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Biomedical subjects

T Amagai

Publications and source records attributed to T Amagai.

At least 37 records · Page 2Linked to original sources
PubMed1988

Immortalization of murine leukocytes by oncogenes. II. Phenotypic characterization of transformants immortalized by v-src or Ha-ras oncogenes: expression of B220, a B-cell lineage specific antigen.

In the course of study to obtain murine dendritic cell lines using oncogenic retroviruses, we have established several immortalized cell lines with characteristics different from those of dendritic cells. The transformants were mainly round nonadherent cells, capable of growing in soft agar, and negative for nonspecific esterase activity. Profiles of cell surface antigens were examined by indirect immunofluorescence technique. The cell lines were positive for Fc receptor (2.4G2), J11d (J11d.2), and B220 (RA3-3A1/6.1) antigens and negative (or dull positive in small percentages) for Ia (M5/144.15.2), IL-2 receptor (3C7), Thy-1 (B5-5), Mac-1 (M1/70.-15.11.5), and macrophage (F4/80) antigens. They were negative for both surface and cytoplasmic immunoglobulins. Several clones were established from these transformant cell lines and cell surface antigens were examined. Antigenic profiles of these clones were very similar to those of the parental cell lines. Some of these clones, however, seemed to increase their Ia antigen expression. The results suggest that the transformants originated from early B-lineage cells.

Animals↗
PubMed1988

[Problems and difficulties in adapting period in infants and children after massive intestinal resection].

In the period of intestinal adaptation following massive resection of intestine, patients have several difficulties and problems. This study was done in 17 survivals of 31 infants and children received massive intestinal resection (residual intestine; less than 75 cm) between 1966-1987 to define these problems and difficulties. 1) Postoperative hepatic dysfunction occurred in 11 of the 17 survivals. The relationship between relapsing frequency of hepatic dysfunction and length of remaining ileal segment suggested that the reduced ileal length may be one of the etiologic factors of the hepatic dysfunction. 2) Evaluation of serum bile acid levels suggested that the disturbance of bile acid reabsorption and its rapid turn over might still remain even after catch-up growth of these patients. 3) Abnormal levels of serum minerals (Ca, InP, Mg, Zn and Cu) were observed in 4 cases during TPN, and these abnormalities disappeared by discontinuation of parenteral nutrition and beginning of oral feeding. 4) Serum vitamin levels (Vit.A, Vit.E, 25-OH-D3, and Vit.B12) still remained in abnormal even after the patients reached school age. These results suggested that very long-term nutritional cares, especially in lipid and vitamin nutrition, are necessary for the children with massive intestinal resection.

Bile Acids and Salts↗
PubMed1988

Suppression of genetic resistance to bone marrow grafts and natural killer activity by administration of fat emulsion.

Graft rejection is one of the major obstacles to successful bone marrow transplantation (BMT). If resistance to marrow grafting could be avoided, BMT could be used widely in treatment of hematological and immunological disorders. There has been evidence that natural killer (NK) cells play a major role in genetic resistance to BMT and that macrophages are also involved in genetic resistance. Agents toxic to macrophages such as silica and carrageenan have been found to have a suppressive effect on genetic resistance to BMT. Parenteral fat emulsions are known to accumulate in macrophages and to impair various functions of macrophages and those of the reticuloendothelial system. We show here that the administration of a fat emulsion, Intralipos 20%, to recipient mice can suppress genetic resistance to bone marrow grafts and NK cell activity probably through the impairment of the macrophage function. The administration of the fat emulsion might be a new tactic in conditioning protocols for human BMT in the future.

Animals↗
PubMed1987

Dysfunction of irradiated thymus for the development of helper T cells.

