david k. c. cooper

In: Recollections of Pioneers in Xenotransplantation … ISBN: 978-1-53613-945-7

Editor: David K. C. Cooper © 2018 Nova Science Publishers, Inc.

Chapter 3

THE NEXT GREAT MEDICAL REVOLUTION:

A CARDIAC SURGEON’S EFFORTS TO DEVELOP

XENOTRANSPLANTATION

David K. C. Cooper* Xenotransplantation Program, Department of Surgery,

University of Alabama at Birmingham, Birmingham, Alabama, US

Keywords: Boston, Cape Town, history, medical, islets, Oklahoma City, organs, pig,

genetically-engineered, Pittsburgh, xenotransplantation

ABBREVIATIONS

Gal galactose-α1,3-galactose;

GTKO α1,3-galactosyltransferase gene-knockout;

IBMIR instant blood-mediated inflammatory reaction;

MSC mesenchymal stromal cell;

NHH National Heart Hospital, London, UK;

NHP nonhuman primate

* Corresponding Author Email: [email protected].

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David K. C. Cooper 28

“Success is the ability to go from one failure to another . . . with no loss of enthusiasm.”

Winston Churchill

INTRODUCTION

As a medical student in the early 1960s I determined that I wished to follow a career

in the relatively new field of cardiac surgery. I had been heavily influenced in this

respect by the fact that at my medical school in London, Guy’s Hospital Medical School,

two of the greatest hearts surgeons of that era were on the staff at the time - Sir Russell

Brock (later Lord Brock) and Donald Ross. After graduation in 1963, I was fortunate to

hold three internships at Guy’s - in general medicine, general surgery, and cardiothoracic

surgery. Immediately following these appointments (in mid-1965), I took a temporary

position as a ship’s surgeon in order to get some rest and recreation after what proved to

be a period of sustained very hard work. I fulfilled this role in 1965 on a short cruise and

on a six-week voyage on a scheduled liner to South Africa and back.

While in Cape Town, at the suggestion of Donald Ross, a South African who had

been an exact contemporary of Christiaan Barnard as a medical student at the University

of Cape Town, I called on Professor Barnard, the senior cardiac surgeon there, and spent

a few hours with him one afternoon. He took me on a ward round with his team, and I

well remember seeing a patient with severe heart failure. When we moved away from the

bedside, Barnard turned to me and said, “Of course, what this patient needs is a new

heart.” At the time, I did not take this suggestion too seriously because I was aware that

the results of kidney transplantation left much to be desired and I felt that it was unlikely

anybody would perform a heart transplant in the near future.

After a year teaching anatomy at Harvard Medical School (where I also gained my

first experience of surgical research at the Peter Bent Brigham Hospital in the department

headed by the legendary surgeon-scientist, Francis Moore), I spent a year in accident and

emergency surgery in Cambridge in the UK. I then decided to pursue a PhD in surgical

research at the University of London.

LONDON AND CAMBRIDGE

By this time (the summer of 1967), I firmly believed that transplantation was the next

major development in cardiac surgery, and so my research at the Institute of Cardiology

(attached to the National Heart Hospital [NHH]), supervised by Donald Ross, primarily

involved methods of resuscitating and storing the heart for purposes of transplantation. I

was also involved with some very early work in heart-lung transplantation in dogs

directed by Donald Longmore, who was in charge of the cardiopulmonary bypass

services at the NHH.

The Next Great Medical Revolution 29

Not long after I had embarked on my research, Christiaan Barnard surprised

everybody by carrying out the world’s first human-to-human heart transplant on

December 3rd, 1967. Some 5 months later, I was privileged to watch the first heart

transplant carried out in the UK (by Donald Ross at the NHH) on May 3rd, 1968.

Having completed most of my experimental studies for the PhD, I spent three months

in India as a volunteer at a hospital in the Punjab, and then returned to clinical surgery for

two years in the UK to fulfill my general surgical requirements before sitting the

examination for the Fellowship of the Royal College of Surgeons of England. I was

fortunate to obtain an appointment at Addenbrooke’s Hospital in Cambridge where I

came under the influence of Roy Calne (later Sir Roy Calne, FRS), one of the foremost

pioneers in organ transplantation. Kidney transplantation was steadily being established

(though the results remained poor), but liver transplantation was in its infancy at the time

(1970-1971). I made a particular effort to participate in some of these operations and

learn something of the immunosuppressive regimens that were being used. I was greatly

impressed by Roy’s energy and drive.

Following this surgical experience, I spent a year completing my PhD dissertation,

and then moved into my chosen field of cardiothoracic surgery. Having completed my

formal training, I was offered a temporary appointment at Papworth Hospital, where the

designated senior registrar (equivalent to chief resident) was planning to spend a year in

the United States. This one year extended to two years, and I was extremely fortunate to

be a member of the team (led by Terence English, later Sir Terence) that carried out the

first four heart transplants at Papworth in 1979, which gave me insight into the clinical

management of these patients.

At the end of my two years at Papworth, I was very keen to obtain an academic

appointment in cardiothoracic surgery (where I would have the opportunity to combine

clinical surgery with research), but this proved very difficult in the UK at that time,

where there were only three academic appointments in the entire country. I therefore

wrote to Christiaan Barnard, asking him whether I could join his team in a research

capacity for a year. He generously offered me a position and, in the event, I remained in

Cape Town for seven years, gradually being promoted to Associate Professor.

CAPE TOWN

When I arrived in Cape Town at the beginning of 1980 (where I was to spend some

of the happiest years of my life), as well as working in the laboratory, Barnard asked me

to take responsibility for the patients undergoing heart transplantation, which I was

pleased to do. This proved immensely interesting, but challenging and stressful because

about 50% of the patients died within a year of the transplant (as they were doing

worldwide). I found Barnard a breath of fresh air as he allowed me considerable


independence and also, although he demanded that the patients should be cared for

conscientiously, he had a sense of humor that made working with him enjoyable. It was

also in Cape Town that I had the great good fortune and pleasure of working with

Winston Wicomb, PhD and Dimitri Novitzky, MD, PhD, two of the most innovative

people I have ever met.

While at the NHH I had written a short article in which I had stated that I thought the

future of heart transplantation might lie in xenotransplantation, with the use of nonhuman

primates (NHPs), such as chimpanzees, as potential sources of organs. I was probably

influenced by the pioneering work of Keith Reemtsma and others in the 1960s [1]. In

Cape Town, I was able to explore the possibility of xenotransplantation personally. When

I arrived, I suggested this as an area of research to Barnard, but he was unenthusiastic.

His opinion was that “we have enough problems in preventing rejection of a human heart

without taking on the added burden of a xenograft.” Nevertheless, as NHPs were so

readily available to us in Cape Town – where a baboon cost me $25 (whereas today, in

2018, in the U.S. the cost is in the region of $8,500) – I embarked on some experiments

in the laboratory, transplanting African green (vervet) monkey hearts into baboons to

immunologically mimic the baboon-to-human situation [2-4] (Figure 3.1). I also carried

out heart allotransplantation in baboons across the ABO barrier, which had some

similarities to xenotransplantation [5].

Figure 3.1. Research colleagues at the University of Cape Town in the early 1980s.

(Left-to-right) Stuart Boyd, Frederick ’Boots’ Snyders, Prescott Madlingozi, Winston Wicomb, Sharon

Smit, and Ferdinand Barends. Not photographed, but also valuable members of the team were Dimitri

Novitzky, Joanna Martin, and John Roussouw.

Although I gave thought to the potential of NHPs as ‘donors’ for humans [6], it soon

became obvious to me that, for various reasons, they would not be ideal. I therefore

switched my attention to the pig as a potential source of organs for humans. To my


knowledge, although John Najarian’s group had investigated transplants between pigs

and dogs (reviewed in [7], there had been no previous studies in the pig-to-NHP model

except Roy Calne’s handful of pig liver transplants in baboons and chimpanzees in the

late 1960s [8-10].

