xenotransplant vasculopathy

8/3/2019 Xenotransplant Vasculopathy

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NORMAN PAUL BRIFFA

DOCTOR OF MEDICINE, UNIVERSITY OF SHEFFIELD

November 2005

TRANSPLANTATION IMMUNOLOGY, DEPARTMENT OF

CARDIOTHORACIC SURGERY, STANFORD UNIVERSITY SCHOOL OF

MEDICINE, STANFORD, CA, USA


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Thesis Contents Page Number

Declaration 8

Acknowledgements 9

Publications/Presentations resulting from this Work. 10

Other Authors Publications on Xenograft Vasculopathy 12

Thesis Abstract 13

1. Chapter 1 Introduction.

1.1The need for organ (heart) transplants epidemiology of heart failure in

the UK 16

1.2Organ Shortage 18

1.3Xenotransplantation 19

1.3.1 Xenograft Rejection 22

1.3.1.1Hyperacute Rejection 23

1.3.1.2Acute Vascular Xenograft Rejection 28

1.3.1.3Xenograft Cellular Rejection 30

1.3.1.4Chronic Xenograft Rejection (Xenograft

Vasculopathy) 30

1.4Chronic Allograft Rejection 32

1.4.1 Alloimmunity 35

1.4.2 Nonimmune Factors 37

1.4.3 Pathophysiology 39

1.4.3.1Endothelial Cell Activation & Initial

Response To Injury. 40

1.4.3.2Alloimmune Response. 42

1.4.3.3Antidonor Antibodies 43

1.4.3.4Chronic Response To Injury 45


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1.4.3.4.1 T Lymphocytes 45

1.4.3.4.2 Macrophages 48

1.4.3.4.3 Cytokines And Growth Factors 48

1.4.3.4.4 Smooth Muscle Cells 49

1.4.3.4.5 Extracellular Matrix 50

1.5Immunosuppressive Agents 53

1.5.1 Structure and mode of Action 53

1.5.1.1T Cell Activation 53

1.5.1.2Cyclosporine A 55

1.5.1.3Leflunomide 57

1.5.2 Mechanisms Of The Effect Of Immunosuppressive Drugs In

Chronic Allograft Rejection (Vasculopathy) 65

1.5.3 Immunosuppressive Drugs In Xenograft Rejection 69

2 Chapter 2 Materials and Methods.2.1Known Models of Xenotransplant Vasculopathy 71

2.2Criteria for choosing model 72

2.3Rodents 73

2.4Organ Transplant Models Of Chronic Rejection

2.4.1 Hearts and Kidneys in Small Animals 73

2.4.2 Transplantation of Arteries 75

2.5Rodent Models Of Xenotransplant Rejection 78

2.5.1

Guinea Pig To Rat 78

2.5.2 Rat To Mouse 79

2.5.3 Mouse To Rat 79

2.5.4 Hamster To Rat 79

2.6Project Aims 84


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2.7Experimental Animals Used 86

2.7.1 The Nude Rat 86

2.8Reasons for Cyclosporine and Leflunomide 87

2.9Surgical Technique Of Chosen Model (Aortic Transplant) 88

2.10 Experimental Groups 91

2.11 Results - Ischaemic Times And Survival 91

2.12 Limitations of the Model 92

2.13 Histology and Computer Morphometry 92

2.13.1 Histology 92

2.13.2 Computer Morphometry 93

2.13.3 Adventitial Counts 94

2.13.4 Statistics 95

2.14 Cellular Response In Xenograft Vasculopathy Phenotypic Analysis

Of Cellular Infiltrate

2.14.1 Immunohistochemistry 97

2.15 Antibody Response To Aortic Xenografts 100

2.15.1 Flow Cytometry 101

2.15.2 Immunofluorescence. 105

2.15.3 Statistics 106

2.16 Complement Involvement in Xenograft Vaculopathy 108

2.16.1 Monoclonal Antibodies used. 109

2.16.2

Anti C3 110

2.16.3 2A1 Anti Rat C5-9 111

3 Chapter 3 Results Histology and Computer Morphometry3.1Literature Review 114

3.1.1 Xenograft vasculopathy 114


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3.1.2 Acute Xenograft Rejection - Hamster To Rat 116

3.1.3 Allograft Vasculopathy In Aortic Transplants 119

3.2Results Histology

3.2.1 Control Day 56 123

3.2.2 Group A (untreated) 123

3.2.3 Group B (athymic) 126

3.2.4 Group C (cyclosporine 10mg/kg) 128

3.2.5 Group D (leflunomide 10mg/kg/day). 131

3.2.6 Group E (cyclosporine 10mg/kg/day and leflunomide

10mg/kg/day) 132

3.3Results Computer Morphometry

3.3.1 Adventitial ratios (days 14 and 56) 134

3.3.2 Neointimal Ratios (days 14 and 56) 135

3.3.3 Adventitial Cell Counts (day 14) 138

3.4Discussion

3.4.1 Histology 139


3.5Limitations

3.5.1 Histology 143


4 Chapter 4 Results Cellular Response In Xenograft Vasculopathy

Phenotypic analysis Of Cellular Infiltrate

4.1Literature Review and Background

4.1.1 Xenograft Vasculopathy 145

4.1.2 Acute Xenograft Rejection 146

4.1.3 Allograft Vasculopathy 151


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4.2Results

4.2.1 Pan T Cells (R73) 159

4.2.2 CD8 Positive Cells (MRC OX-8) 161

4.2.3 CD4 positive cells (W3/25) 163

4.2.4 Macrophages (ED1) 166

4.2.5 B Cells (OX-33 CD45 positive Cells 169

4.2.6 NK Cells (3.2.3) 170

4.2.7 actin positive cells 170

4.3Discussion 174

4.4Limitations 179

5 Chapter 5 - Results - Antibody Response To Aortic Xenografts5.1Literature Review 181


5.1.2 Acute Xenograft Rejection (Hamster to Rat) 182


5.2Results

5.2.1 Antidonor Antibodies (Flow Cytometry)

5.2.1.1Preformed Antidonor Antibodies 191

5.2.1.2Changes over Time 192

5.2.1.314 and 56 day IgM and IgG comparisons 197

5.2.2 Histological/Antibody Correlations 202

5.2.3

Immunofluorescence 203

5.3Discussion

5.3.1 Antidonor Antibodies (Flow Cytometry) 204

5.3.2 Immunofluorescence 213


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5.4Limitations

5.4.1 Antidonor Antibodies (Flow Cytometry) 214

5.4.2 Immunofluorescence 214

6 Chapter 6 - Results - Complement Involvement In Xenograft Vaculopathy6.1Literature Review 216


6.1.2 Acute Xenograft Rejection (hamster to rat) 216


6.2Results

6.2.1 C3 221

6.2.2 C5-9 228

6.3Discussion 233

6.4Limitations 237

7 Chapter 7 General Discussion.

7.1Discussion 240

7.2Future Experiments 243

Appendix 1 244

Appendix 2 256

References 261


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Statement of Originality.

I confirm that this is an original work and all procedures (unless clearly stated in the

acknowledgement section) were performed by the author. The acknowledgement

section recognises those who provided assistance in all aspects of the production of

this thesis whether clerical or intellectual.


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Acknowledgements

Randi Shorthouse who assisted the author with preparing the slides for

immunohistochemistry and immunofluorescence and in preparing the hamster

thymus cells for use in antibody estimation by flow cytometry.

Jason Chan who taught the author and who helped in the flow cytometric

estimations of antidonor antibody

Helio Silva who carried out the High Performance Liquid Chromatography to

estimate the levels of the metabolite of leflunomide.

Margaret Billingham who gave advice on the histological appearance of graft

vascular disease

Timothy Brazelton who taught the author how to use the computer morphometry

system and how to carry out immunofluorescence photography

Randall E. Morris - Principal Investigator of the Project and Director of the

Transplantation Laboratory in the Department of Cardiothoracic Surgery at Stanford

University Medical Center.

Philip Chan - Reader at the University of Sheffield for advice and guidance in

preparation of this thesis.


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Publications/Presentations resulting from this Work.

1. Briffa NP, Shorthouse R, Chan J, Silva H, Billingham M, Brazelton T, etal. Histological and immunological characteristics of, and the effect of

immunosuppressive treatment on, xenograft vasculopathy.

Xenotransplantation 2004 Mar;11(2):149-59. (main paper with the

relevant data)

2. Ikonen TS, Briffa N, Gummert JF, Honda Y, Hayase M, Hausen B, et al.

Multidimensional assessment of graft vascular disease (GVD) in aortic

grafts by serial intravascular ultrasound in rhesus monkeys.

Transplantation 2000 Aug 15;70(3):420-9.

3. Gummert JF, Ikonen T, Briffa N, Honda Y, Hayase M, Perlroth J, et al. A

new large-animal model for research of graft vascular disease. Transplant

Proc 1998;30(8):4023.

