Effect of an anti-C5a monoclonal antibody indicates a prominent role for anaphylatoxin in pulmonary xenograft dysfunction

BACKGROUND: In contrast to renal or cardiac xenografts, the inhibition of complement using cobra venom factor (CVF) accelerates pulmonary xenograft failure. By activating C3/C5 convertase, CVF depletes complement while additionally generating C5a and other anaphylatoxins, to which pulmonary xenografts may be uniquely susceptible. The current study investigates the role of C5a in pulmonary xenograft failure in baboons. METHODS: Left orthotopic pulmonary xenografts using swine lungs expressing human CD46 were performed in baboons receiving: I) no other treatment (n=4), II) immunodepletion (n=5), and III) immunodepletion plus a single dose of mouse anti-human C5a monoclonal antibody (anti-C5a, 0.6 mg/kg administered intravenously) (n=3). The extent to which anti-C5a inhibits baboon C5a was assessed in vitro using a hemolytic reaction involving baboon serum and porcine red blood cells and by ELISA. RESULTS: Baboons in Group III exhibited significantly prolonged xenograft survival (mean=722+/-121 min, P=0.02) compared to baboons in Group I (mean=202+/-24 min) and Group II (mean=276+/-79 min). Furthermore, baboons in Groups I and II experienced pronounced hemodynamic compromise requiring inotropic support whereas those in Group III remained hemodynamically stable throughout experimentation without the need for additional pharmacologic intervention. CONCLUSIONS: These findings indicate that C5a exacerbates pulmonary xenograft injury and compromises recipient hemodynamic status. Moreover, blockade of anaphylatoxins, such as C5a, offers a promising approach for future investigations aimed at preventing pulmonary xenograft injury in baboons.     

Evidence for polyreactive xenoreactive antibodies in the repertoire of human anti-swine antibodies: the 'next' humoral barrier to xenotransplantation?

The xenoreactive nature of anti-Galalpha1-3Gal antibodies, and to a lesser extent, polyreactive antibodies, has been characterized by a number of investigators. With the advent of therapies that avoid hyperacute xenograft rejection due to anti-Galalpha1-3Gal antibodies coupled with the possible development of Galalpha1-3Gal deficient swine, the Galalpha1-3Gal antigen may soon cease to be a barrier to xenotransplantation. With this in mind, the potential xenoreactive nature of polyreactive antibodies was investigated using several approaches. The levels of polyreactive antibodies from the serum of newborn (n = 2) and adult (n = 4) baboons undergoing pulmonary xenotransplantation were evaluated. Depletion of 95% and 94% of total serum IgM, without any decrease in albumin levels, was observed in the newborn baboons. This finding indicates that the IgM present at birth and germ line polyreactive IgM was adsorbed by the xenografts. The depletion of polyreactive antibodies (43-83% reduction of anti-DNP IgM) from adult baboons was also observed following pulmonary xenotransplantation or immunoadsorption therapy plus pulmonary xenotransplantation. Additional experiments using human cord serum indicated that most human polyreactive IgM were adsorbed by pig lung homogenate and that the human polyreactive IgM bound approximately two-fold more to immobilized pig lung antigens than to immobilized human lung antigens. These findings indicate that germline polyreactive antibodies are, for the most part, xenoreactive. These data suggest that polyreactive antibodies, although autoreactive, may be more xenoreactive than autoreactive.     

Depletion of pulmonary intravascular macrophages prevents hyperacute pulmonary xenograft dysfunction

BACKGROUND: Recent years have brought dramatic progress in the field of xenotransplantation, with the development of transgenic swine and various other means of overcoming the rejection mediated by xenoreactive antibodies. Although progress has been rapid with kidney and heart xenografts, progress with pulmonary xenografts has lagged behind. Recent findings have suggested that donor pulmonary intravascular macrophages may play a critical role in the hyperacute dysfunction of pulmonary xenografts. METHODS: The function of pulmonary xenografts from pigs depleted of pulmonary intravascular macrophages was compared with the function of xenografts from normal pigs. RESULTS: Pulmonary xenografts from pigs from which pulmonary intravascular macrophages were depleted survived (23.5+/-0.9 hours) about five times longer than normal (macrophage sufficient) xenografts (4.4+/-1.41 hours) (P< 0.0001). At 21 hours post-reperfusion, the left pulmonary arterial flow was 225.0+/-34 ml/min in lungs depleted of pulmonary intravascular macrophages, whereas all normal xenografts had failed. CONCLUSIONS: These findings indicate that donor macrophages play a critical role in pulmonary xenograft dysfunction. This finding has broad implications for xenotransplantation, suggesting that porcine macrophages might pose a barrier to the engraftment and function of a variety of porcine organ xenografts.     

