Yousuke Nishimura

Sequential mechanisms of cyclophosphamide-induced skin allograft tolerance including the intrathymic clonal deletion followed by late breakdown of the clonal deletion.

The cellular basis of the transplantation tolerance in a model system of BALB/c (Mls-1b) mice rendered cyclophosphamide (CP)-induced tolerant to DBA/2 (Mls-1a) skin allograft was investigated by assessing V beta 6+ T cells. From our results, three major mechanisms that are essential to the CP-induced skin allograft tolerance were sequentially elucidated. The first mechanism was destruction of donor-Ag-stimulated T cells in the periphery by CP treatment. The second mechanism was intrathymic clonal deletion of donor-reactive T cells, such as V beta 6+ T cells, correlating strongly with intrathymic mixed chimerism. The clonal deletion, however, was not always essential for the maintenance of the skin allografts, because DBA/2 skin survived even after the clonal deletion terminated and V beta 6+ T cells reappeared in the periphery of the recipient BALB/c mice. The third mechanism was generation of tolerogen-specific suppressor T cells, especially in the late stage of the tolerance. In contrast, the clonal anergy that is evidenced by the specific suppression of mixed lymphocyte reaction in the recipient BALB/c mice after injecting with DBA/2 spleen cells alone was not considered as a significant mechanism in prolonging skin allograft survival because such anergic mice showed accelerated rejection of the skin allografts. These results may suggest practical hierarchy of the mechanisms of CP-induced allograft tolerance.

Donor-specific prolongation of rat skin graft survival induced by rat-donor cells and cyclophosphamide under coadministration of monoclonal antibodies against T cell receptor alpha beta and natural killer cells in mice.

Because of the recent interest in human xenotransplantation, we investigated the possibility of inducing tolerance in a xenogeneic combination using cyclophosphamide (CP). Donor-specific prolongation of xenogeneic Fisher 344 (F344) rat skin graft survival for up to 60 days was induced in C57BL/6 (B6) mice by giving F344 bone marrow cells and spleen cells on day 0, CP on day 2, and monoclonal antibodies against murine TCR-alpha beta and NK1.1 on days--1 and 3. The inoculation of the xenogeneic cells brought accelerated repopulation of TCR-alpha beta+ T cells, even under the administration of anti-TCR-alpha beta mAb. The quick increase of the host TCR-alpha beta+ T cells caused by the xenogeneic cell injection was deeply suppressed by CP. Mixed lymphocyte reaction, CTL activity, and antibody production against donor F344 were profoundly suppressed for 50 days. Mixed xenogeneic chimerism was observed for 1 month after the inoculation of donor cells in the spleen and peripheral blood of the recipient B6 mice, but was never observed in the thymus. Moreover, when irradiated F344 cells were used in place of viable cells, chimerism was never detected and graft survival was only slightly prolonged. Clonal deletion of V beta 5- or V beta 11-bearing murine T cells was not observed on day 50 in the thymus or spleen of the recipient B6 mice. These results suggest that treatment with viable xenogeneic donor cells, CP, and mAbs against T and NK cells can induce a temporary peripheral mixed chimerism and donor-specific prolongation of xenogeneic skin graft survival. The destruction with CP of T and B cells that are xenoreactive and thus proliferating after antigen stimulation, followed by mechanism other than intrathymic clonal deletion, may be the mechanism of the hyporesponsiveness in the present system.

Evidence of a Vicious Cycle in Mitral Regurgitation With Prolapse Secondary Tethering Attributed to Primary Prolapse Demonstrated by Three-Dimensional Echocardiography Exacerbates Regurgitation

Background— In patients with mitral valve prolapse, nonprolapsed leaflets are often apically tented. We hypothesized that secondary left ventricular dilatation attributed to primary mitral regurgitation (MR) causes papillary muscle (PM) displacement, resulting in this leaflet tenting/tethering, and that secondary tethering further exacerbates malcoaptation and contributes to MR severity. Methods and Results— Three-dimensional transesophageal echocardiography was performed in 25 patients with posterior mitral leaflet prolapse with an intact anterior mitral leaflet (AML) and 20 controls. From 3D zoom data sets, 11 equidistant antero-posterior cut planes of the mitral valve at midsystole were obtained. In each plane, tenting area of nonprolapsed leaflet and prolapse area of prolapsed leaflet were measured. Prolapse/tenting volume of each region was obtained as the product of interslice distance and the prolapse/tenting area. AML tenting volume and whole leaflet prolapse/tenting volume were then obtained. The PM tethering distance between PM tips and anterior mitral annulus was measured from 3D full-volume data sets. The severity of MR was quantified by vena contracta area extracted from color 3D transesophageal echocardiography data sets. AML tenting volume was significantly larger in patients with posterior mitral leaflet prolapse compared with that in controls (1.2±0.5 versus 0.6±0.2 mL/m2; P<0.001). Multivariate regression analysis identified independent contribution to AML tenting volume from an increase in PM tethering distance. Multivariate regression analysis identified independent contributions to MR severity (vena contracta area) from both whole leaflet tenting volume (r=0.44; P<0.05) and prolapse volume (r=0.44; P<0.05). AML tenting volume decreased along with left ventricular volume and PM tethering distance postrepair (n=8; P<0.01). Conclusions— These results suggest that primary mitral valve prolapse with MR causes secondary mitral leaflet tethering with PM displacement by left ventricular dilatation, which further exacerbates valve leakage, constituting a vicious cycle that would suggest a pathophysiologic rationale for early surgical repair.

