C Atkinson

Histopathology of cardiac xenograft rejection in the pig-to-baboon model

BACKGROUNDnThe use of pig organs transgenic for human decay accelerating factor (hDAF) has largely overcome the problems of hyperacute rejection. With improved immunosuppressive protocols, life supporting grafts are showing greater survival times bringing the possibility of clinical xenotransplantation closer. Examination of the histopathology of the rejection process provides insight into the underlying mechanism and may suggest ways in which new immunosuppressive strategies should be directed.nnnMETHODSn44 baboons (Papio anubis) underwent heart transplants of which 39 were from transgenic donors. The transplanted organs were examined histologically and stained for evidence of immunoglobulin and complement deposition as well as cellular infiltrates.nnnRESULTSnIn the transgenic animals survival times were 2 to 99 days (mean 23.5) and the heterotopic group and 1 to 39 days (mean 11.7) in the orthotopic group. There were 3 cases of hyperacute rejection between the 2 groups. Rejected organs showed areas of old and recent myocardial infarction associated with vascular thrombosis. There was widespread deposition within vessels of immunoglobulins IgM and IgG together with complement fractions C3 and C5b to 9 in those organs that were rejected. The amount of complement positive in the longer surviving organs was less than those rejecting early. Cellular infiltate was predominantly macrophage with some later appearing T or natural killer cells.nnnCONCLUSIONSnThe histopathological changes support the importance of immunoglobulin and complement in delayed xenograft or acute vascular rejection. With time there is an increase in cellular infiltrate predominantly macrophages and these findings suggest an increasingly important role for the cells and the rejection process. The presence of areas of infarction and underlying vascular thrombosis is in keeping with endothelial activation and the establishment of procoagulant phenotype which may be due to immunoglobulin, complement, secreted cytokines and direct cellular effects.

The energy metabolism in the right and left ventricles of human donor hearts across transplantation.

OBJECTIVEnBrain death appears to predominantly affect the right ventricle (RV) and right ventricular failure is a common complication of clinical cardiac transplantation. It is not clear to what extent myocardial energy stores are affected in the operative sequence. We aimed to describe the time-dependent variation in high energy phosphate (HEP) metabolism of the two ventricles, and the relationship with endothelial activation and postoperative functional recovery.nnnMETHODSnFifty-two human donors had serial biopsies from the RV and the left ventricle (LV) at (1) initial evaluation, (2) after haemodynamic optimisation, (3) end of cold ischaemia, (4) end of warm ischaemia, (5) reperfusion, and (6) at 1 week postoperatively. HEP was measured by chemiluminescence in biopsies 1-5 and adhesion molecules (P-selectin, E-selectin, VCAM-1) and thrombomodulin were analysed by immunohistochemistry in biopsies 5-6. Seventeen donors and five recipients had RV intraoperative pressure-volume recordings by a conductance catheter. Six patients served as live controls.nnnRESULTSnBrain death did not affect HEP metabolism quantitatively. There was no difference between the RV and LV at any time point, but significant time-dependent changes were observed. The RV was prone to HEP depletion at retrieval, with ATP/ADP falling from 3.89 to 3.13, but recovered during cold ischaemia. During warm ischaemia the ATP/ADP ratio fell by approximately 50%, from 5.48 for the RV and 4.26 for the LV, with partial recovery at reperfusion (P<0.005). Hearts with impaired function in the recipient showed marked variations in HEP levels at reperfusion, and those organs with RV dysfunction failed to replenish their energy stores. However, these organs were not different from normally functioning allografts in terms of endothelial activation and clinical risk factors. There was poor correlation between pressure-volume and HEP data in either donor or recipient studies. Hearts followed-up with HEP and pressure-volume studies showed improvement in the recipient, despite functioning against a higher pulmonary vascular resistance.nnnCONCLUSIONSnHEP are preserved over a wide range of contractile performance in the donor heart, with no metabolic difference between the two ventricles. No correlation with endothelial activation was seen either. Preservation efforts should be directed to the vulnerable periods of implantation and reperfusion.

