Exogenous Immunosuppression Research Articles

Clinical xenotransplantion—how close are we?

Xenotransplantation with pig organs offers a medium-term solution to the shortage of organs available for clinical transplantation. The immunological barriers to xenotransplantation have been, and remain, formidable. In the early 1990s, the identification of Galalpha1,3Gal (Gal) as the main target for human xenoreactive (anti-pig) antibodies and the development of pigs transgenic for a human complement regulatory protein, decay-accelerating factor (hDAF), were major advances. The presence of hDAF on the vascular endothelium of pig organs provided some protection against complement-mediated hyperacute rejection. This protection, however, was short-lived, and, until recently, the longest median time for organ survival that had been achieved (with combinations of biological and pharmacological immunosuppressants) in a series of pig-to-primate organ transplants was under a month. Christopher McGregor and colleagues recently reported to the International Society of Heart and Lung Transplantation (J Heart Lung Transplant 2003; 22: S89) that, by combining the use of organs which express hDAF with the administration of a soluble Gal glycoconjugate and other immunosuppressive agents, the survival of pig hearts in baboons can be extended to a median of 76 days. McGregor's work suggests that immunological barriers to xenotransplantation are not insurmountable. WHERE NEXT? The recent generation of pigs that do not express Gal epitopes (alpha1,3-galactosyltransferase gene-knockout pigs) might remove the need both for the expression of hDAF and the administration of a soluble Gal glycoconjugate. The absence of a natural antibody response will allow investigation of the cellular immune response and of any molecular incompatibilities between pig and primate that may be detrimental to graft survival. Furthermore, the absence of a humoral response may open the way for the induction of immunological tolerance (or unresponsiveness in the absence of exogenous immunosuppression) to a transplanted pig organ.

Immunologic features of porcine circovirus type 2 infection.

Clinical expression of porcine circovirus 2 (PCV-2) infection in swine may result in two distinct high mortality disease syndromes. In North America, postweaning multisystemic wasting syndrome (PMWS), while still sporadic in incidence, predominates. In Europe and elsewhere, both PMWS and a second syndrome, porcine dermatitis and nephropathy syndrome (PDNS), occur in endemic and epidemic forms. PMWS but not PDNS has been reproduced in piglets by inoculations with PCV-2 alone or in PCV-2-infected swine co-infected with porcine parvovirus (PPV) or porcine respiratory and reproductive syndrome (PRRS) virus and also if PCV-2-infected piglets are immunostimulated by injections with an immunogen emulsified in an oil-based macrophage-targeted adjuvant. Subclinical but active infection has been achieved by direct inoculation of piglets with cloned PCV-2 DNA and/or progeny virus derived from cloned DNA. Morphologic changes in lymphoid tissues and preliminary functional data suggest that immunosuppression may occur in PMWS-affected swine. This phenomenon appears to be mediated by generalized lymphoid depletion and replacement by infiltrating and proliferating histiocytes and macrophages. Accumulation of virus in both mononuclear phagocytes and follicular dendritic cells is characteristic of PCV-2 infection. Exogenous immunosuppression of PCV-2-infected gnotobiotic piglets with cyclosporine (Cys), but not corticosteroid (St), potentiates PCV-2 replication and promotes productive virus infection of hepatocytes in Cys-treated piglets, a tropism not previously apparent in experimentally induced PMWS in gnotobiotic swine. In the Cys-treated piglets, inflammatory lesions characteristic of PMWS are absent, even though tissues contain high titers of infectious virus, a finding which suggests that the granulomatous inflammatory lesions characteristic of PMWS are immune mediated.

De novo malignancies after liver transplantation: a major cause of late death.

1. Recurrent and de novo malignancies are the second leading causes of late death in liver transplant recipients, following age-related cardiovascular complications. 2. The increased incidence of de novo malignancies in liver transplant recipients compared with the general population reflects their demographic makeup, known preexistent risk factors for cancer, greater rate of chronic viral infection, and actions of exogenous immunosuppression. 3. The greatest incidence of de novo malignancies is seen in cancers associated with chronic viral infections, such as Epstein-Barr virus-associated posttransplant lymphoproliferative disease, and skin cancers, including squamous cell carcinoma and Kaposi's sarcoma. 4. Although a greater incidence of such malignancies as oropharyngeal malignancy and colorectal cancer was noted, there did not appear to be an increased risk for liver transplant recipients matched for age, sex, and length of follow-up using modified life-table technique and Surveillance Epidemiology End Result data with a similar at-risk group. However, they may present with more advanced stages of disease. 5. An increased incidence of de novo cancers in chronically immunocompromised liver transplant recipients demands careful long-term screening protocols to help facilitate diagnosis at an earlier stage of disease.