The development of cytotoxic T cells and helper T cells in an intact or irradiated thymus was investigated. C57BL/6 (H-2b, Thy-1.2) mice were whole body-irradiated, or were irradiated with shielding over either the thymus or right leg and tail, and were transferred with 1.5 X 10(7) bone marrow cells from B10.Thy-1.1 mice (H-2b, Thy-1.1). At various days after reconstitution, thymus cells from the recipient mice were harvested and a peanut agglutinin low-binding population was isolated. This population was further treated with anti-Thy-1.2 plus complement to remove host-derived cells and was assayed for the frequency of cytotoxic T cell precursors (CTLp) and for the activity of helper T cells (Th). In the thymus of thymus-shielded and irradiated mice, Th activity reached normal control level by day 25, whereas CTLp frequency remained at a very low level during these days. In the thymus of whole body-irradiated mice, generation of CTLp was highly accelerated while that of Th was retarded, the period required for reconstitution being 25 days and more than 42 days for CTLp and Th, respectively. Preferential development of CTLp was also seen in right leg- and tail-shielded (L-T-shielded) and irradiated recipients. Histological observation indicated that Ia+ nonlymphoid cells were well preserved in the thymus of thymus-shielded and irradiated recipients, whereas in L-T-shielded and irradiated recipients, such cells in the medulla were markedly reduced in number. These results suggest strongly that the generation of Th but not CTLp is dependent on radiosensitive thymic component(s), and that such components may represent Ia+ cells themselves in the medulla or some microenvironment related to Ia+ cells.

Animals↗
PubMed1987

Cell surface membrane antigen present on neuroblastoma cells but not fetal neuroblasts recognized by a monoclonal antibody (KP-NAC8).

Neuroblastoma (NB) arises from primitive sympathetic neuroblasts in the adrenal gland or the sympathetic ganglion. NB in situ, sometimes observed in the adrenal glands of autopsied infants, is considered to be a premalignant lesion that may develop into NB. Little is understood about the morphological and biochemical changes that accompany this malignant progression. In this study, a unique monoclonal antibody, KP-NAC8, raised against a human NB cell line is described. This binds to NB cells but not to fetal neuroblasts. The antibody recognizes a Mr 200,000 surface protein on NB cells. KP-NAC8 binds to 15 of 17 human NB cell lines and all 26 fresh NB samples either from tumor tissues or from marrow aspirates involved with tumor. The antibody was found to cross-react with some other tumor cell lines, namely, Ewing's sarcoma (1 of 2), melanoma (1 of 4), lung cancer (3 of 3), and leukemia (2 of 14) cell lines. However, KP-NAC8 did not bind to any rhabdomyosarcoma (0 of 4), Wilms' tumor (0 of 4), retinoblastoma (0 of 2), glioma (0 of 4), and gastric cancer (0 of 2) cell lines examined. Among fetal tissues, KP-NAC8 did not react with normal neuroblasts in the adrenal glands of 5 fetuses. In a further study, the membrane phenotype of fetal adrenal neuroblasts was analyzed by a panel of 12 monoclonal antibodies including KP-NAC8. A comparison of the binding of the same panel of antibodies to fresh NB revealed that antibodies UJ13A, UJ127:11, PI153/3, anti-Thy-1, A2B5, BA-1, BA-2, HSAN1.2, and Leu-7 bound to both fetal adrenal neuroblasts and NB cells. Monoclonal antibodies OKIa-1 and J5 did not bind to either tissues. The only antibody that could distinguish fetal adrenal neuroblasts from NB cells was KP-NAC8. KP-NAC8 may, therefore, define a differentiation-related antigen that may prove helpful in understanding the biological nature of NB and NB in situ.

Adrenal Glands↗
PubMed1987

Production of a monoclonal antibody with specificity for the pellicle of Pneumocystis carinii by hybridoma.

This study reports the establishment of cloned hybridomas between mouse myeloma cells (P3-X63-Ag8, 6.5.3) and spleen cells from BALB/c mice immunized with lung homogenates of nude mice (BALB/c nu/nu) heavily infected with Pneumocystis carinii. A hybridoma subclone, designated 1E12-8, produced antibodies of an IgG1 subclass. Using indirect immunofluorescent antibody techniques, the monoclonal antibody was found to be directed toward the pellicle antigen of air-dried P. carinii both in trophozoite and cyst forms, and to recognize the P. carinii antigen not only from nude mice but also from rats. The antibody did not cross-react with other components of the infected host lung tissue and showed little cross-reactivity with fungi examined. The monoclonal antibody should be useful in the isolation and identification of corresponding antigenic substances of P. carinii and may provide a useful tool in epidemiology, taxonomy, and diagnosis of this organism.

