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Biomedical subjects

T Kuwata

Publications and source records attributed to T Kuwata.

At least 91 records · Page 5Linked to original sources
PubMed1996

Differential induction of helper and killer T cells from isolated CD4+CD8+ thymocytes in suspension culture.

Thymocytes of T cell receptor transgenic mice with nonselecting and RAG-2 -/- backgrounds were developmentally arrested at the CD4+CD8+ stage before positive selection. These thymocytes underwent lineage commitment upon transient stimulation with a combination of ionomycin, a calcium ionophore, and phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, in suspension culture. The effective drug doses were limited within narrow ranges and much lower than those which induce proliferation of mature T cells. The doses corresponded to those which inhibit glucocorticoid-induced apoptosis in these thymocytes. CD4 lineage commitment required longer duration, higher intensity of the stimulation, or both, than CD8 lineage commitment. Functional helper T cells (Th1 and Th2) were induced from the CD4 lineage-committed cells upon secondary stimulation with a combination of ionomycin and PMA followed by lymphokine treatment. Cytotoxic T cells were induced from the CD8 lineage-committed cells upon incubation with concanavalin A and irradiated splenic dendritic cells, but not with the combination of ionomycin and PMA. These results indicate that positive selection is mimicked by the pharmacological stimulation in the absence of other cell types, but that final maturation of CD8 T cells may require a different signal.

Animals↗
PubMed1996

Regulation of T cell apoptosis via T cell receptors and steroid receptors.

Less than 5% of immature CD4/CD8 double-positive (DP) thymocytes are positively selected to survive and differentiate into single-positive CD4 and CD8 T cells, while self-reactive DP thymocytes undergo apoptosis (negative selection). Both positive and negative selection events are active processes that involve signaling through the T cell receptors (TCRs) and through some accessory molecules. The two events differ quantitatively in the strength of the interaction between TCR and peptide/major histocompatibility complex molecules. We established an in vitro model of positive selection that can be analyzed quantitatively. Positive selection is likely to inhibit glucocorticoid-induced apoptosis in DP thymocytes. Proper crosslinking of TCR together with CD4, CD8, or LFA-1 inhibits the death, and its inhibitory activity is mimicked by proper combinations of ionomycin, a calcium ionophore, and phorbol myristate acetate (PMA), a protein kinase C (PKC) activator. The drug concentrations are within narrow ranges, and are lower than those which are required for the proliferation of mature T cells. Transient stimulation with the combinations of ionomycin and PMA induces differentiation and commitment of isolated DP thymocytes to the CD4 or CD8 T cell lineage in suspension cultures. The level of PKC activity appears to determine the lineage to commit. Functional mature T cells are induced from the committed cells upon secondary stimulation. Activation of calcineurin, a Ca2+/calmodulin-dependent protein phosphatase, also appears to be essential for positive selection as well as for the inhibition of glucocorticoid-induced apoptosis. Negative selection and the regulation of mature T cell apoptosis through TCR and steroid receptors are also discussed.

Animals↗
PubMed1996

Genetic complementation between replication-defective mutants of HIV-1 and SIVagm.

To investigate the functional complementation of essential genes for virus growth between HIV-1 and SIVagm derived from African green monkeys, we co-transfected replication-defective molecular clones containing mutations in gag, pol, env, tat or rev, and monitored transient complementation by reverse transcriptase assay (RT), cytopathic effect (CPE) and immunofluorescence assay (IFA). The following results were obtained: 1) No complementation was observed in combinations of the gag and pol mutants. 2) The rev mutant of HIV-1 was minimally complemented by other SIVagm mutants, although the rev mutant of SIVagm was significantly complemented by other HIV-1 mutants. 3) Among all combinations tested, the env mutant of HIV-1 was the most effectively complemented by SIVagm mutants. 4) CPE was mostly absent in combinations of the env mutant of SIVagm and the gag, pol, or tat mutants of HIV-1, although there were significant positive results in RT and IFA assays. These findings provided basic information about the functional compatibility of pathogenic HIV-1 and nonpathogenic SIVagm which will be useful for generating chimeras of these two viruses.

