Tatsushi Onzuka

Bilateral Axillary Arterial Perfusion in Surgery on Thoracic Aorta

Bilateral axillary arterial cannulation for selective cerebral perfusion might minimize cerebral embolic complications during surgery on the ascending aorta and aortic arch. From March 2002 through February 2004, bilateral axillary arterial perfusion was applied in 12 consecutive patients (mean age, 61.3 years). Operative procedures were total arch replacement in 8 patients, hemiarch replacement in 1, and ascending aorta replacement in 3. Antegrade selective cerebral perfusion was established through vascular grafts anastomosed to the bilateral axillary arteries and a perfusion catheter placed directly into the left carotid artery. Bilateral axillary arterial perfusion through the grafts was successful in all patients. There were no early or late deaths and no incidence of neurologic deficit. There were no complications related to cannulation of the axillary arteries. Bleeding, temporary renal failure, acute respiratory distress syndrome, and graft infection occurred in one patient each; all recovered from these complications. Bilateral axillary arterial perfusion is feasible and effective for brain protection during surgery on the ascending aorta and aortic arch.

Role of the Cytokine Profiles Produced by Invariant Natural Killer T Cells in the Initial Phase of Cyclophosphamide-Induced Tolerance

Background. Cyclophosphamide (CP)-induced tolerance is a mixed chimerism-based tolerance induction protocol. Recently, we reported that invariant natural killer T (iNKT) cells were essential for the tolerance induction in this system. In this study, we evaluated the roles of the cytokines produced by iNKT cells. Methods. DBA/2 (H-2d) mice and BALB/c (H-2d) wild-type (WT) or iNKT knock out (KO) mice were used as donors and recipients. WT recipients received three doses (days −7, −4, −1 or 35, 38, 41) or a single dose (day −1 or 0) of &agr;-galactosylceramide (GC) in conjunction with our conditioning regimen that consisted of 108 donor spleen cells on day 0 and 200 mg/kg CP on day 2. To investigate the iNKT cell function, iNKT KO recipients were reconstituted with cytokine (interferon-&ggr;, interleukin [IL]-4, or IL-10) KO iNKT cells and received donor spleen cells and CP. Results. Mixed chimerism was observed in WT recipients, but was reduced in iNKT KO recipients. However, mixed chimerism was absent in WT recipients given GC on days −7, −4, −1, but not in WT recipients given GC on day 35, 38, 41. Donor skin grafts were chronically rejected when mixed chimerism was diminished. Skin grafts were accepted in iNKT KO recipients reconstituted with iNKT cells from interferon-&ggr;, IL-4, or IL-10 KO mice and receiving our conditioning regimen. Conclusions. Invariant NKT cells were required in the initial phase of the induction of chimerism. Our results indicated that known major cytokines produced by iNKT cells were dispensable for the regulatory function of iNKT cells.

The Immunoregulatory Roles of Natural Killer T Cells in Cyclophosphamide-induced Tolerance

Background. Recent studies have indicated that natural killer T (NKT) cells are essential for the establishment of transplantation tolerance. In the present study, we have elucidated the role of recipient and donor NKT cells in cyclophosphamide (CP)-induced tolerance. Method. DBA/2 (DBA; H-2d) mice were used as donors and BALB/c (BALB; H-2d) wild-type (WT) or V&agr;14 NKT-knockout (KO, BALB/c background) mice were used as recipients. Recipients were treated with CP-induced tolerance regimen, which consists of donor spleen cells (SC) on day 0 and CP on day 2. In some experiments, NKT KO mice, which received NKT cells from either WT, inferon-&ggr; KO, or interleukin-4 KO mice, were treated with tolerant regimen. To deplete Ly49 inhibitory receptors on NKT cells in the recipient mice, anti-Ly49 monoclonal antibody cocktails were injected on day −1 when indicated. Results. Donor skin graft was permanently accepted in recipient BALB WT mice with induction of donor mixed chimerism. On the contrary, donor DBA skin allografts were chronically rejected in NKT KO recipient. Lower levels of mixed chimerism were observed in NKT KO recipients comparing to the WT recipients. The production of interferon-&ggr; or interleukin-4 from NKT cells did not affect the induction of tolerance. Depletion of Ly49 positive NKT cells abrogated the induction of skin graft tolerance. Conclusion. Recipient NKT cells, but not donor NKT cells, were dominantly required for the induction of allograft tolerance. Our results indicated that the single cytokine produced by NKT cells did not mediate the regulatory function in the induction of allograft tolerance.

