING ORGAN DONATION

prior to death, and they prefer to retain an altruistic system over commercialism.

Gavin Claire

Cells, tissue and xenotransplantation

Philosophy Department, University of Auckland Aims: New Zealand’s organ donation rates are low by international standards (8pmp) and have remained static for many years. The general public express a willingness to donate that is not reflected in the 50% refusal rate by next of kin to donate in New Zealand Intensive Care Units. Live donors make up some of the shortfall but these donors undergo significant risks to do so. This study investigated for the first time the opinions of New Zealand’s live liver donors, in order to better understand how New Zealand could increase its organ donation rates. Methods: A questionnaire was sent to all 46 of the live liver donors we have had in New Zealand. It asked quantitative and qualitative questions about their experiences pre and post their operation and what motivated them to donate. As they are an informed and experienced group, the questionnaire also asked their opinions on: New Zealand’s current system, why others might refuse to donate organs, and what methods might encourage organ donation in New Zealand. Results: While the numbers are small (21), the results show little concern about pain after surgery (19%) or their own health outcomes (5%). 95% had pain afterwards but despite this all were satisfied with how the procedure went and 86% said they would do it again if possible. There was strong disagreement by participants that they were asked or persuaded to donate. Conclusions: Results indicate that New Zealand’s live liver donors approve of strategies that uphold individual consents made 100 80 60 40 20 0 education

payment

prevent family veto

change to opt out

Figure 1 What would encourage more organ donation?

choice of recipient

EARLY AND/OR LATE DEPLETION OF FOXP3+ REGULATORY T CELLS PREVENTS TOLERANCE TO NICC ISLET XENOGRAFTS INDUCED BY CO-STIMULATORY BLOCKADE Qian Yi Wen1,2, Hawthorne Wayne1, Phillips Peta1, Wu Jingjing1, Liuwantara David1, Burns Heather1, Nan Hai1, Chew Yi Vee1, Alexander Stephen3, Hu Min1, O’Connell Philip1 1Centre

for Transplant and Renal Research, Westmead Hospital, Sydney, of Medicine, University of Sydney, 3Centre for Kidney Research, Westmead Children’s Hospital 2Department

Aim: To determine the role of regulatory Tcells (Tregs) in the induction and maintenance of Tcell mediated tolerance to islet xenografts. Methods: C57BL6-DEREG mice were transplanted with pig NICC (neonatal islet cell cluster). DEREG mice have diphtheria toxin receptor-eGFP attached to their FoxP3 gene. Recipient mice were treated IP with CTLA-4 (500 ug) at day 0, and MR-1 (500 ug) at day 0/2/4/6. Tregs were depleted by diphtheria toxin (DT) (12 ng/gram/ mouse) IP at early stage (day -3) or late stage (day 70). Rejection was determined by histology and function by porcine c-peptide concentration. Results: Without DT, tolerance to NICC xenografts was achieved in DEREG mice with histology at 100-days, showing xenografts with intact islets and positive insulin staining and elevated porcine Cpeptide (mean¼0.44 ng/ml, n¼2). Histology at day 20 and day 90 from DT treated mice showed rejection of the xenograft. CD4-CD8B220+CD25+ Tcells in draining lymph nodes increased to 6.0% of CD4-CD8- Tcells by day 80 in the tolerant group, compared to 2.2% with early depletion group and 3.3% of late depletion group (P¼0.007). Conclusion: Treg depletion at both early or late time points prevents tolerance suggesting they may be important for both the induction and maintenance of tolerance to islet xenografts after co-stimulation blockade. Immunology and Cell Biology

Abstracts A4

GENETICALLY MODIFIED PORCINE NEONATAL ISLET XENOGRAFTS SURVIVE IN BABOONS AND CONTROL DIABETES Hawthorne Wayne J1,2, Hawkes Joanne2, Salvaris Evelyn3, Phillips Peta2, Liuwantara David2, Barlow Helen3, Robson Simon4, Brady Jamie5, Lew Andrew5, Chew Yi Vee2, Nottle Mark6, O’Connell Philip2,7, Cowan Peter3 1Discipline

of Surgery, Sydney Medical School, University of Sydney, for Transplant and Renal Research, Westmead Millennium Institute, Westmead Hospital, Sydney, 3Immunology Research Centre, St Vincent’s Hospital, Melbourne, 4Harvard Medical School, 5Walter and Eliza Hall Institute of Medical Research, Melbourne, 6Department of Obstetrics and Gynaecology, University of Adelaide, 7Discipline of Medicine, University of Sydney 2Centre

Aim: Transgenic expression of human complement regulatory proteins or deletion of aGal (GTKO) can protect neonatal islet cell cluster (NICC) xenografts. The combined effects of these modifications on the outcome of intraportal NICC transplantation in immunosuppressed baboons were investigated. Method: 1–5 day old GTKO piglets transgenic for human CD55, CD59 and H-transferase were used as donors. Recipient baboons received GTKO/CD55-CD59-HT NICC under standard (ATG, tacrolimus, mycophenolate mofetil; n¼5) or costimulation blockade-based immunosuppression (anti CD2, anti CD154, belatacept, tacrolimus; n¼3). The early inflammatory/thrombotic response was compared to that induced by wild type (WT) NICC (n¼4). Graft survival was evaluated by immunohistochemical analysis up to 3 months post-transplant. Results: GTKO/CD55-CD59-HT xenografts exhibited no signs of early thrombosis or infiltrate, and recipient platelet counts, fibrinogen and D-dimer levels were unchanged from baseline. In contrast, WT xenografts triggered widespread thrombosis within 12hrs, with substantial neutrophil and mononuclear cell infiltrate, accompanied by transient decreases in platelet count and fibrinogen and increased Ddimer levels. Analysis of liver biopsies from recipients under standard immunosuppression revealed loss of GTKO/CD55-CD59-HT NICC within one month, with heavy T and B cell infiltrates. However, costimulation blockade-based immunosuppression reduced cellular infiltration, and cells staining positive for insulin, glucagon and somatostatin were present in all GTKO/CD55-CD59-HT xenografts at three months, one animal having reversal of diabetes as quickly as one-month post transplant. Conclusions: Deletion of aGal and expression of human CD55 and CD59 prevent early thrombotic destruction of porcine NICCs in the baboon model. Costimulation blockade-based immunosuppression appears to be more effective than standard immunosuppression in prolonging the survival of genetically modified porcine NICC xenografts. CO-STIMULATION BLOCKADE INDUCED TOLERANCE TO ISLET XENOGRAFTS PRODUCES XENOSPECIFIC TREG THAT ARE CAPABLE OF DOMINANT TOLERANCE Wu Jingjing1, Hu Min1, Hawthorne Wayne1, Phillips Peta1, Burns Heather1, Nan Hai1, Liuwantara David1, Alexander Stephen2, Yi Shounan1, O’Connell Philip1 1Centre for Transplant and Renal Research, Westmead Millennium Institute, University of Sydney, Westmead, NSW, 2Centre for Kidney Research, The Children’s Hospital at Westmead, Westmead, NSW

Immunology and Cell Biology

Aim: 1) Identify the role of regulatory T cells (Tregs) in co-stimulation blockade-induced xenograft tolerance. 2) Define the phenotype and function of xenospecific Tregs. Methods: Balb/c mice transplanted with porcine-NICCs were given a single dose of CTLA4-Fc(500 mg) and 4 doses of MR-1(500 mg). At 4100 days post Transplantation(PTx), CD4+CD25+ Tregs isolated from spleen of tolerised mice(Tol-Tregs) were co-transferred with naı¨ve BALB/c spenocytes(Teff) into NOD-SCID recipients of NICCs xenografts. Tregs from the spleen of naı¨ve BABL/c mice(nTregs) were used as controls. Islet graft function was assessed by histology and C-peptide. Results: Histology of NICC xenografts 100 days PTx in CTLA4-Fc/ MR1 treated recipients showed intact islets and positive insulin staining. The proportion of Tregs in spleen of Tol-recipients at 100 day PTx was higher than in rejecting and non-Tx mice. Both Tol-Tregs and nTregs had diversity of T cell repertoire. Tol-Tregs but not naı¨ve Tregs or CD4+FoxP3- T cells expressed MHC-II and elevated levels of IL-10. Tol-xenografts infiltrating-cells expressed high levels of TGF-b(Po0.001), IFN-g(Po0.05) and IL-10 compared to rejection-group. The xenografts in NOD-SCID mice co-transferred with Tol-Tregs:Teffs at a ratio of 1:10 and 1:20 remained intact up to 70 days and mice were porcine c-peptide positive. In contrast, xenografts in NOD-SCID mice transferred with nTreg at a ratio of 1:20 were rejected. Graft-infiltrating-cells from NOD-SCID mice expressed high levels of IL-10. Conclusions Tregs play an important role in maintaining xenograft tolerance following co-stimulation blockade. Xenospecific Tol-Tregs expressed MHC class II, produced high levels of IL-10 and were capable of transferring dominant tolerance. INTERFERON-GAMMA AND INTERLEUKIN-17A ENHANCE MESENCHYMAL STEM CELLS (MSC) T-CELL SUPPRESSIVE FUNCTION BY MEDIATING AN INCREASE AND INDUCTION OF CD4+CD25HIGHCD127LOWFOXP3+ REGULATORY T-CELLS Sivanathan Kisha N1,2, Rojas-Canales Darling3,4, Hope Christopher M3,4, Carroll Robert3,4, Gronthos Stan5, Coates P Toby3,4 1Department of Medicine, Royal Adelaide Hospital, 2University of Adelaide, 3Centre for Clinical and Experimental Transplantation (CCET), Royal Adelaide Hospital, 4Department of Medicine, University of Adelaide, 5Mesenchymal Stem Cell Group, Department of Hematology, SA Pathology

Aim: MSC modulation of T-cell responses may be enhanced by proinflammatory cytokines. We aimed to identify key cytokines that enhance MSC function. Methods: MSC were treated with cytokines (IFN-g, TNF-a, IL-1b, IL2, IL-12p70 and IL-17A) and gene expression was evaluated by realtime PCR. Treated-MSC ability to increase regulatory T-cells (Tregs) or induce Tregs (iTregs) was determined by flow cytometry after 5 days co-culture with PHA activated total T-cells or CD4+CD25 Tcells respectively. CD4+CD25 T-cell co-culture supernatants were assessed for prostaglandin-2 by ELISA. Results: TNF-a and IL-1b increased IL-6 gene expression in MSC compared to untreated-MSC (UT:MSC). IFN-g upregulated cyclooxygenase expression and induced indoleamine 2,3-dioxygenase in MSC, whilst TGF-b1 expression remained unaffected and IL-10 was undetectable. Phenotypically, IFN-g upregulated B7-H1, but

Abstracts A5

neutralization studies proved it redundant for T-cell immunosuppression. UT:MSC, IFN-g treated-MSC (MSC-g) and IL-17 treated-MSC (MSC-17) all downregulated CD25 expression on CD4+ and CD8+ T-cells to a similar extent, but did not affect CD69. Additionally, co-cultures with PHA-activated T-cells and MSC-g or MSC-17 increased Treg (CD4+CD25highCD127lowFoxp3+) absolute numbers compared to UT:MSC and T-cells alone (Po0.04; n¼3). MSC-g and MSC-17 derived-Tregs promoted an activated Treg phenotype evident by high FoxP3highTreg numbers (Po0.008; n¼3). In subsequent experiments, we demonstrate that MSC-17 induced Tregs from CD4+CD25 T-cells (Po0.02; n¼3). This observation is consistent with high prostaglandin-2 secretion in MSC-17/ CD4+CD25 T-cell co-cultures. Conclusion: MSC-g and MSC-17 enhances MSC efficacy as immunosuppressive agents by promoting the expansion and induction of Tregs. Induction of Tregs by MSC-17 may be mediated through the increased production of prostaglandin-2.

INSTANT BLOOD MEDIATED INFLAMMATORY REACTION SELECT AGAINST PORCINE NEONATAL ISLET CELL CLUSTERS WITH POOR VIABILITY Liuwantara David1, Chew Yi Vee1, Favaloro Emmanuel2, Hawkes Joanne1, O’Connell Philip1, Hawthorne Wayne1,3 1Centre for Transplant and Renal Research, Westmead Millennium Institute, Westmead Hospital, Sydney, 2Diagnostic Haemostasis Laboratory, Haematology Department, Westmead Hospital, Sydney, 3Discipline of Surgery, Sydney Medical School, University of Sydney

The Instant Blood Mediated Inflammatory Reaction (IBMIR) is the initial barrier to the survival of porcine neonatal islet cell clusters (NICC) following xenotransplantation. Whilst IBMIR is a significant cause of islet loss, little is known about the function and viability of the surviving islets. Aim: To investigate islet viability following IBMIR. Methods: NICC from outbred pigs were exposed to human blood plasma to trigger IBMIR and allowed to recover in low glucose culture media. Extracellular flux analyses were performed using an XF24 analyser. NICC were challenged with 20 mM glucose, 5 mM Oligomycin – inhibitor of ATP synthase, 2 mM Trifluorocarbonylcyanide Phenylhydrazone – uncoupler of oxidative phosphorylation in mitochondria and 5 mM Rotenone/ Antimycin-A – inhibitors of the electron transport chain. Results: We found that IBMIR resulted in B62±9.1% (SEM) of NICC death. To our surprise, NICC subjected to IBMIR and survive, have a significantly higher glucose response (150±7.6% vs. 132±3.7% Po0.05) and spare capacity (78±8.7% vs. 43±4.6% Po0.005) compared to control. The Coupling efficiency was comparable between the two groups, but proton leak was significantly higher following IBMIR (47.9±2.0% vs. 41.7±1.5%, Po0.05). Conclusion: IBMIR caused significant loss in cell numbers, but the cells lost appear to be least viable. The NICC which survived showed resilience against IBMIR by increasing proton leak to reduce reactive oxygen species production by the mitochondria. These data supports the utilization of NICC as potential islet replacement therapy for patients with type-1 diabetes.

