Intra-bone Marrow-bone Marrow Transplantation Research Articles

Immunotherapy for malignant tumors using combination of allogeneic intra-bone marrow-bone marrow transplantation, donor lymphocyte infusion and dendritic cells

We have previously shown that the combination of allogeneic intra-bone marrow-bone marrow transplantation (IBM-BMT) and donor lymphocyte infusion (DLI) using CD4+ cell-depleted spleen cells is effective in suppressing tumor growth, but that this does not induce graft-versus-host disease (GVHD) in mice. In this report, we show that formalin-fixed tumor cell-pulsed dendritic cells (FFTCP DCs) have an additive effect with IBM-BMT plus DLI on the suppression of tumor growth, but that the DCs do not augment GVHD. BALB/c mice, which had been subcutaneously inoculated with Meth A (BALB/c-derived fibrosarcoma), were irradiated at a low dose (5 Gy) and were transplanted with bone marrow cells (BMCs) from C57BL/6 (B6) mice into the bone marrow cavity (IBM-BMT). Simultaneously, the mice were intravenously injected with spleen cells from B6 mice, and subcutaneously injected with FFTCP DCs derived from the bone marrow (BM) of B6 mice. At the point of the induction of DCs from BMCs, formalin-fixed Meth A cells were added into the culture. The mice treated with the combination of FFTCP DCs, IBM-BMT and DLI using CD4+ cell-depleted spleen cells showed smaller tumor sizes and longer survival than the mice treated with IBM-BMT plus FFTCP DCs or IBM-BMT plus DLI using CD4+ cell-depleted spleen cells. These results suggest that the combination of FFTCP DCs, IBM-BMT plus DLI using CD4+ cell-depleted spleen cells has potent anti-tumor effects without showing GVHD.

Prevention of Graft-Versus-Host Disease by Intra-Bone Marrow Injection of Donor T Cells

We have recently found that intra-bone marrow-bone marrow transplantation (IBM-BMT) can be used to prevent graft-versus-host disease (GvHD), even when intensive donor lymphocyte infusion (DLI) is carried out. In the present study, in conjunction with IBM-BMT, allogeneic splenic T cells as DLI were also injected into the bone marrow cavity of lethally irradiated (8.5 Gy) recipients. The extent of GvHD was compared with that of recipients that had received allogeneic IBM-BMT plus i.v. injection of allogeneic T cells (intravenous DLI [IV-DLI]). GvHD in recipients treated with allogeneic IBM-BMT plus IBM-DLI was far milder than in those treated with allogeneic IBM-BMT plus IV-DLI. This was confirmed macroscopically and histopathologically. The frequency of regulatory T cells (Tregs) detected as CD4(+)CD25(+) and CD4(+)Foxp3(+) cells was significantly higher in recipients treated with IBM-BMT plus IBM-DLI than in those treated with IBM-BMT plus IV-DLI. Donor-derived helper T (Th) cells polarized to Th2 type in recipients treated with IBM-BMT plus IBM-DLI, whereas Th1 cells were dominant in recipients treated with IBM-BMT plus IV-DLI. Furthermore, the production of transforming growth factor-beta and hepatocyte growth factor from bone marrow stromal cells was enhanced after IBM-DLI. Thus, IBM-BMT plus IBM-DLI seem to preferentially induce Tregs and Th2, resulting in the prevention of GvHD. Disclosure of potential conflicts of interest is found at the end of this article.

Induction of Senile Osteoporosis in Normal Mice by Intra-Bone Marrow-Bone Marrow Transplantation from Osteoporosis-Prone Mice

