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Relationship between MHC II expression on T-lymphocytes and rejection : a model of heterotopic heart.. 2004

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RELATIONSHIP BETWEEN MHC II EXPRESSION ON T-LYMPHOCYTES AND REJECTION A M O D E L OF H E T E R O T O P I C H E A R T T R A N S P L A N T A T I O N R A D E K C T I R A D K A D E R A B E K M . D., Charles University, Prague, Czech Republic, 1993 A THESIS S U B M I T T E D IN P A R T I A L F U L F I L L M E N T OF T H E R E Q U I R E M E N T S F O R T H E D E G R E E OF M A S T E R O F S C I E N C E i n T H E F A C U L T Y OF G R A D U A T E STUDIES D E P A R T M E N T OF S U R G E R Y We accept this thesis as conforming to the required standard The University of British Columbia November, 2004 © Radek Ctirad Kaderabek, 2004 Abstract Relationship between M H C I I expression on T-lymphocytes and rejection (a model of Heterotopic Heart Transplantation) Introduction: The method of choice for monitoring post-transplant rejection status for heart transplant patients is the evaluation of multiple tissue biopsies. The diagnostic technique is time consuming, costly and invasive with possible life threatening complications. The major histocompatibility complex is the most widely distributed and significant determinant for transplantation. We hypothesized that the M H C II expression on peripheral T-lymphocytes is reflective of rejection status in allotransplanted hearts. The objective of this project is to determine if M H C II expression on peripheral T- lymphocytes correlates with histological findings of rejection after transplantation. Materials and methods: The heterotopic heart transplant was performed in 24 female domestic swine, weighing 27-30 Kg. Animals were divided into two groups. Group A (6 donors + 6 recipients) was not treated with immunosuppressive therapy. In group B (6 donors + 6 recipients) immunosuppressive therapy (Cyclosporine 6mg/Kg/BID, Methylpednisolone 1.8mg/Kg/day and Azathioprine 3mg/Kg/day) was used. Cyclosporine levels in blood were maintained at 400 ng/mL. Donor animals were anaesthetisized and the heart was harvested through a median sternotomy. The hearts were antegradely perfused with isoosmolar Tyers's solution and were then preserved in a hypothermic storage for two hours at 4°C. In the postoperative period animals were monitored by E C G , heart-rate, body temperature, blood samples for M H C II expression, n cyclosporine levels, CBC plus differential and creatinine daily . Animals in Group A, survived for a period of five days and animals in Group B, where immunosuppressive therapy was applied, survived for five days also. The endomyocardial biopsies (EMB) were taken from the graft's free wall, apex and septum on day one and five for both groups A and B. The evaluation of #120 EMB (biopsies) was done using modified International Society Heart and Lung Transplant Grading Scale. R e s u l t s : Results of this experimental endeavour demonstrated that M H C II expression on T-lymphocytes significantly correlates with biopsy findings from apex (P<0.01; R = 0.83) for Group A (non-immunosuppressed animals) on day five. The combined data for both Groups A and B (non-immunosuppressed and immunosuppressed animals) at this 2 2 time were also correlated significantly (P<0.01; R = 0.63) for free wall and (P=0.01; R =0.7) for apex despite a significant difference between non-immunosuppressed and immunosuppressed animals. Conclusions: Results of this experimental protocol suggested that there is a significant correlation between M H C II expression on peripheral T-lymphocytes and Grade of rejection assessed by endomyocardial biopsy on day five after surgery. Further studies will be required to establish the long term relationship of M H C II expression on peripheral T-lymphocytes and histological findings, particularly in animals with immunosuppressive therapy. iii Table of Content: Abstract i i Table of Content iv List of Tables vi i i List of Figures vi i i Abbreviations ix Acknowledgment xi I. Introduction 1 1) Historical Review 2 1.1) Transplantation Surgery 2 1.2) The Immunology of Allograft Rejection 3 1.3) Allograft Rejection 5 1.4) H L A System 5 1.5) Immune Recognition and Activation 7 1.6) Class II H L A Antigens 8 2) Prevention and Treatment of Allograft Rejection: The Drugs Used in this Study, their Mechanisms of Action and Toxicology 10 2.1) Azathioprine 11 2.2) Corticosteroids 11 2.3) Cyclosporine 12 iv 2.4) Other Immunosuppressive Agents 14 2.4.1) Tacrolimus (FK 506) 14 2.4.2) Sodium Brequinar (S-BQR) 15 2.4.3) Rapamycin (Sirolimus) 16 2.4.4) Mycophenolate Mofetil (MMF) 16 2.4.5) Mizoribine (MZ) 17 2.4.6) Other therapeutic options 18 2.5) Nonpharmacological therapies 18 3) Pathology of Heart Allograft Rejection 18 3.1) Hyperacute Graft Rejection 19 3.2) Acute Rejection 20 3.3) Chronic Rejection 23 3.4.1) Histopathology of Hyperacute Graft Rejection 24 3.4.2) Histopathology of Acute Rejection 24 3.4.3) Histopathology of Chronic Rejection 24 4) The Diagnosis of Cardiac Allograft Rejection 25 4.1) Cardiac Catheterization 26 4.2) Endomyocardial Biopsy 26 4.3) Classification of Allograft Rejection by 27 Histopathologic Criteria 4.4) Artefacts 27 v II. Hypothesis 30 III. Objectives 30 IV. Specific Aims 30 V . Experimental Design 31 V I . Rationale 33 VII . Materials and Methods 34 1) Surgical Techniques 34 2) Positioning of the Bioptome for Endomyocardial Biopsy 35 3) Technical Considerations 36 4) Parameters of Assessment 36 5) Blood Sampling Protocol 38 6) Intravenous Drug Administration 39 7) Immunosuppression / Drug Regimen 39 7.1) D a y i 39 7.1.1) Immunosuppressive Drugs 40 7.2) Day 2 40 7.3) Day 3 41 7.4) Day 4 41 7.5) Day 5 41 8) Flow Cytometry 42 8.1) Procedure for Cell Preparation 42 8.2) Procedure for Freezing Cells 42 8.3) Antibodies and Dilution Assays 43 vi 8.3.1) Antibodies 43 8.4) F A C S Analysis 43 8.5) Evaluation of Endomyocardial Biopsies 44 8.6) Histopathology of the Transplanted Hearts 44 8.6.1) Specimen Preparation 44 VIII . Statistical analysis 45 IX. Results 46 X . Discussion 55 X I . Conclusion 63 XII . Bibliography 64 vn List of Tables Table 1 I S H L T Scale Table 2 Grade of rejection in non-immunosuppressed animals Table 3 Grade of rejection in immunosuppressed animals Table 4 M H C II expression in non-immunosuppressed animals Table 5 M H C II expression in immunosuppressed animals List of Figures Figure 1 Difference between groups A and B on the day 5 after surgery Figure 2 Difference between rejection status in Groups A and B on day 5 after surgery for apex, free wall and septum Figure 3 Regression analysis - free wall (Groups A and B) Figure 4 Regression analysis - apex (Group A and B) Abbreviations: A P C Antigen Presenting Cell C Complement C P Calcineurin phosphatase C T D R Cardiac Transplant Research Database C R Chronic Rejection D N A Deoxyribonucleotic Acid E T W A Evoked T-wave Amplitude F D A Food and Drug's Administration F ITC Fluorescein Isothyocyanate F K 506 Tacrolimus F K B P Tacrolimus Binding Protein H A Hyaluronan H L A Human Leukocyte Antigen IFN Interferon IL Interleukin ISHLT International Society of Heart and Lung Transplant I V R P Isovolumetric Relaxation Period I M D H Inosine Monophosphate Dehydrogenase Ig Immunoglobulin K D Ki lo Dalton L V Left Ventricle M H C Major Histocompatibility Complex M P Mercaptopurine M M F Mycophenolate Mofetil (MMF) M R I Magnetic Resonace Imaging M Z Mizorbine PE Phycoerythrin PET Positron Emission Tomography R N A Ribonucleotic Acid S - B Q R Sodium Brequinar S M C Smooth Muscle Cell T C R T-cell Receptor T F N Tumor Necrotizing Factor T V D Transplant Vascular Disease V T I Velocity Time Integral x Acknowledgment I would like to express my appreciation to my supervisor, Dr. Karim Qayumi for his guidance, mentoring and support during my Masters program. I also want to say to other friends and colleagues for their help mentoring and support, especially to Dr. Charles Slonecker, Dr. York Hsiang and Dr. John English. My special thank is being addressed to my best friend and wife, Prof. Nancy J. Hermiston for her love, support, and patience. x i I. INTRODUCTION A major challenge in cardiac transplantation is the prevention of acute graft rejection . Surveillance of the transplanted heart must be performed in order to maintain the recipient at an appropriate level of immunosuppression to protect the allograft from the alloimmune response. Surveillance must also be performed to detect early signs of acute rejection in order to institute immediate changes in case management. Histological grading of the rejection process via examination of endomyocardial biopsy specimens is the current gold standard for diagnosis of 2 3 rejection and to monitor efficacy of antirejection therapy . ' Many centers perform routine surveillance biopsies up to eighteen times in the first post- transplant year and annually thereafter. Additional procedures are performed during periods of clinically suspected rejection . 4 Methods that provide immediate, accurate results with limited invasiveness, reduced risk, and are less inconvenient to the patient, are attractive alternatives to numerous endomyocardial biopsies.5'6 To date, surface and intracardiac ECGs , ' echocardiographic parameters, including 2D and Doppler measures of systolic and diastolic function, 9' 1 0> 1 1 , 1 2 and radionuclide imaging including ventriculography and antimyosin imaging, 1 3 ' 1 4 ' 1 5 and MRI 1 6 have demonstrated limited predictive accuracy or practical usage for monitoring and detection of transplant rejection. 4 The method of choice for monitoring post-transplant rejection status for heart transplant patients is the evaluation of multiple tissue biopsies. This diagnostic technique is time consuming, costly and invasive with possible life-threatening complications. Matches between donor and recipient at the M H C locus is the most widely distributed and significant determinant of transplantation success. We hypothesized that de novo M H C class II expression by recipient peripheral T cells, which is a marker of activation, reflects the rejection status of allotransplanted hearts and may 1 beused as an accurate descriptor of the rejection process. The objective of this project was to determine if M H C class II expression on recipient peripheral T cells correlates with histological findings of rejection after cardiac transplantation. We found that there is a significant correlation between the M H C II expression on T- lymphocytes in peripheral blood and Grade of rejection in the allotransplanted hearts in the porcine model. 1. HISTORICAL REVIEW 1.1 Transplantation Surgery Until the 20 th century, transplantation efforts were limited to the grafting of tissues, not organs, because no techniques were available to control bleeding during organ resection or to re- establish circulation after transplantation. Organ transplantation became possible with the development of surgical techniques that 17 permitted blood flow to be interrupted and re-established in the recipient. Dr. Yahoo Carrel, M.D. in France and Father Ditsie Guthrie, M.D. in the USA created the method of anastomosis, joining two blood vessels together to allow blood circulation to "achieve" anastomosis (from Gk, 18 anastomosis: the surgical union of parts, esp. of hollow tubular parts). They developed anarterial clamp to prevent excessive blood loss, as well as techniques to hold arteries in place.The first cadaveric kidney transplant in a human was performed in 1936 in an attempt to treat renal failure caused by mercury poisoning, The patient died two days later 1 9 . Five subsequent attempts during the next decade were surgically successful, but lack of 2 immunosuppression resulted in organ failure due to rejection . In the 1950s, at least eight attempts at renal transplantation between ABO-compatible subjects in France were unsuccessful.21. In 1967, Christian Barnard became fascinated by work with heart transplantation in dogs in the USA. He went to Cape Town, South Africa, and performed the first successful heart transplant in human history. This first recipient died within three weeks due to 22 complications from surgery, but Barnard's second patient lived for seventeen months . At that time, other centres began to perform the procedure. After one hundred and sixty-six heart transplantations had been performed, only 23 recipients were still alive, for an overall mortality rate of greater than 85%, due to complications of rejection23. Although practical aspects of organ transplantation improved as surgical expertise developed, it was clear that the ability to achieve long term organ function would depend on the development of methods to control rejection 2 4 . 