Pulmonary injury in recipients of discordant hepatic and renal xenografts in the dog-to-pig model.

Work in our lab demonstrated that the early post-operative course of discordant hepatic and renal xenotransplantation is complicated by a pulmonary injury. The aim of this study was to characterize the nature of this injury, as well as to determine whether endothelin-1 (ET-1) and inducible-nitric oxide synthase (iNOS) are present in this form of pulmonary injury. Dog-to-pig orthotopic liver and kidney xenografts were performed. Pulmonary artery pressures were monitored throughout all procedures. The lungs were stained with monoclonal antibodies for ET-1, endothelin converting enzyme-1, and iNOS. The lungs from pig recipients of hepatic or renal xenografts were compared to lungs from untreated pigs. Pulmonary artery pressures were elevated in recipients of liver xenografts when the suprahepatic caval cross clamp was placed and continued to rise to systolic levels following unclamping. The mean pulmonary artery pressures in recipients of renal and hepatic xenografts rose significantly following revascularization. Pathology in lungs from kidney and liver recipients was similar, showing congestion with peribronchial and septal edema, with diffuse adhesion of PMN to alveolar endothelium. ET-1, endothelin converting enzyme-1 (ECE-1), and iNOS staining was widespread and intense in alveolar and pulmonary arterial endothelium. Discordant xenotransplantation of livers and kidneys is associated with a significant early pulmonary injury that is associated with early PMN infiltration and expression of ET-1 and iNOS.

Successful treatment of post-transplant lymphoproliferative disorder with interferon-alpha and intravenous immunoglobulin.

We report on the use of interferon-alpha (INF-a) and high-dose non-specific intravenous immunoglobulin (IVIg) in 2 patients (a 60-yr-old female and a 65-yr-old male) who developed post-transplant lymphoproliferative disorder (PTLD) 2 and 8 months after heart and liver transplantation, respectively. Both patients had received immunosuppression with ATG, CsA, azathioprine, and prednisone. The first patient did not receive additional immunosuppression with biological agents. The second patient developed 3 steroid-resistant acute rejection episodes requiring OKT3 (cumulative dose 100 mg) and ATG (cumulative dose 3450 mg). The first patient presented with nodules involving the liver, spleen, lungs and nasophar, ynx. The second patient presented with subcutaneous and liver nodules, as well as pert-portal and para-aortic lymphadenopathies. The histological diagnosis was diffuse B-cell PTLD in both patients. Despite reduction of immunosuppression, a progression of lesions was observed in both patients over 5 months and 2 months, respectively. The first patient received INF-alpha (2 x 10(6) IU, s.c. 3 times/wk) and IVIg (0.5 g/kg i.v. every 15 d) for.4 months, while the second patient received the same therapy for 12 and 7 months, respectively. Complete disappearance of all lesions was observed after 3 months of therapy in the first patient and after 7 months of therapy in the second patient, as assessed by CT scan. PTLD remains in remission 47 and 33 months after therapy, respectively. Our preliminary results suggest that the combination of INF-alpha and IVIg can be an effective therapy for PTLD which does not respond to reduction of immunosuppression.

The pathophysiology of chronic rejection.

The understanding of chronic rejection has been greatly enhanced by the use of genetically controlled experimental models using inbred rats. Models that express all lesions encountered in human transplants are described. Findings of chronic rejection depend on genetic disparity, strength of the immunologic reaction, response to injury, and perpetuation of an ischemic state. Lesions of vasculopathy and parenchymal cell damage may proceed at different rates, but the vasculopathy seems reversible until healing occurs. The experimental models that have led us to significant understanding are described herein.

Irreversible chronic vascular rejection occurs only after development of advanced allograft vasculopathy: a comparative study of a rat cardiac allograft model using a retransplantation protocol.

Indefinitely surviving WF.1L (RT1(1)) cardiac allografts transplanted to LEW (RT1(1)) recipients provide an ideal model for controlled comparative studies of chronic vascular rejection (CVR). To determine the stage of development at which the progressive CVR can be reversed when deprived of an ongoing recipient alloimmune response, WF.1L-LEW cardiac allografts were retransplanted back into syngeneic donor strain WF.1L recipients at specific time periods after initial allogeneic engraftment and were maintained in WF.1L syngeneic hosts for a further 40 days. The vascular changes in the retransplanted allografts were compared with those of nonretransplanted allografts and with nonretransplanted and retransplanted LEW-LEW isografts examined at similar time periods. The early vasculopathic inflammatory changes were consistently reversed by retransplantation of the cardiac allografts back into syngeneic recipients after 20 days and 40 days of allotransplantation. Syngeneic retransplantation of the cardiac allografts at 60 days after allotransplantation did not reverse the essentially nonvasculitic occlusive vasculopathy invariably present in WF.1L-LEW cardiac allografts at this time period. Thus, the vasculitic and minimal subocclusive myointimal changes associated with early CVR in this model are alloantigen dependent and reversible. Irreversible CVR occurs only after advanced proliferative vasculopathy has been established in the allogeneic host.