INVESTIGATING THE EFFECTS OF PURINERGIC RECEPTOR (P2X7R) BLOCKADE ON THE DEVELOPMENT OF GRAFTVERSUS-HOST-DISEASE (GVHD) IN HUMANISED MICE Watson Debbie1, Belfiore Lisa1, Alexander Stephen2, Sluyter Ronald1 1Illawarra Health and Medical Research Institute (IHMRI), The University of Wollongong, 2Centre for Kidney Research, The Children’s Hospital at Westmead, Sydney

P2X7 receptor is an ATP-gated cation channel expressed on a range of immune cells. Extracellular ATP is released upon tissue damage to activate P2X7R and mediate an inflammatory immune response, which has been implicated in mouse models of graft-versus-host disease (GVHD). Aims: To test P2X7R blockade in a humanized mouse model of GVHD to evaluate its potential as a therapeutic strategy to prevent GVHD in humans. Methods: The hu-PBL-SCID model of GVHD is established by injecting NODSCIDIL-2Rgnull (NSG) mice with 10106 human peripheral blood mononuclear cells (hPBMCs). Flow cytometry at 3 weeks post-injection shows all NSG mice engraft with human cells, the majority being CD3+ T cells. Results: Majority of NSG mice injected with hPBMCs develop signs of GVHD based on weight loss, clinical score and survival. GVHD correlated with increased splenic human CD4:CD8T cell ratios and increased serum human IFN-g. Brilliant blue G (BBG) dye is a P2X7R antagonist and in vitro we demonstrate that BBG can block ATPinduced cation uptake in hPBMCs. Intraperitoneal injection of BBG (to block P2X7R) into hu-PBL-SCID mice did not affect engraftment of human cells. There was no difference in weight, clinical scores or survival of Hu-PBL-SCID mice injected with BBG or saline. However there was evidence of a lower splenic human CD4:CD8 T cell ratio and lower human IFN-g in hu-PBL-SCID mice injected with BBG, similar to mice without GVHD. Conclusions: This suggests i.p. injection of BBG does not prevent GVHD but testing of other mechanisms of P2X7R blockade may be required. CAN esRAGE PROTECT PANCREATIC INTEGRITY IN BRAIN-DEAD DONOR MICE? Ghoraishi Tina, Wang Zane, Paul Moumita, Morton Rebecca, Habib Miriam, Bishop G Alex, Sharland Alexandra F Collaborative Transplant Group, University of Sydney Aims: Toll-like receptor signalling is upregulated after brain death, leading to the release of downstream pro-inflammatory cytokines and chemokines. Endogenous secretory (es)RAGE is a naturally-occurring soluble decoy receptor which blocks binding of various ligands to TLRs 2 and 4 and RAGE. Methods: We previously achieved systemic expression by transduction of the mouse liver with an adeno-associated virus vector encoding esRAGE. Brain death was induced by insertion and inflating of an intracranial balloon catheter, and BD mice were maintained for 3 hrs. Experimental groups consisted of BD mice receiving control AAV vector (AAV-HSA), BD mice with AAV-esRAGE, ventilated non-BD

Immunology and Cell Biology

Abstracts A6

and normal mice. Pancreatic tissue was sampled and analysed by RT-PCR and anti-insulin staining. Results: Chemokines including MCP-1, MIP2, IP-10, and KC, C3 and cytokines such as IL-10, IL-6, IL-1b, TNFa were modestly reduced in BD+esRAGE compared with BD+HSA. TLR-4 and RAGE didn’t change. RAE-1 was significantly upregulated in BD+esRAGE. Good preservation of insulin protein was observed in BD+esRAGE. Lower insulin mRNA expression was required to maintain insulin protein levels comparable to normal. Conclusion: A modest reduction in pancreatic expression of proinflammatory genes was noted, concurrent with a significant increase in RAE-1 expression. Expression of this stress ligand may render the pancreas susceptible to immune attack post-transplant. This effect differs from that in kidney, where both inflammatory cytokines and RAE-1 expression were attenuated in the absence of TLR4 signalling. Normal insulin content may relate to protective effects of esRAGE. Further evaluation is necessary. 10-YEAR OUTCOMES OF THE WESTMEAD ISLET TRANSPLANT PROGRAM Hawthorne Wayne1,2,3, Williams Lindy3, Patel Anita3, Davies Sussan3, Hor Katie3, Liuwantara David3, Hawkes Joanne3, Jimenez-Vera Elvira3, Chew Yi Vee3, O’Connell Philip3,4 1Discipline

of Surgery, Sydney Medical School, University of Sydney, Pancreas Transplant Unit, Westmead Hospital, Sydney, 3Centre for Transplant and Renal Research, Westmead Millennium Institute, Westmead Hospital, Sydney, 4Discipline of Medicine, University of Sydney 2National

Aims: Islet transplantation remains a difficult challenge with relatively few units around the world achieving success. The aim of this study was to determine donor characteristics predictive of a transplantable islet preparation and whether enzyme type had an effect on attainment of transplantable islets. Method: Islets were isolated from pancreases of heart beating deceased donors using SERVA NB1 collagenase and NP (SERVA) or Liberase (Roche). Islet preparations were divided into transplanted and Nontransplanted, total islet equivalents (IEQ) and IEQ/gram (IEQ/g) pancreas were compared. Donor age, body mass index (BMI), pancreas weight, cold ischaemic time (CIT) were also compared. The Transplanted group was stratified according to donor age into 10 year cohorts and total IEQ and IEQ/g pancreas compared. Results: 150 islet isolations between 2003 and 2013 were evaluated. A total of 101 isolations were performed using SERVA and 49 using Liberase. 35 were transplanted: 23/101 (1 in 4) SERVA and 12/47 (1 in 4) Liberase. Transplanted preparations had a greater total IEQ and IEQ/g pancreas compared to their non-transplanted counterparts. Donor characteristics predictive of a suitable islet preparation were shorter CIT (342±23vs 421±14 min P¼0.005), larger BMI (31.4±1 vs 27.7± 0.5 kg/m2 P40.001) and greater pancreas weight (101±3 vs 85±2 g P40.001). When stratified for age of donor the 40–49 yr cohort preparations achieved significantly greater number of IEQ (934,297±110,483 P¼0.02), IEQ/g pancreas (9,157±1,047 P¼0.001), those in the 450y having lower numbers of IEQ and IEQ/g pancreas. Conclusions: Donor factors predictive of a transplantable islet preparation were shorter CIT, higher BMI and larger pancreases. The pancreases from the 40–49 years age group provided greater islet yields. Immunology and Cell Biology

Surgery INFERIOR EARLY POST-TRANSPLANT OUTCOMES FOR RECIPIENTS OF RIGHT VERSUS LEFT LIVING DONOR KIDNEYS Clayton Philip1,2,3, Chadban Steven1,2,3, McDonald Stephen1,4, Allen Richard2,3 Registry, Royal Adelaide Hospital, 2Sydney Medical School, University of Sydney, 3Transplantation Services, Royal Prince Alfred Hospital, Sydney, 4Discipline of Medicine, University of Adelaide 1ANZDATA

Aims: Deceased donor transplantation with right kidneys is associated with inferior early transplant outcomes compared with left kidneys due to technical difficulties associated with the shorter right renal vein. The outcomes of transplanting right versus left kidneys from living donors are less well studied. Methods: Using Australia and New Zealand Dialysis and Transplant (ANZDATA) Registry data, we included all recipients of a living donor kidney transplant in Australia/New Zealand over 2004–2012. We used Cox models to compare graft survival between right and left kidneys. Results: The analysis included 2433 left kidneys and 549 (18%) right kidneys. Delayed graft function occurred in 6% of right and 3% of left kidneys (Po0.001). 322 grafts were lost during a median follow-up of 3.9 years. Overall graft survival was 98% (95% CI:98–99%) at 1 month, 97% (96–97%) at 1 year and 88% (87–90%) at 5 years. On unadjusted analysis, right kidneys had inferior graft survival in the first month post-transplant, but beyond the first month rates of graft loss were equivalent (figure). After adjusting for age, sex, race, primary renal disease, co-morbidities, HLA mismatch, peak PRA and donor age, right kidneys remained associated with a significant increase in graft loss in the first month (hazard ratio [HR] 2.14, 95%CI:1.20–3.84, P¼0.01). Beyond one month right kidneys were not associated with graft loss (HR 1.25, 95%CI:0.94–1.68, P¼0.13). Conclusion: Recipients of a right living donor kidney are at higher risk of graft loss in the first month post transplant, although the absolute risk remains low.

Overall graft survival Living donor kidney transplants Australia/NZ 2004-2012 1.00 0.75 0.50 0.25 Left kidney Right kidney

0.00 0

Number at risk Left 2433 Right 549

2

4 Years post transplant

6

8

1829 411

1182 276

631 135

185 39

Abstracts A7

INCIDENCE OF RENAL ALLOGRAFT THROMBOSIS AND OUTCOME OF RE-TRANSPLANTATION IN AUSTRALIA AND NEW ZEALAND: A REGISTRY ANALYSIS Irish Ashley1, McDonald Stephen2, Swaminathan Ramyasuda1 1Renal Transplant Unit, Royal Perth Hospital, 2Central Northern Adelaide Renal and Transplantation Service, Royal Adelaide Hospital

Introduction: Renal allograft thrombosis results in premature graft loss and the need for re-transplantation. The reported incidence of thrombosis varies from 2–7%. We examined the incidence of thrombosis over time and the outcomes for re-transplantation following an episode of graft thrombosis. Methods: Retrospective ANZDATA & ANZOD review of all episodes of allograft arterial or venous thrombosis from 1963–2012. Results: Thrombosis as the cause of graft loss occurred in 450/24,875 (1.81%) of all allografts (2.03% DD, 1.16% of LD) with 376/450 (84%) occurring within 30 days of transplant. 242/450 were arterial (54%) and 85% of all thrombosis occurred in first grafts. There was a reduction in incidence of thrombosis from a peak of 3.39% in 1991 to 0.42% in 2012. In DD, 359 thromboses occurred in 1 of the two paired kidneys and in only 4 cases did both donor kidneys thrombose (0.04%). 257/ 450(57%) patients underwent re-transplantation of whom 13 (5%) suffered graft loss due to thrombosis. In a MLR risks for allograft thrombosis were donor age (OR 1.01 P¼0.006) and era of transplant (2010-12 OR 0.18 P¼0.000) but not cause of death or donor gender. Survival of grafts in patients re-transplanted after an initial allograft thrombosis did not differ from survival after graft loss from all other causes (combined) at 1, 3 5 (62% & 59% respectively) and 10 years. Conclusions: Allograft thrombosis remains a contributor to early graft loss but the incidence has steadily declined since 1991. Most thromboses occur in only 1of the paired deceased donor kidneys suggesting recipient or operative factors contribute. Retransplantation occurred in 57% of patients and graft survival was equivalent to other retransplants with a low risk of recurrent thrombosis. LAPAROSCOPIC KIDNEY TRANSPLANT BY EXTRA PERITONEAL APPROACH: A MODEL FOR SAFE TRANSITION FROM LABORATORY TO THE CLINIC He Bulang1, Mou Lingjun1, Sharpe Kendall1, Hamdorf Jeffrey2, Delriviere Luc1 1WA

Liver & Kidney Transplant Service, Sir Charles Gairdner Hospital, Perth, 2School of Surgery, The University of Western Australia

Aims: Laparoscopic surgery has been increasingly replacing open surgery due to multiple benefits. The aim of this study is to develop a novel laparoscopic technique by extra peritoneal approach for kidney transplant and establish a model of safe transition from laboratory to the clinic. Methods: The study was established to investigate the feasibility and safety for human laparoscopic kidney transplant. During 4 years period, the experiment was firstly conducted on the cadaver animals, then live animals in different phases and human cadavers before the application has been approved for human kidney transplant. The study patient was 49 years old male who received the laparoscopic kidney transplant by extra peritoneal approach. Results: The success was achieved at each step. The patient was hemodynamically stable during surgery. The estimated blood loss

was minimal. The kidney graft function is satisfactory with nadir creatinine level at 111 mmol/L. The incision size is 6 cm by laparoscopic technique with much less analgesia consumption. There is no surgical complication over 5 months follow up. Conclusions: It was demonstrated that laparoscopic kidney transplant by extra peritoneal approach is safe and feasible. This technique has retained the advantages of open kidney transplant, which allows the kidney graft located in the extra peritoneal space without violating intra peritoneal organs. The further study will be needed to improve and refine the technique. This study has established a model of safe transition for a novel laparoscopic surgery from the laboratory to the clinic. DUAL KIDNEY TRANSPLANT: CLINICAL EXPERIENCE AND OVERVIEW OF SURGICAL TECHNIQUES Patel Dhruv, Zwierzchoniewska Monika, Fong Kang, He Bulang WA Liver & Kidney Transplant Service, Sir Charles Gairdner Hospital, Perth Background: Dual Kidney Transplant (DKT) of marginal kidneys otherwise not considered for transplant has allowed utilizing the extended criteria donor (ECD) organs. The aim of this study is to present the outcomes of dual kidney transplant in our unit and review the surgical techniques of DKT in the literature. Material and Method: Between Jan 1999 and Feb 2013, 9 (7 male and 2 female; mean age 48.55 years) cases of DKT were performed in our institute. The surgical techniques used for DKT include single site incision for 8 patients, midline incision for 1 patient. The literature was also searched for overview of surgical techniques for DKT. Result: Two recipients had DKT from paediatric donors (Body Weight o 15 kg) whereas eight recipients had DKT from elderly donors. The mean surgical time was 5 hours 3 min (Range: 3 hours 40 min to 6 hours 24 min). One patient developed lymphocele and subsequent ureteric stricture, which was complete resolved by a surgical procedure. There was no vessel thrombosis and no urine leakage in this cohort. Mean Cr level was 137 (± 37.4) mmol/L at 12 months followup. There was no surgical graft loss. From the literature review, it is inferred that single site DKT is in favour. Conclusion: DKT is an established technique for extended criteria of kidney donors. Surgical complications can be minimized and clinical outcome is satisfactory. Based on our experience, we believe that selective use of DKT can provide excellent outcomes to recipients of kidneys from marginal donors. OUTCOME OF RENAL TRANSPLANTATION IN ADULTS WITH URINARY DIVERSION OR BLADDER AUGMENTATION: A SYSTEMATIC REVIEW Yao Jinna1,2, Lau Howard1, Pleass Henry1,2, Lam Vincent1,2 1Department of Surgery, Westmead Hospital, Sydney, 2Discipline of Surgery, Sydney Medical School, University of Sydney

Aims: End-stage renal failure may occur due to congenital or acquired conditions of the lower urinary tract. Renal transplantation with urinary diversion or bladder augmentation may be a safer option in this subgroup of patients. In this systematic review, we aim to summarise the published evidence of the outcomes of adult renal transplantation in patients with prior urinary diversion or bladder augmentation. Immunology and Cell Biology

Abstracts A8

Methods: A literature search of MEDLINE, EMBASE and Cochrane databases was undertaken to identify studies (Jan 1995 to Dec 2013) of renal transplantation in adults with urinary diversion or bladder augmentation. Results: Five case-control studies and 14 case series were included. A total of 349 renal allografts were transplanted into 316 patients. Urinary diversion was performed in 68% of patients and bladder augmentation was performed in 32% patients. Mean age at time of transplant is 32.2(23 to 52) years. Reasons for urologic surgery were neurogenic bladder(34%) and spina bifida(23%). Complications occurred in 109(50%) of urinary diversion cases and 47(52%) of bladder augmentation cases. Most common surgical complications were ureteric complications(8%), thrombosis(2%), wound infection(2%) and stoma-related complications(2%). Most common non-surgical complications were urinary tract infections (UTI) (34%) and chronic rejection(6%). There were 88 renal graft losses; most common reasons were death with functioning graft(17%), UTI(11%) and chronic rejection(11%). Mean 5-year patient and graft survival were 96(83 to 100)% and 74(34 to 100)%, respectively. Conclusions: Current evidence suggests that renal transplantation in adults with urinary diversion or bladder augmentation is safe and feasible in selected patients.

Complications – cancer, cardiovascular, infections and metabolic CANCER RECURRENCE AFTER KIDNEY TRANSPLANTATION Viecelli Andrea1, Carroll Robert2, Chapman Jeremy3, Clayton Phil4, Craig Jonathan5, Webster Angela3, Lim Wai1, Wong Germaine3,5 1Department

of Renal Medicine, Sir Charles Gairdner Hospital, Perth, Northern Adelaide Renal and Transplantation Service, Royal Adelaide Hospital, 3Department of Renal Medicine, Westmead Hospital, Sydney, 4Department of Renal Medicine, Royal Prince Alfred Hospital, Sydney, 5Centre for Kidney Research, The Children’s Hospital at Westmead, Sydney 2Central

of Surgery, Westmead Hospital, Sydney, 2Discipline of Surgery, Sydney Medical School, University of Sydney

Background: Clinical practice guidelines recommend a waiting time of 2–5 years before transplantation in potential recipients who develop cancer, but little is known about the timing and risk of recurrence in recipients. Aim: We aimed: 1) to determine the timing and risk of cancer recurrence after kidney transplantation in recipients with a prior cancer and 2) to compare the risk of cancer-specific mortality among those who developed cancer recurrence and recipients who developed de novo cancers after transplantation. Methods: Data from the Australian and New Zealand Dialysis and Transplant Registry (ANZDATA) was used to assess the cancer

Aims: Renal transplantation using ‘restored’ kidneys following excision of renal tumours has been proposed to increase the inadequate supply of organs. In this systematic review, we aim to evaluate the published evidence for this novel approach. Methods: A literature search of databases (MEDLINE, EMBASE) was performed to identify studies of renal transplantation after renal tumour excision, focusing on selection criteria, operative strategy, tumour recurrence and survival outcomes. Results: Nine case series and 8 case reports were included. A total of 125 patients underwent renal transplantation using grafts restored following excision of renal tumours. Eighty-nine percent(111/125) of grafts were from live donors. Thirteen of 17 studies reported intraoperative frozen section of the excised renal tumour prior to transplantation. Eighty-four percent(105/125) were proven to be malignant renal cell carcinoma (RCC) on pathology. Ninety-nine percent (104/ 105) of these RCC were smaller than 4 cm. Fourteen studies reported tumour margin status, all of which were negative. Sixteen studies reported follow-up time; the median value was 56(15–138) months. Fourteen studies reported graft outcomes; 5(4%) patients lost their grafts. All 17 studies reported patient survival outcomes, and 13(10%) patients died with a functioning graft. Of the total 105 patients with renal transplants after excision of RCC, there was one recurrence in the graft kidney at 9 years. Conclusions: Current evidence suggests that renal transplantation using kidneys after renal tumour excision is safe and feasible in selected patients. Future studies are required to further define the selection criteria for the donor kidneys and the renal transplant recipients.

