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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.     

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.     

Coagulopathy in α-galactosyl transferase knockout pulmonary xenotransplants

Bush EL, Barbas AS, Holzknecht ZE, Byrne GW, McGregor CG, Parker W, Duane Davis R, Lin SS. Coagulopathy in α‐galactosyl transferase knockout pulmonary xenotransplants. Xenotransplantation 2011; 18: 6–13. © 2011 John Wiley & Sons A/S. Abstract: Background: 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. Methods: 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. Results: 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. Conclusions: 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.     

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

BACKGROUND: 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. METHODS: 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). RESULTS: 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. CONCLUSIONS: 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.     

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-alpha, 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 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.     

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.     

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.     

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.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.     

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

Background

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).

Methods

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%).

Results

Two recipients survived for 3 days; 75 hours (no‐CO) and 80 hours (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 2 hours 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.

Conclusion

Although further investigation is required, CO treatment may provide a beneficial strategy for pulmonary xenografts.     

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

Abstract: Background: 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. Methods: 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 μg/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 Galα1‐3Gal xenoantigen (αGal) from porcine lung which had been perfused and retained for 30 min with human blood was assessed by enzyme‐linked immunosorbent assay using αGal‐binding lectin. Results: 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 αGal after blood retention was significantly lower in the DDAVP group than that of the control group. Conclusion: 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.     

Influence of strain and age differences on the yields of porcine islet isolation: extremely high islet yields from SPF CMS miniature pigs

BACKGROUND: Porcine pancreas is a potential source of material for islet xenotransplantation. However, the difficulty in isolating islets, because of their fragility and the variability of isolation outcome in donor age and breed, represents a major obstacle to porcine islet xenotransplantation. In this study, we compared the islet isolation yield of specific pathogen-free (SPF) Chicago Medical School (CMS) miniature pigs with that of another miniature pig breed and market pigs from a local slaughterhouse. METHODS: Nine adult CMS miniature (ACM) pigs (>12 months), six young CMS miniature (YCM) pigs (6-7 months), four adult Prestige World Genetics (PWG) miniature (APM) pigs (>12 months), and 13 adult market (AM) pigs from a local slaughterhouse were used for islet isolation. RESULTS: The islet yield per gram of pancreas from ACM pigs (9589 +/- 2823 IEQ/g) was significantly higher than that from APM pigs (1752 +/- 874 IEQ/g, P < 0.05), AM pigs (1931 +/- 947 IEQ/g, P < 0.05), or YCM pigs (3460 +/- 1985 IEQ/g, P < 0.05). Isolated islets from ACM pigs were significantly larger than those from AM pigs or YCM pigs. The in vitro and in vivo function of isolated islets showed no difference among experimental groups. The pancreases of ACM pigs contained higher mean islet volume density percentages and larger size of islets than those of AM or APM pigs. CONCLUSIONS: We isolated extremely high yields of well-functioning islets from ACM pigs bred under SPF conditions. SPF CMS miniature pigs should be one of the best porcine islet donors for clinical porcine islet xenotransplantation.     

Herpesvirus infections in xenotransplantation: pathogenesis and approaches

Infectious risk remains an important consideration in the clinical application of xenotransplantation. Vascularized xenografts create unique immunological niches in which bidirectional transmission of pathogens between donor and recipient may occur. Enhanced replication of many pathogens is stimulated by the immune responses induced by transplantation and by the immune suppression used to prevent graft rejection. Herpesviruses are the prototype viruses that are activated during immunosuppression. Quantitative diagnostic molecular assays have been developed for the known herpesviruses causing infection in pigs. Recent data suggest that some herpesviral infections, such as porcine cytomegalovirus, may be excluded from swine used as source animals by careful breeding, while others will require novel strategies for control. This review focuses on porcine and baboon herpesviruses in pig-to-non-human primate solid organ xenotransplantation including direct effects (tissue damage), indirect effects (coagulopathy, rejection), and possible approaches to these infections.     

Cytomegalovirus early promoter induced expression of hCD59 in porcine organs provides protection against hyperacute rejection.