Acute Pancreatitis after Cardiac Transplantation and Other Cardiac Procedures: Case-Control Analysis in 24,631 Patients

Previous series have identified an increased risk of developing acute postoperative pancreatitis in heart transplant recipients and other cardiac surgical patients, and some suggest that mortality is significantly increased when pancreatitis occurs in the transplant setting. We conducted a retrospective case-control analysis of adult patients undergoing orthotopic heart transplant or other cardiac procedures from April 1985 through June 1996 at our medical center. Specific risk factors for outcome were assessed including low cardiac output, intra-aortic balloon pump usage, exogenous calcium repletion, immunosuppression, cytomegalovirus infection, cholelithiasis, prior pancreatitis, and Acute Physiology and Chronic Health Evaluation (APACHE) II scores. There was a 30-fold increase in the incidence of pancreatitis in the heart transplant group [12 of 394 (3%) vs 27 of 24,237 (0.1%); P < 0.01]. Compared with the nontransplant cardiopulmonary bypass patients, the transplant patients experienced a statistically significant increased incidence of immunosuppression and three or more risk factors. Transplant patients with pancreatitis demonstrated a significant increase in APACHE II scores and the incidence of three or more risk factors compared with their transplant control group. Patients undergoing nontransplant cardiac procedures and developing pancreatitis had significantly increased cross-clamp times, incidence of low cardiac output, APACHE II scores, and incidence of three or more risk factors compared with their nontransplant cohort. In conclusion, there is a significant increase in the incidence of pancreatitis after orthotopic heart transplant compared with other cardiac procedures. Analysis demonstrates the additive effect of multiple individual risk factors. Immunosuppression confers significant additional risk for pancreatitis in the orthotopic heart transplant patient.

The gamma interferon gene knockout mouse: a highly sensitive model for evaluation of therapeutic agents against Cryptosporidium parvum.

Cryptosporidiosis is a serious disease in malnourished children and in people with malignancies or AIDS. Current rodent models for evaluating drug therapy against cryptosporidiosis have many limitations, including the need for a high inoculum, the absence of symptoms resembling those seen in humans, and the need to maintain exogenous immunosuppression. We have developed a gamma interferon knockout (GKO) mouse model with which to evaluate therapies against C. parvum and have used paromomycin for evaluation of this model. The GKO model offers considerable improvements over other systems, since it requires no additional immunosuppression and adult mice can be infected with as few as 10 oocysts (compared with 10(7) for SCID mice). Infected mice develop profound gastrointestinal dysfunction due to extensive infection and severe mucosal damage involving the entire small intestine. Clinical symptoms, which include depression, anorexia, weight loss, and wasting, result in death within 2 to 4 weeks. The time of death depends on the oocyst challenge dose. Paromomycin modulated parasitological and clinical parameters in highly predictable and significant ways, including prevention of death. In addition, examination of the extensively infected gut provided an important insight into the dynamics between a specific drug treatment, its impact on the extent and the site of parasite distribution, and clinical outcome. These uniform symptoms of weight loss, wasting, and death are powerful new parameters which bring this model closer to the actual disease seen in humans and other susceptible mammalian species.

Documentation in bone-marrow-augmented organ recipients of the presence of dendritic cell progenitors of donor origin

The ubiquitous presence of donor cell chimerism in the tissues of long-term successful organ allograft recipients1–3 provided quintessential support to the argument that these cells play a seminal role in allograft acceptance and in the induction of donor-specific tolerance. A finding of commensurate significance was the observation that livers (unlike other organs) when transplanted orthotopically across most mouse4 and several rat-strain combinations5,6 and in few outbred pigs7,8 are spontaneously accepted without the necessity for exogenous immunosuppression. This ascertainment complemented by the novel observations made by Lu et al9 has prompted the advancement of the hypothesis that perhaps the comparative tolerogenicity of various organs is contingent on the quantity and quality of resident migratory cells, of which those of dendritic leukocyte lineage conceivably play an auspicious role. Given their functional significance, we initiated a systematic search to establish the presence of progenitors of donor-dendritic cells (DC) in the peripheral blood of bone marrow (BM)-augmented organ transplant recipients. Additionally, the evidence for the multilineage donor cell chimerism was also sought; reported herein is the outcome of these investigations.

