Itsuo Yokoyama

Are N-glycolylneuraminic acid (Hanganutziu-Deicher) antigens important in pig-to-human xenotransplantation?

Abstract:  Background:  N‐glycolylneuraminic acid (NeuGc) epitopes, so called Hanganutziu–Deicher (HD) antigens, which are widely expressed on endothelial cells of all mammals except humans, are considered to be potential targets for natural and elicited anti‐nonGalα1‐3 Gal (Gal) antibodies in humans. We previously reported that anti‐NeuGc antibodies were not detected in healthy humans by enzyme‐linked immunosorbent assay (ELISA) using NeuGc‐GM3‐coated plates, and no antibody production was observed in patients with a history of exposure to pig cells. However, a recent paper has revealed that (i) anti‐NeuGc antibodies to porcine red blood cells (PRBC) are detectable in most healthy humans, and (ii) the majority of anti‐nonGal antibodies are specific for NeuGc epitopes. The purpose of this study was to reassess whether NeuGc is important as an immunogenic nonGal epitope.

Introduction of α(1,2)‐fucosyltransferase and its effect on a‐Gal epitopes in transgenic pig

Abstract: Hyperacute rejection (from pig to human) is thought to result from activation of complement initiated by the binding of host natural antibodies to α‐galactosyl (α‐Gal) epitopes of donor endothelial cells. However, α‐Gal epitope shares a common precursor with H antigen in humans. This means that H antigens as well as α‐Gal epitopes are synthesized in a competitive manner by different enzymes. We thought that it would be possible to convert α‐Gal epitopes into H antigens by introducing cDNA of α(1,2)‐fucosyltransferase (α1–2FT) into porcine cells, and so, pig embryos were microinjected with αl‐2FT cDNA. Transgenic pigs that carried α1–2FT were thus established. Cytotoxicity of fibrocytes derived from skin of transgenic pig was measured by 51Cr release assay, which showed that H antigen‐expressing cells were significantly resistant to a challenge with human sera. These experiments indicate that our method provides a new strategy which contributes to a successful discordant xenotransplantation.

Amlodipine, but not MDR1 polymorphisms, alters the pharmacokinetics of cyclosporine A in Japanese kidney transplant recipients

Background. Cyclosporine A (CsA) is a critical immunosuppressive drug with a narrow therapeutic range and wide interindividual variation in its pharmacokinetics. Many factors, including P-glycoprotein (PGP), influence the oral bioavailability and interpatient variability of CsA. A number of polymorphisms have been identified in the human MDR1 gene, and some of them have been found to be associated with an altered expression of PGP. We have investigated the role of these polymorphisms in CsA absorption from kidney transplant recipients. In addition, we also investigated the effect of amlodipine on CsA absorption. Methods. The area under the time-concentration curve from 0 to 2 hr (AUC0–2) estimated by the trapezoidal rule was used for the evaluation of extent of CsA absorption. The genotypes were identified by a polymerase chain reaction, restriction fragment length polymorphism analysis. Results. No association was found between polymorphisms in the MDR1 and CsA AUC0–2/dose/kg. In contrast, the combination of amlodipine significantly increased CsA AUC0–2/dose/kg (706.2 &mgr;g·hr/L to 819.2 &mgr;g·hr/L, P <0.05). Furthermore, we attempted to compare MDR1 polymorphisms and the absorption of CsA again without patients receiving amlodipine, but there was still no significant difference. Conclusions. There is no relationship between polymorphisms for MDR1 and CsA absorption, suggesting polymorphisms for MDR1 cannot account for the interpatient variability of CsA. Amlodipine, which is the substrate of PGP, significantly increased CsA absorption. These results indicate that PGP plays a significant role in CsA absorption, but its polymorphisms could not influence the CsA absorption.