4. Briffa N, Morris R. Immunosuppressive Drugs for the treatment and

prevention of transplant coronary artery disease. In: Rose M, editor.

Transplant Associated Coronary Artery Vasculopathy.: Landes

Bioscience, 1992.

5. Chronic Xenograft Rejection: Treatment with Cyclosporine and

Leflunomide Prevents IgM and IgG Xenoantibody Formation and Intimal

Thickening in Rat Recipients of Hamster Aortic Xenografts.N.Briffa,

R.Shorthouse, J.Chan, R.Morris THE 4

TH

INTERNATIONAL

CONGRESS OF XENOTRANSPLANTATION Nantes France

September 1997

6. Progressive Graft Vascular Disease (GVD) by Serial Intravascular

Ultrasound (IVUS) in a novel model of combined Auto- and allograft


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Transplantation in non-human Primates. N.Briffa, T.Ikonen, J.Gummert,

Y.Honda, J.Pelroth, M.Hayase, R.C.Robbins, C.Barlow, M.E.Billingham,

B.Hausen, P.G.Yock, R.E.Morris, Stanford University, Stanford,

California. THE ANNUAL MEETING OF THE INTERNATIONAL

SOCIETY OF HEART AND LUNG TRANSPLANTATION Chicago

April 1998.

7. Antibodies are notrequired for chronic rejection of vascularised

xenografts N.P.Briffa, R.Shorthouse, J.Chan, T.Brazelton, R.E.Morris.

THE ANNUAL MEETING OF THE INTERNATIONAL SOCIETY OF

HEART AND LUNG TRANSPLANTATIONSan Francisco, April 1999


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Other Authors Publications on Xenograft Vasculopathy

1. Reemtsma K, Pierson RNd, Marboe CC, Michler RE, Smith CR, Rose

EA, et al. Will atherosclerosis limit clinical xenografting?

Transplantation Proceedings 1987;19(4 Suppl 5):108-18.

2. DP OH, McManus RP, Komorowski R. Inhibition of chronic vascular

rejection in primate cardiac xenografts using mycophenolate mofetil.Ann

Thorac Surg1994;58(5):1311-5.

3. 5. Lin Y, Vandeputte M, Waer M. Effect of leflunomide and cyclosporine

on the occurrence of chronic xenograft lesions.Kidney Int Suppl

1995;52:S23-8.

4. Scheringa M, Buchner B, de Bruin RW, Geerling RA, Melief MJ, Mulder

AH, et al. Chronic rejection of concordant aortic xenografts in the

hamster-to-rat model. Transpl Immunol1996;4(3):192-7.

5. Xiao F, Shen J, Chong A, Liu W, Foster P, Blinder HL, et al. Control and

reversal of chronic xenograft rejection in hamster-to-rat cardiac

transplantation. Transplant Proc 1996;28(2):691-2.

6. Galili U. Significance of anti-Gal IgG in chronic xenograft rejection.

Transplant Proc 1999;31(1-2):940-1.


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Thesis Abstract.

The optimal treatment for patients with severe heart failure remains heart

transplantation because of the improved survival and quality of life of patients who

have undergone this procedure when compared to those on medical treatment. As

the number of available organs diminishes, and the number of potential recipients

increases, an alternative is urgently required.

The use of organs (hearts) from different species (xenotransplantation) is an

attractive option for several reasons. There are also many potential drawbacks to this

option not least of which is xenograft rejection.

If and when acute rejection is avoided, vascularized xenografts are susceptible to a

hitherto unstudied process of chronic rejection. We have used the hamster-to-rat

aortic transplant model to study the immunopathology of this phenomenon and to

determine whether it could be controlled or prevented by immunosuppressive

therapy.

Golden Syrian hamster aortas were transplanted into untreated Lewis rats, athymic

rats, and Lewis rats receiving cyclosporine (10 mg/kg), leflunomide (5, 10 or 15

mg/kg), or 10 mg/kg of both drugs.

Grafts were harvested on days 2, 7, 14, 28 and 56. Grafts were analysed using

computerized morphometry, immunohistochemistry (for phenotype of infiltrating

cells) and immunofluorescence for deposited antibody and complement. Blood was

taken on various days for the measurement of anti-hamster antibodies (flow

cytometry) and of the leflunomide metabolite A77 127. In untreated rats, by day 56,

transplanted aortas developed a cell-free media with a mature neointimal lesion

consisting of actin-positive cells, CD4 T cells, and macrophages.

There were large increases in circulating anti-hamster immunoglobulin M, (IgM),

and IgG, and there was IgG, C3 and C5a deposition in the grafts. In athymic


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recipients, the media architecture was preserved, and the changes in the neointima

and in anti-hamster IgM and IgG production were markedly abrogated. In

cyclosporine treated rats, all changes were markedly variable. In Lewis rats

receiving leflunomide, absence of circulating or deposited IgM did not prevent

neointimal formation by day 14. Antidonor antibody levels rose significantly in this

group after days 14-21. This was not explained by changes in the drug metabolite.

Combination treatment was the most effective at preventing neointimal formation

and humoral changes. Leflunomide monotherapy was the least effective. The

hamster-to-rat aortic transplant model is suitable for the study of xenograft

vasculopathy, the histological and serological changes of which are predominantly T-

cell dependant.


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Chapter 1

Introduction.


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1.1 The need for organ (heart) transplants epidemiology of heart failure in the

UK

Around 900,000 people in the UK today have heart failure - with almost as

many with damaged hearts but, as yet, no symptoms of heart failure1. Both the

incidence and prevalence of heart failure increase steeply with age, with the average

age at first diagnosis being 76 years2. While around 1 in 35 people aged 65-74 years

has heart failure, this increases to about 1 in 15 of those aged 75-84 years, and to just

over 1 in 7 in those aged 85 years and above3. The risk of heart failure is higher in

men than in women in all age groups, but there are more women than men with heart

failure due to population demographics1

.The most common cause of heart failure in

the UK is coronary artery disease - with many patients having suffered a myocardial

infarction in the past. A history of hypertension is also common, as is atrial

fibrillation. Heart damage of unknown cause - such as dilated cardiomyopathy -

accounts for just under 15% of cases under the age of 754. There are few reliable

data for different ethnic groups; it is likely that people of African or Afro-Caribbean

origin are more likely to develop heart failure due to hypertension rather than

coronary artery disease, whereas those of Asian origin have a greater risk of

developing heart failure due to coronary artery disease - often accompanied by

obesity and diabetes mellitus. Heart failure accounts for a total of 1 million inpatient

bed days - 2% of all NHS inpatient bed-days - and 5% of all emergency medical

admissions to hospital. Hospital admissions due to heart failure are projected to rise

by 50% over the next 25 years - largely due to the ageing of the population. It is

estimated that the total annual cost of heart failure to the NHS is around 716

million, or around 1.8% of the total NHS budget: approximately 70% of this total is

due to the costs of hospitalisation1,

5. The costs increase with disease severity, with

the healthcare costs for patients with the most severe symptoms between 8 and 30


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times greater than those with mild symptoms6.

Although the first heart transplant was performed in 1967, it was only after

the introduction of cyclosporine in 1980 that its clinical potential became apparent7,

8. The latest results from the International Society of Heart and Lung Transplantation

demonstrate that 84 percent and 78% of patients are alive one and three years

respectively after heart transplantation. In addition, most surviving patients have a

much improved quality of life9,

10. This contrasts sharply with a dismal prognosis of

patients with terminal heart failure -- albeit some improvements with the introduction

of new medical treatments such as ACE inhibitors, beta-blockers and spironolactone

11,

12,

13,

14,

15,

16,

17,

18.

Just under 40% of patients diagnosed with heart failure die within a year - but

thereafter the mortality is less than 10% per year2. Survival rates are similar to those

from cancer of the colon, and worse than those from cancer of the breast or prostate

19 20. Younger patients do better, as do patients with no other medical problems.

Although drugs have improved the prognosis of patients with heart failure, heart

transplantation remains the best treatment option for appropriate patients with end

stage heart failure.


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1.2 Organ Shortage.

Despite its success, not all suitable patients have been treated with heart

transplantation because of a shortage of human donor organs. This shortage applies

to all forms of solid organ transplants and is getting progressively worse.

Table 1a shows the discrepancy between transplants performed and number

of new patients placed on different organ specific waiting lists in the UK between

April 2002 and March 2003.

ORGANS

TRANSPLANTS

PERFORMED

NEW PATIENTS

GOING ONTO WAITING

LIST.

Cadaveric kidneys 1337 2462

Hearts 147 195

Lungs 117 187

Livers 692 793

Table 1.1

The discrepancy between transplants performed and number of new patients placed

on different organ specific waiting lists in the UK between April 2002 and March 2003.

This discrepancy still exists despite the use in recent years of organs from

marginal donors (older, function less than perfect)21-23

. Clearly an alternative to

allotransplanted organs is required.

