Seizoh Nakata

The mechanism of discordant xenograft rejection.

The mechanism of discordant xenograft rejection using the guinea pig-to-rat heart graft model was studied. In this model, we found that (A) Rejection occurred rapidly, in 17.5 +/- 8.3 min (mean +/- SD) (n = 8). (B) The graft survived longer when the recipient rat was pretreated with cobra venom facter (CVF)

EFFECTS OF TRANSFECTED COMPLEMENT REGULATORY PROTEINS, MCP, DAF, AND MCP/DAF HYBRID, ON COMPLEMENT-MEDIATED SWINE ENDOTHELIAL CELL LYSIS

We established several swine endothelial cell (SEC) lines, expressing human MCP (CD46), DAF (CD55), and MCP/DAF hybrid by transfection of cDNA, and assessed the function of these transfectant molecules on complement-mediated cell lysis as an in vitro hyper-acute rejection model of swine to human discordant xenograft. Discordant organ xenografts are hyper-acutely rejected by complement activation. Amelioration of complement-mediated lysis by these transfectant molecules was tested in each SEC line by lactate dehydrogenase assay. Naive swine endothelial cells were markedly damaged by human complement mainly via the classical pathway, activating only minimally the alternative pathway of human complement. Both MCP and DAF protected SEC from human complement attack in parallel with the expression density, with DAF being more effective than MCP. The MCP/DAF hybrid was more effective than MCP alone, and as effective as DAF in this system. The results suggest that the transfection of DAF or the MCP/DAF hybrid cDNA into organs to be transplanted could protect against hyperacute rejection.

Prolonging discordant xenograft survival with anticomplement reagents K76COOH and FUT175.

The guinea pig heart, when transplanted into the rat heterotopically, is rejected within 30 min via activation of the alternative complement pathway. Natural antibody does not contribute to rejection. This xenotransplantation model was used to assess the effect of anti-complement reagents on discordant xenograft survival. In vivo administration of K76COOH (K76) to rats induced only slight suppression of factors B and D and a marked decrease of C3, leading to the depression of ACH50 (reflecting the potency of the alternative pathway). On the other hand, FUT175 (FUT) reduced C3 activity by about 80% and inhibited factor B activity nearly 100% < 1 hr after the administration, but inhibited factor D activity only marginally. FUT abrogated ACH50 for > 6 hr. Of note, the xenograft beating time was prolonged approximately 3 times by FUT but not by K76, suggesting that direct inhibition of plasma serine protease factor B results in the complete suppression of ACH50 and graft survival. The administration of both K76 and FUT resulted in the longest graft survival, but the effects of these reagents were abolished by additional antigraft antibody. Anticomplement reagents that block factor B and C3 are therefore effective for prolongation of discordant xenograft survival when the graft rejection is associated with the complement alternative pathway.

Evidence That Graft Coronary Arteriosclerosis Begins In The Early Phase After Transplantation And Progresses Without Chronic Immunoreaction: Histopathological Analysis Using a Retransplantation Model

The histopathological features of chronic rejection and its initiation were assessed using rat heterotopic heart transplantation and retransplantation models. Fully allogeneic or minor, non-MHC antigen-mismatch heart grafts transplanted into recipient rats treated with a short course of FK506 showed long-term survival but developed graft atherosclerosis after 40 days posttransplantation. Retransplantation of allografts back into the original donor strain did not prevent graft atherosclerosis if the grafts had resided in the primary recipient for up to 5 days; residence in the primary allogeneic recipient for less than 4 days did not result in graft atherosclerosis in the secondary recipient. Short-course administration of FK506 did not affect the production of these changes. Graft coronary arteriosclerosis begins between 3 and 5 days posttransplantation and progresses without continuous allogeneic immunological drive. The present findings will provide a new means by which to approach the analysis of development of chronic allograft rejection.

Test for ability of decay-accelerating factor (DAF, CD55) and CD59 to alleviate complement-mediated damage of xeno-erythrocytes.

We investigated the susceptibility to human complement (C) of xeno‐erythrocytes into which phosphatidylinositol (PI)‐anchored human C regulatory protein, decay‐accelerating factor (DAF) or CD59 had been incorporated. Erythrocytes of sheep (Esh), swine (Esw), dog (Edg), and guinea pig (Egp), unsensitized with human natural antibody (Ab), were used as xeno‐target. C‐mediated lysis of erythrocytes (E) was induced in both classical and alternative pathways in parallel with the density of the sensitized Ab, except for Egp. The efficacy of DAF/CD59‐mediated protection of the xeno E from human C, however, differed among these E species. In both classical and alternative pathways, Esh or Esw, which are non‐activator surfaces, were protected by the incorporated DAF or CD59, DAF being more effective than CD59. On the other hand, CD59 was more effective than DAF in both pathways in protection of Egp, which is an alternative pathway activator.

