Microsoft Word - Final Report - Xenotransplantation_2003

8/9/2019 Microsoft Word - Final Report - Xenotransplantation_2003

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The w ork desc ribed in this rep ort ent itled XENOTRANSPLANTATION

has been carried out by Miss Trisha Ghosh under my supervision.

She is a bona fide stud ent o f MBA (Pha rm. Tec h), third yea r, Sc hoo l of

Pharma c y a nd Tec hnology Ma nage me nt, NMIMS University, Mumb ai.

Date: Ap ril 16th,2009 Guide:

Plac e:Mumba i Mrs. Reema Thomas,

Lecturer (Clinical Pharmacy)

Schoo l of Pharma cy & Tec hnolog y

Management

Sc hool of Pharmac y and Tec hnology Management,

SVKMs NMIMS University,

Vile-Parle, Mumbai-400056


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As required by university regulation, I wish to state that this work embodied in

this report titled XENOTRNSPLANTATION wa s Comp iled from va rious sources

und er the guida nc e of Mrs. Ree ma Thom as. This wo rk has not b ee n

submitted for any other degree of this or any other university. Whenever

references have been made to previous work of others, it has been clearly

indica ted as such and inc luded in the b ibliog raphy.

Miss Trisha Ghosh

Roll No.:20

Forwarded Through

Guide:

Dr. R. S. Gaud

Profe ssor Pha rmac eut ica l Sc ienc e,

Depa rtment of Pharmac eutic s,

Sc hoo l of Pharma c y and Tec hnologyManagement,

SVKM s, NMIMS University,

Vile Parle (W),

Mumbai 400056


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ACKNOWLEDGEMENTS

It is great pleasure for me to acknowledge all those who have contributed

towards the conception, origin and nurturing of this project.

With a deep sense of gratitude and the respect, I thank my esteemed research

guide Mrs. Reema Thomas , School Of Pharmacy and Technology Management,

Narsee Monjee Institute Of Management and Higher Studies), Mumbai for her

inestimable guidance, valuable suggestions and constant encouragement during the

course of this study. It is with affection and reverence that I acknowledge my

indebtness to her for outstanding dedication, often far beyond the call of duty.

I sincerely thank to Dr. R. S. Gaud , Dean, School Of Pharmacy andTechnology Management for allowing me to work on this project.

At this moment, I thanks with deep gratitude to my classmates andfriends for

their moral support, constant encouragement and patience absolutely needed to

complete my entire study. It was the blessing of them that gave me courage to face the

challenges and made my path easier.

I am indebted infinitely to care, support and trust being shown by my parents

without whom it would not be possible to complete this project.

(Trisha Ghosh)

Roll No. 20


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Contents :

Introduction 6

Disea ses treated by xenotransplantation 15

The need of xenotranspla ntation 17

The risks & hurdles invo lved in xenotransplantation 18

The potential use of allosensitized humans 25

Mod ifica tion of immunosupp ressive regimen 26

Xenotransplanta tion : cultura l, sp iritual, ethica l issues 27

The interest of animals 29

Is xenotransplanta tion intended to be permanent 32

Sc ientific resea rch c arried out in xeno transplanta tion 32

Xenotourism 33

Myths ab out xenotransplantation 35

Future d irec tions : genetic eng ineering 36

US FDA guid elines 37

Guidance to industry 41

Summary 44


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Introduction

Despite practical advances in transplantation medicine, the critical shortage of human

donor organs severely limits widespread clinical use. Developing alternative sources

of cells, organs, and implantable devices for replacement, regeneration, or mechanical

assistance of failing organs is, therefore, paramount. Rather than competing with each

other, these techniques can be complementary, with advances in one type jump-

starting progress in another. One technique can buy time while a patient awaits

application of another, or 2 or more techniques can be combined.

"To solve the transplantation crisis, we need to close the gap between the organ

shortage and the demand for organ substitutes, which will take a while," Robert M.

Nerem, PhD, of the Petit Institute for Bioengineering and Bioscience said at the

Georgia Institute of Technology in Atlanta. Although progress has been greater in

skin substitutes and orthopaedic applications, only replacement of vital organs can

lighten the heavy burden of chronic disease.


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What Is Xenotransplantation?

Xeno means strange, or foreign. The term xenotransplantation (pronounced zeeno-

transplantation) is used to describe a transplant between any two species of animals,

including humans.

Xenotransplantation is any procedure that involves the transplantation, implantation,

or infusion into a human recipient of either (a) live cells, tissues, or organs from a

nonhuman animal source, or (b) human body fluids, cells, tissues or organs that have

had ex vivo contact with live nonhuman animal cells, tissues or organs.i

When only cells are used, the material is often referred to as a cellular xenotransplant

or cellular xenograft. As well, there are certain kinds of xenotransplants which are not

true transplants at all, because the animal organ or cells stay outside the patients

body. These are called extra-corporeal (or outside-the-body) xenotransplants.ii

History:

Xenotransplantation is not a new concept. The idea of transplanting animal parts to

humans has always fascinated man. There are few non documented incidents during

very early

In the 1600s Russians had tried to cure fractures by replacing human bones with dog

bones,obviously unsuccessfully


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Later in the late 1800s frog skin is said to have been extensively used to treat skin

burns and ulcers. One British army surgeon is known to have used this procedure

successfully a plethora of times.

The first scientific reports on xenotransplantation , more specifically renal

heterotransplant appeared appeared early in this century.

In 1905 Princeteau inserted slices of rabbit kidney into nephrotomy on a child with

renal insufficiency. immediate results were excellent he wrote.. volume of urine

increased, vomiting stopped. On 16th

day the child died.

In 1910 Unger described his attempt at transplantation of kidneys from a non-human

primate into man. Patient died 32 hrs after transplantation & autopsy showed venous

thrombosis.

In 1923 Neuhof attemted treatment of a patient with mercury bichloride poisoning by

renal heteotranplant. Patient died 9 days later.

Scientific interest in transplantation died when neurological basis of rejection was

establishediii

.

