Treatment of streptozotocin-induced diabetes mellitus by transplantation of islet cells plus bone marrow cells via portal vein in rats.

BACKGROUND: We have established a new method for the transplantation of allogeneic pancreatic islets (PIs) using sublethal irradiation (9 Gy) plus simultaneous transplantation of PIs and bone marrow cells (BMCs) via the portal vein (PV) followed by intravenous (i.v.) injection of donor BMCs (9 Gy + PV + i.v.). METHODS: Approximately 600 PIs of Brown Norway (BN: RT1An, RT1Bn) rats were transplanted into the liver of streptozotocin-induced diabetic Fischer 344 (F344: RT1Al, RT1Bl) rats via the PV. BMCs (3x108) of BN rats were injected via the PV or i.v. into the recipients simultaneously. In some groups, additional i.v. injections of BMCs from BN rats were given 5 days after the PI transplantation. RESULTS: All the recipients (10 of 10) in the 9 Gy + PV + i.v. group showed normoglycemia for more than 1 year, whereas PIs were rejected within 30 days after transplantation in the group of 9 Gy + i.v. + i.v. CONCLUSIONS: These results suggest that simultaneous transplantation of PIs and BMCs via the PV is effective in inducing persistent tolerance.

Immature dendritic cells (CD11c+ CD3- B220- cells) present in mouse peripheral blood.

It is well known that dendritic cells (DCs) are developed from the peripheral blood of mice when peripheral blood mononuclear cells (PBMCs) are cultured with GM-CSF. We have previously found that immature DCs are present in the blood even in humans. In the present study, we show that CD11c+ CD3- B220- cells in the mouse peripheral blood are immature DCs. The percentage of CD11c+ CD3- B220- cells in the (PBMCs) of normal mice ranges from 0.5 to 2.5%. The CD11c+ CD3- B220- cells in the PBMCs show dendrites, similar in shape to the CD11c+ CD3- B220- cells in the spleen, which are thought to be DCs definitely. However, they have practically no capacity to stimulate the proliferation of allogeneic T cells, and show a lower expression of MHC class II, B7-1 and B7-2 than CD11c+ CD3- B220- cells in the spleen. When the CD11c+ CD3- B220- cells in the PBMCs are cultured with GM-CSF, they show not only the potent ability to stimulate the proliferation of allogeneic T cells but also a higher expression of MHC class II, B7-1 and B7-2. Moreover, they migrate into the spleen when they are injected intravenously. These results suggest that CD11c+ CD3- B220- cells in the PBMCs are immature DCs, and that they migrate into the spleen, where they mature.

Long-term observation after simultaneous lung and intra-bone marrow-bone marrow transplantation.

BACKGROUND: Although lung transplantation is now an established treatment for end-stage lung diseases, allogeneic transplantation of parenchymal organs requires immunosuppressive therapy to prevent rejection. It has been reported that bone marrow transplantation (BMT) induces specific tolerance to donor organs. We have recently discovered a new method for BMT, which is called intra-bone marrow (IBM) BMT, in which bone marrow cells (BMCs) are injected directly into the bone marrow cavity. We demonstrate that IBM-BMT can be used to induce tolerance even in simultaneous lung transplantations in rats without administering any immunosuppressants. METHODS: Allogeneic lung transplantation was carried out from Brown Norway to Lewis rats. Simultaneously, IBM-BMT was carried out. RESULTS: Transplantation of nonirradiated lung or nontreated BMCs (T cell-containing BMCs) induced graft vs host disease. Therefore, the donor lung was irradiated, and T cells in the BMCs were depleted by anti-CD4, anti-CD5, and anti-CD8 antibodies plus anti-mouse antibody-coated magnetic beads. Lung allografts with conventional (intravenous) BMT failed to induce tolerance. However, the recipients treated with lung allografts plus IBM-BMT, which showed either mixed chimerism or full chimerism of hematopoietic cells, did not show symptoms of graft rejection or graft vs host disease, even without the use of immunosuppressants. CONCLUSIONS: These results suggest that simultaneous lung transplantation and IBM-BMT (but not conventional BMT) is effective in inducing persistent tolerance without the use of immunosuppressants.

Bone marrow-derived stem cells can differentiate into retinal cells in injured rat retina.