Increased endothelin expression in a rat cardiac allograft model of chronic vascular rejection.

Endothelins (ET) are potent vasoconstrictors that are directly mitogenic for vascular smooth muscle cells and fibroblasts. It is possible that the vasoconstrictor and mitogenic effects of ET could play a significant role in the vascular remodeling process that occurs in chronic vascular rejection (CVR). We have previously shown that cardiac allografts in the indefinitely surviving major histocompatibility complex identical WF.1L (RT1(1)) to Lewis (LEW) (RT1(1)) inbred rat strain combination provide a highly reproducible model of progressive CVR. The objective of this investigation was to measure endothelin-1 ventricular content of WF.1L-LEW cardiac allografts and to determine the immunohistochemical patterns of ET cellular reactivity at well defined posttransplant time periods. Data were compared with those obtained in similar studies of LEW-LEW syngeneic: heart grafts as well as all recipients' own hearts. The ventricular ET-1 content of the WF.1L cardiac allografts was markedly higher (4.3-, 7.0-, and 4.8-fold at 20, 40, and 60 days, respectively) than in corresponding recipients' hearts. Also, the increase in ventricular ET-1 levels as compared with the recipients' hearts rose significantly only in the allograft group. No comparable differences were observed in the syngeneic heart graft controls. Allografts consistently showed ET staining of intimal myocytes at sites of occlusive and subocclusive intimal proliferation associated with CVR. Allografts also showed ET cellular staining in areas of reparative fibrosis associated with indolent interstitial rejection and ischemic myocardial damage. The results of this study strongly suggest that ET may play a significant role in the pathogenesis of CVR.

Endothelin-1 immunoreactivity and mRNA in the transplanted human heart.

Endothelin-1 (ET-1) is a 21-residue peptide produced by endothelial cells and possesses a wide range of biological activities, including vasoconstriction, mitogenesis, and inotropic effects on the heart. The aim of the present study was to determine the cellular localization of ET-1 immunoreactivity and mRNA in routine endomyocardial biopsy specimens of transplanted human hearts, and to correlate the findings with the associated histological changes. Multiple-step paraffin sections of 72 biopsy samples were immunostained with antiserum to ET-1 and von Willebrand factor (factor VIII) using the avidin-biotin-peroxidase complex method. ET-1 immunoreactivity was localized to vascular and endocardial endothelial cells, as well as to cardiomyocytes. The pattern of endothelial cell immunostaining with the ET-1 antiserum was similar to that of factor VIII. Previous biopsy sites and areas of granulation tissue appeared to have greater ET-1 immunoreactivity, particularly in sections immunostained with the ET-1 antiserum. There was a significant correlation between the presence of ET-1 immunoreactivity and fibrosis or granulation tissue in the biopsy specimens (P < 0.03). There was no correlation between ET-1 immunoreactivity and the presence of cellular infiltrate, definitive rejection, or Quilty effect. In situ hybridization with radiolabeled RNA probes revealed expression of ET-1 mRNA in endothelial cells and myocytes, also in association with granulation tissue and fibrosis. No cellular reactivity was present in control sections stained with the ET-1 antiserum preadsorped with its synthetic peptide. The findings suggests a possible role for ET-1 in vascular regeneration and angiogenesis following myocardial injury.

Cyclosporine and the reversibility of chronic vascular rejection.

A model of chronic vascular rejection of cardiac allografts has been developed in inbred rats using the WF.1L/Gut congenic strain as donor into LEW recipients. The hearts beat for more than 200 days without the need for exogenous immunosuppression. The histopathology is characterized by cellular rejection, vasculitis, and myointimal arterial wall thickening, and by day 60 posttransplant, there are widespread occlusive vascular changes similar to those seen in human cardiac allografts. CsA, at a dose of 15 mg/kg/d, is effective in preventing as well as reversing the vasculopathy. These data (1) confirm other studies of ours on the reliability of the experimental model using this strain combination, (2) establish the time window of days 40 to 60 whereby mechanisms of lesion regression can be studied, (3) prove the MHC class I and class II antigen incompatibility are not a necessary condition for the generation of the vascular lesions, (4) show that CsA is a useful probe for study of the vasculopathy, and (5) suggest that the model is a useful probe of the mechanism of action of CsA.

