PEDIATRIC TRANSPLANTATION

LIVER TRANSPLANTATION: CURRENT MANAGEMENT

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PEDIATRIC TRANSPLANTATION Jorge Reyes, MD, and George V. Mazariegos, MD

The history of clinical transplantation in children has spanned more than 30 years. Marked by a remarkable struggle of both patient and surgeon, this pioneering association was exemplified by the first attempt at human liver transplantation on a 3-year-old boy with biliary atresia in 1963, and then the first successful human liver transplant in 1967 on a 1-year-old child with hepatoma.66,73 Essential components of the success in this field have involved advances in organ preservation, surgical techniques, and preoperative and postoperative care; however, only with the acquisition of deeper insights into transplant immunology did consequent improvements in immunosuppressive regimens and treatment of allograft rejection accomplish acceptable patient survivals with successful engraftment of the kidney,”hliver,hhheart,” lung,I4 pancreas,29 intestine:* multivisceral,7*and bone marrow allograft^.^, 21 The immunologic nature of allograft rejection, elucidated by Medawar (Nobel Laureate, 1960) in 1944,37 demonstrated that disruption of the alloactivated T-cell response could result in prolongation of allograft survival. Observations compatible with this concept were reported in 1963, when combined therapy with azathioprine and prednisone was introduced for kidney tran~plantation.~~ This fundamental therapeutic strategy would then be applied to the grafting of other organs, including the liver, heart, lung, heart and lung, pancreas, and intestines. The ability to subsequently reduce or sometimes even stop treatment was thereafter confirmed in cases of transplantation of the liver and other organs. The Supported in part by research grants from the Veterans Administration and Project Grant No. DK-29961 from the National Institutes of Health, Bethesda, Maryland.

From the Thomas E. Starzl Transplantation Institute (JR, GVM); the University of Pittsburgh School of Medicine (JR, GVM); and Children’s Hospital of Pittsburgh (JR), Pittsburgh, Pennsylvania

SURGICAL CLINICS OF NORTH AMERICA VOLUME 79 * NUMBER 1 * FEBRUARY 1999

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escape from rejection of allografts with or without the aid of immunosuppression has been one of the most fundamental topics in transplantation biology since the description of acquired tolerance by Billingham and colleagues.’, This article examines the basis of successful clinical pediatric transplantation, including advances in surgical technique, control of rejection, and ultimately graft acceptance, examining what the authors believe are the permissive effects of immunosuppressive drugs on a mutual hostgraft leukocyte migration that, in successful cases, leads to mixed longterm microchimerism in the recipient and the transplant. In doing so, the authors consider basic transplant immunology, the evolution of immunosuppressive drugs, the experience with this knowledge in pediatric liver and multiple organ transplantation, and the discovery of the phenomenon of bidirectional cell migration and consequent systemic and graft chimerism, which the authors believe is the essential component for the long-term acceptance of any kind of whole-organ graft. The authors speculate on how the momentum of this progress can be sustained and accelerated and point out how transplantation technology is apt to influence pediatric transplantation in the years ahead. INDICATIONS

The most common indication for liver transplantation in the pediatric population is extrahepatic biliary atresia (Fig. 1). Other indications Malignancy Cholestatic Liver Disease

Congenital Hepatic Fibrosis

Autoir

Acute

Biliary Atresia 54%

Figure 1. Indications for liver transplantation in children. (Data from Belle S, Beringer K, Detre K: Recent findings concerning liver transplantation in the United States. In Cecka JM and Terasaki PI (eds): Clinical Transplants 1996, UCLA Tissue Typing Laboratory, 1997.)

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include metabolic errors of metabolism, such as alphal-antitrypsin deficiency or glycogen storage disease; neonatal hemochromatosis; fulminant hepatic failure; and other cholestatic diseases, such as Alagille’s syndrome. Although the number of patients transplanted for malignancy remains small, transplant offers selected patients with hepatocellular carcinoma or hepatoblastoma limited to the liver the only hope for cure. The classification of diseases that lead to transplantation in children is as follows: Metabolic diseases Alphal-antitrypsin deficiency Tyrosinemia Glycogen storage diseases Type 4 Type 3 Type 1 Wilson disease Perinatal hemochromatosis Acute and chronic hepatitis Fulminant hepatic failure Viral Toxin- or drug-reduced Chronic active hepatitis with cirrhosis Hepatitis B Hepatitis C Autoimmune Idiopathic Intrahepatic cholestasis Idiopathic neonatal hepatitis Alagille syndrome (bile duct paucity syndrome) Familial intrahepatic cholestasis (Byler disease) Obstructive biliary tract disease Extrahepatic biliary atresia Sclerosing cholangitis Traumatic and postsurgical biliary tract diseases Miscellaneous Cryptogenic cirrhosis Congenital hepatic fibrosis Caroli disease Cystic fibrosis Cirrhosis secondary to prolonged total parenteral nutrition Once the diagnosis of liver disease is made, the most important assessment is to determine the severity of the liver disease and its projected outcome. Patients with evidence of end-stage liver disease, including variceal hemorrhage, intractable ascites, hepatorenal syndrome, recurrent infection, and portosystemic encephalopathy, are candidates for immediate listing for transplantation. Selected patients with well-compensated Child’s A cirrhosis and isolated variceal bleeding