In the pig-to-baboon heart transplantation model, I soon found that hyperacute

rejection occurred in every case [11]. I was fortunate to be collaborating with an excellent

cardiac transplant pathologist, (the late) Alan Rose (Figure 3.2), who described the

histopathology of hyperacute rejection in this model [12-14]. To overcome this hurdle,

we perfused the recipient baboon’s blood through the donor pig kidneys (thus adsorbing

anti-pig antibodies) before implanting the heart [15]. This led to some delay in rejection,

but only to a maximum of four days, and was clearly not sufficient.

To my knowledge, the only other person working in this model at that time was Guy

Alexandre in Belgium, who, based on his innovative work in ABO-incompatible clinical

kidney transplantation, was exploring pig renal xenotransplantation by using pre-

transplant plasmapheresis to remove pig antibodies [16].

Figure 3.2. Some major collaborators. (Top, from left) Alan Rose, Egidio Romano, Simon Robson,

Michel Awwad. (Bottom, from left) Henk-Jan Schuurman, David Ayares, Robin Pierson, Rita Bottino.

OKLAHOMA CITY

In 1987, at the recommendation of Christiaan Barnard, who had retired and had

relocated to Baptist Medical Center (now Integris Baptist Medical Center) in Oklahoma

City in an advisory capacity, my Cape Town colleague, Dimitri Novitzky, and I were


invited to join the staff of the hospital, where Nazih Zuhdi was establishing an organ

transplant center. We helped establish a successful heart transplant program and, later,

lung transplantation.

Because of my studies on ABO-incompatible heart allo- and xeno-transplantation, I

was approached by a group from the biotechnology company, Chembiomed, in

Edmonton, Canada, who asked me to collaborate on some studies in this field. Based on

the work of Venezuelan medical scientist, Egidio Romano [17] (Figure 3.2), these

researchers had evidence to suggest that the intravenous infusion of synthetic A or B

oligosaccharides would be bound by the respective anti-A or B antibodies in the blood

and that this antibody-antigen complex would be cleared, thus reducing the antibody

level in the blood and enabling an ABO-incompatible organ graft to be transplanted

without fear of hyperacute rejection. Their theory was that, after an indeterminate period

of oligosaccharide infusion, a state of ‘accommodation’ might develop [18]. In view of

the model I had established of AB-incompatible heart transplantation in baboons, they

invited me to explore the infusion of the relevant oligosaccharides. They had also

developed immunoaffinity columns of the respective A and B saccharides for pre-

transplant extracorporeal immunoadsorption of these antibodies from the blood.

Although relatively skeptical that the continuous infusion of the A or B saccharides

would prevent hyperacute rejection, I readily agreed to their proposal and, together with

Yong Ye, Francisca Neethling, and others, we carried out a series of heterotopic heart

allotransplants in baboons across the AB barrier. To my surprise and satisfaction, their

hypothesis was correct and we were able to prevent antibody-mediated rejection, the

grafts surviving until an adaptive cellular response developed [19].

Figure 3.3. Two of my early and invaluable collaborators - Eugene Koren (far left) and Rafael Oriol (far

right) - with Takaaki Kobayashi and Douglas Smith (who ran the tissue typing laboratory in Oklahoma

City, and had a longstanding interest in swine leukocyte antigens). (Photograph courtesy of Takaaki

Kobayashi.)


With the invaluable financial support of Baptist Medical Center, I was continuing my

studies of pig-to-baboon heart transplantation, and this experience with ABO-

incompatible allotransplantation led me to believe that, if we could identify the

carbohydrate antigenic targets for anti-pig antibodies, then the infusion of the relevant

oligosaccharide (or the extracorporeal immunoadsorption of those antibodies) might lead

to prolonged survival of a pig heart in a baboon. I naively thought that this might be the

only step we needed to take in order to obtain long-term pig graft survival (again because

it was hoped that accommodation would take place).

I was fortunate to begin to collaborate with an experienced medical scientist at the

Oklahoma Medical Research Foundation, Eugene Koren (Figures 3.3 and 3.4). At his

suggestion, we perfused human plasma through pig organs ex vivo, eluted the anti-pig

antibodies that had become bound to the pig vascular endothelium, and sent these to

Chembiomed for testing against a large panel of synthetic oligosaccharides. It was very

exciting when Heather Good (Figure 3.4) reported to me that the major target for anti-pig

antibodies was galactose-α1,3-galactose (Gal) (Figure 3.5), although there were some

minor targets as well [20-22].

Figure 3.4. Members of the collaborative group that in 1991 definitively identified galactose-α1,3-

galactose as a major target for human anti-pig antibodies. (From the left) Francesca Neethling (Baptist

Medical Center [BMC], partly hidden), Yong Ye (BMC), and Marek Niekrasz (University of

Oklahoma). (From the right) Heather Good (Chembiomed), Gene Koren (Oklahoma Medical Research

Foundation), me (BMC), and Andrew Malcolm (Chembiomed). The photograph was taken at the 1st

International Congress on Xenotransplantation held in Minneapolis in August 1991, where this work

was first presented.


Figure 3.5. Diagram of the comparative structures of the human O, A, and B blood group glycans and

the pig α-Gal glycan.

At that time, I had never heard of Gal, but the Chembiomed people told me that they

had carried out some studies for Uri Galili, and that he had demonstrated that all humans

had anti-Gal antibodies and, furthermore, that pigs were among the non-primate

mammals that expressed Gal. I asked them why they had not told me this before we

carried out our experiments, but they were unable to explain this. I rapidly read all I could

of Uri Galili’s work [23], and it became certain to me that we had identified the correct

target for anti-pig antibodies.

We presented the results of our study at the First International Congress on

Xenotransplantation, held in Minneapolis in August of 1991, having submitted the

abstract some months previously [20, 22], which was undoubtedly the first time that the

importance of Gal in xenotransplantation had been reported (discussed in [24]). Others

soon confirmed its importance [25] (which had not been demonstrated by Galili because

xenotransplantation was not an area of interest to him). Rafael Oriol, a consultant for

Chembiomed who was an expert in ABO immunogenetics, elegantly confirmed the

expression of Gal on various tissues of the pig [26] and Francesca Neethling investigated

its role in the primate immune response in vitro [27-29].


Figure 3.6. Photograph taken after the dinner organized by Takaaki Kobayashi in Osaka in 2013 (by

which time some of the attendees had departed). (Seated, from the left) Takaaki Kobayashi, me, Leo

Buhler, Kenji Kuwaki. (Standing, from the left) Minoru Fujita, Rita Bottino, Satoshi Gojo, Kazuhiko

Teranishi, Whayoung Lee, Hidetaka Hara, Yifan Dai, Tadatsura Koshika, Hao-Chih Tai.

It was at the Minneapolis congress that Eugene Koren, Rafael Oriol (Figure 3.3), and

I determined that the ideal approach to xenotransplantation would be to genetically

engineer the pig so that it did not express Gal (an α1,3-galactosyltransferase gene-

knockout [GTKO] pig) [30]. At that time, this was possible in mice and, in fact, was soon

carried out [31, 32], but was not possible in pigs until the development of the technology

that brought about the first cloned mammal, Dolly the sheep [33], and subsequently the

first cloned pig (34) and the first genetically-modified cloned pig [35,36]. A homozygous

GTKO pig was not available until 2003 [37, 38].

In the meantime, with the invaluable help of veterinarian, Gary White, and his

colleagues, particularly Marek Niekrasz (Figure 3.4), at the animal facility at the

Oklahoma University Health Sciences Center [OUHSC], where we learnt of the tether

and jacket system in the management of baboons [39], we spent a great deal of time in

carrying out immunoadsorptions of antibody using immunoaffinity columns of synthetic

Gal oligosaccharides [40, 41] or infusing these Gal saccharides into baboons [42, 43].

