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Biomedical subjects

Y Tomiyama

Publications and source records attributed to Y Tomiyama.

At least 19 recordsLinked to original sources
PubMed1999

Both Stat3-activation and Stat3-independent BCL2 downregulation are important for interleukin-6-induced apoptosis of 1A9-M cells.

A unique subclone of a bone marrow-derived stromal cell line, BMS2.4, produces soluble factors that inhibit proliferation of several types of hematopoietic cell lines. An understanding of these molecules may be informative about negative regulatory circuits that can potentially limit blood cell formation. We used expression cloning to identify interleukin-6 (IL-6) as one factor that suppressed growth of a pre-B-cell variant line, 1A9-M. Moreover, IL-6 induced macrophage-differentiation and apoptosis of 1A9-M cells. During this process, IL-6 downregulated expression of BCL2 in 1A9-M cells and stimulated BCL-XL expression, but had no effect on p53, Bax, or Bak gene expression. Mechanisms for transduction of IL-6-induced signals were then evaluated in IL-6-stimulated 1A9-M cells. Whereas the signal transducer and activator of transcription 3 (Stat3) was phosphorylated and activated, there was no effect on either Stat1 or Stat5. The importance of BCL2 and Stat3 on IL-6-induced macrophage-differentiation and apoptosis was studied with 1A9-M cells expressing human BCL2 or a dominant-negative form of Stat3, respectively. IL-6-induced apoptosis, but not macrophage-differentiation, was blocked by continuously expressed BCL2. A dominant-negative form of Stat3 inhibited both macrophage-differentiation and apoptosis induced by IL-6. However, diminished Stat3 activity did not prevent IL-6-induced downregulation of the BCL2 gene. Therefore, activation of Stat3 is essential for IL-6-induced macrophage-differentiation and programmed cell death in this model. Whereas overexpression of BCL2 abrogates the apoptotic response, Stat3-independent signals appear to downregulate expression of the BCL2 gene.

Apoptosis↗
PubMed1999

Enhanced myocardial glucose use in patients with a deficiency in long-chain fatty acid transport (CD36 deficiency).

UNLABELLED: CD36 is a multifunctional, 88 kDa glycoprotein that is expressed on platelets and monocytes/macrophages. CD36 also has high homology with the long-chain fatty acid (LFA) transporter in the myocardium. Although platelet and monocyte CD36 levels can indicate a CD36 deficiency, they cannot predict specific clinical manifestations in the myocardium of a given person. We examined the hypothesis that a deficiency in LFA transport augments myocardial glucose uptake in patients with a type I CD36 deficiency. METHODS: Seven fasting patients with a type I CD36 deficiency and 9 controls were assessed by cardiac radionuclide imaging using beta-methyl-p-iodophenyl-pentadecanoic acid (BMIPP) as a LFA tracer and by PET with 18F-fluorodeoxyglucose (FDG). RESULTS: None of the patients with a CD36 deficiency showed myocardial uptake of BMIPP. The percentage dose uptake of BMIPP in these subjects was significantly lower than that in normal controls (1.31+/-0.24 versus 2.90+/-0.2; P < 0.005). PET studies revealed that myocardial FDG accumulation was substantially increased in patients with a CD36 deficiency. Quantitative analysis showed that the percentage dose uptake of FDG in patients with a CD36 deficiency was significantly higher than that in normal controls (1.28+/-0.35 versus 0.43+/-0.22; P< 0.01). CONCLUSION: CD36 functions as a major myocardial LFA transporter and its absence may cause a compensatory upregulation of myocardial glucose uptake.

Aged↗
PubMed1998

Association between ligand-induced conformational changes of integrin IIbbeta3 and IIbbeta3-mediated intracellular Ca2+ signaling.

