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S Kusumoto

Publications and source records attributed to S Kusumoto.

At least 73 records · Page 4Linked to original sources
PubMed1997

Specific binding of soluble peptidoglycan and muramyldipeptide to CD14 on human monocytes.

Previously, we were able to show that soluble peptidoglycan (sPG)-induced monokine production in human peripheral monocytes is inhibited by anti-CD14 monoclonal antibodies and by lipid A partial structures. This suggested but did not prove that monocytic surface protein CD14 is involved in the activation of human monocytes not only by cell wall components of gram-negative bacteria such as lipopolysaccharide (LPS) but also by cell wall components of gram-positive bacteria such as sPG. In the present study, we provide experimental evidence that CD14 indeed constitutes a binding site for sPG recognition and activation of human monocytes. The results show that fluorescein isothiocyanate-sPG (FITC-sPG) binds to human monocytes in a saturable, dose-dependent, and specific manner. For maximal binding, 2 to 3 microg of FITC-sPG per ml was sufficient, and this binding is completed within 90 min; about 40% of the binding is completed within the first 3 min. The FITC-sPG binding is considered specific because unlabeled sPG and also muramyldipeptide (MDP), the minimal bioactive structure of sPG, inhibit the binding of sPG to monocytes in a dose-dependent manner. This specific binding was also inhibited by an anti-CD14 monoclonal antibody, LPS, and lipid A partial structure compound 406. Direct evidence for an interaction of sPG with CD14 is provided by experiments involving native polyacrylamide gel electrophoresis that showed a shift of the electrophoretic mobility of CD14 by LPS as well as by sPG. These results allow the conclusion that sPG binds directly to CD14, that MDP represents the active substructure of sPG, and that CD14 may be a lectin-like receptor which plays a key role in cellular stimulation by bioactive components of not only gram-negative but also gram-positive bacteria.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1996

Detection of UDP-D-xylose: alpha-D-xyloside alpha 1-->3xylosyltransferase activity in human hepatoma cell line HepG2.

We previously reported the detection of novel O-linked sugar chains classified as being of the glucosyl-O-serine type [Hase et al. (1988) J. Biochem. 104, 867-868]. The sugar chains are a disaccharide (Xyl alpha 1-3Glc) and a trisaccharide (Xyl alpha 1-3Xyl alpha 1-3 Glc) linked to serine residues in epidermal growth factor-like domains of human and bovine blood coagulation factors. The structures of these sugar chains suggested the presence of an alpha 1-->3xylosyltransferase for their biosynthesis. We report here on the detection of alpha 1-->3xylosyltransferase activity which catalyzes the transfer of xylose to Xyl alpha 1-3Glc in the human hepatoma cell line HepG2. We employed pyridylaminated Xyl alpha 1-3Glc as a fluorescent acceptor and UDP-D-Xyl as a donor. The reaction product was purified by reversed-phase HPLC, and the structure of the transfer product isolated was confirmed to be pyridylaminated Xyl alpha 1-3Xyl alpha 1-3Glc by Smith degradation, mass spectrometry, and alpha- and beta-xylosidase digestions. The apparent K(m) value for pyridylaminated Xyl alpha 1-3Glc was 52 mM and for UDP-D-Xyl 0.28 mM. Optimum pH was 7.2. The enzyme was inactivated by addition of EDTA, and its activity was restored by addition of Mn2+ and Mg2+. These results indicate the presence of a novel enzyme which is able to transfer xylose to Xyl alpha 1-3Glc, forming Xyl alpha 1-3Xyl alpha 1-3Glc in human cells.

Animals↗
PubMed1996

Structural characteristics of peptidoglycan fragments required to prime mice for induction of anaphylactoid reactions by lipopolysaccharides.

Structural characteristics of peptidoglycan fragments required to prime mice for the induction of anaphylactoid reactions by Salmonella abortusequi lipopolysaccharide were examined in endotoxin-resistant C3H/HeJ mice, with special focus on the disaccharide-pentapeptide [N-acetylglucosaminyl-beta(1-4)-N-acetylmuramyl-L-alanyl-D -isoglutaminyl-meso-2,6-diaminopimelyl (DAP)-D-alanyl-D -alanine] and its smaller partial derivatives. The bacterial and synthetic muramyl tripeptides (DAP- and lysine [Lys]-type, respectively) and synthetic muramyl dipeptide primed mice for induction of anaphylactoid reactions accompanied by death within 1 h. The disaccharide-tripeptide exhibited weaker activity, and the disaccharide-tetrapeptide and muramyl tetrapeptide exhibited marginal activity. In contrast, intact peptidoglycans of various bacteria and the disaccharide-pentapeptide lacked the priming activity, although they showed adjuvant activity similar to that of the above components.

