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.

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.

SummaryWe demonstrated in the present study that with bacterial stimulation, an increased number of a/g T cells proliferated in the liver of mice and that even T cells bearing self-reactive T cell receptor (TCR) (or forbidden T cell clones), as estimated by antiV(3 monoclonal antibodies in conjunction with immunofluorescence tests, appeared in the liver and, to some extent, in the periphery. The majority (>80%) of forbidden clones induced had double-negative CD4-8-phenotype. In a syngeneic mixed lymphocyte reaction, these T cells appear to be self-reactive . Such forbidden clones and normal T cells in the liver showed a two-peak pattern of TCR expression, which consisted of u/(3 TCR dull and bright positive cells, as seen in the thymus. A systematic analysis of TCR staining patterns in the various organs was then carried out . T cells from not only the thymus but also the liver had the two-peak pattern of a/a TCR, whereas all of the other peripheral lymphoid organs had a single-peak pattern of TCR . However, T cells in the liver were not comprised of double-positive CD4 *8+ cells, which predominantly reside in the thymus. The present results therefore suggest that T cell proliferation in the liver might reflect a major extrathymic pathway for T cell differentiation and that this hepatic pathway has the ability to produce T cells bearing self-reactive TCR under bacterial stimulation, probably due to the lack of a double-positive stage for negative selection.I t is generally accepted that T lymphocytes differentiate in the thymus and go through a process of positive or negative selection to form the repertoires of mature T cells (1-4) . In earlier studies, several investigators proposed the existence of an extrathymic differentiation pathway of T cells, especially in experiments using congenitally athymic nude mice and in vitro culture systems (5-8) . However, investigators have not reached a consensus, since there is no definite information yet as to where such T cells differentiate outside the thymus. In recent studies, we have shown that both a/(3 and y/b T cells with double-negative (DN) t CD4 -8 -phenotype proliferate in the liver of humans and mice, especially under conditions of autoimmune diseases (9), malignancies (10, 11), and aging (12). These results have led us to consider the possibility that the liver might be a major site for extrathymic differentiation of certain T cells.In the present study, we have applied a recently described ' Abbreviations used in this paper. DN, double negative; DP, double positive, MMC, mitomycin ; MNC, mononuclear cells. 417system for identification of oligoclonal T cells expressing particular VQ of cx/0 TCRs by using mAbs (13,14) to demonstrate the hepatic pathway for cell differentiation . The present results support the possibility that the liver is a major site of extrathymic T cell differentiation and reveal that the hepatic pathway has several unique properties distinct from the intrathymic pathway for T cell differentiation . Materials and MethodsMice and Bacterial ...

Rituximab efficiently reduces anti-ABO antibody titer by selectively eliminating B cells and is safe and effective against humoral rejection after ABO-incompatible liver transplantation.

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.

SummaryEndothelial cell ATP diphosphohydrolases or ATPDases degrade extracellular inflammatory mediators ATP and ADP, thus inhibiting the formation of platelet thrombi, but the modulation of these ecto-enzymes during vascular injury remains largely undetermined. Renal glomerular ATPDase levels were determined in the rat following ische-mia-reperfusion or systemic complement activation, by direct biochemical methods and histochemistry. Ischemia followed by reperfusion times over 30 min were associated with loss of glomerular ATPDase activity. Cobra Venom Factor (CVF) inhibited ATPDase activity and potentiated the deleterious effects of reperfusion. Treatment with either soluble complement receptor type 1 (sCRl), an inhibitor of complement activation, or antioxidants prior to the ischemia-reperfusion was largely protective. Expression of rat glomerular ATPDase activity appears susceptible to the inflammatory injury associated with systemic complement activation and ischemia/reperfusion processes. Oxidative stress could, at least in part, result in the loss of ATPDase activity and thus thrombotic consequences of vascular injury.

In partial-liver transplantation, the use of small grafts sometimes results in graft failure, usually caused by portal hypertension after transplantation (Tx). Portal hypertension after Tx can be decreased with a porto-caval shunt (PCS). The purpose of this study is to clarify the effect of the PCS on extremely reduced-size liver Tx. In a pig model, the posterior segment of 25% of a whole liver was transplanted orthotopically. The pigs were divided two groups: group A, graft with PCS (n = 7), and group B, graft without PCS (n = 7). The PCS was made by means of side-to-side anastomosis of the portal vein and the inferior vena cava. We examined the portal vein pressure, survival rate, regeneration rate of the graft, Ki-67 as an index of cell proliferation, and histological findings, and carried out liverfunction tests. In group A, five pigs survived for more than 4 days and the remaining two died of a perforated gastric ulcer on post-operative day (POD) 2. In group B, all pigs except one died of graft failure within 24 h. Portal vein pressure after reperfusion in group A and group B was of statistically significant difference ( P < 0.05), 14.2 f 3.2 and 18.9 * 4.7 cmH20, respectively.In group A, the regeneration rate of the graft was 94%, 4 days after Tx, and Ki-67 stained remarkably in the parenchymal hepatocytes. In TEM finding, structure of the sinusoid was also well maintained after Tx. From these results we can conclude that the key to success in liver Tx with extremely small grafts lies in the control of the portal vein pressure,

The integrin GPIIbIIIa is known to be crucial to the formation of platelet aggregates and potentiates adhesion to subendothelial matrices via fibrin(ogen), von Willebrand factor, and vitronectin. Given the demonstration by us and others of widespread platelet aggregation during xenograft rejection, we hypothesized that platelet thrombi might contribute to graft dysfunction during development of hyperacute rejection (HAR), as well as during what we have termed delayed xenograft rejection (DXR), e.g., as seen in complement-depleted rat recipients of guinea pig cardiac xenografts. We therefore tested the effects of a specific GPIIbIIIa antagonist (SDZ GPI 562) during xenograft rejection. Lewis rats received heterotopic guinea pig cardiac xenografts and were treated with GPI 562 alone (HAR model) or in combination with cobra venom factor (CVF) (DXR model). A high (0.5 mg/kg) or a low dose (0.1 mg/kg) of GPI 562 was administered perioperatively and then given twice daily in the same dose until rejection. CVF was given daily until rejection. Plasma drawn after the first dose of GPI 562 and at the time of rejection was tested for the ability to inhibit ADP-stimulated platelet aggregation in vitro. Rejected grafts were analyzed by immunohistology. Plasma from animals in the high-dose group completely inhibited platelet aggregation in vitro, whereas plasma from the low-dose group resulted in only partial inhibition. Similarly, whereas low-dose GPI 562 failed to prolong graft survival, high-dose GPI 562 showed a statistically significant increase in graft survival in both HAR and DXR groups. Immunohistologic studies of HAR showed little effect of GPI 562 on platelet aggregation or activation and no effect on fibrin deposition. However, the combination of high-dose GPI 562 and CVF resulted in a significant decrease in intragraft platelet aggregation, P-selectin expression, and leukocyte infiltration compared with CVF alone. In conclusion, GPIIbIIIa antagonist therapy can inhibit platelet aggregation in vitro and prolong xenograft survival. The diminution of intragraft platelet microthrombi formation and leukocyte infiltration suggests an important role for platelet-dependent mechanisms in leukocyte recruitment during DXR.