423: Adenoviral mediated interleukin 10(AdhIL-10) gene therapy in normothermic ex-vivo lung perfusion

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Abstracts

S. Keshavjee,1 K.R. McCurry,2 R.D. Davis,3 J.M. Pilewski,2 P.A. Corris,4 A. Zeevi,2 T.K. Waddell,1 S.M. Arcasoy,5 J.G. Golden,6 M. Strueber,7 R.L. Doyle,8 P. Jaksch,9 A.C. Mehta,10 M.S. Mulligan,11 O.E. Pajaro,12 M.R. Zamora,13 S. Sweet,14 R.N. Woodward,15 R.D. Dedrick,15 H. Wolters,15 K.C. Fang,15 E.P. Trulock,14 1University of Toronto, Toronto, ON, Canada; 2University of Pittsburgh, Pittsburgh, PA; 3Duke University, Durham, NC; 4University of Newcastle, Newcastle Upon Tyne, MO, United Kingdom; 5Columbia University, New York, NY; 6University of California, San Francisco, San Francisco, CA; 7Hannover Medical School, Hannover, Germany; 8 Stanford University, Stanford, CA; 9University Hospital, Vienna, Austria; 10Cleveland Clinic Foundation, Cleveland, OH; 11University of Washington, Seattle, WA; 12Mayo Clinic Jacksonville, Jacksonville, FL; 13University of Colorado, Denver, CO; 14Washington University, St. Louis, MO; 15XDx, South San Francisco, CA Purpose: Indeterminate clinical outcomes, infection risks, and diverse mechanisms of action have engendered controversy about the use of induction therapy (IT) in lung allograft recipients to prevent acute cellular rejection (ACR). T cell immunomodulation using IT involves either cytolytic (e.g. alemtuzumab (ALEM, antiCD52 Ag) and anti-thymocyte globulin (ATG)) or non-cytolytic (e.g. basiliximab (BAS) or daclizumab (DAC), IL2-receptor antagonists (IL2-RA)) agents. To test the hypothesis that a peripheral molecular signature discriminates between the absence (A0) and presence (ⱖA2) of lung ACR, the LARGO Study utilizes gene microarrays and RT-PCR to correlate transcriptional profiles of PBMCs with A0 or ⱖA2 biopsy grades. Methods and Materials: Since IT is given when risks of ACR and infection are highest, gene expression (GE) profiles early post-transplant (p-tx) may also reflect the influence of IT on innate and adaptive host immune responses. To study the effect of IT, we will analyze blood samples from ⬃20 patients each in the cytolytic IT, non-cytolytic-IT or no IT groups, that are matched for steroid dose and infection status, with pathologically confirmed A0 or ⱖA2 rejection grades obtained between 21 and 365 days after transplantation. Both microarrays and RT-PCR quantification of ⬎200 transplant-related genes will be used to identify IT-dependent differential gene expression (fold-differenceⱖ1.2; t-test p⬍0.05). Results: Analysis of the LARGO clinical database derived from 854 patients showed that 43.9% received some IT therapy, while 36.4% did not (19.7% unknown); the majority of patients at 8 of 14 study centers received IT, while 3 centers used no IT and 3 used it selectively. 53.7% of patients received IL2-RA therapy with BAS (34.4%) or DAC (19.3%), while 31.2% or 10.8% received ATG or ALEM, respectively (4.2% other). Conclusions: The data anticipated for presentation at the International Conference may identify novel molecular relationships between induction therapy and host responses to infections and lung allografts.

422 CXCR3 AND CCR5 BLOCKADE PREVENTS ACUTE REJECTION VIA INDUCTION OF REGULATORY CD4 LYMPHOCYTES S. Bastani,1,2 G.T. Schnickel,1 G.R. Hsieh,1,2 C. Garcia,1 M.C. Fishbein,3 A. Ardehali,1,2 1Department of Surgery, Division of Cardiac Surgery, David Geffen School of Medicine, UCLA, Los Angeles, CA; 2Department of Surgery, Division of Cardiac Surgery, West Los Angeles VA Medical Center, Los Angeles, CA; 3Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, UCLA, Los Angeles, CA