Figure 1 Hepatocyte MHCII IAd Expression.

RENAL TRANSPLANTATION USING RESTORED GRAFTS AFTER RENAL TUMOUR EXCISION: A SYSTEMATIC REVIEW Yao Jinna1,2, Lau Howard1, Pleass Henry1,2, Lam Vincent1,2 1Department

Immunology and Cell Biology

Abstracts A9

recurrence risk of all types, excluding non-melanocytic skin cancers, in kidney transplant recipients with a history of cancer prior to transplantation between January 1965 and December, 2012. Results: Of the 21,415 transplant recipients, 651 (3.0%) had a cancer prior to transplantation. Of which, 19 (2.9%) recurred after the first transplant. The median time to first recurrence was 5.8 years [Interquartile range (IQR): 7.8 years]. Of all the cancer recurrences, 7 (37%) occurred in the first two years posttransplant, 6 (31%) between 2–6 years and the remaining 5 cancers (32%) occurred 6 years after the first transplant. The highest recurrence rates were seen in cancers of the urinary tract system (n¼6, 31%), followed by breast (n¼4, 21.5%) and melanoma (n¼4, 15.8%). Over a median follow-up time of 10.4 years, five recipients (26.1%) died from cancer recurrences. There was an increased risk of cancer death among those with a prior malignancy compared to recipients who developed de novo cancers after transplantation (RR: 1.60; 95%: 1.5–1.8, Po0.001). Conclusion: Although the risk of recurrence among those who developed a cancer before the first transplant is low, cancer outcomes are inferior to those without a pre-existing malignancy. RISK OF EARLY AND LATE NON-HODGKIN LYMPHOMA (NHL) IN AUSTRALIAN LIVER AND CARDIOTHORACIC TRANSPLANT RECIPIENTS Na Renhua1, Grulich AE1, Meagher NS1, McCaughan GW2, Keogh AM3, Laaksonen M1, Vajdic CM1 1School

of Medicine, University of New South Wales, Sydney, Morrow Gastroenterology & Liver Centre, Centenary Institute of Cancer Medicine and Cell Biology, Sydney, 3Lung Transplant Unit, St Vincent’s Hospital, Sydney 2AW

Aims: To determine whether risk of NHL is increased in cardiothoracic compared to liver transplant recipients, adjusting for extent of immunosuppression and competing risk of death. Methods: A retrospective cohort study using population-based liver (n¼1924) and cardiothoracic (n¼2631) transplant registries (1984– 2006). NHL incidence was ascertained with the Australian Cancer Database. Adult and paediatric transplant unit medical records were reviewed to ascertain putative risk factor data. Multiple imputation was used to impute missing Epstein-Barr virus (EBV) data. Adjusted hazard ratios (HR) for early (n¼31) and late-onset (n¼69) NHL were estimated using the proportional subdistribution hazards model including organ, age, sex, calendar year of transplant, EBV seronegativity at transplantation, receipt of OKT3 antibody and ATG/ALG, current dose and number of immunosuppressive agents, and for late NHL also time since transplantation. Results: After imputation 22 early (o1 yr) and 45 late (Z1 yr) NHL were available for multivariate analysis. Compared to liver, lung recipients were at increased risk of early NHL (HR 5.18, 95%CI 1.39–19.3). Recipient EBV seronegativity at transplantation (HR 3.33, 95%CI 1.08–10.4), OKT3 (HR 10.0, 95%CI 2.50–40.0), and male gender (HR 3.47, 95%CI 1.44–8.37) were also early NHL risk factors. For late NHL, heart recipients were at increased risk compared to liver (HR 3.66, 95%CI 1.33–10.0). Conclusions: Differences in immunosuppressive regimen do not appear to explain the excess risk of NHL in heart and lung compared to liver transplant recipients. Growing evidence supports heterogeneity in the risk factors for early and late NHL after solid organ transplantation.

HOSPITALISATIONS IN ATSI AND NON ATSI RENAL TRANSPLANT RECIPIENTS IN WESTERN AUSTRALIA Swaminathan Ramyasuda1, Boan Peter2, Irish Ashley1 1Nephrology and Renal Transplant, Royal Perth Hospital, 2Department of Infectious Diseases, Royal Perth Hospital

Introduction and Aim: ATSI renal transplant recipients have increased graft loss and mortality compared with non-ATSI recipients. We aim to compare rates of hospitalisations between ATSI and Non- ATSI de novo renal transplant recipients (RTRs) in Western Australia. Methodology: Retrospective analysis of all consecutive deceased donor transplants (n¼144, 57 ATSI & 87 Non-ATSI) between Jan 2005 and December 2011 in Western Australia. Results: There was no difference in age and gender between the ATSI and Non-ATSI recipients. ATSI had more diabetics (46% vs. 15% Po0.001) and greater residence in remote areas (65 vs. 2%; Po0.0001). ATSI had an increased median length of stay following transplantation [7.5 (IQR 4.5, 10.5) days ATSI vs.6 (IQR 4,7) NonATSI; P¼0.02]. After adjusting for the median follow up period of 47 months, ATSI had an increased number of subsequent hospitalisations (3.75 vs. 3.0 episodes; P¼0.02), duration of hospitalisation (38.1 days ATSI vs 28.0 days Non-ATSI P¼0.01). 61% of hospitalisations in ATSI were attributed to infection compared with 38% in the non-ATSI (Po0.001); In hospital length of stay for an infection-related admission (29.9 ATSI vs. 14.9 Non-ATSI days; Po0.001) and mortality (19.2% ATSI vs. 9.6% NON-ATSI P¼0.002) were greater in the ATSI patients. 90% of deaths in the ATSI patients were due to infection. Conclusion: ATSI had significantly higher rates, hospitalisation and infectious complications post renal transplantation. The greatly increased morbidity and mortality due to infectious complications in ATSI patients is a major impediment to patient and graft outcomes and requires additional study to ascertain contributing factors and suggests the need for re-evaluation of patient selection, screening, prophylaxis and immunosuppressive protocols. HIGH PANEL REACTIVE ANTIBODIES AND GRAFT AND PATIENT SURVIVAL AND INCIDENT CANCER RISK AFTER KIDNEY TRANSPLANTATION Lim Wai1, Wong Germaine2,3 1Department

of Renal Medicine, Sir Charles Gairdner Hospital, Perth, of Renal Medicine, Westmead Hospital, Sydney, 3Centre for Kidney Research, The Children’s Hospital at Westmead, Sydney 2Department

Panel reactive antibody (PRA) level has been used widely to determine the sensitization status of potential kidney transplant candidates. Despite prioritization for highly-sensitised candidates, the waiting time for these candidates remains doubled that of unsensitized candidates. High peak PRA levels have been associated with an increased risk of graft failure, but the association between PRA and all-cause mortality and incident cancer risk remains unknown. Using the Australian and New Zealand Dialysis and Transplant Registry (ANZDATA), we assessed the risk of acute rejection, graft failure, allcause mortality and incident cancer among those with different peak PRA levels using adjusted Cox proportional hazard models. In 7118 kidney transplant recipients between 1997 and 2009 with PRA of 450%, there were more rejection (38.5%, 27.8% and 30.8%; w2¼33.3, Immunology and Cell Biology

Abstracts A10

Po0.001), graft loss (31.4%, 17.3% and 22.6%; w2¼80.7, Po0.001), death (19.3%, 9.5% and 13.3%; w2¼64.4, Po0.001) but not incident cancers (7.5%, 6.2% and 6.4%; w2¼1.7, P-0.428) compared to recipients with PRA of o10% and 10–50% respectively. Compared to recipients with PRA of o10%, those with PRA of 450% were associated with a higher risk of acute rejection (adjusted odds ratio 1.60, 95%CI 1.32–1.93, Po0.001), overall graft failure (hazard ratio [HR] 1.56, 95%CI 1.32–1.85, Po0.001), death censored graft failure (HR 1.53, 95%CI 1.17–2.01, P¼0.002) and all-cause mortality (HR 1.61, 95%CI 1.29–2.00, Po0.001). There was no association between PRA level and risk of incident cancer. Kidney transplant recipients with peak PRA of 450% had significantly higher risk of overall and death-censored graft failure and all-cause mortality independent of acute rejection, age and time on dialysis. Measures to avoid sensitization or to further prioritize highly sensitized kidney transplant candidates should be explored. USE OF TOPICAL IMIQUIMOD IN SKIN MALIGNANCIES AFTER RENAL TRANSPLANTATION Das Gayatri1, Tan Boon1,2, Nicholls Kathy1,3 1Department of Nephrology, Royal Melbourne Hospital, 2Dermatology, Royal Melbourne Hospital, 3Department of Medicine, University of Melbourne

Background: In Australian renal transplant recipients (RTR), nonmelanotic skin cancers, mainly SCC, and precursor lesions, are common. Imiquimod is a cytokine inducer that modifies the innate immune response. It enhances the acquired antitumor immune response and has proven efficacy in non immunosuppressed patients for superficial skin malignancy (SSM). Aim: To evaluate the safety and efficacy of topical imiquimod 5% cream for treatment of SSM, solar keratosis (SK) and warts, in RTR at Royal Melbourne Hospital. Methods: All RTR treated with topical 5% imiquimod cream between its first use in July 2004 and December 2013 at the Royal Melbourne hospital were identified via Pharmacy records. Clinical data was retrospectively collected from hospital records and ANZDATA. Results: 14 lesions in 12 RTR (8 females, 4 males) mean age 56.9±12.0 years (Range 36.9–75 yrs), were treated with topical Imiquimod during this time. Targeted lesions were Bowen’s disease (5 in 4 patient), superficial BCC (5 in 4 patients), and SK (3 in 3 patients). One patient was treated for multiple warts. 2 of the 12 patients had subsequent recurrence of the lesion (1 Bowen’s, 1 SK) at the same site 4 and 12 months later, and 1 SK did not respond to treatment. We judge treatment failure in 3 out of 13 SSM, with a clearance rate of 85%. In no patient did renal function deteriorate during treatment. Conclusion: Topical Imiquimod is a safe and reasonably effective treatment for SSM in RTR.

INFECTIOUS COMPLICATIONS IN ABORIGINAL AND TORRES STRAIT ISLANDER (ATSI) COMPARED WITH NON-ATSI KIDNEY TRANSPLANT RECIPIENTS IN WESTERN AUSTRALIA Boan Peter1, Irish Ashley2, Swaminathan Ramyasuda2 of Infectious Diseases, Royal Perth Hospital, 2Nephrology and Renal Transplant, Royal Perth Hospital 1Department

Immunology and Cell Biology

Aims: To examine infectious complications between aboriginal and torres strait islander (ATSI) versus Non-ATSI kidney transplant recipients. Methods: A retrospective case control study examining microbiological records and electronic discharge summaries. Results: ATSI compared with non-ATSI were significantly more likely to test positive for specific treponemal antibodies (46.7% vs 0%, P¼0.022) and CMV IgG (98.2% vs 73.2%, Po0.001). ATSI compared to controls were less likely to develop bacteruria (61.4% vs 77.4%, P¼0.058), recurrent bacteruria (26.3% vs 35.7%, P¼0.273) or CMV viremia (22.8% vs 33.3%, P¼0.180). However ATSI patients were more likely to develop skin/soft tissue infection (19.3% vs 9.5%, P¼0.131), invasive fungal infection (14.0% vs 8.3%, P¼0.404) or gastrointestinal parasitic infection (12.3% vs 1.2%, P¼0.007) compared to controls. Rates of ESBL producing bacteria, bacteremia, pneumonia and BK nephropathy were similar between the two groups. Conclusions: Less occurrence of high risk CMV donor positive/ recipient negative ATSI kidney transplants and universal CMV prophylaxis may explain lower rates of CMV viremia. Moderate rates of invasive fungal infection in the ATSI population raise the issues of identification of those patients most at risk of this serious complication and consideration of antifungal prophylaxis. Moderate rates of gastrointestinal parasitic infection may be reduced by pre-transplant stool testing and treatment. PRE-TRANSPLANT SCREENING FOR STRONGYLOIDES Gracey David1,2,3, Wyburn Kate1,2,3, Chadban Steven1,2,3, Mawson Jane1,3, Eris Josette1,2,3 1Transplantation

Services, Royal Prince Alfred Hospital, Sydney, of Medicine, University of Sydney, 3Statewide Renal Services, Royal Prince Alfred Hospital, Sydney 2Department

Background: Screening for Strongyloides with serology in the Pre-Renal Transplant setting varies, and is mainly based on a perception of a patient’s clinical risk. Current guidelines from the American Society of Transplantation recommend Strongyloides testing for patients from an area of endemicity or those with gastrointestinal symptoms or eosinophilia pre-transplantation. A recent survey of physicians found that only 9% could recognize a case presentation of a person in need of screening. Strongyloides is most common in the tropics, subtropics, and in warm temperate regions. Studies in immigrant populations have shown up to 46.1% with infection. The highest prevalence of infection today is found in South and Southeast Asia. Methods: We examined all patients screened with Strongyloides serology as part of their Pre-Transplant work up in the past 24 months at out unit. Results: Of 720 patients seen in our Pre-Transplant Clinic between 2008–2013, 15 (2.1%) were screened for Strongyloides. All were of Afro-Caribbean or Asian descent; however not all patients at risk had been screened. In the past 24 months, two patients have developed disseminated Strongyloidisis at our centre. Of these, one was co-infected with Human Immunodeficiency virus and originated from the Caribbean, but had been living in Sydney for years. His pre-transplant Strongyloides serology was negative. The second patient originated from Italy and had positive Strongyloides serology, although this was discovered post-transplant. One potential kidney donor, from Vietnam, also had positive Strongyloides

Abstracts A11

serology and was treated with Albendazole prior to donation. Additionally, all patients from Noumea, who are also transplanted at our unit, are screened routinely. Of the Noumean patients 8/20 (40%) on the deceased renal donor transplant waitlist demonstrate positive serology. Conclusions: Given the recent cases of Strongyloides at our unit and the low frequency of screening at present, we have elected to intensify screening for Strongyloides, and now routinely check Strongyloides serology in all patients as part of their Pre-Transplant work up. THE PRE-RENAL TRANSPLANT ASSESSMENT OF HIV-INFECTED PATIENTS Gracey David1,2, Wyburn Kate1,2, Chadban Steven2,3, Mawson Jane3, Eris Josette1,2,3 1Statewide

Renal Services, Royal Prince Alfred Hospital, Sydney, of Medicine, University of Sydney, 3Transplantation Services, Royal Prince Alfred Hospital, Sydney 2Department

Background: The prevalence of renal disease in Human Immunodeficiency Virus (HIV)-infected patients is increasing. This has led to an increase in the numbers of HIV-infected patients requiring assessment of their suitability for renal transplantation. Methods: We reviewed all HIV-infected patients assessed at our Renal Transplant Unit in the past 12 months (n¼10). Results: This patient group had a high level of co-morbidities, some of which were undiagnosed prior to their review in the Pre-Transplant Assessment Clinic. A minority of the patients assessed were deemed suitable for transplantation. Of the 10 patients, one is active on the deceased donor renal transplant wait list and one has been successfully transplanted. The remaining eight patients had co-morbidities currently preventing them from being activated on the deceased donor renal transplant wait list (see below). All patients reviewed were on combination antiretroviral therapy with undetectable viral loads and normal CD4 T-cell counts. Conclusions: HIV-infected patients require careful Pre-Renal Transplant Assessment. Those patients referred to our unit in the past 12 months demonstrated a high level of infectious and non-infectious co-morbidities compromising their suitability for activation on the deceased donor renal transplant waitlist.

Patient

Contraindication to transplantation

1

Undiagnosed pulmonary TB

Returned to the Philippines.