Animals↗
PubMed1987

Specific cell surface labels in the visual centers of Xenopus laevis tadpole identified using monoclonal antibodies.

Monoclonal antibodies (MAbs) against the optic tectum of Xenopus tadpoles were generated and screened by the immunofluorescent staining of frozen sections of tadpole brains. MAb-A5 stains the 8th and 9th plexiform layers of the optic tectum, whereas MAb-B2 stains all but the eighth and ninth plexiform layers of the optic tectum. MAb-A5 antigen is also detectable in the nucleus of Belonci, the corpus geniculatum thalamicum, the pretectal area, and the basal optic nucleus, all targets of the optic nerve, but is not detectable in the optic nerve or the optic tract. On the other hand, MAb-B2 does not stain any of these visual centers, though many fibers surrounding them are stained. Eye-enucleation experiments showed that MAb-A5 antigen is expressed in the optic tectum even when it is not innervated by optic nerves. Staining of viable brains with these MAbs indicates that these antigens are cell surface molecules. Immunoadsorption followed by SDS-PAGE suggests that proteins are constituents of these antigens. The MAb-A5 antigen in the diencephalon and the mesencephalon is not detectable at stage 35/36, but is detectable at stage 39 when the optic nerves begin to innervate the optic tectum. The spatial as well as the temporal patterns of the expression of the MAb-A5 antigen suggest that this molecule may be involved in the target recognition of optic nerve fibers.

Animals↗
PubMed1987

Concanavalin A-mediated polyclonal helper assay of normal thymocytes and its use for the analysis of ontogeny.

A concanavalin A (Con A)-mediated polyclonal helper assay system was established by using the thymus cells or splenic T cells as a source of helper T cells. When splenic B cells were cocultured with thymus cells or splenic Lyt-2- T cells in the presence of an optimal dose of Con A, B Cells were polyclonally activated and differentiated into immunoglobulin-secreting cells. This Con A-mediated helper activity was completely inhibited by the addition of alpha-methyl-D-mannoside and could not be substituted by culture supernatant of Con A-stimulated thymocytes or splenic T cells. Almost all the activity of the thymus cells was carried by peanut agglutinin low binding population. Genetic restriction between T and B cells was not observed in this helper function. In ontogeny, Con A-mediated helper activity in the thymus was first detected at a few days after birth and reached the adult level at about 1 week of age. The polyclonal helper assay system developed in the present study provides a sensitive system to analyse the helper function of thymus cells and also to delineate the early phase of the differentiation of helper T cell population.

Age Factors↗
PubMed1986

Limiting dilution analysis of the stem cells for T cell lineage.

Stem cell activities of bone marrow, spleen, thymus, and fetal liver cells for T cell lineage were studied comparatively by transferring the cells from these organs through i.v. or intrathymus (i.t.) route into right leg- and tail-shielded (L-T-shielded) and 900 R-irradiated recipient mice, which were able to survive without supplying hemopoietic stem cells. Cells from B10.Thy-1.1 (H-2b, Thy-1.1) mice were serially diluted and were transferred into L-T-shielded and irradiated C57BL/6 (H-2b, Thy-1.2) mice, and 21 days later the thymus cells of recipient mice were assayed for Thy-1.1+ cells by flow cytofluorometry. The percentage of recipient mice possessing donor-type T cells was plotted against the number of cells transferred, and the stem cell activity in each cell source was expressed as the 50% positive value, the number of donor cells required for generating donor-type T cells in the thymuses of 50% of recipient mice. In i.v. transfer experiments, the activity of bone marrow cells was similar to that of fetal liver cells, and about 100 times and nearly 1000 times higher than those of spleen cells and thymus cells, respectively. In i.t. transfer experiments, the number of cells required for generating donor-type T cells was much lower than that in i.v. transfer experiments, although the ratio in 50% positive values between i.v. and i.t. transfers differed among cell sources. In i.t. transfers, the 50% positive value of bone marrow cells was five times, 400 times, and 500 times higher than that of fetal liver cells, spleen cells, and thymus cells, respectively. Our previous finding that stem cells are enriched in the spleens of mice which were whole body-irradiated and marrow-reconstituted 7 days earlier was confirmed also by the present limiting dilution assay carried out in i.v. as well as i.t. transfers.

