Shigeki Taniguchi

Delayed xenograft rejection of pig-to-baboon cardiac transplants after cobra venom factor therapy

BACKGROUND This study sought to (i) investigate the efficacy of cobra venom factor (CVF) in preventing hyperacute rejection (HAR) after pig-to-baboon heart transplantation, (ii) examine the effect of additional splenectomy (Spx) and pharmacologic immunosuppression (IS), and (iii) study delayed graft rejection when HAR is avoided by complement depletion. METHODS Eleven recipient baboons received heterotopic pig heart transplants. Three received either no therapy or IS (cyclosporine + methylprednisolone +/- cyclophosphamide +/- methotrexate) at clinically well-tolerated doses, with graft survival for only 40, 32, and 15 min, respectively. Two received CVF+/-Spx, which extended survival to 5 and 6 days, respectively. Six underwent Spx + CVF therapy + IS; graft survival was 3 hr (technical complication), 6 days (death from sepsis), 10, 12, and 22 days (vascular rejection), and <25 days (euthanized for viral pneumonia with a functioning graft that showed histopathologic features of vascular rejection). RESULTS Dense deposition of IgM and, to a lesser extent, IgG and IgA were seen on the endothelial cells within 1 hr of transplantation, but only trace levels of complement deposition were present in CVF-treated recipients. Within approximately 5-12 days, cardiac xenografts showed progressive infiltration by mononuclear cells, consisting primarily of activated macrophages producing tumor necrosis factor-alpha and small numbers of natural killer cells; T and B cells were absent. CONCLUSIONS We conclude that (i) CVF prevents HAR, (ii) the addition of Spx + IS delays rejection, but (iii) the early deposition of antibody leads to progressive graft injury, resulting in (iv) delayed vascular rejection. Our findings indicate that the features of delayed xenograft rejection described in small animal models also occur in the pig-to-baboon model, and that rejection may occur in a complement-independent manner from the effects of antibody and/or host macrophages.

In vivo immunoadsorption of antipig antibodies in baboons using a specific Gal(alpha)1-3Gal column.

The major role of anti-alphaGal antibodies in the hyperacute rejection of pig organs by humans and baboons has been clearly demonstrated. Spacered alpha-galactose disaccharide (Gal(alpha1)-3Gal) hapten was produced by chemical synthesis and covalently attached to a flexible, hydrophilic polymer (PAA), which in turn was covalently coupled to macroporous glass beads, forming an immunoadsorbent that is mechanically and chemically stable and can be sterilized. The extracorporeal immunoadsorption (EIA) of anti-alphaGal antibodies using this column has been investigated in vivo in 3 baboons. In Baboon 1 (which had hyperacutely rejected a pig heart transplant 4 months previously, was not splenectomized, and did not receive any pharmacologic immunosuppression) the levels of anti-alphaGal antibody and antipig IgM and IgG, as well as serum cytotoxicity, fell significantly after each of 3 EIAs but were not eliminated. Serum cytotoxicity, antipig immunoglobulin and anti-alphaGal antibody rose steeply within 24 hr of the final EIA, suggesting that the return of cytotoxicity was associated with anti-alphaGa1 antibody. In Baboons 2 and 3 (which were immunologically naive and splenectomized, and received triple drug immunosuppressive therapy) serum cytotoxicity was totally eliminated and anti-alphaGal antibody and antipig IgM and IgG levels were greatly reduced by courses of EIA. In Baboon 2, cytotoxicity and all antibody levels remained negligible for approximately one week after the final (fourth) daily EIA. In Baboon 3, cytotoxicity and antibody levels were maintained low by intermittent EIA (over a period of 13 days) for almost 3 weeks, although antipig IgM began to rebound 4 days after the final EIA. We conclude that, in an immunosuppressed, splenectomized baboon, repeated EIA using a specific alphaGal disaccharide column will reduce antipig and anti-alphaGal antibody levels and serum cytotoxicity significantly for several days. This reduction in cytotoxicity will almost certainly be sufficient to delay the hyperacute rejection of a transplanted pig organ, but further studies are required to investigate whether it will be sufficient to allow accommodation to develop.

