異種移植におけるマクロファージ性拒絶抑制効果に対するレシピエント種CD47遺伝子導入の有用性

We have previously proven that the interspecies incompatibility of CD47 is responsible for in vitro phagocytosis of xenogeneic cells by host macrophages. Utilizing an in vivo model in the present study, we investigated whether genetically engineered expression of mouse CD47 in rat insulinoma cells (INS-1E) could inhibit macrophage-mediated xenograft rejection. INS-1E cells transfected with the pRc/CMV-mouse CD47 vector (mCD47-INS-1E) induced SIRPa-tyrosine phosphorylation in mouse macrophages in vitro, whereas cells transfected with the control vector (cont-INS-1E) did not. When these cells were injected into the peritoneal cavity of streptozotocin-induced diabetic Rag2-/-γ chain -/- mice, which lack T, B, and NK cells, the expression of mouse CD47 on the INS-1E cells markedly reduced the susceptibility of these cells to phagocytosis by macrophages. Moreover, these mice became normoglycemic after receiving mCD47-INS-1E, whereas the mice that received cont-INS-1E failed to achieve normoglycemia. Furthermore, injection of an anti-mouse SIRPa blocking monoclonal antibody into the mouse recipients of mCD47-INS-1E cells prevented achievement of normoglycemia. These results demonstrate that interspecies incompatibility of CD47 significantly contributes to in vivo rejection of xenogeneic cells by macrophages. Thus, genetic induction of the expression of recipient CD47 on xenogeneic donor cells could provide inhibitory signals to recipient macrophages via SIPRa; this constitutes a novel approach for preventing macrophage-mediated xenograft rejection.広島大学(Hiroshima University)博士(医学)Philosophy in Medical Sciencedoctora

Gastric Endocrine Cell Carcinoma with Long-Term Survival Developing Metachronous Remnant Cancer

A rare case of primary gastric endocrine cell carcinoma in a 79-year-old man is reported. Upper gastrointestinal endoscopy showed a large Bormann's type 2 tumour located in the middle of the stomach. On computed tomography, the gastric wall was thickened by the large tumour, and there were no distant metastases. Distal gastrectomy, lymph node dissection, and partial resection of the transverse colon were performed because the tumour involved the transverse mesocolon. The final pathological diagnosis was endocrine cell carcinoma, with tumour infiltration up to the subserous layer. Adjuvant chemotherapy was given, but metachronous remnant gastric cancer developed 2 years after surgery. Endoscopic submucosal dissection was performed for the early 0-IIc type gastric cancer, and the surgical margin was preserved. The patient has survived for 5 years after the primary surgery, remaining disease-free so far

Expression of recipient CD47 on rat insulinoma cell xenografts prevents macrophage-mediated rejection through SIRPα inhibitory signaling in mice.

We have previously proven that the interspecies incompatibility of CD47 is responsible for in vitro phagocytosis of xenogeneic cells by host macrophages. Utilizing an in vivo model in the present study, we investigated whether genetically engineered expression of mouse CD47 in rat insulinoma cells (INS-1E) could inhibit macrophage-mediated xenograft rejection. INS-1E cells transfected with the pRc/CMV-mouse CD47 vector (mCD47-INS-1E) induced SIRPα-tyrosine phosphorylation in mouse macrophages in vitro, whereas cells transfected with the control vector (cont-INS-1E) did not. When these cells were injected into the peritoneal cavity of streptozotocin-induced diabetic Rag2(-/-)γ chain (-/-) mice, which lack T, B, and NK cells, the expression of mouse CD47 on the INS-1E cells markedly reduced the susceptibility of these cells to phagocytosis by macrophages. Moreover, these mice became normoglycemic after receiving mCD47-INS-1E, whereas the mice that received cont-INS-1E failed to achieve normoglycemia. Furthermore, injection of an anti-mouse SIRPα blocking monoclonal antibody into the mouse recipients of mCD47-INS-1E cells prevented achievement of normoglycemia. These results demonstrate that interspecies incompatibility of CD47 significantly contributes to in vivo rejection of xenogeneic cells by macrophages. Thus, genetic induction of the expression of recipient CD47 on xenogeneic donor cells could provide inhibitory signals to recipient macrophages via SIPRα; this constitutes a novel approach for preventing macrophage-mediated xenograft rejection

Inhibition of CD47-SIRPα signaling prevents the effect of genetic induction of recipient CD47 in xenografts.

<p>(A) Either P84 or control antibody was injected into the peritoneal cavity of diabetic Rag2^−/− γ chain^−/− mice. After injection, intraperitoneal cells from recipient mice were harvested and SIRPα+ peritoneal cells were counted. (B) Anti-mouse SIRPα mAb (P84) was injected into the peritoneal cavity of Rag2−/− γ chain −/− mice. Expression of mouse SIRPα on mouse peritoneal macrophages was confirmed by FACS analysis. Open and filled histograms represent staining with isotype control and with anti-mouse SIRPα mAb, respectively. (C) Twenty-four hours after the injection of anti-mouse SIRPα mAb (P84), either mCD47-INS-1E or cont-INS-1E cells were injected into the peritoneal cavity of the diabetic Rag2^−/− γ chain ^−/− mice. Blood glucose levels were monitored for 7 days. Data are given as the means ± SD. N.S.: not significant.</p

CD47-SIRPα signaling blockade does not induce phagocytosis of congenic cells.

