Biomedical subjects

Y Hitsumoto

Publications and source records attributed to Y Hitsumoto.

At least 19 recordsLinked to original sources

PubMed1996

Two mechanisms for platelet-mediated killing of tumour cells: one cyclo-oxygenase dependent and the other nitric oxide dependent.

We have tried to identify the cytotoxic effectors in platelet-mediated tumour cell killing, using two tumour cell lines K562 (a chronic myelogenic leukaemic cell line) and LU99A (a lung cancer cell line), which are both sensitive to platelet cytotoxicity. Cyclo-oxygenase inhibitors, acetylsalicylic acid (ASA) and indomethacin, effectively inhibited the platelet-mediated killing of K562 cells, but not that of LU99A cells. In contrast, inhibitors of the nitric oxide (NO) pathway. NG-nitro-1-arginine (L-NA), haemoglobin and methylene blue, reduced the cytotoxic activity of platelets against LU99A, but not against K562. Synthetic analogues of platelet cyclo-oxygenase products thromboxane A2/ prostaglandin H2(TXA2/PGH2) exerted cytotoxicity against K562 cells but not against LU99A cells. Electron microscopic study showed that TXA2/PGH2 analogues induced bleb formation and disruption of the plasma membrane of K562 cells. K562 cells enhanced the production of TXA2 by platelets, as inferred from the accumulation of thromboxane B2 (TXB2), a spontaneous hydrolysis product of TXA2. LU99A cells had no such effects. These results indicate that platelets kill these two tumour cell lines through different mechanisms. In K562, the cyclooxygenase products TXA2/PGH2 possibly play a significant role but in LU99A the NO pathway seems to be involved.

Aspirin↗

PubMed1996

Enhancement of CD3-mediated thymocyte apoptosis by the cross-linkage of heat-stable antigen.

Heat-stable antigen (HSA) is a murine differentiating antigen that is expressed on both CD4-CD8- double-negative and CD4+CD8+ double-positive thymocytes but not CD4+ or CD8+ single-positive thymocytes. Effects of anti-HSA monoclonal antibody, R13, on thymocyte apoptosis induced by various stimulations were investigated by a single-cell suspension culture system. Immobilized R13 enhanced the CD3-mediated DNA fragmentation and killing of thymocytes but not the dexamethasone-induced or phorbol myristate acetate-induced killing of thymocytes. Immobilized R13 by itself could not induce thymocyte apoptosis. Soluble R13 enhanced CD3-mediated apoptosis when HSA and T-cell receptor (TCR)/CD3 were co-cross-linked by a cross-reactive secondary antibody. Even without the cross-reactive secondary antibody, soluble R13 enhanced CD3-mediated apoptosis, although a greater than 100-fold increase in the amount of R13 was needed to give a similar enhancement compared with immobilized R13. Neither R13 by itself nor R13 plus secondary antibody induced cytosolic calcium influx, whereas R13 enhanced CD3-mediated cytosolic calcium increase. These results suggest a functional role of HSA in promoting the activation-induced apoptosis of thymocytes and the involvement of HSA in negative selection.

Animals↗

PubMed1996

Attenuation of human chorionic gonadotropin release by nitric oxide in choriocarcinoma cell lines.

Nitric oxide (NO) is involved in the regulation of endocrine functions, but only a few studies have been reported about its role in placental hormone secretion. We investigated whether NO has any function in the release of human chorionic gonadotropin (hCG) in two different choriocarcinoma cell lines, JEG-3 and BeWo. First, nitric oxide synthase (NOS) was characterized in the choriocarcinoma cells. NOS activity was localized mainly in the particulate fraction and depended on calcium/calmodulin. Activity was inhibited by the presence of the L-arginine analog, NG-monomethyl-L-arginine (L-NMMA; 1 x 10(-4) M). Western blot analysis showed that the choriocarcinoma cells contained an endothelial isoform of NOS. The NO donor, sodium nitroprusside (SNP; 1 x 10(-5) and 1 x 10(-4) M), significantly inhibited hCG secretion in both choriocarcinoma cell lines. The suppression of hCG release by SNP (1 x 10(-5) M) was blocked by the addition of an NO scavenger, hemoglobin (1 x 10(-6) M). L-Arginine (1 x 10(-2) M), a NOS substrate, inhibited basal hCG secretion in JEG-3 cells. Incubation of the cells with L-NMMA (1 x 10(-4) and 1 x 10(-3) M) significantly increased hCG release. Exposure of both cell lines to increasing concentrations of a cyclic GMP analog (8-bromo-cyclic GMP; 1 x 10(-4) to 1 x 10(-2) M) caused a dose-dependent inhibition of hCG release. Cyclic GMP accumulation in response to SNP (1 x 10(-4) M), however, was not detected in either JEG-3 or BeWo cells. These data demonstrated that the endothelial isoform of NOS and a functional L-arginine-NO pathway are present in the choriocarcinoma cell lines. In addition, these findings support the hypothesis that NO produced in these cell lines is involved in the regulation of hCG secretion. We assume that although cyclic GMP is likely to play a role as a second messenger, a cyclic GMP-independent pathway cannot be excluded as a possible physiological mechanism in the attenuation of hCG release by NO.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1996