The Journal of Heart and Lung Transplantation February 2007

Purpose: Blockade of CXCR3 and CCR5 has prolonged allograft survival in experimental studies. Yet, all of the allografts were eventually lost to acute rejection. We hypothesized that combined CXCR3 and CCR5 blockade results in indefinite (⬎100days) allograft survival. Methods and Materials: We used a full MHC mismatched murine cardiac allograft model. In the control group, Balb/c hearts were transplanted into B6:129PF2/J recepients. In the experimental group, Balb/c hearts were transplanted into CCR5 knock-out mice (on B6:129 background). The recepient mice were then treated with anti-CXCR3 antibody everyother day post transplantation. No immunosuppression was given. The donor hearts in the experimental group were harvested at 100 days and analyzed. Results: As expected, the donor hearts in the control group were rejected in 6 ⫾ 1 days post transplantation (n⫽5). Combined blockade of CXCR3 and CCR5 in this model prolonged allograft survival more than 15 folds compared to the control group; all allografts survived more than 100 days (n⫽5). Interestingly, the donor hearts were infiltrated with large number of mononuclear cells. Analysis of these graft infiltrating cells identified them as CD4⫹ CD25⫹ cells expressing Foxp3 gene. Conclusions: This study demonstrates that combined CXCR3 and CCR5 blockade prevents acute rejection in a robust murine model. The mechanism for beneficial effect of combined CXCR3/CCR5 blockade includes induction of regulatory CD4 lymphocytes. 423 ADENOVIRAL MEDIATED INTERLEUKIN 10(ADHIL-10) GENE THERAPY IN NORMOTHERMIC EX-VIVO LUNG PERFUSION M. Cypel, M. Rubacha, M. Sato, S. Hirayama, S. Fischer, K. McRae, M. Liu, T.K. Waddell, M. dePerrot, S. Keshavjee Thoracic Surgery Research Laboratory, Toronto Lung Transplant Program, Toronto General Research Institute, University of Toronto, Toronto, ON, Canada Purpose: Ex-vivo lung perfusion (EVLP) allows time for genetic or other manipulations to repair the donor lung.We have previously shown that IL-10 upregulation leads to inhibition of pro-inflammatory cytokines and improved lung function.We report on 12h EVLP combined with transbronchial AdhIL-10 gene transfection to improve the quality of pig and human lungs. Methods and Materials: 1-Pigs: Lungs were preserved with PerfadexR and then perfused ex-vivo with SteenR solution.After 1 hour of EVLP, lungs were randomly divided into three groups (n⫽3/group): I-1⫻1010 pfu AdhIL-10 transfection, II-1⫻1010 pfu of Ad5BGL2 transfection(Empty vector),and III-normal saline (Control). 2-Human Lungs: Lungs procured for transplantation, but determined to be unsuitable, were similarly transfected with AdhIL-10 4⫻1010 (n⫽2). In both pig and human studies, EVLP was performed for 12h and lung function was assessed. hIL-10 was measured in perfusate, BAL and lung tissue. Results: 1-Pigs: The dynamics of hIL-10 transfection are shown in Table 1. Lung function remained remarkably stable throughout 12h of EVLP in the IL-10 and saline groups, but not in the empty virus group (table 2 p⬍0.05).2-Human Lungs:After 12h of EVLP lung function was excellent: Delta pO2(435mmHg),PVR (291 dyn.sec.cm-5),Compliance(59.5 ml/cmH2O). Mean levels of hIL-10 were: 123.33 pg/ml (perfusate), 1155.5 pg/ml (BAL) and 3.045 pg/mg (tissue). Conclusions: Ex-vivo adenoviral mediated gene transfection of donor lungs is achievable in pigs and human lungs. AdhIL-10 transfection leads to a progressive increase in hIL-10 production in a time dependent manner. This treatment has the potential to repair damaged donor lungs for transplantation.

The Journal of Heart and Lung Transplantation Volume 26, Number 2S

Abstracts

Table 1 hIL-10 Levels (mean, pg/ml, Pig IL-10 group) Hours EVLP

0h

6h

9h

12 h

Perfusate BAL

0 0

0 37.10

13.58 300.33

42.26 1384

Table 2 Pig Lung Function After 12 hours EVLP

IL-10 Empty Control

Compliance (ml/cmH2O)

PVR (dynes ⴛ sec ⴛ cmⴚ5)

Delta PO2 (mmHg)