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benefit from surgical portosystemic shunting. Decisions for management should be made in a collaborative effort by multidisciplinary groups with all options of therapy, including interventional radiology, gastroenterology, and transplant expertise, available. The success of transplantation in patients with sequelae of end-stage liver disease has also heralded an increasing willingness to apply transplantation in patients with lifedisabling complications of liver disease consequent to severe metabolic consequences of chronic liver disease. TECHNICAL ADVANCES IN PEDIATRIC TRANSPLANTATION

Experimental transplantation of the liver in dogs was first reported in 1955 as an auxiliary liver transplant in which the native liver was preserved in its normal position and the new liver placed in a heterotopic site, usually the right paravertebral g ~ t t e r . Clinical ~' applications of this procedure have been inferior to those obtained with orthotopic liver transplantation (OLTx) despite a renewed interest in 1988.77OLTx is the accepted procedure today and involves removal of the native liver with replacement of a liver graft in its normal location.56 The first critical step in preservation of all cadaveric organs involves core cooling in situ by rapid infusion of a chilled solution into the donor aorta, which is done today with variations of the original technique described in 1963 by Marchioro and colleagues.3' Presently, procurement techniques allow removal of all thoracic and abdominal organs, including the with special modifications for unstable and even nonheart beating donors.75Subsequent preservation is then possible using simple refrigeration. Conceptually, this involves minimal dissection of vascular structures and cannulation of abdominal aorta for infusion of preservation solutions (Fig. 2). The abdominal organs can then be removed en bloc with separation of different graft components on the back table, in a bloodless field. This allows the successful transplantation of multiple recipients of various organs, such as liver, pancreas, intestine, or complete multivisceral grafts. Special preservation solutions have been in use since 1976; however, the one currently used was developed at the University of Wisconsin (UW solution) by Belzer and provides safe preservation of the liver for as many as 24 hours. With the UW solution, national organ sharing was made economic and practical.s8In addition to the liver and other organs, the thoracic aorta, iliac vessels, and carotid arteries of the donor are removed to serve as vascular homografts in the recipient. These free grafts may be preserved in the same UW solution and refrigerated for as many as 48 hours. The use of vascular grafts in the recipient must be of the same or compatible blood group. In principle, the recipient procedure demands control of the hilum and the inferior vena cava. The need for venous or arterial grafts is made, after which the liver is devascularized, the infrahepatic and supra-

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Figure 2. The principle of the donor operation is to accomplish rapid in-situ cooling of the abdominal and thoracic organs through cannulation of the aorta, allowing retrieval of the entire abdominal and thoracic solid organs and viscera.

hepatic venae cavae are controlled and clamped, and the liver is removed; however, removal of the retrohepatic vena cava is now only of particular importance in children undergoing transplantation for primary hepatic malignancies. This may involve placing the patient on venovenous bypass; however, children weighing less than 30 kg usually tolerate simple clamping and removal of the retrohepatic vena cava. The standard hepatectomy procedure in the pediatric population uses the "piggyback" technique, which is particularly helpful when venovenous bypass is not feasible. This technique allows the recipient vena cava to remain intact throughout the procedure. The liver is stripped off the vena cava, ligating and dividing venous tributaries, with an ostium created by joining the mouths of the left and middle, or the middle, left, and right hepatic veins to form a common ostium to receive the venous outflow of the liver graft (Fig. 3). If technically demanding, the infrahepatic and suprahepatic

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/ -

A

Figure 3. A, Preservation of the retrohepatic inferior vena cava (IVC) is performed by ligation and division of small venous tributaries, with subsequent clamping and division of the hepatic veins. 13,All three major hepatic veins have been connected to form the outflow for the liver graft. Ostia of varying sizes can be achieved using the middle and left hepatic veins, with or without extensions on the anterior wall of the IVC. (From Tzakis AG, Todo S, Starzl TE: Orthotopic liver transplantation with preservation of the inferior vena cava. Ann Surg 210:649-652,1989; with permission.)

venae cavae may be temporarily clamped and the liver stripped quickly off the vena cava, which is then released after control of venous branches. This technique is more demanding from the technical standpoint; however, maintenance of cardiovascular stability of the recipient is superior to the venovenous bypass technique. This allows for adequate hemostasis and preparation of the recipient field for the implantation of the liver a l l ~ g r a f t . ~ ~

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With the standard OLTx, five anastomoses are present: (1)the suprahepatic vena cava, (2) the infrahepatic vena cava, (3) the portal vein, (4) the hepatic artery, and (5) the biliary reconstruction. With the use of the piggyback technique, a lower caval anastomosis is not performed. The portal vein anastomosis is usually end-to-end; however, in the event of thrombosis or a hypotrophic portal vein, the dissection of the recipient portal vein is taken to the confluence of the superior mesenteric vein and splenic vein, providing a wide venous ostium. The anastomosis can be performed directly to the portal vein or indirectly via an interposition venous graft. Alternatively, a venous jump graft may be placed into the recipient superior mesenteric vein and then anastomosed to the donor portal vein by bringing this vein graft through the transverse mesocolon and in front of the antrum of the stomach (Fig. 4). Variations in the arterial reconstruction are more common because of donor-recipient anomalies (one third of cases). These include anoma-

Recipient superior mesenteric vein Figure 4. The superior mesenteric vein is identified at the base of the transverse mesocolon and inferior edge of the pancreas. Using cadaveric iliac vein, an interposition graft has been placed behind the stomach and anterior to the pancreas. (From Tzakis AG, Todo S, Stieber A, et at: Venous jump grafts for liver transplantation in patients with portal vein thrombosis. Transplantation 48:530,1989; with permission.)

