Transplantation

TRANSPLANTATION RESULTS: In all 4 cases, current MLR showed responsiveness to donor antigen in a magnitude consistent with that seen versus thirdparty antigen.

Nonspecific endothelin receptor blockade with tezosentan protects hepatic viability after ischemia and reperfusion Dean M Anselmo MD, Farin Amersi MD, Xiu-Da Shen MD, Feng Gao MD, Masamichi Katori MD, Charles Lassman MD, Martine Clozel MD, Ronald W Busuttil MD PhD, Jerzy W Kupiec-Weglinski MD PhD, Douglas G Farmer MD Los Angeles, CA

CONCLUSIONS: The reactivity of these animals to both donor and third-party suggests that prolonged allograft survival has been achieved in the absence of tolerance. This result is consistent with a state of immunologic ignorance that allows the allografts to persist without rejection. This theory will be further tested by challenging these animals with skin allografts from their original donors.

INTRODUCTION: Ischemia reperfusion injury (IRI) is an important factor in graft dysfunction after liver transplantation (LT). Endothelin (ET) is a key modulator in the microcirculatory disturbances associated with hepatic IRI, and may contribute to inflammatory mediated injury. Our aim was to investigate the effects of Tezosentan, a competitive ET receptor antagonist, in rat models of hepatic IRI and LT.

Permanent acceptance of cardiac and skin allografts induced by recipient immunization with immunodominant allo-MHC peptide-primed self-DC combined with bone marrow transplantation OO Oluwole MD, HA DePaz MD, MX Jin MD, MA Hardy MD FACS, SF Oluwole MD FACS New York, NY

METHODS: Livers were harvested and stored in UW at 4°C for 24 hours before ex vivo perfusion for 2 hours with or without Tezosentan treatment (10 mg/kg). Impact of Tezosentan treatment (10 mg/kg) on 7-day survival was evaluated in a 24 hours cold ischemia syngeneic orthotopic LT model.

INTRODUCTION: Because operational tolerance is designed to tolerize T cells, we reasoned that pretreatment with adoptive transfer of immunodominant allo-MHC peptide-pulsed self-DC that induces T-cell tolerance might lead to BMC engraftment and robust tolerance.

RESULTS: In ex vivo IRI model, Tezosentan treatment reduced sGOT levels at 60, 90, 120 minute. This correlated with improved portal venous flow, higher bile production, reduced MPO activity and improved oxygen extraction ratio (ERO2). LT survival was significantly improved with Tezosentan treatment (90%; n ⫽ 10) versus untreated controls (50%; n ⫽ 10; p ⬍ 0.05).

METHODS: To evaluate this hypothesis, ACI rats were pretreated with IV. WF Class I (RT1.Au) immunodominant peptide 5 (P5)pulsed self-DC and 0.5 mL ALS. Unresponsive cardiac allograft recipients were given IV. Busulfan 4 mg on days 60, 61 and 62 after heart transplantation and WF BMC (1 ⫻ 107) on day 63.

CONCLUSIONS: ET blockade with Tezosentan reduced hepatocellular damage from IRI, which was associated with improved sinusoidal hemodynamics and tissue oxygenation and reduced neutrophil recruitment as measured by MPO activity. Furthermore, Tezosentan results in a marked improvement in survival after LT. ET blockade may have important clinical relevance by preventing the untoward consequences of IRI in LT.

RESULTS: IV P5-pulsed DC combined with 0.5 mL ALS on day ⫺7 induced 100% permanent donor-specific cardiac graft acceptance (⬎200 days). Whereas the unresponsive cardiac allograft recipients challenged with second-set grafts at 100 days accepted permanently donor-specific cardiac grafts, they rejected donortype skin grafts in a delayed fashion without rejecting the primary cardiac grafts. In contrast, animals pretreated with IV. P5-pulsed self-DC combined with ALS and given unmodified donor BMC 63 days later accepted permanently (⬎100 days) all primary donor specific cardiac grafts and 8 10 skin grafts. In addition, immunization of naı¨ve recipients with allopeptide on day ⫺7 followed by Busulfan on days 0, 1 and 2 and BMT on day 3 led to permanent acceptance of cardiac and skin grafts. FACS analysis at 100 days after BMT showed 2% to 5% of total T cells in PBL, BM and spleen of tolerant animals were of donor origin. Of note is the observation that these animals showed no clinical or microscopic evidence of acute or chronic GVHD.