A P6 substrain of the senescence accelerated mouse (SAMP6) spontaneously develops osteoporosis early in life. These mice show the clinical signs of osteoporosis, such as elevated levels of urinary deoxypyridinoline (Dpd), decreased bone mineral density (BMD), and a significant loss of trabecular and cortical bone thickness at 12 months of age. Here, we describe the transfer of osteoporosis to a normal strain by the injection of bone marrow cells from SAMP6 donors directly into the bone marrow cavity (intra-bone marrow-bone marrow transplantation [IBM-BMT]). More than 1 month after IBM-BMT, hematolymphoid cells were completely reconstituted by donor-derived cells, and bone marrow stromal cells that could differentiate into osteocytes were also found to be of donor origin. In addition, the recipient C57BL/6 mouse showed the features of osteoporosis in the trabecular bone. Decreases in BMD and increases in urinary Dpd were also observed. When the message levels of cytokines (interleukin [IL]-11, IL-6, receptor activator of NF-kappaB ligand [RANKL], osteoprotegerin, macrophage-colony-stimulating factor, and insulin-like growth factor-1) were examined by reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR analysis, IL-6 and IL-11 were reduced to a level similar to that in SAMP6 mice, whereas that of RANKL was increased. These findings indicate that not only the hemopoietic system but also the bone marrow microenvironment are reconstituted as a result of IBM-BMT, and suggest that the development of senile osteoporosis might be attributable to "stem cell disorders." Disclosure of potential conflicts of interest is found at the end of this article.

Allogeneic Intrabone Marrow-Bone Marrow Transplantation plus Donor Lymphocyte Infusion Suppresses Growth of Colon Cancer Cells Implanted in Skin and Liver of Rats

We have recently found that allogeneic intrabone marrow-bone marrow transplantation (IBM-BMT) + donor lymphocyte infusion (DLI) using CD4(+) cell-depleted spleen cells (CD4(-) cells) can prevent graft-versus-host disease (GvHD) but suppress tumor growth (Meth A: fibrosarcoma) in mice. In the present study, we show that allogeneic IBM-BMT + DLI using CD4(-) cells also has suppressive effects on the growth of colon cancer cells implanted not only in the skin but also in the liver of rats. First, we examined the effects of allogeneic IBM-BMT + DLI on the subcutaneously inoculated ACL-15 (rat colon cancer cell line). Lethally irradiated Fischer rats (F344 rats) were transplanted with T-cell-depleted bone marrow cells (BMCs) from Brown Norway (BN) rats. Simultaneously, DLI was performed using whole spleen cells (whole cells), CD4(+) cell-depleted spleen cells (CD4(-) cells) or CD8(+) cell-depleted spleen cells (CD8(-) cells) of BN rats. Although allogeneic IBM-BMT + DLI suppressed tumor growth, a considerable number of rats treated with allogeneic IBM-BMT + DLI using whole cells or CD8(-) cells died due to GvHD. In contrast, allogeneic IBM-BMT + DLI using CD4(-) cells also suppressed tumor growth, but there was no GvHD. Based on these findings, we next examined the effects of allogeneic IBM-BMT + DLI using CD4(-) cells on the cancer cells implanted in the liver. Allogeneic IBM-BMT + DLI using CD4(-) cells via the portal vein significantly prolonged the survival. These results suggest that allogeneic IBM-BMT + DLI using CD4(-) cells could become a new strategy for the treatment of solid tumors.

An Innovative Approach to Bone Marrow Collection and Transplantation in a Patient with β-Thalassemia Major: Marrow Collection Using a Perfusion Method Followed by Intra-Bone Marrow Injection of Collected Bone Marrow Cells

Using small animals (mice and rats) and monkeys, we have found that the combination of bone marrow collection using the perfusion method (PM) and intra-bone marrow-bone marrow transplantation (IBM-BMT) of the collected cells is safe and effective in treating various intractable diseases. Based on these findings, we attempted to apply this method to humans. We report here the first case of a patient (6 years old) with beta-thalassemia major who underwent allogeneic BMT using this new PM + IBM-BMT method. The white blood cell counts of the patient gradually increased to more than 1500/microL by day 47 and continued to increase, reaching the highest level (8600/microL) on day +55. Fluorescence in situ hybridization data on day +33 showed that 98% of the peripheral blood cells were from the donor. Notably, there were no symptoms of graft-versus-host disease (GvHD). However, on day +56, the patient regrettably died of asphyxia resulting from sticky sputum. There was no evidence of infection (in the lung or liver) or GvHD (in the skin) by necropsy. We hope that this case report will help make our new strategies more readily available for the treatment of patients with various intractable diseases.

