[Frontiers in Bioscience 1, d248-265, September 1, 1996]
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CAVEAT LECTOR



XENOTRANSPLANTATION - STATE OF THE ART

D.K.C. Cooper, MA, PhD, MD, MS, FRCS, FACC, FACS

Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, USA

Received 07/16/96; Accepted 08/12/96; On-line 09/01/96

3. CONCORDANT AND DISCORDANT XENOTRANSPLANTATION

3.1. Definitions

Xenotransplantation refers to the transplantation of organs or tissues from an animal of one species into another species. With regard to humans, it clearly refers to the use of a donor other than a human. The terms concordant and discordant xenografting are used loosely to refer respectively to transplantation between closely-related animal species (e.g. baboon-to-human) and between distantly-related species (e.g. pig-to-human) (7).

With regard to the histopathology of the rejection that takes place, we should probably confine our terms to (i) cellular, (iii) antibody-mediated (denoting vascular or humoral), and (iii) mixed rejection (8). Antibody-mediated rejection may be hyperacute (in that it occurs within minutes or a few hours after transplantation) or delayed, occurring some days or even weeks after transplantation.

3.2. Pathogenesis of graft rejection

In general, pre-existing antibodies are not present in humans in high titers against closely-related species (e.g. baboon), but can develop or rapidly increase during the first few days after concordant xenotransplantation. Rejection generally occurs in an accelerated fashion (when compared to that of an allograft) within a few days, and can be of a humoral, cellular, or mixed nature (8).

Significant differences in the rejection process occur in different species combinations and different transplanted organs. For example, cynomolgus monkey hearts transplanted into baboons would appear to be rejected primarily by a cellular mechanism (9,10), not unlike after allografting, whereas African green monkey hearts transplanted into baboons are more likely to be rejected by a humoral (or mixed humoral and cellular) mechanism (11,12). African green monkey livers transplanted into baboons, however, have been reported to be rejected primarily by a cellular mechanism (13).

The presence in humans of relatively high titers of natural pre-formed antibodies against discordant donor species (e.g. pig) leads to immediate hyperacute rejection (HAR) (as may occur when allografting is carried out in a sensitized recipient). The HAR is initiated by the interaction of the antibodies with antigens on the vascular endothelium of the donor organ, resulting in activation of the classical pathway of complement (14) and vascular endothelial cell activation and lysis. In some species combinations, the alternative pathway of complement activation is believed to play a role (15), and evidence has been put forward to suggest that in humans this may be due to dimeric IgA binding to the pig vascular endothelium (16).

If HAR can be avoided (e.g. by depletion of complement by cobra venom factor (17,18)), current evidence is that a delayed form of rejection occurs (often termed 'delayed xenograft rejection' (19)), which leads to more gradual graft failure. The exact mechanism of this delayed xenograft rejection remains uncertain, but appears to be antibody-mediated but complement-independent (19). There is increasing evidence that natural killer cells and macrophages may play significant roles (20).

3.3. Histopathology

The classical histopathologic picture of HAR consists of disruption of the vascular endothelium, with massive interstitial edema and hemorrhage (8) (Figure 1). Intravascular fibrin thrombi are frequently present, and platelet thrombi can be observed. This picture can, however, be considerably attenuated even when early graft failure has occurred.

Figure 1: Photomicrograph of donor pig myocardium following xenotransplantation into a non-immunosuppressed recipient baboon. The donor heart ceased functioning after 4 hours, and histologically shows florid hyperacute rejection with severe interstitial hemorrhage, vascular thrombi, and myocyte necrosis. (Hematoxylin and eosin, x 150)

Immunofluorescence studies demonstrate IgM, IgG, IgA and complement deposition on the vascular endothelium (21, and Kobayashi, T., et al.,submitted for publication) (Figure 2). The histo-pathological features of delayed xenograft rejection vary little from those seen in HAR, although immunohisto-logical studies reveal the presence of cytokines and various cells (21, and Kobayashi, T., et al., submitted for publication).

Figure 2: Immunoperoxidase labeling of a pig-to-baboon cardiac xenograft that was rejected hyperacutely. There is endothelial deposition of IgM, IgG and IgA. The graft also shows endothelial deposition of components of the classical (C1q) and alternate (Factor B, properdin) pathways of complement activation, along with C3d and terminal pathway components (e.g. C6). (Courtesy W.W. Hancock)

3.4. Anti-pig antibodies

Current evidence is that all (or most) human anti-pig antibodies are directed against alpha-galactosyl (alphaGal) epitopes, specifically with a terminal Gal-alpha1-3Gal structure, on the surface of pig vascular endothelium (22-28) (Table 1 and Table 2 and Figure 3). These anti-alphaGal antibodies are also found in apes and Old World monkeys, but not in lower primates (e.g. New World monkeys) or non-primate mammals (including the pig), which, in contrast, express the alphaGal antigen (29). Following the transplantation of a pig organ into a human or baboon, or the extracorporeal perfusion of human blood through a pig organ, there is a marked increase in the titer of anti-alphaGal antibody, increasing by <60-fold over a period of days or weeks (30-32).

