Kikuya Sugiura

A New Method for Bone Marrow Cell Harvesting

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‐host‐disease in allogeneic BM transplantation.

Induction of donor-specific T cell anergy by portal venous injection of allogeneic cells

The mechanisms behind tolerance induction by portal venous (pv) injection of allogeneic cells are investigated. When a hematopoietic stem cell (HSC)-enriched population of BALB/c bone marrow was pv injected into C57BL/6 mice, the response of the T cells in the B6 mice to BALB/c alloantigens in mixed lymphocyte reaction (MLR) decreased until day 4 after the injection. Neither clonal deletion of V beta 11+ T cell nor donor-specific suppressor activity was observed. When recipient T cells were separated into CD4+ and CD8+ cells, only the CD8+ cell population showed donor-specific tolerance. The donor cells were trapped and retained in the host liver. MHC class I antigens were highly expressed on the trapped cells whereas class II antigens or B7 costimulatory molecules were not. The tolerance to BALB/c alloantigens in MLR was obtained also by the pv injection of Meth A, a BALB/c-derived sarcoma cell line. However, tolerance was not induced by the pv injection of B7-transfected Meth A cells. In addition to MLR, tolerance was also observed in DTH responses, and this was also due to the unresponsiveness of CD8+ cells to the donor alloantigens. However, the BALB/c-specific DTH responses were not suppressed after the pv injection of B7-transfected Meth A cells. These results strongly suggest that the tolerance induced by pv injection of allogeneic cells is due to clonal anergy generated by the absence of costimulatory signals in the interaction between donor-specific CD8+ T cells and donor hematopoietic cells trapped in the host liver.

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.

Enhancement of Allogeneic Hematopoietic Stem Cell Engraftment and Prevention of GvHD by Intra-Bone Marrow Bone Marrow Transplantation Plus Donor Lymphocyte Infusion

We examined the effect of intra‐bone marrow (IBM)‐bone marrow transplantation (BMT) in conjunction with donor lymphocyte infusion (DLI) on the engraftment of allogeneic bone marrow cells (BMCs) in mice. Recipients that had received 6 Gy of radiation completely rejected donor BMCs, even when IBM‐BMT was carried out. However, when BMCs were IBM injected and donor peripheral blood mononuclear cells (PBMNCs) were simultaneously injected intravenously (DLI), donor cell engraftment was observed 7 days after BMT and complete donor chimerism continued thereafter. It is of interest that the cells of recipient origin did not recover, and that the hematolymphoid cells, including progenitor cells (Lin−/c‐kit+ cells) in the recipients, were fully reconstituted with cells of donor origin. The cells in the PBMNCs responsible for the donor BMC engraftment were CD8+. Recipients that had received 6 Gy of radiation, IBM‐BMT, and DLI showed only a slight loss of body weight, due to radiation side effects, and had no macroscopic or microscopic symptoms of graft‐versus‐host disease. These findings suggest that IBM‐BMT in conjunction with DLI will be a valuable strategy for allogeneic BMT in humans.

Effects of Administration of Monoclonal Antibodies (anti-CD4 or anti-CD8) on the Development of Autoimmune Diseases in (NZW × BXSB)F1 Mice

(NZW x BXSB)F1 (W/BF1) mice spontaneously develop autoimmune diseases, characterized by lymphadenopathy, lupus nephritis, and immune thrombocytopenia associated with various autoantibodies such as anti-DNA, anti-platelet and anti-cardiolipin antibodies (Abs). In the present study, we investigate the effects of administration of monoclonal Abs (anti-CD4 or anti-CD8 mAb) on the development of autoimmune diseases in W/BF1 mice. MAb was administered from the age of 7 weeks. Prolongation of survival rate and reduction of severity of autoimmune diseases were observed after treatment with anti-CD4 mAb. However, anti-CD8 mAb treatment accelerated the diseases. Serum levels of IFN-gamma and IL-10 in old W/BF1 mice were significantly high, whereas IL-4 levels were low in comparison with those of young W/BF1 mice; the expression of mRNA of IFN-gamma, IL-4 or IL-10 in CD4+ T cells of old W/BF1 mice was parallel to the serum levels of each cytokine. These observations suggest that CD4+ cells are involved in the development of autoimmune diseases in W/BF1 mice, and that CD8+ cells have a suppressive effect on the development of autoimmune diseases in W/BF1 mice.

