Miyoko Yoshida

Estrogen-Mediated Endothelial Progenitor Cell Biology and Kinetics For Physiological Postnatal Vasculogenesis

Estrogen has been demonstrated to promote therapeutic reendothelialization after vascular injury by bone marrow (BM)–derived endothelial progenitor cell (EPC) mobilization and phenotypic modulation. We investigated the primary hypothesis that estrogen regulates physiological postnatal vasculogenesis by modulating bioactivity of BM-derived EPCs through the estrogen receptor (ER), in cyclic hormonally regulated endometrial neovascularization. Cultured human EPCs from peripheral blood mononuclear cells (PB-MNCs) disclosed consistent gene expression of ER &agr; as well as downregulated gene expressions of ER &bgr;. Under the physiological concentrations of estrogen (17&bgr;-estradiol, E2), proliferation and migration were stimulated, whereas apoptosis was inhibited on day 7 cultured EPCs. These estrogen-induced activities were blocked by the receptor antagonist, ICI182,780 (ICI). In BM transplanted (BMT) mice with ovariectomy (OVX) from transgenic mice overexpressing &bgr;-galactosidase (lacZ) regulated by an endothelial specific Tie-2 promoter (Tie-2/lacZ/BM), the uterus demonstrated a significant increase in BM-derived EPCs (lacZ expressing cells) incorporated into neovasculatures detected by CD31 immunohistochemistry after E2 administration. The BM-derived EPCs that were incorporated into the uterus dominantly expressed ER &agr;, rather than ER &bgr; in BMT mice from BM of transgenic mice overexpressing EGFP regulated by Tie-2 promoter with OVX (Tie-2/EGFP/BMT/OVX) by ERs fluorescence immunohistochemistry. An in vitro assay for colony forming activity as well as flow cytometry for CD133, CD34, KDR, and VE-cadherin, using human PB-MNCs at 5 stages of the female menstrual-cycle (early-proliferative, pre-ovulatory, post-ovulatory, mid-luteal, late-luteal), revealed cycle-specific regulation of EPC kinetics. These findings demonstrate that physiological postnatal vasculogenesis involves cyclic, E2-regulated bioactivity of BM-derived EPCs, predominantly through the ER&agr;.

Two-color flow cytometric analysis of thymic lymphocytes from patients with myasthenia gravis and/or thymoma

Phenotypic characteristics of lymphocyte components of the thymus were determined by two-color flow cytometry using monoclonal antibodies to T cell-differentiation antigens and activation antigens, and B cells in eight myasthenia gravis (MG) thymuses and eight thymomas including four associated with MG to clarify the roles of thymus in the diseases. Fifteen normal thymuses and four thymuses from non-MG patients were used as controls. Phenotypes of lymphoid components in thymoma resembled those in normal thymuses, in which the majority of lymphoid cells were immature (common) thymocytes (CD4+CD8+ or CD1+CD3-). The proportions of immature thymocytes, however, were relatively higher and those of mature thymocytes (CD4+CD8-, CD4-CD8+, CD1-CD3+ or CD2+CD3+) were lower in thymomas than normal thymuses. The results speculate that functions which support further differentiations of immature thymocytes into mature thymocytes are deficient in neoplastic epithelial cells of thymoma. In thymomas, therefore, occasional T cells might escape from the thymic surveillance system which eliminates auto-reactive T cells. Between thymomas associated with MG and those without, however, no significant difference was found in the proportions of thymocytes at various stages of maturation. In nonthymomatous thymuses from MG patients, an increase of both B cells (CD2-DR+ or CD2-CD21+) and activated T cells (CD2+DR+ or CD2+IL-2R+) was observed. Furthermore, immature thymocytes were significantly decreased and mature thymocytes were increased in the MG thymuses, as compared with normal thymuses, non-MG thymuses, and thymomas. These alterations were relevant to the histological findings observed in MG thymuses such as thymic involutions and germinal center formations: decreased immature thymocytes are considered to reflect thymic involutions, while germinal centers, in which lymphocytes from peripheral lymphoid organs are activated, are responsible for the increase of B cells, activated T cells, and mature T cells. In conclusions, our results suggest that neoplastic epithelial cells of thymomas are functionally deficient and that MG thymuses are immunologically active and resemble peripheral lymphoid organs.

Clinical significance of LEA-1 expression in adult acute myeloid leukemia

In this study, we examined expressions of several adhesion molecules (AdMs), i.e. leukocyte function antigen-1 (LFA-1: CD11a/CD18), Hermes homing receptor (CD44) and intercellular adhesion molecule-1 (ICAM-1: CD54), on leukemia cells from 51 adult patients with newly diagnosed acute myeloid leukemias (AMLs) to elucidate clinical significance of these AdM expressions. Those expressions in lymphoid malignancies have been correlated with tumor evolutions, but CD44 was detected in all the AML cases examined and CD54 expression did not associate with their clinical characteristics or outcomes. However, we found that LFA-1 expressions significantly correlated with splenomegaly, resistance to induction chemotherapies and short survival periods in AML patients.