Platelet IIbbeta3 is a prototypic integrin and plays a critical role in platelet aggregation. Occupancy of IIbbeta3 with multivalent RGD ligands, such as fibrinogen, induces both expression of ligand-induced binding sites (LIBS) and IIbbeta3 clustering, which are thought to be necessary for outside-in signaling. However, the association between LIBS expression and outside-in signaling remains elusive. In this study, we used various IIbbeta3-specific peptidomimetic compounds as a monovalent ligand instead of fibrinogen and examined the association between LIBS expression and outside-in signaling such as IIbbeta3-mediated intracellular Ca2+ signaling. Using a set of monoclonal antibodies (MoAbs) against LIBS, we showed that antagonists can be divided into two groups. In group I, antagonists can induce LIBS on both IIb and beta3 subunits. In group II, antagonists can induce LIBS on the IIb subunit, but not on the beta3 subunit. Inhibition studies suggested that group I and group II antagonists interact with distinct but mutually exclusive sites on IIbbeta3. Neither group I nor group II antagonist increased intracellular Ca2+ concentrations ([Ca2+]i) in nonactivated platelets. All antagonists at nanomolar concentrations abolished the increase in [Ca2+]i in 0.03 U/mL thrombin-stimulated platelets, which is dependent on both fibrinogen-binding to IIbbeta3 and platelet-aggregation. However, only group I antagonists at higher concentrations dose-dependently augmented the [Ca2+]i increase, which is due to aggregation-independent thromboxane A2 production. This increase in [Ca2+]i was not observed in thrombasthenic platelets, which express no detectable IIbbeta3. Thus, only the group I antagonists, albeit a monovalent ligand, can initiate IIbbeta3-mediated intracellular Ca2+ signaling in the presence of thrombin stimulation. Our findings strongly suggest the association between beta3 LIBS expression and IIbbeta3-mediated intracellular Ca2+ signaling in platelets.

Acetates↗
PubMed1998

Involvement of Na+/Ca2+ exchanger in inside-out signaling through the platelet integrin IIbbeta3.

The platelet integrin IIbbeta3 has become a new target for the treatment of pathological thrombosis. It becomes apparent that the affinity of IIbbeta3 for its ligands is dynamically regulated by inside-out signaling. However, the components that couple diverse intracellular signals to the cytoplasmic domains of IIbbeta3 remain obscure. Employing a chymotrypsin-induced IIbbeta3 activation model, we previously proposed the hypothesis that Na+/Ca2 + exchanger (NCX) may be involved in inside-out signaling (Shiraga et al: Blood 88:2594, 1996). In the present study, employing two unrelated Na+/Ca2+ exchange inhibitors, 3',4'-dichlorobenzamil (DCB) and bepridil, we investigated the role of NCX in platelet activation induced by various agonists in detail. Both inhibitors abolished platelet aggregation induced by all agonists examined via the inhibition of IIbbeta3 activation. Moreover, these inhibitors abolished IIbbeta3 activation induced by phorbol 12-myristate 13-acetate or A23187. On the other hand, neither of these inhibitors showed apparent inhibitory effects on protein phosphorylation of pleckstrin or myosin light chain, or an increase in intracellular calcium ion concentrations evoked by 0.1 U/mL thrombin. These effects of the NCX inhibitors are in striking contrast to those of protein kinase C inhibitor, Ro31-8220. Biochemical and ultrastructural analyses showed that NCX inhibitors, particularly DCB, made platelets "thrombasthenic". These findings suggest that the NCX is involved in the common steps of inside-out signaling through integrin IIbbeta3.

Amiloride↗
PubMed1998

A Gln747-->Pro substitution in the IIb subunit is responsible for a moderate IIbbeta3 deficiency in Glanzmann thrombasthenia.

To clarify a molecular defect responsible for moderate alphaIIb beta3 deficiency, we examined two unrelated patients, MT and MS, suffering from type II and type I Glanzmann thrombasthenia (GT), respectively. Sequence analysis of polymerase chain reaction (PCR) fragments derived from platelet mRNA showed a single A-->C substitution at nucleotide (nt) 2334 leading to a Gln747--> Pro in alphaIIb in both patients. Allele-specific restriction enzyme analysis (ASRA) of genomic DNA demonstrated that patient MT was homozygous for the Gln747-->Pro substitution and patient MS was compound heterozygous for this substitution and for an RNA splice mutation at the consensus sequence of the splice acceptor site of exon 18 (AG-->AA). Furthermore, ASRA showed that, among 17 unrelated Japanese GT patients, this Gln747-->Pro substitution was detected in 4 patients, including MT and MS (homozygous, 2 patients; heterozygous, 2 patients). Cotransfection of Pro747alphaIIb and beta3 constructs into 293 cells resulted in moderate reduction in the amount of alphaIIb beta3 within the transfected cells as well as on the cell surface. However, Pro747alphaIIb beta3 bound the ligand mimetic monoclonal antibody (MoAb) PAC-1 after activation of alphaIIb beta3 by the MoAb PT25-2, suggesting that the mutant alphaIIb beta3 possesses the ligand-binding function. The association between the mutant proalphaIIb and beta3 was not disturbed. Surface labeling and pulse chase study showed that the Gln747-->Pro substitution moderately impaired both intracellular transport of the alphaIIb beta3 heterodimers to the Golgi apparatus and endoproteolytic cleavage of proalphaIIb into heavy and light chains. By contrast, replacement of Gln747 with Ala by mutagenesis did not impair alphaIIbbeta3 expression on the cell surface. These results suggest that the presence of Pro, rather than the absence of Gln, at amino acid residue 747 on alphaIIb is responsible for moderate alphaIIbbeta3 deficiency.