In the early 1960s, renal xenotransplantation from chimpanzees and baboons to

humans was tried as was liver and cardiac xenografting. Preliminary studies

demonstrated the technical feasibility of the procedures. At that time, the ethics of

xenotransplantation was not questioned.iv

In 1984, after Bailey and associates transplanted the heart of a baboon into a

neonate

v

the ethical dilemma of xenotransplantation suddenly struck the medical


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community in the United States. At that time, there was some agreement that research

involving xenografts was ethically defensible because of the shortage of available

organs. However; the case of Baby Fae

vi

raised many ethical questions, notably those

concerning inadequate scientific preparation, the nature of consent for

xenotransplantation and the use of animals from an endangered species.

In the early 1990s, more powerful drugs for preventing rejection of a human organ

by a patients immune system were developed. These drugs provided hope that

xenotransplants might also be more successful. Several patients received animal

hearts or livers but did not survive more than 3 months.

In 1995 one patient in the United States received bone marrow from a baboon.

Although the baboon marrow was rejected by the patients body, the patient is still

alive and appears not to have any harmful side-effects from being treated with the

baboon bone marrow.

In the past few years there have been some encouraging results with cellular

xenotransplants.

In Sweden, 10 patients with diabetes received cells from the pancreas of pigs, to see if

these new cells would produce insulin. None of the pig cells produced insulin in the

long term, and none of the patients got sick from the transplanted pig cells.

In 1999, Maribeth Cook who was suffering from paralysis as an aftermath of stroke

was administered with 30 million fetal porcine cells by an injection to the brain. The

fetal porcine cells were used because they are functionally similar to the human fetal

cells. The procedure was successful and she is now able to walk.


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Currently, a clinical trial in the United States is transplanting specific brain cells from

fetal pigs into patients with Parkinsons disease. Early results indicate improvement in

some patients condition

vii

The different types of xenotransplantation:

When we hear about transplantation, we usually think of organ transplants such as

hearts or kidneys. However, it is important to stress from the outset the sheer variety

of ways of doing xenotransplantation.

Transplantation can also involve tissues, such as bone marrow, or clusters of

specialized cells, such as pancreatic islet cells (which produce insulin). These

transplants are called cell therapies.

Transplants can also involve different types of procedures. Most involve putting

living tissue, cells or an organ into a patient to replace diseased or failing parts of the

body. Less well known are external therapies, which occur outside the body of the

patient. An example is when blood from a patient with liver failure is passed through

a machine containing animal liver cells to remove toxic substances (a procedure

similar to kidney dialysis). Another external therapy involves growing human skin in

the laboratory over a layer of animal cells and later using the skin as a graft to treat

burnsviii

.

Some types of xenotransplantation are relatively advanced and close to moving

beyond research into treatment; others - particularly the transplant of whole organs -

are a long way from being used clinically.


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Some types aim to improve on current treatments, as with diabetes; others aim to

provide treatment where currently there is none, as in external therapies (mentioned

above).

Some types use cells, tissues or organs that are genetically modified; others do not.

Some types use relatively few animals or need not inflict great suffering; others use a

variety of animals, including primates, and may involve considerable suffering for

animals.

Some types use organs that are central to many people's sense of identity, such as

hearts or eyes; others involve parts that are likely to be less central, such as pancreatic

islet cells.ix


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The third type of animal-t

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ransferred into an external device, which is u

xperienced liver failure, and who is waiting fo

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whole organ (eg a kidney), is transplanted into a human to replace a failed kidney. The

patient thus experiences restored kidney function.

Clinical xenotransplantation

The current clinical experience with xenotransplantation is limited to 3 main areas:

tissue xenotransplantation, extracorporeal perfusion of a xenograft for the treatment of

fulminant liver failure and whole-organ xenotransplantation.

Tissue xenografting :

Tissue xenotransplantation with pig-to-human skin grafts and pig heart valve implants

has been used successfully for many years. Xenotransplantation using pig neural cells

has shown promise as a treatment for Parkinson's disease. Pancreatic islet

xenotransplantation offers the potential to cure insulin-dependent diabetes.

Transplanted islets are not initially vascularized: they become vascularized by

recipient vessels over time, thereby bypassing the hyperacute rejection seen in whole-

organ xenotransplantation. Pigs are a good source of donor islet tissue because

porcine and human insulin are structurally similar, pigs and humans have similar

glucose metabolism, and porcine insulin has been used for many years to treat

diabetes.

Xenoislet transplantation has been combined with allograft kidney transplantation in

patients with diabetes and end-stage diabetic nephropathy. Islet cell function was

demonstrated by porcine C peptide found in the urine in some patients; however,

insulin requirements were not affected by the xenotransplant. This work shows that it


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is possible to attain viable islet cells after xenotransplantation, but further

modifications are required to achieve clinical function that allows tapering or

withdrawal of insulin.

3

Extracorporeal perfusion with xenografts

Extracorporeal xenogeneic liver support has been used in patients with fulminant liver

failure as a temporary measure to allow time for the liver to recover function or for an

allograft to become available. A perfusion circuit is established that carries blood

from the patient through the hepatic artery and portal vein of the ex-vivo organ and

then returns the detoxified blood to the patient. Two of 5 patients described in the

recent literature were successfully managed by this technique until allotransplantation

could be performed.viii

Whole-organ xenografting :

There have been sporadic attempts at clinical whole-organ kidney, heart and liver

xenotransplantation.

In the early 1960s, Reemtsma and colleaguesiii

transplanted chimpanzee kidneys into

human recipients before dialysis was widely available. Some of these grafts had

adequate function early, but eventually all of the recipients succumbed to

uncontrollable rejection or infection.

In 1985, Bailey and associates transplanted a baboon heart into a newborn infant who

survived for 3 weeks until the graft was lost to antibody-mediated damage.


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In 1993, Starzl and colleagues

reported 2 cases of baboon-to-human liver

xenotransplantation in patients with end-stage liver disease secondary to chronic

active hepatitis B (1 patient was also HIV positive). Evidence of liver function

included normal coagulation profile, correction of hyperammonemia and clearance of

serum lactate; however, both patients had low serum albumin requiring repeated

transfusions. The first patient lived for 70 days, but the second patient died 26 days

postoperatively. Neither graft had evidence of rejection, and both of these patients

died from sepsis secondary to profound immunosuppression.