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lous branches to the hepatic graft from the aorta directly or from the left gastric or superior mesenteric arteries. Reconstructions of donor arterial abnormalities are usually performed on the back table so that a single arterial anastomosis to a recipient vessel or vascular graft is required. The most common anomaly, a right hepatic artery originating from the superior mesenteric artery, can be reconstructed by anastomosing the right hepatic artery to the take off of the splenic artery on the donor celiac trunk or with an anastomosis of celiac trunk to superior mesenteric artery, which now becomes the inflow artery for the enteric liver graft. With any reconstruction technique, rotational manipulations of these vessels should follow a simple craniad and caudad orientation of the arterial borders to avoid torsion and possible occlusion. The variations in the recipient arterial anatomy are similar, although more often arterial disease or surgical damage to the recipient artery renders this unusable for revascularization. This is overcome easily using free arterial homografts (e.g., iliac artery or carotid arteries) brought from the donor at the time of the organ procurement, which are usually placed to the recipient infrarenal or supraceliac aorta or one of the iliac arteries. The biliary reconstruction in the pediatric population is usually performed end-toside to a defunctionalized Roux-en-Y limb (choledochojejunostomy).In larger pediatric recipients, an end-to-end choledochocholedochostomy with a T-tube is possible. The universal applicability of liver transplantation, which occurred after the early 1980s, has produced a crisis in organ availability. Strategies to increase the supply have included living related donations, donor sharing of split organs, and xenotransplantation. Ideally, a liver graft should be of similar size to the diseased liver being removed; however, this is rarely a clinical reality. Preferences of a close age and weight matching of donor and recipient are impractical. Because so few pediatric donors exist, wide deviations are mandatory and generally involve using larger organs in smaller recipients. Fortunately, children with liver failure present with significant increases in abdominal girth because of hepatosplenomegaly and ascites. This, together with very flexible abdominal wall and lower thoracic cage structures, has allowed for a greater variability in donor size and led to use of reduced-size adult liver grafts. These liver fragments may be from donors up to 10 times larger than the recipient. Because surgical reduction of the allograft liver is a formal hepatectomy, the segments most commonly used are the right lobe (left lobectomy), left lobe (right lobectomy), left lateral segment (right trisegmentectomy), and split liver where the extended right lobe is used on a large recipient, and the left lateral segment is used on the smaller recipient (lateral segmentectomy) (Fig. 5). With a living related donation, the left lobe or left lateral segment is removed from a parent or adult liver donor. In very selected cases, the right lobe is used, usually for a very large pediatric or an adult recipient. The removed fragment is transplanted to a pediatric recipient in the usual orthotopic location using the standard piggyback hepatec-

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Right trisegmentectorny

171

Left lateral segrnentectomy

A

Figure 5. A, The alternatives for hepatic reduction are, in essence, formal hepatectomies. 6, Two recipients can be transplanted by splitting the allograft liver and providing the extended right lobe for an adult recipient and a left lateral segment graft for the pediatric recipient.

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tomy technique. Survival statistics with this procedure have been comparable to those of cadaveric transplantation, and its use has been expanded to many centers throughout the world; however, the risk for donor death exists and raises the sensitive ethical issue of living related d ~ n a t i o nWith . ~ the split liver technique, a large recipient (usually an adult) is given the right lobe of a cadaveric liver and the smaller recipient (usually a child) receives the left lobe or left lateral segment. The division may be carried out ex vivo (on the back table) or in situ at the time of organ procurement. Vascular reconstructions may be required. This technique increases the organ donor pool and is rapidly becoming a standard procedure in many transplant centers, with the added benefit of being able to share segments between centers.53 Transplantation of a liver graft from a significantly disparate species presents an immunologic barrier of preformed xenospecific antibodies or complement activation, which quickly devascularizes the graft by the same mechanism as in ABO-incompatible and presensitized allograft recipients. It is believed that liver allografts and xenoallografts are resistant to such hyperacute humoral rejection; however, human liver xenotransplantation using chimpanzee donors was unsuccessful on three occasions between 1966 and 1973, with deaths occurring after 9, 0, and 14 days post-transplantation. Using the phylogenetically more distant baboon donor in hepatic xenotransplantation attempted between June 1992 and January 1993, two patients survived 7 and 26 days, respectively. Neither antibody nor cell-mediated rejection could be definitively ascribed to the failure of these two cases. Consequently, the current techniques of immunologic control are probably inadequate to permit the acceptance of such grafts. Indirect evidence that inflammatory mediators triggered by preformed xenospecific antibodies (principally IgM) and complement activation suggests that a new generation of complemented inhibitors or the creation of transgenic animal donors whose organs contain transfected human complement regulatory genes may provide 92 for longer survival.35, THE IMMUNOLOGIC BASIS FOR TRANSPLANTATION