Donor-specific tolerance by perioperative intrathymic injection of bone marrow cells in the rat cardiac allograft model : Use of FK506 can shorten the necessary duration of pretransplant intrathymic conditioning

BACKGROUND Many strategies of tolerance induction by intrathymic (IT) injection of donor alloantigens have been reported to date; however, the timing of IT injection is usually 1-3 weeks before transplantation. METHODS To apply IT injection to cadaveric organ transplantation, 1 x 10(8) fully allogeneic bone marrow cells (BMC) of Buffalo (BUF; RT1b) rats were intrathymically injected into Wistar Furth (WF; RT1u) rats at the time of BUF cardiac allografting with short-course therapy of antilymphocyte serum (ALS) and FK506 in our experimental model. RESULTS Allogeneic IT injection of BUF BMC with ALS and FK506 indefinitely prolonged graft survival (mean survival time > 210 days) in all WF rats. On day 130 after grafting, tolerant WF rats accepted donor BUF skin grafts (> 120 days) but not third-party Lewis skin grafts. In control groups, syngeneic IT injection of WF BMC or intravenous injection of donor BUF BMC in combination with ALS/FK506 therapy failed to induce tolerance. In vivo testing was performed during induction (1 month) or during maintenance (6 months of tolerance. In the mixed lymphocyte reaction (MLR), spleen T cells of tolerant rats at 1 month after grafting displayed hyporesponsiveness after stimulation with donor cells. The addition of interleukin (IL)-2 to MLR culture did not restore T-cell responsiveness. Tolerant rats had a significantly decreased frequency of T cytotoxic cell precursors (fTcp) of 1:4,926, and frequency of IL-2-producing T helper cell precursors (fThp) of 1:23,925, compared with naive rats (1: 2,158 and 1:4,266, respectively). By 6 months after grafting, however, the anti-donor MLR proliferative responses of tolerant rats had been restored to the levels of naive splenic T cells. These tolerant rats displayed restoration of the (fTcp) of 1:2,842 and of the (fThp) of 1:5,630, which were comparable frequencies of naive rats. Suppressor T cells did not contribute in this model. In cardiac grafts of tolerant rats induced by IT injection, expression of both Th1 (interferon-gamma and IL-2) and Th2 (IL-4 and IL-10) cytokines was detected in the early phase; thereafter, expression was completely inhibited, except for interferon-gamma in the chronic phase. CONCLUSIONS Perfect donor-specific tolerance was obtained by IT injection of donor BMC at the time of transplantation, while alloimmune responses were maintained at levels similar to those of naive rats.

Canine heart-lung transplantation after 24-hour hypothermic preservation.

We compared the efficacy of Belzer UW solution (UWS) with modified Collins solution (MCS) in a 24 h canine heterotopic heart-lung transplantation model. Nine pairs of mongrel dogs were divided into 2 groups: MCS group (n = 5) and UWS group (n = 4). The donor heart was arrested with cardioplegia. The heart and lung were flushed with MCS or UWS, excised en bloc and immersed in 4 degrees C MCS or UWS for 24 h. Graft function was adequate in 1 of 5 grafts in the MCS group and in all 4 grafts in the UWS group (P less than 0.05). In the UWS group, the prepreservation and posttransplantation cardiac output was 71.2 +/- 21.6 and 96.4 +/- 44.4 ml/min per kg, the PaO2 was 80.8 +/- 12.9 and 74.1 +/- 2.7 mmHg, and the PaCO2 was 25.6 +/- 5.5 and 35.9 +/- 13.3 mmHg, respectively. No difference was significant. In the UWS group, the pulmonary vascular resistance increased significantly to 13.8 +/- 3.7 from 8.8 +/- 5.2 Wood units (P less than 0.05). Post-transplantation myocardial water content in the UWS group (78.6 +/- 2.0) was less than in the MCS group (81.1 +/- 1.4, P less than 0.05). The wet/dry ratio of the lung increased significantly to 8.3 +/- 1.6 from 4.4 +/- 0.6 in the UWS group (P less than 0.01) and to 10.3 +/- 0.8 from 4.1 +/- 0.1 in the MCS group (P less than 0.01), but was less in the UWS group (P less than 0.05). These results suggest that UWS is more effective than MCS in 24 h heart-lung preservation, but does not prevent pulmonary oedema adequately.

Evaluation of an Automatic Fogging Disinfection Unit

A new fogging disinfection method was evaluated as a means of disinfecting ward rooms and operating theaters. A temporary room was established where the disinfection effect of fogging was examined. Based on the results, an automatic fogging disinfection unit was developed. This unit was then used in the disinfection of operating theaters, where its safety and effectiveness were examined.To evaluate the results of disinfection, bacterial culture tests were performed on the floor, walls and other areas of the operating theater, and the number of colony forming units was used as an index of effectiveness. Benzalkonium chloride, alkyldiaminoethylglycine, sodium hypochlorite, glutaral and acidic electrolytic water were used for the operating theaters. The average disinfection effect was 90% or better for all disinfectants, except acidic electrolytic water.The newly developed automatic fogging disinfection unit enables safe and effective disinfection, and may be suitable for disinfecting ward rooms and operating theaters.

Effect of aortocoronary bypass surgery on coronary circulation and myocardial metabolism during atrial pacing

SummaryEleven patients with coronary heart disease, in whom at least one of several bypass grafts to the left coronary artery was patent, were selected for the study. The hemodynamics, coronary sinus blood flow, myocardial oxygen consumption, and myocardial lactate metabolism were evaluated at rest and during atrial pacing stress test before and after surgery.There were no significant improvements in the cardiac index, pulmonary arterial end-diastolic pressure, and left ventricular ejection fraction after aortocoronary bypass surgery. However, significant improvement of coronary sinus blood flow, myocardial oxygen consumption, and myocardial lactate extraction and consumption were found during postoperative atrial pacing compared with the preoperative findings.These results suggest that successful bypass grafting may improve myocardial lactate metabolism in ischemic lesions and contribute to the postoperative relief of angina.