Ignorance rather than tolerance associated with prolonged allograft survival in primates treated with costimulatory blocking antibodies Edwin H Preston MD, He Xu MD, Allan D Kirk MD PhD Bethesda, MD INTRODUCTION: Nonhuman primates treated with costimulation blockade (anti-CD154, or anti-B7) fail to reject MHC-mismatched renal and skin allografts. This rejection-free state lasts in some animals for years after cessation of therapy and appears to represent allograft tolerance in a subset of animals. The immune recognition capabilities of these animals have not been studied, and could offer insight into the mechanism by which they avoid rejection.

CONCLUSIONS: These data show that chimeric animals, unlike unresponsive recipients pretreated with allo-MHC peptideprimed self-DC, accepted permanently both first- and second-set skin grafts. This finding suggests that allo-MHC peptide recipient immunization combined with donor bone marrow infusion induces a stable chimeric state that leads to donor-specific operational tolerance.

METHODS: Alloreactivity was assessed in long surviving rejectionfree allografted rhesus monkeys by Mixed Lymphocyte Reaction (MLR) comparing proliferative responses of recipients to autologous, donor, and third-party cells. Testing was done on 3 renal and one skin recipients with long-surviving allografts (all ⬎ 800 days). Absence of acute or chronic rejection was confirmed by biopsy.

© 2002 by the American College of Surgeons Published by Elsevier Science Inc.

ISSN 1072-7515/02/$21.00

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Non–interleukin-2– dependent immunosuppression increases T lymphocyte apoptosis Kenneth J Woodside MD, Mingdao Hu MD, Wei Song MD, Tao Meng MD, Glenn C Hunter MD, John A Daller MD PhD Galveston, TX INTRODUCTION: Transplant tolerance is dependent on the apoptotic deletion of allospecific T lymphocytes after interleukin-2 (IL2)-dependent T lymphocyte activation. Current immunosuppressive strategies block IL-2 and may prevent T-cell activation. We examined apoptotic alterations in mixed lymphocyte culture (MLC), a model of allospecific lymphocyte activation, by polyclonal antithymocyte antibody thymoglobulin (thymo) and monoclonal anti–IL-2R␣ antibody basiliximab (bas). METHODS: Human lymphocytes were isolated by Ficoll-Paque gradient. Cesium-irradiated (2500 rad) stimulator cells (106 cells/ mL) were cocultured with equal numbers of responder cells. Apoptosis was measured by annexin V staining and propidium iodide exclusion using flow cytometry. Isolated protein was analyzed by Western blotting with densitometry. RESULTS: Apoptosis increases at days 3 and 7 in thymo MLC compared to control and bas MLC. Fas is upregulated in thymo MLC in a dose-dependent manner, although basiliximab does not alter fas. FasL is increased initially and at late time points in thymo MLC. CONCLUSIONS: Thymo increases apoptosis and production of the proapoptotic proteins fas and fasL. In contrast, basiliximab did not change lymphocyte apoptosis or apoptotic protein production. These results suggest that IL-2 pathway blockade may prevent allospecific tolerance and that non–IL-2 pathway blockade may encourage apoptosis of allospecifically activated T cell.