Non-anti-Gal alpha1-3Gal antibody mechanisms are sufficient to cause hyperacute lung dysfunction in pulmonary xenotransplantation

BACKGROUND: Hyperacute lung dysfunction, which is always associated with pulmonary pig-to-primate xenotransplantation is not well understood. The mechanisms associated with its occurrence seem to differ from mechanisms involved in hyperacute xenograft rejection seen in porcine hearts or kidneys transplanted into primates. To determine the contribution of anti-Gal alpha1-3Gal antibodies (alphaGAb) in such a process, we performed a set of orthotopic pig lung transplants into baboons depleted of alphaGAb and compared graft function and survival with those receiving only immunosuppression. STUDY DESIGN: Pigs expressing human membrane cofactor protein served as donors. All baboons received triple immunosuppressive therapy. Depletion of alphaGAb in the experimental group (n = 4) was done by way of immunoadsorption using immunoaffinity membranes. Controls (n = 4) did not undergo immunoadsorption. Orthotopic lung transplants were performed through a left thoracotomy. Main pulmonary artery blood flow and pressure, left pulmonary artery blood flow, and left atrial pressure were recorded. RESULTS: At 1 hour after reperfusion, pulmonary artery graft flows and pulmonary vascular resistances (PVR) were better in animals depleted of alphaGAb than in controls (605 +/- 325.2 mL/min versus 230 +/- 21 mL/min; 27.1 +/- 41.3 mmHg/L/min versus 63 +/- 1 mmHg/L/min). But at 3 hours after reperfusion average graft flows in baboons depleted of alphaGAb had decreased to 277.6 +/- 302.2 mL/min and PVRs had increased 58.3 +/- 42.0 mmHg/L/min. On the other hand, controls maintained stable flows and PVRs (223 +/- 23 mL/min; 61 +/- 3 mmHg/L/min). Survival was ultimately better in control baboons when compared with alphaGAb depleted ones (12.2 +/- 3.3 h versus 4.4 +/- 3.2 h). CONCLUSION: Unlike heart and kidney xenograft transplants, hyperacute lung xenograft dysfunction seems to be mediated by factors other than alphaGAb.     

The role of antibodies in dysfunction of pig-to-baboon pulmonary transplants

OBJECTIVE: Pulmonary transplantation has become the preferred treatment for end-stage lung disease, but application of the procedure is limited because of a paucity of donors. One way to solve donor limitations is to use animal organs as a donor source or xenotransplantation. The current barrier to pulmonary xenotransplantation is the rapid failure of the pulmonary xenograft. Although antibodies are known to play a role in heart and kidney xenograft rejection, their involvement in lung dysfunction is less defined. This project was designed to define the role of antibodies in pulmonary graft rejection in a pig-to-baboon model. METHODS: Orthotopic transgenic swine left lung transplants were performed in baboons depleted of antibodies by one of three techniques before transplantation: (1) ex vivo swine kidney perfusion, (2) total immunoglobulin-depleting column perfusion, and (3) ex vivo swine lung perfusion. Results were compared with those of transgenic swine lung transplants in unmodified baboons. RESULTS: All three techniques of antibody removal resulted in depletion of xenoreactive antibodies. Only pretransplantation lung perfusion improved pulmonary xenograft function compared with lung transplantation in unmodified baboons. CONCLUSIONS: The pathogenesis of pulmonary injury in a swine-to-primate transplant model is different from that in renal and cardiac xenografts. Depletion of antibodies alone does not have a beneficial effect and may actually be detrimental.     