In heart transplantation a possible alternative is the use of mechanical

devices. There are significant underlying problems with the mechanical device

alternative to cardiac allotransplantation. One of the specific problems associated

with the use of mechanical devices is the interaction of the recipients blood with the

artificial surface of the device. This creates a tendency to blood clotting and

thromboemboli. This is particularly devastating when the brain is the organ injured.


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Advances in the understanding of the surface of normal blood vessels and the

biology of performed and protein elements of blood coupled with advances in

materials technology may lead to a solution.

Two other problems associated with the use of mechanical devices are the provision

of an adequate energy supply to power the pumps and as mechanical devices are

foreign bodies, they are susceptible to infection from episodes of bacteraemia that

occur during everyday life.

1.3 Xenotransplantation

The non mechanical biological alternative would be the use of nonhuman

hearts that is xenotransplantation. Apart from the fact that nature has taken care of

the problems that limit the use of mechanical devices, there are many possible

advantages with the use of xenotransplants. Xenotransplantation would solve the

current problems of organ shortage. Figure 1.1 shows the advantages and

disadvantages of using xenotransplants for treatment of end-stage organ disease 24.


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Figure 1.1

Advantages and is advantages of using xenotransplants for treatment of end-stage organ disease

24.

An inexhaustible supply of tissue improves chances of developing

treatments for diseases such as diabetes mellitus or Parkinsons disease. There are

potential benefits over the use of allografts the possibility of manipulating donor

organs before transplantation is exciting as it offers opportunities to develop graft

specific immunosuppressive treatments. There are several drawbacks associated

with a possible clinical use of xenotransplantation. Of these the threat of

transmissible disease is the one that causes major public health concerns. These

fears have been heightened by data showing that co-culture of porcine and human

cell lines allows endogenous porcine retrovirus (PERV) to replicate 25, 26.

Several attempts have been made by US investigators to transplant organs


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from primates to man including highly publicized transplants from baboons27

,28

,29

.

In one case a chimpanzee kidney functioned without rejection for nine months

demonstrating the clinical potential of xenografts30

. Although on purely

immunological grounds primates would be the most suitable donors for human

beings, there are fundamental ethical and practical difficulties with the use of

chimpanzees or baboons. The UK Department of health advisory group on the ethics

of xenotransplantation has recommended that it would the ethically unacceptable to

use primates as source animals for xenotransplantation.

Pigs would make appropriate donors for several reasons (figure 1.2) 24.

Figure 1.2

Comparison of primates and pigs as potential organ donors.24

Although the question of disease transmission still needs to be addressed,

the principal drawback of xenotransplantation of porcine organs is immunological.


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1.3.1 Xenograft Rejection

In 1970, Calne divided xenografts rejection into concordant -- ie similar to that

which occurs unprimed allograft rejection and discordant similar to that which occurs

in primed allograft rejection. The type of rejection of xenogeneic tissue does depend

on whether the tissue is directly vascularised (whole organ) or whether

vascularisation occurs secondarily31

(See figures 1.3 and 1.4) and includes,

hyperacute, acute vascular, cellular and chronic.

Figure 1.3

Types of Xenografts31

Figure 1.4

Types of xenograft rejection 31


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1.3.1.1 Hyperacute Rejection

The hallmark of discordant rejection is hyperacute rejection. Both

hyperacute and acute vascular rejection have been well characterised in both small

animal32

,33

and pig to primate models34

,35

. Whether hyperacute rejection occurs in

xenotransplantation does not just depend on phylogenetic disparity between the two

species involved. Different organs show differing degrees of susceptibility to

hyperacute rejection with liver and lungs being relatively resistant. The two primary

immunological factors, which determine the susceptibility of xenogeneic organs to

hyperacute rejection, are recognition of endothelial cell antigens expressed on the

xenografts by xenoreactive natural antibodies in the circulation of the recipient and

incompatibility of complement regulatory proteins of the donor with the recipients

complement system. Preformed xenoreactive natural antibodies have been

characterized in both the hamster to rat and pig to primate models of

xenotransplantation36

,37

. Xenoreactive natural antibodies in humans and most of

nonhuman primates are predominantly directed against Gal13Gal 38, a

carbohydrate related structurally to blood Group A and B antigens (figure 1.5)39

and

which is present on cell-surface glycoproteins and glycolipids40

,41

.


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Figure 1.5

Molecular structures of blood groups A, B and the Gal Antigen.39

The synthesis of Gal13Gal is catalysed by 1,3-galactosyltransferase

the gene for which is carried by lower mammals and New World monkeys42

,43

.

Human beings, apes and old world monkeys do not express 1,3-

galactosyltransferase and therefore do not make Gal13Gal . This gene was

inactivated in catarrhines millions of years ago and is only present as a functionally

inactive pseudo gene. As humans, apes and old world monkeys do not express

Gal13Gal , they have developed antibodies during infancy and early childhood as

a result of colonization of the GI track by bacteria and viruses and other

microorganisms that express Gal epitopes. The importance of Gal13Gal as a

target of xenoreactive natural antibodies was first suggested by Goode et al44

who

showed that this molecule specifically blocks binding of natural antibodies in human

serum to pig cells. Sandrin45

showed that transfection of a cell line with cDNA

for1,3-galactosyltransferase confers susceptibility to lysis by human xenoreactive

natural antibodies.

Anti gal antibodies represent more than 90 percent of all xenoreactive

antibodies which bind to porcine organs44

. They are members of a broader family of

antibodies that include isohaemagglutinins and antibodies specific for other

Blood Group A Blood Group B Gal1-3Gal


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galactosyl saccharides 46, 47. Analysis of human B cells 48, 49 and circulating

immunoglobulin50

suggests that anti gal antibodies are derived from utilization of

the VH3 immunoglobulin gene family. The importance of these antibodies in

causing hyperacute rejection of xenografts has been demonstrated by showing that

depletion of these antibodies from nonhuman primates prevents hyperacute rejection

of porcine organs even if the complement system is intact51

.

Normal endothelium provides a tight seal and maintains an anticoagulant

environment within the vessel lumen. Anticoagulation is achieved by expression of

thrombomodulin and tethering of coagulation inhibitors such as antithrombin 3 and

tissue factor pathway inhibitor to surface glycosaminoglycans. The principal

paradigm of hyperacute rejection, first proposed by Platt and Bach in 1990 and is

now will widely accepted, places activation of xenograft endothelium at the centre of

the rejection process -- the third component of a triad of factors contributing to

hyperacute rejection, the other two being xenoreactive antibodies and complement

(figure 1.6)

Figure 1.6

The complement system

After revascularisation of xenografts, hyperacute rejection is initiated by

binding of xenoreactive natural antibodies, which activate complement via C1

(classical Pathway). The two most important recipient effector molecules are C5a


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and C5b-9 (MAC) (Figure 1.7), which initiate endothelial cell activation by direct

interaction.

Figure 1.7

Electron microscope images of MAC (membrane attack complex) lesions in liposomes. 52

Several changes immediately follow complement activation which

compromise normal endothelial function. Retraction of individual cells leads to loss

of vascular integrity and leakage of serum components into the tissues. Weibel-

Palade bodies, containing Von Willebrand factor and P selectin fuse with a cell

membrane leading to expression of these two molecules. Circulating leucocytes and

platelets are attracted and activated, and tissue factor, expressed on adluminal layers

of endothelium, is exposed and coagulation is initiated. Thrombosis proceeds in an

uncontrolled manner, partly because luminal coagulation inhibitors are lost as

thrombomodulin and glycosaminoglycan chains are cleaved from endothelial cells as

part of the activation process. (Figure 1.8)


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Figure 1.8

Stages of Hyperacute Rejection with Endothelial Activation. 24

The susceptibility of porcine organs to complement mediated damage is not

entirely a function of the binding of xenoreactive natural antibodies. The rapid

activation of complement in xenografts reflect in part, the failure of porcine decay

accelerating factor (DAF) and membrane cofactor protein (MCP), which ordinarily

regulate complement activation by disassociating and degrading C 3 convertase, and

CD 59 which prevents formation of the C 8 and C 9 complexes to effectively

controlled primate or human complement reactions53

,54

. The failure of these

complement regulatory proteins to control activation of the complement system of

the recipient might make the xenografts more susceptible to hyperacute rejection53

.

The importance of apparent complement control was first demonstrated by studies in

which expression of humans DAF and CD 59 or DAF alone, in transgenic pigs was

sufficient to prevented the hyperacute rejection of porcine organs transplanted into

primates 55, 56, 57. Once seen as the most daunting hurdle to xenotransplantation,

hyperacute rejection can now be circumvented by various therapeutic means

including antibody removal by extracorporeal circulation through columns

containing sugars with -gal residues, use of organs from pigs which are transgenic

for human regulators of complement, various complement inhibitors and the newer

anti B cell immunosuppressive drugs such as mycophenolate mofetil. In addition,


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various genetic manipulations are being tried to create a strain of pig with no or

diminished expression of Gal13Gal on its cells. Accordingly, hyperacute

rejection should no longer be viewed as a special challenge. However when

hyperacute rejection is averted, a xenograft becomes susceptible to a rejection

process known as acute vascular rejection.