Anaphylaxis↗
PubMed1996

[Heparin affinity high-performance liquid chromatography as an alternative to bioassay of CS23 mutein of recombinant human basic fibroblast growth factor].

We investigated whether a chemical assay by high-performance liquid chromatography (HPLC) as an alternative to the complicated and time-consuming bioassay for CS23 mutein of recombinant human basic fibroblast growth factor (rhbFGF-CS23) using the fetal bovine heart endothelial cell line ATCC CRL 1395. Physically, chemically or enzymatically denatured rhbFGF-CS23 was subjected to heparin affinity (HA)-HPLC and the bioassay. Good agreement was observed between the results obtained by these two methods. Moreover, HA-HPLC gave much more reproducible results (RSD = 1.9%, n = 6) than the bioassay (RSD = 7.4%, n = 18). HA-HPLC is therefore a simple, accurate and reproducible alternative to the bioassay for quality control and stability studies for rhbFGF-CS23 preparations. HA-HPLC is also considered to be applicable to assays for FGFs which have heparin affinity and biological activity similar to those of the CS23 mutein.

Animals↗
PubMed1996

[Refractory anemia with excess of blasts (RAEB) with a high level of HbF that preceded apparent pancytopenia].

A 76-year-old male was kept under observation for idiopathic interstitial peumonitis in our hospital from August 1992. Laboratory data revealed a slightly high level of HbF (2.7%) but normal values of other hematological examination. The level of HbF increased slowly, and in April 1994, pancytopenia appeared for the first time. Bone marrow was normocellular with myelodysplasia and 9% blasts. Cytogenetic analysis revealed 46, XY, del (20) (q11;q13). He was diagnosed as having myelodysplastic syndrome (MDS), refractory anemia with excess of blasts. At diagnosis of MDS, the level of HbF was 20.0%. He developed acute myelocytic leukemia 3 months later. It has been reported that approximately 40% of patients with MDS have higher HbF levels than normal, which is considered to be functional abnormality of the MDS clone. It is suggested that the MDS clone had already increased in this patient at presentation, 32 months before pancytopenia appeared.

Aged↗
PubMed1995

Macrophage activation in response to S-form lipopolysaccharides (LPS) separated by centrifugal partition chromatography from wild-type LPS: effects of the O-polysaccharide portion of LPS.

The S-form lipopolysaccharide (LPS) was effectively separated from a native preparation of smooth-type Salmonella abortus equi LPS by means of the centrifugal partition chromatography (CPC). To clarify the mechanisms by which LPS activates macrophages, CPC-separated S-form LPS was assessed for its ability to induce the secretion of tumor necrosis factor-alpha (TNF-alpha) by murine macrophage-like J774.1 cells in comparison with other fractions of LPS which lacks most of O-polysaccharides. LPS dose-response and time-kinetics studies showed that serum factor(s) regulated especially the onset of TNF-alpha secretion in stimulation with S-form LPS. These results strongly suggest that the native (unfractionated) LPS activates macrophages in both O-polysaccharide/serum-dependent and -independent pathways.

Animals↗
PubMed1995

Elevated promotion of prostacyclin production by synthetic lipid A analogs in aged human endothelial cells in culture.

We examined the effects of E. coli lipopolysaccharide (LPS) and synthetic analogs of lipid A, a bioactive moiety of LPS, on the prostacyclin (PGI2) production by young and old human endothelial cells in vitro. PGI2 production by endothelial cells has been shown to decrease during in vitro cellular senescence as well as in vivo. LPS and all the analogs tested in this study did not stimulate PGI2 production by young endothelial cells more than twofold. However, LPS and the majority of the lipid A analogs examined stimulated the PGI2 production by old cells more than twofold (approximately two- to sixfold). These results indicate that the responses to certain stimuli sometimes differ markedly between young and old cells, and this should be carefully considered when evaluating the biological effects of various compounds. Furthermore, these results suggest that certain synthetic lipid A analogs can be used as drugs to prevent some age-related vascular diseases.

Age Factors↗
PubMed1995

Cytokine-inducing glycolipids in the lipoteichoic acid fraction from Enterococcus hirae ATCC 9790.