Animals↗
PubMed1996

Effect of the cholesterol content of a formula on the lipid compositions of plasma lipoproteins and red blood cell membranes in early infancy.

We investigated whether or not a regular formula for full-term infants supplemented with cholesterol (cholesterol-fortified) would increase the plasma cholesterol concentration and alter the red blood cell (RBC) membrane lipid composition in healthy full-term infants compared with their breast-fed counterparts. At 1 mo of age, total plasma cholesterol and low-density-lipoprotein (LDL) cholesterol were significantly higher in the breast-fed infants than in the cholesterol-unfortified, formula-fed infants. At 3 mo of age, total cholesterol and LDL cholesterol were significantly higher in the breast-fed infants than in the two formula-fed infant groups. These significant differences had disappeared by 6 mo of age. Although the cholesterol-unfortified, formula-fed infants had lower proportions of docosahexaenoic acid (DHA, 22:6n-3) and eicosapentaenoic acid (EPA, 20:5n-3) in the RBC membranes compared with the breast-fed group at 6 mo, DHA and EPA concentrations in the cholesterol-fortified, formula-fed infants were not significantly different. The results of the present study suggest that the plasma cholesterol concentration and fatty acid pattern of the RBC membranes in infants fed a cholesterol-fortified formula may be much closer to those in breast-fed infants than in infants fed a cholesterol-unfortified formula.

Aging↗
PubMed1996

In vitro differentiation and commitment of CD4+ CD8+ thymocytes to the CD4 lineage, without TCR engagement.

Thymocyte positive selection is based on protection of immature CD4/CD8 double-positive (DP) thymocytes from apoptosis and their differentiation into CD4 or CD8 single-positive (SP) cells. Intracellular signals essential for positive selection appear to be induced through the TCR and some of the accessory molecules including LFA-1, CD4 and CD8 upon interaction with thymic stromal cells. The signals, however, still remain to be identified. Since physiological levels of glucocorticoids potentially induce or enhance thymocyte apoptosis even in vivo, the signals are likely to inhibit the apoptotic effect of glucocorticoids. We have previously shown that proper cross-linking of TCR-CD3 with LFA-1, CD4 or CD8 inhibited glucocorticoid-induced thymocyte apoptosis in vitro, and that a proper combination of the calcium ionophore, ionomycin and the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), mimicked the inhibitory effect. Here we determined whether this combination of ionomycin and PMA induces differentiation of isolated DP thymocytes from normal and TCR transgenic mice. We found that pretreatment of DP thymocytes with ionomycin and PMA followed by 1 day culture of the cells without the reagents resulted in the differentiation of the cells into CD4 SP and CD4+ CD8lo T cells that have mostly committed to the CD4 lineage. The changes in expression of other differentiation markers were also in good accordance with those associated with positive selection, except the final maturation. The results indicate that moderate and transient increases in intracellular Ca2+ level and PKC activity induce differentiation and commitment of DP thymocytes to the CD4 lineage, and suggested that the biochemical pathway leading to positive selection is based on a similar mechanism.

Animals↗
PubMed1996

Dietary casein phosphopeptides prevent bone loss in aged ovariectomized rats.

The effect of dietary Ca-bound casein phosphopeptides (CaCPP) on the bones of aged ovariectomized (OVX) rats was studied as a model for post-menopausal bone loss. Three groups of ovariectomized rats were fed a control diet or one of two experimental diets, and one group of sham-operated rats (SHAM) was fed the control diet. The experimental diets contained 0.5% Ca and 0.4% P. In one diet, CaCPP was the sole source of calcium and provided 62.5% of dietary phosphorus (CaCPP diet). In the other, Ca-free CPP provided 100% of dietary phosphorus (Ca-free CPP diet). In the control diet, CaCO3 and KH2PO4 were used. During a 17-wk feeding period, there was little change in femoral bone mineral densities (BMD) of ovariectomized rats fed CaCPP and Ca-free CPP, or in the SHAM rats fed the control diet, whereas the bone mineral densities in the control ovariectomized rats decreased with time. Some of the segmental bone mineral densities of the excised femurs from the rats fed CaCPP were significantly higher than those from the control ovariectomized rats, but the values of the Ca-free CPP group were similar to those of the control ovariectomized rats. In the Ca-free CPP group, the discrepancy in bone mineral densities obtained between in vivo results and excised specimens might have been the result of a loss in bone mass due to their significant loss in body weight. There were no significant differences in serum inorganic phosphorus, alkaline phosphatase activity, osteocalcin or 1 alpha-25-dihydroxycholecalciferol concentrations among the ovariectomized groups. In the CaCPP and Ca-free CPP groups, urinary phosphorus excretion decreased and urinary calcium excretion increased significantly with time. The inhibitory effect on bone loss in aged ovariectomized rats could be due to the effects of dietary CaCPP on phosphorus and calcium metabolism.