Initially, there was insufficient synthetic Gal available for us to attempt continuous

intravenous infusion but, from my reading, I realized that melibiose had some similarities

to Gal. Our initial studies, therefore, involved the intravenous infusion of melibiose into

baboons, testing their serum, and also in carrying out pig heart transplants [44]. We did

not measure the level of melibiose in the blood of the baboon, but when urine from the

baboon fell to the floor, the soles of our shoes temporarily stuck to the floor through the

stickiness of the sugar.


Although the extracorporeal immunoadsorption of anti-Gal antibodies clearly

delayed rejection of a transplanted pig organ, this was only temporary, and when

antibodies returned to the blood, a delayed form of antibody-mediated rejection occurred

[45]. Shigeki Taniguchi even investigated hearts from animals that did not express Gal,

e.g., ostriches, without success [46].

With Takaaki Kobayashi (Figures 3.3 and 3.6) and Shigeki Taniguchi, we also

attempted to prolong pig heart graft survival in baboons by the use of cobra venom factor

(kindly provided by David White) and other anti-complement agents, with some success,

achieving wild-type pig heart graft survival of three weeks, which was the world’s

longest xenograft survival at that time [47, 48].

We subsequently demonstrated that sensitization to a xenograft did not seem to

increase the risk of subsequent allotransplantation or vice versa [49-51] (reviewed in

[52]), though others have subsequently shown that prior allosensitization may be

detrimental.

One interesting episode of my time in Oklahoma City followed the studies by our

senior liver transplant surgeon, Luis Mieles, who developed experience of auxiliary liver

allograft and xenograft support in baboons [53]. We believed that temporary support of a

patient in fulminant hepatic failure could be achieved by a baboon liver until a cadaveric

human liver became available. To ensure that the ‘bridging’ of a patient in this way was

not contrary to any government guidelines, I explained our plan over the telephone to an

official of the FDA. “That’s interesting,” he said. “Let us know what happens.” There

appeared to be no guidelines or concerns about transplanting a baboon organ into a

patient in those days. Unfortunately, local opposition within the hospital resulted in this

plan never coming to fruition. If my memory serves me correctly, these plans preceded

Tom Starzl’s clinical attempts at baboon liver transplantation in Pittsburgh in 1993 [54].

BOSTON

As I was no longer completely happy at Baptist Medical Center and had resigned my

position, I was delighted when David Sachs approached me about joining him in the

Transplantation Biology Research Center (TBRC) at the Massachusetts General

Hospital/Harvard Medical School in 1996 (at which time I decided to retire from clinical

surgery). My junior colleague, Francesca Neethling, originally from Cape Town, kindly

moved with me from Oklahoma City, and was an immense help in establishing our

laboratory.

David Sachs was attempting to induce tolerance in baboons to wild-type pig kidney

grafts by hematopoietic cell chimerism and, with Simon Robson (Figure 3.2) and Jay

Fishman, we were fortunate to receive NIH funding to continue these studies. This

involved extensive pre-transplant treatment that included irradiation, which was


associated with considerable morbidity, and so I suggested we explore the induction of

hematopoietic chimerism and kidney xenotransplantation separately. Over the next

several years, I was fortunate to work with a number of excellent research fellows from

Europe and Asia (Figure 3.6).

With regard to the induction of tolerance, our experience was that, even after the

infusion of very large numbers of pig hematopoietic stem cells, chimerism was lost

within minutes, probably from macrophage phagocytosis [55], a problem that still has not

been resolved today.

Through the generosity of David White and his colleagues at Imutran in Cambridge

in the UK, we were fortunate to obtain pigs that expressed a human complement-

regulatory protein, CD55 (DAF). Transplantation of one of these organs delayed

antibody-mediated rejection, but, in our hands, did not prolong graft survival for more

than a few weeks [56].

It was in Boston that I first fully realized the problem of coagulation dysfunction in

the pig-to-NHP model. Surprisingly, I had not seen this in Oklahoma City, possibly

because I just did not recognize it or, most likely, because rejection occurred so rapidly

that consumptive coagulopathy did not have time to develop. In Boston, largely through

the work of Frank Ierino [57] and Tomasz Kozlowski [58], the problem of consumptive

coagulopathy became very clear to us. It was a pleasure to collaborate with Simon

Robson, who had previously provided evidence to suggest that coagulation dysfunction

would be problematic after pig organ xenotransplantation [59].

Figure 3.7. Some of the research team at the Transplantation Biology Research Center at the

Massachusetts General Hospital/Harvard Medical School in the late 1990s. Ian Alwayn is in the left

foreground, with Alan Watts to his right, with Leo Buhler sititng opposite. Leo is flanked by Geoff

Oravec and Anette Wu. (Alan Watts supervised more than 300 antibody immunoadsorptions in

baboons.)


When joined by Leo Buhler (Figure 3.7) and Ian Alwayn (Figure 3.7) (the latter

being the first of several excellent research fellows who joined me from Erasmus Medical

Center in Rotterdam, whose studies I jointly supervised with Jan Ijzermans), it became

clear to us that conventional immunosuppressive therapy consisting of cyclosporine,

mycophenolate mofetil, and corticosteroids, did not prevent an adaptive immune response

(including an elicited anti-pig antibody response) to a pig organ graft [60, 61]. This

stimulated us to explore some of the new co-stimulation-blockade agents that were

becoming available, in particular, anti-CD154 (CD40L) mAb, which successfully

prevented the adaptive immune response [61]. To my knowledge, this was the first use of

costimulation blockade therapy in a pig-to-primate model.

Our impression was that consumptive coagulopathy developed more rapidly after

kidney transplantation than after heart transplantation, and Christoph Knosalla, whose

dedication and persistence did much to get us through this very difficult and frustrating

period, investigated this at the molecular level [62]. Those who followed him in the

laboratory benefited from his immense efforts during this critical period.

Eventually, in 2004, through the genetic engineering work of our colleagues at

Immerge Biotherapeutics and the University of Missouri [38], we were able to test

GTKO pig organ transplants in baboons, using an immunosuppressive regimen based on

anti-CD154mAb. In these studies, Michel Awwad (Figure 3.2) gave us great support

(particularly in identifying baboons with low anti-pig antibody levels) and Henk

Schuurman (Figure 3.2) proved an immensely hardworking and valued collaborator.

Thanks largely to the excellent work of Kenji Kuwaki (Figure 3.6), this combination

of a GTKO pig organ and costimulation-blockade (in baboons selected for low anti-pig

antibody levels) prolonged pig heterotopic heart graft survival for several weeks, and in

one case for six months (the longest survival recorded to that date) [63, 64]. Kaz Yamada

demonstrated that life-supporting GTKO pig kidney graft survival (combined with

thymus transplantation) could be prolonged to a maximum of 83 days [65], which was

comparable to the results obtained by Emanuele Cozzi and his colleagues at Imutran (and

in Padua) where, using a hCD55 transgenic kidney, graft survival had extended to 90

days [66, 67]. Although his work was mainly directed to spleen allotransplantation, Frank

Dor, from Erasmus in Rotterdam, was an invaluable member of our team at the time, and

contributed greatly by mentoring new research fellows and technicians.

Nevertheless, in both the heart and kidney transplants, a thrombotic microangiopathy

developed [63, 64, 68-73], which ultimately resulted in a consumptive coagulopathy.

Rapid excision of the heart graft at this time reversed the coagulopathy, confirming that

its cause was the presence of the graft [74].

Despite the successful production of GTKO pigs and these encouraging results, both

BioTransplant and Immerge went out of business when Novartis ceased its financial

support.