Significance of histochemical expression of Hanganutziu-Deicher antigens in pig, baboon and human tissues

THERE is increasing interest in the clinical application of xenotransplantation. This interest has been sparked predominantly by the severe shortage of donors. In addition, xenotransplantation might be useful for avoiding the recurrence of viral disease in the transplanted organ. The humoral barrier leading to the immune response is well known for clinical practice of xenotransplantation; that is, xenoreactive natural antibodies to the graft endothelium. Human xenoreactive natural antibodies are directed predominantly against Gala1–3Gal (a-Gal antigen), a saccharide expressed in cells of lower mammals and New World monkeys, but not in cells of Old World monkeys, apes, or humans. Anti–a-Gal antibody plays a major role in hyperacute rejection (HAR) after pig-to-human xenotransplantation. Antibody binding to this sugar, expressed as a modification of endothelial cell membrane glycoproteins and glycolipids, activates complement by the classical pathway. Many investigators believe that pig-to-human xenotransplantation will become a common clinical application and that the pig-to-baboon model represents a good experimental design for study. Hanganutziu–Deicher (HD) antibodies have been known as antibodies detectable in patients with serum sickness. In 1924, the Romanian pathologist Hanganutziu noticed that a serum taken from a patient who received antitetanus horse serum abnormally and strongly agglutinated sheep red blood cells. This agglutination was not sheep-specific, as red blood cells from the horse, guinea-pig, rabbit, calf, and pig expressed similar titers. In 1926, Deicher replicated these results. The antibodies reacting broadly with heterologous antigens were called heterophilic antibodies. HD antibodies are heterophilic antibodies as Forssman antibodies and Paul–Bunnell antibodies. Higashi et al and Merrick et al found that antigens recognized by HD antibodies are gangliosides containing N-glucolylneuramic acid (NeuGc). Gangliosides are sialic acid–containing glycophospholipids present on the surface of all mammalian cells. Sialic acids are a group of nine carbohydrates of two main types: N-acetylneuraminic acid (NeuAc) and N-glucolylneuramic acid. NeuAc is expressed ubiquitously, whereas, NeuGc, the hydroxylated form of NeuAc, is not present in birds or humans. HD antigen is widely distributed in mammalian species with the exception of humans. Some antipig antibodies are directed against epitopes other than the a-Gal antigen. Grafting animals into human patients should be followed by an increase in HD antibodies. Therefore, it is possible that non–a-Gal antibody can cause rejection, even if a-Gal epitopes are successfully removed from donor pig organs. The purpose of this study is to elucidate the significance of HD antigen as non–a-Gal epitopes in clinical xenotransplantation.

Changes in intracellular Ca2+ concentrations related to PDT-induced apoptosis in photosensitized human cancer cells

For photodynamic therapy (PDT), human squamous cell carcinoma cells (HSC-2) were treated with 3 microg/ml of photofrin 24 h prior to irradiating the cultures with the excimer dye laser at a dose of 2 J/cm2. Extensive DNA fragmentation was recognized within 2 h of PDT. The proportion of cells with DNA fragmentation on flow cytometric analysis was significantly increased to 44 and 78% at 1 and 2 h after PDT, respectively, compared to control groups. Confocal laser scanning microscopy revealed a slight change in the intracellular Ca2+ concentration occurring at 30 min after PDT and a subsequent marked increase in the Ca2+ concentration 1-2 h after PDT, but no change was observed in the cells exposed to laser irradiation alone and to photofrin alone and in the cells immediately after PDT. These findings suggest that an increase in the intracellular Ca2+ concentration may play an important role in the induction of PDT-induced apoptosis.

Lack of antibody production against Hanganutziu-Deicher (H-D) antigens with N-glycolylneuraminic acid in patients with porcine exposure history.

Abstract: The significance of non‐αgalactosyl antigens remains unclear in pig‐to‐primate xenotransplantation. Hanganutziu‐Deicher (H‐D) antigens with terminal N‐glycolylneuraminic acid (NeuGc) are widely expressed on endothelial cells of mammalian species, with the exception of humans. As baboons and monkeys also express H‐D antigens, a pig‐to‐non‐human primate experimental model cannot resolve the question of whether H‐D antigens can elicit a potent humoral response in human recipients. The purpose of this study was to elucidate the clinical significance of H‐D antigens by examining the sera from patients who have been previously exposed to porcine tissue.

Comparative study of efficacy of removal of anti‐ABO and anti‐gal antibodies by double filtration plasmapheresis

Abstract: Successful clinical ABO‐incompatible renal transplantation has been achieved by the removal of anti‐A or anti‐B antibodies using double filtration plasmapheresis (DFPP). We have compared changes in the levels of anti‐donor antibodies and the histopathology of the renal grafts following human ABO‐incompatible allotransplantation and pig‐to‐baboon xenotransplantation using pretransplantDFPP.

