Effect of repetitive high-dose treatment with soluble complement receptor type 1 and cobra venom factor on discordant xenograft survival.

Hyperacute xenograft rejection may be modified by the activation and depletion of complement (C) using cobra venom factor (CVF). This method of prolonging xenograft survival is toxic and associated with systemic inflammation, which may potentially contribute to the pathologic features of delayed xenograft rejection. Soluble complement receptor type 1 (sCR1) inhibits both the classical and alternative C pathways and thus limits the production of proinflammatory products such as the anaphylatoxins. Hence, we investigated the effects of various sCR1 and CVF regimens, and combinations thereof, in the discordant guinea pig-to-Lewis rat cardiac xenograft model. Mean graft survival time (MST) was significantly prolonged with repetitive dosing (MST=22 hr) or continuous infusion of sCR1 (MST=32 hr) as compared with unmodified controls (MST=15 min). However, sCR1 did not prevent intragraft deposition of C3 or neutrophil infiltration and resulted in only partial inhibition of C-mediated hemolytic activity in vitro. Grafts in rats treated with a single dose of CVF (MST=67 hr) or repetitive doses of CVF (MST=69 hr) survived significantly longer than those treated with sCR1 alone, and lacked C3 deposition or neutrophil accumulation. Sera from these animals were completely depleted of C-mediated hemolytic activity. Animals treated with a single dose of CVF, or sCRI plus a single dose of CVF (MST=64 hr), had similar xenograft survival times. However, immunohistologic studies showed that addition of sCR1 to a single dose of CVF resulted in decreased macrophage activation and reduced levels of cytokines (tumor necrosis factor-alpha and interleukin-1beta) within xenografts as compared with that in recipients treated with CVF alone. Such decreased macrophage activation may result from the binding of C4b by sCR1, since combination therapy was associated with decreased intragraft C4b as compared with either therapy alone. High doses of sCR1 were well tolerated by rats and significantly prolonged discordant xenograft survival (MST=32 hr), although not to the same extent as CVF. The modification of the intragraft immune responses seen with CVF/sCR1 combination therapy may augment further therapeutic manipulations to achieve discordant xenograft survival without the attendant toxicity associated with repeated CVF administration.

Candinas D ，Lesnikoski BA ，Robson SC ，Miyatake T ，Scesney SM ，Marsh HC Jr ，Ryan US ，Dalmasso AP ，Hancock WW ，Bach FH ... -  《TRANSPLANTATION》

Prolongation of cardiac xenograft survival by depletion of complement.

Complement (C) activation is thought to be critical for the hyperacute rejection of xenografts. We investigated the role of C in the rejection of discordant cardiac xenografts by studying outcome in recipients depleted of C, using a highly purified form of cobra venom factor (CVF) in both a small (guinea pig [GP]-to-rat) and large (pig-to-baboon) animal model. A single dose of 30 or 60 units CVF given i.v. to rats completely abrogated hemolytic C activity for up to 72 hr. The lack of hemolytic C activity correlated with nearly undetectable serum levels of C3. Doses of 30 U/kg daily or 60 U/kg every other day over a 7-day period sustained C depletion without morbidity or mortality. Rats receiving GP cardiac xenografts during CVF therapy had significantly prolonged xenograft survival (88 +/- 10 hr in CVF-treated rats vs. 18.6 +/- 7.2 min in control rats, P < 0.001). Rats that rejected GP xenografts at 4 days posttransplant had higher levels of anti-GP antibodies than control rats, without hemolytic C activity at rejection. This rise in xenoreactive Ig reflected an increase in circulating IgG and IgM against GP antigens recognized before transplantation. Histologic analysis of GP cardiac xenografts taken from CVF-treated rats revealed leukocyte and monocyte margination along blood vessels, beginning at 12 hr posttransplant. Progressive cell infiltration, interstitial hemorrhage, and necrosis were observed over the next 72 hr. Rejected GP xenografts showed diffuse deposition of IgM and fibrin within blood vessels but no evidence of C3 deposition. A nonspecific pattern of IgG deposition was noted. CVF was tested in baboons. Complete C depletion was achieved with a dose of 60 U/kg, and was not associated with any morbidity or mortality. Xenotransplantation of a pig heart was performed in one baboon receiving CVF, 60 U/kg/day, for 2 consecutive days. Xenograft survival was prolonged to 68 hr, compared with 90 +/- 30 min in control baboons. Lack of hemolytic activity was noted during engraftment and at rejection. Histology showed evidence of vascular rejection. Immunopathology showed diffuse deposition of IgM, fibrin, and C4, and absence of C3 or membrane attack complex. We conclude that highly purified CVF can achieve marked C depletion with minimal morbidity and no associated fatalities. CVF alone can significantly prolong discordant cardiac xenograft survival. In the GP-to-rat model, the improvement in graft survival achieved with CVF was better than with conventional immunosuppression or isolated acute antibody depletion.(ABSTRACT TRUNCATED AT 400 WORDS)

Leventhal JR ，Dalmasso AP ，Cromwell JW ，Platt JL ，Manivel CJ ，Bolman RM 3rd ，Matas AJ ... -  《TRANSPLANTATION》

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.

Candinas D ，Belliveau S ，Koyamada N ，Miyatake T ，Hechenleitner P ，Mark W ，Bach FH ，Hancock WW ... -  《TRANSPLANTATION》

Prolongation of discordant renal xenograft survival by depletion of complement. Comparative effects of systemically administered cobra venom factor and soluble complement receptor type 1 in a guinea-pig to rat model.

Chrupcala M ，Pomer S ，Staehler G ，Waldherr R ，Kirschfink C ... -  《TRANSPLANT INTERNATIONAL》

Neutrophil adhesion and complement inhibition prolongs survival of cardiac xenografts in discordant species.

Zehr KJ ，Herskowitz A ，Lee PC ，Kumar P ，Gillinov AM ，Baumgartner WA ... -  《TRANSPLANTATION》