1.3.1.2Acute Vascular Xenograft Rejection

Acute vascular rejection (sometimes referred to as delayed xenograft

rejection) can begin within 24 hours of reperfusion and leads to failure of the

xenograft within days to weeks following transplantation58

,59

. The pathological

features of the acute vascular rejection of xenografts, like those of the acute vascular

rejection of allografts, include endothelial swelling, ischaemia, and thrombosis. In

light of recent success in preventing hyperacute rejection, acute vascular rejection

looms as the next major hurdle to the end during survival of xenografts.

Accumulating evidence indicates that acute vascular rejection is initiated by

xenoreactive antibodies. Human subjects and experimental animals exposed to

xenogeneic tissues produce large amounts of xenoreactive antibodies60

. These

xenoreactive antibodies tend to have an increased ability to activate complement 50,

61. Some xenoreactive antibodies in human subjects exposed to porcine tissue appear

to be derived from the same immunoglobulin genes as natural antibodies48

, while

some mayreflect utilisation of gene segments not represented significantly in the

natural antibody repertoire. Of importance however is, that if the individual exposed

to porcine tissue is treated with immunosuppression, only limited amounts of

antibody against new determinants will be produced62

. The importance of anti

donor antibodies was demonstrated in a study in primate by Lin et al, who


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demonstrated that depletion of those antibodies can avert acute vascular rejection63

.

Studies in rodents showed that treatment with immunosuppressive agents that control

production of xenoreactive antibodies also might prevent acute vascular rejection.

The manifestation of acute vascular rejection is thought to be caused by

activation of graft endothelium59

. Acute vascular rejection is associated with

expression of adhesion molecules (i.e. E selectin and P selectin), inflammatory

cytokines64

and procoagulant molecules, such as tissue factor and plasminogen

activator inhibitor type 165

,66

. In addition to any direct effect of xenoreactive

antibodies, the activation of small amounts of the complement on porcine endothelial

cells induces endothelial cell activation. Complement induces the transcriptional

activation of IL-1a, which acts as any paracrine factor that stimulates and the

expression of pro thrombotic and pro inflammatory genes in activated endothelium

67. NK cells can activate xenografts endothelium independently of cytokines and

antibody68

. The function of natural killer cells is normally controlled by receptors

for MHC class 1 antigens 69, 70 and failure of natural killer cells to recognise

disparate MHC class 1 antigens may enhance their activity in xenotransplantation

rejection. Some natural killer cells also express Fc receptors, which may become

involved in the rejection of xenografts with circulating xenoreactive IgG. Consistent

with the potential involvement in the rejection of xenografts, natural killer cells have

been shown to accumulate in organs perfused by xenogeneic blood and in some

rejecting xenografts71

are thought to mediate endothelial cell injury.

Because of the preeminence of antibodies in the aetiology of acute vascular

rejection, it is likely that interventions that will limit the xenoantibody response will

control acute vascular rejection.


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1.3.1.2Xenograft Cellular Rejection

By cellular rejection, one means rejection that involves T-cells and does not require

antibodies. This therefore excludes antibody-dependent cytotoxicity that involves

macrophages and NK cells. Most of our knowledge on xenografts cellular rejection

(T-cell dependant) is based on findings derived from in vitro studies using

endothelial and other antigen-presenting cells and responder T-cells72

,73

. These

experiments have been done with small animal74

,75

and pig to primate models.

Other studies that have contributed to our understanding of xenograft cellular

rejection have been those involving the transplantation of indirectly vascularised

tissue or cells such as skin or islets76

. It was initially thought that in the discordant

pairing of pig to primate, the stimulatory and co-stimulatory molecular

incompatibility between porcine antigen-presenting cells and human responder T-

cells may result in cellular rejection which is less vigorous than the corresponding

allograft rejection. This is not, unsurprisingly, correct. Porcine aortic endothelial

cells are able to present antigen very effectively to responder human T-cells 72. What

differs from allograft rejection is the additional vigorous indirect response whereby

human antigen-presenting cells present a multitude of porcine antigens to human T-

cells. The resulting response is vigorous and resistant to normal doses of known

immunosuppressive drugs77

. The other way the reaction differs from

allotransplantation is that the efferent arm is less dependent on CD 8 cytotoxic T-

cells and seems to require CD 4 T-cells and macrophages78

. It is not known whether

the immunosuppression required to control this vigorous response is incompatible

with a normal life and therefore whether induction of tolerance would be required.

1.3.1.4 Chronic Xenograft Rejection (Xenograft Vasculopathy)

Although consistent long-term survival of primate recipients with life supporting

porcine cardiac or renal transplants has not yet been achieved, there are an increasing


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31

number of reported cases which have survived beyond 30 days79

-- albeit after

several immunomodulatory interventions and on large doses of potent

immunosuppressive drugs. Christopher McGregor and colleagues recently reported

to the International Society of Heart and Lung Transplantation (J Heart Lung

Transplant 2003; 22: S89) that, by combining the use of organs which express human

DAF with the administration of a soluble Gal glycoconjugate and other

immunosuppressive agents, the survival of pig hearts in baboons can be extended to

a median of 76 days. This represents a huge improvement on 5 to 10 years ago when

it was impossible to extend the survival of a pig organ in a primate beyond two

hours. In the most popular small animal model of xenotransplantation i.e. hamster to

rat, there are many series of long-standing survivors after orthotopic liver and kidney

and heterotopic hearts transplants on non-lethal immunosuppressive regimens80

,81

,

82. Because it is only recently that these early stages of xenograft rejection have been

overcome, it is perhaps not surprising that the phenomenon of chronic xenograft

rejection or xenograft vasculopathy is underinvestigated. Chronic xenograft

rejection does undoubtedly occur and has been reported in both small animal83

,82

and pig to primate84

and monkey to baboon concordant xenografts85

,86

. Keith

Reemstma, who is a pioneer of xenotransplantation, believes that xenograft

vasculopathy will severely limit the clinical usefulness of xenotransplantation87

. It is

therefore essential to improve our understanding of this phenomenon if we are to

have any chance of realizing the clinical potential of xenotransplantation. It is

important to understand firstly the clinical impact, immunopathology, and potential

causes of vasculopathy in allotransplantation.


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1.4 Chronic Allograft Rejection

Despite improving early results, it has become clear that clinical

transplantation has not achieved its goal as a long-term treatment. Acute rejection

rates in solid organ transplants have been dropping dramatically, especially in recent

years with the introduction of the newer immunosuppressive drugs. The long-term

outcome or annual rate of graft loss has only marginally improved over the last 20

years. One of the ways this can be expressed is the organs half life or median

survival i.e. the number of years it takes for half of all organs to fail. Figure 1.9

shows the half life for heart transplants performed between 1/1982 and 6/2001.

0

20

40

60

80

100

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Years

Survival(%)

Figure 1.9

Half-life of hearts transplanted between 1982 and 2001 (conditional half-life is calculated by

excluding the deaths that occur in the first year.)

Chronic transplant dysfunction or chronic rejection is a phenomenon in

solid organ transplants displaying a gradual deterioration of graft function more than

1 year after transplantation, eventually leading to graft failure, and which is

accompanied by characteristic histological features namely vasculopathy. It is the

commonest cause of graft loss and patient mortality in heart and lung transplantation,

Half-life = 9.3 yearsConditional Half-life = 12.0 years


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33

after one year.

Cardiac allograft vasculopathy is common after successful heart

transplantation. Figure 1.9 shows the freedom from vasculopathy or chronic

rejection after heart transplantation.

0

10

20

30

40

50

60

70

80

90

100

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5

Years

%FreefromCAVorFreedomfro

SevereRenalDysfunction

Freedom from CAV

Figure 1.10

Freedom from cardiac vasculopathy after heart transplantation.

The prevalence of angiographically detectable CAV out of 1, 3, and five

years after transplantation in cyclosporine treated patients is 14 percent, 37 percent,

50%, respectively, in the Stanford series88

. Cardiac allograft vasculopathy is the

major cause of graft failure and death in patients surviving more than one year after

transplantation89

,90

. The presence of angiographic CAV predicts a five times

greater relative risk of cardiac events91

. Clinical manifestations include myocardial

infarction, congestive heart failure, and sudden death. Survival is directly related to

the extent of CAV, with the poorest outcomes in patients with greater than a 70

percent stenosis by primary or secondary coronary artery or three-vessel coronary

artery disease , 92. In 1963, Porter at Al reported four human cadaveric kidney

allotransplants in which striking obliterative vascular lesions developed a few


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34

months after transplantation93

.

The cardinal histological feature of chronic rejection in all parenchymal

allografts is fibroproliferative endarteritis94

,95

,96

. Figure 1.11 shows in cross

section a human coronary artery in a transplanted heart (and subsequently explanted)

affected by neointimal proliferation which nearly occludes the vessel.