2

Undiagnosed metastatic SCC

TB being treated For surgical intervention

3 4

Undiagnosed atypical TB Severe CCF (EF 15%)

Review after TB treated Considered unfit for surgery

5

Colonic carcinoma (Within 12 months of assessment)

For review after 4 years

6

Melanoma (Within 3 months of assessment)

For review after 5 years

7

Severe peripheral vascular disease, unstable IHD

Preparing for dialysis

8

Undiagnosed HCV

For review by hepatology and possible treatment for HCV

HBA1C FOR THE DIAGNOSIS OF NEW ONSET DIABETES AFTER KIDNEY TRANSPLANTATION Aouad Leyla1, Clayton Philip1,2, Wyburn Kate1,2, Gracey David1,2, Eris Josette1,2, Chadban Steven1,2 1Department 2Department

of Renal Medicine, Royal Prince Alfred Hospital, Sydney, of Medicine, University of Sydney

Aims: New onset diabetes after transplant (NODAT) is common after kidney transplantation. Incidence varies widely depending on patient factors, immunosuppression and diagnostic criteria. Glycosylated haemoglobin A (HbA1c) is an accepted diagnostic test for T2DM and has been proposed for the diagnosis of NODAT. Though more convenient than OGTT, there are limited data on test performance in the early post-transplant period. We compared HbA1c with OGTT for diagnosis of NODAT at 3 months post kidney transplantation. Methods: We performed retrospective analysis of 120 patients without prior diabetes who received a kidney transplant at our institution between 2005–2013 and did not require treatment for NODAT prior to month 3. All underwent OGTT at 3 months posttransplant and had HbA1c measured between 2 weeks before and 6 weeks after the OGTT. Results: Mean age at transplantation was 44(±16) years with 73% on tacrolimus and 100% on prednisone. OGTT classified 45(37.5%) patients as normal, 39(32.5%) as impaired glucose tolerance (IGT) and 35(29%) as NODAT. The area under the receiver operating characteristic (ROC) curve for HbA1c in diagnosis of NODAT was 0.74 (95%CI: 0.65 to 0.84) (figure). HbA1c 46.5% identified NODAT with sensitivity 20%, specificity 94%. Sensitivity of HbA1c for abnormal glucose metabolism (IGT or NODAT) was 74%, specificity 64%. Conclusions: HbA1c 46.5% performs poorly in the diagnosis of NODAT and abnormal glucose metabolism in the early period after kidney transplantation. Changing haemoglobin concentration, acute inflammation and other factors may be contributory and whether HbA1c is a useful test at later timepoints remains to be determined.

Outcome

Immunology and Cell Biology

Abstracts A12

PREVALENCE OF ACTINIC KERATOSIS AND KERATINOCYTE CANCERS IN LIVER TRANSPLANT RECIPIENTS IN QUEENSLAND – RESULTS FROM A PROSPECTIVE STUDY Sinnya Sudipta1,2, Davis Marcia3, Ferguson Lisa3, Burton Sharan3, Pandeya Nirmala4, Fawcett Jonathan2, Soyer HP1,5, Green Adele3 1Dermatology Research Centre, The University of Queensland, School of Medicine, Translational Research Institute, Brisbane, 2Department of Liver Transplantation, Princess Alexandra Hospital, Brisbane, 3Population and Cancer Studies Unit, Queensland Institute of Medical Research, Brisbane, 4School of Population Health, The University of Queensland, Brisbane, 5School of Medicine, Princess Alexandra Hospital, Brisbane

Aims: Keratinocyte Cancer rates in the transplant population in Australia are among the highest in the world, leading to significant morbidity and associated costs to the health care system. While skin disease have been studied quite extensively in kidney and heart transplant patients, data pertaining to liver transplant recipients remain sparse. The aim of this study to prospectively review the prevalence of keratinocyte cancers and actinic keratosis in the Queensland liver transplant population relative to personal, phenotypic, transplant specific and other miscellaneous risk factors. Methods: 170 liver transplant patients were prospectively reviewed at the Princess Alexandra Hospital in Queensland, Australia. Prevalence of actinic keratosis, Bowen’s disease, basal cell carcinoma and squamous cell carcinoma were reviewed relative to risk factors such as age, skin type, previous ultraviolet exposure, reason and length of transplantation, smoking and alcohol. Results: Actinic keratosis and keratinocyte cancers remain a significant problem in the liver transplant recipients in Queensland. Occupational exposure, skin type and length of immunosuppressive significantly increased the risk of these conditions. Conclusions: The findings from this prospective study suggest that the prevalence of keratinocyte cancers and actinic keratoses in liver transplant recipients despite being lower than heart and lung transplantation recipients remains an inherent problem. Regular skin surveillance and careful optimisation of immunosuppressive medication is necessary to reduce the associated morbidity with these conditions. CHANGES IN ANGIOTENSIN CONVERTING ENZYME 2 ACTIVITY FOLLOWING KIDNEY TRANSPLANTATION Crosthwaite Amy1, Velkoska Elena2, Roberts Matthew3, Burrell Louise2, Ierino Francesco1 1Department

of Nephrology, Austin Hospital, Melbourne, 2Department of Medicine, Austin Hospital, Melbourne, 3Department of Nephrology, Eastern Health, VIC Introduction: Angiotensin converting enzyme 2 (ACE2) is a novel regulator of the renin-angiotensin system that counteracts the adverse effects of angiotensin II. ACE2 activity predicts adverse events and myocardial dysfunction in non-transplant patients with heart failure however there is limited data on the role of ACE2 in kidney transplant recipients. Aim: Study the changes in plasma ACE2 activity in patients undergoing kidney transplantation. Method: This is an ongoing prospective cohort study of patients with end-stage kidney disease undergoing kidney transplantation. Blood Immunology and Cell Biology

collection is performed weekly for 12 weeks and then monthly for 12 months. Serum is transported on ice and aliquots frozen at 70 1C. ACE2 enzyme activity was measured using an ACE2-specific quenched fluorescent substrate assay. The rate of substrate cleavage is expressed as pmol of substrate cleaved per mL of plasma per minute. Values below are expressed as mean ACE2 enzyme activity±Standard Deviation. Results: Analysis of pre-transplant ACE2 plasma activity (n¼12) demonstrated a baseline level of 18.4±13.2 which increased significantly at week one (53.0±27.9) (Po0.05). ACE2 activity in subsequent weeks gradually reduced towards the baseline level – Week 2 ¼ 31.8±11.5; Week 3 ¼ 33.2±21.1; Week 4 ¼ 30.0±15.2; Week 6 ¼ 26.2±15.7; and Week 8 ¼ 20.1±8.5. Further analysis of continuing samples are in progress. Conclusion: The present study demonstrates a significant surge in ACE2 during the critical early post-transplant period with physiological and immunological changes. The clinical implications of this early rise in ACE2 and compensatory regulatory role will be the focus of follow up studies.

Immunosuppression and trials IMMUNOSUPPRESSIVE DRUG PHARMACOKINETICS AND IMMUNE MONITORING IN LONG-TERM KIDNEY TRANSPLANT RECIPIENTS Hope Chris1,2, Fuss Alexander1,2, Coates P Toby1,2, Jesudason Shilpanjali1,2, Heeger Peter3, Carroll Robert1,2 1Centre

for Clinical and Experimental Transplantation (CCET), Central Northern Adelaide Renal and Transplantation Service, Royal Adelaide Hospital, 2Department of Medicine, University of Adelaide, 3Department of Nephrology, The Recanati-Miller Transplant Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA Immunosuppression increases Kidney Transplant Recipients (KTR) risk of cancer up to 60-fold compared to the immune-competent. Current immunosuppressive drug doses are monitored by KTR serum trough levels (C0), which may lead over-immunosuppression. Aims: To determine if a variety of immunological parameters can be used as markers of immunosuppression. Methods: Area Under the drug pharmacokinetic Curve (AUC) was measured in 36 KTR. Donor Specific Antibodies (DSA) were measured by Luminex and T cell responses via IFN-g ELISPOT in 50 KTR. Regulatory T cell (Treg) suppression of CD154/CD40 L expression and CFSE dilution was measured in 12 KTR. Lactate Dehydrogenase (LDH) release assay was utilized to determine Natural Killer (NK) cell function in 32 KTR. Results: 70% of KTR with cancer had Tacrolimus 4100 ng.hr/ml and Mycophenolate AUC 455 ng.hr/ml. Median levels of all KTR were 120 ng.hr/ml and 49 ng.hr/ml, respectively. 16–26% of all KTR had DSA (no statistical differences between groups). IFN-g release was lower in KTR with cancer than from KTR with no cancer. One reason may be because Tregs from KTR with cancer were more suppressive than Tregs from KTR without cancer (median% (range), CD154/ CD40 L: 36(13–73) vs. 13(5–54), P¼0.015 and CFSE: 9(2.5–15) vs. 1.6(1–7), P¼0.001, respectively). Additionally, PBMC from KTR with

Abstracts A13

cancer had lower LDH release compared to controls (0(0–5.5) vs. 1.6(0–10.5, P¼0.037), respectively). Removal of NK cells from PBMC from KTR with cancer reconstituted function, P¼0.008. Conclusions: Immune monitoring identifies that KTR with cancer are over-immunosuppressed and may benefit from immunosuppression reduction. PHENOTYPIC ANALYSIS OF LYMPHOCYTE SUBSETS IN RENAL TRANSPLANT RECIPIENTS Dave Vatsa1, Ierino Frank1, Roberts Matthew2, Gregory Debbie1, Paizis Kathy1 of Nephrology, Austin Hospital, Melbourne, 2Department of Nephrology, Eastern Health, VIC 1Department

Introduction: Lymphocyte depletion is a recognized complication of modern era immunosuppression used in renal transplant recipients. Aims: To characterize peripheral blood lymphocyte subsets in renal transplant recipients. Methods: Patients were invited by mail to participate in a study looking at immunological effects of immunosuppression in renal

transplant recipients for whom we were the caring centre in 2013. Flow cytometry for lymphocyte subsets was performed, and the results analyzed in all patients who consented to participate. The data was correlated with baseline characteristics at the time of transplantation and at the time of testing. Results: 148 patients consented to participate, with mean patient age of 55 years at the time of the study, 18% of patients were lymphopenic (total lymphocytes o1000 cells/mL). Majority of patients (128/148) exhibited CD19+ lymphocyte depletion. In addition, the severity of CD19+ lymphocyte depletion was profoundly low, with a median count of 75 cells/mL (range 3–189 cells/mL). Increasing age and time post transplantation were positively correlated with CD19+ lymphocyte depletion. Of the 148 patients, 49% of patients had low CD3 counts (o1090 cells/mL), 51% of patients had low CD4 counts (o650 cells/mL) and 33% of patients had low CD8 counts (o330 cells/mL). Conclusions: This study demonstrates significant depletion in all lymphocyte subsets in renal transplant recipients; in particular, significant depletion of CD19+ lymphocytes suggests B-cell depletion. This data underscores the need for further work to investigate the clinical correlates and sequelae of the lymphocyte depletion. EQUIVALENCE OF LOW-DOSE TACROLIMUS AND STANDARD EVEROLIMUS ON RENAL FUNCTION – LACK OF CHANGE IN RENAL FUNCTION FOLLOWING CONVERSION

CD3+ CD4+ CD8+ CD19+

Average count, cells/mL (+/standard deviation)

Median count, cells/mL

Normal range, cells/mL

Ooi KY1, Hughes P2, Cohney S1,3

1260 (+/630) 724 (+/369)

1090 643

1090–3020 650–2000

of Nephrology, Western Hospital, Melbourne, 2Department of Nephrology, Royal Melbourne Hospital, 3University of Melbourne

497 (+/336) 103 (+/102)

415 75

330–1310 190–550

1Department

Background: Clinical trials of conversion from CNIs to mTORi have failed to show a consistent reduction in IFTA. However, a significant

Table 1 Patient Data

Pre-Cross Over Avg. Creatinine Changeover 1: Tacrolimus-Everolimus Time Since Transplant Pre-Crossover Tac. Level, Creatinine Post-Crossover Eve. Level, Creatinine

Patient 1

Patient 2

Patient 3

124.4±4.67

134.1±5.25

94.3±4.28

3 Months 5.8 12.9

3.5 Months 127 128

5.8 6.8

26 Months 128 124

0.8 3.0

90 86

Changeover 2: Everolimus-Tacrolimus Time Since Transplant Pre-Crossover Eve. Level, Creatinine

6.5 Months 3.5

127

Post-Crossover Tac. Level, Creatinine

6.7

133

Changeover 3: Tacrolimus-Everolimus Time Since Transplant Pre-Crossover Tac. Level, Creatinine Post-Crossover Eve. Level, Creatinine

34 Months 3.7 3.5

110 109

41 Months 5.2 2.6

34 Months 5.0 2.3

79 86

36 Months 97 111

2.3 9.1

86 78

Changeover 4: Everolimus-Tacrolimus Time Since Transplant

38 Months

Pre-Crossover Eve. Level, Creatinine Post-Crossover Tac. Level, Creatinine

4.2 3.3

81 83

Note: Tacrolimus & Everolimus levels in ng/mL Creatinine in mmol/L

Immunology and Cell Biology

Abstracts A14

improvement in renal function could still reduce cardiovascular disease and improve patient outcomes. Trials comparing renal function in patients on maintenance CNI versus those converting to mTORi have had conflicting results; where improvements have been observed, these occur soon after conversion – reflecting removal of the hemodynamic effect of CNIs. In addition, control arms have often maintained comparatively high CNI levels. We sought to evaluate changes in renal function following conversion from various levels of tacrolimus to everolimus and following conversion from everolimus to low-dose tacrolimus. Methods: Changes in renal function were prospectively monitored in patients without rejection who converted from tacrolimus to everolimus or vice-versa. Results: To date, three patients have been evaluated, all having received kidneys from living donors. No patient experienced DGF, rejection or infection. All patients underwent protocol biopsies prior to conversion with no rejection or significant IFTA. As shown in table 1 there was minimal change in renal function following conversion to everolimus after higher pre-conversion tacrolimus levels, and no decline when patients returned from everolimus to low-dose tacrolimus. Conclusions: No significant difference was demonstrated when changing from low-dose tacrolimus to everolimus or vice versa. Further studies need to be undertaken to confirm the equivalence of low-dose tacrolimus and everolimus with respect to renal function. PHARMACOLOGY OF CYCLOSPORINE IN AN OVINE MODEL OF KIDNEY ALLOGRAFT REJECTION Lett Bron1,2, Julie Johnson2, Russell Christine H3, Olakkengil Santosh3, Coates P Toby2,3 of Medicine, University of Adelaide, 2Centre for Clinical and Experimental Transplantation, Royal Adelaide Hospital, 3Department of Renal Medicine, Royal Adelaide Hospital 1Department

Objective: We sought to develop a clinically relevant large animal model of kidney rejection that responds to the application and withdrawal of immunosuppressant drugs in a predictable manner. Methods: 2 year old sheep were MHC mismatched and underwent a kidney transplant and bilateral nephrecotomy. The donor kidney was placed under a skin slap on the neck and anastomosed to the jugular and carotid artery while the ureter was externalized through the skin flap. The sheep was then dosed with cyclosporine for 7 days with the drug being withdrawn on day 8. Blood samples were taken daily and biopsies of the kidney performed every second day. Results: Serum creatinine and urea stayed at an acceptable level for the first 7 days post transplantation, upon withdrawal of immunosuppression there was a rapid increase in both serum creatinine and urea consistent with a failing kidney. Likewise, Banff scores from the biopsies were stable for the first 7 days and saw a rapid spike upon withdrawal of cyclosporine as the kidney underwent rejection. Conclusions: From these initial findings we are confident in the value and practicality of the sheep as a model for kidney allograft rejection due to its ability to be MHC mismatched, the ability to use clinically relevant pharmaceutical intervention, and the predictable and pronounced rejection that is able to be easily monitored. Immunology and Cell Biology

Antibodies ABO INCOMPATIBLE KIDNEY TRANSPLANTATION: A SINGLE CENTRE EXPERIENCE Wyburn Kate1,2, Mansell Ainslie1, Gracey David1,2, Clayton Philip1,2, Verran Deborah3, Chadban Steven1,2, Eris Josette1,2 1Department

of Renal Medicine, Royal Prince Alfred Hospital, Sydney, of Medicine, University of Sydney, 3Department of Surgery, Royal Prince Alfred Hospital, Sydney 2Department

Background: ABO-incompatible (ABOi) kidney transplantation is now an established practice. We report our experience of 42 consecutive ABOi transplant candidates. Results: From July 2007 to October 2013, the 42 consecutive patients underwent immunoadsorption to achieve ABO-Ab titrer1:8, induction with IVIG, steroids and rituximab (in the first 18 patients), then tacrolimus, mycophenolate and steroid maintenance. Forty-one ABOi transplants were performed, one patient was not transplanted due to inability to achieve ABOi titrer1:8. Recipients were (73%)male, median age 43 (range 18–69). Donors were most commonly blood group A(74%) and recipients group O(73%). Median HLA mismatch was 4/6, and CDC T and B cell cross-match negative (except one CDC B cell positive, but flow negative with no DSA). Seven patients had Class I DSA, seven had Class II. Median ABOi titre was 1:16 (range 1–128) pre-immunoadsorption and 1:1(range 1–4) pre-transplant. Protocol biopsies were performed at day 10, 3 months and 1 year for the majority of patients. There were 5 rejection episodes over 6 years of follow-up. At 3 months, 1 and 6 years follow-up the median creatinine was 139 umol/l, 130 umol/l, and 142umol/l respectively. Infective complications occurred in 29% of patients, 3(7%) recipients developed biopsy-proven BK nephropathy. There were 3 basal cell carcinomas and 1 patient developed post-transplant lymphoproliferative disease. Death-censored graft survival was 100%, patient survival was 93%; three patients died, one from infective causes, one from suicide 4 years post-transplant, one from cardiovascular disease. Conclusion: Median-term outcomes indicate ABOi kidney transplantation is a safe and effective means to improve access to living donor kidney transplantation. POST-TRANSPLANT MONITORING OF DE NOVO HUMAN LEUKOCYTE ANTIGEN DONOR-SPECIFIC ANTIBODIES IN RECIPIENTS OF KIDNEY PAIRED DONATION TRANSPLANTS Viecelli Andrea1, Fidler Samantha2, Woodroffe Claudia1, D’Orsogna Lloyd2, Ferrari Paolo1 1Department of Nephrology, Fremantle Hospital, WA, 2Department of Clinical Immunology, Royal Perth Hospital

Aims: De novo donor-specific HLA antibodies (dnDSA) developing post-transplant are a major cause of late graft loss and often precede renal dysfunction. HLA mismatch is a predictor for the development of dnDSA. In the Australian paired Kidney Exchange (AKX) program HLA matching rules are ignored in favour of a virtual crossmatch. Whether this approach is associated with increased incidence of dnDSA is unknown.