The host lymphocyte plays the central role in the immune response to transplanted allografts, bearing the specific capability to differentiate "self" from "nonself." Through a series of developmental processes (under the influence of thymic hormones, cytokine interleukin [ILI-7, or major histocompatibility [MHC] molecules) the pluripotential stem cell in the liver and bone marrow of the fetus undergoes a series of maturation and migration processes through the thymus and peripheral lymphoid tissues (lymph nodes, spleen, and gut) producing CD3+ T lymphocytes. These cells then acquire surface subset differentiation markers (CD4+, DC8+, T-cell receptor) and are responsible for cellmediated immunity.52The B cell is the second lymphocyte subpopulation to develop also under the influence of humoral factors, such as IL-7, IL-

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4, IL-5, and IL-6.12 They are specialized to synthesize and secrete antibody, which can interact with antigen. When an organ is transplanted, stimulation of the resting lymphocyte by histocompatibility antigens present on cells of the transplanted tissue is thought to be presented directly to the host lymphocytes by the host's antigen-presenting cells, the most potent of which is the bone marrow-derived dendritic cells but which also includes macrophages, monocytes, and B cells. These cells express class 2 MHC antigens and secrete cytokines (IL-1, tumor necrosis factor-alpha, and IL-6),26transforming the resting CD4 + T lymphocyte into a large active cell. The T cells, B cells, and antigen-presenting cells operate in a milieu of cytokines to orchestrate the immune response. The amplification of the human response by the CD4+ T cell is produced through the secretion of these cytokines. The CD4+ T cells have two functional subsets on the basis of their pattern of cytokine secretion.3xThus, Thelper-1 cells secrete IL-2 and interferon (1FN)-gamma; T-helper-2 cells secrete IL-4, IL-5, and IL-10; and both types secrete IL-3 and granulocyte macrophage-colony stimulating factor (GM-CSF).B cells synthesize antibody; T cells bearing the CD8+ molecule (which interacts with MHC class 1 antigen bearing cells) can function as cytotoxic cells. T-helper (CD4+ ) and T-suppressor (CD8 ) cells can respectively enhance or suppress the response to antigen.s2The suppressor T cell (CD8+) presents an overamplification of an immunologic stimulus. T-helper cell activation induces secretion of IL-2 (a T cell growth factor), which then binds to IL-2R on resting T cells and stimulates DNA synthesis and cell mitosis. At the present time, effective maintenance clinical immunosuppression (cyclosporine or tacrolimus) focuses on inhibition of antigen induced T-lymphocyte activation (CD4+ T cell) and cytokine production, interruption of the allo-MHC recognition, or effector responses. The use of metabolic inhibitors (e.g., azathioprine or cyclophosphamide) prevents lymphocytic proliferation necessary for the amplification of the activation response. The depletion of circulating lymphocytes during acute rejection has been the focus of corticosteroids or antilymphocyte serum (OKT3). Because effective immunosuppression can weaken the host response to opportunistic infections, the focus of future progress in immunosuppressive therapy is to induce long-lasting, donor-specific unresponsiveness (immunologic tolerance) and at the same time preserve immunocompetence. Early clinical immunosuppression relied heavily on agents or procedures with antiproliferative activity, which included antimetabolites, alkylating agents, toxic antibiotics, and irradiation. By 1960, the possibility of weakening the recipient immune process with corticosteroids, total body irradiation, the cytotoxic 6-mercaptopurine, or its imidazole derivative (azathioprine) had been established in animals.5sThe evolution of conventional immunosuppression for liver transplantation has evolved through successive eras, which can be categorized into periods defined by the immunosuppressive agent available during that era. The era of double-drug therapy was initiated in 1962, with the marriage of

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corticosteroid therapy to baseline therapy with azathioprine. The use of this synergistic drug combination permitted fundamental observations, including the fact that rejection was a reversible process.h8The tripledrug therapy era used the gold standard of transplantation, which was the double-drug regimen with modifications aimed toward attacking the lymphocytes, which had been recognized as mediators of rejection. The most significant addition was the use of antilymphocyte gl~bulin.~" The cyclosporine era, in which cyclosporine and prednisone, with or without azathioprine and antilymphocyte globulin (or OKT3), were used, brought liver transplantation from patient survival rates of 35% at 1 year and less than 25% at 5 years under azathioprine and steroids to survival rates approaching 90% at 1 year.57 In 1984, tacrolimus (FK506), a macrolide produced by Streptomyces tsukubaensis, was discovered in Japan. After 3 years of extensive preclinical laboratory research in Chiba, Pittsburgh, and Cambridge,", 30, 42-44, 81, x2* y4 it was used in February 1989 to successfully salvage liver allograft recipients suffering intractable rejection despite optimal cyclosporine-based imm~notherapy.'~,In patients whose graft dysfunction was attributed to acute or the early stages of chronic rejection, the success rate was as high as 90°/0. With chronic rejection of the liver, salvage approached 50'/0.'~ Benefits of this type of tacrolimus rescue therapy in pediatric recipients of a liver allograft under cyclosporineprednisone immunosuppression are shown in Table 1. In this study, 77 children who presented with chronic rejection (47%) and steroid-OKT3resistant acute rejection (26%) showed a remarkable 4-year patient survival rate (82%)and graft survival rate (74.4%).Also, 84% of the children who were previously on prednisone were weaned completely from steroids, and 58% of .patients who had required antihypertensive medications were completely off their antihypertensive drugs. Such results have given us an ability not only to rescue children with severe rejection but also to enhance their quality of life by improvements in growth and