Aging↗
PubMed1996

Chimeric viruses between SIVmac and various HIV-1 isolates have biological properties that are similar to those of the parental HIV-1.

OBJECTIVE: To examine the biological properties of HIV-1/SIVmac chimeric viruses from HIV-1 isolates that have different replication rates, cell tropisms and cytopathicities. DESIGN AND METHODS: Four chimeric viruses with gag, pol, vif, vpx, nef and long terminal repeats of SIVmax and vpr, tat, rev, vpu and env of various HIV-1 isolates were constructed and compared in vitro. Cynomolgus monkeys were inoculated with two chimeras that were replicative in monkey peripheral blood mononuclear cells (PBMC). RESULTS: The type-specific neutralization of the chimeras by monoclonal antibodies 0.5 beta and mu 5.5, which recognize V3 of HIV-1IIIB and HIV-1MN respectively, was observed to be similar to those of the parental viruses, HIV-1NL432, HIV-1HAN2 and HIV-1SF13. The chimeras constructed from HIV-1SF2 and HIV-1SF13, which were isolates from the same individual but from different disease stages, reflected their parental properties, that is, the isolate from the later stage was rapid-high replicating, was more cytopathic and had a wider host range. Chimeras constructed from HIV-1HAN2' HIV-1SF13 and HIV-1NL432 were infectious to macaque monkeys, although the monkeys infected with the chimera from HIV-1SF13 showed lower virus loads and shorter viremic periods than those infected with the others. CONCLUSIONS: Chimeras have in vitro properties that are similar to those of their parental HIV-1 isolates, but their growth in macaque PBMC was dependent on which HIV-1 isolate was used. Evaluation of a vaccine by challenging with viruses possessing different antigenicities has become possible in macaque monkeys using newly constructed chimeras.

Animals↗
PubMed1996

Replication and cytopathogenicity of human immunodeficiency virus type 1 (HIV-1)/simian immunodeficiency virus agm3 chimeric viruses in human and monkey cells: the 5' half of the HIV-1 genome is responsible for virus cytopathogenicity.

Two chimeric viruses were constructed between human immunodeficiency virus type 1 (HIV-1) and an apathogenic simian immunodeficiency virus (SIVagm3mc) from African green monkeys. One of the chimeras, HE-A391, expressed the HIV-1-derived env, vpu, tat and rev genes and the SIVagm3mc-derived LTR and the gag, pol and vif genes. The other chimera, SE-H13, contained the SIVagm3mc-derived env, tat and rev genes and the HIV-1-derived LTR and the gag, pol, vif and nef genes. Both constructs yielded infectious viruses and their phenotypes (growth-competence and cell-killing capacity) were examined in various CD4+ cells including human and monkey PBMCs. The results indicated that the replicative properties of the chimeras were mainly dependent on the 5'-genomic half of the parental viruses, and the determinant for viral cytopathogenicity was located within the 5' half of the HIV-1 genome.

Animals↗
PubMed1996

Genomic and biological alteration of a human immunodeficiency virus type 1 (HIV-1)-simian immunodeficiency virus strain mac chimera, with HIV-1 Env, recovered from a long-term carrier monkey.