Heat shock protein, inducible nitric oxide synthase and apoptotic markers in the acute phase of human cardiac transplantation.

OBJECTIVEnSolid organ transplantation is associated with activation of apoptotic pathways and other stress markers. We aimed to describe the expression of Bax, Bcl-2, iNOs and Hsp-70 in the endothelium and myocytes of both ventricles and to see if there is any relationship with clinical donor organ failure.nnnMETHODSnTwelve patients undergoing heart or heart-lung transplantation (including three domino cases) were studied with transmural biopsies from the right (RV) and the left ventricles (LV) at the following points: after donor optimisation; at the end of ischaemic time; and after 10 min of reperfusion. The 1-week endomyocardial RV biopsy was also examined. Five donor hearts turned down purely on functional grounds were analysed also.nnnRESULTSnThere was no difference between the RV and the LV for any of the markers at intraoperative assessment. The pattern of expression was not predictive of allograft failure. Donor hearts, however, have a strong pro-apoptotic phenotype, which is largely unopposed by the protective factors Bcl-2 and Hsp-70. Furthermore, the intensity of myocyte staining increases over time for Bax (P<0.001) and iNOs (P=0.02). Domino hearts showed a similar pattern. Compared to usable organs, poorly functioning donor hearts have stronger myocardial staining for Bax (P=0.002) and iNOs (P=0.01).nnnCONCLUSIONSnClinical cardiac transplantation is associated with activation of the Bax and iNOs pathways in both ventricles. The myocardium is affected in time-dependent fashion but this is compatible, to a certain extent, with satisfactory allograft function. Donor hearts turned down on the basis of poor haemodynamic performance have significantly higher expression of Bax and iNOs.

Expression of endothelial adhesion molecules and thrombomodulin in cardiac transplantation

Objective: To describe the pattern of endothelial cell activation (ECA) in the transplanted human heart. Methods: 39 donor hearts had trucut biopsies from the right (RV) and the left ventricles (LV) at: initial assessment of the donor, end of warm ischaemia and after 10 min. of reperfusion. In addition, heart transplant patients had follow up RV biopsies during rejection surveillance at 1 week, 1 month and 3 months postoperatively. Biopsies exhibiting rejection were excluded from the analysis. 6 of the patients were cystic fibrosis domino donors. Another 9 patients undergoing routine cardiac surgery served as controls. Psel, VCAM-1, Esel and thrombomodulin (Thr) were examined by immunohistology. Results: There was no difference between the RV and the LV at any of the intraoperative time points, but important time-dependent variations were seen. Psel, and VCAM-1 (but not Esel) are upregulated in brain-dead and in domino donors (p 0.01 for LV and p 0.005 for RV). Thr is reduced at the baseline in all hearts used for transplantation, and the depletion accentuates postoperatively (p 0.30 for LV and p 0.02 for RV). Psel is present in 85% of vessels throughout transplantation and decreases to cca. 60% post-transplant (p 0.001). VCAM-1 is present in 20% of vessels initially, decreases to 5% during storage (this fall is inversely correlated to the ischaemic time), increases to 47% at reperfusion and gradually decreases thereafter (p 0.001). Esel expression increases progressively from 15% initially to 45% at reperfusion and decreases postoperatively (p 0.001). Patients with donor organ failure did not have any specific pattern of ECA. However, there was a trend towards accumulation of clinical risk factors in this group. Recipients of aprotinin had reduced expression of Esel and VCAM-1 in the LV at reperfusion. Conclusion: Cardiac transplantation (including domino) is associated with marked ECA changes, with no difference between the two ventricles. The changes persist in the postoperative period even in the absence of rejection. Expression of ECA markers is influenced by aprotinin but is not predictive of donor organ failure.