The International Xenotransplantation Association (IXA)

It was during my time in Boston that I helped establish the IXA (in 1998), becoming

its founding honorary secretary. In 1999-2001 had the great privilege of being its second

president (Figures 3.8 and 3.9). The proudest moment of my academic life was when I

was awarded honorary membership of the IXA in 2009 (Figure 3.10), but perhaps the

happiest moment was a dinner in Osaka in 2013, organized largely by Takaaki

Kobayashi, where I was joined by several of my previous research fellows and other

colleagues from Japan and elsewhere (Figure 3.6); several who could not be with us sent

their good wishes in the form of videos or messages. I remain very grateful to Takaaki for

putting this memorable reunion together.

Figure 3.8. With the organizers (Joe Leventhal on the left and Jonathan Fryer on the right) of the 6th

International Congress on Xenotransplantation held in Chicago in 2001. The 9/11 twin towers terrorist

tragedy had just taken place in the USA, and many of those planning to attend the congress cancelled at

the last minute but, thanks to Joe and Jonathan, the congress was very successful. I remain very grateful

to them for overcoming the hurdles they faced.

Figure 3.9. As president of the IXA in 2001, at the Chicago congress I had the privilege of presenting

John Najarian with Honorary Membership (the second person so honored).


Figure 3.10. At the 10th International Congress on Xenotransplantation (held jointly with IPITA) in

Venice in 2009, I was awarded Honorary Membership of the IXA by Robin Pierson (president, left) and

Emanuele Cozzi (organizer of the congress).

PITTSBURGH

Soon after this (in 2004), at the invitation of Tom Starzl (Figure 3.11), I relocated to

the Thomas E. Starzl Transplantation Institute (STI) at the University of Pittsburgh

where, in collaboration with David Ayares (Figure 3.2) and his colleagues at Revivicor in

Blacksburg, VA (which company had received a major venture capital investment from

the University of Pittsburgh Medical Center, a company that owned more than 20

hospitals in the Pittsburgh area), we were able to continue studies in the GTKO pig-to-

baboon model [37] (Figure 3.12). Unfortunately, despite initial assurance of their long-

term commitment, the administration of UPMC did not have sufficient vision and

patience to pursue an interest in xenotransplantation long-term, and Revivicor was

purchased by United Therapeutics (Martine Rothblatt) in July 2011.

As chairman of the Revivicor scientific advisory board (2004-2011), I had

encouraged David Ayares to offer help to several other groups worldwide by providing

them with genetically-engineered pig cells. For example, when the German consortium

established by Bruno Reichart was experiencing difficulty, Revivicor provided them with

GTKO pig cells.

With pigs to be provided by Revivicor, we formed a consortium with Robin Pierson

(Figure 3.2) and his team and applied successfully for a grant restricted to

xenotransplantation studies in NHPs. Fortunately, we have been funded by NIH

continuously for almost the past 15 years.


Figure 3.11. Some members of the Thomas E. Starzl Transplantation Institute (STI) research team at

the University of Pittsburgh with Dr Starzl in 2014. (Seated, from the left) Mohamed Ezzelarab, me, Dr

Starzl, Hidetaka Hara, Hayato Iwase. (Standing, from the left) Hong Liu, Huidong Zhou, Whayoung

Lee, Cassandra Long (who, as lab manager, was key to the team’s smooth functioning, and to whom I

am immensely grateful), and Yuko Miyagawa.

We first explored the detrimental effect of anti-nonGal antibody (i.e., antibody

directed to targets other than Gal) on GTKO pig cells in vitro [75-78]. As first suggested

to me by Simon Robson, we hypothesized (as did others) that binding of anti-nonGal

antibodies, and possibly complement fractions, to the pig vascular endothelium resulted

in low-grade activation that initiated a local procoagulant state [79]. Eventually, this led

to thrombotic microangiopathy and, ultimately, to consumptive coagulopathy.

However, studies by Chih Che Lin, in collaboration with Tony Dorling in the UK,

showed that coagulation dysfunction was more complicated than we had anticipated in

that the mere exposure of primate platelets to pig vascular endothelial cells initiated their

aggregation in the absence of antibody or complement [80, 81].

GTKO pigs expressing a human complement-regulatory protein, CD46 - the CD46

transgenic pigs having been originally produced by Ian McKenzie and his colleagues in

Australia - became available through Revivicor and appeared beneficial in extending

graft survival further [82-84], but did not address the problem of coagulation dysfunction.

When pigs that also expressed a human coagulation-regulatory protein (e.g.,

GTKO/CD46/thrombomodulin pigs) became available, prolonged pig graft survival

became more consistent [85-87].

In both the pig artery patch and heart transplant models, we demonstrated that

CTLA4-Ig (in the form of abatacept or belatacept) was unsuccessful in preventing an

adaptive immune response, but that the combination of anti-CD40mAb and belatacept


was successful [85]. Our colleagues at the National Heart, Lung and Blood Institute (the

NHLBI of the NIH) (using Revivicor pigs and an immunosuppressive regimen originally

based on ours) demonstrated that a high dose of anti-CD40mAb alone was sufficient to

achieve long-term graft survival [88-90]. Indeed, they subsequently reported two baboons

with heterotopic heart grafts that functioned more than one year, in one case for more

than two years, with graft failure only occurring when all immunosuppressive therapy

was discontinued.

These results were highly encouraging, and so in a life-supporting kidney transplant

model, using kidneys from a GTKO/CD46 pig expressing human coagulation-regulatory

proteins, we used a high-dose anti-CD40mAb-based regimen similar, but not identical, to

that used by the NHLBI group. We achieved kidney graft survival of almost five months

– the longest pig kidney graft survival reported to that date [86]. However, soon after, the

Emory/Indiana group reported longer survival of two rhesus monkeys with pig kidney

grafts that functioned, in one case, for almost ten months [91] (Adams AB, personal

communication). There were several differences between the Emory/Indiana and

Pittsburgh experiments, including (i) the use of a monkey versus a baboon, (ii) the use of

anti-CD154mAb rather than anti-CD40mAb, and (iii) the selection of monkeys with low

anti-pig antibody levels. It was notable that the successes at Emory were only achieved

when anti-CD154mAb was used (rather than belatacept), and only when monkeys were

selected with low anti-pig antibody levels. However, surprisingly, this good result was

achieved using a kidney from a pig that did not express a human coagulation-regulatory

protein.

INFLAMMATION

The systemic inflammatory response that develops after pig organ or even artery

patch transplantation in baboons had become increasingly obvious to us, largely through

the astute observations of the very innovative Mohamed Ezzelarab (Figure 3.11) [92, 93].

Inflammation augments the immune response and we believed needed to be controlled if

truly long-term pig graft survival was to be obtained. The inclusion of an anti-IL-6R

blockade agent in our regimen was perhaps the first step towards addressing this barrier

[93], though pigs expressing HO-1 or A20 may be preferable. Hayato Iwase, who proved

meticulous in his care of the baboons, subsequently reported pig kidney graft survival of

almost nine months with no signs of rejection [87].

Our ability to extend life-supporting kidney graft survival for longer periods enabled

us to make observations on pig renal function in the baboon [87,94].


Figure 3.12. Some of the research team at the STI in about 2011. (Left-to-right) Goutham Kumar,

Tadatsura Koshika, Burcin Ekser, Dirk van der Windt, Hidetaka Hara, me, Cassandra Long, Martin

Wijkstrom, Eefjie Dons, Tyler Wilhite, Minoru Fujita.

PIG LIVER XENOTRANSPLANTATION

In Pittsburgh, working with Bruno Gridelli and Burcin Ekser (Figure 3.12) (who

proved immensely productive), we explored GTKO/CD46 pig orthotopic liver

transplantation in baboons using conventional immunosuppressive therapy [95-97]. The

immediate development of thrombocytopenia (in the absence of hyperacute rejection)

indicated that there are additional problems to be overcome if the pig liver is to provide

even a bridge to allotransplantation in patients with fulminant hepatic failure. However,

these studies proved very valuable, and demonstrated that pig hepatic function was

surprisingly good in a baboon [96].