A macaque monkey infected with NM-3, a human immunodeficiency virus type 1 (HIV-1)-simian immunodeficiency virus strain mac (SIVmac) chimeric virus with env, rev, tat and vpu derived from HIV-1 and LTR, gag, pol, vif and vpx derived from SIVmac, became a long-term carrier (more than 2.8 years). This monkey produced neutralizing antibodies to the original NM-3 as well as to the parental HIV-1. The virus recovered at 116 weeks replicated more rapidly and productively in macaque peripheral blood mononuclear cells than the original virus. The recovered virus was not neutralized either by antibodies raised early in the monkey or by a neutralizing monoclonal antibody that recognizes the V3 loop of HIV-1 Env, whereas both the early antibodies and the monoclonal antibody neutralized the original NM-3. Analysis of the virus genomic population revealed a few common mutations in the V3 region that caused amino acid changes. These data are consistent with the hypothesis that the virus escaped from the early antibodies and that the observed mutations contributed to this, as with HIV-1-infected humans. The observed mutations could equally well be the result of adaptation to simian cells. These results suggest that the HIV-1-SIVmac chimeric virus will be useful for investigating genetic variation of HIV-1 env and alteration of biological properties in vivo in relation to the host immune response.

Amino Acid Sequence↗
PubMed1995

Differential expression of protein tyrosine kinases in the cortical and medullary thymic epithelial cell lines.

Expression of protein tyrosine kinase (PTK) was evaluated and compared between cortical and medullary thymic epithelial cell (TEC) lines, TEC 14C18 and TEC1C6, respectively. Reverse transcription-polymerase chain reaction (RT-PCR) methods using degenerate primers for conserved sequence in catalytic domain of PTK revealed the expression of 13 kinds of PTKs in these cell lines. These PTKs included 6 receptor and 7 non-receptor types. In the next step, we compared the intensity of mRNA expression level of these kinases between cortical and medullary TEC lines. Tyrosine kinases of receptor type were differentially expressed between cortical and medullary TEC lines; e.g., mRNA expression of flg was observed more intensively in the cortical TEC line than in the medullary one, while mRNA expression of bek, met and sky was vice versa. On the other hand, expression of non-receptor tyrosine kinases, including four JAK-tyk family members, are comparable in both lines. These results suggested that these PTKs, especially of receptor type, might play an important role in the development of the thymic microenvironment.

Amino Acid Sequence↗
PubMed1995

Trace lipid from whey-mineral complex enhances calcium availability in young ovariectomized rats.

The effects of the trace lipids (L1 and L2) extracted from two kinds of whey-mineral complex on bone metabolism were studied in young ovariectomized (OVX) rats. The concentrations of oestradiol in the L1 and L2 oils were 305 and 1313 pg/ml respectively, while the concentrations of progesterone were 34.3 and 36.9 ng/ml respectively. Trace amounts of 1 alpha,25-dihydroxycholecalciferol (1 alpha,25-(OH)2D) were detected: 56 pg/ml in the L1 oil and 132 pg/ml in the L2 oil. The OVX rats were orally given 0.4 ml each test oil twice weekly. Other animals were given with only a vehicle (control) or an oil mixture with similar levels of oestradiol and progesterone to those in L2 oil (PE oil) for comparison. The Ca absorption rate, bone density of the humerus and femoral Ca content in the rats given L2 were significantly higher than those in the control rats (P < 0.05). Serum calcitonin and 1 alpha,25-(OH)2D from rats given L2 were both significantly higher than those from the control rats, while serum progesterone in the L2 group was slightly lower. It is suggested that the bone metabolism of rats given L2 reflected the compound effect of 1,25-(OH)2D and oestradiol in the L2 oil. In contrast, the relatively low level of these hormones might have been associated with the mediocre bone characteristics of the L1 group. It is suggested from these results that the trace level of hormones could be one of the reasons why whey-mineral complex showed good Ca availability.

Animals↗
PubMed1995

Construction of human immunodeficiency virus 1/simian immunodeficiency virus strain mac chimeric viruses having vpr and/or nef of different parental origins and their in vitro and in vivo replication.