Abstracts A15

Methods: Post-transplant HLA-antibody monitoring in the AKX program has been offered since April 2011. Recipients’ sera collected at 1, 3, 6 and 12 months during the first post-transplant year and yearly thereafter were tested by Luminex Single Antigen Bead (SAB) assay. Results: As of 31 December 2013 there were 60 patients with at least 3 months follow-up. HLA monitoring data was available for 50 patients, sera from 10 patients were not provided to the HLA laboratory. Of the 50 patients whose SAB data were available, 25 had calculated panel reactive antibody 475%, 29 (58%) had preformed DSA (preDSA) 4500MFI and 7 (14%) had preDSA 42000MFI to the matched donor. After transplantation preDSA became undetectable (o500MFI) in 24 patients, persisted in 3 and increased in strength in 2. The onset of dnDSA with MFI 4500 was documented in 5 patients, including the 1 without preDSA, 2 with increase in preDSA and 2 who cleared their preDSA. Conclusions: Among mostly sensitised recipients of a kidney paired donation transplant, development of dnDSA early after transplantation is observed in approximately 10% of cases. The relationship between the course of preDSA and dnDSA and graft function and survival requires further analysis. HLA BROAD ANTIGEN AND EPLET MISMATCHES IN DETERMINING THE RISK OF ACUTE REJECTION AFTER KIDNEY TRANSPLANTATION Hung1,

Wai1,2,

Samantha3,4,

Do Nguyen Lim Fidler Chapman Jeremy R5, Craig Jonathan C5,6, Ferrari Paolo1,7, D’Orsogna Lloyd3,4, Irish Ashley1,8, Wong Germaine5,6 1School of Medicine & Pharmacology, University of Western Australia, Perth, 2Department of Renal Medicine, University of Western Australia, Perth, 3School of Pathology and Laboratory Medicine, University of Western Australia, Perth, 4Department of Clinical Immunology, Royal Perth Hospital, 5Centre for Kidney Research, The Children’s Hospital at Westmead, Sydney, 6School of Public Health, University of New South Wales, Sydney, 7Department of Renal Medicine, Fremantle Hospital, WA, 8Department of Renal Medicine, Royal Perth Hospital

Background: HLAMatchmaker is a computer algorithm that determines structurally-based HLA-antigen compatibility at the epitope level and may better predict the immunological risk of transplant recipients compared to broad HLA antigens matching. Aim: To determine the clinical relevance of HLA-ABDR broad antigen and eplet matching for predicting biopsy-proven acute rejection (BPAR) after kidney transplantation. Methods: Donor-recipient eplet mismatches at HLA-ABDR were calculated for all kidney transplant (n¼644) recipients in Western Australian between years 2000 and 2010. The test performance characteristics of HLA broad antigen and eplet matching for predicting 12-month BPAR were examined using the Receiver Operating Characteristic (ROC) analysis. Results: Of the 644 kidney transplants recipients over a mean followup period of 4 (SD¼3) years, 179 (28%) experienced BPAR. Recipients with higher number of HLA-ABDR broad antigen mismatches (0:17%; 3:35%: 6:30%) or HLA-ABDR eplet mismatches (0–2:19%; 3–9:28%; 10–19:25%; 420:34%) experienced rejection. The area under the ROC curves (95%CI) for broad antigen (ABDR), eplet (ABDR), broad antigen (DR), eplet (DR) and the combined broad

antigen-eplet mismatches were 0.54 (0.49–0.59), 0.54 (0.49–0.60), 0.58 (0.52–0.62), 0.57 (0.52–0.62) and 0.61 (0.56–0.66), respectively. Compared with HLA-ABDR broad antigen matching, the combined HLAbroad antigen-eplet model better predicted BPAR compared to the HLA-ABDR broad antigen model alone (P¼0.01). The optimal balance between test sensitivity and specificity (59% and 44%) of the combined model was achieved using a combined HLA-ABDR (3) and eplet (20) mismatch cut-point. Conclusion: Inclusion of eplet matching to the current allo-cation algorithm may improve the discriminatory value for predicting BPAR in kidney transplant recipients compared with broad antigen matching alone. ATSI RENAL TRANSPLANT RECIPIENTS HAVE HIGHER LEVELS OF HLA AND EPITOPE MISMATCHES COMPARED WITH NON-ATSI RECIPIENTS Fidler Samantha1, Swaminathan Ramyasuda2,3, Irish Ashley2,3, D’Orsogna Lloyd1,4 1Transplantation Immunology Laboratory, University of Western Australia, Royal Perth Hospital, 2Renal Transplant Unit, Royal Perth Hospital, 3Department of Medicine, University of Western Australia, Perth, 4Department of Clinical Immunology, Royal Perth Hospital

Background and Aim: HLA (A, B and DR) typing is conventionally used to allocate deceased donor kidneys. The use of Epitope matching and acceptable mismatch programs have potential to improve immunological compatibility. We evaluated HLA and epitope matches within a cohort of renal transplant recipients to determine if there was a difference between ATSI and non-ATSI recipients. Methodology: Retrospective analysis of all consecutive deceased donor transplants (n¼50, 17 ATSI & 33 non-ATSI) between Jan 2005 and December 2007. HLA class I (A,B,C) and Class II (DP, DQ and DR) mismatches and epitope mismatching using the HLA Matchmaker of all donor recipient pairs were analysed. Results: There was no difference in age and sex distribution between the ATSI and Non-ATSI recipients. However the ATSI had higher number of mismatches for both class I and Class II HLA and by epitope. These patients were followed up for a median of 6 years. 9/17 (53%) of ATSI and 9/33 (27%) of Non-ATSI had graft loss during the follow-up. (P¼0.02). Conclusion: ATSI renal transplant recipients had higher level of epitope mismatches at all loci. Whether the poor graft outcomes in this population could be attributed to higher epitope mismatches, rendering them an immunological disadvantage, warrants further study in a larger study.

Mismatches

ATSI

NON ATSI

P-value

HLA Class I (A,B,C) Epitope Class I (A,B,C)

3.8 18.8

3.1 11.2

NS 0.03

HLA Class II (DP DQ DR) Epitope Class II (DP DQ DR)

4.3 29.2

3 20.2

0.04 0.05

Total HLA I&II Total Epitope I&II

8.1 48

6.1 31.4

0.04 0.03

Immunology and Cell Biology

Abstracts A16

DIAGNOSING AND TREATING ANTIBODY MEDIATED REJECTION IN LIVER TRANSPLANT RECIPIENTS: WHAT CAN LUMINEX TELL US? Newman Allyson1, McCaughan Geoff2, Strasser Simone2, Shackel Nick2, Crawford Michael1, Dilworth Pamela1, Lackey Gavin1, Berberovic Bessie1 Services, Royal Prince Alfred Hospital, Sydney, 2AW Morrow Gastroenterology & Liver Centre, Royal Prince Alfred Hospital, Sydney 1Transplantation

Background: The importance of tissue typing and the potential for antibody mediated rejection (ABMR) in liver transplantation has not always been recognized. With emerging research from international units highlighting its potential role in diagnosis and management of ABMR, we decided to examine the local experience. Aim: The aim of the study was to examine cases of ABMR to determine if the presence of donor specific antibodies (DSA’s) pre and post transplant were of assistance to the diagnosis and management of ABMR. Method: A retrospective analysis of all liver transplants (LT) performed in a single centre between January 1st 2010 and August 31st 2013 was conducted through auditing of databases and medical records. Paediatric patients, multi-organ transplant recipients and those who had previously received a non-liver transplant were excluded from the analysis.

Results: Of the 228 LT performed, 53 grafts have experienced a rejection episode. Two of these were classified as ABMR resulting in acute graft loss in one patient and Chronic ductopenic rejection in the other. The first patient had evidence of ABMR on biopsy and within the failed allograft, which revealed arterial inflammation and fibrin deposition with confluent hepatic necrosis. The second case displayed features of acute rejection, hepatocyte loss and C4d endothelial staining progressing over 12 months to ductopenic chronic rejection with continued endothelial C4d staining. Post transplant DSA’s were measured in these two patients. Both reported the presence of high level Class 2 antibodies, predominately DQ, with mean fluorescence index greater than 10,000 (Table 1). Conclusions: In these 2 patients, post transplant Luminex studies were of assistance in confirming diagnosis and monitoring of antirejection treatments administered. This study highlights the importance of ensuring appropriate and timely post transplant Luminex studies are performed on all LT patients when unusual allograft function is suspected. PROSPECTIVE STUDY OF THE NATURAL HISTORY AND CLINICAL SIGNIFICANCE OF DE NOVO DSA POST RENAL TRANSPLANTATION IN THE FIRST 3 MONTHS Saunders John1, Yin Jianlin2, Wineholt Louise3, Wyburn Kate1, Eris Josette1 1Statewide

Renal Service, Royal Prince Alfred Hospital, Sydney, of Renal Medicine, Royal Prince Alfred Hospital, Sydney, 3Department of Immunology, Royal Prince Alfred Hospital, Sydney 2Department

Table 1 Breakdown of patient results Patient 1

Patient 2

Pre LT Luminex Positive class 1 + 2 DSA present pre LT Cw15(1638)

Positive class 1 Nil

1st LT X-match Mismatch

Not performed 5/6

Negative 4/6

ABO Post LT DSA

Identical DQ7(12119)

Identical DR53(10735)

DQ9(11656) DQ8(4388)

DQ8(16269) DQ7(13724) DQ9(14857) DR9(1437)

Treatment 2nd LT X-match

Rituxumab+Urgent re transplant IVIg- ongoing B-cell positive NA

Mismatch ABO

3/6 (repeat DQ3 mismatch) Identical

NA NA

Post LT DSA

DQ7(7280) DQ9(5533)

NA

DQ8(1654) Cw1(857) Cw15(1085) Cw12(570) Treatment Phoresis and IVIg Repeat Post LT DSA DQ7(4007)

NA NA

DQ9(3445) CW15(725) Status

Alive with functioning graft

Immunology and Cell Biology

Alive with functioning graft

Background: Luminex technology enables identification of anti-HLA donor specific antibodies (DSA) at low levels. The role of DSA monitoring is unclear and the natural history of DSA production is not well delineated. Aims: To examine the development of de novo DSA and correlate with clinical outcomes in the first 3 months post renal transplant. Methods: Single centre prospective observational study of patients transplanted 2010–2012 with follow up to 43 months. Blood was taken at days 0,7,21 and 90 post transplant and analysed for DSA by luminex. Rejection episodes were confirmed histologically and all patients underwent three month protocol biopsy. Results: 73 patients mean age 47.7 yrs were studied; 71% were male; 13% were retransplants. 11(15%) patients developed de novo DSA in samples at day 7, 21 or 90. Of these 3(27%) developed cell mediated rejection and 1(10%) developed antibody mediated rejection. In 62 patients without de novo DSA 9(14%) developed cell mediated rejection and 1(1.6%) developed antibody mediated rejection (AbMR). There was no graft or patient loss. Severity of rejection and graft function at three months were similar. No patients with de novo DSA were C4d positive on protocol biopsy. Conclusions: Development of de novo DSA in the first 3 months post renal transplant was not common and was not associated with increased AbMR. The clinical utility of monitoring for DSA early post transplant remains unclear and warrants larger cohort studies. Study of the behavior and significance of de novo DSA in the longterm is ongoing.

Abstracts A17

de novo DSA (n¼11)

No DSA (n¼62)

Recipient Male Female Age (mean) Donor Deceased Living Age (mean)

9 2 47.8

6

43 19 47.45

35

5 41.2

27 47.9

3 2

2 1

1 2

8 2

58 3

3

1

1

HLA Mismatch (Median) Class 1 Class 2 Graft Number

Cell mediated rejection borderline

1

4

1a 1b

1

1 1

2a 2b

1

2 1

1

1

3 Antibody mediated rejection 3 month creatinine mmol/L (mean)

protein abundance was calculated using DeCyder software and further interrogated by the STRING network program. Results: Delayed graft function occurred in 25% and biopsy proven acute rejection in 17% of patients. From the 3 month urine, 9 proteins including kininogen, MASP2 and cystatin-M correlated to fibrosis (5 with Banff, 4 with CADI) and 2 proteins correlated with delta eGFR. The top KEGG pathway in the STRING network was coagulation and complement cascades. Follow-up 12 month urine analysis based on the same chronic damage groups revealed 11 proteins, 4 identified in the previous 3 month analysis and 7 new proteins, including CD14 and b2 microglobulin. The top KEGG pathway at 12 months was antigen processing and presentation. Conclusions: Using traditional proteomics, we have identified potential non-invasive biomarkers that are predictive of chronic damage. The dominant urine proteome alters between 3 and 12 months. While early urine protein signatures predict subsequent damage, validation in larger and more diverse cohorts is required. THE PREVALENCE OF FRAILTY IN ADVANCED HEART AND LUNG FAILURE Montgomery Elyn1, Hannu Malin1,2, Carter Danielle3, De Tullio Nicole1, Shaw Sara1, Cooper Kerrie1, Wilhelm Kay1,4, Havryk Adrian1, Macdonald Peter Simon1,5 1Heart

165

145.5

Complications – other PROTEOMIC ANALYSIS IDENTIFIES A SPECIFIC EARLY URINE PROTEIN SIGNATURE FOR THE DEVELOPMENT OF CHRONIC ALLOGRAFT INJURY Hor Katie1,2, Diefenbach Eve3, Anderson Patricia4, Alexander Steve5, Nankivell Brian4, Chapman Jeremy4, Rosales Ivy6, Colvin Robert6, Murphy Barbara7, Webster Angela4, O’Connell Philip1 1Centre

for Transplant and Renal Research, Westmead Millennium Institute, Westmead Hospital, Sydney, 2School of Medicine, University of Sydney, 3Proteomics Facility, Westmead Millennium Institute, Westmead Hospital, Sydney, 4Department of Renal Medicine, Westmead Hospital, Sydney, 5Centre for Kidney Research, The Children’s Hospital at Westmead, Sydney, 6Department of Pathology, Massachusetts General Hospital, Boston, 7Department of Nephrology, Icahn School of Medicine at Mount Sinai, New York

