Pulmonary Xenotransplantation Research Articles

Effects of carbon monoxide on early dysfunction and microangiopathy following GalT-KO porcine pulmonary xenotransplantation in cynomolgus monkeys.

Despite progress in the current genetic manipulation of donor pigs, most non-human primates were lost within a day of receiving porcine lung transplants. We previously reported that carbon monoxide (CO) treatment improved pulmonary function in an allogeneic lung transplant (LTx) model using miniature swine. In this study, we evaluated whether the perioperative treatment with low-dose inhalation of CO has beneficial effects on porcine lung xenografts in cynomolgus monkeys (cynos). Eight cynos received orthotopic left LTx using either α-1,3-galactosyltransferase knockout (GalT-KO; n=2) or GalT-KO with human decay accelerating factor (hDAF) (GalT-KO/hDAF; n=6) swine donors. These eight animals were divided into three groups. In Group 1 (n=2), neither donor nor recipients received CO therapy. In Group 2 (n=4), donors were treated with inhaled CO for 180-minute. In Group 3 (n=2), both donors and recipients were treated with CO (donor: 180-minute; recipient: 360-minute). Concentration of inhaled CO was adjusted based on measured levels of carboxyhemoglobin in the blood (15%-20%). Two recipients survived for 3days; 75hours (no-CO) and 80hours (CO in both the donor and the recipient), respectively. Histology showed less inflammatory cell infiltrates, intravascular thrombi, and hemorrhage in the 80-hour survivor with the CO treatment than the 75-hours non-CO treatment. Anti-non-Gal cytotoxicity levels did not affect the early loss of the grafts. Although CO treatment did not prolong overall xeno lung graft survival, the recipient/donor CO treatment helped to maintain platelet counts and inhibit TNF-α and IL-6 secretion at 2hours after revascularization of grafts. In addition, lung xenografts that were received recipient/donor CO therapy demonstrated fewer macrophage and neutrophil infiltrates. Infiltrating macrophages as well as alveolar epithelial cells in the CO-treated graft expressed heme oxygenase-1. Although further investigation is required, CO treatment may provide a beneficial strategy for pulmonary xenografts.

Sustained function of genetically modified porcine lungs in an ex vivo model of pulmonary xenotransplantation

Xenotransplantation could provide a solution to the donor shortage that is currently the major barrier to solid-organ transplantation. The ability to breed pigs with multiple genetic modifications provides a unique opportunity to explore the immunologic challenges of pulmonary xenotransplantation. Explanted lungs from wild-type and 3 groups of genetically modified pigs were studied: (i) α1,3-galactosyltransferase gene knockout (GTKO); (ii) GTKO pigs expressing the human complementary regulatory proteins CD55 and CD59 (GTKO/CD55-59); and (iii) GTKO pigs expressing both CD55-59 and CD39 (GTKO/CD55-59/CD39). The physiologic, immunologic and histologic properties of porcine lungs were evaluated on an ex vivo rig after perfusion with human blood. Lungs from genetically modified pigs demonstrated stable pulmonary vascular resistance and better oxygenation of the perfusate, and survived longer than wild-type lungs. Physiologic function was inversely correlated with the degree of platelet sequestration into the xenograft. Despite superior physiologic profiles, lungs from genetically modified pigs still showed evidence of intravascular thrombosis and coagulopathy after perfusion with human blood. The ability to breed pigs with multiple genetic modifications, and to evaluate lung physiology and histology in real-time on an ex vivo rig, represent significant advances toward better understanding the challenges inherent to pulmonary xenotransplantation.

Attenuation of Hyperacute Dysfunction and Coagulopathy in GalT-KO Pulmonary Xenotransplantation