Animals↗
PubMed1986

Aminoglycoside binding sites in the cochlea as revealed by neomycin-gold labelling.

Neomycin/bovine serum albumin/gold was used as a probe to detect the binding sites of aminoglycosides on the thin sections of the cochlea embedded in Spurr. The binding sites were mainly located on the stereocilia, the cuticular plate of hair cells, the head plates of Deiters' cells, fibrous structures in pillar cells, in the spiral limbus and tectorial membrane and basilar membrane, plasma membranes, mitochondria and the chromatin of various kinds of cells. Triphosphoinositide, acidic glycosaminoglycans, and RNA were considered to be responsible for the binding activity.

Aminoglycosides↗
PubMed1986

Aminoglycoside binding sites in Escherichia coli as revealed by neomycin-gold labeling.

A cytochemical technique for demonstration of neomycin binding sites by electron microscopy was developed and applied to Escherichia coli. Neomycin was conjugated chemically with bovine serum albumin (BSA). Colloidal gold was coated with the conjugated neomycin-BSA. The neomycin-BSA-gold was applied to thin sections of Epon-embedded E. coli and examined. Gold particles were observed on the outer membrane and the cytoplasmic membrane of E. coli. It was probably the ribosomes that were being labeled in the cytoplasm. Different cytochemical controls, including a number of inhibition tests and the use of BSA-gold, proved the specificity of this cytochemical technique and provided the biochemical significance of the observations.

Binding Sites↗
PubMed1985

Regulation of the growth and functions of cloned murine large granular lymphocyte lines by resident macrophages.

Using cloned lines with the morphology of large granular lymphocytes (LGL) from BALB/c mice, we studied the exact requirements for proliferation and their functional characteristics, as well as their regulation. Although these cloned LGL lines were interleukin 2 (IL-2) dependent for growth, experiments using human recombinant IL-2 (rIL-2), known to be active on murine cells, indicated that IL-2 was a necessary but not sufficient factor. Coexistance of normal macrophages in addition to rIL-2 was found to support continuous proliferation of cloned LGL in vitro. This role of macrophages could be replaced by partially purified IL-1 derived from macrophage-conditioned medium. An IL-2 binding assay using 125I-rIL-2 suggested that the role of normal macrophages was to selectively induce and/or maintain high affinity IL-2 receptors (IL-2R) (Kd, 0.2-0.5 nM) without affecting low affinity ones (Kd, 10-30 nM). Functional studies indicated that most of the LGL clones killed various combinations of representative groups of natural killer (NK)-susceptible target cells, including leukemic cells (YAC-1, RL male 1), virus-infected cells (HeLa-measles, HeLa-herpes simplex virus), and normal bone marrow cells (BMC), whereas none of them affected any of NK-resistant target cells, including uninfected HeLa cells. Some of these clones also suppressed in vitro hematopoiesis. Such characteristic cytotoxic spectra, as well as serological phenotypes (Thy-1+, Lyt-1-2-, asialo GM1-positive, T200+, TdT-, Fc receptor-positive) indicated that these LGL clones exactly represent endogenous NK cells, rather than a variety of anomalous killer cells generated in various culture conditions. Although there was significant heterogeneity of cytotoxic spectrum among LGL clones, no clonotypic distribution of specificities was observed. Normal macrophages were found to modulate the functional expression of LGL clones. They augmented the cytotoxic potential of the clones against leukemic and virus-infected targets, but suppressed intrinsic reactivity against normal BMC. Similarly, LGL clones maintained with macrophages showed much less suppressive effect on in vitro hematopoiesis. The present observations on the interaction of cloned LGL and normal macrophages provide a basic explanation for the mechanisms by which the immediate responsiveness to IL-2 of the NK effector system, without exogenous stimulation, and the functional selectivity toward abnormal rather than normal cells, are actively maintained in vivo.

Animals↗
PubMed1985

Early B cell precursors in long-term bone marrow culture: selective development in the bone marrow of irradiated recipients.

The primary site for the growth and differentiation of B cell precursors in irradiated recipient mice was investigated. Bone marrow (BM) cells from lipopolysaccharide (LPS)-responder C57BL/6 mice were transferred into irradiated LPS-nonresponder C57BL/10ScCr mice, and the generation of donor-derived B cells in the recipient was monitored by determining the immunoglobulin-producing cells developed in the LPS-stimulated cultures of recipient's spleen cells as well as BM cells. As previously stated, the transfer of fresh BM cells resulted in the development of LPS-reactive cells both in spleen and BM simultaneously. On the other hand, when long-term cultured BM cells which were shown to be devoid of B cells and pre-B cells were used as the donor cells, the development of LPS-reactive cells was first observed only in BM, and subsequently in both BM and spleen. The failure to detect LPS-reactive cells in the spleen in the early phase, or day 11 after irradiation and reconstitution, was not attributed to the culture condition or the existence of suppressive activity in day 11 spleen cells. These results indicate that B cell precursors lodge only in the BM of irradiated recipients, grow and differentiate in the same place, and then the differentiated progeny migrate to peripheral lymphoid organs.

Animals↗
PubMed1985

Characteristic distribution of noradrenergic terminals on the anterior horn motoneurons innervating the perineal striated muscles in the rat. An immuno-electromicroscopic study.

Dopamine-beta-hydroxylase (DBH) immunohistochemistry was used to demonstrate the noradrenergic fibers and terminals in the anterior column of the rat lumbosacral spinal segments. PAP-positive varicose fibers were widely distributed in the gray matter with preferential accumulation in the nuclear regions containing motoneurons involved in the contraction of perineal striated muscles. Unmyelinated DBH fibers were composed of nodular enlargements (varicosities, 0.4-3.0 microns in diameter) and very fine, short intervals (intervaricose segments, 0.1-0.2 micron in diameter and 1.0-4.0 microns in length). DBH-positive dense products were electron microscopically often confined within small granular particles and less frequently within large granules. Additionally, in order to characterize the innervation pattern of noradrenergic fibers on dendritic bundles organized in the motoneuronal pools innervating the pelvic small muscles, semi-quantitative analysis was done in the area of the dorsolateral nucleus endowed with especially well-developed dendritic bundles. DBH terminals contacting with unreactive dendrites were more common (67.9%) than those with neuronal somata (15.1%), and the remainder (17%) had no contacts with surrounding neuronal elements. Furthermore, specialized synaptic formations were observed in only 20.1% of these nodules. The results suggest that bulbospinal descending noradrenergic neuron systems influence the functioning of pelvic muscles principally via the neuronal contacts with dendritic bundles in the spinal cord.

Animals↗
PubMed1985

Experimental immune complex otitis media: localization of IgG by protein A-gold technique.

Immune complex (IC) was made of bovine serum albumin (BSA) and anti-BSA guinea pig serum, and instilled into the tubotympanic cavity of untreated guinea pigs. The location of IgG was determined by using the protein A-gold technique to trace IC in the tubotympanic mucosa of the otitis media with effusion (OME) caused by the instillation. IgG was found on the effusion, degenerated ciliated cells and on granules of goblet cells. There was no evidence of intense accumulation of IgG on the basement membrane of the mucosal epithelium or the capillaries. It is likely that IC activates complements in the tubotympanic cavity to cause OME.

Animals↗