Chapter 16

Xenotransplantation

David K.C. Cooper* and Robert Wagnery*Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA, USA, yDivision of Laboratory Animal Resources,

University of Pittsburgh, Pittsburgh, PA, USA

No

Co

Chapter Outline

Introduction 391

The Pig as a Potential Source of Organs/Cells for Clinical

Transplantation 391

The Significance of Gal Antigen Expression in Pigs 391

History 392

Pathobiology of Pig-To-Primate Organ Transplantation 393

The Innate Immune Response 393

Coagulation Dysfunction 393

The Adaptive Immune Response 393

The Pig-to-Nonhuman Primate Model 393

nhuman Primates in Biomedical Research. DOI: 10.1016/B978-0-12-381365-7.00016-9

pyright � 2012 Elsevier Inc. All rights reserved.

Intravascular Catheter Placement and Fluid/Drug

Administration 393

Immunosuppressive Therapy 396

Results and Complications 397

Results 397

Complications 397

Genetic Modification of Pigs 399

Summary 400

Acknowledgments 400

References 400

INTRODUCTION

Organ allotransplantation (alloTx) for patients with end-stage organ failure was one of the success stories of thesecond half of the twentieth century. One of the majorlimitations of this form of surgical therapy is the shortageof suitable donor organs, which are largely from deceasedhumans. Furthermore, during the past decade, islet alloTxfor the treatment of type 1 diabetes has become moderatelysuccessful, but the shortage of suitable pancreata fromdeceased humans has limited this form of therapy to lessthan 1000 of the estimated 2 million type 1 diabetics in theUSA during the past decade. In an effort to resolve theproblem of shortage of organs and cells, increasing atten-tion has been directed towards an exploration of the useof animals as potential sources of tissues for clinicaltransplantation.

The Pig as a Potential Source of Organs/Cells for Clinical Transplantation

Xenotransplantation (xenoTx), or the transplantation oforgans, tissues, or cells across species barriers, has some-times been divided into the transplantation of organsbetween concordant or discordant species. Concordant

species, e.g., monkey-to-baboon, are those in which,following an organ transplant, hyperacute rejection doesnot commonly occur, whereas discordant species, e.g. pig-to-baboon, are those in which hyperacute rejection isalmost universal. Despite the problem of hyperacuterejection, for a number of logistic and ethical reasons(reviewed in Cooper and Lanza, 2000 and Cooper et al.,2002) (Table 16.1), the pig has been determined to be themost suitable source of such organs, tissues, or cells forclinical use. Almost all research of direct clinical relevancetoday is being carried out in pig-to-nonhuman primatemodels, although in vitro and small animal studies continueto play important roles in providing initial data.

The Significance of Gal Antigen Expressionin Pigs

Studies in the 1990s determined that, when the recipientwas a nonhuman primate or human, hyperacute rejectionwas largely related to the expression in pigs of a carbohy-drate antigen, Gala1,3Gal (Gal) (Cooper, 1992; Cooperet al., 1993; Oriol et al., 1993), which is not present in OldWorld nonhuman primates and humans (Galili, 1988). Asa result of its absence in these species, they develop natural

391

TABLE 16.1 The Advantages and Disadvantages of the

Pig as a Potential SourceofOrgans andCells forHumans,

in Contrast with those of the Baboon in this Role

Pig Baboon

Availability Unlimited Limited

Breeding potential Good Poor

Period toreproductive maturity

4e8months 3e5 years

Length of pregnancy 114� 2 days 173e193 days

Number of offspring 5e12 1e2

Growth Rapid (adulthuman size within6months)a

Slow (9 years toreachmaximum size)

Size of adult organs Adequate Inadequateb

Cost of maintenance Significantly lower High

Anatomical similarityto humans

Moderately close Close

Physiologicalsimilarity to humans

Moderately close Close

Relationship ofimmune system tohumans

Distant Close

Knowledge of tissuetyping

Considerable(in selected herds)

Limited

Necessity for bloodtype compatibilitywith humans

Probablyunimportant

Important

Experience withgenetic engineering

Considerable None

Risk of transfer ofinfection(xenozoonosis)

Low High

Availability ofspecific pathogen-free animals

Yes Yes

Public opinion More in favor Mixed

aBreeds of miniature swine are approximately 50% of the weight ofdomestic pigs at birth and sexual maturity, and reach amaximumweight ofapproximately 30% of standard breeds.bThe size of certain organs, e.g., the heart, would be inadequate fortransplantation into adult humans.

