Chapter 97: Immunology and Transplantation
Sara K. Rasmussen; Paul M. Colombani
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Key Points
Introduction
Components of the Immune System
Transplantation Immunology
Conclusions
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Key Points
Though nonspecific host defenses are somewhat deficient in the infant and pediatric patient, the effector arm of the specific immunologic response to alloantigens of clinical organ transplants is intact at the time of birth.
Effective organ allograft immunosuppression requires a balance of prevention of rejection of the graft while avoiding the toxicity of excessive immunosuppressing agents.
Immunosuppressive agents can be categorized into those used for induction (rabbit anti-thymocyte globulin, corticosteroids), those for maintenance (mycophenolate mofetil, tacrolimus), and those used to treat acute rejection (corticosteroids).
Introduction
Infections are a leading cause of pediatric morbidity and mortality, particularly in the neonatal period. Explanations for this phenomenon include a lack of previous antigenic experience on the part of pediatric patients; an intrinsic immaturity of lymphocyte functions in this age group; and, for neonates, an active cellular suppression mechanism. In addition, several specific inherited defects of the immune system may manifest themselves in early infancy and continue to plague these patients through childhood and on into adulthood. Acquired defects in the immune defenses occur in transplantation and oncology patients. This chapter will review the specific and nonspecific components of the immune system with particular reference to the pediatric patient's ability to fight infection (see also Chapter 13—“Surgical Infection: Classification, Diagnosis, Treatment and Prevention”). Emphasis will be placed on the differences in these responses in neonates and children compared to the responses in adult subjects. The ability of children to mount an immune response against alloantigens in the transplantation setting will be discussed. In addition, this chapter will review current methods of clinical immunosuppression for the pediatric age group.
Components of the Immune System
Immune responses are generally divided into specific and nonspecific components. Each of these components is further subdivided into humoral and cellular compartments (Table 97-1).
Table 97-1
Summary of Immune System Components
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Nonspecific (Nonimmune) Host Defenses
HUMORAL COMPONENTS
The humoral components of nonspecific host defense systems include the classic complement pathway and the alternate, or properdin, pathway. The complement cascade is made up of 9 serum proteins which are activated during the inflammatory response. The proteins react with immunoglobulins (Ig)—IgM or IgG—and antigen. Activated complement (C′) liberates mediators of the inflammatory response, and the levels of the C3/C5 component correlate with susceptibility to infection. The C3 level determines the rate of phagocytosis or lysis of invading organisms. In the presence of immunoglobulins, the C142 component (the membrane attack complex) activates C3, the binding of immunoglobulins, and eventual opsonization of the organism by the phagocytic system. Complement is detectable at 1% of adult levels at 5 weeks of gestation. By 26 to 28 weeks of gestation there is a rapid increase in complement levels in the serum of the fetus. By term, complement levels are 30% to 50% of adult levels. Complement levels in term infants are subnormal in approximately 50% of neonates, but the mean level of activation may approach 70% to 90% of adult activity following activation.
The properdin or alternate pathway also appears early in gestation during the first trimester. By term, this activity is 30% to 60% of adult levels and is therefore more suppressed in neonates than the levels of proteins in the complement cascade. Quantitatively, preterm infants have a lower level of properdin. Opsonizing activity by the properdin pathway is determined by factor B levels, and because these levels are lower in term and preterm infants overall, opsonizing activity is limited.
CELLULAR COMPONENTS OF THE NONSPECIFIC SYSTEM
The cellular components of nonspecific host defense include polymorphonucleocytes (PMN), monocytes, macrophages, and the modified macrophages of the reticular endothelial system (RES). In response to chemotactic factors, PMN migrate to areas of infection. There is a clear deficiency in chemotaxis because of the decreased elasticity of neonatal PMN, which are also responsible for killing bacteria after phagocytosis. The phagocytic ability of these cells is probably normal at birth, but in vitro assays of phagocytosis are diminished in the cells of term and preterm infants compared to adult cells. The addition of adult serum to these neonatal cells normalizes their activity, which suggests that neonates are simply more deficient in opsonizing factors as alluded to above. The result, however, is that the phagocytic ability of these cells is diminished, particularly in stressed infants and children.
Following engulfment and phagocytosis of organisms, PMNs kill these organisms primarily by peroxidation by oxygen-free radicals produced by the cells. Neonatal PMN clearly have a decreased ability to generate oxygen-free radicals for intracellular killing. Also, under stress conditions, even though phagocytosis may be relatively intact, there is decreased intracellular killing.
Migratory macrophages and monocytes are similarly deficient in chemotactic ability and phagocytosis in the neonatal period. Beyond that, macrophage function appears to be normal.
The RES is a system of fixed macrophages or monocytes in a variety of sites including the liver, spleen, and bone marrow. These cells serve to remove particulates from the bloodstream, including viral, bacterial, and fungal organisms. The RES system is intact in both term and preterm infants. This system is able to respond at maximum levels at term, but serum opsonizing factors are required for optimal function.
Important adjunct components of the nonspecific immune system in neonates are the mononuclear phagocytes in maternal milk ingested during breast feeding. These mononuclear cells comprise 80% of the cells found in colostrum and breast milk. On the first day of breast milk production, there are approximately 2 million cells/mL of breast milk. This number of phagocytes declines to approximately 1.3 million cells/mL by approximately 3 months following birth. These activated cells secrete components of complement and possess immunoglobulin Fc receptors and complement receptors. They also contain and may secrete intracellular IgA. They may also be responsible for the phagocytosis and intracellular killing of swallowed organisms with which neonates have had no previous immune experience. In addition, these cells may elaborate and secrete a number of lymphokines which may be important for the development of optimal function of the gut-associated immune responses in infants.
In summary, there are a number of nonspecific, nonimmune components for host defenses that are all qualitatively present in preterm and term infants as well as in children. In general, the humoral components are deficient in quantity which may lead to a decreased level of serum opsonization factor in neonatal and infant blood. Cellular components of this system are highly dependent on these opsonic factors for optimal function. As a result, overall nonspecific host defenses are somewhat deficient in the pediatric age group, particularly in neonates as compared to adults. This deficiency is primarily due to limiting opsonic activity when infants are under stress, as well as other potential deficiencies in chemotaxis and intracellular killing.
Specific Immune Defenses
Most important in developing specific immunity against infections and mounting an immune response to alloantigens in the transplantation setting are the humoral and cellular components of the immune system.
HUMORAL COMPONENTS
The humoral components of the immune system are primarily the immunoglobulins elaborated by B lymphocytes. In addition to specific immunoglobulin production in response to antigenic stimulus, which is termed active immunity, there is also passive maternally-derived IgG, conferring some immune protection in the first few months of life.
PASSIVE HUMORAL IMMUNITY
The principal immunoglobulin conferring passive immunity in the neonatal period is maternally derived IgG. In neonates, the levels of IgG in the serum correlate well with maternal IgG levels. Immunoglobulin G freely crosses the placental barrier and this transfer of immunoglobulin increases with gestational age. The absolute half-life, on a gram percent basis, of maternally derived IgG is approximately 3 to 4 weeks, but specific protective titers of antibody may last up to 12 months. This duration of protective effect depends on the initial levels at birth. The more premature the infant at birth, the shorter the time duration for maternal transfer of IgG and the result is lower levels of maternally derived IgG in the circulation. The advantage of this maternally transferred IgG is that it provides a transfer of specific immunoglobulin protection against tetanus, diphtheria, pertussis, measles, rubella, and varicella, as well as a wide variety of organisms including streptococci, staphylococci, toxoplasmae, and salmonellae (H-type). These protective immunities reflect the circulating immunoglobulins most adults have in their plasma vascular compartment. Unfortunately there is little transfer of protective antibodies for influenza, poliovirus, and shigella, salmonella (O-type), and Escherichia coli organisms.
ACTIVE HUMORAL IMMUNITY
Besides IgG there are 3 principal antibodies in host defenses: IgM, IgA, and IgE. All 4 immunoglobulins are active in newborn and preterm infants and are synthesized in response to a specific antigenic challenge.
Immunoglobulin M is the first component elaborated in response to a specific antigen or infection. It comprises approximately 15% of the immunoglobulin in adults and is the major immunoglobulin elaborated against gram-negative organisms and viruses. There is no placental transfer of IgM, and therefore any IgM present requires de novo synthesis by the fetus or infant. Immunoglobulin M is first present at 10 to 15 weeks of gestation, and any level of IgM >20 mg/mL signifies an in utero infection.
Immunoglobulin G, the most prevalent component of the immunoglobulin system, as stated above, is primarily transferred from the maternal circulation. Fetal production of IgG begins in the third trimester; low levels are observed at term, followed by a steady increase in endogenous IgG by 3 to 4 months postpartum. By 24 months of age, infants have achieved normal adult production levels of IgG.
The third immunoglobulin component, IgA, also comprises approximately 10% to 15% of adult immunoglobulins. It is the primary immunoglobulin found in secretions, particularly in the gut. Fetal production of IgA begins at 12 weeks of gestation, and there are very low levels throughout the first 2 years of life. As with IgM, there is no placental transfer of IgA, and elevated levels of IgA also indicate de novo synthesis and an in utero infection. In normal term infants at 1 month of age, there are detectable levels of IgA in the circulation. Most children have normal levels of IgA by age 10.
The last humoral component, IgE, is present in infants. As for IgM and IgA, there is no placental transfer, and IgE levels are elevated in skin-sensitizing activity and allergic phenomena.