SUPPRESSION OF THE T CELL RESPONSE

The absence of the expression of Gal and the presence of a human complement-

regulatory protein had additional beneficial effects that we did not anticipate. The first

was that they resulted in a weaker adaptive immune response (as well as protection from

the humoral response) [98, 99], suggesting that this may eventually enable successful

organ xenotransplantation when administering conventional immunosuppressive therapy

rather than costimulation blockade-based therapy, which is not yet clinically available.

Hayato Iwase (Figure 3.11), also found that these genetic manipulations reduced platelet

aggregation to pig aortic endothelial cells [100], indicating an effect in preventing


thrombotic microangiopathy. The expression of a human coagulation-regulatory protein,

of course, reduced platelet aggregation further [100].

The T cell response, which continues to be investigated by my long-time and now

indispensable colleague, Hidetaka Hara (Figures 3.11 and 3.12), can be reduced by more

direct methods, e.g., expression of CTLA4-Ig (or LEA29Y) in the pig [101] or of a

mutant class II transactivator (CIITA-DN) that suppresses SLA class II expression and

prevents its upregulation [102,103]. However, the T cell response is complex; Mohamed

Ezzelarab has demonstrated that human T cells also respond to thrombin-activated pig

cells [104].

NEONATAL B CELL TOLERANCE

Pleunie Rood, who was my first fellow in Pittsburgh and helped me greatly in

establishing the research program at the STI, demonstrated that infant baboons and

humans did not develop anti-pig antibodies until 3-6 months after birth [105, 106],

confirming observations that Francesca Neethling had made in the 1990s in collaboration

with Robert Michler’s group [107, 108]. Furthermore, anti-nonGal antibodies remained

low throughout the first year.

In a very demanding animal model, Eefjie Dons (Figure 2.12) demonstrated that anti-

pig antibody production could be prevented in infant baboons treated with an anti-

CD154mAb [106], suggesting a method by which B cell tolerance to pig antigens could

probably be developed. Unfortunately, we were unable to obtain funding to extend these

observations, which might have enabled successful pig heart transplantation (and even

tolerance induction) in infants and young children, e.g., those with complex congenital

heart disease.

PIG ISLET XENOTRANSPLANTATION

At the TBRC, Leo Buhler had carried out the first pig islet transplant in a NHP using

a costimulation blockade-based regimen [109]. In Pittsburgh, in collaboration with islet

expert, Rita Bottino (Figure 3.2), and Massimo Trucco (now at the Allegheny Health

Network), we carried out several series of experiments of pig islet xenotransplantation in

streptozotocin-induced diabetic cynomolgus monkeys [110-112]. Using islets from CD46

pigs (provided to Revivicor by Ian McKenzie and Bruce Loveland), Dirk van der Windt

(Figure 3.12) and Rita demonstrated normoglycemia for a period >1 year in the absence

of the need for insulin therapy [111], which was the first time such prolonged survival

had been reported. Furthermore, when we obtained GTKO/CD46 pigs expressing human


coagulation-regulatory proteins [113], this excellent outcome was repeated by Rita

Bottino [112]. However, we could not achieve this success on a consistent basis, most

likely through the development of primary graft failure related to the instant blood-

mediated inflammatory reaction (IBMIR). Nevertheless, we were convinced that pig islet

transplantation has an immense clinical potential, and today is close to clinical trials. The

studies by Chung-Gyu Park and his colleagues in Seoul have been particularly

encouraging.

Rita set up an in vitro assay to investigate IBMIR, and provided evidence that, rather

than being simply a non-specific response to exposure of the islets to primate blood, it is

more associated with antibody and complement deposition, suggesting it may be a form

of hyperacute rejection [114, 115].

Mohammed Ezzelarab became interested in the potential of mesenchymal stromal

cells (MSCs) derived from the organ- or islet-source genetically-engineered pigs as a

means of augmenting the survival of a graft [116]. He felt this would be particularly

important in relation to islet transplantation as the MSCs should (i) increase

revascularization of the graft, (ii) have an anti-inflammatory effect, and (iii) reduce the

adaptive immune response. He carried out several in vitro experiments to investigate this

possibility [117-119], but we have not had sufficient grant funding to test genetically-

engineered pig MSC therapy in vivo.

Figure 3.13. The very productive research group at the Shenzhen Second People’s Hospital (First

Affiliated Hospital of Shenzhen University) in about 2016. Seated on the far left is Lisha Mou, who

supervises most of the research by the group. Third from the left is Yifan Dai (Nanjing Medical

University), who set up the collaboration between the Shenzhen and the STI groups. Seated to my left

is Zhiming Cai, the president of the hospital who established the research group, and to his left is

Hidetaka Hara, and then Dengke Pan (Institute of Animal Sciences, Beijing).


One important aspect of pig islet xenotransplantation to which Anna Casu drew

attention was the differences in glucose metabolism between pigs and primates [120,

121].

Through Yifan Dai (formerly of Revivicor and then the University of Pittsburgh,

before he returned to China) (Figure 3.6), we developed a productive collaboration with a

new group based in Shenzhen in China (Figure 3.13), and pursued several lines of

research with them, with a particular interest in islet xenotransplantation. In addition, my

group developed a long-standing collaboration with Professor Yi Wang of the University

of South China in Hengyang, who directed several excellent young research fellows to

work with us.

PIG CORNEAL XENOTRANSPLANTATION

Keen to develop a field that might become ready for clinical trials at an early stage,

Hidetaka Hara and Whayoung Lee (Figure 3.11) began investigations – initially in vitro -

of pig corneal transplantation in NHPs [122-125]. We then carried out full-thickness

corneal transplants (penetrating keratoplasty) as well as endothelial keratoplasty (the first

time this latter technique had been attempted in a pig-to-NHP model), but found that the

development of retrocorneal membranes (but not of rejection) limited success [126]. It is

uncertain why these developed so consistently, but our initial thoughts are that they result

from discrepancies in the thickness between the pig cornea and the recipient cornea; they

are also seen in corneal allotransplants in infants and young children. However, corneal

transplantation has an immense potential and it has only been lack of funding that has

prevented us from exploring this field further [123, 127]. We have been greatly

encouraged by the work of Mee Kum Kim and her colleagues in South Korea (reviewed

in [128]).

TRANSFUSION OF PIG PACKED RED BLOOD CELLS

This is another field we have explored, particularly by Hidetaka Hara. In due course,

I believe that all clinical transfusions of packed red blood cells will be from genetically-

engineered pigs that are housed under isolation conditions, which will provide a safer

source of blood transfusion than humans [129, 130].

BIOPROSTHETIC HEART VALVES

Increasing evidence from our laboratory and from others indicates that valves taken

from GTKO pigs would survive longer in patients than wild-type pig valves [131], a

topic explored by our Canadian collaborator, Rizwan Manji [132, 133]. Valves from


GTKO pigs that express neither N-glycolylneuraminic acid (cytidine monophosphate-N-

acetylneuraminic acid hydroxylase gene-knockout) [134-136] nor the antigen encoded by

β(1,4)N-acetylgalactosaminyltransferase (βGalNT2) [137, 138] will survive even longer

[139]. I feel sure that pigs not expressing these three antigens will form the basis for

clinical trials of organ or cell transplantation.

While the immunological and pathobiological hurdles of xenotransplantation were

being investigated and slowly but steadily overcome [45, 140], consideration had to be

given to three other topics of importance – (i) the potential of xenozoonosis, (ii) the

selection of patients for the first clinical trials, and (iii) the regulatory aspects of this

potential new form of therapy.