Table 1. CONVERSION FROM CYCLOSPORINE TO TACROLIMUS IN PEDIATRIC LIVER TRANSPLANT RECIPIENTS ( n =77) 3 Mo

1Y

2Y

3Y

4Y

Patient survival (%) Graft survival (I%)

91.2 87.2

87.2 84.6

84.6 80.7

83.3 76.9

82.0 74.4

LiverIKidney function*

Pre

1 Wk

4 Wk

25 Wk

50 Wk

Bili mg/dL AST U/L ALT U/L GGTP U/L BUN mg/dL Creat mg/dL

1.3 207 166 298 17 0.8

0.7 136 149 289 27 0.8

0.7 87 97 305 23 0.8

0.7 61 60 120 17 0.8

0.7 70 73 79 16 0.8

~

~~

~~~~~~~

~

*Median values Bill, bilirubin; AST, aspartate aminotransferase, ALT, alanine aminotransferase, GGTP, gammaglutamyl transferase, BUN, blood-urea nitrogen, creat, creatme

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development, as well as resolution of brutalizing facial deformities and hirsutism. Clinical trials using tacrolimus as the primary immunosuppressive agent for recipients of liver, kidney, and thoracic organs were initiated soon thereafter.I9,41, 64, 84 The Pittsburgh randomized trial comparing cyclosporine and tacrolimus was begun in February 1990. A total of 79 low-risk patients were studied under equal treatment variables, including a daily dose of 20 mg prednisone. The patient and graft survivals, according to the originally assigned randomization, reflected better, although not statistically significant, results with tacrolimus.“’ The superiority of tacrolimus to cyclosporine was concluded when it was noted that the composite freedom from rejection, or graft loss (death or retransplantation), was 24% for tacrolimus versus 8% for cyclosporine, and freedom from “adverse events” was 6% for tacrolimus versus 1% for those originally assigned to cyclosporine. Freedom from rejection alone was 33% for patients in the tacrolimus cohort compared with 12% for the cyclosporine cohort. Also, at the end of 1 year, 51 of the 75 patients originally assigned to cyclosporine had been converted to tacrolimus. This trial was discontinued in December 1991 after recommendations from a multi-institutional ”Patient’s Rights Committee,” with the concurrence of the Institutional Review Board (IRB) and the US Food and Drug Administration (FDA). The European4‘ and American’O multicenter trials supported commercial release of tacrolimus by the FDA in June 1994. Both studies showed comparable actuarial patient and graft survivals; however, the use of tacrolimus was associated with a significant reduction in acute, refractory acute, and chronic rejection episodes. The American study delineated substantially more toxic events requiring discontinuation of tacrolimus.1° Although the survival advantage was not statistically significant, it was noted that many of the surviving grafts credited to cyclosporine had been rescued with tacrolimus. Reanalysis of the American study revealed that the composite freedom from refractory rejection, retransplantation, and death was 80% for the tacrolimus arm versus 70% for the cyclosporine The authors’ reported clinical experience in children included 202 consecutive recipients of primary liver allografts treated with tacrolimus between August 1989 and December 1993.86The mean age was 5.0 years, and the mean follow-up was 29.6 months (range, 3-55 months). The overall patient actuarial survival rates in children were 91.1Y0, 89.6%, 88.5%, and 86.2% at 3, 6, 12, 24, and 48 months, respectively. The overall graft survival rates in children were 81.4%, 8O.l%, 79.1%, and 77.0% at 3, 6, 12, 24, and 48 months, respectively. More than 50% of these liver recipients were rejection free. More than 90% of the pediatric recipients were steroid free by 12 months after transplantation, and less than 5% of pediatric recipients were on antihypertensive medication 5 years after transplantation. Retransplantation was required in 9.9% of children. The authors’ experience with drug toxicity, drug interactions, opportunistic infections, and other clinical observations has been published elsewhere.1-3,15, 24. 51, 76

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CHIMERISM AND THE INDUCTION OF DONORSPECIFIC TRANSPLANTATION TOLERANCE

The prevention and treatment of organ rejection by various immunosuppressive agents have been presented with the preceding abbreviated description of the development of allograft immunity and the evolution of transplantation technology. This has been described in terms of manipulations of the recipient immune cell population by destruction of the immunocompetent cells before transplantation, disruption of the alloactivated T-cell response, and suppressing clonal expansion of lymphocytes with the use of immunosuppressive drugs. This experience allowed the development of therapeutic strategies that improved survival after organ transplantation. Interestingly, most patients progressively require less and less immunosuppressive therapy, and some have discontinued therapy a l t ~ g e t h e r . ~ ~ In 1969, it was noted that Kupffer cells and other tissue leukocytes became predominantly of the recipient phenotype within 100 days after transplantation, whereas the hepatocytes retain their donor specificity pe~manently.~~ Twenty-two years later, the authors observed that donor leukocytes from transplanted organs had migrated and survived 70 This throughout the body of the recipient for as long as 3 decades.h(M2, process occurred in all successfully transplanted organs, the liver being the most tolerogenic transplanted organ because of its much larger total load and the lineage profile of the migratory leukocytes. The events following transplantation were then seen as a two-way cellular action-graft versus host and host versus graft. Under the cover of immunosuppressive drugs, the graft and the recipient become genetic composites composed of cells of both parties (Fig. 6). This bidirectional migration has been particularly dramatic in all successfully transplanted intestines and has also been observed to different proportions after liver, kidney, and heart.25,4h, 48, 5y Because neither the recipient nor the graft is leukocyte depleted, it is possible to routinely perform intestinal and multivisceral transplantation without an exorbitant risk for graft versus host disease (GVHD). This spontaneous ”chimerism” after whole-organ transplantation differs from bone marrow transplantation in that the treatment strategy involved empirically leaves both cell populations intact. This reciprocal interaction (mutual natural immunosuppression) may blindfold the MHC effect, thus removing