The use of a liver xenograft as a bridge to allotransplantation has been investigated.ix

Recently, a woman with fulminant hepatic failure received a heterotopic, auxiliary,

pig liver xenograft as a temporary "bridge" in an attempt to stabilize her condition

until an allograft became available.x

The liver showed signs of function but her

neurologic status did not improve and she died 34 hours after xenografting.

What Kinds of Diseases Could Be Treated By Xenotransplantation?

Any disease that is treated by human-to-human transplants could potentially be

treated by xenotransplantation. The most likely candidates for xenotransplantation in

the near future are people with serious kidney, liver or heart disease, diabetes or

Parkinsons disease. People who need bone marrow transplants may also be

candidates for this kind of transplant.

What diseases might be treated by cellular xenotransplants?


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Cellular xenotransplants may be a way to treat people who have diabetes, Parkinsons

disease or certain other diseases. The treatment would involve replacing specific cells

or tissues which do not work properly because of the disease. For diabetics, these are

the islet cells of the pancreas, and for someone with Parkinsons disease they would

be certain brain cells. In both cases, the cells needed for the transplant are difficult to

obtain from human donors.

What diseases might be treated by extra-corporeal xenotransplants?

People with liver failure might be treated with an extra-corporeal (outside-the-body)

xenotransplant using a healthy pig liver. The patients blood circulation would be

connected for a short while to a pig liver that is kept outside the patients body. In

some cases, this might be all that is needed to allow the persons own liver to recover

and start working again. In other cases, the persons own liver might not recover, but

the transplant team would have more time to try to find a suitable human liver. This

kind of short term procedure is sometimes called a bridge to transplant. In either case

the extra-corporeal pig liver would only be temporary.

Other extra-corporeal xenotransplant treatments may be carried out using only a small

number of living animal cells. For example, the animal cells may be included in a

specialized device, such as a filter system. When the blood of a patient with liver

failure is pumped through this filter system, the animal liver cells are able to do the

work of the patients own liver, at least for a short while.


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Why do we need xenotransplantation?

The development of xenotransplantation is, in part, driven by the fact that the demand

for human organs for clinical transplantation far exceeds the supply. It is generally

accepted that there is a shortage of human organs for the purpose of transplantation.

Currently only 5 % of the total organs required for transplantation is available. At

present approximately 6000 and 45000 patients await transplantation in the UK &

USA respectively, almost 3000 Canadians are awaiting an organ transplant.ix

and the

number is steadily increasing by 10-15 % each year. Currently ten patients die each

day in the United States while on the waiting list to receive life-saving vital organ

transplants. Moreover, recent evidence has suggested that transplantation of cells and

tissues may be therapeutic for certain diseases such as neurodegenerative disorders

and diabetes, where, again human materials are not usually available.i

Xenotransplantation offers the potential for an unlimited supply of healthy donor

organs. As waiting lists lengthen, many patients decompensate while waiting for a

transplant. Xenotransplantation could be performed electively and timed so that both

the donor and recipient are in optimal condition before transplantation.

Moreover, the donor animal could be matched or manipulated, or both, to facilitate

long-term acceptance of the graft without the need for maintenance

immunosuppression. Before xenotransplantation can be offered to patients, a number

of hurdles must be overcome, including immunologic barriers, disease transmission,

physiological differences and ethical concerns.xi


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What are the risks/hurdles faced in xenotransplantation?

The immunologic reaction

The immunologic reaction of the recipient to a xenograft is mediated initially by

xenoreactive antibodies, complement and natural killer cells and later primarily by

cellular immune responses. These mechanisms result in hyperacute, acute vascular,

cellular and chronic graft rejection.

Hyperacute rejection is a major barrier to discordant xenotransplantation. Humans

have natural IgM antibodies (xenoreactive antibodies) to 1,3-galactose, a

carbohydrate that is expressed on all nucleated pig cells. After binding of these

preformed antibodies, serum complement is activated, resulting in massive

thrombosis to vascular endothelium with vessel occlusion and graft failure within

minutes to hours of the transplantation.xii

Xenoreactive antibodies can be removed by

adsorption columns, but this is only a temporary solution. A more promising approach

is to create transgenic pigs expressing selected human genes that modify the immune

response. Recently, pigs have been raised that express human complement regulatory

genes, thereby preventing activation of complement and ameliorating hyperacute

rejection.xiii

The next major hurdle is to prevent acute vascular rejection which leads to graft

destruction over a period of days to weeks. Xenoreactive antibodies, macrophages,

natural killer cells and complement appear to play important roles in this process.1


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Later (in days to weeks), xenografts may also be damaged by cellular and chronic

graft rejection. It is not known whether any of these processes can be reliably

prevented by currently available immunosuppressive drugs.

viii

It has been evaluated different combinations of antirejection drugs for

xenotransplantation in a baboon-to-monkey model and the results have been

promising. The combination of cyclosporine, cyclophosphamide and rapamycin

provided long-term survival in concordant kidney xenografts. One monkey with a

baboon liver lived for 3 years, despite withdrawal of all immunosuppression 1 year

after transplantation.viii

Ultimately, the goal of transplantation is to attain a state of tolerance whereby the

recipient's immune system accepts the graft as "self" without the need for

maintenance immunosuppression.viii

The opportunity to genetically manipulate pig

donors provides new ways to induce tolerance to xenografts in humans. Donor bone-

marrow transplantation, radiation and the production of monoclonal antibodies

directed against specific lymphocyte receptors are currently being studied as methods

to induce tolerance.viii

What is immune rejection?

Our immune system protects us from things that are foreign, or not normally part of

our healthy bodies. The most common threats to our health are germs, such as viruses

or bacteria all around us. When foreign materials of any kind gets into our bodies, our

immune system kicks into high gear by developing antibodies and specialized white

blood cells to get rid of them.


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What happens when immune rejection occurs during human-to-human organ

transplants?

Except for identical twins, each persons cells are slightly different from everyone

elses. This is the reason that testing is done to find the right match between an organ

donor and the person who needs the transplant. But a perfect match is rarely possible.

Most times, the transplant patient gets an organ that is only as close as possible under

the circumstances. As a result, the patients immune system recognizes the cells of the

new organ as slightly different than its own and tries to destroy these cells, just as if

they were foreign bacteria or viruses. If the patients immune system is successful,

then so much of the new organ is damaged or destroyed that it cant work properly

and we say its been rejected by the patient.