Figure 1.11

Cross section of a coronary artery from an explanted human heart transplant showing

neointimal proliferation.

The vascular lesions affect the whole length of the arteries in a patchy

pattern. There is concentric of myointimal proliferation resulting in fibrous

thickening and the characteristic onion skin appearance of the intima in small

arteries. Other findings include endothelial swelling, foam cell accumulation,

disruption of the internal elastic lamina, hyalinosis and medial thickening, and

presence of sub-endothelial T-lymphocyte and macrophages. In addition, persistent

focal perivascular inflammation is often seen.

The cause of chronic transplant dysfunction or chronic rejection remains ill

defined. Although it has been proposed that the phenomenon leading to chronic

dysfunction is the result of an ongoing host alloimmune response, nonalloimmune


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35

response is to injury such as ischaemia can cause or aggravate the process. (figure

1.12)

Figure 1.12

A cartoon depicting a summary of the several stages which lead to vascular remodeling in

arteries of a transplanted organ.

1.4.1 Alloimmunity

Several data indicate that chronic transplant dysfunction is the result of the

recipients immune response to incompatible donor tissue antigens. The mechanisms

by which alloimmune responses may lead to chronic transplant dysfunction include

histo incompatibility, acute rejection, sub optimal immunosuppression/non-

compliance and anti donor specific antibodies. Antigenic disparity in humans

between donor and host is associated with the occurrence of chronic transplant

dysfunction, as demonstrated in kidney heart and Lung transplant studies. Long-term

graft survival appears to be strongly correlated with the degree of histocompatibility

matching between donor and recipient97

,98

,99

. The graft survival studies from

single and multicentre studies alike show a strong correlation between acute rejection


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36

episodes and the lifespan of allograft100

,101

,102

. Matas et al showed in a group of

278 cadaveric kidney graft recipients that a single rejection episodes in the first post

transplant here reduces the estimated graft half-life from 33 years to 22 years,

whereas multiple rejections decrease the half life to less than five years103

. Although

acute rejection is not prerequisite for chronic transplant dysfunction in all patients, it

can be stated that at present, acute rejection is the most consistently identified

clinical risk factor for the occurrence of chronic transplant dysfunction.

A low dose of maintenance cyclosporine medication has been associated

with chronic transplant dysfunction in some studies 104, 105. Other studies have

shown no association. At five years post transplant, the percentage of patients who

are free of chronic dysfunction as demonstrated by biopsy was 86% for those using a

cyclosporine dosage of more than 5 mg /day kg per day against 77% for those on less

than 5 mg per kg per day. Additional evidence that chronic dysfunction may be

related to inadequate immunosuppression was provided by the histological studies of

Isoniemi et al 106, 107. They found that lesions were less apparent in patients given

protocols of triple vs double therapy immunosuppressive treatment. Many

experimental animal studies have shown that immunosuppressive agents including

cyclosporine are able to prevent the inflammatory response108

,109

,110

and inhibited

the generation of intima lesions during variable follow of periods.

Non-compliance may also indicate that chronic dysfunction results from

inadequate immunosuppression. In a study by the Minneapolis group, 34 percent of

patients were non-compliant, and this was associated with the late deterioration of

graft function111

.

Many studies have shown that following transplantation, the majority of

patients produce antibodies112

,113

,114

,115

. Both preformed antibodies reactive

against donor tissue and antibodies produced after transplantation against HLA class


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37

1 antigens and other antigens (endothelial cells, smooth muscle cells) are found. The

presence of antibodies correlates with the presence of allograft vascular disease in

clinical116

and preclinical studies. Paul et al demonstrated IgG antibodies against the

glomerular and tubular basement membrane, the mesangial cell, and endothelial cell

antigens in sera of rats with a kidney allograft with chronic dysfunction, whereas

such antibodies were not found in sera from animals that had received a syngeneic

graft.117

,118

. A connection is not however consistently found. Hosenpud et al found

no difference in the presence of IgM antibodies against endothelial cells of cardiac

grafts with or without chronic dysfunction 119.

1.4.2 Nonimmune Factors

In the late 1980s, attention was drawn to the fact that in the pre

immunosuppressive even human kidney transplant between identical twins

developed a late morphological changes. Two-thirds of these kidney isografts

developed glomerular lesions between two months and 16 years after transplantation

120. Nowadays, surgical injury and other non alloimmune specific factors related to

the donor and the graft have been associated with the development of chronic

transplant dysfunction121

,122

,123

. These risk factors include ischaemia, brain-death,

CMV infections, hyperlipidaemia, hypertension, age, gender, race, and the amount of

functional tissue, in kidney transplantation. In clinical transplantation it is still

unclear if ischaemia participates in the development of chronic transplant

dysfunction

124

. The UNOS Registry shows that preservation for more than 24 hours

significantly impaired late kidney graft survival rates compared to cold ischaemic

times between 0 and 24 hours. In cardiac transplants, a prolonged ischaemic time

was a risk factor for transplant arteriosclerosis125

. Experimental transplant studies

have demonstrated that ischaemia can cause chronic rejection like lesions in the


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38

absence of allogenicity126

. Many have demonstrated that rat kidney isografts

developed the same functional and morphological changes as allografts, including

vasculopathy127

,128

, albeit over a much longer time interval. It has been suggested

that in allografts the effect of ischaemia with chronic transplant dysfunction is

indirect by predisposing to acute rejection. Organ grafts with prolonged cold

ischaemia or with delayed graft function experience early acute rejection episodes

more often than grafts that functioned immediately129

,130

,131

,132

,133

.

There is a striking divergence in clinical long-term results between kidney

grafts from cadavers and those from living related and unrelated donors 134. It has

been suggested that brain death activates surface molecules on peripheral organs via

cytokines. In brain dead donors, increased serum cytokine levels are found before

organ procurement135

. In experimental models of brain death, peripheral organs

show increased endothelial cell activation136

,137

and an accelerated tempo of acute

rejection in organs from brain-dead animals is observed138

,139

.

Whilst infections with cytomegalovirus has shown to be related to chronic

transplant dysfunction in cardiac liver and lung transplantation140

,141

,142

,143

,144

, its

association with chronic transplant dysfunction in kidney transplants is not yet clear.

Experimentally, CMV infection has been identified as a promoter of chronic

transplant dysfunction in aorta kidney and heart transplants145

,146

. There are several

explanations as to how the virus may contribute to this process. A protein encoded

by the IE-2 region of human CMV contains sequence homology and shows

immunological cross reactivity with a conserved domain of HLA D-R., which could

potentially enhance the alloimmune response to donor antigens147

. In addition,

CMV encodes a glycoprotein homologous to the heavy chain of MHC class 1

antigen, which has been shown known to bind beta-2 microglobulin, a protein that is

normally associated with class 1 MHC antigens necessary for self not self


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39

recognition in the immune response148

. Active CMV infection is strongly associated

with expression of vascular cell adhesion molecule 1 on capillary endothelium in

heart transplant recipients, suggesting a possible role of this molecule in the adhesion

of lymphocytes and monocytes to vascular endothelium during infection149

.

Increased expression of the cytokines, platelet derived growth factor, a known

stimulant of smooth muscle cell growth factors150

and transforming growth factor

beta-1 have been shown after CMV infection in rat aortic allografts. Finally, smooth

muscle cells migrating to the intima in coronary stenosis express elevated levels of

human CMV immediate early antigen i.e. IE84 and tumour suppressor protein the 53,

suggesting that I E 84 in activates p 53 and predisposes smooth muscle cells to

increase growth as in malignant tumours151

. The importance of hyperlipidaemia in

the pathogenesis of chronic rejection has been demonstrated in both clinical and

animal studies152

,153

,154

,155

.

1.4.3 Pathophysiology

The common denominator of all chronic dysfunction after solid organ

transplant is the development of intimal hyperplasia. Immunohistochemistry of

allografts with chronic dysfunction has shown that T-cells and macrophages are the

predominant graft invading cell types, with an excess of CD 4 over CD 8 T-cells156

,

157,

158,

159,

154. Increased expression of adhesion molecules (ICAM-1, VCAM 1)

160

and MHC antigens161

are seen in allografts with chronic dysfunction. Complement

and IgM deposits are also seen in the area with intimal hyperplasia

161

,

162

. Increased

TGF beta expression is also found163

,164

,165

. The histological lesions, including

intima hyperplasia, infiltrating cells, up regulated adhesion molecules and cytokines

in transplanted organs with chronic rejection do not necessarily reflect an

alloimmune-mediated response.