Alleles↗
PubMed1998

A two-amino acid insertion in the Cys146- Cys167 loop of the alphaIIb subunit is associated with a variant of Glanzmann thrombasthenia. Critical role of Asp163 in ligand binding.

The ligand binding site(s) of the alpha subunit of integrin alphaIIb beta3 (GPIIb-IIIa), a prototypic non-I domain integrin, remains elusive. In this study, we have characterized a Japanese variant of Glanzmann thrombasthenia, KO, whose platelets express normal amounts of alphaIIb beta3. KO platelets failed to bind the activation-independent ligand-mimetic mAb OP-G2 and did not bind fibrinogen or the activation-dependent ligand-mimetic mAb PAC-1 following activation of alphaIIb beta3 under any condition examined. Sequence analysis of PCR fragments derived from KO platelet mRNA revealed a 6-bp insertion leading to a 2-amino-acid insertion (Arg-Thr) between residues 160 and 161 of the alphaIIb subunit. Introduction of the insertion into wild-type recombinant alphaIIb beta3 expressed in 293 cells led to the normal expression of alphaIIb beta3 having the defect in ligand binding function. The insertion is located within the small loop (Cys146-Cys167) in the third NH2-terminal repeat of the alphaIIb subunit. Alanine substitution of each of the oxygenated residues within the loop (Thr150, Ser152, Glu157, Asp159, Ser161, and Asp163) did not significantly affect expression of alphaIIbbeta3, and only Asp163AlaalphaIIb beta3 abolished the ligand binding function. In addition, Asp163AlaalphaIIb beta3 as well as KO mutant alphaIIb beta3 constitutively expressed the PMI-1 epitope. Our present data suggest that Asp163 of the alphaIIb subunit is one of the critical residues for ligand binding.

Adult↗
PubMed1998

Insulin-like growth factor-I augments erythropoietin-induced proliferation through enhanced tyrosine phosphorylation of STAT5.

Insulin-like growth factor (IGF-I) is known to synergistically stimulate the proliferation of hematopoietic cells in combination with other hematopoietic growth factors. However, the precise mechanism underlying the cooperative effects of IGF-I is unknown. In a human interleukin-3 or erythropoietin (EPO)-dependent cell line, F-36P, IGF-I alone failed to stimulate DNA synthesis but did augment the EPO-dependent DNA synthesis of F-36P cells. The treatment of F-36P cells with a combination of EPO and IGF-I (EPO/IGF-I) was found to enhance EPO-induced tyrosine phosphorylation of STAT5, whereas IGF-I alone did not. Furthermore, c-CIS mRNA expression, one of the target molecules of STAT5, was more effectively induced by EPO/IGF-I than by EPO alone. To examine the mechanisms of the EPO- and EPO/IGF-I-induced proliferation of F-36P cells, we expressed dominant negative (dn) mutants of STAT5 and Ras in an inducible system. The EPO-induced DNA synthesis and the cooperative effect of EPO/IGF-I were significantly inhibited by the inducible expression of dn-STAT5 or dn-Ras. In addition, the inducible expression of dn-Ras abolished the IGF-I-enhanced tyrosine phosphorylation of STAT5. These results suggest that IGF-I may augment EPO-induced proliferation by enhancing tyrosine phosphorylation of STAT5 and raise the possibility that Ras may be involved in the augmentation of STAT5 tyrosyl phosphorylation.

Cell Division↗
PubMed1998

Beta-adrenoceptor subtypes in the ureteral smooth muscle of rats, rabbits and dogs.