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Can immune rejection be overcome in human-to-human organ transplants?

The development of new immunosuppressive drugs is one of the reasons that human-

to-human transplants are now so successful. These drugs suppress the patients

immune system, not allowing it to work as well as usual and giving the patients body

a much greater chance of not rejecting the transplanted organ. Patients who receive

human organs must usually take immunosuppressive drugs for the rest of their lives.

How does immune rejection work with whole organ xenotransplants?

When whole animal organs are used as xenotransplants, the problems with immune

rejection are huge because animal and human tissues are so different. In fact, the

organs may be so mis-matched that the xenotransplanted organ may be rejected

within minutes of the transplant.

Is it possible to overcome immune rejection with whole organ xenotransplants?

Until recently it seemed impossible to overcome this. Even immunosuppressive drugs

werent powerful enough to stop rejection in the few patients who had received whole

animal organs. However, recent scientific advances may help solve some of these

rejection problems. One solution may be the development of transgenic animals,

where the animals are bred with specific human genes. The principle behind

developing transgenic animals is this: the human gene present in the animal cells will

help to reduce the chances of immune rejection in the xenotransplant patient. Pre-

clinical studies suggest that this approach might work, at least for short term

xenotransplants.

What about immune rejection of xenotransplants over the long term?


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Although xenotransplants from transgenic animals may reduce the possibility of short

term immune rejection, the longer the xenotransplant remains in the patient, the

greater the chance the immune system will recognize it as foreign and start to reject it.

At this time, even with the combination of transgenic animals and immunosuppressive

drugs, the problems of immune rejection are not entirely solved.

Is immune rejection also a problem for extra-corporeal and cellular

xenotransplants?

Extra-corporeal whole organ xenotransplants may also be affected by immune

rejection and often stop working after a very short time because of widespread tissue

damage. However, some cellular xenotransplants seem better able to resist immune

rejection. For example, cells implanted within the brain are somewhat protected from

immune rejection. Xenotransplants of pancreatic islet cells into diabetic patients may

be encased in a special membrane that helps protect them from immune rejection.

Physiologic incompatibilities

There may be physiologic incompatibilities with some xenografts. For example,

patients with porcine kidney grafts may require supplemental erythropoietin to

maintain normal hemoglobin levels. It is unlikely that pig livers will be able to

provide all of the functions of the more than 2600 proteins and enzyme systems that

are produced in human livers. Finally, the lifespan of pigs is less than 15 years;

whether their organs will work for a human lifetime is unknown. There is limited

information about the function of xenografts in humans. Previous attempts at clinical

xenotransplantation, however, have shown that adequate function may occur early

after transplantation.viii


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ERVs

A group of viruses called endogenous retroviruses (ERVs) are of particular concern.

Instead of actively causing infections like other retroviruses, the endogenous

retroviruses remain dormant in their host - embedded in the genetic material - not

causing any obvious signs of disease. However, they may be activated occasionally,

and it is possible they could then infect other animals, including different species.

Little is known about what might make endogenous retroviruses become active but, if

an animal transplantation product contains an endogenous retrovirus, there is the

potential for it to activate at any time in the future and infect the transplant recipient

.Such an infection could spread to close contacts of the recipient (for example,

medical staff, family, friends) and, in the worst case, to the general population. These

ERVs cannot be screened out.viii

These are known to cause Xenozoonosis, which is

defined as the infection of a pathogen from an animal to a human being due to the

introduction of xenograft.vii


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PERVs

Most pigs have a retrovirus called porcine endogenous retrovirus (or PERV). In

1997 researchers reported that when they mixed pig cells with human cells in the

laboratory, some human cells became infected with PERV. The first evidence of

cross-species transmission of a retrovirus during a transplant occurred in 2000, when

a study found transmission of a PERV from pancreatic pig cells into

immunosuppressed diabetic mice. This raises the possibility that the recipient of a

pig transplant may be infected with PERV, or with another, currently unknown,

infectious disease agent. Some retroviruses have been associated with cancer.ix

It seems likely that the risk of unusual infections will be low since humans and pigs

have lived in close proximity for many years. Moreover, many immunocompromised

patients have been treated with full-thickness pig skin grafts with no evidence of

adverse effects. Nonetheless, xenotransplant recipients, their families and their health

care providers will have to be monitored closely for infectious complications.viii

Public health risks

If the xenotransplant procedure involves a risk not only to the recipient but also to

close contacts - and in the worst-case scenario of an epidemic, a risk to everybody -

individual consent becomes insufficient.

The ethical question then becomes:

is it right to impose risks on people without their consent?


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options include only chronic dialysis or ventricular assist devices. Popma and

colleagues[2]

demonstrated that the presence of allosensitization in 7 individuals was

accompanied by concomitant "cellular xenosensitization" -- ie, higher T-cell

proliferative responses to porcine endothelial cells when compared with T-cell

proliferative responses of nonsensitized individuals. We have previously reported

that nonsensitized human subjects have an increased proliferative response to pig

endothelial antigens in comparison to alloantigens, and that this is related to direct

xenorecognition of pig SLA by CD4 T cells. This suggests that the same

mechanisms that lead to CD4 T-cell sensitization against alloantigens may also

predispose the individual to a state of heightened immune activation directed against

pig xenoantigens. Since the allosensitized state is associated with an increased

incidence of humoral and cellular rejection of allografts, these data suggest that the

highly sensitized allorecipient may not be the most appropriate initial choice for

transgenic pig xenografts using currently available immunosuppressive regimens.