The critical shortage of human donor organs has generated growing interest for porcine to human xenotransplantation. The major immunological barrier to xenotransplantation is the hyperacute rejection (HAR) response that is mediated by preformed xenoreactive antibodies and complement. A promising strategy to control the complement activation, is the expression of human complement regulatory proteins in transgenic animals. We have used the human early cytomegalovirus (CMV) promoter to drive expression of the human complement regulatory protein CD59 (hCD59) in transgenic pigs. A total of eight live transgenic founder animals was born from which five transgenic lines could be established. mRNA analysis and Western blotting revealed high expression of hCD59 in heart, kidney, skeletal muscle, and skin in animals of lines 1 and 5, as well as in the pancreas of four lines. This pattern of expression was confirmed by immunhistological staining. A cell-specific expression in heart and kidney tissue of transgenic lines 1 and 5 was determined. Primary fibroblasts and endothelial cell cultures derived from the aorta of transgenic pigs showed a significantly diminished sensitivity against the challenge with xenoreactive human antibodies and complement whereas non-transgenic control cells were highly susceptible to complement mediated lysis. Ex vivo perfusion of kidneys with pooled human blood revealed a significant protective effect of hCD59 against HAR. The average survival of transgenic kidneys was significantly extended (P<0.05) over nontransgenic controls (207.5+/-54.6 vs. 57.5+/-64.5 min). These data support the concept that hCD59 protects nonprimate cells against human complement mediated lysis and suggest that donor pigs transgenic for hCD59 could play a crucial role in clinical xenotransplantation. Two of five hCD59 transgenic lines showed strong hCD59 expression in several organs relevant for xenotransplantation and a protective effect against HAR. This indicates that the use of the CMV-promoter can facilitate the selection process for optimized transgene expression.     

Reduction of consumptive coagulopathy using porcine cytomegalovirus-free cardiac porcine grafts in pig-to-primate xenotransplantation

BACKGROUND: Xenotransplantation using pigs as the source species for organs carries a potential risk for transmission and activation of porcine herpesviruses. Activation of porcine cytomegalovirus (PCMV) in pig-to-baboon xenotransplantation is associated with xenograft injury and possibly an increased incidence of consumptive coagulopathy (CC). METHODS: To further investigate the role of PCMV activation in the occurrence of CC, a strategy to exclude PCMV from the donor was developed. To exclude PCMV, piglets were early-weaned and raised separated from other swine. These piglets were used as donors in an experimental protocol of pig-to-baboon heart xenotransplantation. RESULTS: Early weaning of piglets was successful in excluding PCMV. Use of PCMV-free cardiac porcine xenografts in baboons resulted in prolonged graft survival and prevented consumptive coagulopathy in all recipients. CONCLUSIONS: The use of PCMV-free cardiac grafts is beneficial in reducing the direct effects of PCMV activation in the graft (tissue damage) and the indirect effects of PCMV activation in the recipient (consumptive coagulopathy).     

Characterization of a pig-to-goat orthotopic lung xenotransplantation model to study beyond hyperacute rejection.

BACKGROUND: A pig-to-goat orthotopic lung xenograft model was developed to test whether depletion of goat xenoreactive antibodies against pig red blood cells would prolong pig lung xenograft survival. METHODS: Adult goats with anti-pig xenoreactive antibodies underwent left pneumonectomy followed by orthotopic transplantation of pig left lung (group 1) or immunodepletion of their xenoreactive antibodies by extracorporeal right pig lung perfusion before transplantation without (group 2) or with (group 3) complete clampage of the right pulmonary artery. In group 4, goat left lungs were orthotopically transplanted into pigs and served as negative controls (pig serum does not have anti-goat xenoreactive antibodies). Each study group included 5 animals. Immunosuppression in surviving recipients included cyclosporine and azathioprine. RESULTS: Group 1 recipients died 7 +/- 3 hours after xenograft reimplantation of severe pulmonary hypertension and dysfunction and vasogenic shock, with little evidence of histologic xenograft injury. Group 2 xenografts had a stable circulatory and respiratory function on reperfusion and survived 9 +/- 4 days. Group 3 animals also tolerated complete occlusion of the right pulmonary artery, and xenografts assured the total respiratory support for 4 +/- 1 days. After immunodepletion, goat serum showed no detectable titers of xenoreactive antibodies, which began to reappear by postoperative day 2, where xenografts showed histologic stigmata of acute (humoral and cellular-mediated) rejection that evolved to a complete xenograft necrose at death. Group 4 xenografts showed scattered features of acute rejection 5 +/- 1 days after the operation. CONCLUSIONS: Pig left lung xenografts can provide prolonged and complete respiratory support after depletion of goat xenoreactive antibodies, but they ultimately necrose once recipient xenoreactive antibodies return to pretransplantation values.     

The immunology of corneal xenotransplantation: a review of the literature

As the worldwide demand for human donor corneas far exceeds supply, there is a need for a new source of corneas for clinical transplantation. Genetically engineered pigs may prove to be that new source, particularly as current evidence indicates that the anatomical and biomechanical properties of human and pig corneas are similar. Experience with clinical and experimental corneal xenotransplantation has been comprehensively reviewed and is summarized. Studies in small and large animal models have documented that both humoral and cellular immune responses play roles in xenograft rejection. Recent progress in the genetic manipulation of pigs has led to the prospect that clinical corneal xenotransplantation, in the absence of exogenous immunosuppressive therapy, may become successful in the foreseeable future.     

