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Biomedical subjects

M Fujihara

Publications and source records attributed to M Fujihara.

At least 19 recordsLinked to original sources
PubMed2005

Heterogeneity of platelet responsiveness to anti-CD36 in plasma associated with adverse transfusion reactions.

BACKGROUND AND OBJECTIVES: Antibodies to CD36 (anti-CD36) are clinically important. As some platelet immunoglobulins produced by transfusion or pregnancy have been shown to induce platelet activation and to play roles in non-haemolytic transfusion reactions (NHTRs), we investigated the in vitro response of platelets to plasma containing anti-CD36. MATERIALS AND METHODS: Plasma containing anti-CD36, implicated in the development of NHTRs and subsequent thrombocytopenia, was incubated with CD36-positive platelets. Plasma-induced platelet activation was examined by evaluating platelet aggregation and RANTES (regulated on activation, normal, T-cell expressed, and presumably secreted) release. RESULTS: Platelet activation was induced by plasma alone in four out of 20 CD36-positive subjects. In seven subjects, platelet activation was synergistically induced by the combination of epinephrine priming and the plasma. The platelets of the nine remaining subjects failed to respond to the plasma. Platelet activation induced by either the plasma alone or by synergy with epinephrine required the involvement of Fc gamma RIIa. The different responsiveness of the platelets was partially associated with the surface levels of CD36 and Fc gamma RIIa, but not with Fc gamma RIIa polymorphisms. CONCLUSIONS: Plasma containing anti-CD36, implicated in the development of NHTRs, exhibited a platelet-activating capability. Additionally, platelets from healthy human subjects exhibited a considerable degree of heterogeneity in their responsiveness to this plasma. The heterogeneity of these responses may determine the occurrence of anti-CD36-related NHTRs.

Antigens, CD↗
PubMed2002

Fatal necrotizing stomatitis due to Trichoderma longibrachiatum in a neutropenic patient with malignant lymphoma: a case report.

Primary invasive mold infection of the oral cavity is a rare but serious complication in immunocompromised hosts. We report a case of fatal Trichoderma longibrachiatum stomatitis in a 66-year-old female patient with malignant lymphoma. The infection rapidly disseminated from the oral mucosa to the lungs during neutropenia. Despite intensive antifungal therapy with amphotericin B and itraconazole, there was a fatal progression of the condition. While Trichoderma species have been recognized to be pathogenic in profoundly immunosuppressed hosts, this is the first report of the primary oral focus causing a fatal infection.

Aged↗
PubMed2001

Invasive Aspergillus stomatitis in patients with acute leukemia: report of 12 cases.

An 8-year retrospective analysis of invasive Aspergillus stomatitis in neutropenic patients with acute leukemia was performed to characterize the epidemiology and clinical features of the infection. Twelve cases of invasive Aspergillus stomatitis were identified with both clinicohistological and microbiological evidence, and the majority of cases were caused by Aspergillus flavus (10 [83%] of 12 patients). The infection was strongly suspected when a neutropenic patient developed persistent fever without a known source, symptoms of gingival pain and facial swelling, and a solitary ulcerating lesion of mucogingiva covered with a gray necrotic pseudomembrane. Aspergillus stomatitis was diagnosed a median 23 days after admission. In all 12 patients, the diagnosis was made during the period of neutropenia. Ten patients (83%) were treated with amphotericin B and surgery and survived with recovery of neutrophils. Two patients died, and disseminated aspergillosis was identified in 1 patient.

Acute Disease↗
PubMed2001

CrkL is an adapter for Wiskott-Aldrich syndrome protein and Syk.

Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia are caused by mutations of the WAS protein (WASP) gene. WASP may be involved in the regulation of podosome, an actin-rich dynamic cell adhesion structure formed by various types of cells. The molecular links between WASP and podosomes or other cell adhesion structures are unknown. Platelets express an SH2-SH3 adapter molecule, CrkL, that can directly associate with paxillin, which is localized in podosomes. The hypothesis that CrkL binds to WASP was, therefore, tested. Results from coprecipitation experiments using anti-CrkL and GST-fusion proteins suggest that CrkL binds to WASP through its SH3 domain and that the binding was not affected by WASP tyrosine phosphorylation. The binding of GST-fusion SH3 domain of PSTPIP1 in vitro was also not affected by WASP tyrosine phosphorylation, suggesting that the binding of the SH3 domains to WASP is not inhibited by tyrosine phosphorylation of WASP. Anti-CrkL also coprecipitates a 72-kd protein, which was identified as syk tyrosine kinase, critical for collagen induced-platelet activation. CrkL immunoprecipitates contain kinase-active syk, as evidenced by an in vitro kinase assay. Coprecipitation experiments using GST-fusion CrkL proteins suggest that both SH2 and SH3 domains of CrkL are involved in the binding of CrkL to syk. WASP, CrkL, syk, and paxillin-like Hic-5 incorporated to platelet cytoskeleton after platelet aggregation. Thus, CrkL is a novel molecular adapter for WASP and syk and may potentially transfer these molecules to the cytoskeleton through association with cytoskeletal proteins such as Hic-5.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Stem cell factor prevents Fas-mediated apoptosis of human erythroid precursor cells with Src-family kinase dependency.

The Fas ligand (Fas-L) expressed on mature erythroblasts may induce apoptosis of more immature erythroid cells that express Fas, whereas stem cell factor (SCF) may prevent Fas-mediated cell death in hematopoietic progenitor cells. The manner in which SCF prevents Fas-mediated cell death still is unclear. Given the essential role of SCF and the potentially important involvement of the Fas/Fas-L system in the development of erythrocytes, we studied mechanisms related to SCF prevention of Fas-mediated apoptosis. We used primary cultured human erythroid colony-forming cells (ECFC) derived from CD34+ cells and enriched glycophorin A positive (GPA+) c-kit+ cells in ECFC. Apoptosis of ECFC was induced by an Fas-L mimetic monoclonal antibody CH11. DNA fragmentation and the activation of caspase-3 and caspase-8 were measured using commercially available kits. Characterization of expanded cells was performed using multiparameter flow cytometry. Lyn kinase activity was measured by enolase kinase assays. SCF inhibited the CH11-induced DNA fragmentation of ECFC as well as enriched GPA+ c-kit+ cells in ECFC, but not those of GPA+ c-kit- cells. SCF also inhibited the activation of caspase-3 and caspase-8, without downregulation of the surface expression of Fas, suggesting that SCF prevents apoptosis through uncoupling of Fas ligation from subsequent caspase activation. PP2, a specific inhibitor of Src-family kinases, antagonized the effects of SCF in preventing Fas-mediated apoptosis. We propose that SCF prevents Fas-mediated apoptosis of erythroid progenitor cells in a manner dependent on the activity of Src-family tyrosine kinases. We also identified active Lyn in erythroid cells. These data suggest the presence of a novel Src-family-dependent function of SCF in the development of erythrocytes.

Antibodies, Monoclonal↗
PubMed2001

Effects of poly(ethyleneglycol)-modified hemoglobin vesicles on agonist-induced platelet aggregation and RANTES release in vitro.

We studied the effects of hemoglobin-vesicles modified with PEG (PEG-HbV), a type of liposome-encapsulated hemoglobin (LEH), on human platelet functions in vitro. The effect of a low concentration of PEG-HbV (Hb; 5.8 mg/dl) was assessed by examining an agonist-induced aggregation response, and that of relatively high concentrations of PEG-HbV (Hb; 0.29, 1 and 2 g/dl) by measuring the release of RANTES (Regulated upon activation, normal T-cell expressed and presumably secreted) from platelets, which is regarded as a marker of platelet activation. The preincubation of platelets with PEG-HbV at 5.8 mg/dl of Hb did not affect platelet aggregation induced by collagen, thrombin and ristocetin. The pretreatment of platelet-rich plasma (PRP) with PEG-HbV at concen trations up to 2 g/dl of Hb had no aberrant effects on the collagen-induced RANTES release. Furthermore, the collagen-induced release of RANTES from PRP was not affected by longer incubation with PEG-HbV at 2 g/dl of Hb. The basal levels of RANTES from PRP were unchanged in the presence of PEG-HbV. These results suggest that PEG-HbV, at the concentrations studied, have no aberrant effects on platelet functions in the presence of plasma.

Chemokine CCL5↗
PubMed2001

Superoxide generation from human polymorphonuclear leukocytes by liposome-encapsulated hemoglobin.

