LOCAL INTRAGRAFT DELIVERY OF ANTIBODY TARGETING E-/L-SELECTIN ATTENUATES POST-TRANSPLANT ISCHEMIA-REPERFUSION INJURY

Abstract

204 Background. E- and L-selectin are responsible for leukocyte rolling on endothelium, which predates leukocytic infiltration of organ grafts. Our laboratory has previously demonstrated that E-selectin is upregulated before other adhesion molecules in human cardiac graft rejection. Purpose. In these experiments, we investigated whether a single intracardiac dose of a unique antibody to both E- and L-selectin (EL-246 mAb, gift of Ligocyte Pharmaceuticals), could prevent subsequent neutrophil infiltration as a measure of transplant ischemia-reperfusion injury. A new heterotopic cervical cardiac allograft model in farm-raised sheep was used to test this hypothesis. Methods. Following dissection, sheep donor hearts were arrested with cold Stanford cardioplegia solution and excised. Donor aortas and pulmonary arteries were anastomosed to the carotid arteries and external jugular veins of recipient sheep, respectively. Prior to reinstitution of blood flow into the graft, the right and left coronary ostia were blindly catheterized through the donor aortic root and perfused with either monoclonal antibody EL-246, 0.2mg/kg, (n=4) or normal saline (n=3). After resuscitation, transplanted hearts were placed in a subcutaneous pocket. Recipient sheep were euthanized at 24 hours and transplanted hearts excised for histology. Neutrophils (PMNs) were assessed on two randomly chosen epicardial, mid-myocardial, and endocardial specimens per transverse section by manual counting with an esterase stain [presented as neutrophils per high power field (PMN/hpf)]. Neutrophil counts were averaged over all specimens in each heart and epicardial, mid-myocardial, and endocardial specimens were analyzed separately and collectively using Student's t test. Results. After 24 hours of reperfusion, the mean neutrophil count (averaged over all myocardial specimens in each heart) was markedly less in the antibody-treated donor hearts (8.38 ± 3.39 PMN/hpf) than in saline-treated donor hearts (17.67 ± 5.48 PMN/hpf), but not statistically significant (p=0.18). Even if the epicardial specimens are eliminated to control for the effects of surgical manipulation, in the remaining mid-myocardial and endocardial specimens, the average number of neutrophils in the antibody-treated hearts (3.25 ±1.06 PMN/hpf) half the average count in control donor hearts (7.17 ± 2.5 PMN/hpf; p=0.07). Conclusions. A single intraoperative coronary infusion of this unique antibody to E- and L-selectin before reperfusion, was effective in attenuating neutrophil infiltration into donor hearts at 24 hours after cardiac transplantation in this large animal model. We are now investigating whether this dual antibody would also reduce early graft-infiltrating lymphocytes through its L-selectin binding. Perfusion of this antibody into graft vasculature following cold preservation solution could be easily applied clinically in all solid organ transplants.