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40

1.4.3.1 Endothelial Cell Activation & Initial Response To Injury.

Under normal conditions contact between leucocytes and vascular

endothelium is random if both cell types are inactive and at rest, the cell touch vessel

walls indiscriminately. In organ transplantation, the endothelial cells are activated by

ischaemia, surgical manipulation, and reperfusion injury, events inherent to the

procedure. After ischaemia and reperfusion endothelial cells produce O2 free

radicals predominantly via the xanthine -- oxidase pathway, which in vitro activates

and damages cells166

. Upon activation, the endothelial cells retract and release

increased amounts of cytokines IL 1, IL-6, IFN-gamma, TNF alpha, the chemokines

IL 8, macrophage chemo attractant protein (MCP 1), macrophage inflammatory

protein 1 alpha (MIP alpha) and MIP 1 beta, colony stimulating factors, and multiple

growth factors such as platelet-derived growth factors (PDGF), insulin growth factor

-- 1 (IGF -- 1), transforming growth factor beta (TGF beta) and pro thrombotic

molecules (tissue factor, plasminogen activator inhibitor). This secretion enhances

migration of neutrophils, monocytes and macrophages, and T-lymphocyte to the site

of injury167

. The release of cytokines also leads to upregulation of adhesion

molecules on the vascular endothelium168

.

The proinflammatory cytokines IL 1 and TNF alpha induce expression of

the adhesion molecules P and E selectin on the endothelium169

,168

, by which

circulating lymphocytes began to adhere to the surface carbohydrates170

,171

,172

.

Leucocytes are then triggered by the chemokines released by the endothelium, which

causes upregulation of the beta-2 integrin receptors LFA-1 and MAC 1 on their

surface. This enables a permanent adherence of leucocytes to the endothelial

adhesion molecules ICAM 1 and VCAM 1173

, the expression of which is induced by

the released cytokines IL 1 beta, IFN-gamma and TNF alpha174

,175

. Activated

complement also plays a role in the adhesion of new neutrophils and monocytes to


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41

endothelium176

. Finally extravasation of leucocytes to the extracellular matrix and

graft tissue occurs177

,139

. The first cells that infiltrate the graft are neutrophils.

They further aggravate inflammatory response through release of O2 free radicals

and inflammatory mediators, including platelet-activating like factors and

leukotrienes. Direct evidence that O2 free radicals, adhesion molecules, and

neutrophils play a role in the pathogenesis of chronic transplant dysfunction has

shown by interference studies178

,179

,180

. One recent study, revealed that carotid

allografts from donor mice deficient in ICAM 1 have a 52 percent reduction of

intimal hyperplasia compared to controls 181.

In addition to the increased expression of adhesion molecules on the

endothelium, after reperfusion of a transplanted organ, a dramatic up regulation of

MHC class I and II antigens on the endothelium occurs182

,183

, which appears to be

induced by release of cytokines IFN-gamma, TNF alpha and TNF beta184

,185

.

Alterations in tissue density of MHC class II antigens are likely to influence the

alloimmune response against the tissue 186. Parenchymal cells are also activated after

ischaemia. In non transplanted kidneys, MHC class I and II antigens are up

regulated on tubular epithelium183

,187

. Epithelial cells in lung autotransplants

showed a mild expression of MHC class II after cold ischaemia185

.

CD 4 T-lymphocyte infiltrate ischaemic allografts, isografts and non

transplanted organs188

183

,127

,128

. In addition, T-cell associated cytokines such as

IFN-gamma and TNF alpha are produced183

and blockade of CD 28 B 7 co-

stimulatory pathway decreased early influx of T-cells and expression of T-cell

associated cytokines189

. Cyclosporine was able to overcome the deleterious effects

of ischaemia in syngeneic transplants with a concomitant decrease in infiltrating CD

4 T-cells. In a liver ischaemia model, CD 4 T-cell deficient mice have significantly

less hepatic damage190

. This response to ischaemic injury is initially independent


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42

from other allogeneicity. Heemann et al have demonstrated that the pattern of

cellular infiltration and cytokine expression in both syngeneic and allogeneic cardiac

grafts was similar is not identical with in the first 48 to 72 hours after engraftment

191. Thus, a complete network of cytokines is already activated before allogeneic

reactions developed as a result of the transplant procedure.

1.4.3.2 Alloimmune Response.

The recognition of histoincompatible MHC Alloantigens will provoke an

alloimmune response. Class I antigens, constitutively expressed on nucleated cells,

interact with CD 8 cells, and class II antigens, constitutively expressed on lymphoid

cells and inducible on endothelial cells, macrophages and fibroblasts are recognized

by CD 4 cells. Intact foreign MHC molecules on donor cells may be directly

recognized by T-cells, either in combination with an allo peptide or a self peptide,

which results in an exceptionally strong immune response. Frequencies of T-cell

precursors that respond to alloantigens are 10 100 fold higher than for other

nominal antigens192

. In draining lymph nodes and spleen, allo reactive T-cells

recognize donor MHC indirectly, presented by self MHC molecules on recipient

antigen-presenting cells193

.

In allo recognition, the MHC antigens are bound to the T-cell receptor. For

activation of T-cells, a co-stimulatory pathway such as the CD 28 receptor on T-cells

with its ligand B7 and CD 40 with its T-cell based ligand CD 40L are necessary for

function and proliferation. The adhesion molecules ICAM 1, VCAM 1 and LFA-1

have also been shown to the stimulate T-cell activation. Once the CD 4 T-cell is

activated, a cascade of events amplifies the alloimmune response. Secreted IL 2

leads to clonal proliferation of alloreactive cells and stimulate CD 8 T-cells to

develop into mature cytotoxic effector cells. Release of cytokines such as IFN-


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43

gamma and TNF alpha may further increase the expression of adhesion molecules,

and MHC antigens on the endothelium, smooth muscle cells, and parenchymal cells.

IFN-gamma is also responsible for the activation of macrophages, which together

with CD 8 cells are cytotoxic to the graft cells, leading to acute graft failure, when no

immunosuppressive drugs are given. Inhibition of T-cell activation by cyclosporine,

FK 506, or anti IL 2 monoclonal antibodies do not prevent the development of

chronic transplant dysfunction in clinical transplantation, possibly due to the fact that

doses are too low to achieve this effect. In experimental models, continuously high

doses of cyclosporine A or blockade of CD 28/B 7 and CD 40/CD 40L costimulatory

pathway, decrease early infiltration and almost completely inhibit intimal

hyperplasia in murine aortic and cardiac allografts108

,194

,110

,195

. Evidence that CD

4 T-cell is involved in the genesis of intimal hyperplasia is elegantly exemplified by

Shi et al. Carotid allografts in mice that were genetically deficient for the CD 4 T-

cell developed intimal thickening to only 40 percent of that seen in controls196

.

1.4.3.3 Antidonor Antibodies

The cytokines IL 4, IL-6 and IL 10 released by activating CD 4 cells are

growth and differentiation factors for B cells. Activation of B cells may result in

maturation into plasma cells with allospecific antibody production. Since

immunoglobulin, complement, and antigen-antibody complexes have been found in

areas of intimal hyperplasia197

,198

,199

, humoral activity has long been thought to be

primarily responsible for chronic transplant dysfunction. A recent finding of up

regulated immunoglobulin J chain in arteriosclerotic lesions suggests the presence of

IgM or IgA producing plasma cells in such grafts200

. Donor specific antibodies are

found against HLA antigens, endothelial cells, mesangial cells, glomerular and

tubular basement membrane, smooth muscle cells and the nucleus201

,202

.


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44

The precise significance of antibody deposition that mitigates over time as

shown in many animal models203

,161

,162

remains to be established. In experiments

with SCID mice, which lacked T and B cell mediated cellular responses, passive

transfer of anti donor specific antibody was sufficient to produce graft

arteriosclerosis with a perivascular mononuclear cell infiltrate in long-standing

cardiac allografts204

. While some investigators found that the degree of intimal

hyperplasia in aortic and cardiac allografts in mice recipients with a defect of

humoral antibody production was comparable to that seen in immunocompetent mice

205, Russell et al showed that cardiac allografts in T-cell deficient mice did not

develop a fibroproliferative arteritis206

. These investigators also demonstrated that

in two donor- recipients mice combinations in which anti donor antibodies are

generally undetectable, intimal fibrosis was uncommon. These recipients became

capable of producing lesions in allograft at hearts when given anti donor class 1

antibody206

. Similar to Russells report, Shi et al showed that CD 4 cells, humoral

antibodies and macrophages together were necessary for intimal hyperplasia in a

mouse carotid allografts model. Arteries allografted into mice, deficient in both T-

cell receptors and humoral antibody, showed almost no neointima proliferation,

whereas those crafted into mice deficient only in humoral antibody, developed a

minimal intimal hyperplasia196

. The mechanism by which antibodies contribute to

chronic dysfunction is speculative. One recent study has shown that anti HLA

antibodies, when attached to the HLA class 1 antigen on cultured endothelial cells,

induced increase gene expression of bFGF receptor and ligand, and a 4 to 6 fold cell

proliferation as it does for smooth muscle cells207

. Marsh et al hypothesized that

IgG induces the accumulation, differentiation and subsequent cytokine production by

intimal macrophages via cross-linking of Fc gamma R thereby preventing apoptosis

of monocytes. Fc gamma R cross-linking induces the production of MCP 1 and IL 8


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45

which can promote both macrophage and lymphocyte accumulation208

,209

.