& Lung Transplant Unit, St Vincent’s Hospital, Sydney, Therapy Department, St Vincent’s Hospital, Sydney, 3Faculty of Medicine, Notre Dame University, Sydney, 4Department of Psychiatry, St Vincent’s Hospital, Sydney, 5Transplantation Laboratory, Victor Chang Cardiac Research Institute, Sydney 2Occupational

Aim: Frailty is common in elderly patients with advanced heart and lung failure. The aim of this study was to assess the prevalence of frailty in younger patients referred for transplantation. Methods: Patients referred for heart (H) or lung (L) transplantation underwent frailty assessment using a modified Share FI instrument (based on the Fried Index). Frailty was defined as three or more of the following; weak grip strength, slowed walking speed, poor appetite, physical inactivity and exhaustion. In addition all patients underwent cognitive assessment and depression screening. Markers of disease severity were also obtained. Results: 105 patients (60 H, 45 L, 59 men, 46 women, mean age 50±1 (range 16–73) years) underwent frailty assessment.: 21 (20%, 16H, 5 L) patients were classified as frail, 54 (51%, 29H, 27 L) pre-frail and 30 (29%, 15 H, 15 L) non-frail. Frail patients were more likely to be anaemic, hypoalbuminaemic and cognitively impaired (all Po0.05 versus other groups). Frail patients had markedly worse survival: 57±17% at 6 months versus 93±3% for pre-frail and 94±5% for

Aim: To correlate early urine proteins with kidney transplant histology and function, to assess biomarker potential. Method: Patients were selected from the GoCAR study who had urine samples at 3 and 12 months and protocol biopsies at 0, 3, and 12 months. Patients were grouped by chronic damage scores using Banff and Chronic Allograft Damage Index (CADI) from 12 month biopsies or delta estimated glomerular filtration rate (eGFR). Urine proteins at 3 and 12 months associated with chronic damage at 12 months were identified by 2D-DIGE and tandem mass spectrometry. Differential Immunology and Cell Biology

Abstracts A18

non-frail patients (Po0.005, Figure 1). Frailty was not associated with age, gender, renal function or disease severity. Conclusions: Although some degree of frailty is common in advanced heart and lung failure, only a minority fulfil criteria for the frailty phenotype. The presence of frailty is associated with a very poor prognosis. Interestingly, the frailty phenotype is unrelated to age or disease severity in this population. SIROLIMUS-INDUCED IMMUNE MEDIATED TOXIC DEMYELINATING POLYRADICULONEUROPATHY IN A RENAL TRANSPLANT PATIENT Muthucumarana Kalindu1, O’Connor Kevin2, Nolan David2, John Mina2, Swaminathan Ramyasuda1, Leong Wai2, Irish Ashley1 1Nephrology and Renal Transplant, Royal Perth Hospital, 2Combined Neuroimmunology Clinic, Royal Perth Hospital

Case: We present a case of severe, late-onset and progressive neuropathy related to mTOR (Sirolimus) exposure. Patient: A 51 year old woman with reflux nephropathy received a deceased donor transplant in 1994 at the age of 30 years. Due to early DGF and BPAR she was managed with OKT3 (14 day course) and maintenance therapy with Cyclosporine, Azathioprine and Prednisolone. She achieved excellent and stable graft function (creatinine 74) and withdrew from steroids after 10 years. In 2005 following biopsy showing CAN grade 2 she converted to Sirolimus 2 mg per day with levels ranging 3.2 to 6.8 and Azathioprine 50 mg. Six years after conversion (2011) the patient developed progressive neurological and systemic symptoms. She exhibited truncal weakness with associated moderate to severe weakness of the proximal and distal legs characterized by inability to roll in bed, to stand from seating and requiring 2 walking sticks to ambulate, with associated upper limb weakness in a similar distribution. The course was subacute and rapidly progressive over a 24 month period, (clinically and neurophysiologically), with associated recurrent night sweats, fever, weight loss and malaise. Thrombocytosis suggested a myeloproliferative disorder but BM was non-diagnostic. An initial diagnosis of CIDP was treated with a sustained trial of high dose IVIG without benefit and after excluding paraneoplastic disorders a superficial radial nerve biopsy demonstrated a chronic neuropathy with focal sparse epineural perivascular inflammation. The patient was commenced on high dose prednisolone with subsequent improvement in overall functional ability and improvement in systemic features. Sirolimus was also discontinued resulting in further dramatic improvement of the neuropathy and other symptomatology, including the persistent 18-month history of night sweats, which abated within days. Repeat testing confirmed a myeloproliferative disorder (thrombocythemia) managed expectantly. After discontinuation of Sirolimus nephrotic-range proteinuria has developed while the graft function remains stable. Conclusion: This is the first case of late-onset inflammatory neuropathy in a renal transplant patient associated with a novel immune mediated toxic neuropathy secondary to Sirolimus. SINGLE CENTRE EXPERIENCE OF TREATMENT OUTCOMES OF RENAL TRANSPLANT RECIPIENTS WITH TRANSPLANT GLOMERULOPATHY Vilayur Adinarayanan Sundareswari1,2, Trevillian Paul1, Mi Choi Young1 1Nephrology 2School

and Renal Transplant, John Hunter Hospital, Newcastle, of Medicine and Public Health, University of Newcastle

Immunology and Cell Biology

Background: Transplant Glomerulopathy (TG) is a manifestation of chronic antibody mediated rejection characterized by re-duplication of glomerular capillary basement membranes. Prognosis of TG is quite poor and treatment options are limited. We have been treating selected patients using a protocol comprising Rituximab, Plasma exchange and IvIg. Aim: To study the clinical characteristics and treatment outcomes of patients with TG. Methods: This is a retrospective cohort study of patients with diagnosis of TG. All patients with a biopsy diagnosis of TG with cg score4¼1 were included. Patients with recurrent MCGN or thrombotic microangiopathy were excluded. The serial eGFRs and protein/ creatinine ratios were collected and pre and post treatment results compared. Results: Since 1983, 38/560 (6.8%) patients developed TG after an average of 112.8 months post-transplant. 22 patients were treated and 16 untreated. Of 22 treated cases (mean age 49 yrs), 12(54%) maintained or improved GFR to a mean of 28.4 ml/min/1.73m2 after 32 months, 9 progressed to ESRD after an average 24months, and 1 person died with good function. Overall the pre-treatment mean slope of GFR (2.66 ml/min/1.73m2/year) reduced by 0.77 ml/min/1.73m2/ year with treatment, an improvement of 29% (NS P¼0.27). The pretreatment mean UPCR was 327 mg/mmol Cr; decreased post-treatment to 187.7 mg/mmol, an improvement of 43% (NS P¼0.10). Out of the 16 untreated patients, 10 have reached ESRD, 4 died and 2 maintain stable eGFR. Conclusion: Diagnosis of TG is associated with poor prognosis; treatment with Rituximab, Plasma exchange and IvIg can stabilize or improve graft function in a significant subset of patients. The predictive characteristics of this group need to be further defined.

RENAL TRANSPLANT IN HIV PATIENTS – A CASE STUDY Aouad Leyla1, Gracey David1,2, Eris Josette1,2, Chadban Steven1,2, Wyburn Kate1,2 1Department 2Department

of Renal Medicine, Royal Prince Alfred Hospital, Sydney, of Medicine, University of Sydney

Background: The prevalence of ESKD in Human Immunodeficiency Virus (HIV)-infected patients is increasing. Kidney transplantation is a viable treatment in carefully selected patients with comparable outcomes to the general transplant population. However transplant poses several challenges including potential increased risk of infection, rejection and drug interactions. A 58-year-old HIV positive Caribbean man with ESKD received a deceased donor kidney transplant in April 2013. He had stable HIV since 1994 on Kaletra (Lopinavir/Ritonavir), Lamivudine and Didanosine with an undetectable viral load and normal CD4 counts. Immunosuppression included induction methylprednisolone and basiliximab and maintenance prednisone, mycophenolate mofetil and significantly reduced dose tacrolimus due to recognized interaction with Ritonavir. Prophylaxis included valgancylcovir, Bactrim, Nystatin and Azithromycin. The immediate post transplant period was complicated by delayed graft function. Despite the low dose, persistently supratherapeutic tacrolimus levels (430 ng/ml) necessitated a change in anti-retroviral therapy from Kaletra to Raltegavir resulting in reduction of tacrolimus levels and initiation of graft function. Later infective complications included disseminated strongyloides, treated with ivermectin and CMV ileitis treated with gancylcovir and CMV IVIG. Severe malnutrition asso-

Abstracts A19

Background: It is recommended that genetic screening in patients with atypical haemolytic uraemic syndrome (aHUS)-related end-stage renal disease be undertaken prior to listing for transplantation. However, cost and limited availability precludes routine screening. Furthermore, up to 30–40% of patients with aHUS do not have identifiable genetic mutations. Previous allograft failure from recurrent aHUS is a strong risk factor for disease recurrence in subsequent grafts, suggesting that retransplantation may not be a viable option for many of these patients. Patient: We report a case of a patient with familial aHUS who underwent successful re-transplantation 30-months following his failed first kidney allograft from recurrent aHUS. He achieved excellent graft function on standard maintenance immunosuppression,

Lean sham Lean IR4h Lean 1d Lean IR3d

500

sh am Le an IR 4h Le an 1d Le an IR 3d

0

Lean

Lean

Lean

TNFa 1.8

TLR4 1.8

2000 1000

N or N mN or m O N sha or m m N 15m or m 1 N or h m 4 N or h m 1d

0

IR 3d

IR 1d

H FD

IR 4h

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sh am

H FD

H FD

IR 3d

IR 4h

IR 1d

H FD

H FD

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500 0

sh am

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HFD sham HFD IR4h HFD IR1d HFD IR3d

1000

IL-6 in serum 5000 HFD fed pg/ml

3000

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4000 3000 2000 1000 0

N or m H FD NO s H ham FD 15 H FD m 1h H FD h 4h H FD 1d

4000

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IL-6 in serum 5000

HFD sham HFD IR4h HFD IR1d HFD IR3d

800

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sh am H FD IR 4h H FD IR 1d H FD IR 3d

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HFD sham HFD IR4h HFD IR1d HFD IR3d

300

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400

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sh am

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IL-6 1.8 HFD sham HFD IR4h HFD IR1d HFD IR3d

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sh am Le an IR 4h Le an 1d Le an IR 3d

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400

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800 Conc

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sh am Le an IR 4h Le an 1d Le an IR 3d

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Chow fed pg/ml

Lean sham Lean IR4h Lean 1d Lean IR3d

Le an

Lean 1d Lean IR3d

400

Introduction: Fatty livers suffer more ischaemia/reperfusion injury (IRI) compared to lean. Influencing mechanisms responsible could have favorable postoperational outcome. Materials and Methods: Fatty liver simulated by feeding mice with a High Fat Diet (HFD-45% of calories from fats) for 14 weeks. The control group received standard chow. Partial warm liver ischaemia

Le an

600

1Collaborative Transplant Group, University of Sydney, 2Bosch Institute, Central Clinical School, University of Sydney

IL-6 1.8

300 Conc

800 Conc

400

Lean sham Lean IR4h

Ganbold Anar1, Habib Miriam1, Cunningham Eithne1, Paul Moumita1, Ghoraishi Tina1, Bishop Alex1, McLennan Susan2, Sharland Alexandra F1

Conc

IL-1b 1.8 1000

AMPLIFICATION OF INNATE IMMUNE RESPONSES IN HIGH FAT DIET-FED MICE UNDERGOING WARM LIVER ISCHAEMIA/ REPERFUSION INJURY

1d Le an IR 3d

and Renal Transplant, Sir Charles Gairdner Hospital, Perth, 2School of Public Health, University of Sydney, 3Centre for Kidney Research, Westmead Hospital, Sydney, 4Centre for Transplant and Renal Research, Westmead Hospital, Sydney, 5Renal & Transplantation Unit, Sir Charles Gairdner Hospital, Perth, 6School of Medicine & Pharmacology, University of Western Australia, Perth

IR 4h

1Nephrology

Ischaemia reperfusion injury

Le an

Chua Samantha1, Wong Germaine2,3,4, Lim Wai5,6

Le an

THE IMPORTANCE OF GENETIC MUTATION SCREENING TO DETERMINE RETRANSPLANTATION FOLLOWING FAILED KIDNEY ALLOGRAFT FROM RECURRENT ATYPICAL HEMOLYTIC UREMIC SYNDROME

with no evidence of clinical or histopathological disease recurrence twelve months after his second deceased donor kidney transplant. Genetic screening prior to re-transplantation identified the patient as having a newly discovered mutation, c.T3566A, within Exon 23 of the Complement Factor H (CFH) gene. Conclusion: The decision for re-transplantation must balance between the risk of disease recurrence and greater risk of death on dialysis. The absence of a more severe CFH genotype assisted in the decision for re-transplantation and suggests the importance of genetic mutation screening in order to stratify the risk of disease recurrence and graft loss versus the benefit of transplantation.

Conc

ciated with recurrent infections required TPN and NG feeding. HIV therapy was modified from didanosine to etravirine, due to development of significant leucopoenia. The patient is now stable with a creatinine of 90umol/L, undetectable HIV viral load, but ongoing low CD4 counts. Conclusions: This case highlights the challenges in transplanting HIV positive patients and the importance of close involvement with immunologists and a multidisciplinary team. We now use a Ritonavir free antiviral regime prior to transplantation in our patients.

Figure 1 PCR results for IL-1b, IL-6, TNFa, TLR4 and serum IL-6 level. Immunology and Cell Biology

Abstracts A20

was induced by ligation of the portal vein branch supplying the left and median lobes for 30 minutes. Serum and tissue samples were harvested after 15 minutes, 1 and 4 hours, 1 and 3 days postreperfusion (n¼5). ALT, HMGB1, and proinflammatory cytokines were measured in serum. Expression of TLR4, RAGE, and various cytokines was assessed using RT-qPCR. Results: IR resulted in greater serum levels of ALT (25906+/1579U/l in HFD against 4694+/1539U/l in lean at 4 h, P¼0.0169), HMGB1 (230.2+/35.13 v/s 35.16+/13.04 at 4 h, P¼0.0318), and IL-6 (3768+/490.3 v/s 625.7+/36.56 at 1 h, P¼0.0282) in serum, IL1 (712.1+/125.7 v/s 271.9+/66.24 at 4 h, P¼0.0159), IL-6 (269.4+/116.5 v/s 10.33+/2.946 at 4 h, P¼0.0286) and TNFa (627.4+/282.8 v/s 76.44+/60.74 at 4 h, P¼0.0286) in liver in the HFD group. TLR4 expression in the HFD group on d1 was 657.7+/479.9, v/s 13.34 +/9.042 in lean, P¼0.057). KC expression was significantly higher in the lean group (143.7+/16.03 lean against 38.99+/9.516 in HFD at 4 h, P¼0.0159). Conclusions: HFD livers post-IR damage was associated with higher HMGB1, TLR4 and downstream cytokines. Competitive antagonism of TLR4 and RAGE may attenuate tissue damage in fatty livers and improve outcome following liver transplantation. IN VIVO CLINICAL ASSESSMENT OF KIDNEY TRANSPLANT MICROCIRCULATION USING SIDESTREAM DARK FIELD IMAGING: AN INNOVATIVE METHOD TO MONITOR EARLY GRAFT FUNCTION Pulitano Carlo1,2, Debiasio Ashe3, Ho Phong1, Hopkins Roy1, Coker David1, Alyami Ali1, Sandroussi Charbel1, Joseph David1, Verran Deborah1, Crawford Michael1, Shackel Nick1,2, Allen Richard1 1Transplantation

Services, Royal Prince Alfred Hospital, Sydney, Institute of Cancer and Cell Biology, University of Sydney, 3Transplantation Services, University of Sydney 2Centenary