A very minor component of the humoral immunity is IgD. It is probably purely a marker of immature B cells because it is usually seen early in B-cell development both intracellularly and on the cell surface. With the development of more mature B cells and isotype switching from IgD to IgM, IgG, IgA, or IgE is produced and IgD is no longer expressed.
Cellular Immune Defenses
The 2 principal specific active components of the cellular immune system are B and T lymphocytes, including natural killer cells.
B LYMPHOCYTES
B lymphocytes are derived from the bursal equivalent which in humans is most likely the fetal liver and bone marrow. The first B cells identified are pre-B cells which contain cytoplasmic IgM only. These cells are present in the fetal liver at 8 weeks of gestation and then are found in the bone marrow at later stages of gestation. The first true B cells that have only surface IgM are found at 10 weeks of gestation, and by 10 to 12 weeks cells containing both IgG and IgM on their cell surface are present, indicating isotype switching from IgM to IgG. Beyond 12 weeks of gestation, both IgA- and IgG-only positive cells are present. By 20 weeks of gestation the fetus can develop specific antibody production in response to infection. Development of the full repertoire of B cells is beyond the scope of this chapter, but it is probably complete by term. This generation of cell lines that react to different antigens (clonal diversity or idiotype generation) is required for the full expression of B-cell immune responses and is present at birth.
Antigen modulation, a peculiarity of fetal B cells, allows a specific antigen to bind to the immunoglobulin on the surface of the B cell. This binding causes capping (internalization) of the antigen-immunoglobulin complex, which produces inactivation of that particular cell. Following this inactivation, there is no reexpression of that specific immunoglobulin on future clonally elaborated B cells, providing a possible explanation for the elimination of forbidden autoreactive clones that react to self cells and also for neonatal tolerance. Antigen modulation is very rare in adults and in children. Its failure in pediatric patients was not appreciated in early organ transplantation and led to severe rejection episodes.
T LYMPHOCYTES
T lymphocytes, which are derived from the thymus (or processed through the thymus), are the effector arm of the cell-mediated immune response. They also provide help for each other and for B cells in differentiating and proliferating through the elaboration of a myriad of cytokines. There are 3 subsets of T lymphocytes: helper, suppressor, and cytotoxic. In addition there is a subset of cytotoxic T cells called natural killer cells, which are nonspecific killer cells. Basically, helper T lymphocytes are primarily responsible for the elaboration of lymphokines which help to induce differentiation and proliferation of other T-cell subsets and B cells. Suppressor T lymphocytes appear to down-regulate immune responses by direct inhibition. Cytotoxic T lymphocytes are stimulated in response to specific antigen activation and are responsible for direct cell cytotoxicity.
The T-cell repertoire is generated and/or matures in the thymus. The thymus gland is derived from the third branchial pouch and is present as early as 6 weeks of gestation. The thymic epithelium is clearly demarcated by 6 weeks of gestation, with the initial invasion of lymphocytes occurring by 8 to 9 weeks of gestation. These lymphocytes have presumably migrated from the fetal liver and bone marrow. By the ninth week of gestation, specific T cells can be identified in the embryonic thymus. And by the 11th week of gestation, these T cells have generated their cell surface markers: CD4 for helper cells and CD8 for cytotoxic and suppressor cells. By 12 to 14 weeks of gestation, the thymus is histologically identical to that in older children and adults. By 15 to 20 weeks of gestation, there are circulating peripheral T cells with mature markers on their cell surface (CD3). T-cells derived either from the thymus or from the circulation of the 18- to 20-week fetus are fully functional T lymphocytes which respond normally to specific antigens as well as to nonspecific mitogenic stimulatory responses.
As a result of this early maturation of T cells, specific cell-mediated immunity is present in term infants. There is an overall decrease in the numbers of T cells in preterm infants who are both small for gestational age and appropriate in size for gestational age. Infants who are small for gestational age, however, may retain decreased proliferative responses up to 12 months after birth.
Of interest is the presence of suppressor cells in the neonatal circulation. These neonatal suppressor cells are probably CD4 suppressors and can cause nonspecific inhibition of the cell-mediated responses of adult T lymphocytes in coculture. There apparently is no in vivo or in vitro inhibition of neonatal cells. These suppressor cells secrete a soluble suppressor factor which is active only against adult cells (both T and B cells). This suppression is not related to the major histocompatibility complex (MHC) and does not cause direct cytotoxicity. It is possible that these circulating cells prevent any maternal immunocompetent cells that may cross the placenta from causing an immune reaction (rejection) in the growing fetus.
Transplantation Immunology
As stated above, both the specific and nonspecific immune systems of infants and children are intact at birth. Qualitatively, all components of these systems are present and functioning. The numbers of cells that can be recruited to respond to any given antigen whether it is infectious or an alloantigen are, however, somewhat deficient in the pediatric age group. In the transplantation situation, pediatric patients, even in the first few months of life, are capable of mounting an immune response to alloantigens. The vigorousness of this response based on absolute numbers of specific responding cells is somewhat decreased, and the younger the child, the slower the response that may develop.
It is well known that the MHC, made up of both class I and class II antigens, is present on virtually all mammalian cells. The various numbers of class I and II antigens present on any cell within an organ determine its antigenicity and lead to various degrees of reactivity by the transplant recipient toward the specific organ transplanted. This explains the variability in rejection rates seen when different vascularized solid organs are transplanted. Pediatric patients have all the cellular and humoral components needed to mount a response to foreign MHC antigens. In addition, they can mount immune reactions to ABO blood groups as well as to minor tissue-specific antigens. The MHC is divided into 2 groups: class I, the A, B, and C loci; and class II, the D and DR loci. In response to an alloantigen, pediatric recipients mount both a humoral and a cell-mediated response.
The humoral response may take the form of a preformed antibody in the case of ABO incompatibility or when the recipient has previously been sensitized to specific MHC antigens (a previous transplant or a blood transfusion). A preformed antibody provides the basis for clinical hyperacute rejection. In addition, first-set reactions occur in an alloantigen situation where B lymphocytes are activated by MHC antigens and produce specific antibodies in response to these antigens.
Cell-mediated responses are also restricted by cell type: CD4 helper cells and CD8 cytotoxic T cells. CD4 cells respond only to class II antigens, and CD8 cells respond to class I antigens. The phenomenon of MHC restriction also applies to these alloantigen responses and basically means that foreign class I and class II antigens must be presented to the responding T cells in relationship to self MHC antigens. For practical purposes monocyte/macrophages process foreign MHC antigens and present these antigens to T or B cells along with self MHC. The result is the activation of CD4 cells in response to class II antigens with the release of cytokines which potentiate cell proliferation, maturation of cells, and recruitment of new T cells into the response. These recruited cells include CD8 cytotoxic T cells and CD8 suppressor T cells which respond to self-class I plus antigen. The theory is that suppressor cells are also elaborated in this immune response and begin to downregulate the overall response and modulate the reaction. In addition to these suppressor cells, there are other varieties of regulatory cells that come into play to downgrade both T- and B-cell responses. These include anti-idiotypic antibodies which are elaborated in response to a large production of a particular IgM or IgG antibody. There are also CD8 T cells with specific T-cell receptors that function as autoregulatory cells to downgrade the overall T-cell responses by direct cytotoxicity.
As stated above, all these components of the effector arm of the specific immune response are present at birth and are involved in the response of pediatric patients to alloantigens of the clinical transplantation situation.
Immunodeficiency
A large number of congenital immunodeficiencies have been described. These clinical entities are usually secondary to point mutations of genes responsible for enzymes or other proteins required for optimal function of the immune system. All components of the immune system can be involved singly for some enzymes or multiply if the proteins affected are critical for common functions of the immune system.
In addition to these inherited or sporadically acquired point mutations immunodeficiency can be acquired after infection with the human immunodeficiency virus.
A detailed description of congenital and acquired immunodeficiency is beyond the scope of this discussion. A summary of the more commonly seen clinical immunodeficiency states appears in Table 97-2.
Table 97-2
Common Immunodeficiencies
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Clinical Immunosuppression
Advances in immunosuppressive therapy have led to the remarkable success of clinical organ transplantation over the past 3 decades. As clinical experience with organ transplantation in children grew, it became apparent that these patients required similar, if not greater, immunosuppression and surveillance for rejection than adult transplant recipients. This requirement for active immunosuppression has also led to a greater frequency of opportunistic infections and secondary malignancies in this population of immunologically naive patients.
Over the years, a variety of agents have been used to provide immunosuppression in transplant recipients. The first clinically used agents were primarily antiproliferative agents developed for cancer chemotherapy and were used with high-dosage corticosteroids. Over the past 2 decades, more-specific agents have become available to provide direct suppression of the responding immune cells actually responsible for rejection.
As our understanding of the specific mechanism of action of these agents has grown, there has been a gradual trend away from the use of high dosages of single or double agents and toward the use of multiple drug combinations in lower dosages to affect different compartments of the humoral and/or cellular immune response to alloantigens. Table 97-3 lists the agents currently used for clinical immunosuppression in the pediatric age group as well as their proposed cellular sites of action.