1.4.3.4. Chronic Response To Injury

1.4.3.4.1 T Lymphocytes

It is not clear why this response to the initial injury does not decrease over

time, as seen in normal healing process. In allografts, it is conceivable that the

alloantigens are responsible for an ongoing cellular and/or humoral response. T-cells

decline to relatively low numbers as the process enters its chronic phase. They and

their products may continue to provide a persisting low-grade immunological

response and ongoing subclinical injury to the grafts endothelium and parenchyma

121. Since there is a continuous supply of donor allopeptides processed and presented

by host professional antigen-presenting cells (dendritic cells, macrophages, B cells),

self MHC restricted T-cells may perpetuate a chronic alloimmune response. Suciu-

Foca and collaborators demonstrated persistent allo-peptide reactivity in patients

developing chronic transplant dysfunction 210, 211. The continued alloimmune

recognition in long-term graft recipients is clear by the presence of graft reactive

cytotoxic T splenocytes in long-term recipients of cardiac allografts212

,213

. The

response of naive CD 4 T-cells is slower than that of memory T-cells and maybe

initiated either by direct presentation of intact foreign MHC molecules by graft

derived dendritic cells or by indirect presentation of peptides derived from

alloantigens by host dendritic cells. Naive cells can only be activated within the

micro environment of the secondary lymphoid organ where they must expand before

migrating to the graft. Therefore alloantigens must travel from the graft to the

lymphoid organs to activate naive T-cells. Naive T-cells cannot reject an allograft in

the absence of secondary lymphoid organs214

Once naive T-cells have undergone

activation and clonal expansion, they can contribute to and may, in some cases


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46

dominate later response. Thus, indirect recognition maybe important for late events

such as chronic rejection reactions215

,216

.

Chronically or repeatedly activated CD 4 T-cells often polarise into subsets

that selectively and exclusively produce DTH (delayed type hypersensitivity)

eliciting cytokines such as IFN-gamma and LT, or a eosinophil activating cytokine

such as IL 4, IL-5, and IL 13 or immunosuppressive cytokine such as IL 10 and TGF

beta217

. Undifferentiated CD 4 effective cells that produce complex mixtures of

cytokines are called Th 0 cells, the IFN-gamma, LT producing subset of effector

cells are called Th 1 cells, the IL 4, IL-5, IL 13 producing subsets are called Th2

cells and the IL 10, TGF beta producing subsets are called Th3 or T regulatory (Tr)

cells. In chronic inflammation, one subset frequently comes to dominate the

response. This competition is partly explained by the fact that certain cytokines

made by one subset can often suppress differentiation of the other subsets215

. In

chronic CD 4 T-cell responses, it is unclear what factors determine whether Th 1,Th

2, or Th3 dominated responses will develop. Much recent focus has been placed on

cytokines in the environment in which naive or Th0 cells and encounter antigens.

This milieu depends in good measure on the innate immune system, comprised of

phagocytes, NK cells and plasma proteins218

. The best understood example of this is

that monocytes and macrophages can produce the cytokine IL 12, which stimulates

naive or Th0 CD 4 T-cells to secrete IFN-gamma and to differentiate into Th 1 cells

that are specialised to produce IFN-gamma and LT219

. Inflammatory cytokines such

as TNF augment IL 12 production. Therefore, the activation of the complement

cascade, which leads to TNF release from mast cells and other sources will favour IL

12 production and promote Th 1 dominated reactions. In transplantation,

endogenous activators of complement, such as those expressed in perioperative

ischaemia/reperfusion injury, may also favour IL 12 production and lead to Th 1


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47

dominant reactions. In other words, local endothelial cell injury can direct the

alloimmune response towards DTH, providing an explanation of the early ischaemia

reperfusion injury within the vessel wall can be a risk factor for DTH and chronic

vascular rejection. This concept is illustrated in figure 1.13.

Figure 1.13

Cartoon depicting the Immune Modulation Model.

Anti donor specific antibodies may also maintain a chronic immunological

injury. Alloantibodies, which can cause acute vascular rejection, similarly cause

complement activation within the vessel wall and may similarly favour development

of DTH against graft vessels. There is good evidence that donor MHC class I and II

antigens play a role in the formation of the neointimal lesion characteristic of chronic

rejection. Carotid allografts from donor mice deficient in MHC class II molecules

showed a reduction of intimal hyperplasia by 33%181

. The absence of such a

continuous allogeneic stimulus in syngeneic transplants explains the much more

rapid development of the lesion of chronic rejection in allografts. The strength of the

initial trigger, the length of the trigger and presence of additional factors determine


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48

the onset and the pace of progress of irreversible chronic lesions.

1.4.3.4.2 Macrophages

Activated T-cells produce, amongst others the cytokine RANTES (regulated

upon activation, normal T-cells expressed and secreted), a macrophage

chemoattractant220

. Other cytokines, such as IL8, MCP 1 and osteopontin released

by interstitial cells and smooth muscle cells are chemotactic for macrophages as well.

Up regulated adhesion molecules contribute to their localisation to areas of injury.

Macrophages invade the graft and become activated by IFN-gamma. The continuous

presence, the activated state and the upregulation of macrophage associated

cytokines in long-term allografts with chronic dysfunction and in other chronic

diseases with fibrotic features, suggest a pivotal role for the macrophage221

,222

,158

.

The importance of macrophages was demonstrated by the prevention of chronic

vascular rejection by treatment with gamma lactone, a synthetic inhibitor of

macrophage activity in a rat renal allograft model 221 and by the observation that

carotid allografts in mice deficient and macrophages, develop only slight intimal

hyperplasia196

. Activated macrophages produce a number of cytokines including

TNF alpha, IL 1 beta, PDGF, the FGF, and TGF beta. These perpetuate and amplify

the fibrogenic signals.

1.4.3.4.3 Cytokines And Growth Factors.

Cytokines and growth factors play an important role in the chronic phase. They have

profound effects on cells of the graft and on the immune system. Cytokines and

growth factors are pleiotropic with biological effects on many cell subpopulations.

They are regulated through autocrine, paracrine or systemic pathways, and there is a

great deal of redundancy in the cytokine networks. The advent of transgenic and


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knockout technology has allowed the dissection of molecular pathways causally

involved in allograft arteriosclerosis223

. The redundancy of the cytokine system has

been stressed by gene knockout experiments. IL-4 is not necessary for the

development of graft coronary arteriosclerosis, nor does it absence augment the

development of vascular lesions. In addition, TNF alpha -- R1 deficiency in either

donor heart or recipient does not abrogate development of graft arteriosclerosis224

.

The increased expression of TGF beta has been linked to transplant arteriosclerosis

both by clinical and experimental studies, and transfection of TGF beta to the kidney

leads to increased accumulation of the extracellular matrix and glomerulosclerosis

225. Interestingly, cardiac allografts in TGF beta deficient recipients developed

significantly more intimal hyperplasia can controls226

. In 1989, IFN-gamma has

already been postulated by Libby et al to play a central role in chronic vascular

rejection because of its effects on T-cells and macrophages227

. The availability of

IFN-gamma deficient mice permitted this group to test the contribution of IFN-

gamma in the development of chronic vascular rejection 228, 229. Cardiac allografts in

IFN-gamma deficient mice develop only minimal transplant arteriosclerosis

compared to controls. In addition similar diminution of graft arteriosclerosis was

found after the administration of IFN-gamma neutralising antibodies in normal rats.

1.4.3.4.4 Smooth Muscle Cells

Once the endothelial cells are injured, the secreted cytokines, IL 1, PDGF,

IGF-I, TGF beta and b FGF and metabolic products such as prostaglandin, the nitric

oxide and oxidised low-density lipoproteins induce smooth muscle cell proliferation,

as suggested by Ross230

. Activated T-cells and macrophages often in close

proximity with the replicating smooth muscle cells, also produce these factors.

Platelets deposited along the injured vascular wall contribute by secreting PDGF,


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EGF, TGF beta and thromboxane-A2. When smooth muscle cells migrated into the

intima, they transform their phenotype from contractile to secretory and the cells

become capable of replication230

,231

. Smooth muscle cells produce many of these

growth factors and may generate similar autocrine or paracrine loops for cell

replication, a seen in classical atherosclerosis230

. These factors also may modulate

extracellular matrix synthesis, angiogenesis, and leucocyte adhesion. Activated

smooth muscle cells can express MHC class I and II and may act as antigen-

presenting cells.