Five high molecular weight glycolipids capable of stimulating human peripheral whole-blood cell cultures to cause interleukin 6 (IL-6) and tumor necrosis factor (TNF)-alpha induction were isolated from one of the lipoteichoic acid fractions (LTA-2) extracted from Enterococcus hirae ATCC 9790 (Tsutsui et al., (1991) FEMS Microbiol. Immunol. 76, 211-218) by a combination of hydrophobic interaction and anion-exchange chromatographies. This purification procedure resulted in a remarkable increase in the cytokine-inducing activities on the weight basis of isolated glycolipids (a maximum of 36- and 17-fold increases of IL-6 and TNF-alpha induction, respectively). The total yield of these bioactive glycolipids amounted to 6 wt% of the parent LTA-2 fraction, while the recovery rate in terms of the cytokine-inducing activities was estimated to be sufficient. The chemical composition and the profile, using SDS-PAGE, revealed that all of the isolated bioactive components were high molecular weight glycolipids, which were distinct from each other and from the parent LTA-2 fraction. These findings suggest that the IL-6 and TNF-alpha-inducing activities previously noted in the parent LTA-2 fraction are not attributable to a chemical entity, the structure of which had been proposed elsewhere (Fischer, W. (1990) in Glycolipids, Phosphoglycolipids and Sulfoglycolipids (Kates, M. ed.) pp. 123-234, Plenum Press, New York), but to the other high molecular weight glycolipids described here.

Animals↗
PubMed1995

Molecular and structural requirements of a lipoteichoic acid from Enterococcus hirae ATCC 9790 for cytokine-inducing, antitumor, and antigenic activities.

Comparison was made between the immunobiological and antigenic properties of two lipoteichoic acid (LTA) fractions (LTA-1 and -2) from Enterococcus hirae ATCC 9790, their glycolipid portions, and synthetic compounds partially mimicking the above bacterial products. The more lipophilic LTA-2 fraction was capable of inducing serum tumor necrosis factor alpha and interleukin-6 in muramyldipeptide-primed mice and serum gamma interferon in those primed with Propionibacterium acnes. The LTA-2 fraction also induced tumor necrosis factor alpha, interleukin-6, and thymocyte-activating factor (essentially interleukin-1) in murine peritoneal macrophage cultures. Consecutive intravenous injections of muramyldipeptide and the LTA-2 fraction in Meth A fibrosarcoma-bearing BALB/c mice caused hemorrhagic necrosis and marked regression leading to complete regression of the tumor with no accompanying weakening or lethal effects. The LTA-2 fraction was at least 10,000-fold less pyrogenic in rabbits than a reference endotoxic lipopolysaccharide. The more hydrophilic LTA-1 fraction, on the other hand, showed at most marginal activity in the in vivo and in vitro assays. Natural glycolipids (NGL-1 and -2) which were prepared from a chloroform-methanol extract of Streptococcus pyogenes and E. hirae cells, and comparable in structure to the lipid moieties of the LTA-1 and -2 fractions, respectively, were practically inactive in all of the assays. None of the test synthetic compounds was immunobiologically active, although synthetic partial counterparts of the structure of LTA proposed by W. Fischer (Handb. Lipid Res. 6:123-234, 1990) reacted with murine monoclonal antibody TS-2, which was raised against OK-432, a penicillin-killed S. pyogenes preparation, and capable of neutralizing the cytokine-inducing activities of the LTA-2 fraction.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1995

Binding of lipopolysaccharide (LPS) to an 80-kilodalton membrane protein of human cells is mediated by soluble CD14 and LPS-binding protein.

Activation of cells by bacterial lipopolysaccharide (LPS) plays a key role in the pathogenesis of gram-negative septic shock. The 55-kDa glycoprotein CD14 is known to bind LPS and initiate cell activation. However, there must be additional LPS receptors because CD14 is linked by a glycosylphosphatidyl inositol anchor to the cell membrane and therefore unable to perform transmembrane signalling. Searching for potential LPS receptors, we investigated the binding of LPS to membrane proteins of the human monocytic cell line Mono-Mac-6. Membrane proteins were electrophoretically separated under reducing conditions, transferred to nitrocellulose, and exposed to LPS, which was visualized with anti-LPS antibody. Smooth- and rough-type LPS, as well as free lipid A, bound to a variety of proteins in the absence of serum. However, in the presence of serum, additional or preferential binding to a protein of approximately 80-kDa was observed. Experiments with differently acylated lipid A structures showed that the synthetic tetraacyl compound 406 was still able to bind, whereas no binding was detected with the bisacyl compound 606. The 80-kDa membrane protein was also detected on human peripheral blood monocytes and endothelial cells. The serum factors mediating the binding of lipid A to the 80-kDa membrane protein were identified as soluble CD14 and LPS-binding protein. From these results, we conclude that this 80-kDa protein is a candidate for the hypothetical molecule for LPS and/or LPS-CD14 recognition and signal transduction.