Expression of mammalian 60-kD heat shock protein in the joints of mice with pristane-induced arthritis.

Previous work has indicated that autoimmunity to the mammalian 60-kD heat shock protein (hsp60) may be necessary for the development of pristane-induced arthritis (PIA), a murine model of rheumatoid arthritis. To characterize the expression of hsp60 in murine joints, immunoblots of joint extracts and frozen histological sections prepared from normal or arthritic mice were probed with the hsp60-specific MoAb 4B989. Hsp60 could be detected in the joints of mice with PIA by both techniques, and was seen to be localized within the inflamed pannus using immunhistochemistry. Immunoblotting revealed that lower concentrations of hsp60 are also present in normal mouse joints, and that the level of expression increases with age, in parallel with greater susceptibility to PIA. In other studies, it was demonstrated that the titres of serum IgG antibodies reactive with the related mycobacterial hsp65, and the in vitro responsiveness of splenic T cells to hsp65, are both elevated in older mice. It is considered that the results are consistent with the hypothesis that PIA develops following environmental priming with mycobacterial hsp65, and the targeting of cross-reactive T cells to self-hsp60 in the joints.

Age Factors↗

PubMed1995

Compositions of very low density lipoprotein subfractions from patients with polydisperse low density lipoproteins.

In some hyperlipidemic patients, low density lipoprotein (LDL) shows several peaks (polydisperse) on polyacrylamide gel disc electrophoreses, though LDL usually shows a single peak (monodisperse). In order to clarify the relationship between the LDL polydispersion and VLDL heterogeneity, LDL and VLDL were prepared from hyperlipidemic patients sera with mono- and polydisperse LDL by sequential ultracentrifugation and fractionated by gradient ultracentrifugation and their compositions were analyzed. Polydisperse LDL was rich in triacylglycerol (TG) and poor in esterified cholesterol (CE) as compared with monodisperse LDL and consisted of the lowest and the medium density subfractions when the LDL was separated into six subfractions. The monodisperse LDL was composed of a single major subfraction of a medium density. VLDL from the patients with polydisperse LDL was relatively rich in the dense and poor in the buoyant subfractions as compared with that from the patients with monodisperse LDL. The subfractions in the former contained more CE and less TG than the corresponding subfractions in the latter. There were no significant differences in the apolipoprotein compositions between those VLDLs. The results suggest that polydisperse LDL might be originated from VLDL that differs in particle sizes, densities and compositions from ordinary VLDL.

Adult↗

PubMed1995

Complement activation by cross-linked B cell-membrane IgM.

The B cell membrane IgM (mIgM) occurs in a monomeric form incapable of activating C. However, when cross-linked by a polyvalent ligand, mIgM may activate C by assuming a polymeric structure like secreted IgM. This possibility was tested with CR2- lymphoma cells, which did not activate C spontaneously. When CR2-deficient mIgM(lambda)+ Ramos cells were treated with F(ab')2 goat anti-lambda, then exposed to human serum, a marked C3 deposition took place, as examined by the flow cytometry. Similarly, C3 deposition on mIgM(kappa)+, mIgD(kappa)+ P32 cells was induced by F(ab')2 of either anti-kappa or anti-mu. Anti-delta was without effect, but the C3 deposition resulting from anti-kappa was markedly enhanced after mIgD was modulated by anti-delta. The mIgM-cross-linked cells bound C1q, and C3 deposition on these cells was abrogated by depletion of C1q, but not Factor B nor D, from serum. The C1-binding step of the mIgM-mediated C activation was inhibited by monomeric Fab' of polyclonal anti-mu containing a blocking Ab to the hemolytic activity of human IgM Forssman Ab. A large proportion of C3 deposits on mIgM-cross-linked cells was found to be associated with mIgM in the form of C3dg or C3d. These results demonstrate that cross-linked mIgM indeed triggers the classical pathway of C.