Background: The results of both heart and kidney GalT-KO (α-1,3-galactosyltransferase gene knockout) swine-to-primate studies have been more encouraging than for lung xenotransplantation. This may be because research in pulmonary xenotransplantation is limited. Here, we evaluated whether GalT-KO lungs could prevent hyper-acute pulmonary xenograft dysfunction, concomitant with activation of the coagulation cascade and consumptive coagulopathy using ex-vivo lung perfusion or in-vivo orthotopic left lung transplant model. Methods: Porcine heart-lung blocks were procured from wild-type (WT) and Japan domestic GalT-KO swine generated at Meiji University. In Group 1 (n=3), WT lungs were perfused with autologous blood to obtain baseline values of pulmonary physiology using this system. Then, either WT (Group 2: n=2) or GalT-KO (Group 3: n=2) lungs were perfused with human blood for 120 minutes. Changes in pulmonary arterial pressure, pulmonary vascular resistance (PVR), gas exchange, complete blood counts and other key variables were followed serially. Following perfusion, the graft tissue was evaluated histologically. Additionally, one cynomolgus monkey received left lung from GalT-KO swine to determine whether the observations ex-vivo predict in-vivo performance (Group 4). Results: WT lungs perfused with autologous blood displayed nearly normal pulmonary function with only 25% increase of PVR (Group 1). Perfusion of human blood through WT lungs led to 100% increase of PVR within 60 minutes (Group 2). In contrast, GalT-KO lungs with human blood demonstrated nearly stable function for 120 minutes with only 30% increase of PVR regardless of initial rise in PVR (Group 3). After 60 minutes of perfusion, platelet counts within the perfusate markedly decreased to 14% of pre-perfusion levels in group 2, compared with 50% in Group 3. In group 2, interstitial and alveolar hemorrhage as well as intravascular thrombosis was observed. In contrast, minimum cellular infiltration and no hemorrhage and intravascular thrombosis with less neutrophil infiltration, and limited IgM and complement deposition revealed by immunohistochemistry were observed in Group 3. Similar to the ex-vivo model, the recipients of orthotopic pulmonary xenografts in Group 4, platelet counts decreased to 54% (60-min) and 41% (120-min) of pre-transplant levels. Although platelet counts gradually decreased to 18% at 16-hour reperfusion, they promptly recovered to 53% at day 3 without development of consumptive coagulopathy (DIC) or hemodynamic instability. The animal was sacrificed for histological examination at day 3. Grossly, the graft exhibited viable with limited area of edematous and hemorrhagic appearance. Conclusion: GalT-KO lungs exhibited stable pulmonary function compared with WT lungs when perfused with human blood in an ex-vivo perfusion model, which was confirmed by the in-vivo transplant model. Although further studies are required in order to understand better the mechanisms of pulmonary xenograft rejection and dysfunction, GalT-KO lungs could be sufficient to further delay or even prevent DIC and thus facilitate prolonged survival of pulmonary xenografts.

294 Prolonged Ex-Vivo Pulmonary Xenograft Function Using Genetically-Modified Porcine Donor Lungs

We explored the unique challenges of pulmonary xenotransplantation, using an ex-vivo perfusion technique that evaluated explanted lungs taken from wild-type (WT) and three strains of genetically-modified (GM) (GalT KO, GalT Ko/CD55-59 transgenic, and GalT KO/CD39/CD55-CD59 transgenic) pigs when perfused with human blood.

Coagulopathy in α-galactosyl transferase knockout pulmonary xenotransplants

After substantial progress on many fronts, one of the remaining barriers still opposing the clinical application of xenotransplantation is a disseminated intravascular coagulopathy (DIC) that is observed in the pre-clinical model of porcine-to-primate transplantation. The onset of DIC is particularly rapid in recipients of pulmonary xenografts, usually occurring within the first days or even hours of reperfusion. In this study, we describe the results of two porcine-to-baboon transplants utilizing porcine lungs depleted of macrophages, deficient in the α-1,3-galactosyltransferase gene, and with the expression of human decay-accelerating factor, a complement regulatory protein. In both cases, evidence of DIC was observed within 48 h of reperfusion, with thrombocytopenia and increases in levels of thrombin-antithrombin complex evident in both cases. Depletion of fibrinogen was observed in one graft, whereas elevation of D-dimer levels was observed in the other. One graft, which showed focal lymphocytic infiltrates pre-operatively, failed within 3 h. The results indicate that further efforts to address the coagulopathy associated with pulmonary xenotransplantation are needed. Further, evidence suggests that resident porcine immune cells can play an important role in the coagulopathy associated with xenotransplantation.