Table 1: Carbohydrate antigens which bound significant levels of human anti-pig heart or human anti-pig kidney antibodies.

ANTIGEN GROUPTRIVIAL NAMECHEMICAL STRUCTURENUMBER OF ANTI-PIG SAMPLES WITH STRONG BINDING (OD³ 0.5)
Anti-pig KidneyAnti-pig Heart
Human O plasmaHuman AB plasmaHuman O plasmaHuman AB plasma
B-likeLinear B type 2alphaGal(1­»3)ßGal(1­»4)ßGlcNAc-R4/44/41/22/2
Linear B type 6alphaGal(1­»3)ßGal(1­»4)ßGlc-R4/44/41/22/2
B disaccharidealphaGal(1­»3)ßGal-R4/44/41/22/2
alpha-D-GalactosidealphaGal-R3/44/4NDND
BB type 4alphaGal(1­»3)ßGal(1­»3)ßGalNAc-R³(1­»2)alphaFuc3/40/40/20/2
B type 5alphaGal(1­»3)ßGal(1­»3)ßGal-R³(1­»2)alphaFuc3/40/41/20/2
A-likeA disaccharidealphaGalNAc(1­»3)ßGal-R2/40/41/22/2
Linear A type 6alphaGalNAc(1­»3)ßGal(1­»4)ßGlc-R2/40/40/21/2
Forssman disaccharidealphaGalNAc(1­»3)ßGalNAc-R2/40/41/22/2
Forssman trisaccharidealphaGalNAc(1­»3)ßGalNAc(1­»3)alphaGal-R3/40/41/22/2
AA trisaccharidealphaGalNAc(1­»3)ßGal-R³(1­»2)alphaFuc3/40/40/20/2
A type 4alphaGalNAc(1­»3)ßGal(1­»3)ßGalNAc-R³(1­»2)alphaFuc2/40/41/20/2
A type 5alphaGalNAc(1­»3)ßGal(1­»3)ßGal-R³(1­»2)alphaFuc2/40/41/20/2
A type 6alphaGalNAc(1­»3)ßGal(1­»4)ßGlc-R³(1­»2)alphaFuc2/40/41/20/2
PP1alphaGal(1­»4)ßGal-R3/40/41/22/2
ParaglobosideßGal(1­»4)ßGlcNAc(1­»3)ßGal(1­»4)ßGlc-R0/40/41/20/2
ßGlcNAc-containingN-Acetyl-ß-D-glucosaminideßGlcNAc-R2/43/41/22/2
ßGlcNAc(1­»4)ßGlcNAc-R0/41/41/22/2
Rhamnose containingalpha-L-Rhamnosealpha-L-Rha-R0/42/41/22/2
alpha-L-Rha(1­»3)ßGlcNAc(1­»2)alpha-L-Rha-R2/42/41/22/2
OtheralphaGalNAc(1­»2)ßGal-R1/41/41/22/2
alphaGalNAc(1­»4)ßGal-R1/40/41/21/2
ßGalNAc(1­»2)ßGal-R0/42/41/22/2
ßGal(1­»3)ßGalNAc-R0/41/41/20/2
alphaGlc(1­»2)ßGal-R1/40/41/21/2
ßGlc(1­»2)ßGal-R1/40/41/21/2
ßGlc(1­»2)alphaMan-R1/41/41/21/2
alphaMan(1­»6)alphaMan-R0/41/41/22/2

R=O-(CH2)8-CO-NH-bovine serum albumin, Gal=galactose, Fuc=fucose, Rha=rhamnose, GlcNAc=N acetylglucosamine, GalNAc=N acetylgalactosamine, Man=mannose, ND=not done.

(Modified from DKC Cooper, et al. (24))

TABLE 2: Structure of the main carbohydrate epitopes exposed at the surface of human and porcine vascular endothelium.

HUMAN
ßGal1-4ßGlcNAca-R*
ABH-ßGal1-4ßGlcNAca-R
alphaNeuAc2-3ßGal1-4ßGalNAca-R
PIG
ßGal1-4ßGlcNAca-R
alphaGal1-3ßGal1-4ßGlcNAca-R
alphaNeuAc2-3ßGal1-4ßGlcNAca-R

*R are glycolipid or glycoprotein carrier molecules anchored in the cell membrane.
Only the epitopes underlined are different between the two species. (From R Oriol, et al. (25))

Figure 3: Three of the major carbohydrate structures that bind human antibodies eluted from pig heart, kidney and red blood cell stroma - alphaGal disaccharide (above), alphaGal trisaccharide type 2 (center), and alphaGal trisaccharide type 6 (below). X- Y = (CH2)8COOHCH3.

Humans are believed to develop anti-alphaGal antibodies during the first few weeks of life through exposure to certain microorganisms that colonize the gastrointestinal tract and which also express alphaGal structures on their cell membranes (33). At birth, anti-alphaGal IgG can frequently be detected in the plasma, presumably passively transferred from the mother, but not IgM (34). As it is predominantly IgM binding that initiates HAR, a pig organ transplanted into a neonatal baboon is not rejected hyperacutely, but does undergo delayed xenograft rejection over the next few days (34).

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