Daniel A. Steinbrüchel

Treatment with total lymphoid irradiation, cyclosporin A and a monoclonal anti-T-cell antibody in a hamster-to-rat heart transplantation model: Graft survival and morphological analysis

Abstract. Treatment with preoperative total lymphoid irradiation and post‐transplant cyclosporin A has been shown to have a synergistic effect on graft survival in alio‐and xenotransplantation. Specific monoclonal antibodies against T cells and T cell subpopulations could offer new ways of preventing graft rejection in xenotransplantation. Graft survival and histology were examined after total lymphoid irradiation plus cyclosporin A treatment versus cyclosporin A plus a monoclonal antibody in a concordant, heterotopic, hamster‐to‐rat heart transplantation model. Preoperative total lymphoid irradiation was given at a dose of 1. 25 Gy, 12 times over a period of 3 weeks. Cyclosporin A at a dose of 12. 5 mg/kg per day was administered perorally and OX‐19, a pan T cell monoclonal antibody, was given as intraperitoneal injections at doses of 100 μg or 500 μg/kg per day from day 0 until graft rejection. While total lymphoid irradiation alone prolonged graft survival to 9. 4 days, total lymphoid irradiation plus cyclosporin A extended graft survival to a mean of 22 days. Cyclosporin alone or combined with the monoclonal antibody could not increase graft survival significantly when compared to untreated animals, which rejected their grafts within 3. 7 days. Vascular rejection was the characteristic morphological finding, even after some weeks of excellent graft function. In conclusion, total lymphoid irradiation and cyclosporin A had a synergistic effect on graft survival in this concordant xenotransplantation model, although recent impressive results from other groups could not be reproduced. Total lymphoid irradiation combined with cyclosporin A appears to delay a primary humoral graft rejection, while the mechanism of rejection, judged by histology, stays the same.

Sequential, morphological, and antidonor antibody analysis in a hamster-to-rat heart transplantation model

Abstract. The pathogenesis and the mechanism of accelerated graft rejection in concordant xenotransplantation are unclear. The histopathological features and kinetics neither fulfill the criteria of classic hyperacute rejection nor resemble an accelerated type of first‐set allograft reaction. The aim of this study was to investigate the mechanism of concordant xenograft rejection in relation to the early morphological changes in hamster hearts transplanted to unmodified rat recipients by sequential, immunohistological analysis of grafts, regional lymph nodes, and spleens and to correlate these results to the production of antidonor antibodies, as determined by a flow cytometric assay. Histopathological features were characterized by a gradually increasing myocytolysis with fragmentation and loss of myofilaments. The first slight signs were observed a few hours after transplantation. Later, vascular changes developed, evolving into a leuko‐cytoclastic type of vasculitis, eventually with thrombosis. No significant interstitial lymphocyte infiltration was present, but neutrophilic granulocytes and macrophages appeared. In addition, a distinct increase in B cells in spleens and lymph nodes was noted. Low levels of preformed antidonor antibodies did not increase during the first 48 h; however, significant amounts of species‐, but not donor‐, specific antibodies were demonstrated at the time of rejection. These data, together with the morphological observations, indicate a primarily humoral xenograft rejection in this model. Minor damage to graft myocytes a few hours after transplantation, progressing to vascular changes within 24–48 h, further suggests that preformed antidonor antibodies directed against endothelial or myocyte determinants may play an initiating role in the pathogenesis of unmodified, concordant xenograft rejection.

Evidence for a primarily humoral rejection mechanism in concordant xenogeneic heart transplantation. A sequential immunohistological study in a hamster-to-rat model

Heterotopic heart transplantations in an unmodified hamster‐to‐rat model were studied sequentially by immunohistochemical analysis. Monoclonal mouse anti‐rat antibodies against B cells, T cells, macrophages and neutrophilic granulocytes (MRC OX‐19, MRC OX‐38, MRC OX‐8, MRC OX‐22, MRC OX‐33, MRC OX‐41 and MRC OX‐42) were used in an indirect immunoperoxidase technique and monoclonal mouse anti‐rat IgM and IgG were used for immunofluorescence. In grafts investigated after 6 h (N = 8) minimal infiltration of macrophages was demonstrated with MRC OX‐41+ and MRC OX‐42+ cells. No T‐ or B cells were seen. In a few cases, deposition of IgG and IgM was seen related to the endothelium of larger vessels. In grafts examined 24 h after transplantation (N = 10) the number of MRC OX‐41+ and MRC OX‐42+ cells had increased and in half of the cases IgM and IgG were located in relation to endothelial cells of larger vessels. In grafts investigated 48 h after transplantation (N = 8) the infiltration with MRC OX‐41+ and MRC OX‐42+ cells had further increased and a few scattered MRC OX‐19+ and MRC OX‐8+ cells appeared. At this time all but one heart had deposition of IgG and IgM in the vessel walls. Upon complete rejection (N = 8) diffuse infiltration of MRC OX‐41+ and MRC OX‐42+ cells was seen, but still only a few scattered T cells could be demonstrated. At this time IgG an IgM deposition appeared in all vessels and was also located in relation to the capillaries. These results further support our hypothesis that acute xenograft rejection in this animal model is primarily of the humoral type.