THE POTENTIAL PROBLEM OF XENOZOONOSIS

Being convinced at a very early stage (the late 1980s) of the clinical potential of

xenotransplantation, in Oklahoma City Yong Ye and I and colleagues began to consider

what microorganisms would need to be eradicated from the pigs [141]. A few years later,

I was fortunate to be a member of a committee set up by Novartis to consider this matter

in detail [142]; the recommendations of this committee remain valid today. While at the

TBRC, my group had the good fortune to collaborate with Jay Fishman and Nicolas

Mueller on aspects of pig microorganisms in relation to xenotransplantation [143-149].

We demonstrated the detrimental effect of pig cytomegalovirus (CMV) on graft outcome,

establishing that all organ-source pigs should be CMV-negative, which can be achieved

by early-weaning of the piglets. These observations have been ‘rediscovered’ by others

more recently.

SELECTION OF PATIENTS FOR THE FIRST CLINICAL

TRIALS OF XENOTRANSPLANTATION

Selection will depend to some extent on how successful the first trials are anticipated

to be, and this in turn will relate to the results achieved in preclinical models. When there

is a perceived moderate risk of failure or complication, selection will need to be

particularly vigorous. If the chance of success is considered to be high (as I believe it

now is), then a wider group of patients could be included. We have considered these

points in a number of publications [150-153]. Based on our experimental data, we have

also considered such topics as to how xenograft function (and impending graft failure)

could be monitored [154].


GUIDELINES AND REGULATORY ASPECTS

OF XENOTRANSPLANTATION

The regulatory aspects to some extent are influenced by the conclusions drawn from

the above two points – potential for infection and the selection of patients [153, 155]. The

ethical aspects of xenotransplantation have long been of interest to me [156, 157].

BIRMINGHAM

Although we were very happy in Pittsburgh, in 2016 Hidetaka Hara, Hayato Iwase,

and I relocated our laboratory to the University of Alabama at Birmingham. The great

attraction was that UAB had recently received funds from Martine Rothblatt (who had

purchased Revivicor) to build a biosecure pig facility that would enable a clinical trial to

be initiated. My hope is that we can transfer xenotransplantation successfully into the

clinic, which would give me the immense pleasure of seeing the research of so many of

us come to fruition.

“History tells us that procedures that were inconceivable yesterday, and barely achievable

today, often become routine tomorrow.”

Thomas Starzl

ACKNOWLEDGMENTS

It has been an immense privilege and pleasure for me to collaborate with so many

research fellows and faculty members at all of the centers at which I have worked. I

would particularly draw attention to Takaaki Kobayashi, who was a recent president of

the International Xenotransplantation Association (IXA), and Leo Buhler, who is the

current editor of Xenotransplantation and president of the IXA. Both have become highly

valued friends.

I had a particularly valuable collaboration with Jan Ijzermans at Erasmus University

Rotterdam, with whom I jointly supervised a number of excellent PhD candidates (Ian

Alwayn, Frank Dor, Pleunie Rood, Dirk van der Windt, and Eefjie Dons). Working with

all of them has been a privilege and I cannot overestimate the contributions they have

made to the work I have briefly reviewed here.

I am greatly indebted to Mohammed Ezzelarab, Hidetaka Hara, and Hayato Iwase,

who, having joined me as research fellows, became my colleagues on the faculty at the

Thomas E. Starzl Transplantation Institute and, in two cases, at UAB, and who have

contributed immensely to our work in the past decade, as has my Pittsburgh colleague,

Rita Bottino.


Little of our recent work would have been possible without the collaboration of

David Ayares, Carol Phelps, and their colleagues at Revivicor, who for several years

have provided us with genetically-engineered pigs not available anywhere else in the

world. Since the early 1990s, my belief has been that genetic engineering of the source

pig is key to the success of xenotransplantation. Until relatively recently, this was not

always realized by others, although they perhaps may not admit it now.

In addition, my group’s long and enjoyable collaboration with Robin Pierson and his

colleagues at the University of Maryland at Baltimore has proved very productive,

particularly in applying successfully together for NIH funding.

I have also had the opportunity of collaborating with colleagues abroad, particularly

with Yifan Dai in Nanjing, with whom I and my colleagues in Pittsburgh helped to

establish a xenotransplantation research program in Shenzhen, headed by Zhiming Cai

and Lisha Mou. I continue to have a valuable collaboration with Yi Wang of the

University of South China in Hengyang, with whom I have jointly supervised several

postdoctoral students (Hao Zhou, Maolin Jiang, Jiang Li, Huidong Zhou, Bingsi Gao,

Tao Li, Qi Li, Juan Li, Liaoran Zhou).

Working in the field of xenotransplantation research has not only been intellectually

stimulating and hugely enjoyable, largely because I have always been totally convinced

of its immense clinical applicability, but has enabled me to make many good friends and

see many parts of the world that I perhaps would not otherwise have visited. The number

of friends is too large to mention, but I would single out Ian McKenzie, Mauro Sandrin,

Tony d’Apice, and Peter Cowan in Australia, Emanuele Cozzi, the late Carl-Gustav

Groth, and Jean-Paul Soulillou in Europe, and Curie Ahn, Chung-Gyu Park, and Mee

Kum Kim in South Korea. In addition, many of my former research fellows have become

good friends.

One of the most frustrating aspects of working in any field of research is insufficient

funding to pursue the studies that you feel will bring eventual success. I was very grateful

to Baptist Medical Center for funding my research in Oklahoma City, and to

Biotransplant and Immerge, as well as the NIH, at the TBRC, and to the NIH and

Revivicor when I was at the STI. I am convinced that, with sufficient funding, we will

solve the remaining problems relating to pig kidney, heart, corneal, and islet

xenotransplantation within the next two years, and those relating to pig liver

transplantation within the next five years, enabling clinical trials to be initiated.

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136 days: longest life-supporting organ graft survival to date. Xenotransplantation.

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[87] Iwase, H., Hara, H., Ezzelarab, M., et al. Immunological and physiologic

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mediated costimulation blockade on non-Gal antibody production and heterotopic

cardiac xenograft survival in a GTKO.hCD46Tg pig-to-baboon model.


[89] Mohiuddin, M.M., Singh, A.K., Corcoran, P.C., et al. One-year heterotopic cardiac

xenograft survival in a pig to baboon model. Am J Transplant. 2014; 14: 488- 489.

[90] Mohiuddin, M.M., Singh, A.K., Corcoran, P.C., et al. Chimeric 2C10R4 anti-CD40

antibody therapy is critical for long-term survival of GTKO.hCD46.hTBM pig-to-

primate cardiac xenograft. Nat Commun. 2016;7: 11138. PMID: 27045379

[91] Higginbotham, L., Mathews, D., Breeden, C.A., et al. Pre-transplant antibody

screening and anti-CD154 costimulation blockade promote long-term xenograft

survival in a pig-to-primate kidney transplant model. Xenotransplantation. 2015;

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[92] Ezzelarab, M.B., Ekser, B., Azimzadeh, A., et al. Systemic inflammation in

xenograft recipients precedes activation of coagulation. Xenotransplantation. 2015;

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[93] Iwase, H., Ekser, B., Zhou, H., et al. Further evidence for a sustained systemic

inflammatory response in xenograft recipients (SIXR). Xenotransplantation. 2015;

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[94] Iwase, H., Klein, E., Cooper, D.K.C. Physiological aspects of pig kidney

transplantation in primates. Comparative Medicine. 2018. In press.

[95] Ekser, B., Long, C., Echeverri, G.J., et al. Impact of thrombocytopenia on survival

of baboons with genetically modified pig liver transplants: clinical relevance. Am J

Transplant. 2010; 10: 273-285.

[96] Ekser, B., Echeverri, G.J., Hassett, A.C., et al. Hepatic function after genetically

engineered pig liver transplantation in baboons. Transplantation. 2010; 90: 483-

493.