1.2. The Immunology of Allograft Rejection Even though the technical/surgical part of transplantation had been mastered, early experiments were obviously doomed to failure because "rejection" was a concept that had not been defined or understood. Histologic analysis performed on some of these early failed allografts demonstrated a pattern of marked inflammation and focal or complete necrosis with occasional thrombosis in rejected tissue, All of these reactions were arising from activation of the host immune response25.In 1944, Sir Peter Medawar began to describe the rejection process as a reaction of the recipient's immune system to antigens on the donated tissue by a series of experiments on skin transplants in rabbits 2 6 . Reflection of immune intolerance to foreign tissue has long been visualized using light microscopic techniques. Medawar's work extended these observations to histolopathological descriptions of rejected tissue where a "black band" of lymphocytes was seen infiltrating the dermis of engrafted skin within 1 to 2 weeks of allograft 3 transplantation. In addition to the formal description of the rejection reaction, Medawar also characterized the decreased time for rejection of a second transplant in an animal sensitized by previous alloantigen sensitization as the "second set response" phenomenon. For these achievements, Sir Peter Medawar has been known as the founder of transplantation immunology27"8. Rapaport applied the results of Medawar's work to study the immunology of skin autografts and allografts in humans. 2 9 These studies provided evidence for the existence of tissue types defined by alloantigens shared between unrelated humans, but on a population basis, differing from individual to individual. Scientists studying different membrane-bound glycoproteins on groups of leukocytes began collaborating with scientists studying tissue typing. The result of this collaboration was the identification of M H C antigens in humans. In humans M H C molecules are known as Human Leukocyte Antigens (HLA antigens). H L A molecules are the most highly polymorphic group of molecules in humans. To date, application of HL A typing determines donor selection in transplantation. Survival of the transplanted organ depends upon the recipient and donor sharing as many HLA antigens as possible. In the classic studies of Mann and colleagues, untreated rejection of heterotopic canineallografts was characterized by a prominent cellular infiltrate in the allograft parenchyma. Studies of canine models by Lower and colleagues had indicated that the histopathologic changes of rejection preceded electrocardiographic evidence of voltage loss, and suggested an important role for heart biopsy and rejection surveillance.26 The most important fact is that to date no indicator of heart allograft rejection in humans has been more sensitive, specific or useful than the endomyocardial biopsy. Rejection, as defined histopathologically, is a very diffuse process. Thus, the likelihood of detecting rejection, when present, has been shown to be 4 95% when three pieces of bioptome -acquired tissue are evaluated microscopically, whereas the "3 1 "JO likelihood rises to 98% with four pieces of tissue. ' The sensitivity is greater in the first few months after transplantation when the prevalence of cellular rejection is highest. 1.3. Allograft Rejection An allograft is an organ or tissue graft from a donor who is of the same species but genetically non-identical to the recipient. An understanding of allograft rejection involves the understanding of the molecular basis of the cells involved in alloantigen recognition and their response to the allograft in an attempt to eliminate it as a foreign invader. In the experiments reported here, the pigs were genetically heterogeneous. The antigens generally capable of eliciting a host rejection response include the HLA, ABO and Lewis blood group antigens, non- H L A monocyte or endothelial cell antigens, and possibly other tissue specific non-HLA antigens34'35. The cell surface expression of these different antigenic systems varies among different organs and cells within the same species. The H L A and A B O blood group antigens are the most widely distributed and significant determinants for successful transplantation. ' There are two major mechanisms and one minor rare mechanism of rejection, each of which involves different immunological effector mechanisms, all of which culminate, in the nonimmunosuppressed host of graft failure. 1.4. The H L A System The H L A expression is controlled by the highly polymorphic gene loci of the M H C mapped to the sixth chromosome in man. H LA system is divided into two major classes: class I (HLA - A , B and C) and class II(HLA DR, DQ, and DP). It is believed that H L A polymorphism is the result of the evolutionary advantage conferred on the species by a diverse antigen- 5 processing system capable of interacting with the widest possible range of invading microbes.35'36 H L A cell surface molecules are important in presenting immunogenic peptides to CD4+ T cells and CD8+ C T L so that foreign cells arising from an allograft can be attacked and eliminated. In allograft rejection, the foreign H L A molecules themselves or H L A bound to processed donor antigens are highly immunogenic and serve as the targets for destruction by host 36 38 T cells sensitized to these foreign alloantigens. ' Class I H L A antigens are present on essentially all nucleated cells, although there is some variation in antigen density among different cells and organs. The class I H L A molecules are composed of a 45 kD a heavy chain non-covalently associated on the cell surface with p 2 microglobulin, a 12kD polypeptide which is not coded by H L A genes. Class II H L A antigens under normal circumstances are expressed primarily on antigen presenting cells (APC's) such as dendritic cells, macrophages, and certain specialized epithelial and endothelial cells. These antigens are made up of a 33-36 kD p chain noncovalently linked to a 24-29 kD a-p chain. The H L A molecules bind foreign peptides for presentation to T-cells (for recognition and activation) in a significantly different way from antibody, which binds much larger conformational antigen epitopes. H L A molecules bind and T-cells recognize and react to antigenic peptides (linear molecules).40 The cell surface expression of H L A class I and especially class II antigens is affected by cell activation or differentiation. Thus, transplanted cells that may not normally express H L A class II antigens can be induced to increase cell surface expression of these antigens by mediators present at the site of inflammatory responses such as rejection (interferon a, p, y, TNF-a, TNF-P, and interleukin-4). The increased expression of H L A molecules on the target cell surface probably serves to increase antigen presentation and thus also increases the potential interactions between T cells and immunogenic target antigen. The H L A molecules themselves are immunogenic as well.41 Before antigen becomes associated with H L A molecules on the surface of cells, it undergoes processing or degradation 6 often within the cell that is to express the antigen. There is a suggested difference between antigens which eventually associate either with Class I or Class II surface molecules.42'43 Endogenous proteins (antigens synthesized within the presenting cell such as viral proteins) areassociated with H L A Class I molecules following intracellular processing. Immunogenic foreign antigens are not necessarily synthesized within the cell but taken up by the endosomal recycling system and are thought to be mainly H L A class II - dependent proteins 4 4 The mature T-cells are represented by two subsets which are phenotypically identified by reaction with different monoclonal antibodies specific for their surface antigens, which are given the cluster designation CD4 and CD8. Both CD4 and CD8 positive T-cells, and are exclusive in terms of their ability to react with class II and class I H L A antigens, a phenomenon known as M H C restriction.45 As suggested by many clinical studies, glycoproteins of the donor H L A system play a pivotal role as allogeneic targets in rejection documenting the long term advantages of H L A matching.46'47 Other studies also suggest that long-term clinical benefits from H L A matching is 48 the reduction of allograft loss during the first five months following transplantation. 1.5. Immune Recognition and Activation Cytokines are a primary signal for the sequential activation and proliferation of T and B cells, both for killing and for antibody production. IL-2 is produced by activated recipient CD4+ Thl cells after they have recognized the foreign peptide: self HL A complexes expressed on the membranes of professional antigen presenting cells, accompanied by other co-stimulatory events delivered by the same cells. Expression of high-affinity receptors for IL-2 on the membranes of CD8+ CTL's activated by foreign peptide: self M H C complexes are a critical first step in the 7 proliferation and differentiation of CD8+ cytotoxic T cell clones that will traffic back to the allograft and cause cells in the engrafted tissue to undergo apoptosis. Antigen presenting cells (APCs) and both helper and cytotoxic T cells play the leading role in the immune recognition or afferent phase of allograft rejection. The normal rate and pattern of lymphocyte migration is disrupted by transplantation, resulting in an increase in the influx rate of white blood cells into the graft.49 The APCs, a group that includes dendritic cells, macrophages, B cells, endothelial and some parenchymal cells, process antigen so that a peptide of eight to ten amino acids (MHC Class I) or ten to twenty-two amino acids (MHC Class II) H L A molecule for presentation to potentially reactive naive, mature T cells.50'51 The T-cell receptor, the associated signal transduction molecular complex CD3 and the CD4 or CD8 co-receptors are all essential in graft rejection reactions. In nearly all T cells the TCR (T-cell receptor) is composed of two polypeptides, a and p, connected to each other by a disulfide bond and closely associated in the cell membrane with another protein group, the CD3 complex, which is made up from at least 4 distinct signal transduction peptides (e, y, 8, and Q. Each molecule in the CD3 complex contains at least one motif, known as an ITAM, which contains tyrosine residues. A number of additional T cell surface molecules such as LFA -1, CD2, and in particular CD28 serve signaling and adhesion functions vital to both the afferent priming phase and the effector phase of T cell activation and clonal expansion. 1.6. Class II H L A Antigens The HLA class II antigens are noncovalently bound heterodimeric glycoproteins but they differ in their function, biochemistry, and genetics. M H C molecules and other molecules encoded in a tightly linked complex of genes involved in the processing and presentation of antigens are encoded within the major histocompatibility locus of the pig both a (heavy) and P 8 (light) chains and are encoded within the major histocompatibility complex.The class II antigens are constitutively expressed on only a limited number of cells in humans, including B cells, monocytes, macrophages, vascular endothelial cells and, dendritic cells. They could be expressed on lymphoid or parenchymal cells during certain inflammatory reactions, especially and typically mediated by y -interferon.53 The class II genes occur in clusters of alpha and beta genes in which the number of each can vary. For the H L A - D R cluster, one invariant a gene is present with several genes. The DRpi gene possesses that account for most of the HLA-DR specificities (DR P-l chain determining specificities DR1, DR2, DR3, DR4, DR5). Other DR P genes are less variable and encode theDRw52 and DRw53 broad specificities. These antigens are analogous to Bw4 and Bw6 in that they are associated with different groups of HLA-DR specificities, but different in that they are not encoded by the same genes as the individual alleles in its cluster. The other major class II loci include DP and DQ, each of which consists of two a and two p genes, not all of which are expressed. Both the a and P genes of DP and DQ express allelic polymorphism.54 Immune recognition requires that the nominal antigen (peptide) be co-recognized with M H C molecules on the cell surface. It is known that M H C heavy and light chains in conjunction with M H C binding peptides are involved in the conformation, trafficking and recognition of the M H C molecules. In allorecognition T cell clones were able to distinguish MHC/peptide complexes arising from different cell types. Both T and B lymphocytes are involved in generating an immune response to alloantigens. Subsequently, antibodies and /or effector T lymphocytes can generate an immune response to allo-MHC antigens resulting in a cascade of events that ultimately leads to destruction or inactivation of the target.55 9 Recruitment of activated T cells into the graft is used as a diagnostic tool for rejection. In fact, even activated T cells circulating in the host periphery after interacting with the graft are shown to be the best markers of acute renal rejection56.Reinke et al have demonstrated that M H C II antigens are upregulated on the surface of activated circulating cells during rejection episode and propose that determination of circulating M H C II-DR+ T cells complements biopsy and is very important for clinicians if histology is equivocal56. Although the precise role of M H C II on T cells is not well understood, it has been shown to be involved in enhancement of cell to cell interaction between T cells themselves and other inflammatory cells. It also enables to present antigens and enhances the magnitude of the inflammatory cascade.57 2. Prevention and Treatment of Allograft Rejection: The Drugs Used in this Study, Their Mechanisms of Action and Toxicology Immunosuppressive drugs that inhibit recipient T lymphocytes or kill T lymphocytestraveling in the graft constitute the principal treatment regimens for graft rejection. Our current success in immunosuppression results largely from our ability to inhibit and control the activation and differentiation of allograft-specific recipient CD4+ and CD8+ T cell clones with TCR's specific for epitopes from the graft following transplantation. Initial attempts at immunosuppression were very nonspecific and involved total body irradiation. These experiments revealed that the immune system could be destroyed and thereby made to accept the transplanted organ. Although each agent has its specific actions, all suppress humoral or cell-mediated immunity or both. Patients undergoing immunosuppression are particularly vulnerable to opportunistic infections such as cytomegalovirus, Pneumocystis 58 carinii, Candida albicans, and Aspergillus, and may rapidly succumb to sepsis. This is the downside of immunosuppression. 