Modification of Immunosuppressive Regimens in an Attempt to Improve Pig to

Nonhuman Primate Xenograft Survival

For the past several years a select group of institutions working in conjunction with

the manufacturers of transgenic pig xenografts have attempted to achieve

prolongation of graft survival beyond days. Unfortunately, this has not yet

consistently been achieved using the currently available transgenic constructs. At

first it was thought that a more aggressive immunosuppressive regimen might lead

to prolonged graft survival. As yet, this has not been achieved and the median


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orthotopic working heart graft survival remains 12 days, with the longest survival

slightly less than 1 month. Interestingly, a small group of animals continue to

hyperacutely reject the transgenic xenograft. Brenner and colleagues

report their

experience with hDAF transgenic heterotopic and orthotopic heart xenografts using

a fairly intensive drug therapy, which in some animals also included

immunoadsorption. Not unlike the previously described morbidity seen by the Loma

Linda group with xenotransplantation between closely related species, these animals

experienced significant morbidity related to the drug therapy without a survival

advantage. In particular, despite this aggressive immunosuppression, these animals

continued to succumb to acute vascular rejection, a second barrier that had to be

overcome for successful pig-to-primate xenotransplantation. These experiments

emphasize the need to better understand the mechanisms involved in acute vascular

rejection, as well as the need to develop additional transgenic constructs that may

better resist these immunologic barriers, such as multiple complement regulators and

approaches that enable reduced expression of pig xenoantigens.

Xenotranplantation : Cultural, spiritual and Ethical Issues

Not all members of the medical profession may be comfortable with

xenotransplantation. Several questions come immediately to mind: Do we really need

xenotransplantation? Does xenotransplantation alter our definition of a human being?

Are we transgressing the laws of nature? Does life need to be prolonged at any price?

What are the psychological and biological effects of xenotransplantation on the


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recipients? What are the implications for society, and for future generations? What are

the effects on animals? Do we care about animal suffering and genetic manipulation

of animals?

iv

Religious viewpoints

As an indication of religious viewpoints, the Australian public consultation on

xenotransplantation received submissions from representatives of the Christian,

Jewish and Islamic religions, who agreed that xenotransplantation does not

contravene the order of creation, and that the use of animals for human benefit is

acceptable. A review of world religions found that xenotransplantation is acceptable

in most of the major religions. Both Islam and Judaism forbid the eating of pork, but

accept xenotransplantation on the basis that humans have a higher place in the world

and therefore have the right to use animals for their welfare, as long as the animals are

treated with respect. A number of religions that do object to transplantation, such as

the Hindu or Buddhist faiths, still allow the individual to make a choice.viii

Identity

Some organs (especially) and tissues are more related to an individual's sense of

personal identity than others. Would - or in fact should - the particular tissue, cells or

organ involved alter our thinking about xenotransplantation?

Consider the transfer of a number of pig pancreas cells to a person suffering from

diabetes. Compare this to a person who has been given a pig's heart, or perhaps even a

pig's eyes.


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Is there a point at which the person receiving the transplant, or other people, would

feel that he or she was now less human? That somehow their personal identity had

been compromised?

viii

The Interests of Animals

Animals likely to be used in xenotransplantation

Non-human primates

Researchers, research sponsors and the wider community generally agree that non-

human primates (such as baboons and other monkeys) are not a suitable source for

any of the proposed animal therapies (external therapies, cell therapies or organ

transplants) because of the risk of infections to the recipient and the wider

community. The US Food and Drug Administration has effectively prohibited the use

of non-human primates in animal-to-human xenotransplantation since 1999.

The use of non-human primates in medical research also raises serious ethical issues.

Non-human primates are highly intelligent animals with complex behavioural and

social needs that are difficult to meet in a medical research environment. However,

baboons are considered the most suitable species for animal-to-animal studies (such

as pig to baboon) to obtain important information on the effectiveness of a procedure

before it can be tested in an animal-to-human trial.


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Pigs

At present, pigs are considered to be the most likely and appropriate non-human

source of organs and tissues. The anatomy and functioning of pigs are very similar to

those of humans. Pigs are domesticated animals that are easy to breed, and,

importantly, pigs are suitable for genetic modification.

Other animals

Animal-to-animal transplantation studies would use a variety of animal species in the

early stages of the research (such as mice, rats and rabbits). If these studies show

promising results, researchers will need to trial the procedure in an animal study that

is as much like the future clinical use of the therapy as possible. This will usually

involve the use of non-human primates (specifically baboons) as transplant recipients,

as noted above, but fish and cattle might also be used for some procedures, such as

helping to grow skin. Researchers are also considering the use of other species (such

as cattle, fish and mice) for cellular transplants.

Which animals work best?

It would seem obvious, when searching for ways to reduce the rejection reaction to

xenografts, to use animals as physiologically close to humans as possible, such as old

world monkeys and the apes (for example, chimpanzees). However, this very

closeness creates complex ethical problems as well as an increased likelihood of

cross-species infection. There are strict regulations in New Zealand about the use of

non-human primates in research, so it is unlikely that these species would be used for

xenotransplantation research in this country.


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At present the pig is the favoured animal for research into xenotransplantation,

because they grow quickly to about the right size, produce large litters and can be

reared in specific pathogen-free conditions (where some but not all micro-organisms

are excluded). In terms of ethical concerns, the fact that pigs have long been used as a

source of meat reduces - but certainly does not eliminate - the concerns of many

people, especially when weighed against the possible benefits. However, recently it

has become clear that there is also the possibility of cross-species infection from pigs.

This takes the issue of whether to perform such a procedure out of the realm of an

individual decision to take a personal risk, usually for the sake of a therapeutic effect,

into the realm of the safety of the xenotransplantation recipient's close contacts and

the community at large. The spectre of HIV and AIDS hangs over discussions on the

possibility of cross-species infection, so we will now turn to look at the risks involved

with xenotransplantation.

Genetic modification of animals

This raises some difficult ethical issues about the rights and welfare of the animals,

such as whether the insertion of human genes may make the animal in some way

'human', or whether inserted genes cause unexpected side-effects in the animals. One

view may be that these issues need to be considered case by case to ensure that the

proposed modification does not alter the animal in any other significant way. The aim

would be to ensure that the animals retain the essential characteristics of their species.

Animal welfare and ethics

Some think it is wrong to cause suffering to or kill animals even if this has major

benefits. Such an argument might be based on the belief that humans and animals


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have the same moral status. It is a matter of controversy how much suffering

xenotransplantation experiments and rearing would cause. For example, some types of

xenotransplantation involve killing young pigs under anaesthesia, whereas others

involve the destruction of a chimpanzee's immune system through chemotherapy and

radiation. To minimise the risk of cross-species infection, source animals for

xenotransplantation would probably need to be bred and raised in monitored,

biosecure facilities. The adverse effects on animals could give rise to particular

concern, especially since these source animals would need to be raised in isolation.