[97] Ekser, B., Burlak, C., Waldman, J.P., et al. Immunobiology of liver

xenotransplantation. Expert Rev Clin Immunol. 2012; 8: 621-634.

[98] Wilhite, T., Ezzelarab, C., Hara, H., et al. The effect of Gal expression on pig cells

on the human T-cell xenoresponse. Xenotransplantation. 2012; 19: 56-63.

[99] Ezzelarab, M.B., Ayares, D., Cooper, D.K.C Transgenic expression of human

CD46: does it reduce the primate T-cell response to pig endothelial cells?


[100] Iwase, H., Ekser, B., Hara, H., et al. Regulation of human platelet aggregation by

genetically modified pig endothelial cells and thrombin inhibition.

Xenotransplantation. 2014; 21: 72-83

[101] Phelps, C.J., Ball, S.F., Vaught, T.D., et al. Production and characterization of

transgenic pigs expressing porcine CTLA4-Ig. Xenotransplantation. 2009; 16: 477-

485.

[102] Hara, H., Witt, W., Crossley, T., et al. Human dominant-negative class II

transactivator transgenic pigs - effect on the human anti-pig T-cell immune

response and immune status. Immunology. 2013; 140: 39-46.

[103] Iwase, H., Ekser, B., Satyananda, V., et al. Initial in vivo experience of pig artery

patch transplantation in baboons using mutant MHC (CIITA-DN) pigs. Transpl

Immunol. 2015; 32: 99-108.

[104] Ezzelarab, C., Ayares, D., Cooper, D.K.C., Ezzelarab, M.B. Human T-cell

proliferation in response to thrombin-activated GTKO pig endothelial cells.


[105] Rood, P.P., Tai, H.C., Hara, H., et al. Late onset of development of natural anti-

nonGal antibodies in infant humans and baboons: implications for

xenotransplantation in infants. Transpl Int. 2007; 20: 1050-1058.

[106] Dons, E.M., Montoya, C., Long, C.E., et al. T-cell-based immunosuppressive

therapy inhibits the development of natural antibodies in infant baboons.


[107] Neethling, F., Cooper, D.K.C., Xu, H., Michler, R.E. Newborn baboon serum anti-

alpha galactosyl antibody levels and cytotoxicity to cultured pig kidney (PK15)

cells. Transplantation. 1995; 60: 520-521.

[108] Minanov, O.P., Itescu, S., Neethling, F.A., et al. Anti-GaL IgG antibodies in sera

of newborn humans and baboons and its significance in pig xenotransplantation.


[109] Buhler, L., Deng, S., O'Neil, J., et al. Adult porcine islet transplantation in baboons

treated with conventional immunosuppression or a non-myeloablative regimen and

CD154 blockade. Xenotransplantation. 2002; 9: 3-13.

[110] Rood, P.P., Bottino, R., Balamurugan, A.N., et al. Reduction of early graft loss

after intraportal porcine islet transplantation in monkeys. Transplantation. 2007;

83: 202-210.


[111] van der Windt, D.J., Bottino, R., Casu. A., et al. Long-term controlled

normoglycemia in diabetic non-human primates after transplantation with hCD46

transgenic porcine islets. Am J Transplant. 2009; 9: 2716-2726.

[112] Bottino, R., Wijkstrom, M., van der Windt, D.J., et al. Pig-to-monkey islet

xenotransplantation using multi-transgenic pigs. Am J Transplant. 2014; 14: 2275-

2287.

[113] Wijkstrom, M., Bottino, R., Iwase, H., et al. Glucose metabolism in pigs expressing

human genes under an insulin promoter. Xenotransplantation. 2015; 22: 70-79.

[114] van der Windt, D.J., Marigliano, M., He, J., et al. Early islet damage after direct

exposure of pig islets to blood: has humoral immunity been underestimated? Cell

Transplant. 2012; 21: 1791-1802.

[115] Nagaraju, S., Bertera, S., Tanaka, T., et al. In vitro exposure of pig neonatal islet-

like cell clusters to human blood. Xenotransplantation. 2015; 22: 317-324.

[116] Ezzelarab, M., Ayares, D., Cooper, D.K.C. The potential of genetically-modified

pig mesenchymal stromal cells in xenotransplantation. Xenotransplantation. 2010;

17: 3-5.

[117] Ezzelarab, M., Ezzelarab, C., Wilhite, T., et al. Genetically-modified pig

mesenchymal stromal cells: xenoantigenicity and effect on human T-cell

xenoresponses. Xenotransplantation. 2011; 18: 183-195.

[118] Kumar, G., Hara, H., Long, C., et al. Adipose-derived mesenchymal stromal cells

from genetically modified pigs: immunogenicity and immune modulatory

properties. Cytotherapy. 2012; 14: 494-504.

[119] Li, J., Ezzelarab, M.B., Ayares, D., Cooper, D.K.C The potential role of

genetically- modified pig mesenchymal stromal cells in xenotransplantation. Stem

Cell Rev. 2014; 10: 79-85.

[120] Casu, A., Bottino, R., Balamurugan, A.N., et al. Metabolic aspects of pig-to-

monkey (Macaca fascicularis) islet transplantation: implications for translation into

clinical practice. Diabetologia. 2008; 51: 120-129.

[121] Casu, A., Echeverri, G.J., Bottino, R., et al. Insulin secretion and glucose

metabolism in alpha 1,3-galactosyltransferase knock-out pigs compared to wild-

type pigs. Xenotransplantation. 2010; 17: 131-139.

[122] Hara, H., Cooper, D.K.C. The immunology of corneal xenotransplantation: a

review of the literature. Xenotransplantation. 2010; 17: 338-349.

[123] Hara, H., Cooper, D.KC. Xenotransplantation--the future of corneal

transplantation? Cornea. 2011; 30: 371-378.

[124] Lee, W., Miyagawa, Y., Long, C., Cooper, D.K.C., Hara, H. A comparison of three

methods of decellularization of pig corneas to reduce immunogenicity. Int J

Ophthalmol. 2014; 7: 587-593.


[125] Lee, W., Miyagawa, Y., Long, C., et al. Expression of NeuGc on pig corneas and

its potential significance in pig corneal xenotransplantation. Cornea. 2016; 35:

105-113.

[126] Lee, W., Mammen, A., Dhaliwal, D.K., et al. Development of retrocorneal

membrane following pig-to-monkey penetrating keratoplasty. Xenotransplantation.

2017; 24(1): doi: 10.1111/xen.12276.

[127] Lamm, V., Hara, H., Mammen, A., Dhaliwal, D., Cooper, D.K.C. Corneal

blindness and xenotransplantation. Xenotransplantation. 2014; 21: 99-114.

[128] Kim, M.K., Hara, H. Current status of corneal xenotransplantation. Int J Surg.

2015; 23: 255-260.

[129] Cooper, D.K.C. Porcine red blood cells as a source of blood transfusion in humans.


[130] Cooper, D.K.C., Hara, H., Yazer, M. Genetically engineered pigs as a source for

clinical red blood cell transfusion. Clin Lab Med. 2010; 30: 365-380.

[131] Cooper, D.K.C. How important is the anti-Gal antibody response following the

implantation of a porcine bioprosthesis? J Heart Valve Dis. 2009; 18: 671-672.

[132] Manji, R.A., Menkis, A.H., Ekser, B., Cooper, D.K.C Porcine bioprosthetic heart

valves: The next generation. Am Heart J. 2012; 164: 177-185.

[133] Manji, R.A., Ekser, B., Menkis, A.H., Cooper, D.K.C. Bioprosthetic heart valves of

the future. Xenotransplantation. 2014; 21: 1-10.

[134] Bouhours, D., Pourcel, C., Bouhours, J.E. Simultaneous expression by porcine

aorta endothelial cells of glycosphingolipids bearing the major epitope for human

xenoreactive antibodies (Gal alpha 1-3Gal), blood group H determinant and N-

glycolylneuraminic acid. Glycoconj J. 1996; 13: 947-953.