Selective endothelial CD62 blockade with P-selectin glycoprotein ligand-Ig reduces ischemia reperfusion injury after rat intestinal transplantation Douglas G Farmer MD, Dean M Anselmo MD, Farin Amersi MD, Xiu Da Shen MD, Feng Gao MD, Sarah Dry MD, Gray Shaw PhD, Ronald W Busuttil MD PhD, Jerzy Kupiec-Weglinski MD PhD Los Angeles, CA INTRODUCTION: Ischemia reperfusion (IR) injury remains a major limitation to successful intestinal transplantation (IT). Although the complex mechanisms of IR are incompletely understood, P-selectin (CD62)-mediated neutrophil (PMN) infiltration is known to play a major role. The aim of this study was to investigate CD62 blockade with soluble P-selectin glycoprotein ligand (rPSLG-Ig) in rat IT models. METHODS: Intestinal grafts were procured, flushed with LR and stored for 6 hours at 4°C before IT. Treated grafts received rPSLG-Ig 0.4 mg/kg intraarterial before storage and IV before reperfusion. Separate groups were used for survival and analysis. Tissue collected from the terminal ileum at 24 hours after IR was evaluated for histologic injury and RT-PCR cytokine analysis.

J Am Coll Surg

RESULTS: Survival at 14 days after IT was 45% in the controls and 100% in rPSGL-Ig–treated rats (p ⬍ 0.05). Histology at 24 hours demonstrated partial to full thickness injury in 61% of untreated grafts and no injury in 81% of the rPSGL-Ig–treated animals (p ⬍ 0.02). This correlated with significantly reduced tissue expression of IL-1b, IFN-␥, IL-4, IL-2, and IL-10 at 24 hours after IR. CONCLUSIONS: Administration of rPSGL-Ig results in markedly improved survival and histopathology after IR injury in IT. These results were associated with reduced intragraft inflammatory and T-cell–derived cytokine expression. Although the mechanism is under further investigation, these findings implicate a significant role for PMN-mediated inflammatory injury after intestinal IR.

T-cell regulation by CD4ⴙCD25ⴙCD45RBLow cells induced by anti-CD45RB treatment PRO Salvalaggio, C Buschenfelde, CE Ariyan, DM Rothstein, GP Basadonna New Haven, CT INTRODUCTION: We have shown that 3 doses of anti-CD45RB mAb induce longterm graft survival and same donor strain tolerance in ⬃50% of C57BL/6 recipients of BALB/c islets. Our data demonstrates that anti-CD45RB treatment promotes tolerance by inducing a shift in CD45 isoform expression from CD45RBHigh to CD45RBLow and upregulating CTLA-4 expression. Here, we examine whether anti-CD45RB treatment induces the expression of regulatory cells. METHODS: Lymphocytes were isolated from Balb/c mice (n ⫽ 3) and phenotyped by immunostaining and flow cytometry analysis after anti-CD45RB mAb treatment (3 doses: 100 ␮g/iv/dose). RESULTS: Flow cytometry analysis showed a ⬃55% increase of CD25 expression on CD4⫹ lymphocytes after treatment with 3 doses of anti-CD45RB. In addition, the CD25⫹CD45RB LowCD4⫹ subset showed a threefold increase after anti-CD45RB treatment. Control untreated allogeneic lymphocytes from C57BL/6 mice (n ⫽ 2) were then used as stimulator cells in mixed lymphocyte reactions (MLR) to demonstrate whether anti-CD45RB–treated T cells regulate the proliferation of untreated lymphocytes. MLR experiments demonstrated that anti-CD45RB treated lymphocytes (unseparated population) do not proliferate when compared to untreated lymphocytes (10% proliferation versus untreated). In addition, when anti-CD45RB treated lymphocytes were cocultured with increasing amounts of untreated T cells (1:1, 1:2, and 1:4 ratios) the proliferation was increased in a dose-dependent manner (⬃50, ⬃75, and ⬃90%, respectively) when compared to the proliferation of untreated cells. CONCLUSIONS: Our data indicates that a short course of treatment with anti-CD45RB mAb induces a significant increase (300%) of CD25⫹CD45RBLowCD4⫹ cells. This cellular subset has been shown to have regulatory properties in both mice and humans. In addition, anti-CD45RB treated lymphocytes inhibit thymidine incorporation and proliferation of allogeneic lymphocytes. Finally, when cocultured with untreated T cells, anti-CD45RB treated lym-

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Transplantation

phocytes regulate the proliferation of allogeneic responder lymphocytes in a dose-dependent manner.