Aurintricarboxylic acid inhibits endothelial activation, complement activation, and von Willebrand factor secretion in vitro and attenuates hyperacute rejection in an ex vivo model of pig‐to‐human pulmonary xenotransplantation

In the xenotransplantation of vascularized organs, such as the lung, a large area of endothelial cell layer is a big hurdle to be overcome. We investigated the potential protective effect of aurintricarboxylic acid (ATA), a known inhibitor of platelet adhesion, on endothelial damage induced by xenogeneic serum. We also assessed its role in hyperacute xenograft rejection using a porcine ex vivo lung perfusion model. Porcine endothelial cells were incubated with human serum and other inflammatory stimuli. For the evaluation of von Willebrand factor (vWF) secretion and tissue factor (TF) expression, we used human endothelial cells. E-selectin expression, complement activation, TF expression and platelet activation were investigated by flow cytometry. In an ex vivo porcine lung perfusion model, the porcine lungs were perfused with fresh human whole blood: unmodified blood (n = 5), ATA-treated blood (n = 5), and ATA and lepirudin-treated blood (n = 5). Aurintricarboxylic acid significantly inhibited TNF-alpha- or lipopolysaccharide-induced endothelial E-selectin expression in a dose-dependent manner. ATA also prevented human serum induced-E-selectin expression and human monocytic cell adhesion to porcine endothelial cells. Moreover, ATA abolished thrombin-induced vWF secretion as well as complement activation. However, ATA induced endothelial TF expression and platelet activation in vitro. In ex-vivo experiments, ATA treatment improved pulmonary function and attenuated sequestration of leukocytes. Although ATA did not influence thrombin generation, we were able to minimize its activity by adding lepirudin to the blood with ATA. Our study demonstrated in vitro protective effect of ATA on the inhibition of endothelial activation and vWF secretion and confirmed detrimental effect of ATA on induction of endothelial TF and platelet activation. The combination of ATA and lepirudin may act beneficially by preventing coagulation perturbation while maintaining improved xenograft survival.

Pre‐treatment of porcine pulmonary xenograft with desmopressin: a novel strategy to attenuate platelet activation and systemic intravascular coagulation in an ex‐vivo model of swine‐to‐human pulmonary xenotransplantation

Von Willebrand factor (vWF) has been proposed as a major contributor to the development of coagulopathy in pulmonary xenotransplantation. Pretreatment of donor swine with 1-deamino-8-d-arginine vasopressin (DDAVP), an analog of vasopressin, can reduce the content of vWF in pulmonary xenografts. Here, we investigate the effects of DDAVP pre-treatment in an ex-vivo perfusion model of pulmonary xenotransplantation. We set up and performed the ex-vivo perfusion using porcine pulmonary accessory lobes and fresh human whole blood (n = 12). Half of the donor swine were given 3 mug/kg DDAVP intravenously for 3 days before ex-vivo perfusion (DDAVP group) and half of them were left untreated (control group). The porcine lung was perfused with fresh blood for 1 h and changes in the following parameters were monitored: pulmonary arterial pressure, pulmonary vascular resistance, blood cell counts, fibrinogen, antithrombin, platelet factor 4, D-dimer, C3a, C4d, and xenoreactive IgM. The release of Galalpha1-3Gal xenoantigen (alphaGal) from porcine lung which had been perfused and retained for 30 min with human blood was assessed by enzyme-linked immunosorbent assay using alphaGal-binding lectin. Both DDAVP and control groups showed typical findings of immediate pulmonary dysfunction: an increase of pulmonary vascular resistance and sequestration of leukocytes and platelets after ex-vivo perfusion. However, in the DDAVP group, the increase of platelet factor 4, C3a, and C4d after perfusion was attenuated compared to that in the control group. The release of alphaGal after blood retention was significantly lower in the DDAVP group than that of the control group. Pre-infusion of DDAVP to the donor swine was beneficial in attenuating platelet activation as well as complement/coagulation activation. These effects of DDAVP are likely to relate to the reduction of porcine vWF content in the xenograft. Therefore, the modulation of vWF secretion in donor lungs could be an additional therapeutic way to reduce systemic coagulopathy in pulmonary xenotransplantation.