Expression of early and late activation markers on peripheral blood T lymphocytes does not reliably reflect immune events in transplanted hearts.

Monoclonal antibodies directed against early (receptors for interleukin-2 and transferrin [IL-2R, TfR]) and late (PTA1, alpha 1 integrin VLA-1) activation antigens were used as probes to monitor cardiac transplant patients for episodes of acute graft rejection. Age- and sex-matched patient control groups consisting of 11 patients awaiting cardiac transplantation and 13 kidney transplant recipients with long-term grafts, respectively, were used to define an upper limit for normal activation antigen expression (mean + 3 SD) in patients. Expression of all cell markers was significantly higher in both patient control groups than in healthy control individuals. Therefore, the level of activation marker expression in heart patients awaiting transplantation was used as comparison for the patient population under study. Sequential monitoring of 24 heart transplant recipients failed to demonstrate a significant correlation of increased activation marker expression with clinical events of immune activation. Subsequently 62 consecutive endomyocardial biopsy scores in 36 patients were compared with the expression of IL-2R, TfR and VLA-1 on peripheral blood T cells. Neither increased cellular infiltration of the endocardium, nor of the myocardium, was associated with increasing proportions of IL-2R, TfR, or VLA-1 positive T cells. Elevated T-cell expression of the three markers combined indicated acute graft rejection with a sensitivity, specificity, and overall accuracy of 38%, 52%, and 43%, respectively. Acute graft rejection in biopsies with associated myofiber damage (biopsy rejection scores 2 and 3A,B) was not associated with a change in the proportion of activated T cells in circulation within the first 6 months after transplantation.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence that recipient CD8+ T cell depletion does not alter development of chronic vascular rejection in a rat heart allograft model.

Progressive chronic vascular rejection is a central feature of indefinitely surviving WF.1L LEW/Gut (RT1(1)) heart grafts transplanted to LEW (RT1(1)) recipients in unmodified donor-recipient combinations. At 70 days posttransplantation, large vessels of the grafts are characterized by the presence of vasculitis, vasculitis with associated variable myointimal thickening, and occlusive myointimal thickening with minimal or absent concomitant vasculitis. To assess the potential role of CD8+ T cells as critical effectors of chronic vascular rejection in this model, LEW recipients of WF.1L heart grafts were effectively depleted of CD8+ T cells as a result of prior thymectomy and anti-CD8 (MRC OX8) monoclonal antibody administration prior to transplantation. WF.1L heart grafts transplanted to LEW recipients that had undergone prior sham thymectomy and MRC OX8 administration, or thymectomy and administration of antibody-free culture supernatant, provided appropriate controls. At 70 days posttransplantation, large vessels of WF.1L heart grafts in all 3 transplantation groups showed similar morphologic features, which were comparable to those observed in heart grafts of long-surviving unmodified donor-recipient pairs. This study has shown that profound selective depression of recipient CD8+ T cells does not alter the characteristic features of chronic vascular rejection in this rat cardiac model, and provides evidence that CD8+ T cells play no critical role in the initiation or development of progressive vascular damage in this setting.

Multiple patterns of MHC class II antigen expression on cellular constituents of rat heart grafts. Lack of correlation with graft survival, but strong correlation with vasculitis.

The patterns of induced major histocompatibility antigen expression on indigenous cellular elements of heterotopic rat cardiac grafts were determined by immunohistologic methods in a variety of donor-recipient combinations. Heart grafts were studied in combined full-MHC- and non-MHC-disparate combinations, isolated intra-MHC-disparate combinations, and non-MHC-disparate combinations. The pattern of class II expression on cellular constituents of the grafts was highly variable and critically dependent upon the nature of the specific unidirectional donor-recipient combination. No uniform pattern of class II expression emerged that was clearly predictive of rapidity of rejection or of protracted survival. However, vasculitis was confined to grafts in combinations in which induced class II expression on graft large vessel endothelium was present. Sites of vasculitis were never encountered in the absence of induced class II expression on overlying endothelium. Vasculitis and associated induced class II expression on large vessel endothelium were present in rapidly rejecting grafts and in grafts with indefinite survival. In the latter, vasculitis was shown to progress to a late phase of occlusive intimal thickening. Induced class I expression on graft cardiac myofibers was present in all the genetically disparate donor-recipient combinations examined in this study, irrespective of the length of graft survival. This investigation has shown that no uniform stereotyped pattern of MHC antigen expression on cellular constituents correlates with the length of graft survival. However, induced class II expression on graft large vessel endothelium is closely associated with vasculitis, which can directly progress to occlusive intimal thickening in grafts with prolonged survival.