Busulfan depletes neutrophils and delays accelerated acute rejection of discordant xenografts in the guinea pig-to-rat model

Complement factor C6 plays a critical role in mediating hyperacute rejection of discordant xenografts. In order to explore the mechanism of discordant xenograft rejection, we investigated kinetics and phenotypes of the cellular infiltrate in xenografts in untreated and leukocyte-depleted recipients, in relation to graft survival. Guinea pig cardiac xenografts were heterotopically transplanted to totally C6-deficient PVG (C-) rats. Grafts were removed after 0, 6, 12, and 24 h ( n = 6). Histological evaluation was performed with hematoxylin and eosin (H & E) and immunoperoxidase staining. The agents fucoidin and busulfan were applied to delay xenograft rejection further. Within 6 h, minimal perivascular edema with isolated infiltrating CD11b/c- and ED1-positive cells were found. An intense infiltration of CD11b/c- and ED1-positive cells with interstitial hemorrhage was present after 24 h, though with little CD161 and CD3 cell infiltration. Inhibition of cell adhesion by fucoidin did not prolong xenograft survival (34 +/- 15 h, n = 4, P<0.47), but the depletion of granulocytes by injection of busulfan did prolong survival of the discordant xenografts, to 62 +/- 22 h ( n = 7, P < or = 0.0039). These results demonstrate a significant effect of specific depletion of granulocytes and macrophages by busulfan therapy on guinea pig cardiac xenograft survival in PVG (C-) rats, suggesting the participation of these infiltrating cells in the xenoreactive rejection process.     

Experimental discordant hepatic xenotransplantation in the recipient with liver failure: implications for clinical bridging trials

BACKGROUND: Clinical xenotransplantation might start with bridge-to-bridge trials. Situations where hyperacute rejection is avoided would provide opportunities for the initiation of bridging trials. Patients with liver failure have a diminished capacity to initiate antibody and complement-induced injury of xenogeneic endothelium. Hyperacute rejection of a liver xenograft manifests as a coagulopathy. We examined the ability of a recipient with liver failure to hyperacutely reject a liver xenograft in the dog-to-pig model in the immediate postoperative period. STUDY DESIGN: Liver failure in pigs was induced with galactosamine. Canine livers were transplanted into pigs with liver failure and into healthy pigs. The postoperative course was monitored for 1 hour for histologic changes in the xenograft, changes in platelet counts, and whole blood clotting with Sonoclot analysis. In vitro assays with pig serum and canine hepatic sinusoidal endothelial cells were used to assess the effect of liver failure on serum cytotoxicity and xenoreactive antibody levels. RESULTS: All untreated pig recipients of liver xenografts died from a coagulopathy. Recipients with liver failure manifested no signs of coagulopathy, and had minimal change in platelet counts or Sonoclot (Sienco Inc., Morrison, CO) tracings. Liver xenograft biopsies from recipients with liver failure showed no evidence of the tissue injury that characterized the biopsies of control recipients. Serum from pigs was less cytotoxic to the canine hepatic sinusoidal endothelium after induction of liver failure. The xenoreactive antibody levels and repertoire were similar in the pig serum before and after liver failure was induced. CH50 (total complement) levels were diminished in pigs after the induction of liver failure. CONCLUSIONS: Liver xenotransplantation used in bridging trials in recipients with liver failure might not face the barrier of hyperacute rejection.     

The role of anti-Galalpha1-3Gal antibodies in acute vascular rejection and accommodation of xenografts

BACKGROUND: A major impediment to the transplanting of porcine organs into humans is the susceptibility of porcine organs to acute vascular rejection, which can destroy a vascularized xenograft over a period of hours to days. Acute vascular rejection of porcine-to-primate xenografts is thought to be triggered by binding of xenoreactive antibodies to the graft. We tested whether antibodies, binding to Galalpha1-3Gal epitopes in porcine tissue, initiate this phenomenon. METHODS AND RESULTS: Specific depletion of anti-Galalpha1-3Gal antibodies from the blood of baboons, using extracorporeal perfusion of separated plasma through columns of Sepharose beads covalently linked to the antigenic trisaccharide, Galalpha1-3Galbeta1-4GlcAc, averted the development of acute vascular rejection in porcine organs transgenic for human decay-accelerating factor and CD59. More importantly, after immunodepletion was stopped and Gala1-3Gal antibodies were allowed to return, these same organs continued to function and remained pathologically normal and thus seemed to achieve a state of accommodation. CONCLUSION: These results demonstrate that anti-Galalpha1-3Gal antibodies cause acute vascular rejection and suggest that depletion of these antibodies leads to accommodation of the donor cardiac xenograft and could supply an important model for additional study.     