The morphology of islets within the porcine donor pancreas determines the isolation result: successful isolation of pancreatic islets can now be achieved from young market pigs

Clinical islet allotransplantation has become an increasingly efficient "routine" therapy in recent years. Shortage of human donor organs leads to porcine pancreatic islets as a potential source for islet xenotransplantation. Yet it is still very difficult to isolate sufficient numbers of intact porcine islets, particularly from young market pigs. In the following study islets were successfully isolated from retired breeders [4806 +/- 720 islet equivalents per gram organ (IEQ/g); n = 25; 2-3 years old; RB] and also from young hybrid pigs [2868 +/- 260 IEQ/g; n = 65; 4-6 months old; HY] using LiberasePI and a modified version of Ricordi's digestion-filtration technique. As expected, isolations from RB showed significantly better results (p < 0.002). A retrospective histological analysis of almost all donor pancreases showed that the majority of organs from RB (80%) contained mainly large islets (diameter > 200 microm), in contrast to only 35% of all pancreases from HY. Remarkably, the islet size in situ, regardless whether detected in RB or HY, strongly determined the isolation result. A donor organ with predominantly large islets resulted in significantly higher numbers of IEQs compared with a donor organ with predominantly small islets [RB(Large Islets): 5680 +/- 3,318 IEQ/g (n= 20); RB(Small Islets): 1353 +/- 427 IEQ/g (n = 5); p < 0.02]. In addition, isolation results were strongly influenced by the quality of the LiberasePI batch, and therefore single batch testing is invariably required. Purification was performed using Ficoll or OptiPrep density gradient centrifugation manually or in the COBE cell processor. Although islet purity was highest when OptiPrep was used, final islet yields did not differ between the different purification methods. Our study demonstrates that islet size in situ is an extremely critical parameter for highly successful islet isolation; consequently, we are now performing a morphological screening of each donor organ prior to the isolation process. Under these conditions highly successful isolations can reliably be performed even from young market pigs.     

Assessment of the potential risk of infection associated with Clostridium difficile from porcine xenografts

Bakri MM, Sutherland AD, Brown DJ, Vesely P, Crossan C, Scobie L. Assessment of the potential risk of infection associated with Clostridium difficile from porcine xenografts.
Xenotransplantation 2009; 16: 472–476. © 2009 John Wiley & Sons A/S. Abstract: There are numerous concerns over the potential for transfer of pathogens between species during clinical xenotransplantation, and although current clinical application is limited, porcine xenografts have been previously used to treat patients with severe burns. Donor animals providing the xenografts are sourced from a healthy commercial herd, however, as pigs are a known source of zoonotic agents, a number of diseases are required to be excluded from pigs used for xenotransplantation purposes. Many studies have indicated the relevance of viral zoonoses, however, little has been done with regard to the potential for transfer of pathogens related to health care associated infections. Clostridium difficile is a major cause of neonatal enteritis in pigs and an important feature of this organism is that pigs can be asymptomatic carriers. This study has examined the incidence of C. difficile PCR ribotypes present in healthy donor pigs to determine if pig faeces, and in particular, contamination of skin with faecal matter, is a potential route for the transfer of C. difficile. Animals were found to have human ribotype 017 present in the faecal matter, however, no C. difficile was isolated from skin samples taken from the same animals. In addition, due to the risk factors associated with C. difficile infection, the antimicrobial susceptibility of the C. difficile isolates has been determined.     

Analysis of potential porcine endogenous retrovirus (PERV) transmission in a whole-organ xenotransplantation model without interfering microchimerism

The question whether porcine xenografts can lead to porcine endogenous retrovirus (PERV) infection of recipients is critical for the evaluation of the safety of pig-to-man xenotransplantation. Unfortunately, polymerase chain reaction (PCR)-based analysis of potential PERV infections in nonhuman-primate whole-organ xenotransplantation models is hampered by false positive results due to chimeric porcine cells. To avoid the inherent analytical problem of xenomicrochimerism, we developed a non-life-supporting pig-to-primate kidney xenotransplantation model: porcine kidneys were transplanted, whereas the functioning recipient kidneys remained in situ. Subsequent to rejection (after 2 hours to 15 days), xenografts were removed, and recipients remained alive for up to 287 days. Immunosuppressive therapy based on cyclophosphamide, cyclosporine, and steroids was maintained for 28 days after transplantation. Using appropriate PCR assays, xenochimerism was found in tissue samples and partly even in peripheral blood leukocytes (PBLs) while the porcine kidneys were in situ. After graft removal, xenochimerism was no longer detectable, thus allowing analysis for possible PERV transmission.