392 Nonhuman Primates in Biomedical Research

anti-Gal antibodies that, following the transplant, bindimmediately to the donor organ, and activate the comple-ment cascade, resulting in hyperacute rejection withinminutes or hours. The mechanism of rejection is verysimilar to that which occurs when ABO-incompatible organalloTx is carried out, though hyperacute rejection occurs inonly about two-thirds of such cases (Stussi et al., 2006).

As NewWorld monkeys and all other mammals expressthe Gal antigen, the experimental animals used in xenoTxhave involved the pig as the organ source and Old Worldnonhuman primates as recipients. As work in the field ofxenoTx has progressed, it has also become realized thatpigs could act as a source of other tissues, such as skin,ligaments, neural cells (for the treatment of conditionssuch as Parkinson’s disease), corneas (for corneal xenoTx),and red blood cells (for clinical transfusion). Xeno-transplantation, therefore, has an immense potential inmedicine, and, if it could be carried out successfully, wouldlead to a revolution in therapy for a multitude of pathologicconditions.

HISTORY

Xenotransplantation has a long history, both in the exper-imental laboratory and in clinical practice (Taniguchi andCooper, 1997; Cooper and Lanza, 2000). As early as theeighteenth century, blood transfusions were carried outfrom animals to man. Corneal Tx was first performed in1838, more than 60 years before the first corneal alloTx in1905 (reviewed in Hara and Cooper, 2011). Early clinicalattempts at organ xenoTx date back to the early 1990s. Ofparticular note are the chimpanzee kidney transplants per-formed in a series of 13 human patients by Reemtsma et al.(1964), and the baboon kidney transplants performed byStarzl et al. in the 1960s (reviewed in Taniguchi andCooper, 1997). The first clinical heart transplant ever per-formed (by Hardy et al. in 1964) involved the Tx ofa chimpanzee heart. Other notable clinical attempts wereby Starzl in relation to liver xenoTx, beginning in the 1960sand continued into the 1990s, using both chimpanzees andbaboons as sources of organs (reviewed in Hara et al.,2008). Although Reemtsma reported that one of his patientsremained well for 9 months supported by a pair ofchimpanzee kidneys, the other trials generally failed withinweeks (Reemtsma et al., 1964). Efforts involving pigorgan transplants were universally unsuccessful withinhours or days.

Experimental models have used varying donor andrecipient species, but, since 1986, the pig-to-nonhumanprimate model has become the standard for research in thisfield (Lexer et al., 1986; Cooper et al., 1988). The mostcommonly used surrogates for humans have been thebaboon (Papio species) and the cynomolgus monkey(Macaca fascicularis). Both of these species mimic humansin producing significant levels of anti-Gal antibody as wellantibodies directed to other antigens, known as anti-nonGalantibodies, the antigenic targets of which remain unknown.Investigations have included both heterotopic and ortho-topic heart or liver xenoTx, kidney xenoTx, ex vivo lungperfusion and lung xenoTx, islet or corneal xenoTx, thexenoTx of neural cells and the transfusion of pig red blood

393Chapter | 16 Xenotransplantation

cells. A more recent introduction is pig artery patch xeno-grafting, which is much simpler than an entire organtransplant, yet which is susceptible to the same immuneresponse.

PATHOBIOLOGY OF PIG-TO-PRIMATEORGAN TRANSPLANTATION

The Innate Immune Response

When the pig organ is unmodified, i.e., not geneticallymodified, and no immunosuppressive therapy is given to therecipient, hyperacute rejection occurs in the majority ofcases (Figure 16.1A) (Rose et al., 1991; Rose and Cooper,1996, 2000). Hyperacute rejection has been defined asantibody-mediated complement activation, leading todestruction of the graft within 24 hours; it frequently occurswithin the first hour. It is known to be associated with thebinding of anti-Gal IgM to the Gal epitopes on the pig organ,with activation of the complement cascade. However, it hasbeen described after the use of organs from pigs in which theGal antigen is not expressed (a1,3-galactosyltransferasegene-knockout [GTKO] pigs), and therefore can be initiatedby binding of anti-nonGal antibodies.