We constructed a series of human immunodeficiency virus 1 (HIV-1)/simian immunodeficiency virus strain mac (SIVmac) chimeric viruses having vpr and/or nef genes of either HIV-1 or SIVmac based on a chimeric virus with LTRs, gag, pol, vif and vpx derived from SIVmac and tar, rev, vpu and env from HIV-1. All of the chimeric viruses replicated in human and macaque peripheral blood mononuclear cells (PBMCs) and in several CD4+ human cell lines, though their growth potentials were slightly different depending on whether vpr and nef were from HIV-1 or SIVmac, or were defective. The presence of nef accelerated replication in all the cells used and the replication of each chimera appeared to reflect that of the parental virus from which nef was derived. The presence of vpr had no clear effect in human and monkey PBMCs, but the replication of each chimera was influenced by the origin of vpr in H9 and A3.01 cells. NM-3rN, which carries HIV-1 vpr and SIVmac nef, was inoculated intravenously into three rhesus monkeys, three cynomolgus monkeys and two pig-tailed monkeys. From 2 to 14 weeks after inoculation, viruses were consistently re-isolated from all the monkeys and virus loads were as high as that of SIVmac reported previously. The results indicate that infection with NM-3rN is more efficient than any of our previous chimeric viruses and suggest that NM-3rN, having HIV-1 Env, will be a useful challenge virus for evaluating AIDS vaccines based on HIV-1 Env in macaque monkeys instead of chimpanzees.

Animals↗
PubMed1995

Targeting of chrolamphenicol acetyltransferase to human immunodeficiency virus particles via Vpr and Vpx.

Vpr and Vpx are the auxiliary proteins of human immunodeficiency viruses (HIVs) selectively incorporated into mature viral particles. We showed that the bacterial chloramphenicol acetyltransferase (CAT) fused to the N-terminus of HIV-1 Vpr, HIV-2 Vpr, or HIV-2 Vpx was incorporated into mature virions in a type-selective manner. By using chimeric proteins between HIV-1 Vpr and HIV-2 Vpx, we found that the N-terminal side of these proteins was mainly important for type-selective virion incorporation. The C-terminal arginine-rich region of HIV-1 Vpr was also found to transport CAT fusion proteins into virions but without any type selectivity. Furthermore, the corresponding regions of HIV-2 Vpr and HIV-2 Vpx had no such activity. This region of HIV-1 Vpr may interact nonspecifically with viral genomic RNA. Collectively, Vpr and Vpx may provide a means to introduce foreign proteins and other molecules into HIV virions for therapeutic purposes.

Amino Acid Sequence↗
PubMed1995

[Identification of motor area by transcranial magnetic stimulation using an "eight-figure" coil in patients with motor paralysis].

In patients with motor paralysis, we tried to identify the functional motor area by transcranial magnetic stimulation using an "eight-figure" coil designed by Ueno. Motor evoked potentials (MEPs) were recorded in 7 patients from 50 to 64 years old, and in 5 normal volunteers 26 to 45 years old. They were stimulated at 49 points over an unilateral hand motor area, and at 21 points over a foot motor area, and surface MEPs were recorded on their contralateral thenar muscle and abductor hallucis brevis muscle. In normal volunteers, the optimal eddy current for stimulating the hand motor area was directed anteriorly parallel to the midline, and for stimulating the foot motor area, it was postero-laterally directed with an angle of 45 degrees towards the midline. MEPs could be induced at their muscle contractions during which their thumbs and middle fingers softly touched each other, and their halluxes slightly flexed. In five patients two kinds of amplitude mappings reconstructed from MEPs were obtained at rest or at muscle contraction. A line connecting these two peaks on an amplitude mapping was regarded as an "MEP-motor area". A geographical difference between the MEP-motor area and MRI-motor area (identified by an MRI surface image) was studied at muscle contraction and at rest. In normal subjects the sites of the MEP-motor area and of the MRI-motor area coincided, whereas, in patients with space-occupying lesions near the central sulcus, the MEP-motor areas were located 1 to 2cm posterior to MRI-motor areas.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1995

Proliferative activity and p53 over-expression of ovarian epithelial tumors.