Transduction of the anti-apoptotic gene survivin protects islets from cell death CE Ariyan, T Dohi, P Salvalaggio, I Millet, D Altieri, G Basadonna New Haven, CT INTRODUCTION: Methods to preserve islets are considered crucial to the success of islet transplant. Survivin, an inhibitor of apoptosis (IAP), regulates the cell cycle, inhibits cell death, and promotes angiogenesis. This study examined the role of survivin in protecting islets from cell death. METHODS: Pancreatic islets harvested from BalbC mice were cultured (1) alone, (2) vector alone (pAd-GFP), or (3) vector with the surviving gene (pAd-survivin). Insulin release was measured by RIA. Cell toxicity was induced with cytokines: TNF-␣, IL-1␤, and IFN-␥. After 48 hours, a colorimetric assay detected cell viability using MTT. Results were read on a spectrophotometer plate reader. RESULTS: Western blotting confirmed the expression of survivin in the islets. Islets transduced demonstrated a normal glucose stimulated insulin release curve, indistinguishable from that of untreated islets. Untreated and pAd-GFP islets had a decrease in cell survival as measured by MTT assay. Islets treated with 2 ⫻ 108 pfu pAd-survivin were completely resistant to cell death.

Table 1. Apoptosis (avg)

Untreated pAd-GFP pAd-surivivin

Alone (OD)

Cytokines (OD)

%

0.401 ⫾ 0.06 0.356 ⫾ 0.1 0.274 ⫾ 0.037

0.188 ⫾ 0.036 0.253 ⫾ 0.063 0.282 ⫾ 0.035

53.2 29.0 0

CONCLUSIONS: Islets transduced with survivin maintained function and are resistant to apoptosis. These data lend support to the use of genetic strategies to prevent islet cell loss in transplantation. Targeting of host ␣␤-TCRⴙ and ␥␦-TCRⴙ T cells reduces the conditioning required to establish chimerism to induce donor-specific tolerance Hong Xu MD, Yiming Huang MD, Paula M Chilton PhD, Suzanne T Ildstad MD Louisville, KY INTRODUCTION: Mixed chimerism induces tolerance to solid organ allografts. Conditioning with 700 cGy total body irradiation (TBI) is required to establish chimerism. We now determined whether the TBI could be reduced by targeting host ␣␤-TCR⫹ and ␥␦-TCR⫹ T cells with monoclonal antibody (mAb). METHODS: Recipient C57BL/10 (H2b) mice were pretreated in vivo with anti–␣␤-TCR and anti-␥␦⫺TCR mAb on day ⫺3. Mice were conditioned with decreasing doses of TBI and trans-