B-Lymphocytes↗

PubMed1995

Murine complement reduces infectivity of Plasmodium yoelii to mosquitoes.

The alternative pathway of complement in the mouse serum significantly reduced, but did not eliminate, the infectivity of Plasmodium yoelii to Anopheles stephensi. The reduction of the infectivity is mainly due to the inability of the zygote to transform into the ookinete in the mosquito midgut.

Animals↗

PubMed1994

Isolation of mouse complement component C7.

Mouse complement component C7 was purified from serum by a sequential procedure of fractionation precipitation by ammonium sulfate, followed by DE-52 anion exchange chromatography. Protein G affinity column chromatography, Mono S cation exchange chromatography and Superdex 200 gel filtration. The final product contained a highly purified mouse C7 component showing a single band on SDS-PAGE at the apparent Mrs of 90 kDa and 100 kDa under non-reduced and reduced conditions respectively. The yield of C7, which was measured by the biological activity, was 7.0%

Animals↗

PubMed1994

Role of membrane-associated lymphotoxin (mLT) in the killing activity of lymphokine-activated killer (LAK) cells towards various tumour cell lines.

Human lymphokine-activated killer (LAK) cells developed by an incubation of peripheral mononuclear cells with IL-2 express the membrane-associated lymphotoxin (LT)-related molecule (mLT). By a further cultivation of mLT expressing (mLT-positive) LAK cells for 24 h without IL-2, mLT disappears (mLT-negative LAK cells). Cytotoxicities of various tumour cell lines by either mLT-positive or -negative LAK cells were compared. Eight out of 12 tumour cell lines, less susceptible to mLT-negative LAK cells than mLT-positive LAK cells, were categorized as group A. Two tumour cells (K562 and Molt-4) had the same susceptibility to both kinds of LAK cells. The others (Daudi and Jurkat) had less susceptibilities only when they were assessed at E:T ratios of less than 5. The four tumour cell lines in the latter two cases, containing K562, Molt-4, Daudi and Jurkat cells, were categorized as group B. The cytotoxicities of group A tumour cells, but not group B tumour cells, by LAK cells were significantly suppressed by the presence of anti-LT antibody. Group A tumour cells had higher LT-binding ability (2.82-16.44 fmol/10(6) cells) than group B tumour cells (less than 1.46 fmol/10(6) cells). Both mLT-positive and -negative LAK cells had similar perforin activities and tumour cell-binding capacities. These results suggest that the mLT-mediated killing mechanism is involved in tumour cell killing by LAK cells. Further, various tumour cell lines can be classified into two large groups according to their susceptibilities to the mLT-mediated killing by LAK cells.

Animals↗

PubMed1993

In vitro degradation of very low density lipoprotein from diabetic patients by lipoprotein lipase.

Fatty acid release by incubation with lipoprotein lipase (LPL) in vitro from very low density lipoproteins (VLDL) obtained from diabetic patients was low compared with that from healthy subjects, though the compositions were similar in both VLDL. Percentages of the large size VLDL decreased and those of the small size VLDL increased after the incubation with LPL. At the same time, on polyacrylamide gel disk electrophoresis, the smaller catabolic products from these VLDL appeared at a similar position to that of low density lipoproteins (LDL) and at the running front where high density lipoproteins (HDL) had migrated. The amount of the small size VLDL and the LDL-like lipoproteins produced from diabetic VLDL were less than those from normal VLDL and inversely correlated with the percent decrease of the large original size VLDL. This fact suggests that VLDL from diabetic patients are poor substrates for LPL compared with normal VLDL.

Diabetes Mellitus, Type 2↗

PubMed1993

Expression of perforin and membrane-bound lymphotoxin (tumor necrosis factor-beta) in virus-specific CD4+ human cytotoxic T-cell clones.