Table 97-3
Classification of Immunosuppressive Drugs by Mechanism of Action
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Corticosteroids (Solumedrol, Medrol)
Corticosteroids, both methylprednisolone and prednisone, have been in clinical use for 4 decades, and they provide potent anti-inflammatory effects by inhibiting transcription of cytokine-encoding genes. At higher dosages there may be a direct cytotoxic effect on lymphocytes. Steroids can be used for both induction therapy and in the treatment of an acute rejection episode. The first dose is given intravenously (Solumedrol 10 mg/kg) during the transplantation procedure, and the dose is decreased with a relatively rapid taper over time. Eventually maintenance therapy is achieved with steroid doses of between 0.1 and 0.3 mg/kg per day by 3 to 6 months posttransplantation. With the availability of more potent immunosuppressive agents, a number of centers have been successful in either reducing steroid use to every-other-day dosing or completely eliminating them from long-term immunosuppressive protocols. The impetus for this trend to eliminate corticosteroid use is the high morbidity and mortality secondary to opportunistic infections, as well as the potent salt and water retention seen with secondary hypertension. The long-term use of steroids also leads to osteoporosis and affects gastric acid production, resulting in gastritis or ulcer formation, and is significantly associated with the development of post-transplant diabetes mellitus. Recent use of induction agents, like thymoglobulin, have allowed for “steroid free” protocols which really comprise a very rapid 4-day taper and discontinuance of steroids post-transplant.
Azathiaprine (Imuran)
Azathiaprine is a purine analog which is metabolized to 6-mercaptopurine by the liver. This agent inhibits DNA synthesis in all mammalian cells. Azathiaprine competes with the enzymes necessary for purine biosynthesis, with the end result that there are fewer purines available for DNA synthesis, a decrease in DNA synthesis, and a decrease in cell division. Azathiaprine can be given intravenously or orally, and the usual starting dose is 1 to 2 mg/kg per day in a single dose. The major toxic side effect of azathiaprine is bone marrow suppression affecting all cell lines in the marrow. Neutropenia is the first evidence of azathiaprine toxicity. Therefore, patients should have their white blood cell counts followed and dose adjustments made for white blood cell counts falling below 5000/mm3. Late toxicity of azathiaprine may include red cell aplasia, hepatitis, and pancreatitis. Mycophenolate mofetil has replaced azathioprine as a first-line immunosuppression agent.
Mycophenolate Mofetil (Cell-Cept)
Mycophenolate mofetil (Cell-Cept) is an antimetabolite similar to azathiaprine. Its potential advantage over azathiaprine is that it competitively inhibits inositol monophosphate dehydrogenase, which is a critical enzyme in the purine salvage pathway. This specific inhibition of the purine salvage pathway is exploited in the clinical transplantation situation because purine biosynthesis through this pathway is the only way that lymphocytes can synthesize purines. Other mammalian cells are able to synthesize purines through a de novo synthetic pathway and are not affected by mycophenolate mofetil. Mycophenolate is well absorbed orally. In the pediatric population, patients are given 600 mg/M2 every 12 hours each day. The principal toxic side effects of mycophenolate mofetil are gastrointestinal upset with anorexia, nausea, vomiting, and diarrhea. As a result, patients may develop crampy abdominal pain secondary to its use. High doses may cause bone marrow suppression with anemia and neutropenia.
MMF has been shown to be effective in the prevention of acute rejection episodes in adult renal transplant patients, as well as improved graft survival in pediatric patients. MMF has an important role in induction as well as maintenance immunosuppression.
Calcineurin Inhibitors
CYCLOSPORINE (SANDIMMUNE, NEORAL)
Cyclosporine is an intracellular fungal metabolite whose immunosuppressive activity was identified and immediately used in clinical transplantation situations. It appears to impart its immunosuppressive effect by inhibiting calcium-dependent T-lymphocyte activation. This inhibition of early activation results in a failure of helper T cells to elaborate cytokines, particularly interleukin 2, a potent inducer of cytotoxic T-cell activity. Cyclosporine can be administered intravenously or orally. It is poorly absorbed from the gastrointestinal tract and must be dissolved in various lipid carriers to facilitate absorption. After transplantation, patients may initially receive 5 mg/kg per day administered intravenously in 3 divided doses or as a continuous intravenous infusion. The usual oral dose for pediatric organ transplant recipients is 10 mg/kg per day divided into 2 doses. Depending on the preparation of cyclosporine used, trough levels are followed on a daily basis until a steady state is achieved. Trough levels between 150 and 500 ng/mL are commonly acceptable in most clinical situations. A microemulsion preparation of cyclosporine (Neoral) offers improved oral absorption and greater ease of dosing. The area under the curve or 2-hour postdose levels appear to be better indications of immunosuppression than trough levels for this preparation. A number of side effects occur secondary to the use of cyclosporine, including neurologic symptoms (tremors, seizures), hypertension, hirsutism, hypercholesterolemia, gingival hyperplasia, hepatotoxicity, and nephrotoxicity. All these side effects can be alleviated by dosage reduction.
TACROLIMUS (PROGRAF)
Tacrolimus is a macrolide antibiotic also derived from a fungus. This drug is 100-fold more potent than cyclosporine on a per-milligram basis. Tacrolimus can be administered intravenously or given orally. Because it is rapidly absorbed in the gastrointestinal tract with good pharmacologic activity, there is little indication for intravenous administration. A typical dose for pediatric patients is 0.15 mg/kg divided into every 12 hour administration. The occasional patient who requires intravenous administration can receive the drug by continuous infusion or by intermittent doses of 0.05 mg/kg per day given every 12 hours. As with cyclosporine, trough levels of tacrolimus are followed daily until stable levels are achieved. Individual pediatric patients may have widely disparate levels when given standard dosing, and doses must therefore be altered based on trough levels. Trough levels between 5 and 10 mg/mL are initially obtained with the higher levels used during rejection episodes. The principal toxic side effects of tacrolimus are neurologic symptoms (tremors, nervousness, sleepiness, and nightmares), hypertension, dyslipidemia, gastric problems (nausea, vomiting, and diarrhea), and development of diabetes mellitus, hyperkalemia, and nephrotoxicity. As in the case of cyclosporine, these toxic side effects are dose-related and usually respond to dose adjustment. There has been an increasing interest in minimizing the use of calcineurin inhibitors, mainly because of the long-term nephrotoxicity and risk for diabetes.
SIROLIMUS
Sirolimus, or rapamycin, is an inhibitor of the mammalian target of rapamycin (mTOR). It is a macrocyclic lactone that is produced by a strain of Streptomyces hygroscopicus. Sirolimus is a potent inhibitor of B- and T-lymphocytes, as well as an inhibitor of smooth muscle proliferation and inhibitor of intimal hyperplasia. Unlike in adults, rapid metabolism of this drug in the pediatric population necessitates drug-level monitoring. Sirolimus does have an adverse effect on wound healing, with a significant incidence of lymphoceles requiring surgical drainage in 1 study. Additionally, use of sirolimus is associated with dyslipidemia. Currently, sirolimus is used as rescue therapy in patients with chronic allograft nephropathy. A major concern regarding usage of sirolimus was a potential increased incidence of PTLD in young, EBV-naïve patients. Currently, sirolimus is mainly used in pediatric liver transplant patients to reduce the nephrotoxicity from calcineurin inhibitor exposure, as well as treatment of rejection episodes.
Anti-CD3 Monoclonal Antibody (Orthoclone OKT3)
Anti-CD3 monoclonal antibody is a murine antibody raised against the T-cell receptors of mature human lymphocytes. Because all mature T lymphocytes have a CD3 receptor, OKT3 monoclonal antibody is extremely effective in eliminating mature T cells from circulation, including activated T lymphocytes in a transplanted organ. The principal toxicity of this agent is directly related to the intravenous administration of foreign protein. There is a known infusion-related cytokine release syndrome that is problematic for patients, causing fever, myalgias, and capillary leakage. This can lead to pulmonary edema and respiratory distress. Furthermore, studies have failed to demonstrate that it has superior efficacy to other agents. Use of OKT3 has fallen out of favor as an induction agent in pediatric solid organ transplantation. It has instead been replaced by newer IL-2 antibody and polyclonal antilymphocyte preparations. The North American Pediatric Renal Transplant Cooperative Study in 2004 reported that none of the pediatric renal transplants performed in 2003 received OKT3 therapy.
Antithymocyte Globulin
Antithymocyte globulin is a polyclonal antibody derived from rabbits that is specific for human T lymphocytes. The polyvalent nature of the antibody eliminates all T lymphocytes from the circulation. CD2 and CD11 are receptors found on immature T lymphocytes, and CD2/CD11 levels are followed in the blood. The agent is given intravenously at doses of 15 to 20 mg/kg per day as a single intravenous dose. There has been a resurgence of the use of polyclonal antibodies as part of an induction regimen for post-transplant immunosuppression. The use of rabbit anti-thymoglobulin has been demonstrated as an effective induction agent for all types of transplants. As with OKT3, there are significant side effects related to foreign protein administration, and patients are pretreated with steroids, diphenhydramine, and acetaminophen to alleviate these effects.
IL-2 Receptor Antibodies
Anti-IL2 receptor blockers target a single antigen on the T-cell, inhibiting the IL-2 induced clonal expansion of activated T cells. Because their target is so narrow, they may offer a more specific suppression than the polyclonal antibodies. Use of these agents is well tolerated, with avoidance of the cytokine release syndrome. Statistically significant lower incidence of graft thrombosis occurred when induction was performed using an IL-2 antibody induction. The main advantage of IL-2 receptor blockers appears to be a decreased incidence of acute rejection episodes; ultimately it is hopeful that this will translate into prolonged allograft survival. The number and severity of acute rejection episodes significantly impacts the incidence of chronic rejection, and hence, graft loss. Use of these antibodies is contraindicated in hepatitis-related liver transplants.
Finally, there is an anti-CD-52 monoclonal antibody, alemtuzumab (Campath H-1) that has seen increasing use. Its administration leads to a profound depletion of peripheral lymphocytes, NK cells, and monocytes. Currently its use is restricted mostly to high-risk transplant recipients.