Potential of an injectable polymer to prevent hyperacute rejection of ex vivo perfused porcine lungs

BACKGROUND: Removal of xenoreactive antibodies in pig-to-human lung transplantation by columns or organ perfusions proofed to be unsatisfactory and associated with adverse effects. In an ex-vivo lung perfusion model, we evaluated the potential of a soluble trisaccharide polymer (GAS914) to bind alpha-Gal antibodies and to protect a pulmonary xenograft from hyperacute rejection (HAR) and pulmonary xenograft dysfunction. METHODS: Porcine lungs were perfused with fresh human blood for 240 min. In the GAS914 treated group (n=6) the polymer was applied in three different concentrations. The control group (n=6) received no GAS914. Survival and function of perfused xenografts were monitored, and alpha-Gal antibodies as well as cytolytic anti-porcine antibodies analyzed. RESULTS: In the GAS-treated group survival of lungs was significantly prolonged, pulmonary vascular resistance reduced, pulmonary edema prevented, and oxygenation improved. On histopathological evaluation application of GAS resulted in minimal graft injury and significantly less deposition of the terminal complement complex C5b-9. Following application of GAS914, up to 89.8% of IgG alpha-Gal, 79.5% of IgM and 73.6% of anti-porcine antibodies in the human blood were bound by the polymer. Subsequent perfusion of porcine lungs resulted in absorption of only 3% of the baseline IgG alpha-Gal antibodies in the GAS914 group, compared to 87% in the controls. CONCLUSIONS: In this ex-vivo lung perfusion model, a trisaccharide polymer prevented immediate HAR, due to effective removal of alpha-Gal antibodies. In combination with additional strategies GAS914 may be a valuable tool in overcoming HAR and dysfunction of pulmonary xenografts.     

Protease inhibitor nafamostat mesilate attenuates complement activation and improves function of xenografts in a discordant lung perfusion model

Tagawa T. Protease inhibitor nafamostat mesilate attenuates complement activation and improves function of xenografts in a discordant lung perfusion model. Xenotransplantation 2011; 18: 315–319. © 2011 John Wiley & Sons A/S. Abstract: Background: Anti‐complement activity of nafamostat mesilate (FUT‐175) is strong including its variety of pharmacological effects. The effect of FUT‐175 for xenografts in an ex vivo guinea pig‐to‐rat lung perfusion model was evaluated. Methods: Heparinized Lewis rat blood was used to perfuse the lungs in three groups (n = 6 each). Group I used Lewis rat left lung for donor, Group X used guinea pig left lung for donor, and Group XF used guinea pig left lung for donor, which was perfused with Lewis rat blood with 0.2 mg/ml of FUT‐175. Complement activity causing 50% hemolysis (CH50) in the perfusion blood and pulmonary function either before or during perfusion were serially measured. Pathological assessments of the lungs were also carried out after perfusion. Results: The duration of satisfactory pulmonary function was significantly increased in Group XF. Complement activity causing 50% hemolysis in Group XF decreased more significantly compared to Group X. FUT‐175 suppressed both the increase in pulmonary arterial pressure and airway resistance, and the decrease in dynamic lung compliance. In Group X, pathology showed intra‐alveolar hemorrhage, perivascular edema, and medial thickening with endothelial swelling of the pulmonary arteries. In Group XF, less changes were observed compared to Group X. Group X showed deposition of IgM, IgG, and C3 at the endothelium of arteries, which was fewer in Group XF, and even fewer in Group I. Conclusions: This study suggests that FUT‐175 inhibited complement activation and improved lung xenograft function. FUT‐175 ameliorates hyperacute rejection in a guinea pig‐to‐rat ex vivo xenogeneic lung perfusion model.     

