Paul Hechenleitner

Accommodation of vascularized xenografts: Expression of “protective genes” by donor endothelial cells in a host Th2 cytokine environment

Organ xenografts under certain circumstances survive in the presence of anti-graft antibodies and complement, a situation referred to as “accommodation.” We find that the endothelial cells (ECs) in hamster hearts that accommodate themselves in rats express genes, such as A20 and bcl-2, that in vitro protect ECs from apoptosis and prevent upregulation in those cells of proinflammatory genes such as cytokines, procoagulant and adhesion molecules. Hearts that are rejected do not express these genes. In addition, vessels of rejected hearts show florid transplant arteriosclerosis whereas those of accommodated hearts do not. Accommodated xenografts have an ongoing T helper cell type 2 (Th2) cytokine immune response, whereas the rejected grafts have a Th1 response. We propose a model for factors that contribute to the survival of xenografts and the avoidance of transplant arteriosclerosis.

T cell independence of macrophage and natural killer cell infiltration, cytokine production, and endothelial activation during delayed xenograft rejection

Rejection of guinea pig cardiac grafts in rats depleted of complement takes place in 3-4 days and involves progressive mononuclear cell infiltration and cytokine expression, fibrin and antibody deposition, and endothelial cell up-regulation of adhesion and procoagulant molecules, a process termed delayed xenograft rejection (DXR). The relative contribution of each effector mechanism and the role of T cells in this complex process are unknown, although small numbers of interleukin (IL) 2 receptor-positive T cells are present at the time of rejection. We investigated the importance of T cells in DXR by comparing discordant xenograft responses of nude rats, which lack T cell receptor (TCR)-alpha/beta+ cells, with those of normal Lewis rats. Nude or Lewis rats receiving guinea pig cardiac grafts were assigned to one of three groups: no therapy, daily administration of cobra venom factor (CVF), or splenectomy plus daily CVF. All untreated rats rejected their xenografts within 10-15 min, whereas grafts in complement-depleted recipients survived a further 3-4 days; splenectomy had no significant additional effect upon graft survival. Immunohistologic analysis in CVF-treated nude recipients with or without splenectomy showed: (1) considerable leukocyte infiltration of xenografts (mean +/- SD, 76+/-14 and 71+/-16 leukocytes/field, respectively, at 72 hr, compared with 68+/-17 in Lewis rats), consisting largely of macrophages (>75% of total leukocytes) plus small numbers of natural killer cells (10-20%) with no detectable B or T cells (TCR-alpha/beta or TCR-gamma/delta); (2) at least 10-fold lower levels of intragraft IgM or IgG deposition than in corresponding Lewis recipients; and (3) considerable cytokine expression by intragraft macrophages (IL-12, tumor necrosis factor-alpha, monocyte chemoattractant protein-1, IL-1beta, IL-6, IL-7, IL-12) and natural killer cells (interferon-gamma), as well as up-regulation of tissue factor expression and dense fibrin deposition. Analysis of recipient sera of both control and nude rats by ELISA, for the binding of IgG or IgM to guinea pig platelets, showed a rapid rise after transplantation in the titers of IgM and IgG antibodies, which was abrogated by prior splenectomy; i.e., data from splenectomized xenograft recipients reflect the presence of only basal levels of IgM and IgG. Thus, our data in nude rats show rejection times and intragraft features of DXR comparable to those in immunocompetent Lewis recipients, despite a lack of detectable host T cells, and, in the case of splenectomized rats, only about one tenth of normal xenoreactive antibody levels. Our data document a new model in which to analyze the immunopathogenesis of DXR.

Apyrase administration prolongs discordant xenograft survival.

Platelet thrombi and vascular inflammation are prominent features of discordant xenograft rejection. The purinergic nucleotides ATP and ADP, which are secreted from platelets and released by injured endothelial cells (EC), are important mediators of these reactions. Quiescent EC express the ectoenzyme ATP-diphosphohydrolase (ATPDase; an apyrase), which exerts an important thromboregulatory function by hydrolyzing both ATP and ADP. We have shown that ATPDase activity is rapidly lost from the surface of the EC following ischemia-reperfusion injury and during xenograft rejection. The aim of this study was to supplement ATPDase activity within xenografts by infusion of soluble apyrases, and thereby validate the importance of local ATPDase activity in the modulation of xenograft rejection. Lewis rats underwent heterotopic cardiac xenografting from guinea pigs and apyrase was administered intravenously (200 U/kg) as a single dose to evaluate effects on hyperacute rejection (HAR). This initial dose was followed by a continuous apyrase infusion (8.0 U/kg/hr) directly into the graft aorta in combination with systemic cobra venom factor (CVF) administration to deplete complement when delayed xenograft rejection (DXR) was studied. Functional apyrase levels in vivo were assessed by the capacity of blood samples taken at the time of surgery and rejection to inhibit platelet aggregation in vitro. Apyrase administration significantly prolonged graft survival in HAR and DXR. Functional assays showed inhibition of platelet aggregation suggesting effective systemic antiaggregatory effects of the administered apyrases. Histologic studies showed that apyrase administration abrogated local platelet aggregation and activation in HAR and DXR. Our data demonstrate that local administration of apyrase prolonged discordant xenograft survival. These observations emphasize the potential importance of purinergic mediators in platelet activation during xenograft rejection.