10 2.1. Azathioprine In 1959, Schwartzand and Damashek, while searching for a less toxic substitute to whole body irradiation, discovered that 6-mercaptopurine (6-MP) suppressed the immune response of antigen-primed rabbits when they received subsequent challenges with the priming antigen.59 Azathioprine, as 6-MP is now called, was first used in clinical organ transplantation in 1963. The compound is a purine anti-metabolite that is converted by the cytochrome oxidase system or hepatic Cytochrome c in the liver to 6-MP. It is a strong but non-specific immunosuppressive agent which inhibits both DNA and RNA synthesis, blocks de novo purine nucleotide synthesis, and causes breaks in chromosomal D N A . 6 0 Azathioprine, therefore, inhibits clonal expansion of lymphocytes activated by antigens expressed by and presented on engrafted cells. The efficacy of azathioprine as an immunosuppressive agent is due to inhibition of lymphocyte proliferation.61 The primary toxicity of azathioprine is evident in the bone marrow, where it causes leukopenia and thrombocytopenia. Other major adverse effects are on the gastrointestinal 62 system, where it produces nausea, vomiting, and symptoms of pancreatitis and hepatitis. Periodic monitoring of blood cells and platelet counts, as well as serum chemistries for 63 pancreatic enzymes and liver function, are recommended for timely detection of toxicity. 2.2. Corticosteroids Corticosteroids are used in the organ transplant regimen at low doses as part of allograft maintenance protocols in combination with other drugs, and at high doses for induction of immunosuppression in the engrafted recipient. 11 Corticosteroids cross the cell membranes, combine with specific binding proteins, and are then transported across the nuclear membrane to sites near cytokine genes. The steroid/protein complex inhibits the transcription of the genes and the production of cytokines.63 Treated macrophages cannot release IL-1 6 4 and IL I also provides a signal for activated T-cells to express the high affinity form of the IL-2 receptor. IL 6 may supply a necessary coordinating signal. Without a high-affinity IL-2 receptor, an antigen-binding T cell will not clonally expand and differentiate. IL-2-activated CD4+ Thl cells also secrete interferon y (IFN-y), which induces macrophages to synthesize more IL-1 and IL-6, so corticosteroids also immunosuppress via feedback inhibition.65 Corticosteroids also inhibit macrophage release of the inflammatory mediators leukotriene B4, thromboxanes and chemokines, which are chemotaxic for neutrophils and effector CD4+ Thl T cells. Cytotoxic responses directed at engrafted cells in a tissue are thus suppressed.66 After an antigen-specific immune response has been initiated, the acute inflammation that produces allograft damage is maintained by non-specific release of cytokines, complement activation, and the activation of the clotting cascade. Therefore, the anti-inflammatory effects of corticosteroids remain crucial even when rejection is ongoing.65 2.3. Cyclosporine Cyclosporine A was introduced for clinical use in 1978. By the mid-1980s' most immunosuppression protocols were cyclosporine-based.62 Derived from a fungus olypocladium inflation, cyclosporine is a cyclic decapeptide.63 Both cyclosporine A and tacrolimus block T-cell proliferation by inhibiting the phosphatase activity of a C a 2 + dependent enzyme called calcineurin at nanomolar concentrations. Calcineurin has a major role in transmitting signals 12 from the T-cell receptor to the nucleus. Both drugs reduce the transcription of several cytokine genes that are normally induced on T-cell activation, including interleukin-2 (IL-2). Cyclophilin, a specific cyclosporine binding protein, has been identified in both the cytoplasm and nucleus of T-cells.65 Cyclophilin is one of the peptidyl-prolyl cis -trans isomerases, a family of cytoplasmic enzymes which plays an important role in normal protein folding during protein synthesis.64'67 The interaction between the drug-binding protein complex and the relevant genes results in the inhibition of IL-2 transcription.68'69 As a result of cyclosporine binding cyclophilin loses its activity. Thus, cyclosporine inhibits a cytoplasmic signal required to initiate normal cytokine gene transcription in the nucleus. Cyclosporine is a potent inhibitor of almost all the known cytokines, including IFN-y and IL-2 through IL -4. 6 5 Although cyclosporine does not directly block macrophage production of IL-1, it may reduce the 70 production of this cytokine by inhibiting T cells through the production of interferon gamma. Thus, cyclosporine, while targeting a different cell and cytokine, produces feedback inhibition similar to that of corticosteroids.65 Therefore, cyclosporine and corticosteroids synergistically inhibit recipient immune responses. This provides a rationale for their combined use in clinical transplantation. Cytokines are a primary signal for the sequential activation and proliferation of T and B cells, both for killing and for antibody production. IL-2 is produced by activated CD4+ Thl and Th2 cells after they have been activated by foreign peptide: self H L A complexes expressed on the membranes of professional antigen presenting cells and other co-stimulatory events. Expression of high-affinity receptors for IL-2 on the membranes of CD8+ CTL's activated by the same peptides on recipient M H C molecules is a critical first step in the proliferation and differentiation of CD8+ cytotoxic T cell clones that will traffic back to the allograft and cause cells in the engrafted tissue to undergo apoptosis (programmed cell death). 13 The most common adverse effects of cyclosporine on organ systems of the body are hyperkalemia, hypertension, hyperuricemia, nephrotoxicity and a variety of infections. Other problems with less incidence (less than 20%) include hepatotoxicity, tremors, hirsuitism, 58 62 gingival hypertrophy, anorexia, gout, paresthesia, and hypomagnesemia. ' 2.4. Other Immunosuppressive Agents Immunosuppressive regimens based on cyclosporine, azathioprin, and corticosteroids are associated with significant drug toxicity, resistant acute rejection and opportunistic infections. Relatively nontoxic alternatives to the cytotoxic class of drugs can now be used for immunosuppression in transplant patients. Fewer complications are associated with tacrolimus, sodium brequinar (S-BQR), rapamycin, mycophenolate mofetil (MMF), and mizoribine. 2.4.1. Tacrolimus (FK506) Discovered in 1984, FK 506 received FDA approval in 1994 for use in the USA. FK 506 is a macrolide lactone, a metabolite extract from the filamentous bacterium Streptomyces tsukubaensis, found in. Japan. It inhibits the proliferation and differentiation (priming) of CD4+ Thl and Th2 T cells and CD8+ CTL's (cytotoxic T lymphocytes). The inhibited lymphocytes include those which are specific for foreign epitopes on the allograft. Tacrolimus penetrates the cell and it specifically inhibits synthesis of interleukins (IL-2 ,-3 and -4), colony stimulating factors, and IFN-y.71 FK 506 binds to a cellular protein. The F K 506 binding protein (FKBP), and the bound complex interferes with the activity of calcineurin phosphatase (CP). CP is an enzyme that is part of the biochemical cascade that transduces activation signals from the surface of a cell to its nucleus, causing the transcription of genes for cytokines. By interfering with CP, 14 FKBP effectively reduces expression of IL -2 and other cytokines, thereby blocking T-cell activation. This is a very selective action, because only the T-cell dependent immune responses are blocked, while the T cell-independent B-cell responses are preserved. T cell stimulation through alternative pathways (CD28) is also-not affected.74'76 The adverse effects profile of FK 506 is similar to that of cyclosporine, including nephrotoxicity, neurotoxicity, and new-onset diabetes. Neurotoxicity extends from mild symptoms of tremor, headaches, and insomnia to syndromes of dysarthria, seizures and coma. Like cyclosporine, there are three clinical settings in which the nephrotoxicity of FK 506 commonly occurs: (1) in the immediate post-surgical period where graft ischemia associated with the vasoconstrictive effect of FK506 may occur; (2) with concurrent administration of other nephrotoxic agents; and (3) with chronic renal insufficiency due to progressive interstitial fibrosis.71 2.4.2. Sodium Brequinar (S-BQR) S- BQR was originally developed as an antimalignancy drug, and was later discovered to have immunosuppressive properties. It has been extremely effective in synergistic combination with cyclosporine in experimental conditions, and whether the synergism exists in clinical transplant situations remains to be evaluated.72 S-BQR inhibits the action of dihydro-oronate dehydrogenase, an enzyme involved in the de novo biosynthetic pathway of pyrimidines involved in the synthesis of DNA and RNA. Inhibition of the de novo synthesis of uridine also blocks the salvage pathway and decreases the function of adhesion molecules, because 73 glycosylation of these molecules requires the pyrimidine nucleotides. Adverse effects of S-BQR include thrombocytopenia, desquamative maculopapular 71 dermatitis, mucositis, and gastrointestinal side effects. 15 2.4.2. Rapamycin (Sirolimus) Rapamycin, also known as sirolimus, was isolated from another Streptomyces hygroscopius, an actinomycete called "Rapa Nui" by the Easter Island natives.60 Rapamycin has a structural similarity to FK 506, being a macrolide, but it does not appear to interfere with cyclosporine A activity. Rapamycin affects both T and B cells directly by preventing cytokines from activating these cells, an action that is uniquely different from that of cyclosporine.74 Rapamycin and F K 506 are reciprocal antagonists of IL-2 secretion and should not be used in combination. There is no evidence to suggest that rapamycin works in synergy with cyclosporine in animal models.71 The full spectrum of activity of rapamycin is not known. Rapamycin can inhibit mitogenic responses in cultured fibroblasts, hepatocytes, endothelial cells, and smooth muscle cells as well as lymphocytes. Because the results of human trials are still pending, we must look at results of trials in experimental animals for a toxicity profile. Dogs are unusually sensitive to the toxicity of rapamycin, whereas in other animals the toxicity has been restricted to weight loss, testicular atrophy, and lethargy. Rapamycin is known to be diabetogenic in rodents, but not in nonhuman primates. 2.4.4. Mycophenolate Mofetil (MMF) M M F , previously identified as RS 61443, is a semisynthetic derivative of the active agent mycophenolic acid (the active agent), isolated from the mold Penicillium glaucum.1* M M F has been used for more than two decades in the treatment of psoriasis and has been proven to be safe. M M F suppresses immunologic rejection responses by inhibiting the enzymes involved in the purine salvage pathway, thereby reducing the availability of purines for DNA synthesis. 