Mechanisms of glomerular injury in experimental immune nephritis. II. Effect of a platelet activating factor receptor antagonist in an autologous nephritis model.

The role of Platelet Activating Factor (PAF) in experimental immune glomerulonephritis was assessed by administering the specific PAF receptor antagonist CV-3988 to inbred LEW rats with the Accelerated Autologous Nephrotoxic Serum Nephritis (AA-NTSN). Intravenous administration of CV-3988 caused a marked and sustained reduction in albuminuria and renal histopathological changes. Conversely, although CV-3988 appeared to modulate the fall in glomerular filtration rate (GFR) in the AA-NTSN, this trend was not statistically significant. Renal glomeruli isolated on day 1 after nephritis induction spontaneously released 16.9 +/- 2.2 ng of PAF per 200 glomeruli, while in glomeruli of healthy rats this secretion was virtually undetectable. The administration of CV-3988 to rats with AA-NTSN did not affect the following: binding of intravenously injected sheep anti-rat glomerular basement membrane (GBM) antibody; levels of autologous antibody to sheep immunoglobulin G; the functional integrity of circulating neutrophils. We conclude that local PAF generation and release is intimately linked with the pathogenesis of glomerular injury in this form of immune disease.

Endocardial infiltrates in human heart transplants: a serial biopsy analysis comparing four immunosuppression protocols.

Endocardial mononuclear cell infiltrates were studied in 2,350 consecutive biopsies from 172 patients over a period ranging from 4 to 16 months post cardiac transplantation. The patients, otherwise unselected, were equally subdivided into four groups based upon the specific type of maintenance immunosuppression used. This was to allow for comparison of the effects of four separate commonly used recipient immunosuppression protocols, which could potentially influence the characteristics of the infiltrates. With azathiaprine-corticosteroid immunosuppression, endocardial infiltrates in otherwise normal biopsies were exceedingly rare, very minor, and invariably unifocal. Mild and moderate rejection were associated with a highly significant stepwise increase in incidence, prominence, and multifocality of endocardial infiltrates. In contrast, with each of the three cyclosporine-based recipient immunosuppression protocols which were evaluated, approximately 15% of biopsies with no evidence of rejection were associated with endocardial infiltrates. There was a wide range of variation in the prominence of the endocardial infiltrates present. Multifocal infiltrates were frequently encountered, the incidence of which was exclusively dependent upon the specific cyclosporine-based immunosuppression protocol used. With mild and with moderate rejection there was a significant stepwise increase in overall biopsy incidence of all endocardial infiltrates in each of the three groups, although there was no variation in relative prominence of the infiltrates, or in incidence of multifocality when biopsies without rejection were compared. The presence of conspicuous vascular spaces within endocardial infiltrates and significant extension of the infiltrates into the adjacent myocardium, with or without associated myofiber necrosis, were characteristic features of the most prominent endocardial infiltrates in all three cyclosporine-based immunosuppression groups. This constellation of changes has sometimes been referred to as "Quilty" effect. The relative incidence with which these particular features were encountered in association with endocardial infiltrates did not vary with rejection category of the biopsies. This study has shown that the presence of all forms of endocardial infiltrates, in the absence of concomitant rejection, is a characteristic manifestation of cyclosporine-based recipient immunosuppression, regardless of the specific protocol and cyclosporine dosage schedule. Under azathiaprine-based immunosuppression, endocardial infiltrates are almost invariably associated with rejection. It is postulated that cyclosporine-related endocardial mononuclear cell infiltration, in the absence of overt rejection, may result from a low level alloimmune response secondary to fluctuations in cyclosporine drug levels or related factors, and that the incidence with these infiltrates occur can be augmented during acute rejection episodes when the strength of the recipient immune response is magnified.

Pathologic features in long-term cardiac allografts.

Pathologic conditions of six long-term orthotopic heart transplant survivors (11 to 17 years) was compared with a group of six similar heart transplant recipients surviving only 2 years. The two groups were matched as far as possible for age and sex of both recipients and donors and for immunosuppressive therapy (azathioprine and prednisone). Ischemic time, HLA-A, -B typing, acute rejection episodes, lipid profiles, and coronary angiograms were investigated in both groups. None of these parameters correlated with survival or disease of the graft. Graft coronary disease was present in 10 of 12 cases and caused graft failure in 8 of 12. All six long-surviving grafts and four of six surviving only 2 years showed occlusive coronary disease. The major difference in the two groups was in the pathologic condition of the coronary arteries in the long-term survivors, which more resembled that of naturally occurring atherosclerosis than the characteristic concentric graft coronary disease present in grafts surviving 2 years. Although the histopathologic features were different in the two groups, no investigated factor was useful in predicting graft disease and survival.