Acute-Phase Proteins↗
PubMed1995

Glycosphingolipids from Sphingomonas paucimobilis induce monokine production in human mononuclear cells.

Glycosphingolipids (GSL) isolated from the gram-negative lipopolysaccharide (LPS)-free bacterium Sphingomonas paucimobilis have remarkable structural similarities with LPS and its hydrophobic part, termed lipid A. Like LPS, but in contrast to the structurally related ceramides and cerebrosides, GSL contain an alpha-linked, negatively charged pyranosidic glycosyl component adjacent to the lipid portion and are capable of forming membranes. Because of these similarities, it was of interest to investigate whether these GSL are also able to induce monokine production in human mononuclear cells (MNC). Our results show that a GSL containing four sugar residues (GSL-4A) induced the release of tumor necrosis factor, interleukin-6, and interleukin-1 in MNC, whereas GSL-1, containing only one glycosyl residue, was inactive. A minimal concentration of 1 microgram of GSL-4A per ml was necessary to induce monokine production in MNC, whereas LPS was as active at a 10,000-fold-lower concentration (0.1 ng/ml). Both GSL-4A-induced monokine production and LPS-induced monokine production were reduced by the bactericidal/permeability-increasing protein and GSL-1. In contrast to LPS, GSL-4A-induced monokine release could be inhibited neither by an anti-CD14 monoclonal antibody nor by lipid A partial structures. We therefore conclude that at the receptor level, different mechanisms are involved in the LPS- and GSL-4A-induced monokine release.

Antigens, CD↗
PubMed1995

Therapeutic efficacy of 5-fluorouracil prodrugs using endogenous serum proteins as drug carriers: a new strategy in drug delivery system.

To establish a novel drug delivery system for 5-fluorouracil (5FU), we have developed a system in which the low-molecular-weight prodrugs of 5FU bound to endogenous serum proteins, thus circulating like those proteins. Subsequently, the prodrugs were slowly hydrolyzed to generate active 5FU in the bloodstream. To examine the therapeutic effect of these prodrugs, we injected them into BALB/c mice previously implanted subcutaneously with Meth A sarcoma. Among the prodrugs, 1-(N-4-chlorophenyl-N-methylcarbamoyl)-5-fluorouracil (5FU-1pCPMC) was effective in reducing tumors and prolonging survival time. The non-hydrolyzable compound, 1-(4-chlorobenzyl)-5-fluorouracil, did not show any therapeutic effect, suggesting that the therapeutic efficacy of 5FU-1pCPMC is due to the sustained release of 5FU from the serum protein-prodrug complex. The data shown here may create a new field in drug delivery system technology.

Animals↗
PubMed1994

Endotoxin and lipid A stimulate proliferation of human T cells in the presence of autologous monocytes.

In this paper we describe a new activity of LPS and partial structures: the induction of DNA synthesis and lymphokine production of human T lymphocytes. The LPS-induced T cell proliferation is dose dependent and requires 100 to 10,000 ng/ml of LPS or synthetic lipid A (compound 506) for optimal stimulation. In contrast, the synthetic lipid A precursor Ia (compound 406) is not active but rather antagonizes LPS-induced proliferation. The proliferation is accompanied by the expression of mRNA for the Th1 cell-derived lymphokines IFN-gamma and IL-2, but not for the Th2 lymphokines IL-4, IL-5, or IL-10. Highly enriched T lymphocyte preparations with less than 0.1% monocytes are not stimulated by LPS, showing that monocytes are required for T cell proliferation. Reconstitution experiments show that only monocytes, but not B lymphocytes, are able to support induction of DNA synthesis. Separating LPS-stimulated monocytes from T lymphocytes by a membrane, permeable for cytokines but not for cells, abolishes T cell proliferation. Fixation of monocytes with paraformaldehyde also abrogates their accessory function for T cells. If the monocytes are preincubated for 2 h at 37 degrees C with LPS and then washed, they still are able to induce T cell proliferation in the absence of additional LPS. Our results indicate that human T cells respond in a monocyte-supported manner to LPS exposure by proliferation and lymphokine production. We hypothesize that this reactivity of T lymphocytes to LPS may be of clinical relevance.