In an attempt to clarify the mechanisms of cytotoxicity mediated by CD4+ cytotoxic T lymphocytes (CTL), the expression of perforin and membrane-bound lymphotoxin (LT) (tumor necrosis factor-beta) in herpes simplex virus (HSV)-specific CD4+ human cytotoxic and noncytotoxic T-cell clones was examined. Three HSV-specific CD4+ human CTL clones that showed HLA-DR-restricted cytotoxicity and proliferative response were established. The cytotoxicity of these clones in 5-hour 51Cr release assays was found to be mediated by the directional target cell lysis and not by the release of cytotoxic soluble factors, ie, "innocent bystander" killing. Northern blot analysis showed that messenger RNAs for perforin and LT, which were both considered to be important mediators for cytotoxicity of CD8+ CTL and natural killer cells, were abundantly expressed in HSV-specific CD4+ CTL clones. Expression of perforin in the cytoplasm of CD4+ CTL clones was also detected by immunohistochemical staining using a monoclonal antibody against perforin. In addition, LT bound to the cell surface of CD4+ CTL clones was detected by flow cytometry. In contrast, little or no expression of perforin and LT was detected in three HSV-specific CD4+ noncytotoxic T-cell clones. Although the cytotoxicity mediated by lymphokine-activated killer cells was partly inhibited by addition of anti-LT antibody, it did not show any effect on the cytotoxicity of HSV-specific CD4+ CTL clones. In addition, it was found that cytotoxicity mediated by these CD4+ CTL clones was Ca2(+)-dependent. These data thus suggest that perforin and membrane-bound LT are both expressed in HSV-specific CD4+ CTL, although perforin might be the more important mediator in short-term culture.

Antibodies, Monoclonal↗

PubMed1993

Inhibition of homologous complement activation by the heat-stable antigen.

The murine heat-stable antigen (HSAg) is of particular interest due to its unique tissue distribution. HSAg is expressed on most thymocytes, bone marrow cells, immature B cells, and erythrocytes, but not on peripheral T and mature B cells. Although HSAg has been thought to be a differentiation antigen, its actual biological significance remains unknown except for the HSAg on antigen presenting cells. Recently, a new rat anti-HSAg mAb, R13, has been developed. Here it has been found that the mouse complement activation on mouse erythrocytes, but not the human, guinea pig or rabbit complement activations, was enhanced in the presence of the Fab fragment of R13. Affinity-purified HSAg derived from mouse erythrocytes could be passively incorporated into rabbit erythrocytes because of its molecular characteristic of glycosylphosphatidyl inositol-anchored protein. Mouse complement activation, but not guinea pig complement activation, was partially suppressed on the HSAg-incorporated rabbit erythrocytes. These findings suggest that HSAg has a homologous complement regulating activity.

Animals↗

PubMed1993

Membrane-associated lymphotoxin expression and functional analysis of lymphokine-activated killer cells derived from tumor-infiltrating lymphocytes.

The expression of a membrane-associated lymphotoxin molecule (mLT) on lymphokine-activated killer (LAK) cells obtained from 18 patients with malignant tumors and its role in the tumor cell killing mechanisms were investigated. LAK cells from tumor-infiltrating lymphocytes (TIL-LAK cells) were mainly composed of CD3-positive cells, whereas LAK cells from peripheral blood lymphocytes (PBL-LAK cells) were mainly composed of CD16- and CD56-positive cells. However, mLT was found to be expressed on TIL-LAK cells as well as PBL-LAK cells. The degree of mLT expression correlated with the killing activity of LAK cells towards L929 cells (r = 0.806, P < 0.01, n = 15), but not with that towards Daudi or K562 cells. Although the degree of mLT expression correlated with the amount of secreted lymphotoxin (LT) in the supernatant of LAK cell culture, the secreted LT itself could not account for the tumor cell killing activity of LAK cells. Polyclonal rabbit anti-LT antibody partially inhibited the killing activities of LAK cells towards L929 cells and this inhibition was found in the combination of autologous tumor cells and PBL-LAK cells. These findings suggest the possibility that the mLT-related cytotoxicity is involved in the tumor cell killing mechanisms of TIL-LAK cells as well as PBL-LAK cells.

Adult↗

PubMed1992

Purification of the murine heat-stable antigen from erythrocytes.