Figure 6. A, Transplantation as a unidirectional immune reaction results in rejection (HVG) after whole organ transplantation (in this case the intestine), and graft versus host (GVH) after bone marrow transplantation. B, Transplantation seen as a bidirectional migration of immunocytes. Under the cover of immunosuppression, a mutual nullifying of the immune reaction would control HVG and GVH reactions. ( f r o m Reyes J: Small bowel and liver transplantation in children. In Kelly D (ed): Pediatric Liver Disease. London, Blackwell Science Ltd., 1999; with permission.)

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tissue matching as a crucial requisite for success, and largely eliminates the threat of GVHD.hY Evidence supporting chimerism was found in retrospective studies of long-term survivors of kidney allografts (30 y post-transplantation), liver allografts (10-21 y post-transplantation), and recipients of thoracic organs.5y,y3 The identity of donor and recipient cells was established after special staining procedures (immunostaining or sex identification after fluorescence in situ hybridization [FISH], and polymerase chain reaction [PCR; DNA fingerprinting]). The observation of surviving donor multilineage passenger leukocytes being associated with organ graft acceptance permits us to review the engraftment of any whole organ in the same context as “mini”-bone marrow transplantation. The acceptance of this concept inherently brings about the issue of eventually stopping immunosuppressive medication altogether. Many noncompliant patients have stopped their medications sporadically and then completely when all liver functions remained normal. Other patients have had their immunosuppression withdrawn because of infectious complications.50 The long-term complications following pediatric liver transplantation are well known and include growth failure; renal failure; hypertension; infection; skin lesions; neurologic complications; and malignancies, such as post-transplant lymphoproliferative disease.6,28, 54 In fact, patients’ long-term quality of life is most affected by the lifelong immunosuppression that they are thought to require. The morbidity established from the chronic use of immunosuppression is an incentive to establish the lowest possible level of immunosuppression necessary to maintain stable graft function. The authors’ prior finding that complete freedom from immunosuppression or significant withdrawal of immunosuppression was possible in long-surviving recipients of liver allografts59 prompted a prospective trial of drug weaning.33,47 The drug weaning protocol established for pediatric liver transplant patients at the University of Pittsburgh includes long-term survivors (>5 years status post-OLTx) who are medically compliant and have normal liver function without recent acute rejection episodes within the past 2 years. In this group of 43 patients prospectively followed up, the recipient diagnosis included biliary atresia in 27 and alpha,-antitrypsin deficiency and other diagnoses in 8 patients, respectively. Immunosuppression was based on azathioprine and prednisone in 4, cyclosporine in 28, and tacrolimus in 11. Drug withdrawal was begun at a mean time of 6 years after liver transplantation. The baseline immunosuppressant of tacrolimus or cyclosporine was decrementally weaned at 2-month to 3-month intervals as long as hepatocellular enzyme tests remained stable. Liver injury tests of aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma glutamyltransferase (GGTP), and bilirubin are monitored weekly after changes in drug dosage. Tacrolimus or cyclosporine levels are not used in monitoring because baseline levels in this patient population are frequently low or undetectable. Liver biopsy is done for sustained alterations in liver tests. Preexisting compli-

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Rejection (n=6)

Weaning in Progress (n: =20) 46%

Off Medication

1=17)

40% Figure 7. Weaning results of immunosuppression in children.

cations included infections, skin lesions or tumors, neurologic abnormalities, renal failure, hypertension, and growth failure. Significant weaning is ongoing in 46% of these patients, with a reduction in baseline immunosuppression by 36% of their cyclosporine level, 83% decrease in the tacrolimus level, and 62% decrease in their prednisone dosage. Forty percent (17 of 43) of the patients are off immunosuppression, with a mean time from weaning of 1.7 years. The current follow-up is 3.5 years off drugs, with a range of 0.8 to 6.4 years (Fig. 7). No patient or graft loss has occurred. Rejection has occurred in 14% of patients (6 of 43), being mild in two patients and mild to moderate in four patients, prompting switch to tacrolimus from cyclosporinebased immunosuppression. All patients currently have normal liver function follow-up, and chronic rejection has not been seen. All preexisting complications of infections, skin lesions, tumors, and neurologic sequelae have resolved; however, renal insufficiency and hypertension have not yet been significantly altered by weaning. From these results, it is clear that most long-term survivors after pediatric transplantation can tolerate a lower baseline of immunosuppression than is currently routinely practiced. Some patients may be able to be weaned completely. It is imperative that such weaning be accompanied by careful surveillance and prompt intervention to accurately diagnose not only rejection but also the other causes of late graft dysfunction that are uncovered over time.45Pediatric patients have the greatest potential benefit from the significant dosage reductions or complete drug withdrawal that can be potentially realized.

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LIVER PLUS OTHER ORGANS The Intestine