Is a xenotransplant intended to be permanent?

Not always. While organ transplants are generally intended to be permanent, some

kinds of transplanted cells may need to be replaced regularly in order to function well

and remain vital. Also, certain kinds of xenotransplants are not really true transplants

at all, because the animals organ or cells stay outside the patients body and are used

only in the short term, often as a bridge to transplant

How is scientific research on xenotransplantation carried out?

Research on xenotransplantation follows the same steps as research into any other

treatment for human disease. The first step involves studies that are carried out in the

laboratory and/or on animals. These kinds of studies are called pre-clinical trials.

They do not involve human patients.


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Many xenotransplantation studies have been carried out using laboratory animals. As

well as increasing scientists understanding of how well xenotransplantation works or

does not work, they have helped scientists understand how xenotransplantation might

be made safer and more successful for treating patients.

When pre-clinical studies show that the treatment is safe and effective in animals, the

new treatment or therapeutic product is tested on patients, under very controlled

conditions. This kind of testing is called a clinical trial or a clinical study. Usually,

only small numbers of patients are involved. They volunteer to take part in the

research and consent only after the potential risks and benefits of the study are fully

explained to them. In Canada and in many other countries, clinical studies must be

approved by a regulatory authority, usually a government health department or related

agency.vii

In Canada, Health Canada regulates clinical trials involving xenotransplants. As of

March 2000, no clinical trials have been approved by Health Canada.vii

Xenotourism

Xenotourists are people who, in desperation, travel overseas to countries that do

allow xenotransplantation, undergo the procedure and then return to their country

where xenotransplantation is prohibited. Because this may be done covertly there

would be no possibility of even minimal monitoring of such people.


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As a result, the management of these risks needs to be considered as these

xenotransplantation could be performed perhaps without the careful selection and

husbandry of source animals needed to reduce the risk of cross-species infection.

The kinds of public health measures needed to manage the movement of

xenotourists would be ethically controversial, and could include:

a requirement to disclose information about xenografts to medical authorities

a register of recipients

regular checks on the health of recipients, including taking blood samples

informing the recipient's relations and close contacts that they have had

xenografts

restricting the travel of recipients

(in extreme cases) quarantining recipients.

And, of course, anyone who has previously received an animal transplant will need

to be excluded from donating any of their organs or tissues in the future. This rule

will need to apply in all countries, including those that operate a presumed consent

system for organ donation. These measures might be justifiable if the recipients

accepted these as conditions for undergoing xenotransplantation.

Some believe that, given the difficulties of regulating xenotourism, it would be

better to have well-regulated xenotransplantation here than to take the risk of people

going to less well-regulated countries for xenotransplantation and bringing disease

back.


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Myths of Xenotransplantation

The myths of xenotransplantation as explored by Jeffrey Platt, MD, of the Mayo

Clinic in Rochester, Minnesota.The myths that were explored included

(1) xenotransplantation is a new idea,

(2) xenotransplantation will only happen far into the future,

(3) the major problem in xenotransplantation is infectious disease, and

(4) the safest approach to xenotransplantation is continued bench research without

clinical trials.

Dr. Platt contended that xenotransplantation is not a new idea and it is not only for the

future. Xenotransplantation was first attempted in the early years of the 20th century.

On a limited scale, xenotransplantion is occurring today. Among the xenotransplants

currently used are fetal porcine central nervous system tissue for the treatment of

Parkinson's disease and porcine skin for the treatment of burns. Porcine livers are also

used via ex vivo perfusion to cleanse the blood of human patients with liver failure.

While it is commonly thought that the major problem in xenotransplantation is

infectious disease, it may well be that xenotransplantation will contribute a solution to

the problem of infectious disease. As opposed to an allograft, it may be possible to

define the infectious disease risk of transplantation with a xenograft. More to the

point, it is possible that a xenograft could be used to avert reinfection of a transplanted

organ that would inevitably occur if a human organ were used.

Finally, Dr. Platt argued that the safest approach to xenotransplantation is not

continued research without clinical trials. First, bench research may have its own


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risks. Xenogeneic tissues, even human tissues, are commonly transplanted into

immunodeficient rodents, and it is possible that recombination of viral genes could

lead to emergence of a human pathogen. Yet, this possibility is not monitored, as it

would be in clinical xenotransplantation research. In addition, clinical

xenotransplantation could be viewed as a model system to study the potential

pathogenicity of animal viruses. The assessment of such pathogenicity could allow the

development of measures to prevent entry of pathogenic virus into humans through

domestic or commercial endeavors.

Future Directions: Genetic Engineering

Genetic engineering could be used to generate sources of xenografts able to resist

tissue injury and rejection. Typically, xenoreactive antibodies, complement,

macrophages, and the natural killer cells of a xenograft recipient may react with the

xenograft to trigger activation of endothelial cells. Instead of inhibiting thrombosis

and inflammation, the endothelial cells that are activated promote thrombosis and

inflammation, and undergo apoptosis. Most efforts in the genetic engineering of pigs

as a source for xenografts have focused on abrogating the initiation of xenotransplant

rejection, thus decreasing the expression of antigen and bringing about the expression

of complement regulatory proteins. However, Dr. Soares asserted that the use of

genetic engineering to modulate endothelial cell activation could potentially be of

value.

A common event in the activation of endothelial cells is the induction of apoptosis.

Accordingly, efforts might be made to inhibit apoptosis. Several genes believed to


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function in this regard, ie, A20 and heme oxygenase-1 (HO-1), have been explored.

The potential contributions of HO-1 were discussed.

HO-1 degrades heme, a prosthetic group of proteins such as hemoglobin, to yield

carbon monoxide and iron. The organs from knockout mice lacking HO-1 are rapidly

rejected by xenogeneic recipients. Similarly, treatment of recipients with tin

protoporphyrin, which inhibits HO-1, leads to rapid rejection of xenografts. These

results suggested that HO-1 is protective against xenograft injury, especially

reperfusion injury because the active product of HO-1 may be carbon monoxide.

Treatment of xenograft recipients with carbon monoxide inhibits platelet aggregation

and monocyte activation, and appears to prolong the survival of xenografts.

