Intractable autoimmune diseases in chimeric resistant MRL/lpr mice were treated by a new bone marrow transplantation (BMT) method consisting of fractionated irradiation, 5.5 Gy ؋ 2, followed by intrabone marrow (IBM) injection of whole bone marrow cells (BMCs) from allogeneic normal C57BL/6 (B6) mice (5.5 Gy ؋ 2 ؉ IBM). In MRL/lpr mice treated with this method, the number of donor-derived cells in the bone marrow, spleen, and liver rapidly increased (almost 100% donor-derived cells by 14 days after the treatment), and the number of donorderived hemopoietic progenitor cells concomitantly increased. Furthermore, donorderived stromal cells were clearly detected in the cultured bone pieces from MRL/lpr mice treated with 5.5 Gy ؋ 2 ؉ IBM. All the recipients thus treated survived more than 1 year (> 60 weeks after birth) and remained free from autoimmune diseases. Autoantibodies decreased to almost normal levels, and abnormal T cells (

Abstract-Heme oxygenase-1 (HO-1) is a stress protein that has been suggested to participate in defense mechanisms against agents that may induce oxidative injury, such as heme and inflammatory molecules. Incubation of endothelial cells in a high-glucose (33 mmol/L) medium for 7 days resulted in a decrease of HO activity by 34% and a decrease in HO-1 and HO-2 proteins compared with cells exposed to low glucose (5 mmol/L) (PϽ0.05) or cells exposed to mannitol (33 mmol/L). Overexpression of HO-1 was coupled with an increase in HO activity and carbon monoxide synthesis, decreased cellular heme, and acceleration in all phases of the cell cycle (PϽ0.001). Key Words: cell cycle Ⅲ oxidative stress Ⅲ superoxide anion production Ⅲ gene transfer Ⅲ heme oxygenase E xposure of endothelial cells to elevated glucose levels causes glucose oxidation, resulting in the generation of excess reactive oxygen species (ROS) in endothelial cells. A reduction in antioxidant reserves has been attributed to endothelial cell dysfunction in diabetes, even in patients with well-controlled glucose levels. 1-3 Hyperglycemia-mediated local formation of ROS is considered to be the major contributing factor to endothelial dysfunction, including abnormalities in cell cycling 1,4,5 and delayed replication, and these abnormalities can be reversed by antioxidant agents 6,7 and an increased expression of antioxidant enzymes. 8 Du et al 9 have demonstrated that hyperglycemia stimulates the induction of apoptosis in endothelial cells by a mechanism that involves the generation of ROS and superoxide anion formation. Moreover, high glucose conditions facilitated the susceptibility of various serum proteins to oxidation, which contributes to the inhibition of endothelial cell proliferation. 10 Wolf et al 11 have reported that high glucose stimulates mitogen-activated protein kinase, which was associated with an enhancement in p27 Kip1 protein and growth arrest.We have previously shown that overexpression of the human heme oxygenase-1 (HO-1) gene in rabbit and rat endothelial cells renders the cells resistant to oxidative stress-causing agents 12 and enhances cell growth 13,14 and angiogenesis, 15 which highlights the important metabolic and cytoprotective role of the HO-1 gene. 12,15-17 Inhibition of HO activity has been shown to exacerbate the inflammatory response in the arterial wall in animal models of atherosclerosis model. 18 HO-1 is expressed, under basal conditions, at low levels in endothelial cells 12,15,19,20 and can be induced in these cells in response to oxidants, including heme, H 2 O 2 , and tumor necrosis factor. [21][22][23] It is conceivable, then, that upregulation of HO activity could function to attenuate the glucosemediated inhibition of cell-cycle progression.The objectives of this study were to determine the effects of glucose on HO activity and the expression of HO-1 and HO-2 proteins and DNA distribution and to examine the role of heme metabolism by HO on cell-cycle progression. We also examined the effect of overexpression and un...

Transplantation of whole bone marrow cells may be a better and more readily available treatment for Achilles tendon rupture than cultured mesenchymal stem cells.

The purpose of this study was to compare anterior cruciate ligament (ACL) regeneration between animal groups subjected to intra-articular injection of fresh whole bone marrow cells (BMCs), cultured mesenchymal stem cells (MSCs), or saline. Partially transected ACLs in Fischer 344/Nslc rats were prepared, followed by injection of BMCs, MSCs, or saline into the articular cavity at 1 week after transection. Donor cells expressing green fluorescent protein were detected in the recipient's transected ACLs at 4 weeks in the BMC and MSC groups, and their ACLs appeared almost normal histologically. Further, there were significantly more mature spindle cells in the BMC group than in the saline group at 4 weeks. Biomechanically, the tensile strength in the BMC group reached near normal levels at 4 weeks after injection. The levels of transforming growth factor-β1 in the ACL tissue and knee joint fluid in the BMC group were increased significantly compared with that of the saline group at 4 weeks as detected by immunohistochemical analysis. In conclusion, intra-articular bone marrow transplantation using fresh whole BMCs is an effective treatment for ACL partial rupture. This therapy is easy to apply in a clinical setting because no culture system is required for collecting MSCs.

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Summary. Thrombocytopenia is typically observed in patients undergoing cord blood transplantation. We hypothesized that delayed recovery of the platelet count might be caused by defects in the megakaryocytic differentiation pathway of cord blood progenitors. To test this hypothesis, we compared the features of in vitro megakaryocytopoiesis between cord blood progenitors and those in bone marrow cells after isolation of CD34 cells as progenitors. The proliferative responses of the progenitors in cord blood are higher than those in bone marrow cells in the presence of interleukin (IL)-3, stem cell factor (SCF) and thrombopoietin (TPO). However, the ability to generate mature megakaryocytes was higher in bone marrow progenitors than in cord blood in the same in vitro culture system, when examined by the expression of CD41, polyploidy and proplatelet formation. Furthermore, an earlier induction of c-mpl protein, a receptor for TPO, was observed in the progenitors from bone marrow than in those from cord blood in the presence of SCF and IL-3. Therefore, the ability to generate mature megakaryocytes in bone marrow progenitors is superior to that in cord blood, and the delayed engraftment of platelets after cord blood transplantation might be attributed to the features of cord blood megakaryocyte progenitors.

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To minimize contamination of bone marrow cells (BMCs) with T cells from the peripheral blood, a new "perfusion method" for collecting BMCs is proposed using cynomolgus monkeys. Two BM puncture needles are inserted into a long bone such as the humerus, femur, or tibia. One needle is connected to an extension tube and the end of the tube is inserted into a culture flask to collect the BM fluid. The other needle is connected to a syringe containing 30 ml of phosphate-buffered saline. The solution is pushed gently from the syringe into the medullary cavity, and the medium containing the BM fluid is collected into the culture flask. There is significantly less contamination with peripheral blood, determined from the frequencies of CD4 + and CD8 + T cells, when using this method (<6%) than when using the conventional method (>20%) consisting of multiple BM aspirations from the iliac crest. Furthermore, the number and progenitor activities of the cells harvested using this "perfusion method" are greater than those harvested using the conventional aspiration method. This perfusion method was carried out 42 times using 15 cynomolgus monkeys, and no complications such as pulmonary infarction or paralysis were observed. These findings suggest that the "perfusion method" is safe and simple and would be of great advantage in obtaining pure BMCs, resulting in a less frequent occurrence of acute graft-versus-hostdisease in allogeneic BM transplantation.