Pathogenesis and mechanisms of graft rejection in concordant xenotransplantation with special reference to hamster-to-rat cardiac transplantation.

During the last decade research in xenotransplantation has experienced a hndamental transformation from a scientific curiosity to an exciting area of biologic inquiry. Considerable efforts to address the problem of interspecies organ transplantation are urged by the increasing worldwide shortage of donor organs. Accumulating knowledge, increasing numbers of publications and an accelerated rate of research progress reflect this intensified scientific interest. I hope this summary of current knowledge, based on our own 10 years’ experience with respect to cardiac xenotransplantation in rodents will advance hture research and help in the discovery of the “missing links” in xenotransplantation.

Treatment of hamster heart to rat xenotransplantation

Hamster-to-rat cardiac transplantation is an intensively studied model of primarily vascularized organ xenotransplantation. Spontaneous graft rejection is rapid compared to allografts, and has a graft histology that is characterized by morphological features decisively different from those observed in acute allogeneic rejection. However, the type of rejection can not be classified as hyperacute, according to rejection tempo and different morphology. Cardiac graft rejection occurs within three to four days in untreated recipients, which makes the model very suitable for the study of different immunosuppressing and immunomodulating treatment strategies. Inbred donor and recipient animals are commercially available and the use of rodents offers many advantages from a practical point of view, while microsurgical techniques allow highly reproducible results to be achieved in terms of surgical success and minimal failure rates. However, the impact of surgical trauma on graft survival and the influence of altered cardiac haemodynamics due to heterotopic transplantation, with a functional elimination of the left ventricle and subsequent reduced coronary blood flow, are essentially unknown. Graft survival data must therefore be interpreted cautiously with respect to possible graft outcome in other species--species combinations with a heart graft in an orthotopic position. A few publications from the 1970s and 1980s demonstrated that rejection in this model probably cannot be controlled by conventional immunosuppression. 1-4 Some years later Knechtle et al.~-7 achieved impressive long-term graft survival by combining total lymphoid irradiation (TLI) and cyciosporin A (CsA), but these results proved to be difficult to reproduce by others. This challenge, together with a general increased interest in xenotransplantation, has resulted in several studies during the last three to four years which have focused on reproducible control of rejection by different

The histology at various stages of acute rejection in concordant xenogeneic heart transplantation

In an attempt to describe early morphologic changes in heterotopic xenogeneic heart transplantation a sequential study was performed in a hamster‐to‐rat model. Mild morphologic changes observed after four to six h were characterized by slight interstitial edema and focal myocyte damage with fragmentation and loss of myofibrillar elements. No lymphocytic infiltration appeared. Moderate morphologic changes observed after 12–24 h were characterized by moderate interstitial edema, and the appearance of mild hemorrhage and scattered extravasated neutrophilic granulocytes. The myocardium had more widespread areas with myocyte damage, sometimes with small foci of necrotic cells and adjacent neutrophilic granulocytes and macrophages. Vascular changes with perivascular edema and swelling of the endothelium were seen and a few neutrophilic granulocytes could be found in the vessel walls. No lymphocytic infiltration appeared. Severe morphologic changes observed after 44–48 h or at the time of complete rejection were characterized by severe interstitial hemorrhage, appearance of widespread necrosis and marked vascular changes with development of leukocytoclastic‐like vasculitis, possibly with thrombosis. Only a few lymphocytes appeared. The findings were essentially different from those observed in allogeneic heart transplantations, where classical first‐set allograft rejection was seen. In normal donor hearts and syngeneic transplanted hearts used as controls, no significant morphologic changes were demonstrated. On the basis of this study we consider xenogeneic acute rejection to be primarily of the humoral type.

Hope for successful xenografting by immunosuppression with monoclonal antibody against CD4, total lymphoid irradiation and cyclosporine: six months' survival of hamster heart transplanted into rat

Hamster hearts were transplanted to rats, and the effects of combinations of total lymphoid irradiation (TLI), cyclophosphamide, cyclosporine A (CyA) and monoclonal antibodies (MAB) were investigated. Controls not immunosuppressed rejected their xenograft in 3 to 5 days, while combination immunosuppression including MABs against CD4 or IL-2-receptors extended graft survival significantly. In one case, the graft was still functioning 180 days after transplantation, which is the longest survival seen in this model. The use of specific MABs may open a new era for both xeno- and allo-transplantation.