Immunomodulatory effects of the alkaloid sinomenine in the high responder ACI-to-Lewis cardiac allograft model.

Extracts of the plant Sinomenium acutum have been used safely since ancient times in Chinese medicine for treatment of rheumatic diseases, and the purified alkaloid, sinomenine, was recently shown to have anti-inflammatory and antirheumatic effects. This study describes the effects of sinomenine in the high responder ACI-->Lewis cardiac transplant model in which allograft rejection occurred at 5 days posttransplant. Treatment with sinomenine (15-30 mg/kg/day i.p.) or a subtherapeutic dose of cyclosporine (CsA, 1.5 mg/kg/day, i.m.) prolonged allograft survival only marginally (mean survival of 5.4 and 7.8 days, respectively). In contrast, the combination of sinomenine and CsA had a statistically significant synergistic effect, with a mean survival of 42.2 days (P < 0.001). Allografts harvested at day 5 from recipients treated with either sinomenine or CsA showed dense mononuclear cell infiltrates with widespread subepicardial infarcts, edema, and microvascular platelet and fibrin deposition. Immunohistologic analysis showed that intragraft leukocytes consisted of >75% macrophages with approximately 10-20% T cells and <5% B or NK cells. Mononuclear cell activation was shown by expression of IL-2R (CD25, 10-20%) and labeling for IL-2 (approximately 10%), and IFN-gamma (10-20%), as well as TNF-alpha (>50%) and iNOS (>50%), but only low levels of IL-4 or IL-10 (<5%). Intragraft endothelial cells were activated, as shown by upregulation of MHC class II antigen and ICAM-1 (CD54) compared with only basal levels in normal donors hearts. Combined sinomenine/CsA therapy significantly enhanced graft morphology, resulting in only mild mononuclear cell infiltration, and an absence of infarcts, platelets, or fibrin deposition. Though residual intragraft mononuclear cells at day 5, as in control grafts, consisted primarily of macrophages plus small numbers of IL-2R+ T cells, these cells lacked expression of IL-2, had only low levels of IFN-gamma, but showed dense labeling for IL-4 and IL-10. In addition, TNF-alpha and iNOS were reduced to basal levels and no endothelial cell activation was observed, despite high titers of endothelium-bound IgM, IgG, and C3. Mitogen-induced in vitro proliferation of rat thymocytes was also more effectively decreased by the sinomenine/CsA combination than by either agent alone. These studies demonstrate the therapeutic value of sinomenine in transplantation, and indicate that this agent has novel and interesting antimacrophage, T cell, and endothelial effects that warrant further evaluation.

Protective genes expressed in endothelial cells of second hamster heart transplants to rats carrying an accommodated first graft

Abstract: Accommodation refers to survival of a xenograft despite the presence of anti‐donor organ antibodies and complement. We have recently shown that accommodation of a hamster heart transplanted to a rat receiving short‐term cobra venom factor (CVF) and continuing cyclosporine A (CyA) therapy is associated with i) the expression in the endothelial cells (EC) and smooth muscle cells of the graft of a number of “protective” genes, ii) a prominent intragraft Th2 cytokine profile, and iii) the relatively heavy deposition of IgG2c antibodies on the EC of the graft. In contrast, rejecting grafts do not express the protective genes, have a Th1 cytokine profile, and apparently have lesser amounts of IgG2c. These findings are consistent with host factors (Th2 cytokines and IgG2c) contributing to xenograft accommodation. To test whether these host factors may predispose to the development of accommodation, we placed a second hamster heart into each of 12 rats carrying a surviving first heart; recipients were, at the time, receiving only CyA. Whereas first grafts transplanted to rats receiving only CyA survive for 3 to 4 days, 11 out of 12 second transplants survived more than 20 days, and the other survived for 7 days. Nine of the twelve were not rejected: of these, four were removed between day 35 and 132 for study, and the remainder are still beating at 35 to 52 days. The surviving second hearts we studied had accommodated in that the picture on immunopathology was the same as for surviving first hearts. We suggest that the Th2 cytokines and perhaps the IgG2c response are factors in allowing prolonged survival of the second grafts and, further, that these factors contribute to the expression in the EC and smooth muscle cells of the surviving second hearts of the protective genes.

Duodenal histology for monitoring treatment of acute rejection in pancreaticoduodenal allografts in rats.