If hyperacute rejection is prevented, either by a geneticmanipulation of the pig, or by immunoadsorption of anti-pig antibodies from the recipient, acute humoral xenograftrejection (sometimes known as acute vascular rejection ordelayed xenograft rejection), usually develops within days.This is again usually associated with antibody binding andcomplement activation, but there is probably a greater roleof innate immune cells, such as neutrophils and macro-phages, but the end result is largely the same (Ezzelarab2009).

Coagulation Dysfunction

In both these phenomena, it is increasingly clear thatabnormal coagulation within the vessels of the graft playsa significant role, and this may well be associated withmolecular incompatibilities between the pig and primatewith regard to the coagulation systems (Bach et al., 1994;Robson et al., 2000; Chen and Dorling, 2005). Forexample, pig tissue factor pathway inhibitor does notsuccessfully inhibit primate factor Xa, pig thrombomodulindoes not catalyze primate protein C, and pig von Wille-brand factor is associated with excessive primate plateletaggregation.

Even if early rejection is avoided, these incompatibili-ties can result in the development of a thrombotic micro-angiopathy, in which the vasculature of the organ is steadilyoccluded by thrombus, resulting in ischemic necrosis ofthe tissues (Figure 16.1B) (Houser et al., 2004). Theconsumption of clotting factors in this process can lead to

a consumptive coagulopathy in the nonhuman primaterecipient.

The Adaptive Immune Response

If both hyperacute and acute humoral xenograft rejectionare prevented, but immunosuppressive therapy is inade-quate, there is likely to be a T-cell-dependent elicitedantibody response, resulting in high levels of anti-pig IgG(Dorling and Lechler, 1998; Mirenda et al., 2005). Bindingof these antibodies to the vascular endothelium initiateshistopathological changes indistinguishable from acutehumoral xenograft rejection. Surprisingly, acute cellularrejection, as seen in the majority of allotransplants, hasvirtually never been recorded after pig-to-nonhumanprimate organ xenoTx. This is most likely because thehumoral response overwhelms the cellular response,though T and B cells may well be seen in the graft.

In pig heart grafts that have survived for more thanapproximately 3 months, graft vasculopathy develops(Figure 16.1C) (Kuwaki et al., 2005; Tseng et al., 2005), asseen in human allografts that have survived for manymonths or years (chronic rejection). This form of chronicrejection is as poorly understood in xenoTx as it is inalloTx.

With the introduction of genetically engineered pigs thatare partially resistant to the primate antibody-mediatedresponse, it is becoming clear that the coagulation dysfunc-tion between the two species is playing an increasing role.Indeed, aberrant coagulation may be a muchmore importantcausative factor in the development of acute humoral xeno-graft rejection than previously anticipated (Chen et al.,2004). However, it is likely that it is initiated by immunefactors, such as antibody binding to the vascular endothe-lium, leading to endothelial activation (Bach, 1994).

THE PIG-TO-NONHUMAN PRIMATEMODEL

The pig-to-nonhuman primate model is very demandingand requires considerable resources in both personneland facilities. In the majority of cases, a tether system isused to enable indwelling intravascular catheters to beavailable throughout the course of the experiment formonitoring blood pressure, blood draws, and drug infusion(e.g., inotropic or immunosuppressive agents) (Figure 16.2)(Cooper et al., 1994).

Intravascular Catheter Placement andFluid/Drug Administration

At operation, under full inhalational anesthesia, Tygoncatheters are placed in the carotid artery and jugular veins

FIGURE 16.1 Summary of the known immunologic barriers to pig-to-primate heart transplantation. (Reproduced with permission from Zhu X et al.,

J Heart Lung Transplant 2007;26:210e218.)

394 Nonhuman Primates in Biomedical Research

FIGURE 16.2 Drawing showing jacket and tether system allowing

continuous or intermittent hemodynamic monitoring, drug and fluid

infusion, and blood sampling in a nonhuman primate without the need for

sedation. Neck or femoral venous and/or arterial cannulae are inserted,

tunneled subcutaneously to the middle of the back, and brought out

through a protective flexible metal tube (held in place by a strong jacket).

The tether system is flexible, allowing the animal free movement within

the cage. (Reproduced with permission from Handbook of Animal Models

in Transplantation Research, Cramer DV, Podesta L, Makowka L (eds).