Historically, the evolution of experimental and clinical intestinal transplantation has distantly paralleled other organs; however, clinical success was limited to days because of a high incidence of graft loss from technical complications and rejection. Indeed, the therapeutic strategies established through experimentation5** 71 and with clinical transplantation of other organs did not have impact on grafts containing an intestinal component. This struggle in exemplified by a series of human applications of the modified form of this operation under cyclosporine-based immunosuppression that was begun in 1987 with a 3-year-old girl who received a multivisceral abdominal graft that contained the stomach, duodenum, pancreas, small bowel, colon, and liver. She survived for 6 months with good intestinal graft function without developing either rejection or GVHD.22Four other patients achieved functional cadaveric intestinal 34 or grafts transplanted a10neF3 as liver and intestinal composite as multivisceral allografts.'3 The only survivor with a functioning graft is the intestine-alone recipient of G o ~ l e tThe . ~ ~liver and intestinal recipi34 survived for 58 and 66 months. A living related donor ents of Grant32, intestinal segment was transplanted by Deltzso in February 1988 and supported nutrition for 61 months. The accumulated experience with other organs and advances in transplantation immunology and technology, as described in previous sections, have brought us to the forefront of intestinal transplantation. The demonstration in clinical liver transplantation of the greater efficacy of tacrolimus was a major determinant in the renewal of efforts to transplant the intestine. Laboratory and clinical observations with intestinal transplantation have played a critical role in establishing a generic bidirectional paradigm of transplantation and immunology that is relevant to all organ^.^^,^^ Between June 1990 and January 1996, the authors performed 58 intestinal transplantations in 55 children under immunosuppression with tacrolimus and low-dose steroids, to which prostaglandin E l was added briefly during the early postoperative stage.*5There were three types of intestinal allografts, which are shown in Figure 8: (1) isolated intestinal ( n = 17), (2) combined liver and intestine ( n = 33), and (3) multivisceral ( n = 8) (Fig. 9). Patients with satisfactory liver function without evidence of portal hypertension received an isolated intestinal allograft. Patients with inborn errors and also total parenteral nutrition (TI")-induced cholestatic liver disease received a liver plus intestinal allograft. The multivisceral allograft was indicated in patients who had extensive abnormalities of the entire gastrointestinal tract, which included absorptive/secretory, motility, or vascular disorders. The principles and various modifications of these procedures have been described h5, 7y, x7 The colon was included as part of the allograft elsewhere.lh<

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Figure 8. Intestinal transplantation can be performed as an isolated intestine graft (A), as a component of composite grafts such as liver/small bowel (B),and as multivisceral grafts (C). (From Reyes J, Bueno J, Kocoshis S, et al: Current status of intestinal transplantation in children. J Pediatr Surg 33:243-254,1998; with permission.)

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midway through the authors’ experience with intestinal transplantation and distributed among all three graft cohorts. This was promoted by high postoperative stoma1 outputs requiring frequent admissions for dehydration. The authors also included donor bone marrow transplantation at the time of intestinal transplantation. Bone marrow cells are recovered from the same donor as the intestine and were infused intravenously into the recipient in the immediate postoperative period in nine cases (eight primary and one retransplantation). The rationale for this approach of including simultaneous bone marrow infusion after solid organ transplantation was derived from experimental and clinical evidence that the existence of ”chimerism” is critical for graft acceptance.*O,39 A total of 27 boys and 28 girls were studied, with a mean age of 3.2 years (range, 0.5-18.0 y). The original diseases leading to transplantation are listed in Table 2. All patients were followed up through March 1997. Median follow-up was 462 days. All donors were cadaveric of the identical ABO blood type as the recipients. Matching of human leukocyte antigen (HLA) was random and uniformly poor. A history of normal intestinal function in a potential liver donation referral is adequate for possible intestinal donation. The procurement of multivisceral organs focuses on the isolation and cooling of these organs, preserving their vascular and parenchymal anatomy. The organs are flushed with UW solution.8s The cold ischemic time averaged 7.83 hours (range, 2.8-14.8 h). During the potential follow-up of 1 to 6 years, 30 patients are still alive. The actuarial survival rates for the 55 patients at 1, 3, and 5 years were 72%, 55%, and 55%, respectively. The estimated actuarial survival rates for all types of grafts were 66%, 4870, 62.8%, and 48% at 1, 3, and 5 years, respectively (Fig. 9). Isolated intestinal transplants provided the best patient and graft survival at all follow-up times. Of the four patients in whom retransplantations were performed, three had composite liver plus intestinal grafts and died, and one had isolated intestinal retransplantation and is alive. Inclusion of the allograft colon or bone marrow had no effect on survival or immunosuppressive management. The incidence of rejection of the intestinal allograft was go%, with a mean of Table 2. INDICATIONS FOR INTESTINAL TRANSPLANTATION IN CHILDREN Volvulus Gastroschisis Necrotizing enterocolitis Intestinal atresia Pseudo-obstruction Microvillus inclusion disease Intestinal polyposis Hirschsprung’s disease Trauma