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planted with 15 ⫻ 106 allogeneic B10.BR (H2k) bone marrow (BM) cells on day 0. RESULTS: 100% of recipients pretreated with mAb engrafted with 400 and 500 cGy TBI 1 month after transplantation. Engraftment occurred in 0, 60%, 82%, and 90% of mice conditioned with 0, 100, 200, and 300 cGy TBI, respectively. The level of chimerism correlated with the amount of TBI. However, only the group conditioned with 500 cGy TBI maintained durable chimerism up to 6 months. The engraftment was multilineage, with production of donor T cells, B cells, NK cells, macrophages and granulocytes. Recipients with durable chimerism accepted donor-specific skin grafts, but promptly rejected major histocompatibility complex disparate third party BALB/c (H2d) skin grafts. CONCLUSIONS: Pretreatment of recipients with anti–␣␤TCR and anti–␥␦-TCR mAb significantly reduces the TBI requirement for establishing mixed chimerism, confirming a major role for T cells in mediating alloresistance. The development of targeted conditioning protocols with minimal toxicity may expand the application of BM transplantation to tolerance induction. Facilitating cells: FCp33/TCR␤ heterodimer expression and the absence of GVHD effector activity Yolonda L Colson MD PhD, Renee D Wright Boston, MA INTRODUCTION: The facilitating cell (FC), a CD8⫹ donor bone marrow cell that expresses a novel FCp33/TCR␤ heterodimer distinct from conventional ␣␤⫹ T cells, enables hematopoietic stem cell (SC) alloengraftment and induction of donorspecific transplantation tolerance. However, the graft versus host disease (GVHD) potential of the FC has not been aggressively studied. METHODS: Using a Parent 3 F1 transplantation model (B6 3 B6D2F1) to highlight GVHD potential, lethally irradiated (1100 cGy) B6D2F1 were reconstituted with purified SC (Scal⫹/Lin⫺) and FC (CD8⫹/␣␤␥␦TCR⫺) populations isolated from donor B6 marrow using rare-event flowcytometric cell sorting. Splenic ␣␤TCR⫹ T cells were sorted as positive GVHD effector controls. Recipients of 2000 donor SC alone, SC plus 1–2 ⫻ 105 FC or SC plus 1–2 ⫻ 105 T cells were assessed weekly for survival and severity of GVHD. The five-parameter GVHD clinical scores range from ⱕ2 for no GVHD to 7–10 in lethal disease. RESULTS: All recipients of SC alone or SC⫹FC survived ⬎6 weeks and exhibited similar peak clinical scores (1.6 ⫾ 0.2 versus 2.0 ⫾ 0.4). In contrast, recipients of SC⫹T cells exhibited decreased survival (36.4% mortality at 21 days) and more severe GVHD than SC⫹FC (4.0 ⫾ 0.7, p ⬍ 0.01 Mann-Whitney). CONCLUSIONS: These findings demonstrate that, unlike ␣␤TCR expression on T cells, TCR␤ expression with the FCp33 protein on the FC does not possess GVHD effector activity. Further characterization of FCp33 and its function(s) might offer

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insight into opposing mechanisms of GVHD, rejection, and transplantation tolerance.

Role of endothelin-1 in microvascular dysfunction caused by cyclosporin A Chumpon Wilasrusmee MD MSc, Monica Da Silva MD, Josephine Siddiqui BS, Skuntala Wilasrusmee MD, David Bruch BS, Dilip S Kittur MD ScD Syracuse, NY INTRODUCTION: Endothelin-1 (ET-1) is a patent vasoconstrictive peptide that plays a significant role in cyclosporin A (CyA)-associated allograft vasculopathy. Previously we have demonstrated in an in vitro model of endothelialitis that CyA induces endothelial dysfunction and leads to disruption of capillaries. The present study addresses the effects of ET-1 on CyA-induced endothelial dysfunction. METHODS: Endothelial cells (ECs) were cultured on laminin-rich matrix, Matrigel, to form capillarylike networks. Treatment of ECs with CyA was performed at the onset of the experiment before capillary tube formation or after capillary tubes had matured. ET-1 antibody was incubated with CyA to prevention disruption of capillaries and the results were quantified by counting the number of capillary tubes in triplicate well. Statistical significance was determined with ANOVA followed by Bonferroni method. ppET-1 gene expression was studied by RT-PCR. RESULTS: ppET-1 was expressed in ECs during capillary tube formation but disappeared once capillary tubes matured (24 hours after the onset of experiment). ppET-1 gene expression was significantly induced in dysfunctional tubes caused by CyA. Inhibiting protein synthesis by cycloheximide inhibited capillary tube formation; exogenous ET-1 partially reversed this inhibition and allowed initiation of tube formation. Anti ET-1 antibody (0.1 to 1.5 mg/mL) prevented CyA mediated capillary disruption (p ⬍ 0.001). CONCLUSIONS: Endothelin-1 is an important molecule in CyAinduced endothelial dysfunction and, thus, in allograft vasculopathy. Blocking of ET-1 action may pave the way to prevent endothelial dysfunction caused by CyA.