We investigated the beta-adrenoceptor subtypes mediating ureteral relaxation in rats, rabbits and dogs. The relaxing effects of beta-adrenoceptor agonists were evaluated on KCl-induced ureteral contractions. The rank order of potency of the catecholamines tested was isoprenaline > noradrenaline > adrenaline in rat ureter; isoprenaline > adrenaline > noradrenaline in rabbit ureter; only isoprenaline was effective in canine tissues. The beta1-adrenoceptor agonist, dobutamine, produced relaxation of rat ureter. The beta2-adrenoceptor agonist, procaterol, produced more significant relaxation of rabbit ureter than did dobutamine. CL-316243 [(R,R)-5-[2-[[2-(3-chlorophenyl)-2-hydroxyethylamino]propyl]-1,3-b enzodioxole-2,2-dicarboxylate] and CGP-12177A [(+/-)[4-[3[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-+ ++benzimidazol-2-one hydrochloride], beta3-adrenoceptor agonists, were more effective in relaxing canine ureter than were dobutamine and procaterol. Isoprenaline-induced relaxation was antagonized by a beta1-adrenoceptor antagonist, CGP-20712A [2-hydroxy-5(2-((2-hydroxy-3-(4-((1-methyl-4-trifluoromethyl)1H-imidazol e-2-yl)phenoxy)propyl)amino)ethoxy)-benzamide monomethane sulphonate], in rats and by a beta2-adrenoceptor antagonist, ICI-118,551 [(+/-)-1-[(2,3-dihydro-7-methyl- 1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol hydrochloride], in rabbits. The non-selective beta-adrenoceptor antagonist, bupranolol, antagonized isoprenaline-induced relaxation in all species tested. In conclusion, beta-adrenoceptor agonists may relax ureter by stimulating mainly beta1-adrenoceptors in rats, beta2-adrenoceptors in rabbits and mainly beta3-adrenoceptors in dogs.

Adrenergic beta-Agonists↗
PubMed1998

Growth-supporting activities of fibronectin on hematopoietic stem/progenitor cells in vitro and in vivo: structural requirement for fibronectin activities of CS1 and cell-binding domains.

Fibronectin (FN) is supposed to play important roles in various aspects of hematopoiesis through binding to very late antigen 4 (VLA4) and VLA5. However, effects of FN on hematopoietic stem cells are largely unknown. In an effort to determine if FN had a growth-supporting activity on hematopoietic stem cells, human CD34(+)/VLA4(bright)/VLA5(dull) hematopoietic stem cells and a murine stem cell factor (SCF)-dependent multipotent cell line, EML-C1, were treated with or without FN in a serum and growth-factor-deprived medium, and then subjected to clonogenic assay in the presence of hematopoietic growth factors. The pretreatment of the CD34(+) cells with FN gave rise to significantly increased numbers of granulocyte-macrophage colony-forming units (CFU-GM), erythroid burst colony-forming units, and mixed erythroid-myeloid colony-forming units. In addition, the numbers of blast colony-forming units and CFU-GM that developed after culture of EML-C1 cells with SCF and the combination of SCF and interleukin-3, respectively, were augmented by the pretreatment with FN. The augmented colony formation by FN was completely abrogated by the addition of CS1 fragment, but not of GRGDSP peptide, suggesting an essential role of FN-VLA4 interaction in the FN effects. Furthermore, the effects of various FN fragments consisting of RGDS-containing cell-binding domain (CBD), heparin-binding domain (HBD), and/or CS1 portion were tested on clonogenic growth of CD34(+) cells. Increased colony formation was induced by CBD-CS1 and CBD-HBD-CS1 fragments, but not with other fragments lacking CBD or CS1 domains, suggesting that both CS1 and CBD of FN were required for the augmentation of clonogenic growth of hematopoietic stem/progenitor cells in vitro. In addition to the in vitro effects, the in vivo administration of CBD-CS1 fragment into mice was found to increase the numbers of hematopoietic progenitor cells in bone marrow and spleen in a dose-dependent manner. Thus, FN may function on hematopoietic stem/progenitor cells as a growth-supporting factor in vitro and in vivo.

Amino Acid Sequence↗
PubMed1998

Endogenous adenosine inhibits P-selectin-dependent formation of coronary thromboemboli during hypoperfusion in dogs.