We investigated the interactions between liposome-encapsulated hemoglobin (Neo Red Cells: NRC) and human polymorphonuclear leukocytes as assessed by superoxide generation. NRC triggered superoxide generation from neutrophils in a dose-dependent manner. Empty liposomes also induced superoxide production of neutrophils. Superoxide generation of neutrophils induced by phorbol myristate acetate (PMA) was delayed but intensified both by NRC and empty liposomes. The intensity of superoxide generation induced by NRC was smaller than that by the empty liposomes. As NRC contained superoxide dismutase (SOD) that was copurified with hemoglobin from red blood cells and its activity remained, SOD contained in NRC may partially eliminate superoxide.

Blood Substitutes↗
PubMed2001

Virus inactivation in hemoglobin solution by heat treatment.

To increase the safety of stroma-free hemoglobin solution (SFH) as an artificial oxygen carrier source, we investigated the effect of heat treatment on virus inactivation in hemoglobin solution. The hemoglobin solution spiked with vesicular stomatitis virus (VSV) was treated at 60 degrees C for 1 hr under either an air or CO atmosphere. VSV was inactivated at >5.8 log10 and >6.0 log10 under the air and CO atmosphere, respectively. Although the methemoglobin rate increased after the heat treatment under the air atmosphere, no methemoglobin formation was observed by the treatment under the CO atmosphere. Isoelectric focusing analysis revealed the denaturation of hemoglobin after the heat treatment under the air, while hemoglobin banding was not altered in the carbonylated condition. Some protein bands other than hemoglobin were weakened or disappeared on SDS-PAGE after the heat treatment under both conditions. In addition, the hemoglobin concentration in the SFH was higher after the heat treatment than before the treatment. These findings indicate that the heat treatment under the CO atmosphere inactivates viruses without hemoglobin denaturation, and hence, this method is a promising approach to prepare a safer SFH as artificial oxygen carriers.

Blood Substitutes↗
PubMed2001

Effects of poly(ethyleneglycol)-modified hemoglobin vesicles on N-formyl-methionyl-leucyl-phenylalanine-induced responses of polymorphonuclear neutrophils in vitro.

[Poly(ethyleneglycol)]-modified hemoglobin vesicles (PEG-HbV), a type of encapsulated hemoglobin, have been developed as artificial oxygen carriers and it is important to evaluate their blood compatibility. We studied the effects of PEG-HbV on human polymor phonuclear neutrophils (PMNs) in vitro, focusing on the functional responses to N-formyl-methionyl-leucyl-phenylalanine (fMLP) as an agonist. The pretreatment of the PMNs with PEG-HbV up to a concentration of 60 mg/dl Hb did not affect the fMLP-triggered chemotactic activity. In parallel to these results, the fMLP-induced upregulation of CD11b (Mac-1) levels on the PEG-HbV-pretreated PMNs was comparable to that of untreated cells. Furthermore, the pretreatment of the PMNs with the PEG-HbV even at 600 mg/dl Hb did not affect the gelatinase B (Matrix methalloproteinase-9 (MMP-9)) release, suggesting that the fMLP-induced release of secondary and tertiary granules was normal. In addition, the fMLP-triggered superoxide production of the PMNs was unchanged by the pretreatment with the PEG-HbV at 600 mg/dl Hb. Thus, these results suggest that PEG-HbV, at the concentrations studied, have no aberrant effects on the fMLP-triggered functions of human PMNs.

Blood Substitutes↗
PubMed2001

Characterization of bovine angiogenin-1 and lactogenin-like protein as glycyrrhizin-binding proteins and their in vitro phosphorylation by C-kinase.

Angiogenin-1 (p15, an angiogenesis inducer with RNase activity) and lactogenin-like protein (p17) isolated from partially purified bovine lactoferrin (bLF) preparations were characterized as glycyrrhizin (GL)-binding proteins (gbPs). As expected, bLF-affinity column chromatography confirmed these two gbPs to be bLF-binding proteins. These two purified gbPs exhibited RNase activities when incubated with poly(C) as a substrate. Both GL and glycyrrhetinic acid (GA) at 100 microM significantly inhibited RNase activities of these two gbPs, both of which functioned as phosphate acceptors of C-kinase in vitro. Phosphorylation of p15 and p17 by C-kinase was inhibited by GA in a dose-dependent manner with the 50% inhibition dose (ID50) of approx. 10 microM, whereas GL required a relatively high dose (300 microM) to inhibit significantly it. A GA derivative (oGA, ID50=approx. 0.3 microM) was found to be a potent inhibitor of the C-kinase-mediated phosphorylation of these two gbPs in vitro. In addition, a possible physiological significance of C-kinase on the physiological interaction between bLF and two bLF-binding proteins (p15 and p17) is noted.