Reversed activity of mitochondrial adenine nucleotide translocator (ANT) in ischemia reperfusion Andrey E Belous MD PhD, C Wright Pinson MD MBA FACS, Ravi S Chari MD FACS Nashville, TN INTRODUCTION: Ischemia-reperfusion injury may be because altered cytoplasmic Ca⫹⫹. Mitochondrial Ca⫹⫹ (mCa⫹⫹) uptake attenuates cytoplasmic Ca⫹⫹, but increase of mCa⫹⫹ may trigger apoptotic pathways. Ischemic depression of electrochemical gradient across the inner mitochondrial membrane (⌬⌿) triggers reversal of F0F1 ATP synthase, but it is not known, how cytoplasmic ATP becomes available to be hydrolyzed by the intramitochondrial domain of this enzyme. We hypothesized that mitochondrial adenine nucleotide translocator (ANT) reverses its activity during ischemia,

J Am Coll Surg

thereby making cytoplasmic ATP available for F0F1 ATP synthase hydrolysis. METHODS: Rat livers perfused with 4°C UW through the portal vein were processed immediately (no isch) or after 24 hours cold storage (24 hours isch). Mitochondria were separated by differential centrifugation. ATP-dependent mCa⫹⫹ uptake was determined by scintillation counting in the presence of ATP (5 mmol/L), with variable extramitochondrial [Ca⫹⫹] with or without 15 ␮M of bongkrekic acid (BA) (ANT blockade); nonhydrolysable analog of ATP (AMP-PNP) served as negative control. All measurements were performed in triplicate. Student’s t-test, with p⬍0.05 was taken as significant. RESULTS: (1) inhibition of ANT by BA prevents mCa⫹⫹ accumulation in presence of ATP even at high Ca⫹⫹ concentrations; (2) 24 hours cold ischemia is associated with increased ATP-dependent mCa⫹⫹ uptake; (3) increased extramitochondrial Ca⫹⫹ stimulated mCa⫹⫹ uptake in presence of ATP but not AMP-PNP or BA. CONCLUSIONS: These data demonstrate that ANT plays an important role in mCa⫹⫹ uptake under ischemic conditions by reversing its activity and allowing for the outer ATP to be transported into the matrix to be hydrolyzed by reversed F0F1 ATP synthase. The diabetic milieu inhibits pancreatic precursor differentiation Hiroyuki Kobayshi MD, Troy Spilde MD, Amina Bhatia MD, Julie Marosky MD, Mark Hembree BA, Barry Preuett BA, Zhixing Li PhD, Krishna Prasadan PhD, Charles Snyder MD, George Gittes MD Kyoto, Japan INTRODUCTION: We previously showed that isolated embryonic pancreatic epithelia placed under the renal capsule develops into mainly islets. We hypothesized that the diabetic milieu may enhance islet formation, with implications for future islet/stem cell transplantation. METHODS: Embryonic day 13.5 rat pancreases were harvested and, in half of these, the epithelium was separated from surrounding mesenchyme by mechanical dissociation. Pancreases were transplanted under the renal capsule of adult syngeneic rats using 4 experimental groups: (1) nondiabetic host transplanted with whole embryonic pancreas, (2) nondiabetic host with isolated embryonic pancreatic epithelium, (3) streptozotocin-induced diabetic host with whole pancreas, (4) diabetic with epithelium. After 2 weeks, standard histology and quantitative immunohistochemistry for insulin-, glucagon-, and amylase-positive cells was performed. RESULTS: Surprisingly, there were less insulin-positive cells in pancreases that developed in a diabetic milieu (p⬍0.05), and also surprising was that there were fewer insulin-positive cells in the absence of mesenchyme (p⬍0.005). Blood glucose was not affected in any groups. CONCLUSIONS: The diabetic environment inhibits differentiation of pancreatic precursor cells into insulin-positive cells, which may present yet another hurdle in diabetic treatment strategies using stem cells. Also, because the surrounding pancreatic mesenchyme is necessary for optimal insulin differentiation, such factors may be applicable to optimal stem-cell engineering for transplantation.