Generation of tumor-specific cytotoxic T lymphocytes in vivo by combined treatment with inactivated tumor cells and recombinant interleukin-2.

In order to search for a new therapy that would maximize the effect of interleukin-2 (IL-2) in evoking antitumor immunity in vivo, the therapeutic effect of a combination of mitomycin-C(MMC)-treated tumor cells and recombinant IL-2 was examined for its induction of antitumor activity against established melanoma metastasis. In C57BL/6 mice intravenously (i. v.) injected with B16 melanoma cells on day 0, the combined treatment with an intraperitoneal (i. p.) injection of MMC-treated melanoma cells on day 6 and 2500 U rIL-2 (twice daily) on days 7 and 8 markedly reduced the number of pulmonary metastases. This antitumor activity was more effective than that in untreated controls and mice that were injected with MMC-treated melanoma cells alone or rIL-2 alone. When the i. p. injection of MMC-treated tumor cells was replaced by other syngeneic tumor cells, antitumor activity against metastatic melanoma was not induced. The antitumor activity induced by this treatment increased in parallel with an increase in the dose of rIL-2 injected. In contrast, an i. p. injection of soluble tumor-specific antigens alone could induce only a marginal level of antitumor activity, and this activity was not augmented by subsequent i. p. injections of rIL-2. In vivo treatment with anti-CD8 monoclonal antibody (mAb), but not with anti-CD4 mAb or anti-asialo-GM1 antibody, abrogated the antitumor activity induced by this combined therapy. This suggests that the antitumor effect was dependent on CD8+ T cells. Lung-infiltrating lymphocytes from mice that had been i. v. injected with melanoma cells 11 days before and were treated with this combined therapy, showed melanoma-specific cytolytic activity. This combined therapy also showed significant antitumor activity against subcutaneously inoculated melanoma cells. These results demonstrate that the combined therapy of an i. p. injection of MMC-treated tumor cells and subsequent and consecutive i. p. administration of rIL-2 increases antitumor activity against established metastatic melanoma by generating tumor-specific CD8+ CTL in vivo.

Prolongation of Kidney Graft Survival by Cyclophosphamide-induced Tolerance in Rats

In this study, we have extended a cyclophosphamide (CP)-induced tolerance system to kidney transplantation in rats to examine whether or not we can overcome fully allogeneic (major histocompatibility complex plus minor histocompatibility) antigen barriers in organ transplantation. In the recipient Lewis (LEW, RT1(1)) rats that were primed intravenously with 4 x 10(8) spleen cells plus 2 x 10(8) bone marrow cells from Brown-Norway (BN, RT1n) rats and treated intraperitoneally with 100 mg./kg. of cyclophosphamide (CP) 2 days later, the survival of kidney allografts, but not skin allografts, from BN was prolonged as compared with that in the untreated LEW rats. Some of the kidney allografts survived for more than 100 days without further immunosuppressants. The tolerant state induced was tolerogen specific, and the suppression of tissue damage of the grafted kidney in such tolerant rats was also confirmed by the histopathological findings of the grafted kidney. These results indicate that considerable levels of tolerance can be induced, at least in organ transplantation, across fully allogeneic antigen barriers in rats by a CP-induced tolerance system. We believe that the present study is the first step in applying our CP-induced tolerance system using skin grafting in the murine model to clinical organ transplantation.