Treatment Strategies
The agents described above are usually prescribed in combination to provide immunosuppression adequate to maintain a graft in a transplant recipient without rejection or significant toxicity. Each of these agents is used to provide effective immunosuppression at 3 different stages in the posttransplantation period. The first stage is the induction stage at the time of implantation of the organ, followed by a maintenance period. The treatment of any rejection episodes that may occur also demands a change in treatment strategy. Common immunosuppression protocols are listed in Table 97-4.
Table 97-4
Immunosuppression Protocols
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INDUCTION THERAPY
Currently, most kidney, heart, pancreas, intestine, and lung-transplant recipients receive induction therapy. Induction therapy begins at the time of implantation of the graft and continues for the first few weeks after transplantation. The most common induction therapy utilized is rabbit-anti-thymocyte globulin combined with intravenous steroids. In contrast, there is little use of induction antibodies for liver transplantation.
MAINTENANCE THERAPY
Maintenance therapy involves maintaining a steady level of conventional immunosuppression while diminishing the overall dosage to obtain the minimal immunosuppressive dosage necessary to achieve a rejection-free patient. Increasingly, there have been attempts to avoid steroids in the maintenance therapy. With the introduction of cyclosporine- and tacrolimus-based therapies, there has been significant ability to withdraw steroids in transplant recipients. While steroid-minimizing regimens have shown improvement in growth markers, longer term follow-up demonstrates a significant decrease in graft function and an increase in rejection episodes. Newer studies that utilize antibody-based induction regimens have shown more promise. The best results with steroid withdrawal have been based on extended induction with IL-2 antibodies and complete steroid avoidance. Currently, most maintenance regimens target various T-cell activation mechanisms.
Tacrolimus trough levels are maintained at 10 ng/mL for the first 3 months following transplantation with a gradual reduction to approximately 5 to 7 ng/mL after that time. Long-term maintenance levels of tacrolimus are gradually decreased, and many patients receive very low detectable levels, 1 to 3 ng/mL, on a long-term basis.
Mycophenolate mofetil dosing continues for the first-year post-transplantation with dose adjustments made for any gastrointestinal toxicity or bone marrow suppression. The use of a mycophenolate/tacrolimus combination is the most frequently used discharge regimen in kidney and liver recipients. More rarely, mycophenolate/sirolimus, tacrolimus/sirolimus, and mycophenolate/cyclosporine combinations have been used. Tacrolimus has virtually replaced the use of cyclosporine in most centers. Discharge without steroid therapy is more common in liver than kidney recipients; 20% of liver recipients and 27% of intestinal recipients in 2004 were discharged without steroids.
ANTIREJECTION STRATEGY
Despite induction therapy, a significant number of patients develop rejection episodes during the first few weeks following transplantation. Fortunately, the incidence of rejection has been decreasing overall. Most episodes of acute kidney and liver rejection are treated with corticosteroids, administered at a dose of 10 mg/kg/day. Some centers are using antithymocyte globulin with success.
Complications of Immunosuppression
The clinical immunosuppressive strategies currently used attempt to strike a balance between rejection prevention and the infectious or toxic complications of immunosuppressive drugs. The individual immune response of pediatric patients to the antigen load with a transplanted organ unfortunately establishes the degree of immunosuppression necessary to achieve freedom from rejection. For a small number of patients who respond vigorously to the transplanted organ, this converts to more severe complications and toxicities from higher doses of immunosuppressive drugs. Table 97-5 summarizes the common complications of immunosuppression. The problem seen most often in pediatric patients undergoing significant chronic immunosuppression involves opportunistic infections, which translates into more frequent problems with bacterial and fungal sepsis often related to indwelling catheters. In addition, children lack specific immunity to a number of parasitic and viral pathogens. Pediatric transplant recipients, therefore, must be aggressively assessed for opportunistic infection in the post-transplantation period; they commonly require prophylactic agents to prevent these infections. Table 97-6 summarizes the prophylactic regimen used at the Johns Hopkins Hospital for pediatric patients during the first 6 months posttransplantation. In general, prophylaxis for Pneumocystis carinii pneumonia, as well as for opportunistic herpes virus infections including cytomegalovirus and Epstein–Barr virus (EBV), is used in patients who are at risk for these infections. This population of pediatric patients includes those who undergo induction or receive OKT3 or antithymocyte globulin therapy, as well as those who receive a cytomegalovirus- or EBV-mismatched organ from a viral positive adult donor. One potential advantage of the IL2-receptor antibodies is a decrease in the incidence of PTLD, as well as overall better tolerance of its infusion compared to OKT3 and ATG.
Table 97-5
Complications of Immunosuppression
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Table 97-6
Infection Prophylaxis
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In addition to developing a primary mononucleosis syndrome secondary to EBV activation or infection, pediatric transplant recipients are at higher risk for EBV associated post-transplantation lymphoproliferative disorder. A primary mononucleosis syndrome characterized by lymphadenopathy, hepatosplenomegaly, pharyngitis, and fever may progress to frank lymphoma or to a leukemic state secondary to unchecked proliferation of transformed B lymphocytes. In an immunologically normal individual, cytotoxic T cells are generated, which presumably eliminates these clones of transformed B cells and downregulates the mononucleosis syndrome. With significant immunosuppression, pediatric transplant recipients are unable to mount an effective T-cell response to these EBV-transformed B lymphocytes and essentially develop Burkitt-type lymphoma or leukemia. These patients must be aggressively treated with withdrawal of immunosuppression. If they have significant bulky lymphomatous disease or leukemia, they may require cytoreduction therapy to eliminate the majority of proliferating B cells. Withdrawal of immunosuppression following adequate cytoreduction presumably allows for recovery of the T-cell arm of the immune system and provides long-term cellular control of the transformed B lymphocytes.
Effects of Immunosuppression
As increasing numbers of pediatric transplant recipients survive and require long-term treatment with immunosuppressive agents, we will see more evidence of significant long-term toxicity. The primary late effects of corticosteroids include Cushing syndrome, osteoporosis, impaired growth, avascular necrosis of the femoral head, cataracts, glaucoma, cardiovascular disease, and gastritis or peptic ulcer disease. Long-term use of azathioprine can cause hepatitis, pancreatitis, and red cell aplasia. Cyclosporine used on a long-term basis can lead to hypercholesterolemia, arteriosclerosis, hypertension, nephrotoxicity, neurotoxicity, hyperkalemia, and hyperglycemia. The effects of long-term use of tacrolimus include hypertension and nephrotoxicity, diabetes mellitus, and dyslipidemia as described above. Adverse effects of sirolimus include impaired wound healing and dyslipidemia. It is minimization of these adverse effects which drives investigation into immunosuppression minimization.
Secondary malignancies also are a fact of life for long-term immunosuppressed transplant recipients. A small number of patients may develop Kaposi sarcoma, which is presumably related to an EBV-like virus. Posttransplantation lymphoproliferative disorder secondary to EBV infection also can occur in chronically immunosuppressed patients. Patients are also at higher risk for the common malignancies seen in non-immunosuppressed patients, such as Hodgkin disease, non-Hodgkin lymphoma, and skin cancer. Our pediatric transplant recipients at the Johns Hopkins Hospital undergo yearly cancer surveillance including a chest radiograph and a general physical examination to screen for skin lesions and any other problems. Teenaged female recipients should undergo yearly pelvic examinations and Papanicolaou tests when appropriate.
Conclusions
Fortunately for pediatric surgeons and their patients, neonates and children have a competent immune system at birth that provides significant responses in the face of infection and surgical stress. All the features of the immune system are qualitatively present in newborns and in older children, but there are significant quantitative differences between children and adults.
A variety of congenital and acquired immunodeficiency states may occur in the pediatric age group that complicates their treatment.
In the transplantation clinical population an intact immune system requires significant immunosuppression to prevent rejection. The overall naivete of the pediatric immune system and the difference in how medications are metabolized in the transplantation clinical situation results in a higher incidence of secondary complications of immunosuppression than in adults, and therefore greater vigilance for complications of immunosuppression must be maintained. There have been great strides in long-term transplant patient and graft survival, which has led to increasing interest in producing immunosuppression regimens that minimize adverse effects on the patients. Ultimately, the goal is to induce a tolerant state for the transplanted organ, but until this is achieved, careful tailoring of the medications to the individual patient must be maintained.
Selected Readings
Abbas AK, Lichtman AH, Prober JS Cellular and Molecular Immunology, Updated Edition. Philadelphia, PA: WB Saunders Co; 2009.
Agarwal A, et al Immunosuppression in pediatric solid organ transplantation. Semin Pediatr Surg 2006;(15):142–152.
Colombani PM. Clinical immunosuppression. In: Oldham KT, Colombani PM, Foglia RP, eds. Surgery of Infants and Children: Scientific Principles and Practice. Philadelphia, PA: Lippincott-Raven; 1997:671–700.
Hannet I, Erkeller-Yuksel F, Lydyard P, Deneys V, DeBruyere M. Developmental and maturational changes in human blood lymphocyte subpopulations. Immunol Today 1992;13:215–218.
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Meier-Kriesche H-U, et al Immunosuppression: evolution in practice and trends, 1994–2004. Am J Transpl 2006:(6, part 2):1111–1131.
Schonder K, Mazareigos G, Weber R. Adverse effects of immunosuppression in pediatric solid organ transplantation. Pediatr Drugs 2010;12(1):35–49.
CrossRef
Yang I, et al Immunosuppressive strategies to improve outcomes of kidney transplantation. Semin Nephrol 2007;27(4):337–392.