LYMPHOCYTE SUBSETS OF THE PERIPHERAL BLOOD IN MYASTHENIA GRAVIS DETERMINED BY TWO-COLOR FLOWCYTOMETRY

Lymphocyte subsets of the peripheral blood in 43 patients with myasthenia gravis (MG) were determined by two-color flow cytometry using a number of monoclonal antibodies. In the MG patients without thymectomy (Tx) and prednisolone (PSL) treatment, lymphocyte counts, B-cells, CD4+ cells and their subsets were normal, but numbers of T-cells, CD8+ cells and CD8+ CD 11-subsets were significantly decreased. Furthermore, proportions of activated cells in T-cells, CD 16+ Leu7- and CD16+ Leu7+ NK subsets were significantly high in the patients. The changes in T-cells, CD8+ cells and activated T-cells were less marked in the MG patients than Sjögrens syndrome (SS) used as a disease control. Contrary to MG patients, lymphocyte counts, CD4+ cells and their subsets were decreased, and the proportions of B-cells were high in SS patients. These results suggest altered immunologic conditions, immunologically active and deficient conditions, in both diseases, although the alterations were more prominent in SS than MG. PSL treatments and Tx significantly altered the lymphocyte profiles: PSL decreased lymphocytes, B-cells, T-cells, CD4+ cells and their subsets, while the proportions of CD8+ cells were increased. The changes were compatible with the known immunosuppressive effects of PSL. After Tx, lymphocytes and B-cells decreased, but the proportions of T-cells, CD8+ cells and their subsets, and NK cells subsets returned toward normal. CD4+ CD8+ cells were not increased in MG patients, and the cells did not decrease after Tx. Some of these observations might be relevant to clinical effects of Tx, although the mechanism responsible for these changes is still unknown.

A Novel Variant of B-Lymphoid Leukemia Expressing Kappa/Lambda Light Chains

We studied a patient with an indolent leukemia which behaved similarly to chronic lymphocytic leukemia (CLL). Leukemic cells, however, showed larger cell diameters and lower nuclear/cytoplasmic ratios than typical CLL cells, and contained numerous cytoplasmic vacuoles. The cells also demonstrated some morphologic characteristics of hairy cell leukemia. Furthermore, flow-cytometric analysis demonstrated a distinct population of κ/λ double-positive tumor cells, as well as κ single and λ single populations. Southern blot analysis confirmed rearranged bands for both light chains with a monoclonal heavy chain rearrangement. Despite a decision not to treat this asymptomatic patient, disease progression was not observed. This case may represent a unique variant of B lymphoid leukemia. Possible mechanisms of abnormal light chain expression are discussed.

Flow Cytometric Analysis of T-Cell-Rich B-Cell Lymphoma

Flow Cytometric Analysis of T-Cell-Rich B-Cell Lymphoma H. Hiroshi Kawada S. Shigeki Watanabe M. Miyoko Yoshida R. Ryuki Fukuda N. Nobumasa Kobayashi A. Akira Masumoto Y. Yoshiaki Ogawa Y. Yoshiaki Ohbayashi S. Shuji Yonekura Y. Yukinobu Ichikawa Fourth Department of Internal Medicine, Tokai University School of Medicine, and Blood Transfusion Center of Tokai University Hospital, Bohseidai, Isehara, Kanagawa, Japan

Spontaneous and anti-Fas antibody-mediated apoptosis of the peripheral blood lymphocytes in Sjögren’s syndrome, rheumatoid arthritis and systemic lupus erythematosus

The objective of this study was to clarify possible roles of lymphocyte apoptosis in autoimmune rheumatic diseases. Spontaneous and anti-Fas antibody-mediated apoptosis of peripheral blood (PB) lymphocytes were measured by a flow cytometric method using propidium iodide in primary Sjögren’s syndrome (SS), rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Spontaneous apoptosis levels were significantly higher in 26 SLE patients than in 19 healthy controls. The apoptosis levels were not significantly different between 19 primary SS patients or 28 RA patients and the controls, but high apoptosis levels were observed in some of the patients. The apoptosis of PB lymphocytes was also enhanced in seven SS patients with RA and 16 SS patients with SLE. Anti-Fas antibody could induce apoptosis of PB lymphocytes from both healthy controls and patients’ groups. The antibody-mediated apoptosis levels were higher in RA, SLE and secondary SS patients with RA or SLE, but not in primary SS patients. Each patients’ group was further divided into two groups according to their mean apoptosis levels: patients with high and those with low spontaneous or antibody-mediated apoptosis levels. Clinical variables reflecting disease activity for each disease were then compared between the two groups. Serum rheumatoid factor (RF) and anti-SS-A/Ro antibody titers were higher in the RA or primary SS patients with high spontaneous apoptosis levels than those with low apoptosis levels, respectively. When all the patients’ groups were evaluated together, the spontaneous apoptosis levels negatively correlated with PB lymphocyte counts. In addition, the spontaneous apoptosis levels were decreased by the co-culture of PB lymphocytes with interleukin-2 (IL-2: 100 U/ml) in most individuals including patients and healthy controls. We conclude that spontaneous apoptosis of PB lymphocytes was enhanced in SLE and secondary SS patients. Production of RF or anti-SS-A/Ro antibodies was associated with enhanced apoptosis of PB lymphocytes in RA or primary SS patients. The apoptosis levels were related with lymphocytopenia observed in the patients examined, although various factors including IL-2 seemed to be protective against the apoptosis of circulating lymphocytes. Furthermore, anti-Fas antibody induced apoptosis of PB lymphocytes from the healthy controls and patients, and the antibody-mediated apoptosis levels were high in RA, SLE and secondary SS patients.