Animals↗
PubMed2001

[Evaluation of non-surgical therapy for the malignant lymphomas located mainly in the stomach and duodenum].

We evaluated the effects of the conservative treatment to 29 patients with non-Hodgkin lymphomas located mainly in stomach and duodenum. We could induce complete remission in all the cases of stage I, II, and III including MALT lymphomas by the combination therapy of Helicobacter pylori (H. pylori) eradication, chemotherapy, and radiation, and the complete remission persisted in all except for the three cases who died of other causes. Even in stage IV lymphomas mainly located in stomach and duodenum, we could induce complete remission in 50% of them. Considering the quality of life of patients with lymphomas located mainly in stomach and duodenum, the conservative treatment may be of benefit more than the surgical approach.

Adult↗
PubMed2000

Thrombopoietin and interleukin-2 induce association of CRK with STAT5.

Crk (Crk I and II) proteins and closely related CrkL are adapters which are commonly involved in various signaling processes in various cells, and these proteins share many ligands. Whether they have redundant or distinct physiologic roles is unclear. By coprecipitation and far Western blotting analysis, we demonstrate that Crk (I/II) binds to tyrosine phosphorylated STAT5 in cells stimulated by cytokines such as thrombopoietin (TPO) and interleukin-2 (IL-2). The association did not require nuclear elements and can be observed in primary cells as this was also demonstrated in TPO-stimulated platelets. Using a beta-casein promoter STAT5 binding site as a probe, we have also demonstrated that CrkL (a close relative of Crk) antiserum, but not Crk antiserum, supershifted the STAT5-DNA complex by an electrophoretic mobility shift assay, suggesting that CrkL, but not Crk, is the major component of the complex. Thus, Crk and CrkL may have distinct roles in the regulation of STAT5.

Base Sequence↗
PubMed2000

Comparison of sensitivity to ultraviolet B irradiation between human lymphocytes and hematopoietic stem cells.

To investigate the clinical applicability of prophylaxis of post-transplant graft-versus-host disease by UV-B irradiation of stem cell preparations, the UV-B sensitivities of human lymphocytes and primitive hematopoietic progenitors were compared. The mononuclear cell fractions (MNC) derived from human cord blood and granulocyte-colony-stimulating factor-mobilized peripheral blood were used. After UV-B irradiation, lymphocyte proliferation ability, hematopoietic colony-forming cells, and apoptotic cells were analyzed. At a dose of 33 J/m(2), significant differences were observed in the residual percentages of hematopoietic progenitors and lymphocyte functions [ANOVA, F (5,46) = 19.4; P <.0001], and the difference between CFU-C (85.2% + 24.0%; n = 8) and MLR (12.7% + 12. 6%; n = 10) was significant (P <.0001). There were no significant differences in the residual percentages of CFU-C, HPP-CFC, and LTC-IC. Percentages of annexin V-positive cells in the total MNC and the CD34(+) cell population in MNC after UV-B irradiation were 69.8% and 18.7%, respectively. In conclusion, there was a range of UV-B doses over which T lymphocytes were inactivated but hematopoietic progenitors, including HPP-CFC and LTC-IC, were preserved.

Annexin A5↗
PubMed2000

Stromal cell-independent differentiation of human cord blood CD34+CD38- lymphohematopoietic progenitors toward B cell lineage.

To study the cytokine regulation of early stages of human B-lymphopoiesis, we developed a stroma-free two-step culture system. Single human cord blood CD34+CD38- cells were individually cultured by micromanipulation with interleukin (IL)-3, stem cell factor (SCF), fIt3 ligand (FL), IL-6 and granulocyte colony-stimulating factor (G-CSF). About 10% of the cells formed primary colonies, which were individually tested for myeloid and B-lymphoid potentials by reculturing aliquots of the primary colony cells into secondary myeloid and B-lymphoid cultures. One third of the primary colonies proved capable of differentiation into CD19+IgM+ cells, as well as into myeloid lineage cells. RT-PCR analyses revealed that some cells in the primary culture had already matured to express B cell-specific transcripts. Thus, the combination of IL-3, SCF, FL, IL-6 and G-CSF supported the differentiation of CD34+CD38- lymphohematopoietic progenitors toward B cell lineage in addition to myeloid lineages. Screening of cytokines to identify the minimum requirement of cytokines in the primary culture revealed that IL-3 and SCF were essential and that the addition of FL, and to a lesser extent IL-6 or G-CSF, to the combination of IL3 and SCF remarkably enhanced the primary colony formation and the generation of CD19+ cells in the secondary B-lymphoid culture.