Sara K. Rasmussen; Paul M. Colombani
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Key Points
Introduction
Components of the Immune System
Transplantation Immunology
Conclusions
Selected Readings
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Key Points
Though nonspecific host defenses are somewhat deficient in the infant and pediatric patient, the effector arm of the specific immunologic response to alloantigens of clinical organ transplants is intact at the time of birth.
Effective organ allograft immunosuppression requires a balance of prevention of rejection of the graft while avoiding the toxicity of excessive immunosuppressing agents.
Immunosuppressive agents can be categorized into those used for induction (rabbit anti-thymocyte globulin, corticosteroids), those for maintenance (mycophenolate mofetil, tacrolimus), and those used to treat acute rejection (corticosteroids).
Introduction
Infections are a leading cause of pediatric morbidity and mortality, particularly in the neonatal period. Explanations for this phenomenon include a lack of previous antigenic experience on the part of pediatric patients; an intrinsic immaturity of lymphocyte functions in this age group; and, for neonates, an active cellular suppression mechanism. In addition, several specific inherited defects of the immune system may manifest themselves in early infancy and continue to plague these patients through childhood and on into adulthood. Acquired defects in the immune defenses occur in transplantation and oncology patients. This chapter will review the specific and nonspecific components of the immune system with particular reference to the pediatric patient's ability to fight infection (see also Chapter 13—“Surgical Infection: Classification, Diagnosis, Treatment and Prevention”). Emphasis will be placed on the differences in these responses in neonates and children compared to the responses in adult subjects. The ability of children to mount an immune response against alloantigens in the transplantation setting will be discussed. In addition, this chapter will review current methods of clinical immunosuppression for the pediatric age group.
Components of the Immune System
Immune responses are generally divided into specific and nonspecific components. Each of these components is further subdivided into humoral and cellular compartments (Table 97-1).
Table 97-1
Summary of Immune System Components
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Nonspecific (Nonimmune) Host Defenses
HUMORAL COMPONENTS
The humoral components of nonspecific host defense systems include the classic complement pathway and the alternate, or properdin, pathway. The complement cascade is made up of 9 serum proteins which are activated during the inflammatory response. The proteins react with immunoglobulins (Ig)—IgM or IgG—and antigen. Activated complement (C′) liberates mediators of the inflammatory response, and the levels of the C3/C5 component correlate with susceptibility to infection. The C3 level determines the rate of phagocytosis or lysis of invading organisms. In the presence of immunoglobulins, the C142 component (the membrane attack complex) activates C3, the binding of immunoglobulins, and eventual opsonization of the organism by the phagocytic system. Complement is detectable at 1% of adult levels at 5 weeks of gestation. By 26 to 28 weeks of gestation there is a rapid increase in complement levels in the serum of the fetus. By term, complement levels are 30% to 50% of adult levels. Complement levels in term infants are subnormal in approximately 50% of neonates, but the mean level of activation may approach 70% to 90% of adult activity following activation.
The properdin or alternate pathway also appears early in gestation during the first trimester. By term, this activity is 30% to 60% of adult levels and is therefore more suppressed in neonates than the levels of proteins in the complement cascade. Quantitatively, preterm infants have a lower level of properdin. Opsonizing activity by the properdin pathway is determined by factor B levels, and because these levels are lower in term and preterm infants overall, opsonizing activity is limited.
CELLULAR COMPONENTS OF THE NONSPECIFIC SYSTEM
The cellular components of nonspecific host defense include polymorphonucleocytes (PMN), monocytes, macrophages, and the modified macrophages of the reticular endothelial system (RES). In response to chemotactic factors, PMN migrate to areas of infection. There is a clear deficiency in chemotaxis because of the decreased elasticity of neonatal PMN, which are also responsible for killing bacteria after phagocytosis. The phagocytic ability of these cells is probably normal at birth, but in vitro assays of phagocytosis are diminished in the cells of term and preterm infants compared to adult cells. The addition of adult serum to these neonatal cells normalizes their activity, which suggests that neonates are simply more deficient in opsonizing factors as alluded to above. The result, however, is that the phagocytic ability of these cells is diminished, particularly in stressed infants and children.
Following engulfment and phagocytosis of organisms, PMNs kill these organisms primarily by peroxidation by oxygen-free radicals produced by the cells. Neonatal PMN clearly have a decreased ability to generate oxygen-free radicals for intracellular killing. Also, under stress conditions, even though phagocytosis may be relatively intact, there is decreased intracellular killing.
Migratory macrophages and monocytes are similarly deficient in chemotactic ability and phagocytosis in the neonatal period. Beyond that, macrophage function appears to be normal.
The RES is a system of fixed macrophages or monocytes in a variety of sites including the liver, spleen, and bone marrow. These cells serve to remove particulates from the bloodstream, including viral, bacterial, and fungal organisms. The RES system is intact in both term and preterm infants. This system is able to respond at maximum levels at term, but serum opsonizing factors are required for optimal function.
Important adjunct components of the nonspecific immune system in neonates are the mononuclear phagocytes in maternal milk ingested during breast feeding. These mononuclear cells comprise 80% of the cells found in colostrum and breast milk. On the first day of breast milk production, there are approximately 2 million cells/mL of breast milk. This number of phagocytes declines to approximately 1.3 million cells/mL by approximately 3 months following birth. These activated cells secrete components of complement and possess immunoglobulin Fc receptors and complement receptors. They also contain and may secrete intracellular IgA. They may also be responsible for the phagocytosis and intracellular killing of swallowed organisms with which neonates have had no previous immune experience. In addition, these cells may elaborate and secrete a number of lymphokines which may be important for the development of optimal function of the gut-associated immune responses in infants.
In summary, there are a number of nonspecific, nonimmune components for host defenses that are all qualitatively present in preterm and term infants as well as in children. In general, the humoral components are deficient in quantity which may lead to a decreased level of serum opsonization factor in neonatal and infant blood. Cellular components of this system are highly dependent on these opsonic factors for optimal function. As a result, overall nonspecific host defenses are somewhat deficient in the pediatric age group, particularly in neonates as compared to adults. This deficiency is primarily due to limiting opsonic activity when infants are under stress, as well as other potential deficiencies in chemotaxis and intracellular killing.
Specific Immune Defenses
Most important in developing specific immunity against infections and mounting an immune response to alloantigens in the transplantation setting are the humoral and cellular components of the immune system.
HUMORAL COMPONENTS
The humoral components of the immune system are primarily the immunoglobulins elaborated by B lymphocytes. In addition to specific immunoglobulin production in response to antigenic stimulus, which is termed active immunity, there is also passive maternally-derived IgG, conferring some immune protection in the first few months of life.
PASSIVE HUMORAL IMMUNITY
The principal immunoglobulin conferring passive immunity in the neonatal period is maternally derived IgG. In neonates, the levels of IgG in the serum correlate well with maternal IgG levels. Immunoglobulin G freely crosses the placental barrier and this transfer of immunoglobulin increases with gestational age. The absolute half-life, on a gram percent basis, of maternally derived IgG is approximately 3 to 4 weeks, but specific protective titers of antibody may last up to 12 months. This duration of protective effect depends on the initial levels at birth. The more premature the infant at birth, the shorter the time duration for maternal transfer of IgG and the result is lower levels of maternally derived IgG in the circulation. The advantage of this maternally transferred IgG is that it provides a transfer of specific immunoglobulin protection against tetanus, diphtheria, pertussis, measles, rubella, and varicella, as well as a wide variety of organisms including streptococci, staphylococci, toxoplasmae, and salmonellae (H-type). These protective immunities reflect the circulating immunoglobulins most adults have in their plasma vascular compartment. Unfortunately there is little transfer of protective antibodies for influenza, poliovirus, and shigella, salmonella (O-type), and Escherichia coli organisms.
ACTIVE HUMORAL IMMUNITY
Besides IgG there are 3 principal antibodies in host defenses: IgM, IgA, and IgE. All 4 immunoglobulins are active in newborn and preterm infants and are synthesized in response to a specific antigenic challenge.
Immunoglobulin M is the first component elaborated in response to a specific antigen or infection. It comprises approximately 15% of the immunoglobulin in adults and is the major immunoglobulin elaborated against gram-negative organisms and viruses. There is no placental transfer of IgM, and therefore any IgM present requires de novo synthesis by the fetus or infant. Immunoglobulin M is first present at 10 to 15 weeks of gestation, and any level of IgM >20 mg/mL signifies an in utero infection.
Immunoglobulin G, the most prevalent component of the immunoglobulin system, as stated above, is primarily transferred from the maternal circulation. Fetal production of IgG begins in the third trimester; low levels are observed at term, followed by a steady increase in endogenous IgG by 3 to 4 months postpartum. By 24 months of age, infants have achieved normal adult production levels of IgG.
The third immunoglobulin component, IgA, also comprises approximately 10% to 15% of adult immunoglobulins. It is the primary immunoglobulin found in secretions, particularly in the gut. Fetal production of IgA begins at 12 weeks of gestation, and there are very low levels throughout the first 2 years of life. As with IgM, there is no placental transfer of IgA, and elevated levels of IgA also indicate de novo synthesis and an in utero infection. In normal term infants at 1 month of age, there are detectable levels of IgA in the circulation. Most children have normal levels of IgA by age 10.
The last humoral component, IgE, is present in infants. As for IgM and IgA, there is no placental transfer, and IgE levels are elevated in skin-sensitizing activity and allergic phenomena.
