Shinryu Lee

Major Histocompatibility Complex Restriction Between Hematopoietic Stem Cells and Stromal Cells In Vitro

We have previously found that a significant number of hematopoietic progenitors accumulate in engrafted bones with the same major histocompatibility complex (MHC) as the transplanted bone marrow cells. In the present study, to further clarify the MHC restriction between hematopoietic stem cells (HSC) and microenvironment, we carried out cobblestone colony formation assays by culturing HSCs with MHC‐matched or ‐mismatched stromal cell monolayers. The formation of cobblestone colonies under MHC‐mismatched stromal cells significantly decreased in comparison with MHC‐matched stromal cells. However, the decrease in cobblestone colony formation under MHC‐mismatched stromal cells was not significant when using MHC class I‐deficient HSC or stromal cells. Taken together with the results using B10 congenic strains, it is suggested that the MHC preference is restricted by MHC class Ia molecules. Treatment with monoclonal antibodies (mAbs) against MHC class Ia molecules of stromal cell phenotypes significantly enhanced the cobblestone colony formation, whereas treatment with mAbs against HSC phenotypes significantly inhibited it. The expression of cytokines to promote hematopoiesis was enhanced by the mAbs against stromal cell phenotypes. The enhancement of cytokine expression was also observed when stromal cells and HSCs were MHC‐matched. These results suggest that signaling via the MHC molecules augments stromal cell activity and elicits the MHC restriction.

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.

Magnetic Resonance Determination of Tumor Thickness as Predictive Factor of Cervical Metastasis in Oral Tongue Carcinoma

Objectives To establish an accurate and reproducible means of measuring tumor thickness as a preoperative prognostic factor for cervical metastasis in oral tongue carcinoma.

Prevention of accelerated presbycusis by bone marrow transplantation in senescence-accelerated mice

A substrain of the senescence-accelerated mouse (SAM), the SAMP1 mouse, is an animal model for accelerated senescence including the age-related acceleration of both immunological dysfunction and hearing loss caused by the impairment of spiral ganglion cells. In the present study, we examine whether the accelerated presbycusis can be prevented by allogeneic BMT. Young SAMP1 (H-2k) mice were irradiated with 9 Gy and then reconstituted with bone marrow cells from normal BALB/c (H-2d) mice. Allogeneic BMT was found to prevent the development of immunological dysfunction, hearing loss, and apoptosis of spinal ganglion cells in SAMP1 mice. These findings indicate that some types of accelerated presbycusis do not result from defects in the cochlea, but do from defects in the hematopoietic stem cells (HSC) and immunocompetent cells derived from the HSC. If this is the case, either allogeneic BMT, which replaces abnormal HSC with normal HSC and reconstructs a normal immune system in the recipients, or autologous BMT using genetically modified bone marrow cells, could become a new strategy for the treatment of presbycusis. Bone Marrow Transplantation (2001) 28, 323–328.

Correlation between accelerated presbycusis and decreased immune functions

The aim of the current study is to analyze the relationship between presbycusis and the immune system, which is affected by pathogenic environments, and to devise a strategy for the prevention of presbycusis using the SAMP1 mouse, an animal model for accelerated senescence that shows both immunological dysfunction and hearing loss caused by the impairment of spiral ganglion cells in the cochlea. When these mice were bred in different pathogenic environments, we found that the development of age-related diseases such as presbycusis was delayed in the mice bred under clean conditions. Prednisolone administration showed no significant prevention of the development of presbycusis in the mice, suggesting that autoimmune mechanisms are not involved in the acceleration of presbycusis. It is conceivable that pathogen-induced infections impose a severe stress on the host, impairing the hosts immune functions. A reduction in the number of pathogens may therefore prevent the acceleration of the aging process. These findings suggest that not only the gene backgrounds but also immune functions affect the development of presbycusis in SAMP1 mice. Further studies into the relationship between systemic immune functions and the neuro-generation system may provide additional information about the treatment for age-related diseases.

Maintenance of systemic immune functions prevents accelerated presbycusis.

There is no effective therapy for progressive hearing loss such as presbycusis, the causes of which remain poorly understood because of the difficulty of separating genetic and environmental contributions. In the present study, we show that the age-related dysfunctions of the systemic immune system in an animal model of accelerated presbycusis (SAMP1, senescence-accelerated mouse P1) can be corrected by allogeneic bone marrow transplantation (BMT). We also demonstrate that this presbycusis can be prevented; BMT protects the recipients from age-related hearing impairment and the degeneration of spiral ganglion cells (SGCs) as well as the dysfunctions of T lymphocytes, which have a close relation to immune senescence. No donor cells are infiltrated to the spiral ganglia, confirming that this experimental system using BMT is connected to the systemic immune system and does not contribute to transdifferentiation or fusion by donor hematopoietic stem cells (HSCs), or to the direct maintenance of ganglion cells by locally infiltrated donor immunocompetent cells. Therefore, another procedure which attempts to prevent the age-related dysfunctions of the recipient immune system is the inoculation of syngeneic splenocytes from young donors. These mice show no development of hearing loss, compared with the recipient mice with inoculation of saline or splenocytes from old donors. Our studies on the relationship between age-related systemic immune dysfunctions and neurodegeneration mechanisms open up new avenues of treatment for presbycusis, for which there is no effective therapy.