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2.9 k 2.7 episodes per graft, whereas the rejection rate of the liver allograft when it formed part of a composite graft was 43%. Rejection was more frequent and more severe in recipients of isolated intestinal grafts, thereby supporting the authors' belief that in recipients of composite intestinal grafts, the liver graft is protective of the intestinal component. Infection as a complication attributable to the immunosuppressive therapy was the major cause of morbidity and mortality. Bacteria and fungi were accountable for many such complications; however, viral infections with cytomegalovirus (CMV) in 16 patients and Epstein-Barr virus-associated post-transplant lymphoproliferative disease (PTLD) in another 16 children were responsible for significant morbidity. Fourteen of the 16 patients who developed PTLD lost their grafts and lives to this complication. Therapy for CMV was successful in more than 90% of cases, with no adverse effect on survival, whether the donor or recipient was CMV positive. Analysis of these cases has yielded six statistically significant risk factors for graft loss and death: (1)high tacrolimus blood trough levels, (2) bolus steroid therapy, (3) OKT3 use for treatment of allograft rejection, (4) length of operation, (5) CMV positive status of donor and recipient, and (6) the development of PTLD. Evidence of donor cells was detected in all bone marrow augmented bowel recipients by either PCR or flow cytometry. In the female recipients who received male allografts, presence of donor cells was confirmed by FISH for the Y chromosome. Mild evidence of GVHD was found in only one of the nine recipients who were given bone marrow cells. The authors believe that intestinal transplantation is a valid therapeutic option for patients with intestinal failure suffering complications of TPN. The complex clinical and immunologic course of these patients is reflected in a higher complication rate and patient and graft loss than seen after heart, liver, and kidney transplantation, although better than after lung transplantation. The Kidney

Results of simultaneous and sequential pediatric liver and kidney transplantation in children with end-stage liver disease or subsequent end-stage renal disease have varied. Between 1984 and 1995, 12 children underwent liver transplantation with simultaneous or sequential kidney transplantation. There were six male and six female patients, with a mean age of 10.3 years (0.15-17.2 y). There were 7 simultaneous kidney and liver transplants; five patients had kidney transplantation following successful liver transplantation (one patient after liver and small bowel transplantation). A total of 12 liver and 15 kidney grafts were placed. Indications for liver transplantation were oxalosis (4), congenital hepatic fibrosis (2), cystinosis (l),alpha,-antitrypsin deficiency (l),TPN-induced (l),polycystic liver disease (l),cryptogenic cirrhosis (l),and hepatoblastoma (1). Indications for kidney transplantation were oxalosis (4), poly-

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cystic kidney disease (3), drug-induced (3), cystinosis (l), renal failure following previous liver and kidney or liver and small bowel transplantation (2), and rejection of previous kidney transplant (2). Actuarial patient survival rates following most recent transplantation were 66% at 1 year and 58% at 5 years. Actuarial liver graft survival rates at 1 year were 83% and 58% at 5 years. All liver graft loss resulted from patient mortality rather than inherent graft dysfunction. One-year and 5-year actuarial kidney graft survival rates were both 60%. Of six lost kidney grafts, two failed secondary to rejection, and four were lost to patient mortality. The 1-year patient survival rate was 100% for United Network for Organ Sharing (UNOS) status-3 patients, but only 33% for status-2 and status-1 patients. All ultimately lost kidney and liver grafts were in patients who were either status 1 or 2 at the time of initial transplant. The mean number of biopsy-documented acute cellular rejection episodes per number of grafts was 1.1 for liver transplants and 0.9 for kidney transplants. This was independent of simultaneous or sequential transplantation, degree of HLA mismatch, panel-reactive antibody, or immunosuppressive regimen. The mean renal and hepatic function (at a mean follow-up time of 57 months) is as follows: blood urea nitrogen, 31 mg/dL; creatinine, 2.0 mg/dL; bilirubin, 1 mg/dL; AST, 41 U/L; ALT, 31 U/L; alkaline phosphatase, 193 mg/dL; and GGTP 168 mg/dL. The authors concluded that patient and graft survival with simultaneous or sequential pediatric liver and kidney transplantation seems to correlate strongly with medical status at the time of initial transplantation. A flexible clinical staging approach may allow for more appropriate distribution of kidney grafts. SUMMARY

Advances in organ preservation, surgical technique, and postoperative care have permitted the rapid development of liver transplantation in children. Consequently, the applicability of this procedure has gone beyond the treatment of life-threatening complications of chronic liver disease and now includes disabling morbidities and quality-of-life issues. The use of hepatic segments for transplantation with reduced or split cadaveric grafts and living-related donors has decreased the mortality of children awaiting liver transplantation. We are presently armed with a new potent immunosuppressive drug, tacrolimus, and an understanding that the migration and grafting of passenger leukocytes of bone marrow origin is the seminal explanation for allograft acceptance. The next forefront will involve manipulation of the process not only for the transplantation of already successful whole organs-such as the liver, kidney, pancreas, and heart-but also in the development of the intestinal transplantation program. Thus, augmentation of leukocyte traffic in unconditioned recipients of cadaver allografts with concomitant intravenous infusion of donor bone marrow cells under the same immunosuppressive management of tacrolimusprednisone treatment will be the path into the future.

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