A very minor component of the humoral immunity is IgD. It is probably purely a marker of immature B cells because it is usually seen early in B-cell development both intracellularly and on the cell surface. With the development of more mature B cells and isotype switching from IgD to IgM, IgG, IgA, or IgE is produced and IgD is no longer expressed.
Cellular Immune Defenses
The 2 principal specific active components of the cellular immune system are B and T lymphocytes, including natural killer cells.
B LYMPHOCYTES
B lymphocytes are derived from the bursal equivalent which in humans is most likely the fetal liver and bone marrow. The first B cells identified are pre-B cells which contain cytoplasmic IgM only. These cells are present in the fetal liver at 8 weeks of gestation and then are found in the bone marrow at later stages of gestation. The first true B cells that have only surface IgM are found at 10 weeks of gestation, and by 10 to 12 weeks cells containing both IgG and IgM on their cell surface are present, indicating isotype switching from IgM to IgG. Beyond 12 weeks of gestation, both IgA- and IgG-only positive cells are present. By 20 weeks of gestation the fetus can develop specific antibody production in response to infection. Development of the full repertoire of B cells is beyond the scope of this chapter, but it is probably complete by term. This generation of cell lines that react to different antigens (clonal diversity or idiotype generation) is required for the full expression of B-cell immune responses and is present at birth.
Antigen modulation, a peculiarity of fetal B cells, allows a specific antigen to bind to the immunoglobulin on the surface of the B cell. This binding causes capping (internalization) of the antigen-immunoglobulin complex, which produces inactivation of that particular cell. Following this inactivation, there is no reexpression of that specific immunoglobulin on future clonally elaborated B cells, providing a possible explanation for the elimination of forbidden autoreactive clones that react to self cells and also for neonatal tolerance. Antigen modulation is very rare in adults and in children. Its failure in pediatric patients was not appreciated in early organ transplantation and led to severe rejection episodes.
T LYMPHOCYTES
T lymphocytes, which are derived from the thymus (or processed through the thymus), are the effector arm of the cell-mediated immune response. They also provide help for each other and for B cells in differentiating and proliferating through the elaboration of a myriad of cytokines. There are 3 subsets of T lymphocytes: helper, suppressor, and cytotoxic. In addition there is a subset of cytotoxic T cells called natural killer cells, which are nonspecific killer cells. Basically, helper T lymphocytes are primarily responsible for the elaboration of lymphokines which help to induce differentiation and proliferation of other T-cell subsets and B cells. Suppressor T lymphocytes appear to down-regulate immune responses by direct inhibition. Cytotoxic T lymphocytes are stimulated in response to specific antigen activation and are responsible for direct cell cytotoxicity.
The T-cell repertoire is generated and/or matures in the thymus. The thymus gland is derived from the third branchial pouch and is present as early as 6 weeks of gestation. The thymic epithelium is clearly demarcated by 6 weeks of gestation, with the initial invasion of lymphocytes occurring by 8 to 9 weeks of gestation. These lymphocytes have presumably migrated from the fetal liver and bone marrow. By the ninth week of gestation, specific T cells can be identified in the embryonic thymus. And by the 11th week of gestation, these T cells have generated their cell surface markers: CD4 for helper cells and CD8 for cytotoxic and suppressor cells. By 12 to 14 weeks of gestation, the thymus is histologically identical to that in older children and adults. By 15 to 20 weeks of gestation, there are circulating peripheral T cells with mature markers on their cell surface (CD3). T-cells derived either from the thymus or from the circulation of the 18- to 20-week fetus are fully functional T lymphocytes which respond normally to specific antigens as well as to nonspecific mitogenic stimulatory responses.
As a result of this early maturation of T cells, specific cell-mediated immunity is present in term infants. There is an overall decrease in the numbers of T cells in preterm infants who are both small for gestational age and appropriate in size for gestational age. Infants who are small for gestational age, however, may retain decreased proliferative responses up to 12 months after birth.
Of interest is the presence of suppressor cells in the neonatal circulation. These neonatal suppressor cells are probably CD4 suppressors and can cause nonspecific inhibition of the cell-mediated responses of adult T lymphocytes in coculture. There apparently is no in vivo or in vitro inhibition of neonatal cells. These suppressor cells secrete a soluble suppressor factor which is active only against adult cells (both T and B cells). This suppression is not related to the major histocompatibility complex (MHC) and does not cause direct cytotoxicity. It is possible that these circulating cells prevent any maternal immunocompetent cells that may cross the placenta from causing an immune reaction (rejection) in the growing fetus.
Transplantation Immunology
As stated above, both the specific and nonspecific immune systems of infants and children are intact at birth. Qualitatively, all components of these systems are present and functioning. The numbers of cells that can be recruited to respond to any given antigen whether it is infectious or an alloantigen are, however, somewhat deficient in the pediatric age group. In the transplantation situation, pediatric patients, even in the first few months of life, are capable of mounting an immune response to alloantigens. The vigorousness of this response based on absolute numbers of specific responding cells is somewhat decreased, and the younger the child, the slower the response that may develop.
It is well known that the MHC, made up of both class I and class II antigens, is present on virtually all mammalian cells. The various numbers of class I and II antigens present on any cell within an organ determine its antigenicity and lead to various degrees of reactivity by the transplant recipient toward the specific organ transplanted. This explains the variability in rejection rates seen when different vascularized solid organs are transplanted. Pediatric patients have all the cellular and humoral components needed to mount a response to foreign MHC antigens. In addition, they can mount immune reactions to ABO blood groups as well as to minor tissue-specific antigens. The MHC is divided into 2 groups: class I, the A, B, and C loci; and class II, the D and DR loci. In response to an alloantigen, pediatric recipients mount both a humoral and a cell-mediated response.
The humoral response may take the form of a preformed antibody in the case of ABO incompatibility or when the recipient has previously been sensitized to specific MHC antigens (a previous transplant or a blood transfusion). A preformed antibody provides the basis for clinical hyperacute rejection. In addition, first-set reactions occur in an alloantigen situation where B lymphocytes are activated by MHC antigens and produce specific antibodies in response to these antigens.
Cell-mediated responses are also restricted by cell type: CD4 helper cells and CD8 cytotoxic T cells. CD4 cells respond only to class II antigens, and CD8 cells respond to class I antigens. The phenomenon of MHC restriction also applies to these alloantigen responses and basically means that foreign class I and class II antigens must be presented to the responding T cells in relationship to self MHC antigens. For practical purposes monocyte/macrophages process foreign MHC antigens and present these antigens to T or B cells along with self MHC. The result is the activation of CD4 cells in response to class II antigens with the release of cytokines which potentiate cell proliferation, maturation of cells, and recruitment of new T cells into the response. These recruited cells include CD8 cytotoxic T cells and CD8 suppressor T cells which respond to self-class I plus antigen. The theory is that suppressor cells are also elaborated in this immune response and begin to downregulate the overall response and modulate the reaction. In addition to these suppressor cells, there are other varieties of regulatory cells that come into play to downgrade both T- and B-cell responses. These include anti-idiotypic antibodies which are elaborated in response to a large production of a particular IgM or IgG antibody. There are also CD8 T cells with specific T-cell receptors that function as autoregulatory cells to downgrade the overall T-cell responses by direct cytotoxicity.
As stated above, all these components of the effector arm of the specific immune response are present at birth and are involved in the response of pediatric patients to alloantigens of the clinical transplantation situation.
Immunodeficiency
A large number of congenital immunodeficiencies have been described. These clinical entities are usually secondary to point mutations of genes responsible for enzymes or other proteins required for optimal function of the immune system. All components of the immune system can be involved singly for some enzymes or multiply if the proteins affected are critical for common functions of the immune system.
In addition to these inherited or sporadically acquired point mutations immunodeficiency can be acquired after infection with the human immunodeficiency virus.
A detailed description of congenital and acquired immunodeficiency is beyond the scope of this discussion. A summary of the more commonly seen clinical immunodeficiency states appears in Table 97-2.
Table 97-2
Common Immunodeficiencies
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Clinical Immunosuppression
Advances in immunosuppressive therapy have led to the remarkable success of clinical organ transplantation over the past 3 decades. As clinical experience with organ transplantation in children grew, it became apparent that these patients required similar, if not greater, immunosuppression and surveillance for rejection than adult transplant recipients. This requirement for active immunosuppression has also led to a greater frequency of opportunistic infections and secondary malignancies in this population of immunologically naive patients.
Over the years, a variety of agents have been used to provide immunosuppression in transplant recipients. The first clinically used agents were primarily antiproliferative agents developed for cancer chemotherapy and were used with high-dosage corticosteroids. Over the past 2 decades, more-specific agents have become available to provide direct suppression of the responding immune cells actually responsible for rejection.
As our understanding of the specific mechanism of action of these agents has grown, there has been a gradual trend away from the use of high dosages of single or double agents and toward the use of multiple drug combinations in lower dosages to affect different compartments of the humoral and/or cellular immune response to alloantigens. Table 97-3 lists the agents currently used for clinical immunosuppression in the pediatric age group as well as their proposed cellular sites of action.