US FDA Guidelines

Purpose

To provide a comprehensive approach for the regulation of xenotransplantation that

addresses the potential public health safety issues associated with xenotransplantation

and to provide guidance to sponsors, manufacturers and investigators regarding

xenotransplantation product safety and clinical trial design and monitoring.

Regulatory Policy and Guidance Development

Xenotransplantation products are subject to regulation by the FDA (e.g., under section

351 of the Public Health Service Act (42 U.S.C. 262) and the Federal Food, Drug and


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Cosmetic Act (21 U.S.C. 321 et. seq.)). In accordance with the statutory provisions

governing premarket development, xenotransplantation products are subject to FDA

review and approval. Investigators of such products should obtain FDA review of

proposed xenotransplantation clinical trials before proceeding.

FDA intends to publish guidance documents to assist sponsors and investigators

interested in conducting clinical trials in the field of xenotransplantation. These

documents will provide reasonably detailed and timely pragmatic guidance to

sponsors regarding xenotransplantation product safety and clinical trial development,

including specific recommendations for the procurement and screening qualification

of source animals, the manufacture and testing of xenotransplantation products,

preclinical testing, clinical trial design, and post-transplant monitoring/surveillance of

recipients of xenotransplantation products. FDA will provide notice and invite public

comment on these draft documents.

Application Review

Currently, several xenotransplantation clinical trials are under FDA oversight. In

order to respond efficiently to the data submitted to the agency in xenotransplantation

product applications, CBER has developed a mechanism for the systematic and

regular evaluation of the scientific and clinical literature relevant to

xenotransplantation and submissions to xenotransplantation product files. A

Xenotransplantation Product Reviewer Working Group, consisting of the review staff

responsible for the review of xenotransplantation submissions (clinical, product, and

pharm-tox reviewers, and veterinary staff) meets regularly to:


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discuss application of the principles set forth in relevant FDA regulations,

review and discuss current scientific and medical data and literature relevant

to xenotransplantation

review and discuss the current status of xenotransplantation applications

submitted to the agency

discuss the unique issues that these products may present

highlight areas of concern where further expert advice may be needed

This provides for a consistent review of xenotransplantation applications and should

facilitate the recognition of patterns, trends, and/or common problems that may be

associated with xenotransplantation products. This data evaluation and management

process is linked to the regulatory process and is applied during regulatory decision

making and policy design.

Scientific Investigations and Research

Research conducted at CBER has been instrumental to the understanding of safety

issues associated with xenotransplantation. CBER is engaged in scientific

investigations of known and emerging infectious agents and immunological hurdles

that will need to be overcome for the safe and effective use of xenotransplantation

products. The results of these studies have helped CBER in its safety assessment

including assessment of risk and the development of diagnostic methods and

standards. CBER researchers continue to develop assays appropriate for safety

monitoring and are working with sponsors and collaborating with other government

scientists in this development.


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These research scientists apply their unique expertise in performing regulatory review

and to the development of regulatory policy for xenotransplantation. Working groups

analyze data and events, develop and propose strategies for appropriate studies, risk

assessment, prevention, communication and agency response or regulatory action

(e.g., request for more data from sponsors, request for particular product assays,

placement of clinical hold) and discuss these proposals and strategies with advisory

committees when needed or at public meetings as appropriate.

This process provides improved coordination of efforts to address the potential safety

issues associated with xenotransplantation and provides a mechanism whereby

infectious agents and diseases that may be associated with xenotransplantation can be

rapidly recognized and an appropriate and timely regulatory response generated.

Public Discussion and Consultation

FDA has sponsored, planned or participated in numerous open public meetings and

orkshops, both domestic and international that focused in whole or in part on

xenotransplantation. These activities are essential for both sharing information and

receiving public input on issues relevant to xenotransplantation.i

In 1997, FDA formed a Xenotransplantation Subcommittee of the Biological

Response Modifiers Advisory Committee (BRMAC) as an ongoing mechanism for

open discussions of the scientific, medical, social, and ethical issues and the public

health concerns raised by xenotransplantation, as well as specific ongoing and

proposed protocols. Open public meetings update the committee and the general


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public on the issues associated with xenotransplantation and the development of FDA

policy regarding the regulation of xenotransplantation products.i

DHHS Xenotransplantation Committee

The DHHS Xenotransplantation Committee oversees departmental initiatives to

address the public health issues raised by xenotransplantation. It is comprised of

members from FDA, CDC, NIH and HRSA and is administered through the Office of

the Assistant Secretary for Planning and Evaluation/Office of Science Policy.i

DHHS has established the Secretary's Advisory Committee on Xenotransplantation

(SACX), which will consider the full range of complex scientific, medical, social,

ethical, and public health concerns raised by xenotransplantation, and make

recommendations to the Secretary on policy and procedures.i

Guidance for Industry

Source Animal, Product, Preclinical, and Clinical Issues Concerning the Use of

Xenotransplantation Products in Humansi

I. REGULATORY RESPONSIBILITYII. SOURCE ANIMAL CHARACTERIZATION

A. General ConsiderationsB. Animal Welfare ConcernsC. Animal Origin


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D. Animal Health and HusbandryE. Harvest of Nonhuman Live Cells, Tissues or Organs for Use in

Producing Xenotransplantation Products

F. Source Animal History for Xenogeneic Cell LinesG. Disposal of Animals and Use of Byproducts

III. CHARACTERIZATION OF XENOTRANSPLANTATION PRODUCTSA. General ConsiderationsB. Considerations for Classes of Xenotransplantation Products

IV. MICROBIOLOGICAL TESTING OF XENOTRANSPLANTATIONPRODUCTS

A. General ConsiderationsB. Considerations for Classes of Xenotransplantation ProductsC. Assay Design for the Detection of Infectious Agents

V. MANUFACTURING AND PROCESS-RELATED GMPCONSIDERATIONS FOR HARVEST AND PROCESSING OF

XENOTRANSPLANTATION PRODUCTS

A. General ConsiderationsB. Contamination/Cross-Contamination PrecautionsC. Validation and Qualification

VI. PRECLINICAL CONSIDERATIONS FOR XENOTRANSPLANTATIONA. General ConsiderationsB. Issues Related to Infectious AgentsC. Xenotransplantation Product-Host InteractionsD. Considerations for the Use of Heterogeneous Xenotransplantation

Products


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E. In Vitro and In Vivo Tumorigenicity Models for Xenotransplantation

Products Intended for Transplantation

F.