CRC Press, Boca Raton, 1994.)

395Chapter | 16 Xenotransplantation

(Figure 16.3A) (or branches of the femoral artery and vein).They are tunneled under the skin and brought out in theback, where they are connected to similar catheters withina flexible metal tether (Figure 16.3B). The flexible tether isconnected to the roof of the animal’s cage (Figure 16.3C),allowing the catheters to be connected to pumps(Figure 16.3D). This allows blood withdrawal and druginfusion without the need to sedate the animal. A very lowdose of heparin is infused continuously through eachcatheter to ensure that they remain patent. (Heparin is alsobelieved to be beneficial in inhibiting the coagulationdysfunction that develops after xenoTx.)

During surgical procedures and in the early post-operative period (e.g., 24 hours), the intravascular cathetersallow monitoring of arterial and central venous pressures

and heart rate. Easy access to arterial blood also facilitatesmonitoring of blood gases and electrolytes.

The administration of fluids and/or drugs through eachcatheter is controlled by a pump, which can be pro-grammed to administer fluids and agents either continu-ously or intermittently. Up to four different infusions canbe administered through each pump. Therefore, if twovenous catheters are present, up to eight different infu-sates can be administered simultaneously, if required(which is quite often necessary in baboons with organxenografts). This ensures that the animal is neither over-loaded with fluid nor dehydrated, and enables drugs to beadministered at intervals without the need for personnel tobe present on all occasions. In our experience, thesepumps have proved reliable and have required littlemaintenance.

Occasionally, indwelling gastric catheters have beenused to allow oral medications to be administered and tofacilitate treatment of postoperative anorexia. Anorexia isrelatively common in these animals, often associated withimmunosuppressive agents (or radiation), graft inflam-mation and/or cytokine release, or, more rarely, pain.Although providing significant advantages in themanagement of the primate, the tether system can beassociated with complications, which include cathetererosion through a vein wall, infection (particularly as theanimal is immunosuppressed), and hemorrhage followingdisconnection of the lines.

Some drugs are available in soluble forms, and cansometimes be administered orally, particularly to cyn-omolgus monkeys, though we have found this approachless successful in baboons. Most drugs and supportivemedications (fluids, electrolytes, glucose) must be givenintravenously in the postoperative period. Indeed, in manyrespects care of these animals, following a complexprotocol, is comparable to that of a patient in an intensivecare unit. (More details of critical care of laboratoryanimals are included in Volume 1, Chapter 17.)

As rejection can occur rapidly, monitoring of graftfunction is necessarily intensive, requiring frequent blooddraws for measurement of complete blood count, metabolicfunctions, and immunosuppressive drug levels; monitoringfor infection or sepsis also has to be meticulous. Immu-nological monitoring includes lymphocyte subset counts(e.g., T and B cells), anti-pig antibody levels, complement-dependent cytotoxicity assays, and mixed lymphocytereactions.

Following heterotopic heart xenoTx, cardiac function ismonitored by palpation of the graft (2e3 times weekly)through the abdominal wall, electrocardiographic moni-toring, and measurement of troponin levels at intervals.Following kidney xenoTx, changes in serum creatinine andblood urea nitrogen provide most information on renalfunction, though urine analysis and urine enzyme assay

(A) (B)

(C) (D)

FIGURE 16.3 (A) With the baboon lying supine, with the head turned slightly to the left (under the towel at the left of the figure), the right carotid artery

and internal jugular vein have been identified and isolated. The external jugular vein is isolated separately (not shown), and all three vessels are can-

nulated. (B) The three catheters are tunneled subcutaneously and brought out through the skin of the back, where they are attached to similar catheters

within the flexible metal tether (lower right in figure). The catheters are securely sutured to the skin to ensure they do not protrude below the jacket. (C)

When the baboon is returned to the cage, the tether is connected to the roof of the cage, and (D) the three catheters connected to pumps. (Courtesy of

Burcin Ekser, MD.)