16 Lymphocytes activated by engrafted antigens have a high proliferative index and this drug 71 inhibits their proliferation and differentiation into effector cells. M M F in vivo is phosphorylated to become mycophenolic acid, which noncompetitively inhibits the enzyme inosine monophosphate dehydrogenase (IMDH). IMDH is involved in the de novo synthesis of guanine.76 The drug can be administered orally, with high bio-availability, and it is eliminated in the bile.71 However, because the drug is very effectively inactivated, it has a low potency, and it binds with low affinity to IMDH. 7 6 For effectiveness, it is important for patients to maintain immunosuppressive levels of the drug in the blood. Furthermore, inadequate doses could lead to 76 T- and B- cell proliferation because they have been held in check at a late stage of activation. The majority of side effects are minor gastrointestinal ones. Upper respiratory tract infections and herpes viruses (HHV's) infections also have been reported.71 Animal studies reveal that side effects include anemia in rats; leukopenia, diarrhea and anorexia in monkeys; and gastrointestinal hemorrhage in dogs.70 2.4.5. Mizoribine (MZ) Mizoribine, also known as Bredinin, has been used in Japan, but not widely. Like M M F (mycophenolate mofetil), mizoribine is a pro-drug that needs to be phosphorylated to be active. Mizorbine, like MMF, competitively inhibits IMDH. So far no studies have proved any kind of advantages of mizoribine over drugs in the current therapeutic armamentarium. Toxicity of mizoribine is especially evident in dogs, who developed hemorrhagic enteritis and erosive mucosal lesions and, when on high doses, endotoxemia. 17 2.4.6. Other Therapeutic Options Those include prostaglandin E , lipoxygenase inhibitors, and the older, but still effective, antithymocyte globulins. Still another therapeutic option available is leflunomide, which suppresses lymphocyte activation by inhibiting Lck, Fyn and ZAP-70, all tyrosine kinases involved in signal transduction after phosphorylation of ITAM's and signal transduction which results in the transcriptional activation of the IL-2 gene and the a chain of the high-affinity IL-2 receptor. 2.5. Nonpharmacological Therapies Among the non-pharmacological options available are techniques such as total lymphoid irradiation, photo-chemotherapy, thoracic duct lymphocyte depletion and pre-treatment of the graft with a monoclonal antibody (like OKT3) specific for the extra-cellular aspects of the CD3 complex followed by complement. 3. Pathology of Heart Allograft Rejection The immune intolerance and its reflections had been visualized for many years with the light microscope. Later on, with interest in biopsies of the human heart in the 1950's, and the invention of the transvenous bioptome in Japan, the possibility of monitoring heart allograft rejection became feasible in the 1970s'. Among the many pioneering contributions of the Stanford Heart Transplant Program, the most important was the modification of the transvenous bioptome by Philip Caves and its application to human heart allograft rejection.77 Secondly, the biopsy became the Gold standard in the management of heart allograft recipients, and is still being used today. Thanks to Dr. Margaret Billingham,78 the classification for the heart allograft rejection status was established 18 and is being used to guide clinical management of heart allograft recipients. The high sensitivity of the endomyocardial biopsy (generally 85%-100% for three to five pieces of endomyocardium) in detection of cellular rejection relates to the universality of alloantigens in every region of the endomyocardium. The sensitivity is greater in the first few months after transplantation when the 77 prevalence of cellular rejection is highest. 3.1. Hyperacute Graft Rejection Hyperacute rejection is characterized by thrombotic occlusion of the graft vasculature that begins within minutes to hours after host blood vessels are anastomosed to graft vessels. This reaction is mediated by pre-existing antibodies in the host's circulation that bind to the donor's endothelial antigens. Hyperacute rejection is very rare. Vascularized organs (especially heart and kidney) are at risk for hyper-acute rejection if the patient has in his blood preformed donor-specific alloantibodies. This humoral presensitization (or alloimmunization) may be caused by a previous transplant, blood transfusion or pregnancy. The primary target of the donor-specific antibodies is the vascular endothelium of the transplanted organ. The most common target antigens are HLA class I, ABO system, but other less well defined antigens have also been considered. HLA class II antigens seem less relevant in hyper-acute rejection because they are not strongly expressed on the vascular endothelium. Antibodies mediating hyper-acute rejection are almost always complement-fixing and they could be IgG or IgM type or both. Alloreactive T lymphocytes are occasionally involved. A critical step in alloantibody-mediated rejection is the initiation of the complement cascade. This involves the binding to and activation of Clq , which requires at least two complement-fixing sites on the antigen/antibody complex. This requires a close proximity 19 between antigens and no complement activation will occur if the antigen density is too low on the cell surface. Activation of the complement cascade leads to the release of various inflammatory mediators and the initiation of the coagulation and fibrinolytic systems. Hyper- acute rejection is manifested by rapid vascular constriction, edema and thrombotic occlusion. Endothelium stimulation and exposure of subendothelial basement membrane proteins activate platelets which then adhere and aggregate in the vasculature. Soon afterward, polymorphonuclear leukocytes (PMN) adhere to the vascular endothelium. 3.2. Acute Rejection Acute rejection is a process of vascular and parenchymal injury mediated by T cells and antibodies that usually begins after the first week of transplantation. Acute allograft rejection remains one of the leading causes of death and morbidity in cardiac transplantation.78'79 The incidence of rejection reported in a cohort of seventeen hundred patients transplanted between 1992 and 1994 in the Cardiac Transplant Research Database (CTDR), was 1.4 - 1.5 rejections per patient in one year. Forty-two percent of the patients were free of rejection, 31 % had only one rejection episode, and 27 % had more than one rejection at one year. The incidence of hemodynamic compromise was 8%. Twelve percent of patients received cytolytic antibody therapy for the treatment of rejection. Significantly, there is no simple biochemical marker of cardiac rejection, and over 90% of episodes of acute cellular rejection are not accompanied by any cardiac symptoms or measurable change in graft function. This is, in part because biopsies are performed on a routine protocol basis in an attempt to detect rejection before it induces cardiac dysfunction. The entireapproach of using protocol biopsies to diagnose rejection in cardiac transplantation is based on the very high mortality associated with the development of hemodynamic compromise or 20 evidence of significant graft dysfunction in heart transplant recipients . Data from a review of three thousand three hundred and thirty-seven patients transplanted between1990-1994 showed that patients with hemodynamic compromise had a 40% mortality in the six months following such an episode and up to 50% by one year. Ten percent of patients treated with cytolytic therapy for hemodynamic compromise in this series had no histologic evidence of rejection on the biopsy81. Acute rejection is mediated by lymphocytes that have become alloactivated against donor transplantation antigens. In vivo stimulation of alloreactive T cells takes place primarily in the peripheral lymphoid tissues of the recipient although intragraft sensitization may occur. The strongest stimulus is provided by donor dendritic cells (also referred to as passenger leukocytes) present in the allograft which will enter the circulation and end up in the lymphoid tissues of the recipient. These dendritic cells function as antigen presenting cells and provide a strong stimulus to M H C class II-restricted CD4+ cells, which can stimulate the growth and differentiation of H L A class I reactive CD8+ cytolytic lymphocytes. Alloreactive lymphocytes enter the circulation and react with antigens on allograft vascular endothelium, the primary target of the initial stage of cellular rejection. The lymphocyte- endothelial interactions depend on the expression of appropriate target antigens on the endothelium. HLA class I antigens are expressed at the cell surface whereas H L A class II antigens expression must be induced by various cytokins, especially gamma interferon. Furthermore, adhesion molecules (such as integrins, selectins) play an important role in the cell surface interactions between lymphocytes and endothelium cells, and in their activation and release of cytokines and inflammatory mediators. These processes lead to the migration (extravasation) of lymphocytes through the vascular wall. 21 Graft-infiltrating lymphocytes mediate various effector mechanisms of allograft immunity. Besides having a direct cytotoxic effect on graft parenchymal cells, lymphocytes may mediate a delayed type hypersensitivity mechanism of graft rejection. The latter involves therecruitment of macrophages and NK cells. Varying proportions of CD4+ and CD8+ lymphocytes are found in cellular infiltrates of rejecting allografts. Many of them express T cell activation markers, like IL-2 receptors and through release of gamma -interferon and other cytokines; there is an increased expression of H L A antigens (especially class II) on the vascular endothelium and other target cells in the graft parenchyma. Consideration must be given to additional immunological mechanisms such as graft injury secondary to infection, especially those caused by viruses. This means that other types of lymphocytes may infiltrate the graft and could mediate immune effector responses against virus- infected cells within the graft. Another possibility is that graft-infiltrating lymphocytes mediate the recurrent autoimmune disease process. Severity of rejection is defined histopathologically by the number of infiltrating inflammatory foci associated with the increasing amount of parenchymal, vascular, and myocyte damage. In very severe forms of rejection there are found phenomena such as edema, necrosis and hemorrhage. The classification of acute rejection was originally proposed by Dr. Billingham in the 1970's and later simplified and unified by ISHLT. Besides the usual development of a rejection, defined as acute rejection, there could also develop conditions which are not as well understood called hyper-acute rejection and chronic rejection. When rejection is due to preformed alloantibody (presensitization), it can result in very rapid graft loss in what has been called hyper-acute rejection.82 The role and importance of alloantibodies directed primarily against M H C class I has been studied using animal models and in clinical studies as well. 8 3' 8 4 Antibodies reactive with MHC II class antigens have been found in transplant recipients and have been shown to cause acute graft loss.85 Both CD4 (MHC class II 22 reactive and CD8 (MHC class I reactive) T cells are important to rejection and each have over- lapping or exclusive effector roles. Our current success at immunosuppression results largely from our ability to inhibit and control the changes within rejection reaction effector cells following immune stimulation. The initial attempts were very non-specific and involved total body irradiation. Those experimentsrevealed that the immune system could be destroyed and thereby made to accept the transplanted organ; however, the recipient remained defenseless to infection and rapidly succumbed to sepsis.86 Current methods to prevent graft rejection primarily involve the induction of energy in potentially alloreactive T cells through specific immunosuppressive agents.61 3.3. Chronic Rejection Transplant vascular disease (TVD) is one of the major causes of morbidity and mortality in long-term survivors of heart transplantation. T V D is the most serious clinical manifestation of chronic rejection process. Chronic rejection (CR), a process involving progressive developmentof fibrosis and vascular stenosis, is responsible for the majority of late allograft failures. The pathogenesis of CR is believed to be a cycle of recurrent immunologic injury followed by an injury repair process. During the injury repair process, vascular smooth muscle cells (SMCs) proliferate, migrate to form a neo-intima. The generation of myofibroblasts and synthesis of matrix lead to a scar, which, after repeated cycles of injury and repair, enlarges and compromises vascular flow. These SMCs preferentially express a number of genes that cause the secretion of extracellular matrix proteins. Some of these regulate cell motility. One of these is the hyaluronan (HA) receptor R H A M M . Interference with R H A M M : H A interactions after vascular injury may limit SMC proliferation and migration, thereby reducing the vascular narrowing associated with chronic rejection. 