Rejection of porcine islet xenografts mediated by CD4+ T cells activated through the indirect antigen recognition pathway

We have previously demonstrated that human T cells responding to porcine islets are primarily CD4+ and recognized porcine major histocompatibility complex class I molecules through the indirect pathway of antigen presentation. To determine whether this mechanism is responsible for rejection of adult porcine islets xenografts, porcine islets from adult pigs were transplanted under the kidney capsule of streptozotocin-treated CD4-knockout (KO), CD8-KO, Ig-KO and normal C57BL/6 mice. Islet xenografts were acutely rejected with similar kinetics when transplanted into normal C57BL/6 (MST=17.6 +/- 3.5 days) and Ig-KO (MST=19.0 +/- 1.7 days) mice. Interestingly, islet xenografts were rejected significantly earlier when transplanted into CD8-KO mice as compared with normal C57BL/6 (MST=7.0 +/- 0.01 days, P=2 x 10-4). Histopathological analysis revealed classical acute cellular rejection with severe diffuse interstitial cellular infiltrates in all rejected islet xenografts. In contrast, islet xenografts were not rejected when transplanted into CD4-KO mice (MST >/= 100 days, P=1 x 10-9). Histopathological analysis revealed no cellular infiltrates and intact islet xenografts. CD4+ T cells from both normal C57BL/6 and CD8-KO xenograft recipients showed detectable proliferative responses to porcine islets in the presence but not in the absence of syngeneic antigen-presenting cells. In addition, the anti-islet proliferative responses observed in normal C57BL/6 mice were significantly lower than those observed in CD8-KO mice. IgG anti-porcine antibodies were readily detected in C57BL/6 and CD8-KO xenograft recipients but not in Ig-KO or CD4-KO recipients. These results indicate that indirectly activated CD4+ T cells mediate acute rejection of adult porcine islet xenografts and that xenoreactive CD8+ T cells and antibodies are not necessary in this process.     

The Role of Antibodies and Von Willebrand Factor in Discordant Pulmonary Xenotransplantation

Pulmonary xenotransplantation is one potential solution to the paucity of donors but is currently limited by rapid failure of the graft. Unlike cardiac and renal xenotransplants, pulmonary xenografts release large quantities of swine von Willebrand factor (vWF). Swine vWF binds xenoreactive antibodies and is capable of activating primate platelets. The contribution of swine vWF to lung xenograft dysfunction is not entirely clear. To probe the role vWF plays in xenograft dysfunction, we traced the fate of xenoantibodies in vWF+ and von Willebrand factor-deficient (vWFD) swine lungs. These studies showed that the vast majority of xenoantibodies bind the vWF released from the vWF+ swine lung, and thus do not remain bound on lung endothelium. The vWF complexed to xenoantibody remained capable of aggregating primate platelets. With this information, we performed swine-to-baboon lung transplants using vWF+ and vWFD donors. Without vWF present to complex xenoantibodies, a picture of hyperacute rejection more typical of heart and kidney xenografts, with antibody deposition along the graft endothelium, interstitial hemorrhage, and edema occurred. These findings suggest that porcine vWF plays a major role in the pathogenesis of pulmonary xenograft dysfunction, and suggests promising strategies to treat lung xenograft dysfunction.     

Serum anti-pig antibodies as potential indicators of acute humoral xenograft rejection in pig-to-cynomolgus monkey kidney transplantation

BACKGROUND: Hyperacute rejection of solid organ pig xenografts in nonhuman primates has been overcome by using donors transgenic for human complement regulatory proteins, but grafts are still susceptible to humoral (antibody-mediated) rejection. We investigated whether circulating xenoreactive antibodies are a useful indicator of this xenograft rejection. METHODS: Five assays were employed in a retrospective analysis on 20 selected cynomolgus monkey recipients of renal xenografts transgenic for human decay-accelerating factor, with survival between 4 and 60 days. The assays included hemolytic and hemagglutination assays and the measurement of immunoglobulin (Ig)G and IgM binding to porcine endothelial cells and leukocytes, and to the Gal alpha 1-3Gal trisaccharide (Gal) antigen. To assess non-Gal-directed antibodies, sera were absorbed with a Gal-coated resin. A predictive value was defined as an increase in antibody levels before a decline in graft failure (>20% increase in creatinine levels) and humoral rejection in graft pathology. RESULTS: Data on hemolytic anti-pig antibody correlated with those on IgM antibody to endothelial cells, leukocytes, and Gal. In absorbed sera IgM and IgG antibody to endothelial cells and leukocytes correlated with each other, indicative for an elicited antibody response to non-Gal antigens. Sixteen animals showed humoral rejection, and in all but two animals one or more assays was considered of predictive value. On the other hand, increased antibody levels were noted in two animals without signs of rejection in graft pathology and in two cases with cellular xenograft rejection. CONCLUSIONS: It is recommended to use multiple assays (preferably hemolytic, anti-Gal, and anti-endothelial cell) to be able to fully monitor the peripheral antibody responses in pig-to-primate xenograft recipients.     