396 Nonhuman Primates in Biomedical Research

(particularly gamma glutamyltransferase [GGT]) can behelpful. In addition, ultrasound studies have been founduseful in determining whether the heart remains contract-ing, or whether blood flow persists through the heart orkidney. Liver function after xenoTx can be monitored byfrequent measurement of protein synthesis, detoxification,and coagulation. (Pre-Tx liver failure in the nonhumanprimate can be induced by a variety of toxins [aflatoxins,alcohol, D-galactosamine] or radiation, although nativehepatectomy often suffices for the needs of the experi-ment.) In all cases, tissue biopsies are carried out atintervals, either through an open operative procedure ofby percutaneous transvenous (heart) or percutaneousfine needle (kidney or liver) techniques; these biopsyprocedures can all be associated with complications,particularly bleeding.

When islet xenoTx is performed, which is usually intothe portal vein so that the islets reside in the liver, theintravascular lines are frequently maintained in situ onlyduring the phase of initiation of diabetes by streptozotocinuntil the islet graft is clearly functioning, at which time they

are removed (van der Windt et al., 2009). Monitoring of pigislet function is by frequent measurement of blood glucoseand porcine C-peptide (which can be clearly differentiatedfrom monkey C-peptide); insulin levels can also bemeasured. To measure blood glucose, monkeys can bemonitored by skin prick, and can be trained to cooperate inthis form of monitoring.

Immunosuppressive Therapy

Immunosuppressive therapy, directed against both thehumoral response, e.g., anticomplement agents, such ascobra venom factor, and the cellular response, e.g., standardpharmacologic immunosuppressive agents, such as tacro-limus and mycophenolate mofetil, or the newer co-stimu-latory blockade agents, such as anti-CD154mAb orCTLA4-Ig, is usually initiated pre-Tx and continuedthroughout the course of the experiment (Kuwaki et al.,2005; Tseng et al., 2005). Table 16.2 provides an exampleof an immunosuppressive regimen with the supportivetherapy required. Increasingly, anticoagulant or antiplatelet

TABLE 16.2 Representative Immunosuppressive and

Supportive Drug Regimen used in Pig-to-Nonhuman

Primate Heart or Kidney Transplantation Experiments at

the University of Pittsburgh

Agent/Dose

1. Antithymocyte globulin (ATG) 1e10mg/kg x2 i.v. (days e3and e1) to reduce the CD3þ T cell count to <500/mm2.

2. Human antihuman CD154mAb 25mg/kg i.v. (on days e1,0, 4, 7, 10, 14, 19, and weekly) to maintain a trough level of>400 mg/ml.

3. Mycophenolate mofetil (MMF) by continuous i.v. infusion tomaintain a trough level of 3e6 mg/ml.

4. Methylprednisolone 5mg/kg/day i.m. on the days of ATGadministration and on day 0 (day of Tx) and day 1, taperingto 0.5mg/kg/day i.m. by day 6, and to 0.1mg/kg/day i.m. byday 10 (maintained throughout period of follow-up)

5. Prostacyclin 20 ng/kg/min by continuous i.v. infusion for48 hours.

6. Dopamine 2e7 mg/kg/min by continuous i.v. infusion for48 hours.

7. Ganciclovir 5 mg/kg/day as prophylaxis against cytomega-lovirus infection for at least the first month.

8. Cimetidine 10mg/kg x2 daily while intravascular cathetersare in situ to prevent peptic ulceration.

9. Heparin to maintain the aPTTat 150 s throughout the periodof follow-up.

10. Buprenorphine 0.01mg/kg will be given 6e12 hourly i.v. forat least the first 72 hours after any surgical procedure.

11. Cefazolin 10mg/kg/x2 days i.v. for 48 hours after anysurgical procedure.

397Chapter | 16 Xenotransplantation

agents are also administered (Kuwaki et al., 2005;Ezzelarab et al., 2009), frequently making the post-Txprotocol a complicated one (an example is illustrated inTable 16.3). A prolonged state of immune suppression canobviously be associated with complications, particularlyinfection.

Efforts are being made by at least one group to inducea state of ‘immunological tolerance’ in which, after initialintensive immunosuppressive therapy, possibly includingwhole body and/or thymic irradiation, all therapy can beslowly withdrawn (Yamada et al., 2005; Griesemer et al.,2009). Based on results in allogeneic models, the theory isthat, when the immune system recovers, it will no longerrespond to donor-specific antigens, but will respond nor-mally to other antigens, e.g., those expressed on bacteriaand viruses. This is clearly the ultimate goal of both alloTxand xenoTx, but in xenoTx it would appear that there areseveral hurdles, such as coagulation dysregulation, thatneed to be surmounted before this approach is likely to besuccessful. Furthermore, the intensive induction therapyrequired can increase the risk of infectious complications.