23 3.4.1. Histopathology of Hyperacute Graft Rejection The histopathology of hyperacute rejection is manifested by the following findings: 1. Thrombosis and edematous changes in the rejected organ 2. No pronounced infiltration of mono-nuclear cells 3. Deposits of immunoglobulin, complement C3 and fibrinogen (immunostaining) Antibody -mediated rejection may also occur without thrombotic occlusion. In this case, the histological pattern is a vasculitis which may lead to necrosis of individual cells of graft blood vessels. Because lymphocytes may be also involved, this process is referred to as acute vascular rejection. 3.4.2. Histopathology of Acute Rejection Histopathology of acute cardiac rejection is fully described and presented by histopathologic criteria summarized in ISHLT grading table (Grade 0 - Grade 4). Table No.l 3.4. Histopathology of Chronic Rejection The histopathology of chronic rejection is characterized by following findings: 1. Progressive fibrosis of endocardium, myocardium, epicardium, and epicardial coronary intima. 2. Increasing frequency of B-cells and plasma cells in endocardium, epicardium, and epi-cardial coronary intima. 3. Myopathic myocyte enlargment, myocyte nuclear prominence and hyperchromatism, and myocyte atrophy with interstitial and replacement fibrosis. 24 4. The Diagnosis of Cardiac Allograft Rejection Until 1978, the diagnosis of cardiac allograft rejection was made largely by summated R- wave voltage on the electrocardiogram.78 The presumption was that rejection was associated with the development of myocardial edema causing a loss of voltage. The introduction of transvenous biopsy of the heart by Caves in the mid -1970s, significantly increased the sensitivity and specificity of making the diagnosis of rejection, and has become the Gold standard for 87 RR establishing the diagnosis of rejection in cardiac transplant recipients. ' On the other hand, there have been a number of non-invasive markers of rejection that have been suggested over time for non-invasive diagnosis of rejection.78 This includes placement of catheters into the heart at the time of transplantation to directly record intramyocardial electrograms which can be read by telemetry while the patient is asleep to measure any decrease in voltage in the heart.89"91 This approach has been particularly applicable in children where a biopsy may be technically difficult, and has been shown to have high sensitivity and specificity in the limited trials to date.91 The second area of significant investigation has been the use of echocardiography to measure a number of parameters of systolic and diastolic function92. This approach has not been broadly adapted due to the impact of changes in preload and heart-rate on these measurements. Echocardiography is a primary method of following children, where alterations in posterior wall thickening are usually the primary indication to warrant invasive biopsy. A number of nuclear techniques have also been evaluated, including radio labeled 93 antimyosin antibodies, PET scanning, and MRI imaging to assess myocardial water edema . These tests have also not had significant clinical utility. Most recently, the rise in plasma concentration of Troponin T, a cardiac-specific isoform used to diagnose acute myocardial infarction, has been shown to have some correlations with cardiac rejection.94 25 The problem with all of these immunologic markers is that they were highly sensitive and specific when correlated with biopsy evidence of rejection only within the first three months 80 post-transplant, limiting the utility and diagnostic accuracy of these tests . 4.1. Cardiac Catheterization Cardiac catheterization is the general name for a group of procedures in which long, thin tubes (catheters) are placed in the heart and its adjacent blood vessels. The tubes may be hollow or solid. The hollow tubes allow measurements of pressures, injection of fluids such as X-ray dyes, and or withdrawal of blood. The solid tubes contain wires for recording electrical activity and or for pacing the heart. Specialized instruments, such as bioptomes, permits one to obtain a tiny piece of heart muscle for laboratory study. 4.2. Endomyocardial Biopsy Endomyocardial biopsy involves the use of a flexible bioptome to obtain tissue samples from the right (and occasionally left) ventricle of the heart. The heart is approached via a transarterial or transvenous heart catheterization. Endomyocardial biopsy results in a small tissue sample (average size 1 to 2 mm); multiple samples (usually 4 to 6) are required because pro- nounced topographic variations may be found within the myocardium. Endomyocardial biopsiesare typically performed for the monitoring of cardiac allograft rejection and anthracycline cardiotoxicity, and in detecting infiltrative disorders of the myocardium (i.e. myocarditis). 26 4.3. Classification of Allograft Rejection by Histopathologic Criteria The International Society of Heart and Lung Transplantation has established a biopsy grading classification, that is based on the amount of cellular (lymphocytic) infiltration and the presence or absence of myocyte necrosis.This classification divides biopsies into mild [(la and b) Grade 1], focal (Grade 2), diffuse moderate[(3a)Grade 3] or diffuse severe rejection [(3B and 4) Grade 4]. In essence, rejection severity is measured histopathologically by increasing numbers of infiltrating inflammatory cells and increasing number of inflammatory foci, associated with an increasing amount of parenchymal and vascular damage and, in most severe forms, often accompanied by an appearance of edema and evidence of hemorrhage. As such, the histopathologic severity of inflammation in acute rejection follows a logical geographical algorithm, progressing from perivascular and interstitial aggregates and, ultimately, to widespread mixed cellular infiltrates. 4.4. Artefacts The standard range of bioptome and biopsy-related artefacts apply to the study of specimens from heart allografts. Recollection of the normal constituents of the endomyocardium is essential to the process of evaluating biopsy specimens from allografts. These normal features include the endothelial lining, stromal fibrous proteins, like collagen and elastin, smooth muscle cells, fibroblasts, and pericytes, Thebesian venous channels, small arteries, arteriols and capillaries,myocardial fibres including Purkinje fibres, and resident must cells, lymphocytes and monocytes. Biopsy sites-the histopathology of biopsy site reflects the acute injury of the bioptome followed by healing and repair. Fresh microthrombus may be found overlying areas of acute 27 myocyte necrosis, focal hemorrhage and leukocytic infiltration when a repeated biopsy is performed within a few days of the previous biopsy. Endocardial fibrous tissue-may ensue from several healing processes including acute rejection, biopsy sites and mural thrombus. Adipocytes-are associated with the myocytes and connective tissue of the endomyocardium. The adipose tissue may be extensive at times and may masquerade as a site of biopsy-induced epicardial perforation. The extent of endomyocardial adipose tissue is determined in part by the use of corticosteroids in immunosuppressive therapeutic regimens, in part by a tendency to cardiac adiposity in some people, and in part by the process of healing upon repeated injury. Myocardial calcification may be found in biopsy specimens, or on autopsy examination of heart allografts. The significance of such calcification is unclear, although global myocardial ischemia at the time of harvest or later during hemodynamic instability may be a factor. Opportunistic infections - they may"masquerade"as alloreactive inflammatory process. Bacterial, fungal, viral, and protozoal infections may involve more than 50% of the transplant population. Systemic infections derived from respiratory and urinary tracts or skin are most common. Bacterial and fungal infections typically elicit a mixed inflammatory infiltrate pre- dominated by polymorphonuclear leukocytes. The infiltrate associated with viral or protozoal infections may be quite reminiscent of rejection. Quilty effect- the prominent, nodular, endocardial infiltrates described by Margaret Billingham are well known in cyclosporine treated patients95. These infiltrates are primarily mononuclear (B-lymphocytes) with occasional prominent p-lasma cells. Typically, there are well vascularized with blood vessels lined by prominent endothelium. The size of infiltrates varies 28 from tiny to very large and the border between the endocardial infiltrate and the underlying myocardium may be smooth and distinct or may suggest an "invasive" endocardial infiltrate. 29 II. H Y P O T H E S I S We hypothesize that M H C II expression on T-lymphocytes in peripheral blood is an indicator of rejection status in the allotransplanted heart The objective of this study was to investigate if M H C II DR - beta expression on, peripheral T lymphocytes correlates with rejection status in a porcine heterotopic allotransplanted heart model. 1. To design an experimental model for heterotopic heart transplantation and to identify 2 groups of animals (A - non-immunosuppressed; B- immunosuppressed) in this experiment. 2. To identify an accredible method for quantitative evaluation of cardiac biopsies. 3. To quantify the expression of M H C II DR - beta lymphocytes in peripheral blood. 4. To develop less invasive diagnostic method for early determination of rejection status in organs after the transplantation. 5. To identify the relationship between the grade of rejection and M H C II DR - beta lymphocytes in peripheral blood. III. O B J E C T I V E IV. S P E C I F I C AIMS 30 V. EXPERIMENTAL DESIGN Twenty-four 3-months old female domestic swine Sus scrofa domesticus,L., each weighing approximately 27 to 30 kg's, were used in this study. The animals were randomly subdivided into two experimental groups. The aim of the first experiment (group A)was to establish what correlation, if any, exists between acute allograft rejection and the upregulation of the expression of the M H C Class II histocompatibility molecule DR -beta on peripheral T lymphocytes in recipient animals that had not received immunosuppressive treatment. This control group (A) consisted of 6 age-matched donors and 6 age-matched recipients and did not receive immunosuppressive drugs prior to or post-transplantation.The aim of the second experiment (group B) was to determine what correlation, if any, exists between graft rejection and the expression of the M H C Class II molecule DR-beta on peripheral T lymphocytes in recipient animals that had received immunosuppressive treatment prior to and during the post- transplantation period until the time of sacrifice. Pilot study The heterotopic heart transplantation was performed in both immunosuppressed and non- immunosuppressed groups with ex vivo preservation times of the donor hearts between one and half to two hours at 4° C in hypothermic storage. Although surgical procedures are routinely performed in our laboratories, our project was proceded by a pilot study using eight experimental animals, four donors and four recipients. During the pilot study we had been developing the most reliable transplant procedure examinig following options: First approach. Upper -middle laparotomy the graft was implanted into the recipient's retroperional space, with the donor ascending aorta anastomosed end to side with the recipient's abdominal aorta and the donor pulmonary artery anastomosed end to side with the recipient's inferior vena cava. Second approach. The incision was made in the right flank of the abdomen between the iliocostal muscle 31 and external oblique abdominal muscle in order to prepare a „pocket" between the external and internal abdominal muscles for the graft. The donor-ascending aorta was anastomosed end to side with the recipient's abdominal aorta and the donor pulmonary artery was anastomosed end to side with the recipient's inferior vena cava. The second approach was chosen as a standard surgical procedure for our experiment. The reasons were: 1. Less invasive and therefore less risk for the postoperative complications for the animal. 2. Better conditions for postoperative care and monitoring of the animal. Assessment parameters included biopsies, blood sampling and monitoring o f vital and physiological signs. A l l animals were maintained in accordance with the guidelines of the Canadian Council of Animal Care under the supervision of the Animal Care Committee of the University of British Columbia. 32 VI. RATIONALE The rationale for the use of a large animal model were: 1. This was considered as a preclinical trial; hemodynamic and functional measurements are easier in large animals; 2. The amount of tissue and blood required for biochemical and pathophysiological analysis of this project can not be acquired from a small animal; 3. Only in the large animal, according to this type of project, is the evaluation and correlation of functional, biochemical, and morphological analyses possible. The rationale for using swine included: Swine have enzymatic similarities, such as the presence of tissue xanthine oxidase, amount of red and white blood cells, blood volume, to humans. 