Prevention of autoimmune hearing loss in MRL/lpr mice by bone marrow transplantation

We examined the effects of bone marrow transplantation (BMT) on immune-mediated inner ear diseases in MRL/Mp-lpr/lpr (MRL/lpr) mice, which manifest not only lupus nephritis but also sensorineural hearing loss (SNHL) at the age of 20 weeks. These mice were treated with cyclophosphamide (CY) and irradiation (5 Gy × 2), followed by the transplantation of bones plus bone marrow cells from allogeneic normal C57BL/6 mice at the age of 12 weeks. Hematolymphoid cells were reconstituted with donor-derived cells 3 months after BMT. Thus-treated MRL/lpr mice showed neither lupus nephritis nor SNHL even 24 weeks after BMT. No pathological findings were observed in either glomeruli or cochleae. These findings suggest that BMT can be used to prevent the development of autoimmune SNHL in MRL/lpr mice. Bone Marrow Transplantation (2000) 26, 887–892.

Induction of immune-mediated hearing loss in SCID mice by injection of MRL/lpr mouse spleen cells

Induction of immune-mediated hearing loss in SCID mice by injection of MRL/lpr mouse spleen cells The MRL/lpr mouse, which has a mutation in the Fas gene encoding a cell-surface receptor for apoptosis, shows an accumulation of abnormal immunocompetent cells and SLE-like disease. It has recently been reported that this mouse also manifests sensorineural hearing loss (SHL) with cochlear pathology at 20 weeks of age. We examined the effects of injecting MRL/lpr spleen cells on the development of SHL in severe combined immunodeficient (SCID) mice, which originally develop neither SHL nor cochlear pathology. Immune-mediated SHL and cochlear pathology were, indeed, transferred to the SCID mice by the injection of spleen cells from the MRL/lpr mice. These findings suggest that cell-mediated immunity is involved in the development of SHL and cochlear pathology.

Persistent tolerance induced after portal venous injection of allogeneic cells plus cyclophosphamide treatment.

The injection of allogeneic cells via the portal vein (p.v.) is known to reduce responses to donor-alloantigens. In the present study, we have obtained persistent tolerance across Mls and multiple minor histocompatibility complexes by p.v. preimmunization followed by the administration of cyclophosphamide (CY). A hundred percent survival of (BALB/c x DBA/2)F1 (CDF1) skin grafts for more than 200 days was observed when BALB/c mice were preimmunized with spleen cells of CDF1 (3 x 10(7)) via the p.v. and administered 300 mg/Kg CY 2 days after the p.v. injection. Comparable survival of the skin graft was observed when bone marrow cells instead of spleen cells were p.v. preimmunized. However, the survival rate was significantly decreased when LPS-stimulated blastic cells were p.v. preimmunized. Microchimerism has been observed in the liver, thymus, bone marrow and peripheral blood of recipients. V beta 6+ cells decreased in CD4+ cells of recipients of the p.v. preimmunization plus CY treatment. However, there was no difference in the decrease in V beta 6+ cells between recipients accepting the CDF1 skin grafts and recipients that had rejected the skin grafts. Furthermore, no intrathymic depletion of the V beta 6+ cells was observed. From these results, it is suggested that, rather than clonal deletion, other mechanisms such as clonal anergy or suppression may be involved in the induction of persistent tolerance after the p.v. preimmunization plus CY treatment.

Bone marrow transplantation as a strategy for the treatment of autoimmune hearing loss in MRL/Mp-lpr/lpr mice.

Sensorineural hearing loss (SNHL) has been reported to develop as a main part of or in combination with systemic and organ-specific autoimmune diseases. The aim of the current study is to treat autoimmune SNHL in MRL/Mp-lpr/lpr (MRL/lpr) mice, a murine model of systemic autoimmune disease, using allogeneic bone marrow transplantation (BMT), which replaces recipient bone marrow cells with bone marrow cells from a non-autoimmune-prone donor. The results indicate that BMT can be used to treat SNHL; cochlear pathology, serum autoantibodies and lupus nephritis are ameliorated. Therefore, it is conceivable that the autoimmune SNHL in the MRL/lpr mice results not from defects in the cochlea, including the stria vascularis, but from defects in the bone marrow, and BMT would therefore provide a curative effect on inner ear autoimmune dysfunction associated with systemic autoimmune diseases.