Intravenous infusion of Galα1-3Gal oligosaccharides in baboons delays hyperacute rejection of porcine heart xenografts

BACKGROUND Hyperacute rejection (HAR) of pig-to-primate discordant xenografts is caused by the deposition of preexisting natural antibodies that recognize Galalpha1-3Gal (alphaGal)-terminating oligosaccharides on glycoproteins and glycolipids, followed by complement-mediated lysis of the grafts endothelium. In vitro, these natural xenoantibodies can be blocked by alphaGal-containing oligosaccharides. We undertook in vivo pig-to-baboon cardiac xenotransplantation experiments to evaluate free oligosaccharides as inhibitors of HAR. METHODS Initial 15-min intravenous infusions of alphaGal-oligosaccharides into baboons were used to measure pharmacokinetic parameters, and to assess the extent of neutralization of anti-alphaGal antibody activity. AlphaGal trisaccharide (Galalpha1-3Galbeta1-4GlcNAc) or pentasaccharide (Galalpha1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc ) was administered at 0.5 mmol/kg into baboons. Next, two baboons that received porcine heterotopic heart xenografts were continuously infused with alphaGal pentasaccharide for 4-5 hr, maintaining the serum oligosaccharide concentration in the millimolar range. RESULTS Pharmacokinetic analysis indicated that the oligosaccharides were rapidly cleared from the blood, with a serum half-life of 50 min. In the period during which blood oligosaccharide concentration was above 1 mM, as determined by high-pressure liquid chromatography, the serum cytotoxic activity against porcine cells was completely abolished. HAR of the xenograft was inhibited during the infusions, although there was some histological and immunohistological evidence of antibody-mediated injury on biopsies taken at the end of this period. CONCLUSIONS Intravenous alphaGal oligosaccharides, by inhibiting anti-alphaGal antibody activity, delay but do not abolish the onset of HAR.

Effects of hormonal supplements on the maintenance of cardiac function in potential donor patients after cerebral death.

It is well-known that cardiac function in cerebrally dead patients rapidly deteriorates, leaving the organ unfit for donation. This study investigated whether or not cardiac function in patients with cerebral death can be maintained in a desirable condition with hormonal supplementation. In studies of changes in hormones before and after cerebral death, insulin, glucagon, triiodothyronine, thyroxine, cortisol, vasopressin, epinephrine, and norepinephrine values were measured with a lapse of time after cerebral death. Among them, triiodothyronine and cortisol levels were markedly reduced after cerebral death; therefore, these two hormones were selected as hormonal supplements. The average period from the judgment of cerebral death to cardiac arrest was 4.3 days in 12 patients with no hormonal supplement (group I) and more than 11.5 days in 4 patients with hormonal supplement (group II). This period for patients in group II was significantly longer (p less than 0.05). In 2 of the group II patients the hormonal supplementation was discontinued at the familys request, and in the other 2 patients, it was discontinued because of proposed renal donation. Hemodynamic comparisons between the two groups showed that the mean arterial pressure and the left ventricular maximum dp/dt were significantly higher (p less than 0.01) as was the cardiac index (p less than 0.05) on the 3rd day after cerebral death in members of group II. Thereafter, in group II, an excellent hemodynamic state was maintained until hormonal supplements were discontinued. We conclude that the triiodothyronine and cortisol supplements were effective in the maintenance of cardiac function in patients after cerebral death.

Internal thoracic artery grafting for congenital coronary malformations

We report 2 patients with congenital coronary anomalies (atresia of left main coronary artery and anomalous origin of the left coronary artery from the pulmonary artery) successfully treated with single or double internal thoracic artery grafting. Because the internal thoracic artery has a potential for circumferential as well as longitudinal development, and because of the uncertainty of ultimate vein graft function, we believe that the internal thoracic artery is the best graft material for the treatment of congenital coronary malformations requiring bypass operation in children, adolescents, or even in adults.

Major carbohydrate epitopes in tissues of domestic and African wild animals of potential interest for xenotransplantation research

Oriol R, Candelier J‐J, Taniguchi S, Balanzino L, Peters L, Niekrasz M, Hammer C, Cooper DKC. Major carbohydrate epitopes in tissues of domestic and African wild animals of potential interest for xenotransplantation research. Xenotransplantation 1999; 6: 79‐89. ©Munksgaard, Copenhagen.