Reply: The pulmonary first-pass effect, xenotransplantation and translation to clinical trials

Sir, we appreciate Dr Harting and colleagues for their praise and interests in our paper. The pulmonary first-pass effect of injected stem cells (lung trapping) has been issued in designing various clinical trials of intravascular stem cell transplantation (Palmieri et al. , 2004). The intravenously injected stem cells are trapped initially in the lungs and seem to escape from them to be redistributed in the systemic organs later. In rodents, intravenously injected mesenchymal stem cells (MSCs) or peripheral blood stem cells (PBSCs) accumulated initially in the lungs when observed at 5, 15 or 60 min after the injections (Gao et al. , 2001; Daldrup-Link et al. , 2004; Schrepfer et al. , 2007), and gradually moved to liver, spleen, kidney and bone marrow for 48 h (Gao et al. , 2001; Daldrup-Link et al. , 2004). Pulmonary accumulation of MSCs was 50–60% in 1 h after the injection and decreased afterward to about 30% even at 3 …

Prolonged Function of Macrophage, von Willebrand Factor-Deficient Porcine Pulmonary Xenografts

Porcine von Willebrand factor (vWF) activates human and primate platelets. Having determined the importance of pulmonary intravascular macrophages (PIMs) in pulmonary xenotransplantation, we evaluated whether, in the absence of PIMs, vWF might play a role in pulmonary xenograft dysfunction. Utilizing a left single-lung transplant model, baboons depleted of anti-alphaGal antibodies received lungs from either vWF-deficient (n = 2); MCP-expressing (n = 5); MCP PIM-depleted (n = 5); or vWF-deficient PIM-depleted swine (n = 3). Two out of three of the PIM-depleted, pvWF deficient grafts survived longer than any previously reported pulmonary xenografts, including PIM-depleted xenografts expressing human complement regulatory proteins. Depletion of PIM's from vWF-deficient lungs, like depletion of PIM's from hMCP lungs, resulted in abrogation of the coagulopathy associated with pulmonary xenotransplantation. Thus, in terms of pulmonary graft survival, control of adverse reactions involving pvWF appears to be equally or even more important than is complement regulation using hMCP expression. However, based on the rapid failure of PIM-sufficient, pvWF-deficient pulmonary xenografts, pVWF-deficient pulmonary xenografts appear to be particularly sensitive to macrophage-mediated damage. These data provide initial evidence that vWF plays a role in the 'delayed' (24 h) dysfunction observed in pulmonary xenotransplantation using PIM depleted hMCP organs.

Pre-treatment of donor with 1-deamino-8-d-arginine vasopressin could alleviate early failure of porcine xenograft in a cobra venom factor treated canine recipient☆

Unlike cardiac or renal xenotransplants, the depletion of complement using cobra venom factor (CVF) does not improve pulmonary xenograft survival. Several cases suggest that the swine von Willebrand factor (vWF) may play a major role in presenting a different pathogenesis of pulmonary xenograft dysfunction from other organs. To evaluate the role of vWF and the complement system in mediating hyperacute vascular injury of pulmonary xenografts and elucidate pathogenesis of the injury, we performed swine-to-canine orthotropic single lung xenotransplantation after pre-treatment of 1-deamino-8-d-arginine vasopressin (DDAVP) and CVF. We set up three groups for lung xenotransplantation: group I served as the control group; group II, recipients pre-treated with CVF; group III, donors pre-treated with DDAVP (9 mg/kg, 3 days)/recipients pre-treated with CVF (60 u/kg). Hemodynamic data, coagulation and complement system parameters, and grafted lung pathologies were examined serially for 3h after transplantation. DDAVP infusion reduced the vWF content in swine lung tissue in vivo (7.7+/-2.4 AU/mg vs 16.0+/-5.6 AU/mg, P < 0.0001). Infusion of CVF 24 h prior to transplantation effectively depleted the recipient's serum C3 and complement hemolytic activity below the detectable range. Regardless of the use of CVF, both groups I and II transplanted with unmodified grafts showed an immediate drop in leukocytes and platelet counts after transplantation. However, in group III, in recipients transplanted with DDAVP pre-treated swine lung, the platelet count did not decrease after transplantation (P = 0.0295). The decrease of plasma antithrombin and fibrinogen tended to be attenuated in group III. Light microscopic examination revealed extensive vascular thromboses in both capillary and larger vessels, as well as early pulmonary parenchymal damage in groups I and II, but were rarely observed in group III. Complement inhibition alone was not enough to alleviate intravascular thrombosis, the main pathology in pulmonary xenotransplantation. Pre-infusion of DDAVP to the donor animal was effective in preventing platelet sequestration and attenuated intravascular thrombosis. It is suggested that the strategies targeting vWF would be promising for successful pulmonary xenotransplantation.