1. It was simple to control sex, age, type, and size for optimal modeling. 2. Our laboratory have had extensive experience with the use of swine for our other experimental models. The induced expression of M H C II -DR-beta molecules in peripheral T lymphocytes was used as a measure of T cell activation because in our previous studies, using the swine model of single lung transplantation, we observed that M H C II DR -beta molecules increase their surface expression in the host peripheral T-lymphocytes97. Other studies proved that increased expression of M H C class II antigen occurs during cardiac and lung allograft rejection.96'98 The expression of this antigen in T -cells influences diverse aspects of the immune response system including stimulation of the mixed lymphocyte reaction99, interaction of T cells - T cells and T cells-monocytes100"1, and the ability of T cells to present antigen in alloantigen-specific immune response136 . M H C II antigen has been shown to be upregulated on the surface of activated 33 circulating T cells during the rejection episode102 . Determination of circulating M H C II - D R + T cells are proposed to complement biopsy in renal transplantation and is shown to be very 102 important for clinicians if histology is equivocal. In solid organ transplantation the histological analysis of allograft biopsies remains the "gold standard" for diagnosing rejection.103 Therefore we have chosen the endomyocardial biopsy evaluation as a tool for the assessment of grade of rejection. V I I . MATERIALS AND METHODS 1. S u r g i c a l T e c h n i q u e s In the donor procedure using the midline longitudinal mediastinal sternotomy, the heart is harvested with large vessels including the aortic arch, pulmonary arteries up to the entrance to the lungs (hilum), pulmonary veins and their branches, inferior and superior vena cava, and azygos and hemiazygos veins had been ligated. After the vessels were mobilized, the place for aorta cross-clamping was prepared. Heparine sodium (300 IU/kg) was administered intravenously. Following the procedure of anterograde crystalloid cardioplegia, the cannula was inserted into the ascending aorta and supplied with cardioplegic Tyer's solution. After cross-clamping the aorta, IVC/SVC were ligated immediately and 1000 mL of cold Tyer's solution was administered to arrest the heart. All the vessels were ligated. The heart was removed from the chest and placed in 1000 ml Tyer's solution maintained at 4° C. In the recipient procedure the incision was made in the right flank of the abdomen between the iliocostal and external oblique abdominal muscle in order to prepare a "pocket" between the external and internal oblique abdominal muscle, for the graft. Through the retro- peritoneal approach, IVC and the descending (abdominal) aorta were mobilized. After the 34 donor's heart was prepared, all the branches of the aorta and pulmonary artery were ligated, leaving the main stump of the aorta. The animal was heparinized (300 IU/Kg) and the descending aorta of recipient was partially clamped and anastomosed to the ascending aorta of the donor heart. The sidewall clamp was removed and air was evacuated from the hole in theascending aorta of the donor heart. The hole was ligated. The same procedure was repeated with the anastomosing pulmonary stump to the IVC. The cross clamp was removed, then the heart was reperfused and subsequently defibrillated. A 6 mm diameter Hortex prosthesis was anastomosed to the right atrium of the donor heart and to the subcutaneous tissue of the posterolateral abdominal wall area. It served as a "tunnel" for the introduction of the bioptome into the right side of the engrafted heart. The wound is" drained through the contra-aperture underneath. The neck approach was taken in the external jugular vein introducing the 2-3 lumen catheter for blood sampling and administration of drugs. The tip of the catheter was tunneled just under the skin to the posterior triangle of the neck near the ear where it was externalized. 2. Positioning of the Bioptome for Endomyocardial Biopsy This procedure consists of the following steps. The bioptome is inserted via the external jugular vein, the ima vein, and the superior vena cava into the right atrium with the tip pointed toward the lateral wall of the right atrium. At the level of the mid-right atrium, the bioptome is rotated anteriorly approximately 180° and advanced through the tricuspid valve apparatus toward the right ventricle. The bioptome is advanced to the interventricular septum with the jaws opened. 35 3. Technical Considerations Proper handling of endomyocardial biopsy specimens obtained by standard bioptome catheters from the right ventricular septum is imperative in efforts to issue accurate diagnoses. Three to six samples of endomyocardium, each with a minimum dimension of 1.5 to 2 mm, should be obtained and fixed in 10% neutral buffered formalin. Tissue is processed for paraffin embedding and serially sectioned at 4 microns thickness throughout the block. Five slides with three to four levels each are stained with H&E; with two complementary slides stained with Masson's trichrome. This is the minimum recommendation for adequate assessment of rejection. Fibrotic samples, fatty samples, and clots can be readily distinguished from endocardium with considerable myocardial tissue. Electron microscopy and immunohistochemistry are not necessary for the pathologic diagnosis of rejection, although they may be included in certain research protocols. The nature of the specimens available for diagnostic pathologic examination will change considerably over time in a given patient. It will include many chronic changes. Such changes may indicate that the pathologist will stay in constant contact with transplant physicians regarding each patient's clinical progress. 3. Parameters of Assessment The investigational methods included blood sampling, endomycocardial biopsy collections and physiological monitoring. The peripheral blood samples were centrifuged to separate white blood cells for the further measurements which were done by two-color flow- cytometry analysis obtaining M H C II DR-beta intensity on peripheral T-Lymphocytes. This was done to assess cyclosporine levels in blood, using fluorescence polarization, and competitive binding radioimmunoassay, to evaluate CBC and differential using cell counter, and to assess creatinine levels in peripheral blood by calorimetric analysis using picric acid. Endomyocardial 36 biopsies were taken from the free wall, apex and septum in the right ventricle. Physiological monitoring included E C G , heart rate, respiratory rate, temperature, behavior, food and water intake. Both blood sampling and physiological monitoring were performed on daily basis and EMBs were taken on day 1 (day of transplant) and day 5 (last day after transplant). 37 5. Blood Sampling Protocol 1. The catheter tip was cleaned with an alcohol swab before and after each insertion. 2. Blood was obtained from the distal lumen of the brown-topped catheter. 3. 20 cc and 10 cc syringe with an 18-gauge needle were used. In the event of a problem, i.e.,an agitated pig, a short extension catheter (butterfly) was used for safe blood sampling to avoid personal injury or the injury to the pig. 4. Approximately 15 cc of heparinized saline (Heparine and 0.9% NaCl physiological solution) was drawn and used to flush the catheter. 5. A 20 cc syringe was used for withdrawing blood, once inserted into the catheter. Clear the syringe with one or two withdrawals and reinsertion of blood. Fill three 7 cc's yellow- topped ACD tubes for MHC II -T-lymphocyte monitoring. 6. Six to eight cc of blood was withdrawn. Approximately 2 cc of this blood was placed into two EDTA 3cc purple-topped tubes. These blood samples were used for determining CBCD (differential) and cyclosporin A levels. 7. The remaining blood was placed into 10 cc SST tube (golden-topped). This sample was used for the determination of urea, bilirubin, ALT, AST, and creatinine (for liver and kidney function) content.. 8. After the blood was drawn, catheter was flushed with a bolus of heparinized saline under positive pressure, using the 20 cc syringe. 9 . 2 cc of pure heparin was used as a heparin "lock" to prevent occlusion of the catheter from thrombi or coagulation. After harvest, all peripheral blood samples were centrifuged through a Ficoll Hypaque gradient in order to crudely separate white blood cells for further measurements. 38 6. Intravenous Drug Administration 1. The catheter tip was cleaned with an alcohol swab after each use. 2. The proximal (white topped) lumen of the catheter was flushed with 5 to 10 mL heparinized saline after each drug administration. 3. A heparin "lock" was placed after the last drug administration (2 to 3 mL of pure heparin in the 5cc syringe). 7. Immunosuppression / Drug Regimen 7.1. Prophylaxis Commencing both donor and recipient, Ceftin (Cefuroxime acetyl) 500 mg, BID, p.o. was administered 2 to 3 days before surgery as a prophylactic treatment. 7.1.1. Day 1 On day 1 (day of surgery) antibiotics such as Ancef, Flagyl and Gentacidin had been administered in the following order: Ancef (Cefazolin sodium) 15 mg /Kg, BID,I.V. First dose was administered after induction of anesthesia. The second dose of the identical amount, I.V., was administered at 11:00 p.m. Optionally, this dose was administered post-surgically via L M . route. Flagyl (Metronidazole) 20 mg/Kg - commencing on the day of surgery. Gentacidin (Gentamycin sulfate) 2-4 mg/Kg (LM. or LV). This antibiotic was used as an adjunct only if infection was suspect/occured. 39 7.1.2. Immunosuppressive Drugs Imuran (Azathioprine) 3 mg/Kg, p.o. was administered prior to induction of anesthesia. Infusion time for administration of CS-A (6 mg/Kg) had been over 2 to 4 hours. Blood sample was taken 30 minutes after completion of CS-A administration to determine peak level of CS-A in peripheral blood.Solumedrol (Methylprednisolone): TD = 14.3 mg/Kg x Mr ( TD = total dose; Mr = the mass of the recipient animal) had been administered according to following regimen on the day 1 (day of surgery): • Vi TD administred LV. at induction of anaesthesia • lA TD administred LV. at reperfussion of the implanted organ • lA TD administred LV. 8 hours after surgery; approximately 11:00 p.m. 7.2. Day 2 07:00 a.m. the following drugs had been administered: • Cefazolin sodium, 15 mg/Kg, LV. • Flagyl, 20 mg/Kg, p.o. • CS-A, 6 mg/Kg, LV. (infusion time over one hour; glass bottles) • Methylprednisolone, 1/8 TD, LV. • Azathioprine, 3 mg/Kg, p.o. 03:00 p.m. • Methylprednisolone, 1/8 TD, LV. 40 07:00 p.m. • CS-A, 6 mg/Kg,I.V. (infusion over an hour, glass bottle) • Cefazolin sodium, 15 mg/Kg, I.V. 7.3. Day 3 07:00 a.m. the following drugs had been administered: • CS-A : 5.5 mg/Kg; I.V. • Azathioprine : 3 mg/Kg; P.O. • Methylprednisolone : 1 mg/ Kg; I.V. • Metronidazole : 20 mg/Kg, P.O. • Cefazoloin sodium : 15 mg/Kg; I.V. 07:00 p.m. Cyclosporine in infusion over an hour and Cefazoline sodium had been administered: • CS-A : 5.5 mg/ Kg, I.V. • Cefazolin sodium : 15 mg/Kg; I.V 7.4. Day 4 On the day 4, the drug regimen protocol was identical with drug regimen for day 3, by which the levels of CS-A were maintained at the concentration of 350 - 450 ng/mL in plasma. 7.5. Day 5 Day of sacrificing the experimental animals (no drug administration was used) 41 8. Flow Cytometry 8.1. Procedure for Cell Preparation Blood samples (18 mL) were collected in an acid citrate dextrose (ACD) vacutainers and diluted with equal volume of phosphate buffered saline (PBS). The diluted blood was slowly added to 10 mL of Ficoll-hypaque (Gibco/BRL) in 15 mL sterile tube. Samples were centrifuged for 30 min. at 1500 rpm and washed twice with T C 199 mixed with 2% Fetal calf serum (FCS) for 15 min. at 1500 rpm. 8.2. Procedure for Freezing Cells Sterile technique was used throughout the procedure. The minimum cell concentration for freezing was 5x 105 cells/mL. The maximum was 1x10x8 cells/mL. For our purpose the amount 2x106 of cells/mL was chosen. Cell suspension were frozen in 1ml aliquots. FCS and DMSO were added as 20% and 10% of the total volume. Example: cell count 2.25 x 107 cells /mL volume 0.98 mL Total cells 2.21xl07 cells If the volume of the cell suspension was increased to 2 mL, the cell concentration would be 1.1 x 107 cells/ml. Since cell suspensions were frozen in 1 mL aliquots: 20% FCS 0.4 mL 10% DMSO 0.2 mL Cell suspension 1.4 mL Total 2.0 mL (i.e. 2 aliquots) 42 The cell suspension was mixed and placed on ice. Sterile vials were stored at -70 C. When the cell suspension was chilled to 4° C, DMSO was added drop-wise while mixing into the cell suspension. The cells were dispensed into the vials. The vials were placed into cryo-container and placed in -70° C freezer. 