RETRANSPLANTATION IN MINIATURE SWINE

In miniature swine, one-haplotype class I disparate renal allografts are accepted without exogenous immunosuppression by approximately 35% of recipients. Alternatively, transplants bearing a two-haplotype class I mismatch are always rejected acutely. However, long-term acceptance in the latter animals can be achieved uniformly with a 12-day course of cyclosporine. In vitro studies of recipient cell-mediated lymphocytotoxicity responses have shown donor-specific cytotoxic T lymphocyte clones in tolerant animals, suggesting that tolerance may be a local phenomenon or a central phenomenon activated in the milieu of the graft. Six animals were retransplanted with kidneys MHC-matched to their original allograft to determine whether (1) tolerance is a central phenomenon; (2) host tolerance can be broken with a fresh challenge of donor antigen and antigen-presenting cells; and (3) graft adaptation is required for maintenance of tolerance. Four of the retransplanted animals had been spontaneous acceptors of one-haplotype class I-disparate grafts and two had been rendered tolerant to two-haplotype class I-mismatched kidneys with CsA induction. All six explanted allografts showed no histological evidence of rejection and all six retransplants were accepted without exogenous immunosuppression. These findings suggest that in miniature swine tolerance of class I-disparate kidneys is a stable, centrally mediated phenomenon that cannot be broken with a challenge of fresh donor antigen and donor-type APCs. Furthermore, successful retransplantation without immunosuppression in animals receiving CsA induction therapy for their first transplant suggests that graft adaptation is not necessary for the maintenance of tolerance.

Selective breeding of miniature swine leads to an increased rate of acceptance of MHC-identical, but not of class I-disparate, renal allografts.

Previous work from this laboratory demonstrated that tolerance to MHC-identical or class I-disparate renal allografts develops in approximately one third of miniature swine without exogenous immunosuppression. A back-cross study indicated that rejection of MHC-identical transplants due to minor Ag was controlled by one or possibly two non-MHC-linked, autosomal dominant Ir genes. According to this hypothesis, and assuming complete penetrance, graft acceptors would be homozygous recessive at the relevant Ir loci, as would their offspring. Alternatively, if the gene(s) were incompletely penetrant, then two acceptors could give rise to a rejector. However, a high rate of MHC-identical graft acceptance would still be expected in the offspring of acceptors even if the Ir gene(s) were incompletely penetrant. To test this hypothesis and to obtain a higher frequency of acceptor animals for studies of tolerance, a program of selective breeding of renal allograft acceptors was begun. In the present paper, we assess the effect of selective breeding on renal graft acceptance. The analysis indicates a marked increase in the rate of MHC-identical graft acceptance, from 27.3% (n = 24) for the earliest of the four chronologic subgroups assessed to 64.5% (n = 33) for the most recent subgroup (p less than 0.0001). Calculations of kinship revealed that the increased acceptance of MHC-identical grafts was not the result of differences between acceptors and rejectors in donor/recipient consanguinity. Class I-disparate grafts (n = 128) were similarly stratified chronologically and compared. Unlike MHC-identical grafts, the rate of acceptance of class I-disparate grafts has not changed over time. We conclude that rejector/acceptor status with respect to class I MHC incompatibility is determined by genetic factors in addition to those that control responses to minor antigen incompatibilities only.

Post-transplant lymphoproliferative disorders: a morphologic, phenotypic and genotypic spectrum of disease.

The post-transplant lymphoproliferative disorders represent a spectrum of life-threatening, generally Epstein-Barr virus-associated lymphoid proliferations which occur in the setting of exogenous immunosuppression following organ transplantation. Histopathological, phenotypic, genotypic and Epstein-Barr virus studies have revealed a broad range of abnormalities. At one end of the spectrum, they are polymorphic polyclonal proliferations with many features of a florid viral infection and, at the other end, they are monomorphic and monoclonal which would fulfill the criteria for a conventional non-Hodgkin's lymphoma, usually of B-cell type. Some patients have both polyclonal and monoclonal lesions and some have two or more monoclonal populations. Finally, because of the varied appearances, different classification schemes have been developed with reported prognostic implications.