<p>(A) Twenty-four hours after injection of either P84 or control antibody, CFSE-labeled congenic T cells were injected into the peritoneal cavity of mice. After 6 h, the intraperitoneal cells from recipient mice were harvested. Mouse macrophages counterstained with allophycocyanin-conjugated anti-mouse CD11b and phagocytosis of CFSE-labeled congenic T cells were determined by FACS analysis. Representative FACS profiles are shown. Regions representing non-phagocytosing macrophages are shown in the upper left quadrants, regions representing phagocytosing macrophages are shown in the upper right quadrants, and regions representing residual congenic T cells are shown in the lower right quadrants. (B) Phagocytic activity was calculated by using the following formula: phagocytic activity = (percentage of engulfing macrophages/percentage of total harvested macrophages)×100. Data are presented as means ± SD.</p

Mouse CD47-expressing rat INS-1E cells attenuate phagocytosis by mouse macrophages.

<p>(A) CFSE-labeled pRc/CMV-mouse CD47-transfected rat INS-1E cells (mCD47-INS-1E) and control vector-transfected rat INS-1E cells (cont-INS-1E) were injected into peritoneal cavity of streptozotocin-induced diabetic Rag2^−/− γ chain ^−/− mice. After 6 h, the intraperitoneal cells from the recipient mice were harvested. Mouse macrophages counterstained with allophycocyanin-conjugated anti-mouse CD11b and phagocytosis of CFSE-labeled targets were measured by FACS analysis. Representative FACS profiles are shown. Regions representing non-phagocytosing macrophages are shown in the upper left quadrants, regions representing phagocytosing macrophages are shown in the upper right quadrants, and regions representing residual targets are shown in the lower right quadrants. (B) Phagocytic activity was calculated by the following formula: phagocytic activity = (percentage of engulfing macrophages/percentage of total harvested macrophages) ×100. Data are given as the means ± SD.</p

Generation of mouse CD47-expressing rat cell line.

<p>(A) Structure of pRc/CMV-mouse CD47. The entire coding region of the mouse CD47 cDNA was PCR-amplified. The amplified PCR product was digested and full-length mouse CD47 cDNA was inserted into the expression vector pRc/CMV. (B) Expression of mouse CD47 on a transfected rat insulinoma cell (INS-1E) was confirmed by FACS analysis. Representative histograms obtained by FACS analysis for mouse PBMCs, pRc/CMV-transfected rat INS-1E cells (cont-INS-1E), and pRc/CMV-mouse CD47-transfected rat INS-1E cells (mCD47-INS-1E) are shown. Open and filled histograms represent staining with isotype control and with anti-mouse CD47 mAb, respectively. (C) Tyrosine phosphorylation of SIRPα in mouse macrophages was induced by incubation with pRc/CMV-mouse CD47-transfected rat INS-1E cells (mCD47-INS-1E), but not with control vector-transfected rat INS-1E cells (cont-INS-1E). Differentiated mouse macrophages were incubated with mCD47-INS-1E or cont-INS-1E at 37°C for 30 min. The cells were lysed, and the lysates were mixed with mouse anti-mouse SIRPα antibodies and 50% slurry of protein G-sepharose beads by rotation at 4°C for 8 hrs. Precipitated proteins were separated by 8% SDS-PAGE, followed by blotting to a nitrocellulose membrane. Rabbit immunoaffinity-purified anti-phosphotyrosine IgG and goat anti-rabbit HRP-conjugated IgG were used as primary and secondary antibodies, respectively. Mouse CD47-transfected INS-1E (mCD47-INS-1E) alone (lane 2), mouse macrophages incubated in medium alone (lane 3) or mouse macrophages incubated with cont-INS-1E (lane 4) or mCD47-INS-1E (lane 5) are shown. Immunoblotting with anti-mouse SIRPα was used as loading control. IP, immunoprecipitation; IB, immunoblotting; anti-pY, anti-phosphotyrosine.</p

Incidental renal cell carcinoma presenting in a renal transplant recipient with autosomal dominant polycystic kidney disease: a case report

Abstract Introduction We report an instructive case of incidental renal cell carcinoma in a patient with autosomal dominant polycystic kidney disease who underwent simultaneous bilateral native nephrectomy and living donor renal transplantation. Case presentation A 57-year-old Asian man with end-stage kidney disease due to autosomal dominant polycystic kidney disease received a living kidney graft from his brother. Because of recurrent infection, chronic pain and enlarged kidneys, he underwent a bilateral nephrectomy with concomitant renal transplantation. The total weight of the removed kidneys was 6kg; the maximal diameter of the larger kidney was 28cm. His left kidney had a 1cm diameter tumor. Pathology indicated papillary renal cell carcinoma. At the time of this report, the transplant kidney function was normal with no evidence of local recurrence or distant metastasis. Conclusion This case shows and reinforces the importance of considering the possibility of an occult malignancy in the native kidneys of patients with autosomal dominant polycystic kidney disease. Simultaneous bilateral native nephrectomy should be considered in these renal transplant recipients not only for preventing the development of adverse symptoms but also for detecting an occult malignancy.</p

Surface study of organopalladium molecules on S-terminated GaAs

Organopalladium species ({Pd}) immobilized on an Sterminated GaAs substrate (S/GaAs) effectively catalyzes C-C bond formation in the Mizoroki-Heck reaction with cycle durability. However, the immobilizing mechanism of {Pd} is unknown. In this study, we deposited Pd(OCOCH3)2 on S/GaAs in two different methods, namely dry-physical vapor-deposition and wetchemical deposition, and compared the catalytic activities in the Mizoroki-Heck reaction. Also, S-termination and {Pd}-immobilization on GaAs grains were performed by the wet-chemical method to monitor the change in the surface chemical structure during the preparation process with diffuse reflectance Fourier transform infrared spectroscopy (FT-IR). FT-IR measurements implied that the immobilization of catalytic active {Pd} was related to the OH groups on the S-terminated surface. {Pd}-S/GaAs prepared dryphysically showed poor catalytic activity, because {Pd} was not immobilized under absence of OH groups. (© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim