Biomedical subjects

M Inaba

Publications and source records attributed to M Inaba.

At least 37 records · Page 2Linked to original sources

PubMed1992

[Vitamin D3].

With the application of biochemical technique came the discovery that vitamin D is metabolized sequentially to 25-hydroxyvitamin D in liver and then to 1.25-dihydroxyvitamin D (1.25D), the hormonal form of vitamin D, in kidney. The latter process is strictly controlled with the demand of calcium and phosphorus. The produced 1.25D binds its specific receptor probably located in the nuclei. With the application of modern tools of molecular biology came the discovery that vitamin D receptor (VDR), which belongs to steroid receptor family, acquires increased affinity for DNA when it binds to 1.25D and then VDR complexes bind to DNA with its two zinc-finger projections. VDR complexes recognize vitamin D responsive element on DNA, two tandemly repeated hexanucleotide sequences separated by three base pairs. Furthermore, to interact with the VDRE, VDR complexes require nuclear accessory factor, which might be retinoid X receptor (RXR). As above, molecular biology helps us to extend our knowledge in the field of vitamin D.

Calcitriol↗

PubMed1992

Detection of equine immunoglobulin-secreting cells by a plaque assay.

A protein A-hemolytic plaque assay was applied to detect immunoglobulin (Ig)-producing cells in horse peripheral blood, using pokeweed mitogen as a B lymphocyte activator. A maximum number of Ig-secreting cells was obtained when horse peripheral blood lymphocytes were cultured in a medium containing horse serum. The number of Ig-secreting cells in young horses (2 years old) was lower than that in adult horses (6 to 23 years old). In addition, the plaque formation was unchanged from blood samples kept at 4 degrees C for 24 hours, while blood samples kept for 72 hours did not yield plaques. These results indicate that the plaque assay is a reliable and useful method for detecting Ig-secreting cells in the peripheral blood of the horse.

Animals↗

PubMed1992

Enrichment of murine bone marrow natural suppressor activity in the fraction of hematopoietic progenitors with interleukin 3 receptor-associated antigen.

Natural suppressor (NS) activity has been identified in several sites of active hematopoiesis. In this study we characterized NS activity in murine bone marrow (BM) using monoclonal antibodies (mAbs) to interleukin 3 (IL-3) receptor-associated antigen (IL-3RAA) and various cytokines that exert a strong influence on hematopoiesis or lymphocyte interaction. NS activity of BM cells of relatively low density was enhanced by IL-3 or granulocyte-macrophage colony-stimulating factor (GM-CSF). When the BM cells were separated into IL-3RAA+ cells and IL-3RAA- cells, the IL-3RAA+ cells demonstrated potent NS activity, whereas IL-3RAA- cells had either no or weak NS activity. The IL-3RAA+ cells showed non-T- and non-B-cell phenotype and had high affinity to wheat germ agglutinin (WGA), a marker for hematopoietic progenitors. In assays for hematopoietic activity, it appeared that the early differentiating progenitors (day 8 spleen colony-forming units [CFU-S], granulocyte-macrophage colony-forming units [CFU-GM]) were enriched in the IL-3RAA+ cell population, whereas more immature multipotent progenitors (day 12 CFU-S, granulocyte erythrocyte macrophage megakaryocyte colony-forming units [CFU-GEMM]) were contained in the IL-3RAA- cell population. Both suppressor cells and IL-3RAA+ cells spontaneously developed from the IL-3RAA- cell population. These findings suggest that NS cells in murine BM are early hematopoietic progenitors and are probably committed to the myeloid lineage. Hybridoma cells established between the IL-3RAA+ cells and BW5147 cells produced suppressor factor(s). This finding suggests that the NS cells produce soluble mediator(s) that may be responsible for their suppressive action.

Animals↗

PubMed1991

Analyses of acute graft-versus-host-like reaction in [MRL/lpr----MRL/+] chimeric mice using MRL/lpr-Thy-1. 1 congenic mice.

When MRL/Mp(-)+/+(MRL/+) mice are lethally irradiated and then reconstituted with MRL/Mp-lpr/lpr (MRL/lpr) bone marrow and/or spleen cells, these MRL/+ mice develop "lpr-GVHD" which is similar to acute graft-versus-host disease (GVHD). Using a Thy-1 congenic strain of MRL/lpr mice (MRL/lpr-Thy-1.1), we analyzed T cell subpopulations in the thymus and spleen of MRL/+ mice suffering from lpr-GVHD. lpr-GVHD was induced in MRL/+ mice by transplantation of bone marrow cells (BMC) from MRL/lpr-Thy-1.1 mice; severe lymphocyte depletion associated with fibrosis was observed in the spleens after 7 weeks of bone marrow transplantation (BMT). Thymocytes of the host MRL/+ thymus were replaced with donor-derived cells from the early stage of lpr-GVHD, whereas in the spleen, a small number of host T cells (Thy-1.2+) (4-5%) were retained until the late stage of lpr-GVHD. Donor-type (Thy-1.1+) T cell subsets were not different from those of nontreated MRL/+ mice in the thymus, whereas in the spleen. CD8+ T cells (Thy-1.1+) reached a peak at 5 weeks after BMT, and CD4+ T cells (Thy-1.1+), a peak at 6 weeks. The elimination of T cells from MRL/lpr BMC had no evident effect on the prevention of lpr-GVHD. T cell subpopulations showed a similar pattern to GVHD elicited by MHC differences. Analyses of autoreactive T cells expressing V beta 5 or V beta 11 revealed that autoreactive T cells were deleted from the peripheral lymph nodes. Interestingly, the levels of IgG anti-ssDNA antibodies markedly increased, and both IgM and IgG rheumatoid factors slightly increased 5 to 7 weeks after BMT. These findings are discussed in relation to not only GVHD elicited by MHC differences but also autoimmune diseases.

Animals↗

PubMed1991

Analyses of T-cell differentiation from hemopoietic stem cells in the G0 phase by an in vitro method.

Using differential radiation sensitivity of components of mouse embryonal thymus, an in vitro method for studying T-cell differentiation from hemopoietic stem cells (HSCs) in the G0 phase was established. Intrathymic T-cell precursors present in embryonal thymus were found to be quite radioresistant (up to 20 Gy), and consequently 25-Gy-irradiated embryonal thymic lobes were used. Thymic lobes (25-Gy irradiated) taken from mouse fetuses (gestation day 15) were placed in Millipore-HA culture plates supported on squares of gelatin foam sponge in 24-well culture plates in which neonatal thymus stromal cells were cultured. HSCs (10(5) cells per well) in the G0 phase were added to these thymic lobes and cocultured at 37 degrees C in a 5% CO2/95% air incubator. Half the culture medium was changed every week. After 3 weeks, a large number of colonies had formed. Immunohistochemical studies and fluorescence-activated cell sorter analyses revealed that the colonizing cells regularly develop and exhibit surface markers characteristic of T cells (Thy-1, IL-2R, L3T4, Lyt-2, etc.). In situ hybridization analyses revealed that mRNA expression for T-cell receptor (TCR) beta chains occurred within colonizing cells. Using a monoclonal antibody (F23.1), expression of TCR beta-chain variable domain (V beta 8) on the surface of these developing T cells was demonstrated. These cells responded to interleukin 2 and/or anti-CD3 monoclonal antibody, indicating functional T cells. This method will be useful in studying T-cell differentiation pathways from pluripotent HSCs and in clarifying the mechanisms involved in negative and positive selection of T cells within the thymus.

Animals↗

PubMed1991

Distinct mechanisms of neonatal tolerance induced by dendritic cells and thymic B cells.

To assess the role of different types of antigen-presenting cells (APC) in the induction of tolerance, we isolated B cells, macrophages, and dendritic cells from thymus and spleen, and injected these into neonatal BALB/c mice across an Mls-1 antigenic barrier. One week after injection of APC from Mls-1-incompatible mice or from control syngeneic mice, we measured the number of thymic, Mls-1a-reactive, V beta 6+ T cells and the capacity of thymocytes to induce a graft-vs.-host (GVH) reaction in popliteal lymph nodes of Mls-1a mice. Injection of thymic but not spleen B cells deleted thymic, Mls-1a-reactive V beta 6+ T cells and induced tolerance in the GVH assay. The thymic B cells were primarily of the CD5+ type, and fluorescence-activated cell sorter-purified CD5+ thymic B cells were active. Injection of dendritic cells from spleen or thymus also induced tolerance, but the V beta 6 cells were anergized rather than deleted. Macrophages from thymus did not induce tolerance. Dendritic cells and thymic B cells were also effective in inducing tolerance even when injected into Mls-, major histocompatibility complex-incompatible, I-E- mice, but only thymic B cells depleted V beta 6-expressing T cells. Therefore, different types of bone marrow-derived APC have different capacities for inducing tolerance, and the active cell types (dendritic cells and CD5+ thymic B cells) can act by distinct mechanisms.

Animals↗

PubMed1991

Characteristics of the 25-hydroxyvitamin D3- and 1,25-dihydroxyvitamin D3-24-hydroxylase(s) from HL-60 cells.

1,25-Dihydroxyvitamin D3 induces the human promyelocyte leukemia cell line, HL-60, to differentiate into macrophages/monocytes via a steroid-receptor mechanism. This system is a relevant one for an investigation of the molecular mechanism of 1,25-dihydroxyvitamin D3. We have now examined the effect of 1,25-dihydroxyvitamin D3 on the induction of 1,25-dihydroxyvitamin D3- and 25-hydroxyvitamin D3-24-hydroxylase activities in HL-60 cells. The hydroxylase activities were measured by a periodate-based assay, which was validated by comparison with well-established HPLC analysis. HPLC analysis also suggested that 1,25-dihydroxyvitamin D3 induces a 23-hydroxylase in addition to the 24-hydroxylase. 1,25-Dihydroxyvitamin D3- and 25-hydroxyvitamin D3-24-hydroxylase activities were stimulated as early as 4 h after the addition of 10(-7) M 1,25-dihydroxyvitamin D3 and became maximal by 24 h. 1,25-Dihydroxyvitamin D3 stimulated both activities in a dose-dependent manner up to 10(-6) M. The Km of 24-hydroxylase for 1,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 were 2 x 10(-8) M and 5.2 x 10(-7) M, respectively. Cycloheximide (5 microM) inhibited 1,25-dihydroxyvitamin D3-mediated stimulation of 24-hydroxylase activity. Other differentiation inducers, such as retinoic acid and phorbol ester, did not induce either activity. 1,25-Dihydroxyvitamin D3-24-hydroxylase in HL-60 mitochondria was solubilized with 0.6% cholate and reconstituted with NADPH, beef adrenal ferredoxin, and beef adrenal ferredoxin reductase, each component being essential for 24-hydroxylase activity. These results strongly suggest that the 24-hydroxylase in HL-60 cells is a three-component cytochrome P450-dependent mixed-function oxidase.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗

PubMed1991

Antitumor activities and schedule dependence of orally administered MST-16, a novel derivative of bis(2,6-dioxopiperazine).

We studied bioavailability, treatment schedule dependence, and therapeutic efficacy of orally administered MST-16, a novel derivative of bis(2,6-dioxopiperazine), against murine tumors and human tumor xenografts. The rate of its intestinal absorption was about 50%, and it was immediately metabolized to its parent compound, ICRF-154. Therapeutic efficacy of MST-16 was heavily dependent on the treatment schedule: 9 daily oral administrations and treatment every 4 h on day 1 only were much more effective against s.c.-implanted L1210 leukemia than a single dose or five daily administrations giving the same total dose. Orally administered MST-16 showed potent life-prolonging effects (196%, 219% and 148%) in mice inoculated i.p. with P388, L1210 leukemia, and C-26 colon adenocarcinoma, respectively, but had no effect on B16 melanoma inoculated in the same way. MST-16 inhibited more than 80% growth of Lewis lung carcinoma, B16 melanoma, and C-38 colon adenocarcinoma implanted s.c., but had only a minor effect on M5076 fibrosarcoma. Lung metastasis of Lewis lung carcinoma was also effectively suppressed. Furthermore, MST-16 significantly inhibited growth of human colon, lung and breast cancers implanted s.c. in nude mice. We also made a kinetic analysis of the in vitro cell-killing effect by ICRF-154, the active form of MST-16 in vivo. It demonstrated a cell cycle phase-specific and time-dependent action, providing a reasonable explanation for the schedule-dependent therapeutic effect of MST-16.

Administration, Oral↗

PubMed1991

Ontogeny of thymic B cells in normal mice.

Ontogeny of thymic B cells and their surface characteristics were analyzed using monoclonal antibodies (mAbs) against B220 molecules (CD45, CD45R). A small number of B cells were detected in fetal thymus on Gestation Day 14 (approximately 3.5% of the low-density fraction). Similarly, the percentage of B cells in the low-density fraction was 3.2% on Gestation Day 18, and 3.5% on Day 1 after birth. These were the same level as that of adult mice. CD5+ B cells, which form the major population of thymic B cells, were also found in the fetal life (0.5% on Day 14 and 2.2% on Day 16 in the low-density cells). The percentage of CD5+ B cells in B cell-enriched fraction was about 65% on Day 1 after birth, which is the same level as that in adult mice. These results indicate that a small number of B cells or cells in the B-cell lineage are present in the fetal thymus and also suggest the importance of these thymic B cells in the negative selection of T cells during early developmental stages.

Animals↗

PubMed1991

Neonatal tolerance induction in the thymus to MHC-class II-associated antigens. IV. Significance of intrathymic chimerism of blood-born Ia+ cells in Mls tolerance.

The significance of thymus cell chimerism in the induction and maintenance of tolerance was investigated. Mls-1b BALB/c mice were neonatally tolerized by the intravenous administration of either bone marrow (BM) cells or peritoneal cavity (PerC) cells from Mls-1b/a (BALB/c x AKR) F1 mice. Tolerance was long-lasting in the BM cell group, but transient in the PerC cell group, probably because PerC cells lack hemopoietic stem cells required for a continuous supply of tolerance-inducing cells. The degree of anti-Mls-1a responsiveness of these BALB/c thymus cells was correlated with the degree of intrathymic distribution of the inoculated F1 cells. The effect of BM cell inoculation, resulting in a year-long deletion of Mls-1a-reactive V beta 6-bearing T cells is in marked contrast to that of PerC cell inoculation which causes only a transient loss of V beta 6+ mature thymocytes (for about 1 week after birth). This functional profile of the tolerant state correlates well with the degree and persistence of the intrathymic presence of F1 type Ia+ cells. The long-lasting presence of donor-derived cells throughout the thymus tissue in the BM cell group is also in marked contrast to the early disappearance of Ia+ cells (within 2-3 weeks) from the cortex and then from the medulla in the PerC cell group, although these Ia+ cells were once spread throughout the thymus tissue 4 days after the tolerance-inducing cell inoculation. Taken together with a failure to induce consistent unresponsiveness to Mls-1a determinants in Mls-1b thymocytes regenerating in Mls-1a-thymic epithelial environments, all the above data indicate that intrathymic chimerism caused by hemopoietic stem cell-derived MHC-class II-bearing cells is a requisite for the induction and maintenance of unresponsiveness by means of clonal deletion in experimentally as well as naturally induced tolerance to Mls determinants.

Animals↗

PubMed1991

Hereditary high-potassium erythrocytes with high Na, K-ATPase activity in Japanese shiba dogs.

The sodium (Na) and potassium (K) concentrations and Na, K-ATPase activity were examined in erythrocytes from 24 Japanese shiba dogs and 79 dogs of 24 other breeds. Eleven of the shibas had erythrocytes with high K and low Na concentrations, together with high Na, K-ATPase activity (HK RBCs), while red cells from the remaining shibas and all of the other breeds examined showed low K and high Na concentrations, with no enzyme activity (LK RBCs). The concentration of reduced glutathione in HK RBCs was about five times that in LK RBCs. All the findings from HK shibas were in good agreement with those from HK mongrel dogs found in Japan previously. Since the shiba is a Japanese breed of dog, the results of the present study strongly suggest that the gene for HK RBCs may be inherent in dogs indigenous to Japan, particularly in shiba dogs.

Animals↗

PubMed1991

Effect of alpha 1-blockade on diminished forearm blood flow in diabetics.

An increased risk of atherosclerotic disease has been reported in patients with diabetes mellitus. The present study was therefore designed to determine forearm blood flow (FBF) in patients with essential hypertension or those with diabetes mellitus with or without hypertension. FBF determined by venous occlusion plethysmography decreased with age in controls as well as in patients with essential hypertension, whereas FBF in diabetics was significantly lower irrespective of age or blood pressure. As a result, vascular resistance was significantly higher in diabetics than in controls or patients with essential hypertension. Glycemic control in normotensive diabetics during 3 weeks significantly augmented a diminished FBF. alpha 1-Blockade by oral administration of 1 mg of prazosin also augmented the diminished FBF in diabetics, in association with a significant decrease in mean blood pressure and vascular resistance. These results suggest that FBF may be a simple and useful index for determining arterial and/or venous distensibility, and that alpha 1-blocker therapy, in addition to glycemic control, may be a first-line antihypertensive treatment for diabetics with associated hypertension.

Administration, Oral↗

PubMed1991

Effect of dietary calcium on renal prostaglandins.

The present study was designed to clarify the possible role of renal prostaglandins (PGs) on blood pressure (BP) regulation during calcium (Ca) restriction or supplementation. Twelve normotensive women with a mean age of 21.2 years participated in the study. After 1 week of normal Ca intake (mean +/- SE, 536 +/- 2 mg/day), a low-Ca diet (163 +/- 1 mg/day) was given for a further 1 week. Additional asparagine Ca (3 g as Ca/day) was also given to half of the subjects. BP, heart rate, and serum total and ionized Ca concentrations were measured at the end of each period. Levels of Ca, sodium, PGE2, 6-keto-PGF1 alpha and thromboxane (TX) B2 excreted into urine were also determined. The plasma level of ionized Ca was significantly increased without any change in total Ca in both groups. Low and high Ca intake decreased and increased urinary Ca excretion by 28% and 56%, respectively. BP was not altered after Ca deprivation or loading. However, urinary PGE2 excretion was significantly augmented from 668.9 +/- 68.1 to 959.7 +/- 183.1 ng/day by Ca loading, whereas Ca deprivation decreased PGE2 excretion (695.4 +/- 108.1 to 513.2 +/- 55.2 ng/day). No changes were observed in 6-keto-PGF1 alpha or TXB2 urinary excretion. These results suggest that renal PGE2 synthesis is stimulated or decreased by 1-week Ca loading or deprivation, indicating a possible antihypertensive role of renal PGE2 during high-Ca intake in hypertensives.

6-Ketoprostaglandin F1 alpha↗

PubMed1991

Characterization of B cells in human thymus.

The surface characteristics of B cells present in the human thymus were investigated. Cytofluorometrical and immunohistological studies, using anti-human IgM or anti-B cell monoclonal antibodies (mAbs; anti-Leu 12Ab, anti-Leu 16Ab, or L26), revealed that a small number of B cells are present in the human thymus. The thymic B cells were detected only in a low-density cell population, whereas in a high-density cell fraction, only T cells were found. In 15 cases, all of which the thymi were histologically normal, the percentages of B cells in the low-density fraction were 0.28% to 50% (6.8% in average), and Leu 1+ (CD5+) B cells in the low-density fraction were 0.1% to 26% (3.5% in average); approximately 50% of the thymic B cells were Leu 1+ B cells. These results indicate that B cells, especially Leu 1 (CD5)+ B cells, are also present in the human thymus, as suggested from our previous reports on mice.

Adolescent↗

PubMed1991

Monoclonal antibodies against a preadipose cell line (MC3T3-G2/PA6) which can support hemopoiesis.

Monoclonal antibodies against a mouse preadipose cell line (MC3T3-G2/PA6:PA6), which can support hemopoiesis by direct cell-to-cell interaction, were produced and characterized. The antibodies react with PA6 but not PA6-M (a mutant cell line) which has the capacity neither to contact with hemopoietic stem cells (HSCs) nor to support hemopoiesis. Endosteal cells in the bone marrow show positive staining to these antibodies. They inhibit pseudoemperipolesis of PA6 to HSCs, resulting in a significant decrease in hemopoietic cell number. These findings suggest that the monoclonal antibodies bind to the stromal cell receptors for HSCs and block the binding of HSCs to stromal cells leading to suppression of hemopoiesis.

Adipose Tissue↗

PubMed1991

Provirus integration at the 3' region of N-myc in cell lines established from thymic lymphomas spontaneously formed in AKR mice and a [(BALB/c x B6)F1----AKR] bone marrow chimera.

Among 18 thymic leukemia cell lines which have been established from spontaneous thymic lymphomas in AKR mice as well as in bone marrow chimeras which were constructed by transplanting allogeneic bone marrow cells into irradiated AKR mice, three proviral integration sites were identified; near c-myc, N-myc and pim-1 loci. No integration site specific for chimeric leukemia cell lines was found. In three thymic leukemia cell lines which contained rearranged N-myc genes, insertions of long terminal repeats (LTRs) of murine leukemia viruses were detected at 18 or 20 bp downstream of the translational termination codon. These results demonstrate that the 3' region of the N-myc gene is one of the integration targets for murine leukemia viruses in spontaneous thymic lymphomas. In these three cell lines, N-myc mRNA was stably transcribed and transcription of c-myc mRNA was down-regulated. The integrated murine leukemia viruses in AKR thymic leukemia were most likely AKV, though the DNA sequence of the LTR inserted in the genome of a leukemic cell line from [(BALB/c x B6)F1----AKR], CAK20, was different from LTRs of murine leukemia viruses so far reported.

Antigens, Surface↗

PubMed1991

Uremic serum contains humoral factor(s) larger than fifty kilodaltons which suppresses endothelin production in cultured endothelial cells.

Direct effects of human uremic serum on the production of endothelin-1 in cultured porcine endothelial cells were examined in this study. Uremic serum decreased the level of monomeric endothelin-1 secreted into the culture medium by endothelial cells. This effect occurred at a transcriptional step because uremic serum decreased the endothelin-1 mRNA level in those cells. For the partial characterization of this inhibitory activity, uremic serum was fractionated with a centricut column. Uremic serum contains humoral factor(s) larger than 50 kD which suppress the endothelin-1 mRNA level in cultured endothelial cells.

Animals↗

PubMed1991

Significance of proteolytic activity in 1,25-(OH)2D3-induced differentiation of HL-60 cells.

Two types of HL-60 cells, 1,25-(OH)2D3-responsive ATCC HL-60 cells and 1,25-(OH)2D3-resistant LG HL-60 cells were used. Despite the presence of enough amounts of normal 1,25-(OH)2D3 receptors, only 22% of LG cells matured after a 4-day treatment with 10(-7) M 1,25-(OH)2D3, while 80% of ATCC cells differentiated. However, 1,25-(OH)2D3 inhibited the proliferation of LG cells to the same degree as that of ATCC cells. 1,25-(OH)2D3 also induced (1) the ability to metabolize 1,25-(OH)2D3 to 1,24,25-(OH)3D3, and (2) up-regulation of the 1,25-(OH)2D3 receptor in LG as well as ATCC cells. Furthermore, the proportion of mature LG cells was 78% after treatment with 10(-7) M 1,25-(OH)2D3 for the first 48 h and 10(-7) M dbcAMP for the second 48 h, which was greater than that when treated only with 10(-7) M dbcAMP for the second 48 h (24.2%). These results indicate that 1,25-(OH)2D3 receptor complexes function normally in LG cells at commitment step in cell differentiation. ATCC cells had a serine proteinase to destroy specific 1,25-(OH)2D3-binding activity of the unoccupied receptor and digest 53-kD receptor to a small fragment with a MW of 16.3 kD, while not affecting the level of the specific binding of the occupied receptor. Other cells, such as murine leukemia cells, M1, and human chronic myeloid leukemia cells, the differentiation of which is induced by 1,25-(OH)2D3, seemed to have the same type of proteinase, suggesting the physiological significance of this proteinase in 1,25-(OH)2D3-induced cell differentiation.

Bucladesine↗