Background. Although the value of duodenal histology as a means to diagnose acute rejection in pancreaticoduodenal allografts has been validated, it is not known how the duodenum responds to antirejection treatment in comparison with the pancreas. Methods. Diabetic Lewis rats received a pancreaticoduodenal allograft. Cyclosporine was given for 5 days and then discontinued for 2 days (group 1), for 4 days (group 2), for 6 days (group 3), for 8 days (group 4), for 9 days (group 5), and for 10 days (group 6). Two animals of each group were killed for histology at the end of immunosuppressive-free intervals. In the remaining rats, rejection was treated with methylprednisolone on 3 consecutive days. Duodenal histology was compared with pancreatic morphology before and after treatment of rejection. Results. Duodenal histology had a positive and negative predictive value of 100% for detection of acute rejection in the pancreatic portion of the graft. After antirejection treatment, duodenal morphology was however less accurate (positive predictive value, 96%; negative predictive value, 67%). The Spearman correlation coefficient (p) of duodenal and pancreatic rejection grades was higher before antirejection treatment (p =1.0) than thereafter (p =0.724). Considering interstitial and vascular changes separately, vascular rejection correlated to a higher extent than interstitial rejection between the two portions of the graft (p =0.725 vs. p =0.677). Conclusions. Duodenal histology accurately predicts the initial diagnosis of rejection of the pancreas. However, after treatment of acute rejection, duodenal morphology is more likely to recover from rejection than the pancreas. Awareness of this phenomenon might be important for the interpretation of duodenal follow-up biopsies.

At what stage does pancreas allograft rejection become irreversible? An experimental study

BACKGROUND It is commonly believed that abnormal blood glucose levels indicate irreversible rejection. We were interested in determining the stage at which rejection remains reversible. METHODS A total of 54 Lewis rats were rendered diabetic with 55 mg/kg streptozocin and were then given a pancreas transplant from Brown Norway donors. Pancreatic juice was collected in a subcutaneous reservoir. All recipients received 15 mg/kg cyclosporine A (CsA) for 5 days. CsA was then discontinued for 2 days (n=7, group 1), 4 days (n=7, group 2), 6 days (n=9, group 3), 8 days (n=9, group 4), 9 days (n=11, group 5), and 10 days (n=11, group 6). Two animals of each group were euthanized at the end of the immunosuppressive-free interval, for histological assessment of the grade of rejection (G0, GI, GII, GIII). Rejection was treated with methylprednisolone (7 mg/kg body weight) and CsA (15 mg/kg body weight). The volume of pancreatic juice, together with juice cytology (C0, CI, CII) and blood glucose levels, was assessed daily. RESULTS Blood glucose remained normal throughout the observation period in animals with GI and GII rejection. The numbers of animals that became diabetic were as follows: 5 of 9 (group 4), 7 of 11 (group 5), and 8 of 11 (group 6). Decreased amounts of pancreatic juice were observed in all animals, except those in group 1. The histology returned to normal after anti-rejection therapy in four animals (57%) of group 1, in two animals (28%) of group 2, and in one animal (11%) of groups 3 and 4, respectively. Although there was no animal in groups 5 and 6 with normal graft histology after treatment, there were still four (36%) and three (27%) animals, respectively, that were normoglycemic and that had pancreatic grafts with well-preserved islets. CONCLUSIONS From these data, we conclude that even GIII rejection with severe endothelialitis and isleitis can be reversed. Therefore, we suggest that a trial of enhanced immunosuppression is justified in patients with advanced pancreas allograft rejection.

Improved technique for infrahepatic vena cava reconstruction in guinea pig–to–rat liver transplantation

Guinea pig–to–rat orthotopic liver transplantation is associated with serious technical problems contributing to impaired graft perfusion and primary graft failure. In order to shorten the procurement procedure and thereby minimize liver damage before flushing, a simplified technique for infrahepatic caval reconstruction was developed. Dissection of the infrahepatic vena cava (IHVC) from adrenal glands and renal and lumbar veins represents the most difficult and time‐consuming part of the donor operation, which is often not well tolerated by the animal; we avoided this step by using an isogeneic vena cava interposition graft (VCIG) following in situ perfusion. This graft is connected with the IHVC transsected just below the liver with a cuff technique. Donor operations lasted 15 to 20 minutes with the new technique (n = 7) compared to 52 to 76 minutes with conventional technique (n = 7). Reduced operating time was associated with markedly improved graft perfusion and significantly better graft survival. This modification of the donor procedure for the guinea pig–to–rat liver xenograft using a VCIG significantly reduces operating time and improves reperfusion and recipient survival.

Eine neue Technik der orthotopen Lebertransplantation vom Meerschweinchen auf die Ratte

Aufgrund des enormen Wissenszuwachses auf dem Gebiet der Xenotransplantation und der Moglichkeit, diskordante xenogene Organe als „bridging“ bei fulminantem Leberversagen einzusetzen, werden dringend entsprechende in vivo-Forschungsmodelle fur die diskordante Lebertransplantation benotigt.

CD28/B7 : EIN ELEMENTARES, COSTIMULATORISCHES SIGNAL DER T-ZELL-AKTIVIERUNG NACH ORTHOTOPER LEBERTRANSPLANTATION

Infektiologische Komplikationen sowie die chronische, mit Ductopenie einhergehende Transplantatabstosung stellen nach wie vor das Hauptproblem nach klinischer orthotoper Lebertransplantation (OLT) dar, so das innovative Strategien in der Modulation der Immunantwort des Transplantatempfangers erforderlich sind.