Immunological monitoring in organtransplantation and autoimmune disease

The clinical manifestations of graft rejection or autoimmune disease represent the terminal phase in a complex sequence of inflammatory events. Although the spectrum of response to injury within each tissue is normally narrowly defined, the immune mechanisms involved may be heterogeneous, influenced by the immune status of the host, the nature of the stimulus (i.e. viral antigen, allostimulation, or aberrant recognition of self), and the use of exogenous immunosuppression. Within this framework, immunological monitoring is employed to distinguish the causative effector mechanisms, to characterize the disease course, to tailor therapeutic intervention, and to monitor treatment impact in individual immunological disorders.

THE FAILURE OF SKIN GRAFTING TO BREAK TOLERANCE TO CLASS I-DISPARATE RENAL ALLOGRAFTS IN MINIATURE SWINE DESPITE INDUCING MARKED ANTIDONOR CELLULAR IMMUNITY

Long-term specific tolerance to one haplotype class I plus minor antigen disparate renal allografts develops without exogenous immunosuppression in approximately 35% of miniature swine (n = 128). Previous studies have suggested that this phenomenon is related to limited class I-specific helper T cell activity as evidenced by the failure of antibody class switching in vivo and the ability of exogenous interleukin 2 to elicit antidonor responses in vitro. To determine whether tolerance could be broken by inducing antidonor reactivity with donor antigen and a source of T cell help, multiple skin grafts bearing donor class I plus third-party class II antigens were placed on tolerant animals. Skin grafts were placed at least 3 months after the kidney transplant, at which time all recipients had normal renal function as measured by blood urea nitrogen and serum creatinine. First-set rejection of skin grafts by SLAad and SLAdd hosts occurred in 11.8 +/- 1.1 days (mean +/- SEM, n = 6) and in 9.3 +/- 0.9 days (n = 4), respectively. Coincident with skin rejection, most animals developed a transient rise in BUN to 62 +/- 11 mg/dl (n = 10) and a similar rise in Cr to 4.9 +/- 1.2 mg/dl (n = 10), with normal levels returning in all animals within two weeks. Subsequent skin grafts with the same disparity did not undergo second-set rejection and did not induce BUN or Cr elevations. Prior to skin grafting, animals showed no antidonor activity in mixed lymphocyte reaction or cell-mediated lymphocytotoxicity assays. After two skin grafts, all animals developed donor-specific CML and secondary MLR responses, and additional skin grafts amplified this cellular immunity. Development of marked antidonor immunity without a break in tolerance suggested that either graft adaptation or local suppression might be involved in maintaining tolerance to class I MHC antigens. In preliminary studies, an immunized SLAad animal and an immunized SLAdd animal were retransplanted with kidneys MHC matched to their first allografts. In both cases, the second graft was accepted permanently without immunosuppression, suggesting that graft adaptation is not necessary for the maintenance of tolerance to renal allografts in miniature swine.

INDUCTION OF KIDNEY TRANSPLANTATION TOLERANCE ACROSS MAJOR HISTOCOMPATIBILITY COMPLEX BARRIERS BY BONE MARROW TRANSPLANTATION IN MINIATURE SWINE

Previous studies from our laboratory have shown that permanent lymphohematopoietic chimerism can be induced in MHC-disparate miniature swine by bone marrow transplantation after lethal total-body irradiation. The purpose of the present study was to determine in this large animal model whether such chimerism would lead to permanent tolerance to a vascularized allograft without a requirement for exogenous immunosuppression. Eight miniature swine that had received MHC-mismatched BMT more than five months earlier underwent kidney transplantation (KTx) from a donor MHC matched (n = 5) or MHC mismatched (n = 3) with the BMT donor. All animals had regained in vitro responsiveness to third-party MHC antigens, as measured by mixed lymphocyte reaction (MLR), before KTx but remained nonresponsive to MHC antigens of the BMT donor and self. All three animals that received KTx mismatched for BMT donor MHC rejected promptly (mean survival time 7.0 days). Of the five animals that received KTx matched for BMT donor MHC, four showed no evidence of rejection and have functioning KTx greater than 200 days after KTx. The fifth animal had excellent renal function for 60 days but then developed a slowly rising BUN and serum creatinine, and died 75 days after KTx. The course of this animal's rejection is consistent with that previously described for rejection due to minor antigen disparities. The difference in survival of KTx matched or mismatched for the MHC of the BMT donor was statistically significant (P = 0.0062). The survival of KTx matched for the MHC of the BMT donor was significantly different from that of control animals without BMT receiving KTx mismatched for MHC (P = 0.0018). We therefore conclude that BMT is an effective means for induction of tolerance to an MHC mismatched KTx in this large animal model.