Introduction: Ischaemia reperfusion is known to affect microcirculation. Imaging of the human kidney microcirculation in real-time has the potential to assess injuries and risk of early graft failure. The aim was to evaluate the feasibility of studying the kidney microcirculation in vivo using, a new infrared technology, Sidestream dark-field (SDF) imaging. Methods: Twenty patients undergoing living (N¼11) and deceased (N¼9) kidney transplantation were studied. Images were obtained using SDF imaging 5 min after kidney revascularization in 2 different areas of upper and lower poles. Microcirculation measurement included vessel density, and two indices of vessels flow. Using these parameters, a microvascular damage score (MDS) was created. The MDS was correlated with Patient Hemodynamics and anesthetics parameters. Graft injury was determined by serial measurement of creatinine, eGFR, Cystatin C, and endothelin-1 for the first week. Results: MDS correlated significantly with all markers of renal function and damage between day 1 and 5. Microvascular perfusion was 38% lower in deceased kidneys compared with living donor. Cadaveric grafts had significantly higher MDS (Po0.0001) and worse renal function. Patients requiring dialysis within 5 days had higher MDS (Po0.0001), associated with smaller vessel diameter and heterogeneous perfusion pattern with oscillating or absent flow. Conclusions: SDF provides a reliable technology for measurement of in vivo microcirculation of the transplant kidney. MDS correlated with

Immunology and Cell Biology

subsequent graft function and need for dialysis. This technology will be an important research and clinical tool to select patients that will benefit from pharmacologic agents targeting kidney transplant microcirculation. THE NOVEL ADENOSINE RECEPTOR AGONIST VCP746 REDUCES RENAL ISCHAEMIA REPERFUSION INJURY Seibt Benjamin1,2, Lu Bo1, Fang Doreen1,2, Roberts Veena1,2, May Lauren3, Cowan Peter1,2, Dwyer Karen1,2 1Immunology

Research Centre, St Vincent’s Hospital, Melbourne, of Medicine, University of Melbourne, 3Department of Pharmacy, Monash University, Melbourne 2Department

Background: The activation of the adenosine A2B receptor (A2BR) is protective in renal ischaemia reperfusion injury (IRI). Clinically the use of adenosine/adenosine analogues is limited by unwanted systemic effects. VCP746 is adenosinergic compound. Aim: To characterise VCP746 in vitro and assess its efficacy in a mouse model of renal IRI. Methods: Calcium mobilisation studies were performed in stably transfected CHO-A2BR cells. C57Bl/6 wild type mice (n¼3–5) underwent right nephrectomy and 22.0 minutes left renal ischaemia or no IRI (Sham) at 37 1C. Mice received 100 mg/kg VCP746 (10% DMSO/ 90% Saline) or Vehicle (10% DMSO/90% Saline) intravenously 5 min before IRI. 24 hours post IRI mice were sacrificed; kidney harvested for histological analysis and serum creatinine (SeCr) assayed. Results: VCP746 activated the A2BR with similar potency to adenosine (EC50 1290 nM vs. 813 nM, ns). Severe renal injury was induced following 22 minutes ischaemia (SeCr 150±27 mmol/L) which was significantly attenuated with VCP746 treatment (SeCr 83.8±4.9 mmol/L) Fig 1. No overt unwanted effects were observed with VCP746 pre-treatment. Conclusion: VCP746 is a novel adenosinergic compound with A2BR activity which is well tolerated and attenuates renal IRI.

Figure 1 IRI versus Sham and VCP versus Vehicle: Creatinine levels after right nephrectomy and left IRI for 22.0 min or no IRI (Sham) in C57Bl/6 wild type mice. 100 mg/kg VCP (10% DMSO/90% Saline) or Vehicle (10% DMSO/90% Saline) was intravenously injected (via penile vein). n¼3-5; ttest: *Po0.05; **Po0.01; ***Po0.0001.

Abstracts A21

MESENCHYMAL STROMAL CELLS (MSC) DO NOT PROTECT AGAINST RENAL ISCHAEMIA REPERFUSION INJURY (IRI) IN AN IN VIVO MURINE MODEL Clark Carolyn J1,2,3, Lu Bo4, Gobe Glenda C3, McTaggart Steven J2,3,5 1Department of Renal Medicine, Sunshine Coast Hospital and Health Service, 2Solid Organ Transplant Team, Mater Institute, Brisbane, 3School of Medicine, University of Queensland, Brisbane, 4Immunology Research Centre, St Vincent’s Hospital, Melbourne, 5Department of Medicine, Royal Children’s Hospital, Brisbane

Aim: This study was designed to confirm efficacy of MSC in an in vivo model of renal IRI. Methods: (1) A murine model of bilateral renal IRI was optimised with MSC administered at the time of reperfusion. GFP+ MSC were administered by intravenous (IV), intra-peritoneal or sub-capsular injection. Renal structure and function was assessed at 48 hours and tissues were analysed for GFP+ DNA and stained for immunohistochemistry to assess MSC presence. (2) A novel method of murine intra-arterial (IA) administration of MSC was developed by cannulation of the carotid artery and deposition of the MSC into the aortic arch, after unilateral renal IRI. Results: (1) MSC were preferentially found in the kidney after all modes of administration except IV, where they were predominantly found in the lung. However, there was no difference in serum creatinine, urea or histological score at 48 hours between the vehicle and MSC groups, regardless of the MSC administration method. (2) MSC were found in the lung, kidney, heart and spleen 24 hours after IA administration. The mean serum creatinine was 80 mmol/l±35.4 in the vehicle group and 81.6 mmol/l±34 in the MSC group. There was also no difference in serum urea, lactate dehydrogenase or renal histological score. Conclusion: These studies found that MSC did not protect against renal IRI, regardless of the route of MSC administration. MESENCHYMAL STROMAL CELLS (MSC) DO NOT PROTECT AGAINST HYDROGEN PEROXIDE INDUCED EPITHELIAL CELL DAMAGE IN AN IN VITRO HUMAN MODEL OF RENAL ISCHAEMIA REPERFUSION INJURY (IRI) Clark Carolyn J1,2,3, Gobe Glenda C3, McTaggart Steven J2,3,4 Coast Hospital and Health Service, 2Solid Organ Transplant Team, Mater Institute, Brisbane, 3School of Medicine, University of Queensland, Brisbane, 4Department of Medicine, Royal Children’s Hospital, Brisbane 1Sunshine

Aim: Studies have shown nephroprotective effects of MSC, which are known immunomodulatory cells. This work investigated the effect of MSC on human proximal tubular epithelial cells (PTEC) in the presence or absence of dendritic cells (DC) and CD4 cells. Methods: An in vitro model of IRI using hydrogen peroxide exposure was developed using an immortalised cell line of PTEC. Human MSC, DC and CD4 cells were applied to the model and PTEC viability and cytotoxicity was assessed at 24 hours by flow cytometry and lactate dehydrogenase (LDH) respectively. Results: In both direct and indirect cell culture, bone marrow derived MSC did not increase the proportion of live HK2 cells (53.8±3.9% vs 41.8±10.8% with no MSC). There was no difference in cytotoxicity as measured by LDH (91.8±13.2% vs control 100%). CD4 cells and DC did not increase the proportion

of live cells in the presence of MSC (56.3±6.5% for CD4 cells and 52±9.4% for DC vs 47.6±5.3% with HK2/MSC alone) or alter cytotoxicity. The results were not altered by application of placental derived MSC, MSC conditioned media or MSC pre-stimulated with interferon gamma and TNF alpha. Conclusion: These studies found that MSC did not protect against renal IRI, either alone or in the presence of immune cells. FOLLISTATIN ATTENUATES RENAL ISCHAEMIAREPERFUSION INJURY AND IMPROVES RENAL FUNCTION IN MICE Fang Doreen1,2, Lu Bo1, McRae Jennifer1, Roberts Veena1,2, Hayward Susan3, De Kretser David4, Cowan Peter1,2, Dwyer Karen1,2 1Immunology

Research Centre, St Vincent’s Hospital, Melbourne, of Medicine, University of Melbourne, 3Monash Institute of Medical Research, Monash University, Melbourne, 4Monash Institute of Medical Research, Prince Charles Hospital, Brisbane 2Department

Background: Ischaemia-reperfusion injury (IRI) accompanies organ transplantation causing inflammation and potentially contributing to poor graft function. Activin is a key driver of inflammation and it is regulated by follistatin. Aim: To investigate the level of activin and the effect of follistatin treatment in renal IRI. Methods: Mice received follistatin or vehicle prior to right nephrectomy and unilateral renal ischaemia for 20 or 22 mins. A sham group underwent right nephrectomy only. Mice were sacrificed at 24 hrs. Serum was collected to measure activin A and B, and creatinine. H&E-stained renal sections were scored to evaluate tubular injury. Results: Renal IRI increased serum activin A and B, creatinine, and tubular injury score. In the 20 mins cohort, follistatin treatment reduced serum activin A and B, and creatinine. There was a trend to improvement in tubular injury score. Serum creatinine and tubular injury score were reduced in the 22 mins cohorts, but had no significant change in the serum activin levels. Renal injury was more severe in the 22 mins cohort. (Table 1) Conclusions: Serum activin A and B levels increase during renal IRI and is accompanied by significant reduction in renal function. Follistatin treatment prior to ischaemia confers renal protection in both mild and severe injury, although there was no concomitant reduction in serum activin A and B in severe renal injury. Whether there is a change in the expression of activin in the kidneys requires further investigation. Table 1 Summary of activin levels and renal injury parameters in sham and renal IRI with vehicle or follistatin treatment. All values are expressed as mean±SEM

Sham, n¼4

Vehicle, n¼8

Follistatin,

P value

P value

n¼10

(sham versus

(follistatin

vehicle)

versus vehicle)

20 mins renal ischaemia Activin A (pg/ml) Activin B (pg/ml)

84.9±6.2 1666.5±157.4

Creatinine (mmol/l)

44.3±0.8

Tubular injury score

0

254.5±42.3 3553.7±85.5

156.2±42.3 1438.5±329.6

Po0.01

Po0.05

Po0.0001

Po0.001

82.8±0.7

56.7±4.9

Po0.0001

Po0.01

2.9±0.1

2.2±0.4

Po0.0001

P¼0.11

22 mins renal ischaemia Activin A (pg/ml)

74.4±3.6

167.7±21.8

Activin B (pg/ml)

1161.0±180.7

6049.0±979.6

Creatinine (mmol/L)

18.5±0.5

217.4±16.8

Tubular injury score

0.2±0.1

3.4±0.2

183.5±25.7 5685.0±1264.0 157.7±12.7 2.9±0.1

Po0.05

P¼0.65

Po0.01

P¼0.83

Po0.0001

Po0.05

Po0.0001

Po0.05

Immunology and Cell Biology

Abstracts A22

Immunobiology HIGH-LEVEL EXPRESSION OF AN ALLO-MHC II TRANSGENE CAN BE ACHIEVED IN MOUSE HEPATOCYTES, BUT LACK OF CHAPERONES AND ACCESSORY MOLECULES MAY PREVENT TOLERANCE INDUCTION Moawadh Mamdoh1, Cunningham Eithne1, Paul Moumita1, Wang Chuanmin1, Tay Szun S2, Hu Min3, Alexander Stephen3, Logan Grant J4, Alexander Ian E4, Bertolino Patrick D2, Bowen David G1,2, Bishop G Alex1, Sharland Alexandra F1 Transplant Group, University of Sydney, 2Liver Immunobiology Laboratory, Centenary Institute of Cancer Medicine and Cell Biology, Sydney, 3Centre for Kidney Research, The Children’s Hospital at Westmead, Sydney, 4Gene Therapy Research Unit, Westmead Hospital, Sydney 1Collaborative

High-level expression of an allo-MHC II transgene can be achieved in mouse hepatocytes, but lack of chaperones and accessory molecules may prevent tolerance induction. Aims: Previous studies have shown specific tolerance and long term survival of cardiac grafts following donor MHCII gene transfer to recipient bone marrow. In our published data, expression of donor MHC class I (Kb) in recipient liver induced tolerance to Kb–expressing skin grafts. We wanted to investigate the effect of liver-directed, AAVmediated expression of MHC class II on fully allogeneic heart transplants. Methods: We injected C57BL/6 (H-2b) WT and Foxp3GFPmice with varying doses (5108–51011) of an AAV vector encoding H-2IAd (rAAV-IAd). Hepatocyte expression of IAd and costimulatory molecules was assessed by FACS, whilst Invariant chain (Ii) and H-2M expression was determined by RT-PCR. Fully-allogeneic DBA/2 (H-2d) hearts were transplanted into C57BL/6 mice at d7 or d100 post-transduction.

Immunology and Cell Biology

Results: Treatment with doses of 51010 or 51011 rAAV-IAd produced high-level IAd expression on hepatocytes (Figure 1). Foxp3+ CD4+ T cells were increased in livers of mice treated with 51011 rAAV-IAd (7.7±2.2%, Po0.03), compared to normal controls (2±0.76%) and 51010 IAd-treated mice (2.4± 0.6%). Heart graft survival in IAd-treated mice was not prolonged, compared to normal controls (MST 8d and 7d respectively). Hepatocytes from IAd-injected mice did not express CD40, CD80, CD86 or PD-L1 and minimal expression of H2-M and Ii suggested impaired peptide editing of IAd. Conclusion: High level expression of MHCII IAd can be achieved in recipient liver using rAAV vectors. However, initiating tolerance or immune response to grafts bearing IAd might require supplying H2-M and invariant chain (Ii) genes for hepatocyte-biosynthesis of fully functional MHCII IAd. DISRUPTION OF CD8-CORECEPTOR BINDING ABROGATES TOLERANCE INDUCTION VIA LIVER-DIRECTED EXPRESSION OF DONOR MHC CLASS I – A ROLE FOR PD-1/PD-L1? Paul Moumita1, Cunningham Eithne1, Bunker Daniel1, Wang Zane1, Wang Chuanmin1, Bremner Kate2, McGuffog Claire2, Tay Szun S2, Logan Grant J3, Alexander Ian E3, Bertolino Patrick2, Bishop G Alex1, Bowen David G1,2, Sharland Alexandra F1 1Collaborative Transplant Group, University of Sydney, 2Liver Immunobiology Laboratory, Centenary Institute of Cancer Medicine and Cell Biology, Sydney, 3Gene Therapy Research Unit, The Children’s Hospital at Westmead, Sydney

Aims: rAAV-mediated expression of donor MHC (H-2 Kb) in recipient liver results in tolerance to Kb-bearing skin grafts and leads to impaired IFN-g production in response to Kb, but no substantial

Abstracts A23

deletion of CD8+ T cells. To determine the role of direct recognition of class I by CD8-dependent T cells in the functional silencing implicated in this model, we mutated AAV-Kb (rAAV-D227 K), replacing the a3 domain Asp 227 with Lys, and abrogating CD8 binding. Methods: Kb-bearing 178.3 skin was grafted onto uninjected recipients and mice injected with rAAV-D227 K or rAAV-Kb. Proliferation of Kb-reactive Des and OT-1 transgenic T cells was assessed following adoptive transfer into mice expressing Kb or D227K-Kb. Results: Grafts onto rAAV-Kb-injected B10.BR mice survived long term (MST4250d, n¼5), while uninjected and rAAV-D227K-injected mice rejected 178.3 skin (MST¼16d and 27d, respectively, n¼6). B10.BR hepatocytes from Kb –transduced mice expressed PD-L1 but this was not seen following Kb-D227 K transduction (Figure 1B), or Kb transduction of syngeneic C57BL/6 mice. Des T cells (moderate affinity) proliferated in mice expressing Kb and expressed CD69 and PD-1, while Des transferred to mice expressing D227K-Kb did not (Figure 1A,C&D). However, D227K- Kb was capable of stimulating OT-1 T cells (high affinity). Conclusions: Following their interaction with Kb on hepatocytes, alloreactive T cells are activated and express PD-1. Cross-talk between activated alloreactive CD8+ T cells and hepatocytes promotes hepatocyte expression of PD-L1, enabling silencing of PD-1-expressing T cells. The D227 K mutation disrupts direct recognition by CD8dependent T cells abolishing this interaction and subsequent tolerance induction.

Lewis. CD4+CD25T cells proliferated less to PVG than to Lewis. Tolerant CD4+CD25+ cells did not respond to PVG, and were similar to self but proliferated to Lewis similar to naı¨ve CD4+CD25+T cells proliferation to PVG and Lewis. Proliferation of tolerant CD4+CD25+ T cells was enhanced to PVG but not to Lewis or self, by IL-5 or interferon-gamma but not by IL-10 or IL-13. IL-2 and IL-4 induced non-Ag specific proliferation. Tolerant CD4+CD25+T cells had enhanced expression of the receptor for interferon-gamma (ifngr) and IL-5 (il5ra) compared to naı¨ve CD4+CD25+T cells. After culture with IL-5, il5ra expression was retained but not ifngr. After culture with IFN-g, ifngr expression was retained and il5ra lost. Conclusions: Tolerant hosts have activated antigen specific CD4+CD25+Treg that express receptors for IFN-g and IL-5 and require these cytokines to support their survival. These in vitro assays may be developed further to diagnose transplant tolerance.