Antibacterial and antiviral prophylactic agents aretherefore administered routinely (Table 16.2). Antibiotics

are usually administered for 48 or 72 hours after anyinvasive procedure, such as introduction of the intravas-cular catheters or the actual organ transplant. In contrast,antiviral agents, such as ganciclovir, are maintained for atleast 1 month and frequently throughout the course of theexperiment as activation of recipient cytomegalovirus(CMV) has been associated with an increased incidence ofgraft failure (Mueller et al., 2004).

RESULTS AND COMPLICATIONS

Results

XenoTx has made significant progress during the past25 years (Cooper et al., 2007; Zhu et al., 2007; Piersonet al., 2009), though not to the extent that clinical trials oforgan xenoTx are likely to take place within the next yearor two, though tissue xenoTx is already underway andcorneal and islet xenoTx trials are likely to occur in theforeseeable future. With regard to organ xenoTx, hetero-topic heart transplants have functioned for up to 6 months(Kuwaki et al., 2005; McGregor et al., 2005) and orthotopicheart transplants for nearly 2 months (Vial et al., 2000;McGregor et al., 2009). Kidney transplants have beenslightly less successful, largely to an increased incidence ofconsumptive coagulopathy as a result of coagulationdysfunction; graft survival has extended close to 3 months(Cozzi et al., 2003; Yamada et al., 2005; Griesemer et al.,2009). Orthotopic liver xenoTx has been associated with animmediate fall in platelet count to dangerously low levels,resulting in internal hemorrhagic complications within thefirst week despite adequate hepatic function (Ekser et al.,2010). Pig lungs appear to be particularly susceptible toinjury, and graft survival generally remains less than24 hours (Cantu et al., 2007; Nguyen et al., 2007).

More successful results have been obtained in the fieldof islet xenoTx, with diabetic monkeys being maintained ina normoglycemic state for >6 months (Cardona et al.,2006; Hering et al., 2006), with one for >12 months (vander Windt et al., 2009). Very few pig corneal transplantshave been carried out in nonhuman primates, but the resultshave been encouraging with grafts from unmodified pigsremaining viable for several months with local corticoste-roids (Pan et al., 2007). Red blood cells from geneticallymodified pigs have been demonstrated to be preferable toABO-incompatible allo-transfusions, but not yet compa-rable to ABO-compatible transfusions (Long et al., 2009).

Complications

Complications include those associated with intravascularcatheters, as mentioned above. Wound healing has not beena significant problem, even when the recipients have

TABLE 16.3 Recipient Protocol for Orthotopic Pig Liver Transplantation in Baboons

Day Procedure/Therapy

e27 Jacket training

e20 Tether training

e14 Mixed lymphocyte reaction (MLR) versus WT, GTKO, and GTKO/CD46 pig PBMC. (Same pigs have to be usedas sources of PBMC after liver Tx)Store serum and plasma for complement-dependent cytotoxicity assay (CDC), antibody binding assays (by FACS),and coagulation studiesT and B cell count

e6 Intravascular catheter insertions (Two venous [IJV and EJV] and one arterial [carotid artery])Blood samples for CBC, PT, aPTT, ACT, INR, fibrinogen, LFTs,RFTs, electrolytes, total protein, albumin, cholesterol, triglyceride, amylase, lipase, iron, CH50Buprenorphine 0.01mg/kg i.v. or ketoprofen 2mg/kg i.v. x 2 daily for 3 daysCefazolin 25mg/kg i.v. x 2 daily for 2 daysBegin famotidine 0.25mg/kg i.v. x 2/day and continue dailyBegin mycophenolate mofetil (MMF) 110mg/kg by continuous i.v. infusion to maintain blood level of3e6 mg/ml

e5 Begin tacrolimus 0.05e0.2mg/kg x 2 daily i.m. to maintain a blood trough level of 10e20 ng/ml

e4 MMF and tacrolimus levels every day until the day of TxT and B cell count

e3 Begin ganciclovir prophylaxis 5 mg/kg/day i.v., and continue dailyBlood cultureAntithymocyte globulin (ATG) 2e10mg/kg i.v., depending on the WBC count (to reduce the CD3þ T cellcount to <500 cells/mm3)

e2

e1 CBC þ lymphocyte subset countsAntithymocyte globulin (ATG) 2e10mg/kg i.v., depending on the WBC count (to reduce the CD3þ T cellcount to <500 cells/mm3)Cobra venom factor 1e3mg i.v. daily for 4 days (to maintain the CH50 <10%)NPO after noon e fluids only for 18 hours before surgeryCheck infusion pumps and cassettes (see A below)Ketorolac 0.5mg/kg i.v. daily (half-life 3.5e9 hours)