Combinations of Xenotransplantation Products with Devices

VII. CLINICAL ISSUES IN XENOTRANSPLANTATIONA. General ConsiderationsB. Clinical Protocol ReviewC. Xenotransplantation SiteD. Criteria for Patient SelectionE. Risk/Benefit AssessmentF. Screening for Infectious AgentsG. Patient Follow-upH. Archiving of Patient Plasma and Tissue SpecimensI. Health Records and Data ManagementJ. Informed Consent

K. Responsibility of the Sponsor in Informing the Patient of NewScientific Information


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Summary

The tremendous need and urgency for donor organs have provided a great stimulus to

creative scientific investigation in the field of xenotransplantation. The success in

overcoming the immediate barrier of hyperacute rejection has driven some

investigators to consider clinical application of transgenic pig-to-human

xenotransplantation. The lessons learned from the series of presentations at this

meeting include a cautionary note regarding the use of allosensitized humans as

appropriate recipients of pig xenografts; the need for further understanding of the

immune mechanisms involved in acute vascular rejection, cellular rejection, and

chronic rejection; and the complexities in immunomodulation of the xenograft. Every

animal model has its limitation, and the need to proceed to the clinical arena with

novel therapies must take into consideration the limitations imposed by the particular

animal model tested. Some questions will eventually only be answered by

appropriately selected clinical application.


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G l o s s a r y o f W o r d s a n d P h r a s e s

Allosensitization - Exposure to an alloantigen that induces immunologic memory

cells.

Bridge to transplant - a short term procedure using an extra-corporeal

xenotransplant that could buy a patient time until a human organ becomes available.

Clinical trials, clinical studies - research studies on new drugs or treatments which

are carried out with human patients. The patients volunteer for this research and

consent only after the potential risks and benefits of the study are fully explained to

them.

Endogenous retrovirus - many species of mammals, including humans, have certain

kinds of viruses or fragments of viruses in their cells. These are embedded in their

DNA and are passed from one generation to the next, usually without causing any

harm in the host species, for example PERVs.

Extra-corporeal - outside the body; sometimes called ex vivo. The term refers to

certain kinds of xenotransplants which are not true transplants at all, because the

animal organ or cells are connected to but stay outside the patients body.

Immunosuppressive drugs - drugs which reduce the effectiveness of a patients

normal immune system. They are given to patients who have received (human)

transplants so that their immune system wont reject the new, slightly mis-matched

organ.


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Immune rejection - a patients immune system will normally accept a transplanted

organ (from either a human or an animal donor) only if its a fairly close match with

its own tissue type. Antibodies and special white blood cells will attack the foreign

cells in the transplant, and damage it so much that it cannot work properly. When this

happens, doctors say that the patients immune system has rejected the new organ. It

usually has to be removed.

Medical device - a combination of biological materials such as animal cells, and other

materials such as an artificial membrane or filter system.

Microorganisms - very small living organisms such as viruses, bacteria or fungi that

may cause infection and disease. Commonly known as germs or infectious agents.

Non-human primates - the group of animals which is biologically most similar to

humans, including chimpanzees, baboons and monkeys.

PERVs- stands for pig (or porcine) endogenous retroviruses. PERVs have been a part

of all pig cells for thousands of years. They are not active and are normally harmless

to the pigs.

Pre-clinical studies - research studies on new drugs or treatments which do NOT use

human patients. Pre-clinical studies are confined to laboratory or animal studies.

Risk to third parties - refers to the indirect risk that people other than the

xenotransplant patient might be exposed to. (The pig and the xenotransplant patient

are considered to be the first two parties in the risk equation.) The risk to third parties

refers to the possibility that a xenotransplant patient might be infected with an


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infectious agent, such as a virus, from the xenotransplant, and that this infection might

be passed on to other people through intimate or daily contact.

Species specific microorganisms that can only infect one species of animalfor

example pigs or humans.

Transgenic animals - animals that contain a gene (or genes) from another kind of

living organism, for example, pigs which contain a human gene.

Xeno - is used as a prefix and means strange or foreign.

Xenotransplant or xenograft - the living animal material that is transplanted into

humans in xenotransplantation.

Xenotransplantation - a transplant procedure in which a human patient receives an

organ (such as a kidney or liver) or living cells (such as brain cells) that come from a

healthy animal instead of from a human donor. The same term can be used to describe

a transplant between any two different species of animals.

Xenozoonosis (plural xenozoonoses) - zoonotic diseases that might be passed from

animals to people by means of a xenotransplant.

Zoonoses, zoonotic infections - microorganisms carried by animals, which can also

infect and/or cause diseases in humans under normal (non-transplant) conditions.


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1

U.S. FDA 2006 action plan for xenotransplantation

2Health Canada, Therapeutics Products Programme, December 2000

3Reemtisma K, MC Cracken BH, Schlegel JU.Renal heterotransplant in man.Annals

of Surgey 1964 ;160:384-408

4Beauchamp, Gilles. Ethics and xenotransplantation. Canadian Journal of Surgery,

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5Bailey LL, Nehlsen-Cannarella SL, Concepcion W, Jolley WB. Baboon-to-human

cardiac xenotransplantation in a neonate.JAMA 1985;254(23):3321-9.

6Jurkiewicz MJ. Nobel Laureate: Joseph E. Murray, clinical surgeon, scientist,

teacher.Arch Surg 1990:125(11):1423-4.

7Public consultation on xenotransplantation

8Xenotransplantation Discussion document [ Bioethics Committee, New Zealand ]

9Ye Y, Luo Y, Kobayashi T, Taniguchi S, Li S, Kosanke S, Secondary organ

allografting after a primary "bridging" xenotransplant. Transplantation1995;60(1):19-22.

10Makowka L, Cramer DV, Hoffman A, Breda M, Sher L, Eiras-Hreha G, et al. The

use of a pig liver xenograft for temporary support of a patient with fulminant hepatic

failure. Transplantation 1995;59:1654-9.

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13Shigeki Taniguchi, David KC Cooper, Clinical Xebotransplantation: past, present

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