Intra-bone Marrow-Bone Marrow Transplantation: A New Strategy for Treatment of Stem Cell Disorders

We have established new bone marrow transplantation (BMT) methods for the treatment of stem cell disorders such as autoimmune diseases. The methods include the perfusion method (PM) and intra-bone marrow (IBM)-BMT. PM, in comparison with the conventional aspiration method, can minimize the contamination of bone marrow cells (BMCs) with T cells from the peripheral blood. Therefore, without removing T cells, no graft-versus-host disease develops in the case of PM. Because BMCs collected using the PM contain not only hemopoietic stem cells (HSCs) but also mesenchymal stem cells (MSCs), the injection of both cells directly into the bone marrow cavity (IBM-BMT) facilitates the engraftment of donor hemopoietic cells. With IBM-BMT, no graft failure therefore occurs even if the radiation dose is reduced. IBM-BMT is applicable to regeneration therapy and various age-associated diseases such as osteoporosis, because it can efficiently recruit donor-derived normal MSCs into the bone marrow. We believe that this strategy heralds a revolution in the field of transplantation and regenerative therapy.

Intra-bone marrow-bone marrow transplantation facilitates hemopoietic recovery including dendritic cells

In this report, we provide evidence using a serial bone marrow transplantation (BMT) protocol that intra-bone marrow (IBM)-BMT (IBM-BMT) can efficiently reconstitute the hemopoietic system with cells of donor origin, in contrast to conventional intravenous (IV)-BMT (IV-BMT). Furthermore, the hematolymphoid system of secondary recipients that had received bone marrow cells (BMCs) from primary recipients treated with IBM-BMT recovered earlier than that of the secondary recipients of BMCs from primary recipients treated with IV-BMT. This was the case when the Lin −/c-kit + progenitor cells of the secondary and tertiary recipients were examined. These findings indicate that IBM-BMT can facilitate the development of not only cells of various lineages but also the effective generation and, more importantly, the maintenance of the progenitor cells. Furthermore, we show that IBM-BMT can reconstitute the dendritic cell (DC) subsets (myeloid and lymphoid DCs), which are critical for the initiation of both adaptive and innate immune responses. The frequency of both myeloid and lymphoid DC subsets was approximately equal to that of normal age-matched untreated controls and, after second and third BMT, this ratio was close to that observed in the normal controls. However, the lymphoid DCs were clearly reduced in the secondary and tertiary recipients of BMCs from mice that had received IV-BMT. Therefore, the development of DC subsets is also normally maintained in the IBM-BMT group.

A New Strategy for Treatment of Malignant Tumor: Intra-Bone Marrow–Bone Marrow Transplantation Plus CD4− Donor Lymphocyte Infusion

Donor lymphocyte infusion (DLI) is clinically used for the treatment of malignant tumors. We have found recently that intra-bone marrow-bone marrow transplantation (IBM-BMT) can be used to treat various autoimmune diseases, even when radiation doses are reduced. In addition, recently we have found that IBM-BMT can prevent not only graft failure but also graft-versus-host disease (GvHD). Based on these findings, we attempted to prevent and treat the progression of a tumor (Meth-A cell line: BALB/c-derived fibrosarcoma) by DLI plus IBM-BMT. When the tumors had grown to approximately 10 x 10 mm, the tumor-bearing BALB/c (H-2(d)) mice were irradiated with 5 Gy, and whole spleen cells from C57BL/6J (B6) (H-2(b)) mice (as DLI) were then intravenously injected into the BALB/c mice. Simultaneously, bone marrow cells (BMCs) from B6 mice were injected directly into the bone marrow cavity of the BALB/c mice (IBM-BMT). The tumors decreased in size, but the mice died of GvHD. However, when CD4(+) T-cell-depleted spleen cells were used for DLI, the recipients showed only mild GvHD and survived longer, due to the slow growth of the tumor. In contrast, when CD8(+) T-cell-depleted spleen cells were used for DLI, the recipients showed more severe GvHD than those injected with whole spleen cells. These results suggest that IBM-BMT plus DLI (the depletion or reduction of a certain cell population like CD4(+) T cells) could be helpful to suppress both GvHD and tumor growth.

Tolerance induction in lung transplantation with intra-bone marrow-bone marrow transplantation

Tolerance induction in lung transplantation with intra-bone marrow-bone marrow transplantation