The rat anti-mouse erythrocyte (MRBC) monoclonal antibody (mAb), R13, has been developed. The MRBC membrane protein recognized by R13 (R13-Ag) can be purified by loading the butanol-extracted MRBC membrane solution on a R13-conjugated Cellulofine column in the presence of 0.1% CHAPS followed by elution with 1% CHAPS. The amino acid sequence of the affinity-purified R13-Ag corresponded to that predicted from the cDNA for the murine heat-stable antigen. It was revealed that the actual heat-stable antigen was composed of 27 amino acids.

Amino Acid Sequence↗

PubMed1992

Correlation between killing activity towards the murine L929 cell line and expression of membrane-associated lymphotoxin-related molecule of human lymphokine-activated killer cells.

Human lymphokine-activated killer (LAK) cells expressed a membrane-associated lymphotoxin-related molecule (mLT) which was detected by flow cytometric analysis with anti-lymphotoxin antibody. Upon removal of exogenous interleukin-2 from LAK cell culture medium and another 24 h cultivation, the expression of mLT was decreased. Corresponding to the decrease of mLT expression, the killing activity of LAK cells towards L929 cells was remarkably reduced and killing of MIA PaCa-2 and U937 cells was moderately reduced, whereas killing of Daudi and K562 cells was fully restored. The supernatant of mLT-expressing LAK cells had no cytotoxic activity towards L929 cells in the absence of actinomycin D. Moreover, not only the killing of L929 cells but also that of human tumor cell lines (MIA PaCa-2, U937) by mLT-expressing LAK cells was partially inhibited in the presence of anti-lymphotoxin antibody. These results suggest an involvement of mLT in the killing of some tumor target cells by LAK cells.

Animals↗

PubMed1992

Agalactosyl IgG in pristane-induced arthritis. Pregnancy affects the incidence and severity of arthritis and the glycosylation status of IgG.

The effect of pregnancy on the incidence and severity of pristane-induced arthritis was examined along with the glycosylation status of IgG during the ante-natal and post-partum periods. It was found that pristane-induced arthritis is prevented by pregnancy. In addition, the levels of agalactosyl IgG fall during pregnancy but rise to greater than normal within a few days of parturition, before resetting towards the norm shortly afterwards. Interestingly, the level of agalactosyl IgG correlates with the severity of arthritis. As previously reported IL-6 may be an important factor, not necessarily the only one, in the production of agalactosyl IgG. Here it is clearly demonstrated that the kinetics of IL-6 activity post-pristane injection parallels the kinetics of agalactosyl IgG production. In addition, the overshoot in agalactosyl IgG levels immediately post-partum coincides with a burst in IL-6 activity. It is considered that these changes in IgG glycoform levels, or the factors which control them, may be related to the mechanisms underlying prevention/remission of arthritis during pregnancy.

Animals↗

PubMed1992

Relationship between interleukin 6, agalactosyl IgG and pristane-induced arthritis.

IL-6 titres in sera and peritoneal exudate fluids (PEF) derived from pristane injected DBA/1 and CBA/Igb mice were measured. Arthritic DBA/1 mice had significantly higher serum IL-6 titres than nonarthritic or normal mice at 160 days post pristane injection. By contrast, although both arthritic and non-arthritic CBA/Igb mice had higher serum IL-6 titres than normal mice, there was no significant difference in serum IL-6 titre between these two groups at day 200-230. In both strains of mice, the IL-6 titres in PEF were more than 10 times serum levels regardless of arthritis. As previously reported for CBA/Igb mice, agalactosyl IgG levels are raised in pristane injected DBA/1 mice and the percentage is higher in arthritic animals than that in non-arthritic mice. An association between serum agalactosyl IgG levels and PEF IL-6 in pristane injected DBA/1 was demonstrated. Moreover, the injection of recombinant IL-6 into normal mice increased their serum agalactosyl IgG levels. However, it is considered that IL-6 is not the only factor involved in the production of agalactosyl IgG.

Animals↗

PubMed1991

Expression of membrane-associated lymphotoxin/tumor necrosis factor-beta on human lymphokine-activated killer cells.

A membrane-associated lymphotoxin (LT)-related molecule was detected on human lymphokine-activated killer (LAK) cells by flow cytometric analysis. Kinetic analysis revealed that the LT antigenicity on LAK cells appeared at 9 h after the beginning of culture and was maintained thereafter. By autoradiography, the molecular weight of membrane LT was estimated to be 31 kD and/or 62 kD.

Cell Membrane↗