Proliferative activity (PA) and p53 over-expression were assessed in 68 cases of primary ovarian epithelial tumors of variable grades of malignancy by immunohistochemical methods using antibodies to PCNA (proliferative cell nuclear antigen) and p53 molecule. First, we compared the values in benign tumors, tumors of borderline malignancy and malignant tumors. Malignant tumors possessed higher PA value (26.8 +/- 14.7%) than benign tumors (1.6 +/- 1.4%), and tumors of borderline malignancy exhibited an intermediate value (11.4 +/- 5.1%). Intranuclear accumulations of p53 product were more frequently observed in malignant tumors (16/32, 50%) than in tumors of borderline malignancy (3/9, 33%). None of the benign tumors exhibited p53 over-expression. In malignant tumors, PA correlated well with the histologic grade of tumors, although the histologic type and stage of the diseases did not correlate significantly with PA. Then, we focussed on the cases of malignant ovarian cancer composed of heterogeneous lesions showing a different grade of malignancy; invasive lesion (IL), non-invasive lesion (NIL), and benign appearing lesion (BAL). PA of NIL was almost identical with that of IL (19.6 +/- 13.5% vs. 23.3 +/- 12.6%), while BAL showed significantly lower PA (2.1 +/- 3.0%, p < 0.001) than the above two lesions and was similar to that of benign tumors. Furthermore, p53 over-expression was never observed in BAL, even in cases in which IL and/or NIL showed p53 over-expression. Thus, the results indicated that histologically benign-appearing lesions of malignant ovarian epithelial tumors possessed a biologic character similar to benign tumors. Collectively, all these findings would support the concept that a part of malignant ovarian epithelial tumors would be derived from benign tumors through progressive transformation.

Adenocarcinoma, Clear Cell↗
PubMed1994

Immunolocalization of platelet-derived growth factor, transforming growth factor-beta, and fibronectin in acute megakaryoblastic leukemia manifesting tumor formation.

Acute megakaryoblastic leukemia (AMKL) manifesting myelofibrosis and tumor formation in the liver with marked increase of reticulin is described. The megakaryoblastic nature of the leukemic cells of the bone marrow and the hepatic tumor nodule was established by positive immunohistochemical stains for CD41a and CD41b on frozen tissue sections. Immunolocalization of the platelet-derived growth factor (PDGF) protein and transforming growth factor (TGF)-beta protein also was demonstrated in the leukemic cells of the bone marrow and the hepatic tumor. Further, the deposition of fibronectin that has been known as the ligand of CD41a molecule and collagen types I and IV were recognized in the extracellular matrix of the bone marrow and the hepatic tumor. These results suggest that specific expression of growth factor proteins by the leukemic cells may selectively regulate the fibrosis of the bone marrow as well as the tumor formation of AMKL. The expression of adhesion molecules and growth factor proteins by the leukemic cells and the deposition of extracellular matrix are discussed in relation to the myelofibrosis as well as the tumor-forming nature of AMKL.

Collagen↗
PubMed1994

Persistent infection with SIVmac chimeric virus having tat, rev, vpu, env and nef of HIV type 1 in macaque monkeys.

A chimeric human and simian immunodeficiency virus carrying the tat, rev, vpu, env, and nef genes of human immunodeficiency virus type 1 was generated. The chimeric virus, NM-3n, grew competently in peripheral blood mononuclear cells from cynomolgus monkeys like the parental SIVmac. Two cynomolgus monkeys and one rhesus monkey inoculated with NM-3n raised antibodies to SIVmac Gag and HIV-1 Env. The antibodies raised in the cynomolgus monkeys persisted for at least 1.7 years. The antibodies contained virus neutralizing activity not only to the original chimeric virus but also to the parental HIV-1. Infectious viruses were isolated from one of the cynomolgus monkeys 37 and 63 weeks after inoculation and from the rhesus monkey continuously from 6 weeks after infection onward. The recovered virus maintained its chimeric structure but included several clones with mutations in the env V3 region. When the recovered virus was inoculated to another rhesus monkey, no difference in the frequency of virus recovery was seen from the originally infected monkeys. These carrier monkeys have so far shown no sign of the disease.

Animals↗