Gene therapy-mediated CD40L and CD28 co-stimulatory signaling blockade plus transient anti-xenograft antibody suppression induces long-term acceptance of cardiac xenografts

BACKGROUND: The authors have previously demonstrated that inhibition of CD28 and CD40 ligand (CD40L) co-stimulatory signals by adenovirus-mediated cytotoxic T-lymphocyte-associated (CTL) antigen 4 (A4) immunoglobulin (Ig) and CD40Ig gene therapies induces tolerance or long-term acceptance in rat liver and heart allograft transplantation. In this study, the authors examined whether co-stimulation blockade with a brief course treatment of FK779, a novel leflunomide derivative, would be an ideal strategy for controlling xenograft rejection. METHODS: Hamster hearts were transplanted into Lewis rats. Adenovirus vector coding (Ad) CD40Ig, CTLA4Ig, or LacZ gene (1 x 10(9) plaque-forming units) was administered intravenously to recipient rats 2 days before or immediately after xenografting. FK779 (10 mg/kg/day) was administered orally to recipients for 7 days beginning on day -1. Graft survival, graft histology, and xenoreactive antibodies were examined. RESULTS.: Both untreated and AdLacZ-treated control rats rejected cardiac xenografts, with a median survival time (MST) of 3 days. Co-stimulatory blockade alone by AdCTLA4Ig, AdCD40Ig, or both could not overcome such delayed xenograft rejection (DXR) (MST, 3-4 days). Under a short-course FK779 treatment that suppressed T-cell-independent xenoreactive antibodies, administration of AdCD40Ig (MST, 30.5 days) but not AdCTLA4Ig (MST, 9 days) significantly prolonged xenograft survival as compared with the FK779 monotherapy (MST, 7 days). In contrast, DXR and cellular rejection were controlled successfully and all xenografts were accepted for over 100 days when AdCTLA4Ig and AdCD40Ig were administered under FK779 induction therapy. However, chronic rejection was present in all long-term surviving xenografts. CONCLUSIONS.: Gene therapy-based co-stimulation blockade with FK779 induction treatment seems to be an attractive strategy with which to control xenograft rejection.     

Isolated lung perfusion with melphalan prolongs survival in a rat model of metastatic pulmonary adenocarcinoma.

INTRODUCTION: Survival after isolated lung perfusion (ILuP) with melphalan was tested in a model of unilateral pulmonary adenocarcinoma. METHODS: On day 0, rats were randomized into four groups: Group 1 (n = 9) received tumor cells intravenously for induction of bilateral lung metastases, whereas groups 2-4 (n = 21) underwent a 10-min occlusion of the right pulmonary artery during tumor cell injection for induction of unilateral left lung metastases. On day 7, groups 1 and 2 received no treatment. Group 3 underwent left ILuP with melphalan (2.0 mg/kg) while group 4 received melphalan intravenously (0.5 mg/kg). The end point of the study was death from metastatic disease. RESULTS: Median survival of ILuP-treated animals (81 +/- 12 days) was significantly longer compared to group 1 (18 +/- 1 days; p = 0.0001), group 2 (28 +/- 3 days; p = 0.0002) and group 4 (37 +/- 6; p = 0.0004). CONCLUSIONS: ILuP with melphalan prolongs survival in the treatment of experimental metastatic pulmonary carcinoma.     

Hyperacute lung rejection in the pig-to-human model 4: evidence for complement and antibody independent mechanisms