27 ⫾ 2 13 ⫾ 5.6 24.3 ⫾ 5.5

631.7 ⫾ 166 1219 ⫾ 140 791.1 ⫾ 140

460 ⫾ 71 113 ⫾ 9.8 404 ⫾ 138

424 ANTI-CD20 PREVENTS ALLOANTIBODY PRODUCTION AND ATTENUATES CARDIAC ALLOGRAFT VASCULOPATHY IN MONKEYS TREATED WITH ANTI-CD154 S.S. Kelishadi,1 T. Zhang,1 B.N. Nguyen,1 G. Wu,1 E. Welty,1 C.J. Avon,1 S. Pfeiffer,1 C. Schroder,1 A. Azimzadeh,1 R.N. Pierson, III,1 1Cardiac Surgery, University of Maryland School of Medicine and Baltimore VAMC, Baltimore, MD Purpose: Blockade of CD40-154 prolongs cardiac allograft survival in cynomolgus macaques, but cardiac allograft vasculopathy (CAV) causes graft failure, and is closely associated with appearance of anti-donor antibody (Ab). We hypothesized that depletion of CD20⫹ B-cells might interfere with formation of anti-donor plasma cells and alloantibodies, and thus prevent CAV. Methods and Materials: Thirty MLR-mismatched heterotopic cardiac cynomolgus allograft recipients were treated with anti-CD154 high intensity monotherapy (␣CD154; n⫽17, 6 with ATG) or ␣CD154 with additional ␣CD20 (20mg/kg q wk for 4 weeks: ␣CD154⫹␣CD20; n⫽13, 11 with ATG). Some animals received an additional donor thymus graft or bone marrow infusion. Acute rejection was usually treated with steroids; graft survival was censored at 90 days. Results: 10 animals died with beating grafts, mainly with ATG-associated lung pathology, and are excluded from the survival analysis. Graft survival with ␣CD154⫹␣CD20 (median ⬎90d) and proportion of grafts surviving to 90 days (5/5) was significantly increased relative to ␣CD154 (median 43d, IQR(25-75) 35-82d, p⬍0.007; 3/15 ⬎90d). With ␣CD154, 15/15 (100%) developed anti-donor IgM and 14/15 (93%) IgG, usually weeks before graft failure, whereas no IgG was detected in samples analyzed to date from the ␣CD154⫹␣CD20 group, and weak IgM was transiently detected in only one animal. All ␣CD154 grafts had severe CAV (score ⱖ3 on 0-4 scale), in many instances with fibrosis and unscorable vessels due to infarction. In stark contrast, ␣CD154⫹␣CD20 was associated with CAVⱕ 1 in 4 of 5 grafts, and CAV 2 in the 5th, and preserved myocardial architecture. Conclusions: Using ␣CD20 with ␣CD154 is associated with decreased elaboration of alloAb, prolonged graft survival to ⬎90 days, and significant attenuation of CAV. Concomitant thymus transplant or ATG induction did not appear to be necessary to this effect; ATG is associated with significant morbidity. These findings suggest that ␣CD20 may reduce the incidence of CAV, perhaps by inhibiting alloAb. 425 VAD-ASSOCIATED THROMBOEMBOLISM: POSSIBLE ROLE FOR INTRINSIC COAGULATION CASCADE S. Kallam,1 R.N. Sangrampurkar,1 Z. Kon,1 R.N. Pierson,1 B.P. Griffith,1 R.S. Poston,1 1Cardiac Surgery, University of Maryland Medical Center, Baltimore, MD

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Purpose: Thromboembolism (TE) in ventricular assist device (VAD) patients often follow acute bursts in thrombin production. The purpose of this study was to further investigate the factors that lead to this dysregulated thrombin metabolism. Methods and Materials: Blood from 20 consecutive VAD patients was assayed for factor XIIa activity (intrinsic cascade), reaction time and maximum amplitude of tissue factor activated thrombelastography (TEG-r and TEG-MA, extrinsic cascade and platelet activity), F1.2 level (thrombin generation) and plasminogen activator inhibitor-1 (PAI-1) levels (fibrinolysis). TE was defined as clinical stroke or asymptomatic ⬎2-fold rise in S100 level. Each assay was compared: 1) before and after VAD implantation and 2) for 24 hours preceding TE vs. other postoperative timepoints. Results: PAI-1 was the only marker that was elevated chronically after VAD implantation. Just prior to TE, significant elevations were noted in F1.2 (1.20 vs. 0.34nmol/mg, p⫽0.0001) and XIIa (2.48 vs. 1.45ng/ml, p⫽0.002). ROC analysis identified discrete cutoff values for both F1.2 (0.5nmol/L/mg) and XIIa (2ng/ml) that strongly predicted TE (p⬍0.0001, Fischer’s exact test). Factor XIIa activity significantly correlated with F1.2 (R ⫽ 0.59, p ⫽ 0.0003) and S100 (Figure 1) (R ⫽ 0.52, p ⫽ 0.003) but not with PAI-1 (R ⫽ 0.07). Conclusions: VAD recipients demonstrate compromised fibrinolysis in the setting of acute bursts in thrombin generation that are associated with increased risk of TE. The significant correlation of factor XIIa activity with both TE and thrombin production suggests that the intrinsic cascade may prove to be a novel antithrombotic target in these high risk patients.

426 VASCULAR ENDOTHELIAL GROWTH FACTOR-C ENHANCES EXPERIMENTAL OBLITERATIVE BRONCHIOLITIS BY INDUCING LYMPHANGIOGENESIS R. Krebs,1 J.M. Tikkanen,1 A.I. Nyka ¨ nen,1 S. Yla ¨ -Herttuala,2 1 1 1 P.K. Koskinen, K.B. Lemstro ¨ m, Transplantation Laboratory, University of Helsinki, Helsinki, Finland; 2A.I.Virtanen Institute for Molecular Sciences, University of Kuopio, Kuopio, Finland Purpose: The role of vascular endothelial growth factor-C (VEGF-C) in the development of obliterative airway disease (OAD) as a manifestation of chronic rejection was investigated in the rat tracheal allograft model. Methods and Materials: Tracheal allografts were transplanted heterotopically from DA- to WF-rats into the greater omentum. Syngeneic controls were performed from DA to DA rats. Expression of VEGF-C and VEGFR-3 protein and lymphatic vessel marker LYVE-1 was analysed