0 Hold morning tacrolimus and MMF before surgeryCH50 daily (give CVF if indicated)Begin methylprednisolone i.v. x 2 daily as per protocol (see B below)Prostaglandin I2 by continuous i.v. infusion for 3e5 daysCefazolin 25mg/kg i.v. x 2 daily for 3 daysImipenem 500mg i.v. x 3 daily (depending on baboon weight) for 3 daysAmikacin 150mg i.v. daily for 3 daysHeparin 100U/kg i.v.Recipient hepatectomy þ splenectomyOrthotopic liver transplantationLiver biopsy before abdominal closure (formalin, OCT, EM)Blood samples for CBC; LFTs, PT, aPTT, INR, fibrinogenBuprenorphine 0.01mg/kg i.v. 2x daily for 3 days or ketoprofenHold afternoon tacrolimus dose after TxBegin continuous i.v. heparin infusionBlood pressure monitoring through arterial line

1 Hold morning tacrolimus dose (until level known)Blood samples for CBC, PT, aPTT, ACT, INR, fibrinogen, chemistry, electrolytesVitamin K (5 mg i.v. daily)Blood sample for tacrolimus level dailyBlood sample for MMF level alternate daysAdjust ACT to 160 s by increasing rate of heparin infusionBlood culture (on alternate days)Store serum and plasma samples daily for coagulation studies, Western blot, antibody binding assays, etc.

398 Nonhuman Primates in Biomedical Research

TABLE 16.3 Recipient Protocol for Orthotopic Pig Liver Transplantation in Baboonsdcont’d

Day Procedure/Therapy

2 Blood samples for CBC PT, aPTT, INR, fibrinogen, LFTs, T and B cell countAdjust ACT to 180 s by increasing rate of heparin infusion

3 Adjust ACT to 200 s by increasing rate of heparin infusion

5 T and B cell count

7 Store serum (5ml) for CDCT and B cell countMLR

14 Consider percutaneous liver biopsyStore serum (5ml) for CDC

21 Consider percutaneous liver biopsyMLRStore serum (6ml)Store serum (5ml) for CDC

28 Euthanasia

A: Infusion pumps

Line A: Heparinized normal saline

Line B: Normal saline

Line C: Dopamine

Line D: Phenylephrine

B: Methylprednisolone regimen

Day 0: 10mg/kg i.v.

Day 1: 2.5mg/kg i.v. x 2

Day 2: 1.25mg/kg i.v. x 2

Days 3e6: 0.5mg/kg i.v. x 2

Days 7e13: 0.25mg/kg i.v. x 2 daily

Days 14e20: 0.125mg/kg i.v. x 2 daily

Days 21e27: 0.1 mg/kg i.v. x 2 daily

Days 28 onwards: 0.2mg/kg i.v. daily

399Chapter | 16 Xenotransplantation

received high doses of steroids (though it might becomemore of a problem if rapamycin were to be used morecommonly in this model). Infection remains a complicationof immunosuppressive therapy, and requires constantmonitoring. It can take the form of a positive blood cultureor sepsis around the intravascular catheters. Some unusualinfectious agents have been reported. Lymphoma is rela-tively rare, but is likely to be reported more commonly asthe period of graft and recipient survival increases. In ourown experience, self-mutilation has not been seen, butcomplications such as acute gastric dilatation, collagenouscolitis, and intussusception have occurred.

The development of a consumptive coagulopathy asa direct result of coagulation dysfunction is one of the

major complications of organ xenoTx seen today, evenwhen evidence for an immune response is minimal (Buhleret al., 2000; Lin et al., 2008). It can be reversed if the graftis excised before the condition has become too advanced.Aggressive medical management, however, has routinelyfailed to correct consumptive coagulopathy.