Table 97-3
Classification of Immunosuppressive Drugs by Mechanism of Action
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Corticosteroids (Solumedrol, Medrol)
Corticosteroids, both methylprednisolone and prednisone, have been in clinical use for 4 decades, and they provide potent anti-inflammatory effects by inhibiting transcription of cytokine-encoding genes. At higher dosages there may be a direct cytotoxic effect on lymphocytes. Steroids can be used for both induction therapy and in the treatment of an acute rejection episode. The first dose is given intravenously (Solumedrol 10 mg/kg) during the transplantation procedure, and the dose is decreased with a relatively rapid taper over time. Eventually maintenance therapy is achieved with steroid doses of between 0.1 and 0.3 mg/kg per day by 3 to 6 months posttransplantation. With the availability of more potent immunosuppressive agents, a number of centers have been successful in either reducing steroid use to every-other-day dosing or completely eliminating them from long-term immunosuppressive protocols. The impetus for this trend to eliminate corticosteroid use is the high morbidity and mortality secondary to opportunistic infections, as well as the potent salt and water retention seen with secondary hypertension. The long-term use of steroids also leads to osteoporosis and affects gastric acid production, resulting in gastritis or ulcer formation, and is significantly associated with the development of post-transplant diabetes mellitus. Recent use of induction agents, like thymoglobulin, have allowed for “steroid free” protocols which really comprise a very rapid 4-day taper and discontinuance of steroids post-transplant.
Azathiaprine (Imuran)
Azathiaprine is a purine analog which is metabolized to 6-mercaptopurine by the liver. This agent inhibits DNA synthesis in all mammalian cells. Azathiaprine competes with the enzymes necessary for purine biosynthesis, with the end result that there are fewer purines available for DNA synthesis, a decrease in DNA synthesis, and a decrease in cell division. Azathiaprine can be given intravenously or orally, and the usual starting dose is 1 to 2 mg/kg per day in a single dose. The major toxic side effect of azathiaprine is bone marrow suppression affecting all cell lines in the marrow. Neutropenia is the first evidence of azathiaprine toxicity. Therefore, patients should have their white blood cell counts followed and dose adjustments made for white blood cell counts falling below 5000/mm3. Late toxicity of azathiaprine may include red cell aplasia, hepatitis, and pancreatitis. Mycophenolate mofetil has replaced azathioprine as a first-line immunosuppression agent.
Mycophenolate Mofetil (Cell-Cept)
Mycophenolate mofetil (Cell-Cept) is an antimetabolite similar to azathiaprine. Its potential advantage over azathiaprine is that it competitively inhibits inositol monophosphate dehydrogenase, which is a critical enzyme in the purine salvage pathway. This specific inhibition of the purine salvage pathway is exploited in the clinical transplantation situation because purine biosynthesis through this pathway is the only way that lymphocytes can synthesize purines. Other mammalian cells are able to synthesize purines through a de novo synthetic pathway and are not affected by mycophenolate mofetil. Mycophenolate is well absorbed orally. In the pediatric population, patients are given 600 mg/M2 every 12 hours each day. The principal toxic side effects of mycophenolate mofetil are gastrointestinal upset with anorexia, nausea, vomiting, and diarrhea. As a result, patients may develop crampy abdominal pain secondary to its use. High doses may cause bone marrow suppression with anemia and neutropenia.
MMF has been shown to be effective in the prevention of acute rejection episodes in adult renal transplant patients, as well as improved graft survival in pediatric patients. MMF has an important role in induction as well as maintenance immunosuppression.
Calcineurin Inhibitors
CYCLOSPORINE (SANDIMMUNE, NEORAL)
Cyclosporine is an intracellular fungal metabolite whose immunosuppressive activity was identified and immediately used in clinical transplantation situations. It appears to impart its immunosuppressive effect by inhibiting calcium-dependent T-lymphocyte activation. This inhibition of early activation results in a failure of helper T cells to elaborate cytokines, particularly interleukin 2, a potent inducer of cytotoxic T-cell activity. Cyclosporine can be administered intravenously or orally. It is poorly absorbed from the gastrointestinal tract and must be dissolved in various lipid carriers to facilitate absorption. After transplantation, patients may initially receive 5 mg/kg per day administered intravenously in 3 divided doses or as a continuous intravenous infusion. The usual oral dose for pediatric organ transplant recipients is 10 mg/kg per day divided into 2 doses. Depending on the preparation of cyclosporine used, trough levels are followed on a daily basis until a steady state is achieved. Trough levels between 150 and 500 ng/mL are commonly acceptable in most clinical situations. A microemulsion preparation of cyclosporine (Neoral) offers improved oral absorption and greater ease of dosing. The area under the curve or 2-hour postdose levels appear to be better indications of immunosuppression than trough levels for this preparation. A number of side effects occur secondary to the use of cyclosporine, including neurologic symptoms (tremors, seizures), hypertension, hirsutism, hypercholesterolemia, gingival hyperplasia, hepatotoxicity, and nephrotoxicity. All these side effects can be alleviated by dosage reduction.
TACROLIMUS (PROGRAF)
Tacrolimus is a macrolide antibiotic also derived from a fungus. This drug is 100-fold more potent than cyclosporine on a per-milligram basis. Tacrolimus can be administered intravenously or given orally. Because it is rapidly absorbed in the gastrointestinal tract with good pharmacologic activity, there is little indication for intravenous administration. A typical dose for pediatric patients is 0.15 mg/kg divided into every 12 hour administration. The occasional patient who requires intravenous administration can receive the drug by continuous infusion or by intermittent doses of 0.05 mg/kg per day given every 12 hours. As with cyclosporine, trough levels of tacrolimus are followed daily until stable levels are achieved. Individual pediatric patients may have widely disparate levels when given standard dosing, and doses must therefore be altered based on trough levels. Trough levels between 5 and 10 mg/mL are initially obtained with the higher levels used during rejection episodes. The principal toxic side effects of tacrolimus are neurologic symptoms (tremors, nervousness, sleepiness, and nightmares), hypertension, dyslipidemia, gastric problems (nausea, vomiting, and diarrhea), and development of diabetes mellitus, hyperkalemia, and nephrotoxicity. As in the case of cyclosporine, these toxic side effects are dose-related and usually respond to dose adjustment. There has been an increasing interest in minimizing the use of calcineurin inhibitors, mainly because of the long-term nephrotoxicity and risk for diabetes.
SIROLIMUS
Sirolimus, or rapamycin, is an inhibitor of the mammalian target of rapamycin (mTOR). It is a macrocyclic lactone that is produced by a strain of Streptomyces hygroscopicus. Sirolimus is a potent inhibitor of B- and T-lymphocytes, as well as an inhibitor of smooth muscle proliferation and inhibitor of intimal hyperplasia. Unlike in adults, rapid metabolism of this drug in the pediatric population necessitates drug-level monitoring. Sirolimus does have an adverse effect on wound healing, with a significant incidence of lymphoceles requiring surgical drainage in 1 study. Additionally, use of sirolimus is associated with dyslipidemia. Currently, sirolimus is used as rescue therapy in patients with chronic allograft nephropathy. A major concern regarding usage of sirolimus was a potential increased incidence of PTLD in young, EBV-naïve patients. Currently, sirolimus is mainly used in pediatric liver transplant patients to reduce the nephrotoxicity from calcineurin inhibitor exposure, as well as treatment of rejection episodes.
Anti-CD3 Monoclonal Antibody (Orthoclone OKT3)
Anti-CD3 monoclonal antibody is a murine antibody raised against the T-cell receptors of mature human lymphocytes. Because all mature T lymphocytes have a CD3 receptor, OKT3 monoclonal antibody is extremely effective in eliminating mature T cells from circulation, including activated T lymphocytes in a transplanted organ. The principal toxicity of this agent is directly related to the intravenous administration of foreign protein. There is a known infusion-related cytokine release syndrome that is problematic for patients, causing fever, myalgias, and capillary leakage. This can lead to pulmonary edema and respiratory distress. Furthermore, studies have failed to demonstrate that it has superior efficacy to other agents. Use of OKT3 has fallen out of favor as an induction agent in pediatric solid organ transplantation. It has instead been replaced by newer IL-2 antibody and polyclonal antilymphocyte preparations. The North American Pediatric Renal Transplant Cooperative Study in 2004 reported that none of the pediatric renal transplants performed in 2003 received OKT3 therapy.
Antithymocyte Globulin
Antithymocyte globulin is a polyclonal antibody derived from rabbits that is specific for human T lymphocytes. The polyvalent nature of the antibody eliminates all T lymphocytes from the circulation. CD2 and CD11 are receptors found on immature T lymphocytes, and CD2/CD11 levels are followed in the blood. The agent is given intravenously at doses of 15 to 20 mg/kg per day as a single intravenous dose. There has been a resurgence of the use of polyclonal antibodies as part of an induction regimen for post-transplant immunosuppression. The use of rabbit anti-thymoglobulin has been demonstrated as an effective induction agent for all types of transplants. As with OKT3, there are significant side effects related to foreign protein administration, and patients are pretreated with steroids, diphenhydramine, and acetaminophen to alleviate these effects.
IL-2 Receptor Antibodies
Anti-IL2 receptor blockers target a single antigen on the T-cell, inhibiting the IL-2 induced clonal expansion of activated T cells. Because their target is so narrow, they may offer a more specific suppression than the polyclonal antibodies. Use of these agents is well tolerated, with avoidance of the cytokine release syndrome. Statistically significant lower incidence of graft thrombosis occurred when induction was performed using an IL-2 antibody induction. The main advantage of IL-2 receptor blockers appears to be a decreased incidence of acute rejection episodes; ultimately it is hopeful that this will translate into prolonged allograft survival. The number and severity of acute rejection episodes significantly impacts the incidence of chronic rejection, and hence, graft loss. Use of these antibodies is contraindicated in hepatitis-related liver transplants.
Finally, there is an anti-CD-52 monoclonal antibody, alemtuzumab (Campath H-1) that has seen increasing use. Its administration leads to a profound depletion of peripheral lymphocytes, NK cells, and monocytes. Currently its use is restricted mostly to high-risk transplant recipients.