Pulmonary xenotransplantation: Rapidly progressing into the unknown

As one approach to circumventing the dire shortage of human lungs for transplantation, a handful of investigators have begun to probe the possibility of pulmonary xenotransplantation. The immunologic and perhaps physiologic barriers encountered by these investigators are considerable and progress in pulmonary xenotransplantation has lagged behind progress in cardiac and kidney xenotransplantation. However, during the last few years there have been substantial advances in the field of pulmonary xenotransplantation including, most noticeably, significant progress in attenuating hyperacute dysfunction. Progress has been made in understanding the barriers imposed by xenoreactive antibodies, complement, coagulation incompatibility and porcine pulmonary intravascular macrophages. Although our understanding of the barriers to pulmonary xenotransplantation is far from complete and the clinical application of pulmonary xenotransplantation is not yet in sight, current progress is fast paced. This progress provides a basis for future work and for a hope that the shortage of human lungs for transplantation will not always be a matter of life and death.

Preface: Future approaches to replacement of organs

Preface: Future approaches to replacement of organs

Should lungs from donors with severe acute pulmonary embolism be accepted for transplantation? the Hannover experience

Lung transplantation has evolved to an accepted treatment modality for patients suffering from end-stage lung disease. The number of listed patients waiting for a lung transplant is dramatically higher than the number of available donor organs, which contributes to a 1-year mortality on the waiting list of approximately 20% and of 40% after 2 years of listing. Many strategies aiming for an increase of donor organs have been discussed controversially and are partially under clinical examination, such as the utilization of lungs from non–heartbeating donors or the concept of living-related lung transplantation. Other approaches to the problem are even more experimental, such as pulmonary xenotransplantation. During the 2003 annual meeting, The International Society for Heart and Lung Transplantation discussed the issue of redefining current lung donor criteria and the concept of using marginal donor organs for selected recipients in a main session. Many groups presented their outcomes following lung transplantation using lungs from donors who were heavy smokers, from donors with onset of fatal asthma, or from elderly donors up to 77 years of age. One major consensus of this intense discussion, however, was that some donors are still considered as inadequate for lung donation, such as patients who show signs of severe pneumonia or aspiration as well as those who died from fulminant pulmonary embolism. Here, we report on 3 cases in which lung grafts from patients with fatal severe acute pulmonary embolism were successfully transplanted. We further describe a novel strategy of in situ retrograde lung flush perfusion that we are routinely using in our clinical program now, which is of special importance for lung graft retrieval from donors with acute pulmonary embolism.

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.

Prevention of acute lung injury in swine: depletion of pulmonary intravascular macrophages using liposomal clodronate

Background. Swine contain large numbers of pulmonary intravascular macrophages (PIMs) that mediate the physiological response observed in acute lung injury (ALI). As the hyperacute dysfunction observed in pulmonary xenotransplantation is similar to endotoxin-induced ALI, PIMs may play a critical role in pulmonary xenograft dysfunction. We used liposomal clodronate to eliminate the PIM population in a model of acute swine lung injury. Materials and methods. Experimental swine ( n = 6) received liposomal clodronate (1.25 g/10 kg) and control swine ( n = 5) received saline containing liposomes before infusion of lipopolysaccharide (450 ng/kg). Results. Control swine demonstrated higher peak pulmonary artery pressures (41.8 ± 2.2 versus 16.8 ± 1.2 mm Hg; P < 0.0001) and higher peak pulmonary vascular resistances (1405 ± 209 versus 353 ± 81 dynes · s · cm −5; P = 0.0016) in response to lipopolysaccharide infusion. Clodronate treated swine also had significantly lower serum levels of tumor necrosis factor-α, interleukin-6, and thrombin. Conclusions. Liposomal clodronate effectively attenuates acute swine lung injury induced by endotoxin. This method of depletion of the PIM population presents a promising new treatment of swine lungs before xenotransplantation.