Lower extremity ischemia secondary to internal thpraciccoronary artery bypass grafting

Vascular complications associated with the use of the internal thoracic artery as a conduit for coronary artery bypass are infrequent. However, acute limb-threatening ischemia can occur after the use of the internal thoracic artery for myocardial revascularization when this vessel forms an important collateral to an obstructed aortoiliac artery system. Although this is a rare complication, the consequences are devastating. Due to the risk of peripheral vascular insufficiency, we now perform simultaneous revascularization of the myocardium and the lower extremity when an internal thoracic artery that appears to be a major collateral to the ipsilateral aortoiliac system is used.

Epithelial regeneration and preservation of tracheal cartilage after tracheal replacement with cryopreserved allograft in the rat

OBJECTIVE We investigated the origin of the epithelium in transplanted cryopreserved tracheal allografts in rats and tried to clarify the mechanism by which immunogenicity is reduced in this procedure. METHODS Tracheal transplantation was performed with PVG rats (allele at the RT1 locus: c) used as donors and ACI rats (allele at the RT1 locus: a) as recipients. After resection of a 5-ring segment of the cervical trachea of an ACI rat, the trachea was reconstructed with the cryopreserved tracheal segment of a PVG rat (n = 6). No immunosuppressive agents or steroids were given. Histologic changes were determined and immunohistochemical staining was performed to investigate major histocompatibility complex class I antigens of the transplanted tracheal segment. RESULTS Two months after tracheal transplantation, 6 surviving ACI rats were killed. Histologically, the epithelium and tracheal cartilage of the transplanted cryopreserved segment displayed normal structure. Immunohistochemical staining showed that the major histocompatibility complex class I antigen of the ACI rat was expressed in the epithelium of the transplanted segment and that the class I antigen of the PVG rat was expressed in the cartilage of the transplanted segment. CONCLUSIONS After transplantation of the cryopreserved trachea, the epithelium of the transplanted cryopreserved segment originated from the recipient epithelium whereas the cartilage retained the structure of the donor trachea. We hypothesize that transplantation of a cryopreserved trachea leads to the growth of the recipients epithelium over the donor trachea, thereby reducing the antigenicity of the transplant.

Cobra venom factor stimulates anti-alpha-galactose antibody production in baboons. Implications for pig-to-human xenotransplantation.

Cobra venom factor (CVF) depletes complement and may therefore be of use in preventing the hyperacute rejection that follows discordant organ xenotransplantation. In two baboons studied, the intramuscular injection of CVF (0.25 mg/kg) was followed by a marked reduction in serum C3 and CH50, and serum cytotoxicity to pig kidney (PK15) cells. There was, however, a very rapid rise in the level of anti-alpha-galactose (alpha Gal) antibody, and a slower rise in anti-CVF antibody. A second intramuscular injection of CVF on day 14 was ineffective in reducing C3, CH50, and serum cytotoxicity. The major oligosaccharide of CVF is known to contain alpha Gal residues, which we suggest stimulate the major increase in anti-alpha Gal antibody level seen in the present study. In the clinical situation, this might lead to an increased immune response to a concomitantly transplanted pig organ.

Glycans derived from porcine stomach mucin are effective inhibitors of natural anti-alpha-galactosyl antibodies in vitro and after intravenous infusion in baboons.

The current shortage of donor organs has stimulated investigation of pig-to-human xenotransplantation as a practical alternative to allotransplantation. However, a major obstacle to this xenotransplantation is hyperacute rejection, which is believed to be initiated by the interaction of natural anti-alpha-galactosyl (alphaGal) antibodies with alphaGal epitopes on pig vascular endothelium. Previously, we reported that neutral oligosaccharides derived from porcine stomach mucin (PSM) are effective inhibitors of human anti-alphaGal IgG in vitro. We now report that O-glycans derived from PSM by beta-elimination (PSMO) reduce the cytotoxicity of both baboon and human sera to pig kidney (PK15) cells in vitro. Crude PSM had some inhibitory effect in vitro, but PSMO were more than 100 times more potent. Moreover, 1 microg/ml of beta-eliminated PSMO that bound to an immunoaffinity column of anti-alphaGal antibodies were four times more efficient than total PSMO in protecting PK15 cells from the cytotoxic effect of baboon or human sera. Blood recovered from baboons after intravenous infusion of PMSO also showed significant protection of PK15 cells. We conclude that PSMO eluted from an anti-alphaGal immunoaffinity column demonstrate potent inhibitory effects against baboon and human serum cytotoxicity to PK15 cells in vitro and when administered intravenously. PSM may provide a cheap and readily available source of glycans that will be of therapeutic value in the prevention of hyperacute rejection.