Adult↗
PubMed1994

Trilineage recovery by combination therapy with recombinant human granulocyte colony-stimulating factor and erythropoietin in patients with aplastic anemia and refractory anemia.

Ten patients with aplastic anemia (AA) and seven patients with refractory anemia (RA) were treated with recombinant human granulocyte colony-stimulating factor (rhG-CSF) and erythropoietin (rhEpo) in combination. rhG-CSF (5-20 micrograms/kg) and rhEpo (120-720 U/kg) were administered by s.c. injection three times a week for at least six months, and the administration was continued as maintenance therapy for as long as possible when hematological responses were observed. Six (60%) of the ten AA patients and four (58%) of the seven RA patients showed multilineage responses. Of these responders, six patients achieved trilineage recovery. While all of the responders were dependent on red blood cell transfusions and eight of them required platelet transfusions before treatment, they now no longer need transfusions of either red blood cells or platelets. A median treatment duration of 9 (range 1 to 28) months was required to achieve multilineage recovery. The responders showed an ability to maintain the multilineage recovery for 9+ to 47+ months and to tolerate long-term treatment. These results indicate that the long-term treatment with rhG-CSF and rhEpo may benefit a substantial percentage of patients with AA and RA and provide an optional therapy for these patients.

Adolescent↗
PubMed1994

The significance of the hydrophilic backbone and the hydrophobic fatty acid regions of lipid A for macrophage binding and cytokine induction.

Natural partial structures of lipopolysaccharide (LPS) as well as synthetic analogues and derivatives of lipid A were compared with respect to inhibit the binding of 125I-labelled Re-chemotype LPS to mouse macrophage-like J774.1 cells and to induce cytokine-release in J774.1 cells. LPS, synthetic Escherichia coli-type lipid A (compound 506) and tetraacyl precursor Ia (compound 406) inhibited the binding of 125I-LPS to macrophage-like J774.1 cells and induced the release of tumor necrosis factor alpha (TNF alpha) and interleukin 6 (IL-6). Deacylated R-chemotype LPS preparations were completely inactive in inhibiting binding and in inducing cytokine-release. Among tetraacyl compounds, the inhibition-capacity of LPS-binding was in decreasing order: PE-4 (alpha-phosphonooxyethyl analogue of 406) > 406 >> 404 (4'-monophosphoryl partial structure of 406) > 405 (1-monophosphoryl partial structure of 406). In the case of hexaacyl preparations, compounds 506, PE-1 (alpha-phosphonooxyethyl analogue of 506) and PE-2 (differing from PE-1 in having 14:0 at positions 2 and 3 of the reducing GlcN) inhibited LPS-binding and induced cytokine release equally well, whereas preparation PE-3 (differing from PE-2 in containing a beta-phosphonooxyethyl group) showed a substantially lower capacity in binding-inhibition and cytokine-induction. The conclusion is that chemical changes in the hydrophilic lipid A backbone reduce the capacity of lipid A to bind to cells, whereas the number of fatty acids determines the capacity of lipid A to activate cells. These results indicate that the bisphosphorylated hexosamine backbone of lipid A is essential for specific binding of LPS to macrophages and that the acylation pattern plays a critical role for LPS-promoted cell activation, i.e. cytokine induction.

Acylation↗
PubMed1994

Soluble peptidoglycan-induced monokine production can be blocked by anti-CD14 monoclonal antibodies and by lipid A partial structures.

We have investigated the interaction of soluble peptidoglycan (sPG), in comparison with lipopolysaccharide (LPS), with human mononuclear cells (MNC) by determining the capacity of sPG to induce interleukin-6 (IL-6) and IL-1 release. In addition, we investigated the modulation of their interaction by anti-CD14 monoclonal antibody and by partial structures of LPS. We found that sPG, like LPS, was able to induce IL-6 and IL-1 production by MNC. However, dose-response experiments revealed that at least 3,000 ng of sPG per ml was necessary for induction, whereas the optimal LPS concentration was 1 ng/ml. Anti-CD14 monoclonal antibody reduced sPG- and LPS-induced IL-6 and IL-1 production. Moreover, partial structures of LPS were able to reduce monokine production induced by sPG and LPS. We conclude that sPG constitutes, like LPS, an inflammatory cytokine inducer and that CD14 is involved in the activation of human monocytes not only by LPS but also by sPG.

Acute-Phase Proteins↗