Oki Takeuchi

Pro-Carboxypeptidase R Cleaves Bradykinin following Activation

Arginine carboxypeptidase (CPR) is a labile enzyme present in human serum which is unrelated to carboxypeptidase N. In this study we demonstrate that CPR exists in a precursor form in plasma and can be converted to the active form by trypsin and presumable trypsin-like enzymes. The trypsin-generated active form can not only cleave a small synthetic substrate, hippuryl-L-arginine, but can remove terminal arginine from bradykinin.

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.

Complement fragment C4d deposition in peritubular capillaries in acute humoral rejection after ABO blood group-incompatible human kidney transplantation.

Background. Acute humoral rejection (AHR) is the most important risk factor for early graft loss in ABO-incompatible (ABO-i) kidney transplantation (RTx). The pathogenesis and diagnostic criteria for AHR after ABO-i RTx remain unclear. Complement fragment C4d deposition in peritubular capillaries (PTC), which is a sensitive indicator for activation of the classical complement pathway, was studied to establish the pathologic diagnostic indicator of AHR. Methods. Forty-four graft biopsy specimens from 19 patients with ABO-i living donors were analyzed within 90 days after RTx. Nineteen biopsy specimens with acute rejection after ABO-compatible (ABO-c) living-related RTx were used as controls. Diffuse and bright C4d deposition in PTC was considered significantly positive. Results. All of 8 recipients with AHR showed significantly positive C4d in PTC in the ABO-i group, but 9 of 11 recipients without AHR were negative. In the ABO-c RTx group, 16 of 19 recipients were negative for C4d in PTC. The prevalence of C4d in PTC was significantly higher in ABO-i RTx (P <0.05). Conclusions. C4d deposition is valuable as a specific and sensitive indicator for AHR, even of mild severity, in ABO-i RTx.

Potential value of high‐dose mizoribine as rescue therapy for ongoing acute humoral rejection

Mizoribine (MZ) inhibits the proliferation of lymphocytes selectively via inhibition of inosine monophosphate dehydrogenase, like mycophenolate mofetil (MMF). The clinical dosage of MZ (2–5 mg/kg) is much lower than that of MMF (20–60 mg/kg). The purpose of this study was to examine whether high‐dose MZ would be effective for treatment of acute humoral rejection. Renal transplantation was performed in a different pig strain combination. Group 1 (n = 2) received no treatment. Group 2 (n = 4) received cyclosporine microemulsion (6 mg/kg) and prednisolone (0.1 mg/kg) as baseline immunosuppression. Groups 3 (n = 4), 4 (n = 4) and 5 (n = 4) were additionally treated with MZ for rescue therapy, 30, 10 and 3 mg/kg, respectively, immediately after rejection was observed. All pigs developed acute vascular rejection between days 4 and 8. Complete reversal of acute rejection including reduction of elevated serum creatinine, suppression of anti‐donor antibody production and pathological finding, was obtained in 3/4 (group 3), 1/4 (group 4) and 0/4 (group 5). Rescue with high‐dose MZ (30 mg/kg) reversed ongoing acute humoral rejection. Such a high dose of MZ was tolerable for pigs. However, leukocytopenia was observed when MZ trough level was maintained over 10 μg/ml. Treatment with high‐dose MZ would be applicable to a clinical trial, if blood level is carefully monitored.

Comparative study of antibody removal before pig-to-baboon and human ABO-incompatible renal transplantation

THE MECHANISM of rejection following pig-to-baboon xenotransplantation (XenoTx) has been compared with that following ABO-incompatible allotransplantation (ABO-Tx), because both involve natural antibody (Ab) against carbohydrate epitopes. Recently, ABO-Tx has shown excellent outcomes following pretreatment with double filtration plasmapheresis (DFPP). We compared changes in anti-donor Ab and histopathology of the renal graft between ABO-Tx and XenoTx. Before renal transplantation (Tx), DFPP was performed on days 26, 24, 22, and 21 in ABO-Tx (n 5 25) and on days 22 and 0 (immediately before reperfusion) in XenoTx (n 5 4). Cyclosporine (or tacrolimus), steroid, and cyclophosphamide were administered to both patients and baboons. Baboons were divided into two groups according to the extent of Ab removal: XenoTx Group I, Ab removal to the same extent as in ABO-Tx (n 5 2); XenoTx Group II, nearly total removal of Ab (n 5 2). Anti-A/B and anti-pig IgM (IgG) Abs were measured by flow cytometry. Twenty-five patients underwent ABO-Tx. All grafts remain functioning, except in two cases where the grafts were lost from recurrence of the original disease after 6 months and from chronic rejection after 5 years, although 3 had reversible humoral rejection. In pig-to-baboon renal transplantation without DFPP pretreatment, hyperacute rejection (HAR) was observed 60 and 90 minutes after transplantation. In XenoTx Group I, no HAR was observed. Grafts were rejected on days 6 and 7, and showed coagulative necrosis caused by vascular rejection; biopsies on days 1, 4, and 5 showed only minimal changes. In XenoTx Group II, the baboons could not tolerate the extra DFPP and died 4 hours and 2 days after transplantation. Immunohistochemical study of the grafts showed IgM, IgG, and C3 binding to endothelial cells within 1 hour post-Tx in XenoTx Group I, and IgM and C3 binding as early as 1 hour post-Tx in XenoTx Group II, while no Ab binding was detected at any time (1 hour to 2 years) after ABO-Tx. The same extent of Ab removal in XenoTx Group I as in ABO-Tx inhibited HAR but could not prevent delayed vascular rejection. Even when anti-pig Ab was depleted by 98% (XenoTx Group II), the small amount of remaining Ab still had the ability of binding to the pig graft within 1 hour. For successful xenografting, we suggest that the concentration of xenoantigen epitopes expressed on pig grafts may need to be reduced by genetic engineering to the level of the ABO antigens on human kidneys.

A case report of a renal transplant recipient developing chronic glomerular rejection with a weak antibody against anti‐donor T‐cell, only detected by flow‐cytometry crossmatch

Abstract:  The pathogenesis of antibody‐mediated rejection has been investigated, but the precise mechanism of chronic glomerular rejection remains unclear. We have followed the clinicopathological course of a patient with pre‐existing anti‐donor antibody only detected by flow‐cytometry crossmatch for over 3 years. Glomerular endothelial injuries and peculiar glomerular lesions were noted in biopsy specimen of postoperative year 3; however, both typical chronic vascular rejection lesions and peritubular capillary multilayered lesions were not revealed. We consider that the presence of weak anti‐donor antibody leading early onset of acute humoral rejection played a role in the pathogenesis of early onset of chronic transplant glomerulopathy.