It has recently been shown that bone marrow cells can differentiate into various lineage cells including neural cells in vitro and in vivo. We therefore examined whether bone marrow stem cells can differentiate into retinal neural cells in adult rats. PKH-67-labeled stem cell-enriched bone marrow cells (BMCs) were injected into the vitreous space of eyes in which the retinas had been mechanically injured using a hooked needle. Two weeks after the injection of these cells, immunohistochemical examinations were carried out. The stem cell-enriched BMCs had been incorporated and had differentiated into retinal neural cells in the injured retina. The stem cell-enriched BMCs had accumulated mainly in the outer nuclear layer around the injured sites. The incorporated cells expressed glial fibrillary acidic protein, calbindin, rhodopsin, and vimentin. These results raise the possibility that stem cell-enriched BMCs have the ability to differentiate into retinal neural cells, and that the injection of stem cell-enriched BMCs into the retina would help repair damaged retinal cells.

Comparison of bone marrow cells harvested from various bones of cynomolgus monkeys at various ages by perfusion or aspiration methods: a preclinical study for human BMT.

Using cynomolgus monkeys, we have previously established a new method for harvesting bone marrow cells (BMCs) with minimal contamination of the BMCs with T cells from the peripheral blood. We originally conducted this new "perfusion method" in the long bones (the humerus, femur, and tibia) of cynomolgus monkeys. Here, we apply the perfusion method to obtain BMCs from the ilium of cynomolgus monkeys, since BMCs are usually collected from the ilium by the conventional aspiration method in humans. The perfusion method consists of two approaches: transverse iliac perfusion and longitudinal iliac perfusion. BMCs harvested by the perfusion method from the long bones and ilium were compared with those collected from the ilium by the aspiration method. The contamination of BMCs with peripheral blood, determined by the frequencies of CD4+ and CD8+ T cells, was significantly lower in BMCs obtained from the ilium or long bones by the perfusion method (CD4+ plus CD8+ T cells <4%) than in those obtained by the iliac aspiration method (CD4+ plus CD8+ T cells >20%). However, the numbers of immature myeloid cells, such as myeloblasts, promyelocytes, myelocytes, and metamyelocytes, were higher in BMCs obtained by the iliac perfusion method than in those obtained by the iliac aspiration method. The assays for in vitro colony-forming unit in culture revealed that progenitor activity was significantly higher in BMCs obtained by the perfusion method than in those obtained by the aspiration method. These findings suggest that the contamination of BMCs with peripheral blood is much less when using the perfusion method than when using the aspiration method. To determine the best site for harvesting BMCs by the perfusion method, age-dependent changes in BMCs harvested by the perfusion method from the long bones and ilium were examined. The numbers of BMCs varied in the long bones (humerus > femur > tibia) and showed age-dependent decreases, whereas they remained similar in the ilium of cynomolgus monkeys from 3 years to 6 years of age. However, in cynomolgus monkeys, BMC harvesting by the perfusion method from the ilium (but not from the long bones) is found to involve the risk of fat emboli, particularly when the BMCs are quickly perfused under high pressure. These findings suggest, even in humans, that the perfusion method is better than the aspiration method, and that the best site for collection of BMCs is the humerus.

Marked increase in number of dendritic cells in autoimmune-prone (NZW x BXSB)F1 mice with age.

Here, we report that the number of CD11c(+)CD3(-) B220(-) cells increases in autoimmune-prone male (NZW x BXSB)F1 (W/BF1) mice with age. The CD11c(+)CD3(-)B220(-) cells from W/BF1 mice show a typical stellate shape and induce the proliferation of T cells. In the CD11c(+)CD3(-)B220(-) cells from W/BF1 mice, CD11b (Mac-1alpha), NK 1.1, and CD95 (Fas) are upregulated in comparison with normal mice, while the expression of CD8alpha, CD117 (c-kit), CD135 (Flk-2/Flt-3), and Sca-1 decreases. There is a significant increase in Flt-3L (FL) mRNA in the bone marrow of W/BF1 mice with age. Moreover, activated hemopoietic cells express high levels of FL. The injection of CD11c(+)CD3(-)B220(-) cells from old W/BF1 mice to young W/BF1 mice transiently induces autoimmune disease (thrombocytopenia). These results suggest that hyperproduction of FL from activated hemopoietic cells induces a dramatic increase in the number of dendritic cells in aged W/BF1 mice, followed by the acceleration of autoimmunity.