8.3. Antibodies and Di lu t ion Assays 8.3.1. Antibodies The primary monoclonal antibodies were obtained from V M R D Inc., Pullmann, Washington. Four different types of antibodies were used: 1. anti-CD3 antibody (8E6;i.e. T-lymphocyte marker) 2. anti M H C II -DR-beta (MSA30) 3. anti-CD 45 (leukocytes) 4. antimonocytic antibody [Swine workshop cluster (SWC 1)]. The Gout anti-mouse (GAM) were obtained from Caltag, Inc.,and included: 1. FITC labelled IgG 1 (used for CD3 staining) 2. PE labelled IgG 2a (used for M H C II staining) 3. FITC labelled IgG and IgM ( used for leukocyte and monocyte staining). 8.4. F A C S Analysis : Two million cells were used for each antibody staining. Cells were incubated with primary antibodies at 4° C for 30 minutes, and then stained with secondary antibodies at 4 degree 43 Celsius for 30 minutes. After each incubation, cells were washed with 2% TCI99 for 15 minutes and centrifuged twice at 1500 rpm. A maximum of 10,000 cells were counted by the flow cytometer. Leukocyte population was identified using swine anti-CD45 and anti-monocytic antibodies. Two secondary goat-anti mouse antibodies labeled with phycoerythrin (PE) and fluorescein isothyocyanate (FITC) were used for staining the lymphocytes bound to the primary antibodies. 8.5. Evaluation of Endomyocardial Biopsies ISHLT modified evaluating criteria for determining the Grade of rejection in histopathological specimens. 8.6. Histopathology of the Transplanted Hearts 8.6.1. Specimen Preparation 1. Three to six EMB samples were obtained from the right ventricle samples of EMB were obtained from right ventricle (septum, free wall, apex) 2. The minimum dimension of the sample were 0.15 to 0.2 cm 3. Samples were fixed in 10% neutral buffered formalin (formaldehyde) 4. Tissue was processed for paraffin embedding and serially sectioned at 4 microns thickness 5. Slides were stained with H&E and two complementary slides are stained with Masson's trichrome (This was a minimum recommendation for adequate assessment of rejection). 6. Slides were evaluated at the pathology laboratories at Saint Paul's Hospital, Vancouver. 7. The specimens were evaluated by two independent pathologist, both blinded, according to modified ISHLT scale. (Table 1). 44 Table N o . 1 ISHLT Grade Histologic Appearance Interpretation 0 No cellular infiltrate No acute rejection 1 a 1 focus cellular infiltration without myocyte necrosis Mild acute rejection lb > 1 focus of cellular infiltration without myocyte necrosis Mild acute rejection 2 1 focus of cellular infiltration with myocyte necrosis Focal moderate acute rejection 3a 2 foci of cellular infiltration with myocyte necrosis Diffuse moderate acute rejection 3b/4 >2 foci of cellular infiltration with myocyte necrosis and myocardial edema Severe acute rejection VIII. Statistical Analysis Statistical analysis was performed by non-parametric analog of unpaired t-test (Mann Whitney U-test) and multiple regression analysis for between a comparison of groups and correlation of different variables, respectively. The data are presented as Mean (plus - minus) standard deviation. P<0.05 is considered to be significant. The use of parametric analysis requires the data to have normal distribution and equal variances ion groups. Mean and standard deviation are the key elements of parametric statistics. Since our data consisted of six observations per group only, one can not rely on parametric statistics because such a small sample of data does not necessarily demonstrate whether the distribution is normal or skewed. If the data deviates from normality, interpreting the mean and 45 standard deviation in terms of normal distribution produces a misleading picture. Therefore, the type of analysis such as non-parametric statistics, which use the ranks of observations rather than the actual measurements become suitable. Non-parametric statistics retain much of the information about the size of responses without making any assumptions about the distribution of the data. Non-parametric methods provide one with the opinion of assuming as little as possible when analyzing the data. Non-parametric statistical methods are 95% as powerful as parametric methods when the data are from normally distributed populations and more reliable when the data are not from normally distributed populations. Multiple regression analysis is always being used under the circumstances that not allow the scientist/biostatistician to predict the type of correlation and its development. I.e. there is no possibility to predict if the further relationship will be linear, logarithmic, or exponential.142 46 IX. Results In the control group (A), results of our investigation of non-immusuppressed animals demonstrated no significant difference in the first two days (first and second day) after transplantation in comparison to the time before the transplant. However, the M H C II expression on peripheral T - lymphocytes increased on days 4 and 5 indicating a significant difference in comparison to pre-transplantation time and the first two days after transplantation. On the other hand, M H C II expression on peripheral T-lymphocytes in animals with immunosuppressive therapy showed a different feature. The M H C II expression on peripheral T-lymphocytes had a tendency to decrease on day 3 after transplantation and demonstrated a significantly different level on day 5 after transplantation in comparison to pre- transplantation period of time. Figure 1 depicts the comparison between the non-immunusuppressed and immuno - suppressed groups (A and B) on day 5. The M H C II expression on peripheral T -lymphocytes manifests a significant difference [(P< 0.03) T-test] between groups A and B on day 5 after the transplantation. (Figure 1). 47 50 40 c 0) 30 c 20 o X 10 0 Comparison of MHC II expression between Groups A and B - •— non-immunosupp. - » immunosupp. P-ifJ.03 Pretransplant Day Figure No . l Results of the morphological assessment of the heart tissue obtained from ventricular wall, septum and apex are depicted in figure 2. These demonstrate a significant difference [septum: (P<0.05); apex: (P<0.03); free wall: (PO.01)] in the grade of rejection between the groups A (red) and B (blue) [immunosuppressed and non- immunosuppressed animals] in all three regions of heart. Histological findings (rejection grades) for groups A and B are shown in the tables 2 and 3. 49 Figure No. 2 C .2 3 o 0 'a? tc 2 o <1) TJ «J CD ' 0 P<0.01 P<0.05 T P<0.03 13 Immunosuppressed G . • Non-immunosuppressed G . FW Septum Apex Table No. 2 (Grade of rejection in non-immunosuppressed animals- Group A on Day 5) Group A Apex Free wall Septum Mean 4.033 4.006 3.752 Stdeviation 0.582 0.321 0.594 St. error 0.238 0.131 0.242 Biopsy 1. 5.000 4.000 4.500 Biopsy 2. 4.000 3.400 3.375 Biopsy 3. 4.000 4.125 3.166 Biopsy 4. 3.500 4.350 4.500 Biopsy 5. 3.400 4.000 3.570 Biopsy 6. 4.300 4.180 3.400 Table No. 3 (Grade of rejection in immunosuppressed animals- Group B on Day 5) Group B Apex Free wall Septum Mean 1.500 1.117 1.883 Std. Deviation 0.593 0.204 0.884 Std. Error 0.242 0.183 0.361 Biopsy No. 1 1.000 1.000 3.000 Biopsy No. 2 2.600 1.000 3.000 Biopsy No. 3 1.600 1.000 1.500 Biopsy No. 4 1.000 1.000 1.000 Biopsy No. 5 1.500 1.500 1.500 Biopsy No. 6 1.300 1.200 1.300 Since the aim of the study was to evaluate the relationship between the expression of M H C II on the peripheral T-lymphocytes and the grade of rejection in the transplanted organ, we performed the regression analysis in order to define this relationship.The regression analysis for both A and B groups had shown significant correlation between the histological findings from free wall and apex and M H C II expression on peripheral T-lymphocytes in our study. Regression analysis between the histological findings from septum and M H C II expression on peripheral T- lymphocytes did not reveal any significant correlation at all. Values (Groups A and B together) of M H C II expression on the peripheral T - lymphocytes (on the Y axis) were plotted against the grade of rejection from the free wall heart tissue (X axis). Results of this regression analysis demonstrated a significant correlation between the expression of M H C II on the peripheral T-lymphocytes and degree of rejection ongoing in the free wall of the heart. (R2 = 0.683; PO.01); Figure 3; Table 4,5. Figure No.3 M H C I I R e g r e s s i o n P l o t 1.5 2 Free Wall 2.5 3.5 52 Results of regression analysis performed on combined data (both groups A and B) where M H C II expression on the peripheral T-lymphocytes (Y axis) had been plotted against the grade of rejection from apex (X axis) demonstrated a significant correlation between the expression of M H C II on peripheral T-lymphocytes and degree of rejection. (R2 = 0.734; R = 0.857; PO.01 ).Figure 4; table 4,5. Figure N o . 4 Regression Plot 53 Table No. 4 MHCII expression in non-immunosuppressed animals - Group A Group A Day I Day II Day III Day IV Day V Mean 21.383 18.567 20.450 27.932 39.550 Std. deviation 6.667 8.347 11.011 15.383 16.288 Std. Error 2.722 3.408 4.495 6.280 6.650 Animal No. 1 30.700 26.400 11.500 54.800 69.300 Animal No. 2 18.100 22.600 39.100 30.900 38.700 Animal No. 3 28.200 28.100 28.500 30.800 39.550 Animal No. 4 20.800 8.300 16.800 23.993 28.500 Animal No. 5 14.100 10.500 14.000 16.300 21.700 Animal No. 6 16.400 15.500 12.800 10.800 39.550 Table No. 5 M H C II expression in immunosuppressed animals - Group B Group B D a y l Day 2 Day 3 Day 4 Day 5 Mean 24.833 23.529 25.278 22.960 18.542 Std. deviation 13.776 10.937 10.155 16.193 4.201 Std. Error 5.624 4.465 4.146 6.611 1.715 Animal No. 1 46.000 23.500 24.200 8.200 17.900 Animal No. 2 14.000 2.600 25.400 22.600 23.800 Animal No. 3 12.000 30.200 30.400 53.600 14.000 Animal No. 4 29.000 33.800 15.210 21.500 15.900 Animal No. 5 34.000 26.180 41.820 10.770 23.650 Animal No. 6 14.000 25.270 21.090 16.000 14.640 X . Discussion Survival after cardiac transplantation has improved steadily over the past decade, with mortality at 1 year falling to as low as 10% in many centres.103"4 This improvement in survival is due to better, more specific immunosuppressive agents such as cyclosporine and antilymphocyte antibodies. 9 5 ' 1 0 4 " 5 Furthermore, early diagnosis of rejection by surveillance of endomyocardial biopsy, contributed to this improvement.106"7 This early diagnosis is exceedingly important, and most clinicians experienced in transplantation are aware of the problem (very high mortality in patients with acute rejection associated with consequent allograft dysfunction). Unfortunately, current procedures for this early diagnosis involves the purchase of up to twenty biopsy procedures for each patient, within the first year after cardiac transplantation. Furthermore, patient discomfort and potential risk result from the multiple invasive procedures. Accordingly, the search for non-invasive techniques for the diagnosis of cardiac allograft rejection continues in order to invent a reliable non-invasive technique for graft rejection diagnosis with full benefit for patients and health care professionals. A perfect screening test for cardiac allograft rejection should be easy to administer repetitively, and should be non-invasive, low cost, and most importantly, it should have a high sensitivity. The need for specificity varies, depending on implications for further diagnosis. In the diagnosis of cardiac allograft rejection, the screening test must be nearly 100% sensitive, given the potentially fatal implications of missing acute rejection. Even if specificity is only 103 50%, this would result in a decrement of one half of the biopsies performed on a routine basis. Three general classes of possible screening tests for cardiac rejection are cardiac functional assessment, biochemical/immunological assays and myocardial imaging. It has long been believed that systolic function as assessed by ejection phase indexes is neither sensitive nor specific for allograft rejection.108"9 This has led to several studies investigating the use of diastolic indexes of left ventricular function as being potentially more sensitive in this 55 diagnosis.110 It was first demonstrated that echocardiographic derived indexes of diastolic function were abnormal in patients with acute allograft rejection. Later Derumeaux et a / 1 1 0 proved that Doppler tissue imaging, using ejection fraction and diastolic myocardial wall velocity as diagnostic variables, could be a new tool for non-invasive acute rejection diagnosis. Stork et a/" 1 proved that sonographic evaluation of L V diastolic function PEV (peak velocity), PHT (pressure half-time), VTI- (velocity time integral) of early mitral flow, and IVRP (isovolumetric relaxation period) helps to detect cardiac rejection early and decreases the frequency of myocardial biopsy. In transplant recipients, significantly higher values than in 22 age-matched healthy controls were found for PEV (71 versus 56 cm/s; P< 0.01), PHT (51 versus 43 ms; PO.001), VTI-E (72 versus 57 mm; PO.001), and IVRP (90 versus 73 ms; PO.001). During rejection, heart rate increased significantly from 78 to 91 beats per minute (PO.01). Furthermore, a significant decrease was found for PEV from 73 to 63 cm/s (PO.01), for PHT from 52 to 40 ms (P< 0.001), for VTI-E from 75 to 61 mm (PO.001), and for IVRP from 90 to 74 ms (PO.001) during cardiac rejection. Thus, sonographic evaluation of L V diastolic function helps to early detect cardiac rejection and to decrease the frequency of myocardial biopsy but can not be used for a detection for certain grade of rejection. One exclusive study suggested that pacemaker evoked T-wave amplitude (ETWA) may be a sensitive non-invasive marker of cardiac allograft rejection. This clinical study conducted on 45 recipients with median duration of follow-up of 129 days showed the sensitivity value of 55% and specificty of 62%. 1 1 2 Another study showed that there are ECHO detectable differencesbetween rejection and non-rejection hearts. Since variations of ECHO variables are large, rejection may not be determined by ECHO alone. Thus a biopsy may still be required for 113 final diagnosis of rejection especially in patients with confusing clinical findings. Subsequently, several scientists114"16 demonstrated, that diastolic functions were prolonged in rejecting patients. These groups reported sensitivities from 78% to 88%).'14 Another 56 study, using radionuclide ventriculographic indexes of diastolic function reported similar results. 1 1 8 In addition, contaminating factors such as donor-recipient size matching may play an important role in diastolic properties of the allograft. 1 1 8 - 1 9 120 Few animal studies had been carried out. Everett had conducted two different studies. The first study demonstrated that intramyocardial unipolar peak-to-peak amplitudes obtained from plunge electrodes in the canine orthotopic heart transplant model are highly sensitive and specific (100%) for diagnosing rejection. The second study showed that R-wave amplitude analysis remains an accurate non-invasive means for the early detection of cardiac allograft rejection and should allow more selective use of EMB in the heterotopic heart transplant canine model.1 2 1 Imaging and characterization of the myocardium to diagnose rejection have been 122 123 attempted including echocardiography tissue characterization, backscatter analysis, indium labeled white cells, 1 2 4 and magnetic resonance imaging.125 FW Smart126 reported that serial magnetic resonance imaging with monitoring of change in signal intensity is a potential, non- invasive method of assessing clinically significant tissue rejection in patients with heterotopic hearts that are stable more than one month after transplantation. Although preliminary results in animal models have been promising, the results in patients have yielded sensitivities that are inadequate. Detection of myocardial rejection is difficult in patients with heterotopic heart transplantation because of the complex vascular anatomy present after transplant surgery. To determine whether magnetic resonance imaging might be useful for the assessment of heart rejection, eight patients with heterotopic heart transplantation were serially studied on 27 occasions In 30% instances a significant change in the magnetic resonance imaging signal occurred without clinical or biopsy evidence of rejection and vice versa. Thus, this study could be evaluated inadequate because of high degree of false positive or false negative results. 57 The development of hybridoma technology by Kohler and Milstein 1 2 7 in 1975 not only led to their receiving of Noble prize but also to the development of monoclonal antibodies currently in clinical use.103"4 One of the antibodies is the monoclonal antibody to cardiac myosin developed by Khaw and colleagues. The antibody itself or its Fab fragment has been found to myocytes in which the sarcolema is no longer intact and when labeled with Indium 111 has been demonstrated to be useful in the diagnosis of myocardial infarction128 and myocarditis. Yasuda T. Ballester, and Frist 1 2 9 - 3 1 have extended their experience using indium labeled antimyosin antibodies in the diagnosis of acute cellular cardiac rejection. Average values for sensitivity in this studies were 90% and for specificity only 60%. Frist et al.131 conducted 20 studies of 7 days to 9 years after transplantation, to evaluate antimyosin imaging as non-invasive means of detecting human cardiac rejection, the Fab fragment of murine monoclonal antimyosin antibodies was labeled with Indium-Ill and given intravenously to 18 patients (age 45 +/- 12 years). The sensitivity, specificity, and overall accuracy of the technique were 80%. Ballester and colP2 in their initial experience of 53 studies in 21 patients, an abnormal antimyosin uptake ratio (1.55) yielded a sensitivity of 95% for the diagnosis of rejection requiring treatment (moderate or severe rejection). The specificity was quite low (29%), with a majority of patients with no rejection also exhibiting antimyosin uptake ratios out of the normal range. In a follow up study of patients at least one year after cardiac transplantation, a negative antimyosin antibody scan assured the absence of rejection requiring treatment (sensitivity=100%), with four of the eleven patients with positive scans demonstrating clinically significant rejection (specificity=33%). Specificity, as anticipated, increased with an increasingly abnormal scan. Thus, both early and late biopsies after cardiac transplantation, appears to be nearly 100% sensitive but not specific. Based on these data, approximately one third of all biopsies on stable patients could therefore be avoided.133 58 The third major area of investigation has attempted to directly assay for increases in allo- immunologic activity. Study conducted by RG Masters^4 and colleagues suggested that brain natriuretic peptide (BNP) plasma levels might be a potential screening test for acute cardiac rejection. Generally, the plasma level associated with a rejection period is higher than 400 pg/mL 133 35 at the stage of acute mild rejection (lymphocytic infiltrate without myocyte necrosis). " With the further understanding of immunologic activation, lymphocytes subsets, and activation markers, along with the development of flow cytometry techniques, several groups have investigated the possibility that the expression of activated antigens on circulating lymphocytes 136 8 might predict rejection. Although Garner and his colleagues reported a sensitivity of 94% for the detection of rejection using cytoimmunologic monitoring, his results have not been confirmed by Roodman and May. McGhie 1 4 0 conducted the canine study using 111 Indium labeled antibody. This study proved that radiolabeled monoclonal antibodies to M H C II antigen can detect cardiac allograft rejection in this large animal model. Carlquist and his colleagues140 in their study on humans found that there is not highly significant correlation between class II antigen and interleukin -2-induced lymphocyte proliferation during acute cardiac rejection (35%-65%). In 1998 Winkler and col. 1 4 1 conducted a study analyzing M H C II expression on circulating T cells by flow cytometry in patients with acute rejection after organ transplantation. Peripheral lymphocytes obtained during serial blood drawings from 206 patients after kidney, heart or liver transplantation were analyzed for H L A - DR expression on CD8+T cells by two-color cytometry. Patients were investigated during stable course as well as during episodes of graft rejection or infection. The immunosuppressive the HLA-DR expression pattern observed. The significant increase in HLA-DR expression was observed during acute biopsy-proven graft rejection. In contrast to patients after liver transplantation, in kidney or heart transplant recipients with acute graft rejection the increase in 59 HLA-DR expression failed to reach statistical significance. In addition to this, an increase in HLA-DR+CD8+T cells was also detectable during bacterial or viral infections. As mentioned above, in the past two decades many animal experiments investigating the relationship between the immune response to rejection and the grade of rejection have been conducted. The purpose of these studies was to find a method of early detection of acute, subacute and chronic stage rejection of transplanted organs. Current detection methods still depend on readings done by transplant pathologists. Three general classes of possible screening tests for cardiac rejection are cardiac functional assessment, biochemical/immunological assayes and myocardial imaging. Few studies had proven that increased level of M H C II antigen can detect cardiac allograftbeta antigens can up-regulate after the transplantation . In our research project, the goal was to find a method to detect early stage of rejection, i.e. within the first five days, which was less invasive than current practices, caused less danger and discomfort to the patient and was more economical for medical institutions to perform.In our study we have proven that: 1. The M H C II expression on peripheral T-lymphocytes between the non- immunosuppressed and immunosuppressed groups (A and B) demonstrates a significant difference (P<0.03) on day 5. 2. Results of the morphological assessment of the heart tissue's ventricular wall, septum and apex demonstrate a significant difference in the grade of rejection between the non-immunosuppressed and immunosuppressed groups (A and B) on day 4 after transplantation [free wall: (P<0.01); septum: (P<0.05); apex: (P<0.03)]. 3. There is a significant correlation between the grade of rejection and M H C II expression on T-lymphocytes in peripheral blood: 60 A. Regression analysis of combined data (Groups A and B) between M H C Ilexpression and grade of rejection from the free wall [ R =0.683; P<0.01]demonstrated a significant correlation B. Regression analysis between M H C II expression and grade of rejection in non immunosuppressed animals (Group A) demonstrated a significant correlation [PO.01; R 2 =0.827] C. Regression analysis of combined data (Groups A and B) between the M H C II expression and the grade of rejection in apex demonstrated a significant correlation as well [PO.01; R 2 =0.734]. In summary, with this information provided by a simple blood test we were able to determine the grade of rejection and hence the quantity of immunosuppressive medication required to treat the patient. We were also able to easily monitor the effectiveness of that medication and the change in the grade of the rejection of the transplanted organ. The animal study has been conducted for a relatively short period of time (5 days per animal - 24 animals in total). Because of financial constraints we were not able to keep the animals for more than a five- day period. The results of this short study were so encouraging that a longer study of either small or large animals followed by clinical trials would be required to investigate the long term effectiveness and feasibility of this new diagnostic tool. This new technique is less invasive and very safe for the patient. Based on our results the technique is highly sensitive and specific. The current diagnostic techniques have, in average, an 80% sensitivity and specificity. Our results predict the possibility of almost 90% sensitivity and specificity for this new technique. It would be possible to extend this study for detection of rejection in transplanted organs such as liver, kidneys and pancreas. In addition the cost effectiveness of this technique could be of great 61 interest to medical institutions. For the above reasons we would highly recommend continuation of this study. 6 2 XI. Conclusion During the early stages after transplantation (day one to day five) there is a significant correlation between the grade of rejection and M H C II expression on peripheral T-lymphocytes. Based on the results of this study it can be speculated that: 1. This method can be used clinically as a less invasive indicator of rejection for patients after transplantation. 2. This method can be used to modulate immunosuppressive therapy more vigorously and cost- effectively. 3. Early deduction of rejection and modulation of immunosuppressive therapy with this less invasive and less costly method may be beneficial for the transplant patients and costless for the health care system. 63 XII . Bibliography 1. Balk A H et al. Effect of adopting a new histological grading system of acute rejection after heart transplantation. Heart. 1997; 78 : 603-60 2. Billingham M E et al. A working formulation for the standardization of nomenclature in the diagnosis of heart and lung rejection: Heart Rejection Study Group: the International Society For Heart Transplantation. J Heart Transplant. 1990; 9:587-593 3. Winters GL, McManus B. 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