[135] Zhu, A., Hurst, R. Anti-N-glycolylneuraminic acid antibodies identified in healthy

human serum. Xenotransplantation. 2002; 9: 376-381.

[136] Lutz, A.J., Li, P., Estrada, J.L., et al. Double knockout pigs deficient in N-

glycolylneuraminic acid and galactose alpha-1,3-galactose reduce the humoral

barrier to xenotransplantation. Xenotransplantation. 2013; 20: 27-35.

[137] Byrne, G.W., Du, Z., Stalboerger, P., Kogelberg, H., McGregor, C.G. Cloning and

expression of porcine beta1,4 N-acetylgalactosaminyl transferase encoding a new

xenoreactive antigen. Xenotransplantation. 2014; 21: 543-554.

[138] Estrada, J.L., Martens, G., Li, P., et al. Evaluation of human and non-human

primate antibody binding to pig cells lacking GGTA1/CMAH/beta4GalNT2 genes.


[139] Lee, W., Hara, H., Ezzelarab, M.B., et al. Initial in vitro studies on tissues and cells

from GTKO/hCD46/NeuGcKOpigs. Xenotransplantation. 2016; 23: 137-150.

[140] Cooper, D.K.C, Ekser, B., Ramsoondar, J., Phelps, C., Ayares, D. The role of

genetically-engineered pigs in xenotransplantation research. J Pathol. 2016; 238:

288-299.


[141] Ye, Y., Niekrasz, M., Kosanke, S., et al. The pig as a potential organ donor for

man. A study of potentially transferable disease from donor pig to recipient man.


[142] Onions, D., Cooper, D.K.C., Alexander, T.J., et al. An approach to the control of

disease transmission in pig-to-human xenotransplantation. Xenotransplantation.

2000; 7: 143-155.

[143] Mueller, N.J., Barth, R.N., Yamamoto, S., et al. Activation of cytomegalovirus in

pig-to-primate organ xenotransplantation. J Virol. 2002; 76: 4734-4740.

[144] Mueller, N.J., Sulling, K., Gollackner, B., et al. Reduced efficacy of ganciclovir

against porcine and baboon cytomegalovirus in pig-to-baboon xenotransplantation.

Am J Transplant. 2003; 3: 1057-1064.

[145] Gollackner, B., Mueller, N.J., Houser, S., et al. Porcine cytomegalovirus and

coagulopathy in pig-to-primate xenotransplantation. Transplantation. 2003; 75:

1841- 1847.

[146] Mueller, N.J., Kuwaki, K., Dor, F.J., et al. Reduction of consumptive coagulopathy

using porcine cytomegalovirus-free cardiac porcine grafts in pig-to-primate

xenotransplantation. Transplantation. 2004; 78: 1449-1453.

[147] Mueller, N.J., Livingston, C., Knosalla, C., et al. Activation of porcine

cytomegalovirus, but not porcine lymphotropic herpesvirus, in pig-to-baboon

xenotransplantation. J Infect Dis. 2004; 189: 1628-1633.

[148] Mueller, N.J., Kuwaki, K., Knosalla, C., et al. Early weaning of piglets fails to

exclude porcine lymphotropic herpesvirus. Xenotransplantation. 2005; 12: 59-62.

[149] Mueller, N.J., Ezzelarab, M., Buhler, L., Haeberli, L., Ayares, D., Cooper, D.K.C.

Monitoring of porcine and baboon cytomegalovirus infection in

xenotransplantation. Xenotransplantation. 2009; 16: 535-536.

[150] Ibrahim. Z., Ezzelarab, M., Kormos, R., Cooper, D.K.C Which patients first?

Planning the first clinical trial of xenotransplantation: a case for cardiac bridging.


[151] Ekser, B., Gridelli, B., Tector, A.J., Cooper, D.K.C. Pig liver xenotransplantation

as a bridge to allotransplantation: which patients might benefit? Transplantation.

2009; 88: 1041-1049.

[152] Cooper, D.K.C, Teuteberg, J.J. Pig heart xenotransplantation as a bridge to

allotransplantation. J Heart Lung Transplant. 2010; 29: 838-840.

[153] Cooper, D.K.C., Wijkstrom, M., Hariharan, S., et al. Selection of patients for initial

clinical trials of solid organ xenotransplantation. Transplantation. 2017; 101: 1551-

1558.

[154] Knosalla, C., Gollackner, B., Bühler, L., et al. Correlation of biochemical and

hematological changes with graft failure following pig heart and kidney

transplantation in baboons. Am J Transplant 2003; 3: 1510-1519.


[155] Cooper, D.K.C., Pierson III, R.N., Hering, B.J., et al. Regulation of clinical

xenotransplantation – time for a reappraisal?. Transplantation. 2017; 102: 1766-

1769. Doi:10.1097/TP.

[156] Cooper, D.K.C. Ethical aspects of xenotransplantation of current importance.

Xenotransplantation 1996;3:264-274.

[157] Smetanka, C., Cooper, D.K.C. The ethics debate in relation to xenotransplantation.

Rev Sci Tech. 2005; 24: 335-342.

DAVID K. C. COOPER - BRIEF BIOGRAPHY

David Cooper is a Professor of Surgery in the Department of Surgery at the

University of Alabama at Birmingham (UAB) in the USA.

He studied medicine at Guy’s Hospital Medical School in London (now merged with

King’s College London), and subsequently trained in general and cardiothoracic surgery

in Cambridge and London, interrupting this training to carry out research for the PhD

degree. He was present at the first heart transplant in the UK in 1968, and was a member

of the team that initiated the heart transplant program at Papworth Hospital in Cambridge

in 1979. Between 1972 and 1980, he was a Fellow and Director of Studies in Medicine at

Magdalene College, Cambridge.

In 1980 he took up an appointment in cardiac surgery at Groote Schuur Hospital at

the University of Cape Town where, under Professor Christiaan Barnard (who, in 1967

had carried out the world’s first heart transplant), he had responsibility for patients

undergoing heart transplantation. With Winston Wicomb and Dimitri Novitzky, he

developed a hypothermic perfusion device to store donor hearts (which was used

clinically), and investigated the detrimental effects of brain death on donor organs before

establishing thyroid hormone therapy in the management of potential organ donors.


In 1987, he relocated to the Oklahoma Transplantation Institute in the USA where he

continued to work in both the clinical and research fields. With colleagues, he identified

the importance of the Gal antigen in xenotransplantation. After 17 years as a surgeon-

scientist, he decided to concentrate on research, initially at the Massachusetts General

Hospital/Harvard Medical School in Boston, subsequently at the Thomas E. Starzl

Transplantation Institute at the University of Pittsburgh, and now at UAB. His major

interest is the xenotransplantation of organs, islets, and corneas in pig-to-nonhuman

primate models, with the aim of using genetically-engineered pigs as a solution to the

organ shortage for clinical transplantation.

The Royal College of Surgeons of England appointed him to a Hunterian

Professorship (1983), Arris and Gale Lectureship (1988), Amott Lectureship (2002), and

awarded him the 1997 Jacksonian Prize and Medal. Professor Cooper has published

almost 900 medical and scientific papers and chapters, has authored or edited 11 books,

and has given more than 300 invited presentations worldwide.

Between 1994-1997, he was Honorary Secretary/Treasurer of the International

Society for Heart and Lung Transplantation. He was the Founding Honorary Secretary of

the International Xenotransplantation Association IXA) in 1997, its President in 1999-

2001, and the Editor-in-Chief of Xenotransplantation from 2001-2007. In 2009, he was

elected to Honorary Membership of the IXA.

He has received several awards for his research, and in 2016 he was made a Fellow

of his alma mater, King’s College London.

david k. c. cooper

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