The role of the porcine von willebrand factor: baboon platelet interactions in pulmonary xenotransplantation

Porcine von Willebrand factor (pvWF) has been shown to bind to human glycoprotein Ib (GPIb) and cause activation of human (or primate) platelets in the absence of shear stress. Pulmonary xenografts develop disseminated intravascular coagulation (DIC) and microvascular thrombosis within hours of reperfusion, and the aberrant interaction between pvWF and human platelets may be a possible cause of xenograft-associated DIC. Experimental baboons (n=3) received mouse anti-human GPIb monoclonal antibody before undergoing orthotopic pulmonary xenotransplantation with porcine lungs expressing human membrane cofactor protein (CD46). Blocking the pvWF-GPIb interaction with a monoclonal antibody to GPIb prevented the agglutination of human and baboon platelets by pvWF in vitro. In vivo, the anti-GPIb antibody prevented platelet deposition and prevented the increases in D-Dimers (P=0.011) seen in control xenograft recipients (n=5). However, there was no difference in elevations of prothrombin times (PT) or improvement in the vasoconstriction associated with the loss of xenograft function. This study indicates that the DIC associated with the hyperacute dysfunction of pulmonary xenografts is a complex phenomenon that is affected by, but not solely dependent on, activation of platelets. Aberrant interactions between pvWF and GPIb play a significant role in DIC associated with pulmonary xenotransplantation.

Disseminated intravascular coagulation in association with pig-to-primate pulmonary xenotransplantation.

Profound coagulopathy has been proposed as a barrier to xenotransplantation. Disseminated intravascular coagulation (DIC) has been observed with the rejection of renal and bone marrow xenografts but has not yet been described in pulmonary xenografts. This study examined the coagulation parameters in five baboons that received pulmonary xenografts and one baboon that was exposed to porcine lung during an extracorporeal perfusion. Platelet counts, prothrombin times (PT), and levels of fibrinogen, D-dimers, and thrombin-antithrombin III complex (TAT) were analyzed. In addition, serum levels of plasminogen activator inhibitor-1 (PAI-1), thrombomodulin (TM), tissue plasminogen activator (tPA), and tissue factor (TF) were determined. Hyperacute pulmonary xenograft dysfunction, which occurred within 0-9 hr of graft reperfusion, was associated with clinically evident DIC. This coagulopathy was characterized by thrombocytopenia, decreased fibrinogen levels, elevations in PT, and increases in D-dimers and TAT. Furthermore, transient increases in PAI-1, increases in TM, and increases in tPA were observed in the serum of some but not all recipients. None of the baboons demonstrated measurable increases in soluble TF. Although DIC in renal or bone marrow xenotransplantation develops over a period of days, DIC associated with hyperacute pulmonary xenograft dysfunction develops within hours of graft reperfusion. Thus, the DIC in pulmonary xenotransplantation may represent a unique and/or accelerated version of the coagulopathy observed with renal and bone marrow xenotransplantation.

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

The xenoreactive nature of anti-Galα1–3Gal 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-Galα1–3Gal antibodies coupled with the possible development of Galα1–3Gal deficient swine, the Galα1–3Gal 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.

Immune Complex Formation after Xenotransplantation: Evidence of Type III as well as Type II Immune Reactions Provide Clues to Pathophysiology

Rejection of renal and cardiac xenografts is initiated when natural antibodies of the recipient bind to donor endothelium, activating complement on the surface of endothelial cells. Pulmonary xenotransplants, however, reveal less evidence of antibody binding and complement activation and, in contrast to other xenografts, fare worse when the complement of the graft recipient is depleted. Accordingly, we asked whether distinct immunochemical reactions might occur after xenotransplantation of the lung and what implications such reactions might have for pulmonary pathophysiology. Analysis of serum from baboons after transplantation with porcine lungs revealed complexes containing baboon IgM and porcine von Willebrand factor. The baboon IgM in these complexes was specific for Galalpha1-3Gal. Immune complexes were also seen, albeit to a lesser extent, in the serum of kidney and heart xenotransplant recipients. Deposits of porcine von Willebrand factor and baboon C3 were detected in livers and spleens of transplanted baboons. These results indicate pulmonary xenotransplantation eventuates in formation of immune complexes and in the deposition of those complexes at distant sites. Immune complex formation could explain the peculiar fate of xenoreactive antibodies after pulmonary xenotransplantation and might contribute to the pathophysiology of the lung and systemic changes not previously considered a complication of xenotransplantation.

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. (J Thorac Cardiovasc Surg 2000;120:29-38)