Efficacy and Limitations of Natural Killer Cell Depletion in Cyclophosphamide-Induced Tolerance

PurposeWe previously developed a cyclophosphamide (CP)-induced tolerance protocol, consisting of an intravenous injection of 1 × 108 donor spleen cells (SC) given on day 0 and an intraperitoneal injection of 200 mg/kg CP given on day 2. In the present study, we modified this protocol with natural killer cell (NK) depletion in recipient mice, and evaluated the efficacy of tolerance induction.MethodsWe used B10.D2 (H-2d; IE+) and B10 (H-2b; IE−) mice as both donors and recipients. The recipient mice were treated with donor SC, CP, and donor bone marrow cells (BMCs) with or without NK depletion.ResultsA higher level of mixed chimerism was achieved in the NK-depleted recipients. Survival of both the skin and heart donor grafts was significantly prolonged in the NK-depleted recipients. Donor reactive Vβ11+ T cells were found at the same level as in untreated control mice. Pretreatment with recipient NK cell depletion was effective in inducing higher levels of donor mixed chimerism; however, permanent engraftment of donor bone marrow was not achieved.ConclusionSurvival of donor grafts was remarkably prolonged in the NK cell-depleted group, but transplantation tolerance could not be induced. Our results suggest that NK cell depletion in CP-induced tolerance conditioning has some effect on the induction of donor-specific tolerance.

Application of Chimerism‐based Drug‐induced Tolerance to Rat into Mouse Xenotransplantation

The current critical shortage of human donor organs has stimulated the feasibility of the xenogenic transplantation, such as swine to primate. We have previously reported the induction of donor‐specific tolerance in MHC‐disparated recipient mice by using our cyclophosphamide (CP)‐induced tolerance conditioning. In this study, we examined the efficacy of our CP‐induced tolerance conditioning in xenogenic transplantation model. F344 rats and B10 mice were used as donors and recipients. Recipient mice were treated with donor spleen cells, CP, Busulfan and bone marrow cells, with or without prior NK‐cell depletion. Donor mixed chimerism, and the presence of donor reactive T‐cell population were analysed by flow cytometry. The survival of the donor skin grafts were observed after the conditioning. Donor mixed chimerism was temporary induced but terminated at 10 weeks after treatments. Donor‐specific prolongation of the skin graft survival was observed after the treatments, however, grafts were rejected in the long term. NK‐cell depletion, prior to the treatments, did not affect the levels of the mixed chimerism or graft prolongation. The donor‐reactive recipient T‐cell population was remained the same level as the untreated mice, suggesting the failure of the induction of the central T‐cell tolerance. Thus, partial efficacy of our CP‐induced tolerance treatments in the rat to mice xenotransplantation was observed. Our results suggested that the additional treatments were required to establish the stable xenogenic tolerance.

Effects of Lipopolysaccharide on the Induction of Mixed Chimerism in Cyclophosphamide-Induced Tolerance

Cyclophosphamide (CP)‐induced tolerance is a mixed chimerism‐based tolerance and is one of the strategies used to induce transplant tolerance. Toll‐like receptor (TLR) agonists are reportedly able to abrogate the induction of tolerance by activating alloreactive T cells, or by inhibiting Treg cells. However, little is known about the effect of the immune response mediated by TLR on mixed chimerism‐based tolerance protocols. In this study, we evaluated the influence of lipopolysaccharide (LPS), which is best known as an TLR4 agonist, on CP‐induced tolerance. BALB/c (H‐2d) mice received a conditioning regimen consisting of 108 donor DBA/2 (H‐2d) spleen cells (SC) on day 0 and 200 mg/kg CP on day 2. A single dose of 20 μg LPS was injected on day −2, 0, 7, or 35. Our results showed that LPS infusion at any time point resulted in chronic rejection of donor skin grafts and the abrogation of mixed chimerism in 33–60% of recipients. We found a correlation between skin graft acceptance and higher levels of mixed chimerism. Flow cytometric analysis revealed that donor‐reactive T cells were permanently eliminated, regardless of LPS infusion. In conclusion, LPS‐infusion had little influence on the immune response of donor‐reactive T cells, but had a significant effect on the induction and maintenance of mixed chimerism in CP‐induced tolerance.