THE TEST FOR TOLERANCE: TEST OF TRANSPLANT TOLERANCE BY IN VITRO ASSAY OF CD4+CD25+T CELLS

Introduction: Donor brain death (BD) adversely affects organ quality. Inflammation mediated by engagement of TLR4 and RAGE may contribute to inferior outcomes. Endogenous secretory (es)RAGE is a soluble decoy receptor which sequesters HMGB1 and other RAGE ligands blocking binding to TLR-2, TLR-4 and RAGE. Aim: To determine whether in vivo expression of esRAGE can reduce inflammation in syngeneic KTx from BD donors. Methods: Donor and recipient C57BL/6 mice received 51011 VG rAAV-esRAGE or control vector 7 days pre-transplant. Donor kidneys were grafted 1hr after BD induction, and sampled at d1 and 4 posttransplantation. Cellular infiltration, proliferation and gene expression were determined. Serum and kidneys were also collected 3hrs post-BD. Results: Serum HMGB1 levels in esRAGE-BD mice (B10 ng/ml) were significantly less than untreated BD mice (425 ng/ml, P¼0.02). There was a trend towards reduced expression of CXCL10, MCP-1, MIP-2, CCL22, IL-10, IL-6, IL-1b and TNF-a in the kidneys of esRAGE-BD mice, but this was not consistently observed after KTx. Neutrophil and macrophage infiltrate density was substantially reduced by esRAGE (neutrophil 9.1±2.0/HPF cf 23.1±6.0, P¼0.03; F4/80 27.3±3.2/HPF cf 65.1±9.1, Po0.01, Figure 1A). Proliferation of parenchymal cells

Robinson CM1, Hall BM1, Plain KM2, Tran GT1, Verma ND1, Boyd R1, Nomura M1, Hodgkinson SJ1 1Immune

Tolerance Group, University of New South Wales, Sydney, of Sydney

2University

Aim: To compare the reactivity of tolerant and naı¨ve CD4+, CD4+CD25+ and CD4+CD25 T cells. Antigen(Ag) specific Treg require Ag stimulation and cytokines to survive, thus we examined which Th1 and Th2 cytokines were required. Methods: Cells from DA rats tolerant to fully allogeneic PVG hearts for 4100 days were assayed in MLC. Different Th1 or Th2 cytokines were added to MLC and cytokine receptor expression assayed by RT-PCR. Results: Peripheral T cells subsets were similar in naı¨ve and tolerant: CD4+(49v42.5%), CD8+(16v15.5%), CD4+CD25+(6.1v5.6%) and CD25+Foxp3+(10.9v9.1%). Serial dilution showed similar proliferation of both naı¨ve and tolerant CD4+T cells to PVG and to third party

MODULATION OF INFLAMMATORY RESPONSES AFTER BRAIN DEATH AND SYNGENEIC RENAL TRANSPLANTATION BY SYSTEMIC EXPRESSION OF ESRAGE Wang Zane Z, Wang Chuanmin, Habib Miriam, Paul Moumita, Cunningham Eithne C, Wu Huiling, Chadban Steven, Allen Richard DM, Bishop G Alex, Sharland Alexandra F Collaborative Transplant Group, University of Sydney

Immunology and Cell Biology

Abstracts A24

(PCNA staining) was enhanced in esRAGE-treated mice (7.6±0.5/ HPF cf 4.2±1.2, P¼0.05, Figure 1B). Conclusions: esRAGE significantly reduced neutrophil and macrophage infiltration following syngeneic renal transplantation from BD donors. RAGE is a binding partner for the integrin MAC-1, and reduced leucocyte infiltration may reflect inhibition of this interaction. esRAGE increased proliferation of renal parenchymal cells after transplantation from BD donors, similar to documented effects of soluble RAGE in liver injury. INTERLEUKIN-12 PROMOTES INDUCTION OF POTENT TH1-LIKE CD4+CD25+T REGULATORY CELLS THAT INHIBIT ALLOGRAFT REJECTION IN UNMODIFIED HOSTS Hall BM1, Verma ND1, Plain KM2, Robinson CM1, Boyd R1, Tran GT1, Wang C3, Bishop GA3, Hodgkinson SJ1 1Immune

Tolerance Group, University of New South Wales, Sydney, Science, University of Sydney, 3Collaborative Transplant Laboratory, University of Sydney 2Veterinary

Aims: To examine if CD4+CD25+Foxp3+Treg activated with alloAg and IL-2, which express IL-12 receptor, can be further activated by IL-12 and specific alloAg. Methods: Naı¨ve DA CD4+CD25+T cells cultured for 3 days(3d) with PVG alloAg and IL-2 (Ts1 cells), were re-cultured with PVG alloAg and either IL-12(p70), IL-2 or IL-12(p70)+IL-2 for another 4d. These re-cultured Treg were tested for their capacity to suppress. Results: After culture for 4d with IL-12(p70) alone, but not with IL-12(p70)+IL-2, the Treg were induced to express t-bet and ifn-g and continued to express foxp3, ifngr, il-12r2. Il2 was not induced. Yield was 30% of original population. These IL-12 activated Th1-like Treg suppressed proliferation of CD4+T cells in MLC at o1:1024, whereas Treg after cultured with IL-2 and alloAg suppressed at 1:32. 5106 IL-12p70 alloactivated Th1-like Treg given i.v. to normal non-immunosuppressed DA rats markedly delayed PVG heterotopic heart allograft rejection to 44d(14–70)(median (range) (n¼5) compared to rejection in normal: 8d(7–9)(n¼5)(Po0.01) and in hosts given Treg re-cultured with IL-2: 6d(n¼5)(Po0.05). Th1-like Treg did not delay Lewis graft rejection 8d(7–9)(n¼5) vs no cells 8d(7–9)(n¼5). Conclusion: Highly suppressive Th1-like Treg were induced from naı¨ve CD4+CD25+Treg in 7d by culture with alloAg, first with IL-2 then IL-12. These Th1-like Treg suppressed in vitro at 1:1000 and delayed specific-donor allograft rejection at a ratio of about 1:100 to naı¨ve CD4+CD25 effector lineage T cells. The induction of potent alloAg specific Treg by culture of natural Treg in vitro for a week, has major therapeutic potential.

TLR4 DEFICIENCY ATTENUATES ACUTE KIDNEY ALLOGRAFT REJECTION BUT DOES NOT PROLONG SURVIVAL Wu Huiling1,2, Chadban Steven1,2 1Department

of Renal Medicine, Royal Prince Alfred Hospital, Sydney, Transplant Group, University of Sydney

2Collaborative

Aim: TLRs play an important role in innate immunity. TLR4 deficiency is protective in kidney ischaemia-reperfusion injury. TLR

Immunology and Cell Biology

signalling provides a link between innate and adaptive immunity and we have reported that absence of MyD88 (a TLR signal adaptor) induced donor-specific kidney allograft tolerance. Here we investigated the role of TLR4 signalling in kidney allograft rejection. Methods: Kidney transplants were performed in nephrectomised mice: BALB/c to B6(WT-allografts), BALB/c.TLR4/ to B6.TLR4/(TLR4/allografts) and B6 to B6(isografts). Results: Survival to 100 days was observed in all isografts, 4 of 20 TLR4/allografts(MST¼60 days) and 12 of 31 WT-allografts(MST¼ 40days). At day 14 post-transplant, WT allografts developed kidney dysfunction with increased serum creatinine(Scr) compared to isografts (Po0.001) while TLR4/ allografts had better graft function than WT allografts (Po0.05). Both WT and TLR4/ recipients developed histological evidence of acute rejection with similar degrees of tubulitis and infiltrates of CD4, CD8 and CD68 cells in allografts, while an increase in CD11c+ cells was evident in TLR4/ compared to WT allografts (Po0.05). iNOS mRNA expression was decreased but IL2 was increased in TLR4/ compared to WT allografts (Po0.05). IDO mRNA expression was increased in TLR4/ compared to WT allografts (Po0.01). At day100 post-transplant, surviving WT and TLR4/ allografts exhibited similar degrees of kidney dysfunction. Conclusion: Acute kidney allograft rejection was modestly attenuated in TLR4/ mice, however long-term allograft survival and function were not affected in this fully-MHC-mismatched allograft rejection model. Protection against acute rejection may involve increases of DC11c+ cells and IDO expression. ANALYSIS OF TFH CELLS AND TREGS IN PERIPHERAL BLOOD OF PAEDIATRIC RENAL TRANSPLANTS AND PAEDIATRIC VASCULITIS Lijic Natasha Anne1, Wang Yuan Min2, Hu Min1, Zhang Geoff Y2, Sawyer Andrew1, Zhou Jimmy Jianhen2, Wyburn Kate3, Alexander Stephen I1 1Centre

for Kidney Research, The Children’s Hospital at Westmead, Sydney, 2Centre for Reproductive Health, The Children’s Hospital at Westmead, Sydney, 3Department of Medicine, University of Sydney Background: Antibody mediated disease in transplantation and in vasculitic renal disease is of increasing interest. T follicular helper cells (TFH) are the newly described subset of CD4 T cells that assist in antibody production. Aim: To measure the proportion of TFH and Treg cells in patients with transplants and autoimmune renal disease to see if the disease is due to a failure of self tolerance or exacerbation of antibody inducing TFH cells. Method: We measured subsets of T cells including TFH cells and Tregs in the peripheral blood of children with renal transplants, a patient with Henoch Schonlein Purpura and control subjects. Peripheral blood was ficoled and PBMC cells were stained for CD4; for the TFH markers CxCR5, ICOS, HLA-E and PD-1, and for the Treg markers CD4, CD25, CD127low and intracellular FOXP3 before flow cytometric analysis. Results: There was an increase in peripheral TFH cells as measured by CD4/CXCR5+ in patients with transplants and in the child with HSP compared with controls. Tregs were equivalent between controls and the child with HSP but reduced in the transplant patients. Conclusion: Peripheral TFH cells may be an indicator of antibody formation in renal disease and in transplant recipients.

Abstracts A25

INTERLEUKIN-5 THERAPY PREVENTS CHRONIC ALLOGRAFT REJECTION BY INDUCTION OF T REGULATORY CELLS Hodgkinson SJ1, Hall RM1, Tran GT1, Robinson CM1, Wang C2, Sharland A2, Hall BM1 1Immune

Tolerance Group, University of New South Wales, Sydney, Transplant Laboratory, University of Sydney

2Collaborative

Aim: Naı¨ve CD4+CD25+Foxp3+Treg activated by specific alloantigen (alloAg) and IL-4, not IL-2, express the specific IL-5 receptor (Ts2 cells). Ts2 cells are more potent alloAg specific Treg than nTreg. Here we examined if IL-5Rx activated alloAg specific Ts2 cells to prevent chronic rejection. Methods: F334 recipients with Lewis heart grafts received 5000units rIL-5 ip daily for 10d from 7d after grafting. Rejection was scored on a semi-quantitative scale. Results: Sham Rx rats developed rejection at 18d (mean score of +++) and all rejected by 28d(n¼5). IL-5Rx prevented rejection, with all grafts ++++-+++ until cessation of IL-5Rx at day 18(Po0.01 vs sham Rx). After 18d, there was rejection (mean score ++) until 50d(n¼5)(Po0.01). Another group continued IL-5Rx 3x/week from 18(n¼5), had transient rejection a few days after changing to 3x/week, but by 30d grafts recovered with 3 of 5 ++++, one +++ and one ++ at 60d (Po0.05 vs short IL-5Rx; Po0.01 vs sham Rx). IL-5Rx had increased CD4+CD25+T cells to 6–8% vs 3–4% in controls and normal. At 18d after 10d of IL-5Rx, CD4+CD25+T cells responded to Lewis but not to F344 or third party PVG, showing activation of alloAg specific Treg by IL-5Rx. CD4+CD25+T cells from rats treated 50d with IL-5 showed response to Lewis was enhanced by IL-5. Conclusion: IL-5 is known to promote growth of Ag specific Ts2 cells that may prevent rejection. IL-5Rx induced Ag specific Ts2 cells and long term treatment ensured good graft function. This therapy may have potential to prevent chronic allograft rejection.

Irish Swaminathan Kong Wai Yew4

Ramyasuda1,

Fidler

Outcome measures HEARTS FROM AGED RATS SHOW INCREASED SUSCEPTIBILITY TO BRAIN DEATH AND ISCHAEMIAREPERFUSION INJURY

DETERMINANTS OF FREE LIGHT CHAINS IN RENAL TRANSPLANT RECIPIENTS Ashley1,2,

dependent FLC regulation in renal transplant recipients (RTR) is unknown. Aim: To explore determinants of FLC in RTR and their relationship with measures of immune regulation (sCD30 and hsIL6), allograft function and tubular injury and fibrosis markers (serum and urine TGFb, urine NGAL). Methods: Single Centre cross-sectional study of 129 stable RTR (76M) a median of 6.7 (IQR 2.9–12.7 years) following transplant. 93 (72%) received a CNI. Median creatinine 123 (93–163 mmol/l) BP (130/77) and uPCR 22 (10.5–53.5 mg/mmol). Results for FLC and their ratios and clearance are shown (median and IQR or mean±SD). Only 2 patients had a FLC ratio above the reference range (1.8) and no patient had a defined monoclonal disorder. Serum FLC were significantly correlated with serum hsIL6, sCD30, creatinine and uNGAL and increased in patients receiving a CNI, ACEi/ARB and diuretics. Urine FLC were significantly correlated with serum sCD30, creatinine, urine and serum TGFb, serum hsIL6, uNGAL and systolic BP. Multiple regression identified creatinine, sCD30 and CNI use as predictors of both serum FLC. Urine l was predicted by serum l and sCD30 and urine k by serum k only. Conclusions: Both serum and urine FLC are increased in RTR and this is correlated with measures of increased regulation/production of B-cells (sCD30 and hsIL6) and excretion (creatinine). There is greater fractional clearance of kFLC than lFLC. Urine FLC excretion is correlated with markers of tubular injury and fibrosis. FLC are potential markers of B-cell activation and tubular injury and warrant further study.

Kumarasinghe Gayathri1,2, Gao Ling1, Doyle Aoife1, Chew Hong1, Jabbour Andrew1,2, Hicks Mark1, Macdonald Peter1,2

Samantha3,

1Renal

1Transplantation Laboratory, Victor Chang Cardiac Research Institute, Sydney, 2Department of Cardiology, St Vincent’s Hospital, Sydney

Background: Free Light Chains (FLC) are secreted from B-cell lineage under immune regulation. Excretion of FLC is renal dependent and in excess FLC are nephrotoxic. The role of immune and renal

Aim: Registry data show a higher rate of primary graft failure, oneyear and long-term mortality associated with transplants from older donors; however, whilst organ donation rates remain low, we are forced to accept organs from older ‘marginal’ donors. These hearts are hypothesized to function poorly due to increased susceptibility to brain death (BD), ischaemia and reperfusion injury

Transplant Unit, Royal Perth Hospital, 2Department of Medicine, University of Western Australia, Perth, 3Transplantation Immunology Laboratory, Royal Perth Hospital, 4Department of Nephrology, Faculty of Medicine University of Malaya

Serum mg/l

RTR Reference Range

Urine mg/l

k

l

ratio

k/ l Clearance

k

l

ratio

17 12-28

21 14-32

0.88

9.4 (6.7-13.1)

12.7 (5.7-26.3)

0.26-1.65

3.0

5.6 1.8-10.0 3.2 ± 3.3

7.9 (5.9-12.2)

7.3 (3.3-19.4)

49.9 14.4-99.4 5.4 ± 5.0

1.9 (0.46-4)

Immunology and Cell Biology

Abstracts A26

ns

***

% Recovery

150

**

100

50

d ol th on m 18

on m 12

12

w

ee

th

k

ol

ol

d

d

0

Figure 1 Comparing estimated glomerular filtration rate (eGFR) between DCD and DBD kidney recipients. PreTx ¼ pre-transplantation. ***Po0.001, *Po0.05.

** p