Treatment Strategies
The agents described above are usually prescribed in combination to provide immunosuppression adequate to maintain a graft in a transplant recipient without rejection or significant toxicity. Each of these agents is used to provide effective immunosuppression at 3 different stages in the posttransplantation period. The first stage is the induction stage at the time of implantation of the organ, followed by a maintenance period. The treatment of any rejection episodes that may occur also demands a change in treatment strategy. Common immunosuppression protocols are listed in Table 97-4.
Table 97-4
Immunosuppression Protocols
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INDUCTION THERAPY
Currently, most kidney, heart, pancreas, intestine, and lung-transplant recipients receive induction therapy. Induction therapy begins at the time of implantation of the graft and continues for the first few weeks after transplantation. The most common induction therapy utilized is rabbit-anti-thymocyte globulin combined with intravenous steroids. In contrast, there is little use of induction antibodies for liver transplantation.
MAINTENANCE THERAPY
Maintenance therapy involves maintaining a steady level of conventional immunosuppression while diminishing the overall dosage to obtain the minimal immunosuppressive dosage necessary to achieve a rejection-free patient. Increasingly, there have been attempts to avoid steroids in the maintenance therapy. With the introduction of cyclosporine- and tacrolimus-based therapies, there has been significant ability to withdraw steroids in transplant recipients. While steroid-minimizing regimens have shown improvement in growth markers, longer term follow-up demonstrates a significant decrease in graft function and an increase in rejection episodes. Newer studies that utilize antibody-based induction regimens have shown more promise. The best results with steroid withdrawal have been based on extended induction with IL-2 antibodies and complete steroid avoidance. Currently, most maintenance regimens target various T-cell activation mechanisms.
Tacrolimus trough levels are maintained at 10 ng/mL for the first 3 months following transplantation with a gradual reduction to approximately 5 to 7 ng/mL after that time. Long-term maintenance levels of tacrolimus are gradually decreased, and many patients receive very low detectable levels, 1 to 3 ng/mL, on a long-term basis.
Mycophenolate mofetil dosing continues for the first-year post-transplantation with dose adjustments made for any gastrointestinal toxicity or bone marrow suppression. The use of a mycophenolate/tacrolimus combination is the most frequently used discharge regimen in kidney and liver recipients. More rarely, mycophenolate/sirolimus, tacrolimus/sirolimus, and mycophenolate/cyclosporine combinations have been used. Tacrolimus has virtually replaced the use of cyclosporine in most centers. Discharge without steroid therapy is more common in liver than kidney recipients; 20% of liver recipients and 27% of intestinal recipients in 2004 were discharged without steroids.
ANTIREJECTION STRATEGY
Despite induction therapy, a significant number of patients develop rejection episodes during the first few weeks following transplantation. Fortunately, the incidence of rejection has been decreasing overall. Most episodes of acute kidney and liver rejection are treated with corticosteroids, administered at a dose of 10 mg/kg/day. Some centers are using antithymocyte globulin with success.
Complications of Immunosuppression
The clinical immunosuppressive strategies currently used attempt to strike a balance between rejection prevention and the infectious or toxic complications of immunosuppressive drugs. The individual immune response of pediatric patients to the antigen load with a transplanted organ unfortunately establishes the degree of immunosuppression necessary to achieve freedom from rejection. For a small number of patients who respond vigorously to the transplanted organ, this converts to more severe complications and toxicities from higher doses of immunosuppressive drugs. Table 97-5 summarizes the common complications of immunosuppression. The problem seen most often in pediatric patients undergoing significant chronic immunosuppression involves opportunistic infections, which translates into more frequent problems with bacterial and fungal sepsis often related to indwelling catheters. In addition, children lack specific immunity to a number of parasitic and viral pathogens. Pediatric transplant recipients, therefore, must be aggressively assessed for opportunistic infection in the post-transplantation period; they commonly require prophylactic agents to prevent these infections. Table 97-6 summarizes the prophylactic regimen used at the Johns Hopkins Hospital for pediatric patients during the first 6 months posttransplantation. In general, prophylaxis for Pneumocystis carinii pneumonia, as well as for opportunistic herpes virus infections including cytomegalovirus and Epstein–Barr virus (EBV), is used in patients who are at risk for these infections. This population of pediatric patients includes those who undergo induction or receive OKT3 or antithymocyte globulin therapy, as well as those who receive a cytomegalovirus- or EBV-mismatched organ from a viral positive adult donor. One potential advantage of the IL2-receptor antibodies is a decrease in the incidence of PTLD, as well as overall better tolerance of its infusion compared to OKT3 and ATG.
Table 97-5
Complications of Immunosuppression
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Table 97-6
Infection Prophylaxis
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In addition to developing a primary mononucleosis syndrome secondary to EBV activation or infection, pediatric transplant recipients are at higher risk for EBV associated post-transplantation lymphoproliferative disorder. A primary mononucleosis syndrome characterized by lymphadenopathy, hepatosplenomegaly, pharyngitis, and fever may progress to frank lymphoma or to a leukemic state secondary to unchecked proliferation of transformed B lymphocytes. In an immunologically normal individual, cytotoxic T cells are generated, which presumably eliminates these clones of transformed B cells and downregulates the mononucleosis syndrome. With significant immunosuppression, pediatric transplant recipients are unable to mount an effective T-cell response to these EBV-transformed B lymphocytes and essentially develop Burkitt-type lymphoma or leukemia. These patients must be aggressively treated with withdrawal of immunosuppression. If they have significant bulky lymphomatous disease or leukemia, they may require cytoreduction therapy to eliminate the majority of proliferating B cells. Withdrawal of immunosuppression following adequate cytoreduction presumably allows for recovery of the T-cell arm of the immune system and provides long-term cellular control of the transformed B lymphocytes.
Effects of Immunosuppression
As increasing numbers of pediatric transplant recipients survive and require long-term treatment with immunosuppressive agents, we will see more evidence of significant long-term toxicity. The primary late effects of corticosteroids include Cushing syndrome, osteoporosis, impaired growth, avascular necrosis of the femoral head, cataracts, glaucoma, cardiovascular disease, and gastritis or peptic ulcer disease. Long-term use of azathioprine can cause hepatitis, pancreatitis, and red cell aplasia. Cyclosporine used on a long-term basis can lead to hypercholesterolemia, arteriosclerosis, hypertension, nephrotoxicity, neurotoxicity, hyperkalemia, and hyperglycemia. The effects of long-term use of tacrolimus include hypertension and nephrotoxicity, diabetes mellitus, and dyslipidemia as described above. Adverse effects of sirolimus include impaired wound healing and dyslipidemia. It is minimization of these adverse effects which drives investigation into immunosuppression minimization.
Secondary malignancies also are a fact of life for long-term immunosuppressed transplant recipients. A small number of patients may develop Kaposi sarcoma, which is presumably related to an EBV-like virus. Posttransplantation lymphoproliferative disorder secondary to EBV infection also can occur in chronically immunosuppressed patients. Patients are also at higher risk for the common malignancies seen in non-immunosuppressed patients, such as Hodgkin disease, non-Hodgkin lymphoma, and skin cancer. Our pediatric transplant recipients at the Johns Hopkins Hospital undergo yearly cancer surveillance including a chest radiograph and a general physical examination to screen for skin lesions and any other problems. Teenaged female recipients should undergo yearly pelvic examinations and Papanicolaou tests when appropriate.
Conclusions
Fortunately for pediatric surgeons and their patients, neonates and children have a competent immune system at birth that provides significant responses in the face of infection and surgical stress. All the features of the immune system are qualitatively present in newborns and in older children, but there are significant quantitative differences between children and adults.
A variety of congenital and acquired immunodeficiency states may occur in the pediatric age group that complicates their treatment.
In the transplantation clinical population an intact immune system requires significant immunosuppression to prevent rejection. The overall naivete of the pediatric immune system and the difference in how medications are metabolized in the transplantation clinical situation results in a higher incidence of secondary complications of immunosuppression than in adults, and therefore greater vigilance for complications of immunosuppression must be maintained. There have been great strides in long-term transplant patient and graft survival, which has led to increasing interest in producing immunosuppression regimens that minimize adverse effects on the patients. Ultimately, the goal is to induce a tolerant state for the transplanted organ, but until this is achieved, careful tailoring of the medications to the individual patient must be maintained.
Selected Readings
Abbas AK, Lichtman AH, Prober JS Cellular and Molecular Immunology, Updated Edition. Philadelphia, PA: WB Saunders Co; 2009.
Agarwal A, et al Immunosuppression in pediatric solid organ transplantation. Semin Pediatr Surg 2006;(15):142–152.
Colombani PM. Clinical immunosuppression. In: Oldham KT, Colombani PM, Foglia RP, eds. Surgery of Infants and Children: Scientific Principles and Practice. Philadelphia, PA: Lippincott-Raven; 1997:671–700.
Hannet I, Erkeller-Yuksel F, Lydyard P, Deneys V, DeBruyere M. Developmental and maturational changes in human blood lymphocyte subpopulations. Immunol Today 1992;13:215–218.
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Meier-Kriesche H-U, et al Immunosuppression: evolution in practice and trends, 1994–2004. Am J Transpl 2006:(6, part 2):1111–1131.
Schonder K, Mazareigos G, Weber R. Adverse effects of immunosuppression in pediatric solid organ transplantation. Pediatr Drugs 2010;12(1):35–49.
CrossRef
Yang I, et al Immunosuppressive strategies to improve outcomes of kidney transplantation. Semin Nephrol 2007;27(4):337–392.
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