Porcine α-1,3-galactosyltransferase: full length cDNA cloning, genomic organization, and analysis of splicing variants

Full length cDNA and genomic DNA of porcine α-1,3-galactosyltransferase were isolated, and their structures were analysed. The coding region was encoded by six exons as in the mouse, and the length of each exon was conserved between the two species. The porcine exons were designated Exon 4–9, since in the mouse coding exons started from Exon 4. Introns tended to be longer in the porcine gene; the distance from Exon 4 to the 3′-end of Exon 9 was 24 kb, while this region was 18 kb in the mouse gene. The cDNA structure was extended from the previous data to the 3′-end and to the 5′ side of the cDNA. In addition to a cDNA clone with all coding exons, clones lacking parts of these exons were isolated and their structures were determined. One variant lacked Exon 5; the second, Exons 5 and 6; and the third, Exons 5, 6 and 7. The last variant was not found in the mouse, and cDNA transfection of this variant yielded scarcely any enzymatic activity using asialo α1-acid glycoprotein as a substrate, and decreased activity using N-acetyllactosamine as a substrate.

Liberation of vasoactive substances and its prevention with thromboxane A2 synthase inhibitor in pig liver transplantation

There are multiple causes of liver graft nonfunction in the early post-transplant period. Since a severe microcirculatory disturbance based on ischemia-reperfusion liver injury is considered to be the main underlying pathophysiology, it is suspected that various vasoactive substances are liberated after reperfusion of the graft. In order to investigate this matter, we conducted an experimental study with pig liver allotransplantation. Two groups of animals received donor grafts with or without thromboxane synthase inhibitor (sodium ozagrel), 1.25 mg/kg body weight intravenously, given at the time of liver harvesting. All of the recipient animals in the treatment group (n=10) survived longer than 7 days whereas three of ten animals in the control group died within 7 days. Serum lactate dehydrogenase (LDH) in the recipient serum at 1 h after reperfusion was significantly lower in the treatment group (915.1±167.3 U/l) than in the control group (1264.4±134.7 U/l). Serum thromboxane B2 (2261.7±1055.7 pg/ml) and endothelin-1 (6.3±2.2 pg/ml) after reperfusion in the treatment group were significantly lower than those in the control group (4220.0±1711.0 pg/ml and 11.2±3.1 pg/ml, respectively). Although serum angiotensin II after reperfusion tended to be lower in the treatment group than in the controls serum renin activity was less than 3 ng/ml in both groups of animals. There were no differences in the plasma endotoxin levels between the two groups. We conclude that the administration of sodium ozagrel to the donor animals provided better graft function in recipients than no such treatment. We speculate that the inhibition of thromboxane A2 production suppresses the liberation of other vasoconstrictive substances, preventing microcirculatory disturbance and, thereby, contributing to improved graft function after liver transplantation.

Prevention of experimental hepatic metastasis with thromboxane synthase inhibitor.

To investigate the effectiveness of thromboxane (Tx) synthase inhibitor in the prevention of experimental hepatic metastasis, an in vivo study was designed. Hepatic metastasis was brought about by injection of 1×105 cells of colon 38 tumor into the portal vein of C57 B1/65 mice. Seven groups (n=16 in each group) received different treatments: with TxA2 synthase inhibitor (sodium ozagrel), 5, 10 or 15 mg/kg BW before tumor inoculation, and daily for the following 3 days, (groups A, B and C, respectively); with acetyl salicylic acid (aspirin), 1.0, 1.5 or 2.0 mg/kg BW (groups C, D, and E, respectively); a control group, inoculated with vehicle only. Serum TxB2, a stable metabolite of TxA2, and prostaglandin F1α were measured. Labeling index for tumor proliferation by bromodeoxy-uridine radioimmuno-assay was also studied. Incidence of metastasis in groups A (60.5%), B (49.5%), C (43.0%), D (80.5%), E (66.0%) and F (58.4%) was less than that in the control group (100%). Tumor size, number or labeling index did not differ among the groups. Serum TxB2 (pg/ml) levels were significantly lower in all of the groups than in the control. Serum PGF1α levels in the groups with aspirin were lower than those in sodium ozagrel. Tx synthase inhibitor is effective in the prevention of experimental hepatic metastasis when it is given before and immediately after tumor inoculation. As Tx synthase inhibitor leaves metabolic pathway to PGI2 productionintact, it is more effective in the prevention of metastasis than aspirin since aspirin inhibits both thromboxane and PGI2.