GENETIC MODIFICATION OF PIGS

The steadily increasing success of experimental xenoTxhas largely been due to the development of geneticallymodified pigs using nuclear transfer (cloning) tech-nology. Our ability to knock-out a gene, such as thea-1,3galactosyltransferase gene, which is responsible for

400 Nonhuman Primates in Biomedical Research

the production of Gal oligosaccharides, has clearly beenimportant (Phelps et al., 2003; Kolber-Simonds et al.,2004). The insertion of a human gene to provideprotection of a pig organ has also played a major role,particularly in regard to the introduction of a humancomplement-regulatory protein, e.g., CD46 (membranecofactor protein), CD55 (decay-accelerating factor), orCD59, to protect against the effects of complementactivation (Cozzi and White, 1995; Loveland et al.,2004). Today, major efforts are being directed towardsexpressing human anticoagulant, antiplatelet, anti-inflammatory genes, such as tissue factor pathwayinhibitor, thrombomodulin, endothelial cell protein Creceptor, CD39, or hemeoxygenase-1 (HO-1).

An effort is also being made to provide local, rather thansystemic, immune suppression to the graft by, for example,expression of pig or human CLTA4-Ig on graft cells.Expression of this agent results in costimulatory blockade,thus preventing T cell activation, which has been clearlydemonstrated by in vitro assays. However, when pCTLA4-Ig was expressed constitutively, the pigs were found to bepartially immuno-incompetent and susceptible to infectiouscomplications (Phelps et al., 2009). Techniques are nowavailable to overcome this problem in some cases byexpressing the gene only in the tissues to be transplanted,e.g., in the pig islets alone using an insulin promoter.‘Anticoagulant’ or ‘anti-inflammatory’ genes, e.g., tissuefactor pathway inhibitor, CD39, that might similarly beproblematic if expressed widely in the pig, have also beenintroduced only into the islets.

Techniques of genetic engineering that allow a gene tobe ‘switched on’ or ‘off’, such as those using the Cre-Loxsystem, are currently being explored, as this would allowthe gene to be expressed functionally only in the organ afterTx, but not in the pig before organ ‘donation’.

SUMMARY

Wild-type pig tissues, such as small intestinal submucosa(SIS, which is rendered void of cells and is repopulatedwith recipient cells) are already being used extensively inclinical surgical practice. GTKO pig tissues, to which thereis a reduced inflammatory response, will probably be thenext innovation in clinical practice. In view of the world-wide shortage of human corneas for Tx, the pig cornea islikely to find a significant role in the treatment of cornealblindness, particularly in less-developed countries; thegenetically modified pigs can be bred and housed ina country with the necessary resources, and the corneasshipped to the centers where they will be transplanted.

Clinical trials of islet xenoTx will probably take placewithin the next few years (Hering, 2009). The need is vast,and the supply of human islets limited. The xenoTx ofdopamine-producing neural cells for the treatment of

neurodegenerative conditions, such as Parkinson’s disease,underwent clinical trials more than a decade ago, and thesemay be resumed if current experiments prove encouraging(Badin et al., 2010). With both islet and neural cell trans-plantation, the risks to the patient are small, but the benefitscould be considerable.

XenoTx of whole organs requires further significantadvances in the laboratory before it can be ethically intro-duced into the clinic, but bridging of patients in fulminantliver failure by a pig liver while they await a human organmay well be feasible within a few years (Ekser et al., 2009),as may bridging with a pig heart in patients who areawaiting human heart transplantation (Cooper et al., 2000).

The potential of xenoTx is so great that experimentalresearch is likely to continue and expand. However, it ispersonnel-intensive, time-consuming, and expensive. Asthe only relevant model is the pig-to-nonhuman primatemodel, progress will inevitably be relatively slow. Ourincreasing ability to genetically engineer pigs for thispurpose, however, provides optimism that the remainingproblems will be overcome.

ACKNOWLEDGMENTS

We are most grateful to Burcin Ekser, MD, for kindly providing

Figure 16.3. Work on xenotransplantation in the Thomas E. Starzl

Transplantation Institute of the University of Pittsburgh is supported

in part by NIH grants U01 AI068642 and R21 A1074844, and by

Sponsored Research Agreements between the University of Pittsburgh

and Revivicor, Inc., Blacksburg, VA, USA.

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