Expression of estrogen receptor α and β genes in the mediobasal hypothalamus, pituitary and ovary during the canine estrous cycle

Estrogen receptor α (ERα) and ERβ mRNA levels were measured in the mediobasal hypothalamus, the anterior pituitary and the ovary of beagle bitches at various stages of the estrous cycle. With polymerase chain reaction analysis we detected ERβ gene transcripts in all tissue samples. The levels of hypothalamic and pituitary ERα and β mRNAs increased from mid anestrus to proestrus and declined thereafter. In the ovary, ERα mRNA levels increased from proestrus to diestrus and were positively correlated with plasma progesterone levels (r=0.62, P<0.01), whereas ERβ mRNA levels increased from mid anestrus to proestrus and were positively correlated with plasma estradiol-17β levels (r=0.73, P<0.001). These results suggest that the rise in hypothalamic and pituitary ERα and β mRNAs is associated with termination of anestrus, and that increases in ovarian ERα and β mRNAs may be involved in initiating development of the follicle or corpora lutea.

Isolation of murine pluripotent hemopoietic stem cells in the Go phase

A method to purify pluripotent hemopoietic stem cells in the Go phase from mouse bone marrow was established. Bone marrow cells from 5-fluorouracil (5-FU)-treated mice were fractionated by Percoll density gradient. The cells with density between 1.063 and 1.075 were further separated into wheat germ agglutinin (WGA)-positive and -negative cells using fluorescent-activated cell sorter (FACS) after staining with fluorescein isothiocyanate-conjugated WGA (FITC-WGA). An assay for spleen colony-forming units (CFU-S) revealed that the WGA-positive cells (1 X 10(6)) produced 1380 CFU-S (about 150 times of the number in the original bone marrow cells) on day 12 (but no CFU-S on day 8), whereas the WGA-negative cells produced no CFU-S. Thus, the stem cells in the Go phase are found to be enriched 150 times in 5-FU-treated WGA-positive cells.

Evaluation of transplantation of autologous bone marrow stromal cells into the cerebrospinal fluid for treatment of chronic spinal cord injury in dogs.

OBJECTIVE To evaluate effects of transplantation of bone marrow stromal cells (BMSCs) into the CSF for the treatment of chronic spinal cord injury in dogs that had not responded by 1 month after decompressive surgery. ANIMALS 23 dogs. PROCEDURES Dogs with paraplegia and loss of nociception in the pelvic limbs for at least 1 month after decompressive surgery were assigned to transplantation or control groups. Dogs in the transplantation group received BMSCs injected into the CSF 1 to 3 months after decompressive surgery. Dogs in the control group did not receive additional treatments. Improvements in gait, proprioceptive positioning, and nociception were evaluated by use of the Texas Spinal Cord Injury Scale for ≥ 6 months after BMSC transplantation. RESULTS 6 of 10 dogs in the transplantation group regained the ability to walk, whereas only 2 of 13 dogs in the control group regained the ability to walk. Scores for the Texas Spinal Cord Injury Scale in the transplantation group were significantly higher than scores in the control group at the endpoint of the study (6 months after BMSC transplantation or after decompressive surgery for the transplantation and control groups, respectively). Only 1 dog (transplantation group) recovered nociception. All dogs from both groups had fecal and urinary incontinence. No complications were observed in relation to BMSC transplantation. CONCLUSIONS AND CLINICAL RELEVANCE Injection of BMSCs into the CSF caused no complications and could have beneficial effects on pelvic limb locomotion in dogs with chronic spinal cord injuries.

Analyses of Dendritic Cell Subsets in Pregnancy

PROBLEM: Changes in the frequency of dendritic cell (DC) subsets in the peripheral blood were analyzed as pregnancy progressed, and the effects of human chorionic gonadotropin (hCG) on myeloid and lymphoid DC subsets were phenotypically and functionally examined.

Marked Increase in Number of Dendritic Cells in Autoimmune-Prone (NZW × BXSB)F1 Mice with Age

Here, we report that the number of CD11c+CD3− B220− cells increases in autoimmune‐prone male (NZW × 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‐1α), NK 1.1, and CD95 (Fas) are upregulated in comparison with normal mice, while the expression of CD8α, 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.