1.4.3.4.5 Extracellular Matrix

As the endothelium is damaged, the underlying extracellular matrix can

become activated and molecules can act as co-stimulators for leucocytes to facilitate

recruitment and extravasation. Exposed collagen and fibronectin may act as

costimulators for activated CD 4 T-cells232

,233

. After activation by antigens, T-cells

synthesise heparanase, which facilitates cellular migration through tissue 234. The

cleavage of heparin and sulphate by this enzyme also activates and releases

fibrogenic growth factors, such as basic fibroblast growth factor in the extracellular

matrix235

. TGF beta, produced by the activated T-cells and macrophages, stimulates

the production of extracellular matrix molecules and inhibits the matrix degrading

enzymes (metalloproteinases). The predominance of fibrogenic over antifibrogenic

influences in the formation of the extra cellular matrix is illustrated in figure 1.14


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Figure 1.14

The Predominance Of Fibrogenic Over Antifibrogenic Influences In The Formation Of The

Extra Cellular Matrix


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Figure 1.15

A simplified cartoon showing how all the systems and networks described in the text interact

with one other.


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1.5 Immunosuppressive Agents.

1.5.1 Structure and Mode of Action

1.5.1.1 T cell Activation

The cellular arm of the immune response mediates both acute and chronic allograft

rejection. The antigen-specific T-cell receptor (TcR)-CD3 complex on the surface of

T lymphocytes recognizes antigen (peptide) binding in the groove formed by major

histocompatibility complex (MHC) proteins. T-cell activation results in T-cell

recognition of either foreign antigen or foreign MHC proteins, both of which may be

expressed by allografted cells 236. Both CD4+ T cells that secrete lymphokines and

CD8+ T cells that mediate cytotoxicity participate in the immune response to

allografted cells. T-cell immunosuppression, therefore, is one necessary component

of effective transplantation regimens.

Ligation of the TcR-CD3 complex, which may be mimicked by

crosslinking with anti-TcR or anti-CD3 monoclonal antibodies (mAbs), results in a

number of early biochemical signals 237. These signaling pathways involve a number

of intermediates and result, finally, in the induction of gene transcription, including

those genes encoding lymphokines and their receptors238

. While the final pathways

leading to lymphokine gene transcription are not fully delineated, a number of

required steps are known. At least two pathways, one leading to the activation ofras

and one involving a calcium-dependent event leading to the activation of the serine-

threonine phosphatase calcineurin, appear to cooperate for initiation of lymphokine

gene transcription.

Calcium is mobilized from the extracellular environment and a rise in intracellular

calcium concentration is a well-characterized event following TcR ligation. A rise in

intracellular calcium stimulates a number of calmodulin-dependent events; calcium is

a necessary cofactor in the activation of the calmodulin-dependent serine/threonine


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phosphatase calcineurin, which appears, in turn, to be required for the

dephosphorylation (and activation) of specific substrates. In T cells one substrate of

calcineurin appears to be the cytoplasmic component of the heterodimeric complex

of nuclear factor of activated T cells (NF-AT), a transcription factor found in the

promoter of a number of lymphokine genes. The other component of NF-AT is a

heterodimer comprised of fos and jun transcription factor family members, which are

activated by ras. Therefore, the cooperative activation ofras and of calcium-

calmodulin dependent calcineurin activity, both impact directly on the transcriptional

regulation of lymphokine-gene expression . ( see Figure 1.16)

Figure 1.16

Molecular Pathway of T cell Activation and Site of CsA action. 239


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1.5.1.2 Cyclosporine

The signal transduction pathways recruited following TcR-CD3 ligation

have been elucidated in part by studies directed at understanding the molecular

mechanism of immunosuppressant action.

Cyclosporine A (CsA) is a cyclic undecapeptide (See Figure 1.17), which

completely inhibits lymphokine gene activation, including interleukin IL-2, IL-3, IL-

4, TNF alpha, and GM-GSF among others240

,241

,242

,243

,244

.

Figure 1.17

The cyclic undecapeptide structure of Cyclosporine A239

Exogenous lymphokine added to mitogen-activated CsA T-cell cultures

restores proliferation, suggesting that inhibition of T-cell proliferation by the drug is

proximal to this step. CsA therefore inhibits T-cell pathways associated with an early

rise in intracellular calcium concentration and T-cell proliferation.

The ability of CsA to inhibit lymphocyte proliferation and lymphokine

production require drug binding to specific intracellular receptors known as

immunophilins245

,240

,246

. The biological function of immunophilins in the absence


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of drugs has been the subject of recent study, and they appear to play a role in protein

folding and protein trafficking in the cell

The immunophilins that CsA binds to is a family of receptors termed

cyclophilins (CyPs)246

.CyP catalyzes cis-trans intraconversion of peptidyl-prolyl

bonds, a property termed rotamase or isomerase activity. Inhibition of this common

enzymatic activity is not sufficient to explain the drugs effect of inhibition of T-cell

function247

,248

,249

.The complex formed by drug binding to immunophilin

(CsA/CyP complex) behaves as a novel biological entity that associates with high

affinity with a molecular target(s) within the cell, found to be calcineurin 250, 251

In vitro, the complex of CyP/CsA inhibites calcineurin phosphatase activity251

, and

in vivo treatment of T cells with CsA led to a dose-dependent inhibition of

calcineurin activity which correlates with inhibition of lymphokine production in

these cells252

,253

,254

. Over-expression of native or constitutively active calcineurin

modified the ability of CsA to inhibit lymphokine gene induction255

,256

,257

,258

,259

,

260. In addition to inhibiting T-cell lymphokine gene transcription CsA has been

shown to inhibit other T-cell activation processes. CsA inhibits apoptosis, or the

induction of activation-induced cell death in vitro, a process that is also regulated by

calcineurin261

,244

,252

,262

,263

. In addition, CsA inhibited degranulation of mast ce1ls

264,

265,

266,

267,

268,

269and of cytotoxic T lymphocytes (CTL)

254,

270,

271. Calcineurin

phosphatase activity is also necessary for degranulation254

. A number of B-cell

functions are sensitive to inhibition by CsA ; apoptotic B cell death, for instance, is

regulated by calcineurin

272

. Neutrophil chemokinesis on vitronectin (but not on

fibronectin or albumin) is inhibited by FK506273

. Calcineurin has recently been

shown to enhance the inactivation of I274, a cytoplasmic inhibitor of NF.

Practically, calcineurin increases the DNA binding activity of NFB, a ubiquitous

transcription factor, which may relate to the toxicity observed with these drugs.


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Stimulation of resting T cells requires not only engagement of the TcR

(signal 1) with antigen/MHC complexes but also additional signals (signal 2); CD28

appears to be an important costimulatory pathway in T-cell activation275

,276

. In the

absence of a costimulatory signal in concert with engagement of the TcR, the T cell

may become tolerized to antigen and persistent antigen-unresponsiveness (anergy)

may result. Engagement of CD28 during antigen stimulation prevents the induction

of antigen unresponsiveness. The ligands for CD28 include members of the B7

molecules. Anti-CD28 mAb and CHO (Chinese Hamster Ovary) cells transfected

with B7-1 provide co-stimulatory signals that prevent the induction of anergy 275, 277,

278,

279,

280, and analogs of a high-affinity B7- 1 ligand have been successfully tested

in animal models for the ability to induce allograft tolerance281

,282

. In addition,

CD28 ligation in the presence of activators of protein kinase C induces human T-cell

proliferation283

,284

and is resistant to inhibition by CsA284

,285

,286

,261

. It is therefore

possible that episodes of CsA or FK506 resistant transplant rejection may be

mediated by calcium-independent, CD28-dependent pathways of T-cell activation,

and effective treatment may require immunosuppressive regimens capable of

targeting pathways such as CD28.

1.5.1.2 Leflunomide

Considerable research on leflunomide (LFM) has been accomplished since

it was first recognized as an immunosuppressive agent by its ability to inhibit the

development of arthritis in rats by Bartlett and Schleyerbach at Hoechst

pharmaceuticals in the early 1980s287

. In vivo, LFM is rapidly converted to A77

1726, the only known immunosuppressive metabolite, which is believed to be

responsible for the drugs immunosuppressive effects. (see Figure 1.18.)


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Figure 1.18

Molecular Structure of Leflunomide and its active Metabolite A77 1726 288

A771726 noncytotoxically and reversibly inhibits proliferation of both T

and B cells stimulated by a variety of mitogens and inhibits antibody production (see

below)289

,290

,291

,292

,293

.

A77 1726 is able to directly inhibit the proliferation of stimulated smooth

muscle cells in vitro294

. This might explain why in addition to its effectiveness in

several animal models of arthritis, autoimmune disease, and allograft and xenograft

rejection, particularly those in which antibody plays a prominent role289

,291

. LFM

also inhibits chronic rejection (vasculopathy) (see below).

The clinical experience with LFM in Phase III trials for the treatment of

rheumatoid arthritis has been remarkably free of toxicity-related events other than

mild gastrointestinal symptoms, with prolonged treatment295

. Several new LFM

analogs, the malononitriloamides, have in vitro biological effects that appear to be

identical to those of A77 1726 293, 296 . Malononitriloamides are structurally very

similar to A77 1726. As the malononitriloamides are expected to have shorter half-

xenotransplant vasculopathy

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