Antigens, CD34↗
PubMed2000

Characterization of bovine and human lactoferrins as glycyrrhizin-binding proteins and their phosphorylation in vitro by casein kinase II.

The binding ability of bovine and human lactoferrins (bLF and hLF; LFs) to a glycyrrhizin (GL)-affinity column and their phosphorylation by casein kinase II (CK-II) in vitro were biochemically investigated. It was found that (i) both bLF and hLF are GL-binding proteins; (ii) purified both proteins function as phosphate acceptors of CK-II; and (iii) this phosphorylation is completely inhibited by two polyphenol-containing anti-oxidant compounds (quercetin and epigallocatechin gallate) at I microm, whereas a glycyrrhetinic acid derivative (oGA) inhibits it at one tenth the concentration of GL. The DNA-binding affinity of hLF was reduced by GL in a dose dependent manner. However, no significant effect of the CK-II-mediated hLF phosphorylation on its DNA-binding affinity was detected. These results suggest that the GL-induced inhibition of the DNA-binding affinity and the CK-II-mediated phosphorylation of hLF may be closely correlated with the anti-inflammatory effect of GL in the human body.

Animals↗
PubMed2000

Biochemical characterization of bovine lactoferrin as a glycyrrhizin-binding protein in vitro.

Lactoferrin (LF) from bovine colostrum was biochemically characterized as a glycyrrhizin (GL)-binding protein (gbP) in vitro. It was found that (i) bovine LF (bLF) and a synthetic bovine lactoferricin (bLFcin, the N'-terminal region of bLF at the positions 17--41) had a high affinity to a GL-affinity column; (ii) approximately 1.8 moles of GL were bound to a molecule of bLF with a binding constant of approx. 1.20x10(4) M(-1) at pH 6.8; and (iii) GL, but not glycyrrhetinic acid (GA), induced a conformational change of bLF. In addition, the glucuronic acid moiety of the GL molecule was found to be responsible for binding to bLF, because (i) no binding of GA and two glucoses-GA (Glc-Glc-GA) to bLF was detected; and (ii) a synthetic fluorinated GL (GlcA-GlcF-GA) and mono-glucuronyl-GA (mono-GlcA-GA) were bound significantly to bLF. A similar binding of GL to human LF (hLF) was also observed under the same experimental conditions. Data provided here suggest that (i) bLF contains plural GL-binding sites; and (ii) the specific binding of GL to bLF may modulate the physiological activity of bLF in vivo.

Amino Acid Sequence↗
PubMed2000

Lipopolysaccharide-triggered desensitization of TNF-alpha mRNA expression involves lack of phosphorylation of IkappaBalpha in a murine macrophage-like cell line, P388D1.

Activation of nuclear factor kappaB (NF-kappaB) is thought to be required for cytokine production by lipopolysaccharide (LPS)-responsive cells. Here, we investigated the contribution of NF-kappaB in preventing LPS-induced transcription of the tumor necrosis factor alpha (TNF-alpha) gene in a murine macrophage cell line, P388D1, when tolerance was induced in the cells with a short exposure to a higher dose of LPS. Electrophoretic mobility shift assays with the kappaB elements of the murine TNF-alpha promoter and enhancer revealed that nuclear mobilization of heterodimers of p65/p50, c-rel/p50 and p65/c-rel, and homodimers of p65 was markedly reduced in LPS-tolerant cells, whereas that of p50 homodimers was only slightly increased. Western blot analysis showed that the phosphorylation of Ser32 on IkappaBalpha and its transient degradation did not occur in LPS-tolerant cells. These results thus suggest that desensitization of TNF-alpha gene expression in this LPS-tolerant state is closely associated with down-regulation of transactivating NF-kappaB and may involve a defect in the LPS-induced IkappaBalpha kinase pathway.

Animals↗