Anti-CD4 Monoclonal Antibody Reduces the Dose of Cyclophosphamide Required to Induce Tolerance to H-2 Haplotype Identical Skin Allografts in Mice

Abstract Cyclophosphamide (CP)-induced tolerance which consists of a single i.p. injection of 200 mg/kg CP 2 days after priming with 1 × 10 8 donor spleen cells (SC), leads to long-lasting donor-specific skin allograft tolerance in H-2 compatible, multiminor antigen incompatible, murine strain combinations. In this system, the optimal dose of CP has been suggested to be 200 mg/kg, however, such a dose of CP causes strong myelosuppression. In the present study, we therefore attempted to reduce the dose of CP by administering anti-CD4 monoclonal antibody (mAb) before donor cell priming in this toleranceinducing system. When C3H/He (C3H; H-2 k , Mls-1 b ) mice were injected i.p. with 200 ltg anti-CD4 mAb on day -3, 1 × 10 8 AKR/J (AKR; H-2 k , Mls-1 a ) SC plus 3 × 10 7 bone marrow cells (BMC) i.v. on day -2 and then 100 mg/kg CP i.p. on day 0, a long-lasting donor-specific skin allograft tolerance was established; furthermore, the decreases in the number of leukocytes and the concentration of hemoglobin (Hb) in the peripheral blood were all less in the C3H mice treated with this new combined protocol than in the C3H mice injected with 200 mg/kg CP following the previous protocol. In the periphery of these tolerant mice, the number of donor-reactive Vβ6 + CD4 + T cells decreased and mixed chimerism was observed on both days 14 and 80. On the other hand, in the mice injected with AKR SC, BMC plus 100 mg/kg CP without anti-CD4 mAb, the number of Vβ6 + CD4 + T cells decreased on day 14, and then recovered by day 80 when the mixed chimerism disappeared. These results therefore suggest that the combined use of anti-CD4 . mAb with CP can reduce the dose of CP without affecting the efficiency of inducing donor-specific tolerance, probably due to the enhancement of the destruction effect of donor-reactive T cells by CP.

Morphometric study of the human mitral annulus: guide for mitral valve surgery.

Background during mitral valve surgery, it is important for surgeons to understand the anatomy of the mitral valve annulus to prevent surgical complications. This study aimed to perform morphometry of the mitral annulus to facilitate secure suturing during ring annuloplasty or replacement of the mitral valve. Methods an anatomical study was carried out on 7 human hearts. We divided the mitral valve annulus into sections containing 8 different points. It was noted that the annulus was a complex structure which has fibrous continuity with the mitral leaflets, and with or without the aortic annulus. Results there was always a segment of the annular fibrous structure which was facing directly toward the left ventricular cavity. The length of the segment ranged from 1.0 to 3.4 mm. In terms of the size of the annulus, there were large variations within the subject and among the subjects. The shortest distance between the mitral annulus and left circumflex coronary artery was at the anterolateral commissure, and the length was 3.3 mm. Conclusion this study has improved understanding of the anatomy of the mitral annulus, which could help surgeons to avoid operative complications. Based on this study, several suggestions are made for placing sutures for mitral valve replacement and mitral ring annuloplasty.

The induction of skin xenograft tolerance in rat-to-mouse combination could be affected by DFR mediating cells and antibodies against rat bone marrow cells as well as NK cells in the cyclophosphamide-induced tolerance system

We investigated whether the prolongation of skin xenograft survival was obtained by a tolerance-inducing method using cyclophosphamide (CY), by which long-lasting skin allograft tolerance could be induced. The long-lasting skin allograft survival could be obtained in the recipient C3H/HeN (C3H) mice which were given 100 micrograms of anti-CD4 mAb on day -3, 1 x 10(8) spleen cells (SC) plus 3 x 10(7) bone marrow cells (BMC) derived from C57BL/6 (B6) mice on day -2,200 mg/kg CY on day 0, and which were grafted with allogeneic B6 skin on day 14. When the C3H mice were treated with anti-CD4 mAb, 1 x 10(8) s.c. plus 5 x 10(7) BMC derived from F344 rat and CY, the F344 skin grafts survived slightly longer (about 15 days) than those in untreated recipients (about 8.4 days). Such a prolongation of skin xenograft survival was considered donor-specific because rejection of 3rd party skin grafts from BN rats occurred significantly earlier than that of F344 skin grafts. In the recipient C3H mice treated with anti-CD4 mAb, F344 s.c. plus BMC and CY, mixed chimerism in the periphery was detected for a few days after CY administration, although intrathymic chimerism was not detected throughout this study. In these recipient C3H mice, cytotoxic T lymphocytes (CTL) against F344 antigens were completely abrogated through the delayed footpad reaction (DFR) remained at a low but significant level. Moreover, though antibody (Ab) activity against F344 s.c. was completely abrogated, neither Ab activity against F344 BMC, which seemed to have a background common to natural Ab activity, nor NK activity were abrogated by this treatment. These results suggested that DFR mediating cells directly mediated skin xenograft rejection in the recipient mice treated with anti-CD4 mAb, F344 cells, and CY. Such cells may interfere with establishment of mixed chimerism and long-lasting skin xenograft tolerance, presumably in cooperation with CY-resistant Ab activity and NK cells.