Table 1.
Recipient and donor information, immunosuppressive regimen and biopsy time-points.
Table 2.
Liver immunohistopathology at 2 h and at necropsy.
Table 3.
Primary and secondary antibodies used for immunofluorescence staining of liver xenografts and native organs.
Table 4.
Extent of immunofluorescence staining of liver xenografts and native organs.
Figure 1.
Histopathology (H&E) of (A) healthy (non-transplanted) pig liver, (B) baboon liver 31 days after allotransplantation, and (C–F) WT pig liver after pig-to-baboon xenotransplantation.
A) Histomorphological appearance of a normal pig liver (×100). Distinct envelope of connective tissue surrounding hepatic lobules. B) Baboon-to-baboon allotransplantation (B3408) on post-operative day 31 (×200). Patchy, mild-to-moderate periportal lymphocytic infiltrates. C) WT pig-to-baboon liver xenoTx at 30 min (B16907) (×100). Significant hepatocellular vacuolar change, patchy sinusoidal congestion, and interlobular septal edema. D) WT pig-to-baboon liver xenoTx at 1 h (B16907) (×200). Severe hepatocellular vacuolar change, focal hepatocyte necrosis, and few thrombi. E) WT pig-to-baboon liver xenoTx at 3 h (B16907) (×100). Early coagulative necrosis, significant sinusoidal congestion, and large thrombus. F) WT pig-to-baboon liver xenoTx at 5 h (just before euthanasia) (B16907) (×100). Acute hemorrhagic coagulative necrosis, extensive hemorrhage, and large fibrin thrombi.
Table 5.
Macroscopic observations at necropsy of hemorrhage in native organs and body cavities after genetically-engineered pig liver xenotransplantation in longest-surviving baboon recipients.
Figure 2.
Macroscopic and microscopic appearance of GTKO and GTKO/CD46 pig livers 2 hours after perfusion of the graft and at necropsy.
A) B18508 (2-hour post-reperfusion). Macroscopic appearance of well-perfused pig liver. Lobular dark areas were noted in the middle of the liver (arrows) suggesting an arterio-embolic event. B) B18908 (2-hour post-reperfusion). Prominent increase in neutrophils (H&E ×200). Small panel at top left (H&E ×100). C) B18508 (2-hour post-reperfusion). Intra-hepatocellular vacuolar change. The pig liver in B18508 demonstrated the most severe degree of involvement (although the change was still relatively mild and subtle in extent) (H&E ×200). D) B18908 (light area at necropsy). Vacuolar hepatocellular cytoplasmic change (H&E ×200). E) B18508 (dark area at necropsy). Extensive hemorrhage and hemorrhagic necrosis, sparing only focal groups of hepatocytes in the portal regions (H&E ×200). Solid black bar indicates 0.1 mm for figures 2B, 2C, 2E, and 2F. Macroscopic appearance of livers at necropsy (day 7), with dark patchy areas and a yellowish color suggesting cholestasis in some cases, has been already shown [14].
Figure 3.
Immunohistological assessment of liver xenografts.
Cellular infiltrates (CD3, CD20), antibody (IgG and IgM) and complement (C3, C4d, C5b-9) deposition were obvious in the WT pig liver at 2 hours, but were minimal or absent in GTKO and GTKO/CD46 pig livers. Fibrin and platelets were present in WT or GTKO and GTKO/CD46 pig livers regardless of post-Tx time, immunosuppression, and genetic modification of the organ-source pig. For details, see Table 2. (g) = green, (r) = red. Solid white bar indicates 0.1 mm for all figures.
Figure 4.
Electron microscopic appearance of liver xenografts.
A) Ultrastructure of GTKO/CD46 liver before excision from the pig. B) (2 h post-reperfusion). Normal appearance of hepatocytes (H) with widespread platelet (P) aggregation and deposition along liver sinusoidal endothelial cells. C) (necropsy, dark area). Red blood cells (R) with fibrin deposition (F) indicating fibrin clots in the sinusoids. D) (necropsy, light area). Platelet, platelet/monocyte and platelet/necrotic cell aggregates along liver sinusoidal endothelial cells, and patchy endothelial cell death. Dashed lines indicate endothelial cells lining the sinusoids. AB = apoptotic body, EC = endothelial cells, F = fibrin, H = hepatocytes, P = platelets, R = red blood cells. The line at the bottom right indicates 2 µm.
Table 6.
Immunohistopathological features of thrombotic microangiopathy in recipient native organs at necropsy.
Figure 5.
Macroscopic and microscopic appearances of native organs.
A) Heart: Left panel, macroscopic appearance of heart (B18908). Arrow indicates macroscopic bleeding in the myocardium (B18908). Right panel, subendocardial hemorrhage within the myocardium as well as on the epicardial surface (H&E ×100). B) Lungs: Left panel, macroscopic appearance of lung in which there was patchy bleeding (B3208) (Table 5). Right panel, acute congestion, focal atelactasis, multiple thrombi with no features of inflammation (H&E ×200). C) Staining for platelets (CD41+) was positive in lungs, suggesting platelet aggregation and migration. This phenomenon was observed in lungs regardless of bleeding, but was present in other native organs only when bleeding had occurred (Table 6) (×100). D) Small intestine: Left panel, macroscopic hemorrhage in the wall of small intestine. Right panel, although the mucosa appeared normal, prominent submucosal hemorrhage was noted (B18908) (H&E ×100). E) Kidneys: Left panel, kidneys were macroscopically normal, except in one case (B18908) which showed small, patchy petechiae. Right panel, acute glomerular congestion associated with tubular and interstitial hemorrhage (H&E ×100). F) Lymph nodes: Although the macroscopic appearance was normal, lymph nodes in one case (B18508) showed hemorrhage in the hilar and medullary regions (H&E ×200).
Figure 6.
Average anti-nonGal antibody levels (IgM and IgG) in 4 baboons.
Serum from the longest-surviving recipient baboons (n = 4; B7708, B7808, B18508, and B18908) was drawn daily (pre-Tx to postoperative day 7), and stored at −80°C until processed. Binding of (i) anti-nonGal IgM and IgG antibodies to GTKO and GTKO/CD46, and of (ii) anti-pig antibodies (Gal and nonGal), to WT pig peripheral blood mononuclear cells (PBMCs) was measured by flow cytometry [29]. A) IgM binding against WT, GTKO, and GTKO/CD46 pig PBMCs: Significantly increased binding to WT pig PBMCs. However, GTKO and GTKO/CD46 pig PBMCs showed decreased binding throughout the study. B) IgG binding against WT, GTKO, and GTKO/CD46 pig PBMCs: Although IgG binding against WT pig PBMCs was slightly increased; there was no significant difference between bindings against GTKO and GTKO/CD46 pig PBMCs.