HAMSTER TO RAT KIDNEY TRANSPLANTATION : TECHNIQUE, FUNCTIONAL OUTCOME AND COMPLICATIONS

Hamster to rat kidney transplantation has only recently been introduced as model of concordant xenografting. The kidney model offers unique possibilities for studying both immunological and functional aspects of xenografts as opposed to the widely used heterotopic heart model. This article provides a detailed description of surgical technique as well as data on functional outcome and complications. The renal artery with a small segment of the aorta is sutured end-to-side to the abdominal aorta of the recipient, and the renal vein is anastomosed end-to-side to the inferior vena cava. The urinary system is reconstructed by bladder-to-bladder anastomosis. Xenografts will maintain close to normal serum-creatinine levels for 2–3 days, after which they are rejected. Complications occurred in 22% of xenografts. Postrenal obstruction due to severe hematuria or ureter stenosis was the most frequent problem encountered.

An ultrastructural analysis of concordant and discordant cardiac xenografts in unmodified recipients

Ultrastructural changes in endothelial cells in a hamster‐to‐rat and guinea pig‐to‐rat heart transplantation model are described. In the hamster‐to‐rat model, changes were minimal with an increase in cytoplasmic vesicles 4–6 h after transplantation. 16–24 h after transplantation larger vesicles appeared and the basement and cell membranes were less well defined. 42–48 h after transplantation the changes had progressed with destruction of cell membranes and the appearance of extravasated erythrocytes. At the time of complete rejection, changes had further progressed with widespread endothelial cell destruction and infiltration of neutrophilic granulocytes and macrophages, and large amounts of fibrin were present. In the guinea pig‐to‐rat model, changes were characterized by the appearance of platelets in close contact with the endothelium of the capillaries 1–3 min after transplantation. 4–6 min after transplantation the basement membranes as well as the cell membranes were affected with indistinct borders and interruption. Occasionally fusion of platelets and endothelial cell membranes was demonstrated. In the grafts examined 7–9 min after transplantation, changes had further progressed. Massive aggregation of platelets now appeared in relation to remnants of endothelical cells. Signs of microvascular damage appeared in both models, but with different morphology. In hamster grafts, endothelial cell activation is indicated by gradual changes in the cell membranes resulting in vascular damage and infiltration of the grafts by macrophages and neutrophilic granulocytes. In the guinea pig grafts, activation of endothelial cells results in platelet aggregation, formation of microthrombi, and subsequent tissue damage. Even though antibody and complement are involved in both types of rejection the basic mechanisms are different.

Survival, function, morphology and serological aspects of rat renal allografts. Effect of short‐term treatment with cyclosporine A, anti‐CD4 and anti‐interleukin‐2 receptor monoclonal antibodies

The aim of this study was to investigate the effect of short‐term treatment with cyclosporine A (CyA) combined with anti‐CD4 (OX‐38) and anti‐interleukin‐2 receptor (OX‐39) monoclonal antibodies (MAbs) on graft survival, graft function, morphology, and anti‐donor antibody levels in a BN‐to‐LEW rat kidney transplantation model. Spontaneous rejection occurred at 9.3 days (range 9–10 d). Administration of CyA (12.5 mg/kg/d) for 14 days prolonged graft survival to 33 days (range 23–40 d, P<0.02). Supplementing with OX‐38 and OX‐39 100 μg/kg/d, given i.p. from days 0 to 7, further prolonged graft survival to 70 days (range 38‐> 100 d, P<0.02 vs controls and CyA group). One of seven recipients had good graft function for more than 100 days. A three‐fold increase of the MAb dosage did not improve mean graft survival (53.5 d), but three of eight recipients had well functioning grafts for > 100 days. Kidney function was characterized by reduced creatinine clearance, also in the recipients with long‐term graft survival, and a defect in concentrating urine creatinine with subsequent pronounced increase in urinary output. Graft histology showed a complex pattern of interstitial alterations including mononuclear cell infiltration, fibrosis, tubular atrophy and vascular damage with intimal/endothelial cell hyperplasia and perivascular inflammation. In nine of 10 MAb‐treated recipients with graft survival > 60 days, granular deposits of immunoglobulins and C3 were found by immunofluorescence microscopy (IFM). The deposits were localized in the glomerular capillaries and mesangium. IFM in MAb‐treated control animals could not demonstrate any deposits. Flow cytometric evaluation of posttransplant serum samples against donor target cells showed increasing amounts of anti‐donor antibodies until the time of rejection, while recipients with long‐term graft function had moderately positive cross‐matches up to two months after transplantation. Hereafter antibody titres decreased and cross‐matches at the time of sacrifice were again negative. The morphological findings and the flow cytometric cross‐match results seem to indicate a postponed antibody‐mediated type of rejection. The reason why some kidney recipients showed decreasing antibody titres and stable long‐term graft function is unclear.