MECHANISM OF TOLERANCE FOLLOWING CLASS I-DISPARATE RENAL ALLOGRAFTS IN MINIATURE SWINE

Previous studies utilizing a recombinant MHC haplo-type in our partially inbred miniature swine herd have demonstrated that some recipients matched only for SLA class II show long-term acceptance of renal allografts without exogenous immunosuppression. Such animals have been shown to develop systemic tolerance as evidenced by prolonged rejection times of subsequent donor-specific, but not third-party, skin grafts. In the present studies in vitro cellular responses of long-term tolerant animals and of 7 animals studied sequentially are presented. Long-term tolerant animals demonstrated responses consistent with the absence of the class I reactive helper populations normally present in naive controls. Animals studied sequentially segregated into two groups based on cellular reactivity and survival. All animals showed complete loss of antidonor class I cell-mediated lymphocytolytic (CML) reactivity by postoperative day 10. However, animals surviving less than 20 days maintained CML reactivity to donor class I plus third-party class II in the posttransplant period, while animals surviving greater than 40 days lost such reactivity. Addition of exogenous interleukin 2 could not reverse this loss. These studies suggest that tolerance induction to a renal allograft across a class I only difference involves effects on both helper and killer class I reactive cell populations.

Exogenous immunosuppression with methylprednisolone does not reactivate a type D retrovirus in rhesus monkeys

To determine whether immunosuppression would result in retrovirus expression in previously infected rhesus monkeys, chronic high dose methyl prednisolone therapy was administered to two groups of animals for 4 weeks. One group was antibody positive for a type D retrovirus, designated Type D/3/wisc. The second group of animals had no known exposure to Type D/3/wisc and was antibody negative to this virus. The monkeys were evaluated for immunosuppression and retrovirus re-expression following the corticosteroid therapy. Although this treatment induced a marked cellular immunosuppression in all animals, as measured by in vitro assays, in none of the animals was retrovirus viremia or retrovirus-associated disease detected.

The Risk of Cytomegalovirus Infection in Seronegative Transfusion Recipients Not Receiving Exogenous Immunosuppression

We studied 637 transfusion recipients seronegative for cytomegalovirus (CMV) in the following categories: neonates; pregnant women; and patients experiencing trauma, burns, cardiovascular surgery (adult or pediatric), major surgery, or gastrointestinal hemorrhages. Cultures and serological tests were used to follow up subjects for evidence of CMV infection for a period of three months after their last transfusion. Six (0.9%) developed CMV infection. No significant differences in risk among patient categories were observed. Infected patients received a significantly larger mean number of units of cellular blood products (CBP; 50.0 +/- 38.9 vs. 6.2 +/- 8.5; P less than .001) and plasma (23.7 +/- 15.3 vs. 2.6 +/- 4.6, P less than .001) than did uninfected patients. This result represents a risk per unit of CBP transfused of 0.14%, or approximately 0.38% per unit of seropositive CBP transfused. We observed, however, that patients exposed to CBP from greater than 30 donors had a higher risk of acquiring CMV infection than would be predicted if infectious units were randomly distributed among all donors (P less than .01).

Opportunistic mycotic infections in pregnant women

Immunosuppression was the major factor associated with the deaths of 44 women who died from opportunistic mycotic infections. The patients were immunosuppressed by malignancies, irradiation, cytotoxic drugs, and metabolic diseases. An additional four patients may have developed candidiasis as a result of antibiotic therapy. Coccidioidomycosis was the only mycosis solely associated with pregnancies not altered by exogenous immunosuppression.