BACKGROUND: We assessed whether the combination of complement regulation and depletion of xenoreactive antibodies improves the outcome of pulmonary xenografts compared with either strategy alone. METHODS: Lungs from pigs heterozygous (hDAF(+/-)) or homozygous (hDAF(+/+)) for the human decay accelerating factor transgene (hDAF) or their nontransgenic litter mates (hDAF(-/-)) were perfused with heparinized whole human blood. In additional groups, xenoreactive natural antibodies (XNA) were depleted by pig lung perfusion (hDAF(-/-)/AbAbs, hDAF(+/-)/AbAbs) before the experiment. This combined approach was augmented by adding soluble complement receptor 1 (sCR1) to the perfusate in one further group (hDAF(+/-)/AbAbs/sCR1). RESULTS: HDAF(-/-) lungs perfused with unmodified human blood were rejected after 32.5 min (interquartile range, IQR 5 to 210). HDAF(+/-) lungs survived for 90 min (IQR 10 to 161, P = 0.54). Both groups showed a rapid rise in pulmonary vascular resistance (PVR), which is a characteristic feature of hyperacute rejection (HAR). This phenomenon was blunted in the hDAF(+/+) group, although survival (48 min, IQR 14 to 111) was not further prolonged. Antibody depletion (AbAbs) led to a significant increase in survival time (hDAF(-/-)/AbAbs: 315 min, IQR 230 to 427; hDAF(+/-)/AbAbs: 375 min, IQR 154 to 575), reduced PVR and less complement production. Addition of sCR1 reduced complement elaboration but did not further improve survival (200 min, IQR 128 to 580) and surprisingly tended to increase PVR. CONCLUSIONS: Depletion of xenoreactive antibodies is more effective than membrane-bound complement regulation to blunt hyperacute rejection of pulmonary xenografts, but even the combined approach including soluble-phase complement inhibition is not sufficient to reliably prevent organ failure within hours. It therefore seems likely that other factors independent of antibody and complement contribute to HAR in this model.     

Intrauterine creation and repair of pulmonary artery stenosis in the fetal lamb. Weight and ultrastructural changes of the ventricles

Fetal lamb experimental models were employed for intrauterine creation and repair of pulmonary artery stenosis. The study group was composed of 51 fetal lambs including 29 models of pulmonary artery stenosis and 22 control lambs. Gestational age was 89 days at creation of pulmonary artery stenosis. Fourteen fetal lambs (Group A) were studied after creation of the stenosis at 131 days of gestation and compared to normal age-matched control lambs. The systolic right ventricular pressure was significantly higher after creation of pulmonary artery stenosis (76.6 +/- 17.8 versus 50.3 +/- 23.5 mm Hg), but the systolic pulmonary artery pressure was unchanged. The mean right ventricular weight and the mean right ventricular/left ventricular weight ratio were significantly greater after pulmonary artery stenosis than in normal control animals. The transverse myocyte diameter was not modified by pulmonary artery stenosis, but on electron microscopic study the myocytes appeared mature. Ten lambs (Group B) underwent intrauterine repair of pulmonary artery stenosis at 131 days of gestation without cardiopulmonary bypass. The pulmonary artery was clamped and patched. Immediately after repair the right ventricular pressure fell significantly from 85.8 +/- 18.9 to 62.2 +/- 14.6 mm Hg. At birth, 7 +/- 6 days after repair, Group B was compared to Group C (unrepaired pulmonary artery stenosis, five fetuses) and to normal control lambs. The mean right ventricular weight and the mean right ventricular/left ventricular weight ratio were not statistically different in Group B and in the control group. There were no ultrastructural changes after intrauterine repair. We conclude that intrauterine creation of pulmonary artery stenosis causes right ventricular hypertrophy with more mature myocytes. Intrauterine repair of pulmonary artery stenosis is feasible without cardiopulmonary bypass and rapidly abolishes the preponderance of right ventricular weight over left ventricular weight.     

Single lung transplantation in rats with chemically induced pulmonary hypertension.

Physiologic effects of single lung transplantation on pulmonary hypertension were studied in rats with monocrotaline-induced pulmonary hypertension. Inbred rats treated with monocrotaline (40 mg/kg) received a left lung isograft from a normal donor 2 weeks later, when pulmonary hypertension became significant (transplant group; n = 6). These rats and control rats treated with monocrotaline (mediated control group; n = 11) or vehicle alone (normal control group; n = 9) were followed up weekly by metabolic treadmill testing for exercise tolerance and oxygen consumption up to 6 weeks after monocrotaline (4 weeks after transplantation), when all rats underwent hemodynamic and histologic examinations. Whereas maximal oxygen consumption and exercise tolerance consistently deteriorated in the medicated control group of rats, indices in the transplant group stopped deteriorating 2 weeks after lung transplantation and remained at levels similar to those of normal control rats. Severe pulmonary hypertension (68 +/- 19 mm Hg) and right ventricular hypertrophy (right ventricular/left ventricular weight ratio, 0.95 +/- 0.19) were confirmed in medicated control rats in contrast to transplant animals, in which these two indices remained at normal control levels. Whereas left-to-right lung perfusion ratio was constant among rats not receiving transplants (0.69 +/- 0.16), it was significantly elevated (2.27 +/- 0.65; p less than 0.001) in those receiving transplants, suggesting preferential flow through the lung isograft. The results suggest that, in the early phase of pulmonary hypertension, single lung transplantation shifts pulmonary perfusion to the grafted lung, avoiding right ventricular pressure overload and thereby preserving exercise tolerance at a nearly normal level in rats with monocrotaline-induced pulmonary hypertension.     