Application of cyclophosphamide-induced tolerance in α1,3-galactosyltransferase knockout mice presensitized with Galα1-3Galβ-4-GlcNAc antigens

PurposeHyperacute rejection (HAR) mediated by the natural antibody (nAb) against Galα1-3Galβ-4-GlcNAc (αGal) is the major obstacle in xenogeneic organ transplantation. Previously, we reported the acceptance of donor heart grafts in anti-αGal nAb-producing galactosyltransferase knockout (GalT KO) mice after cyclophosphamide (CP)-induced tolerance conditioning. In the present study, we applied our tolerance induction conditioning in presensitized recipient mice.MethodsGalT KO (αGal−/−, H-2b/d) recipient mice were presensitized with αGal+ rabbit red blood cells (RRBCs). Presensitized or nonsensitized recipient mice were treated with CP-induced tolerance conditioning, consisting of AKR (αGal+/+, H-2k) spleen cells (SC), CP, busulfan (BU), and AKR bone marrow cells (BMC). We assessed the survival of donor hearts and skin grafts and analyzed the production of anti-αGal Abs by flow cytometry.ResultsDonor mixed chimerism was achieved in the presensitized GalT KO mice treated with CP-induced tolerance conditioning. In parallel with the disappearance of anti-αGal Abs, permanent acceptance of donor heart grafts and skin grafts was observed in presensitized and GalT KO mice treated with CP-induced tolerance conditioning.ConclusionsBoth B-cell and T-cell tolerance was achieved in the presence of a higher titer of anti-αGal Abs after treatment with CP-induced tolerance conditioning.

Concealed Left Ventricle to Right Atrium Fistula Complicating Infective Endocarditis

We report a case of aortic prosthetic valve endocarditis presenting with subaortic stenosis without perivalvular leakage and vegetations in the left ventricular outflow and right atrium, the latter being attached to the atrioventricular septum. Intraoperatively, an abscess that had formed on the aortic annulus and perforated to the right atrium was unexpectedly found, the fistula being occluded by vegetations. Even when no left-to-right shunts are detected by imaging, vegetations adjacent to the atrioventricular septum may conceal a left ventricle-right atrium fistula, resulting in prosthetic valve endocarditis presenting clinically as subaortic stenosis without perivalvular leakage.

Antibody-mediated T-cell reduction or increased levels of chimerism overcome resistance to cyclophosphamide-induced tolerance in NKT-deficient mice.

We reported that invariant NKT‐cell knockout (iNKT KO) mice are resistant to the induction of intrathymic chimerism and clonal deletion in the cyclophosphamide (CP)‐induced tolerance system (CPS). However, another report shows that clonal deletion with chimerism may be intact in iNKT KO recipients in a bone marrow transplantation model. We also reported that pretreatment with anti‐Thy1.2 mAb, which reduces the number of T cells and iNKT cells, promotes allograft tolerance across H‐2 barriers in the CPS. In this study, we evaluated the efficacy of T‐cell depletion in the CPS, and the relationship between the role played by iNKT cells in central tolerance and mixed chimerism. BALB/c (H‐2d) wild‐type, or iNKT KO (Jα18−/−) mice were pretreated with 20–100 μg of anti‐Thy1.2 mAb and given 108 donor DBA/2 (H‐2d) spleen cells on Day 0, and 200 mg/kg CP on Day 2. Pretreatment with T‐cell depletion resulted in higher levels of mixed chimerism, increased intrathymic clonal deletion of donor‐reactive cells, and the induction of skin graft tolerance in iNKT KO recipients in CPS. This suggests that the high levels of mixed chimerism overcame the resistance to CP‐induced tolerance in iNKT KO mice. Consistently, the enhancement of mixed chimerism by injection of tolerant donor spleen cells (SC) rendered iNKT KO recipients susceptible to CP‐induced tolerance. These results suggest that iNKT‐cell‐mediated immunoregulation of central tolerance is evident at low levels of peripheral mixed chimerism in the CPS.