Combination of hDAF-transgenic pig hearts and immunoadsorption in heterotopic xenotransplantation of immunosuppressed baboons

INTRODUCTION: Hyperacute xenograft rejection (HXR) and acute vascular rejection (AVR) after xenotransplantation are triggered by xenoreactive antibodies (XAb) and an activated complement cascade. In a heterotopic (abdominal) xenotransplantation model we combined immunoadsorption (IA, Ig-Therasorb column) and a quadruple immunosuppressive drug therapy in recipient baboons with donor pig hearts transgenic for human decay accelerating factor (hDAF). METHODS: According to XAb titers between 6 and 14 cycles of IA were performed preoperatively in 4 recipient baboons (18.6 +/- 2.5 kg). Hearts of hDAF-transgenic donor pigs (6.1 +/- 1.1 kg, Imutran Ltd., a Novartis Pharma AG Company, Basel, Switzerland) were heterotopically transplanted using the abdominal technique in baboons. Immunosuppression consisted of cyclophosphamide (CyP) induction therapy, ERL080 (Novartis Pharma AG), cyclosporin A (CyA, Neoral), and steroids. Blood levels of mycophenolate, CyA, immunoglobulins (Ig), anti-pig-antibodies, complement factors, and cardiac enzymes were determined. Abdominal electrocardiography (ECG), echocardiography, and palpation were used for monitoring of the pig hearts. Myocardial tissue specimens were examined using immunohistochemistry, light microscope (LM), and electron microscope (EM). RESULTS: Ten cycles of IA alone removed 78% of XAb and accordingly IgM, IgG, IgA, complement C3, and C4. None of the xenografts was hyperacutely rejected, but xenograft failure occurred after 5.0 +/- 1.3 days (range, 2.4-8.0 days) because of an AVR associated with a rapid XAb increase within 24 hours. White blood cell count (10.3 +/- 2.2 G/L) showed a maximum of 13.1 +/- 2.1 (day 1) and constant levels (1.4 +/- 0.3-2.1 +/- 1.3 G/L) between day 3 and 6. Histology (LM/EM) showed massive hemorrhage, necrosis, and vascular thrombi as signs of AVR. CONCLUSION: Although HXR was prevented by using IA and hDAF-transgenic donor hearts, AVR was not avoided due to insufficient immunosuppressive regimen used and a missed postoperative IA treatment as a result of an inefficient control of XAb production.     

Pulmonary Root Replacement With the Freestyle Stentless Aortic Xenograft in Growing Pigs

Background. The stentless xenograft with its favorable hemodynamic performance on the left side of the heart seems an attractive, readily available alternative for the reconstruction of the right ventricular outflow tract in children.

Methods. To assess its function in a preclinical animal investigation, we replaced the pulmonary root with a Freestyle stentless aortic xenograft in 18 piglets of 26.6 ± 3.2 kg weight. The animals were allowed to grow as much as possible and slaughtered when symptoms of heart failure developed or body weight reached more than 160 kg. All valve explants were analyzed by gross examination and photography and, in 4 representative pigs, by histologic examination.

Results. Fourteen animals died prematurely after 2 weeks to 11 months. Twelve xenograft explants showed thick, immobilized, large nodular structures as cuspal remnants causing significant stenosis. At microscopy, large cuspal masses of degenerating collagen and fibrin and various inflammatory cells were frequently found. In the growing pig, most of the xenografts implanted in the pulmonary position showed early degeneration causing severe stenosis.

Conclusions. Use of this valve for right ventricular outflow tract reconstruction in children cannot be recommended.