The activation of platelets and the formation of neutrophil- platelet conjugates may lead to the development of thromboemboli. We studied whether blockade of adenosine receptors during coronary hypoperfusion may cause thromboemboli via P-selectin-dependent mechanisms in 30 open-chest dogs. When coronary blood flow was reduced to 20% of the control, it was stable at low levels with increases in adenosine levels. When 8-p-sulfophenyltheophylline, an adenosine receptor antagonist, was infused during coronary hypoperfusion, coronary blood flow decreased gradually and approached almost zero 20 min after its administration. Histological examination revealed thromboemboli in the small coronary vessels. During hypoperfusion in the presence of 8-p-sulfophenyltheophylline, the mAb against P-selectin attenuated both the reduction in coronary blood flow and the formation of thromboemboli, and improved contractile and metabolic dysfunction of the myocardium. Flow cytometric analysis indicated that the expression of P-selectin on platelet and neutrophil-platelet adhesion were increased during coronary hypoperfusion, and that both were further augmented by 8-p-sulfophenyltheophylline. Immunohistochemical examination showed no staining of P-selectin in the ischemic myocardium. Adenosine inhibited the thrombin-induced expression of P-selectin on platelet and neutrophil- platelet adhesion via adenosine A2 receptors. Adenosine appears to inhibit the formation of thromboemboli during coronary hypoperfusion by suppressing the expression of P-selectin on platelets and neutrophil-platelet adhesion.

Adenosine↗
PubMed1998

Effect of the interaction between fibronectin and VLA-4 on the proliferation of human B cells, especially a novel human B-cell line, OPM-3.

Very late antigen (VLA)-4 integrin has been suggested to play an important role in haemopoiesis. However, little is known concerning the roles of the fibronectin (FN)/VLA-4 interaction in the proliferation of human B cells. In this study we investigated the effect of immobilized FN on the proliferation of various B-cell lines, including a newly-established B-cell line, OPM-3, and human tonsillar B cells, that primarily express VLA-4 but not VLA-5. Immobilized FN significantly promoted the proliferation of OPM-3 cells and normal B cells via VLA-4. The cross-linking of beta1 integrins of OPM-3 cells resulted in the phosphorylation of the focal adhesion kinase (FAK) associated 90 kD protein, an increase in FAK-associated kinase activity, and the phosphorylation of Raf-1. Furthermore, the MEK1 inhibitor, PD98059, inhibited the FN-promoted proliferation of OPM-3 cells. These results demonstrate that the FN/VLA-4 interaction transmits the growth signal(s) which may be mediated by Ras pathway in OPM-3 cells, and suggest that OPM-3 cells may be of great value in studying the roles of the FN/VLA-4 interaction in human B-cell growth.

B-Lymphocytes↗
PubMed1998

Decreased nitric oxide-mediated natural killer cell activation in chronic fatigue syndrome.

BACKGROUND: L-Arginine (L-Arg), one of the essential amino acids, has been reported to have an immunomodulatory effect. The precise mechanism of the L-Arg-induced natural killer (NK) cell activation remains unresolved,and the effect of L-Arg on NK cells in chronic fatigue syndrome (CFS) patients has not been estimated. METHODS: NK cell function was evaluated in 20 subjects with CFS and compared with that in 21 healthy individuals. RESULTS: In healthy control subjects, NK activity was significantly increased after treatment with L-Arg, an NK function enhancer, for 24 h, whereas the same treatment failed to enhance NK activity in the CFS patients. We thus focused on L-Arg metabolism, which involves nitric oxide (NO) production through NO synthase (NOS). The expression of inducible NO synthase (iNOS) transcripts in peripheral blood mononuclear cells was not significantly different between healthy control subjects and CFS patients. The L-Arg-mediated NK cell activation was abolished by addition of NG-monomethyl-L-arginine, an inhibitor for iNOS. Furthermore, incubation with S-nitroso-N-acetyl-penicillamine, an NO donor, stimulated NK activity in healthy control subjects but not in CFS patients. CONCLUSION: These results demonstrate that the L-Arg-induced activation of NK activity is mediated by NO and that a possible dysfunction exists in the NO-mediated NK cell activation in CFS patients.

Adjuvants, Immunologic↗
PubMed1998

Oxidized LDL increases and interferon-gamma decreases expression of CD36 in human monocyte-derived macrophages.

CD36 is a glycoprotein with an Mr of 88 kDa that is expressed on platelets, monocytes/macrophages, capillary endothelial cells, and adipocytes. We previously demonstrated that CD36 is involved in the uptake of oxidized low density lipoprotein (OxLDL) by using CD36-deficient macrophages (J Clin Invest. 1995;96:1859). However, the regulation of CD36 expression in human monocyte-derived macrophages has not been fully elucidated. The current study attempted to clarify the effect of OxLDL and cytokines, both of which are present in atherosclerotic lesions and may play an important role in atherogenesis, on the expression of CD36. A cell enzyme-linked immunosorbent assay and flow cytometry were used to detect CD36 protein. A ribonuclease protection assay was used to measure CD36 mRNA in human monocyte-derived macrophages. The expression of CD36 was increased during the differentiation of monocytes to macrophages. Incubation of macrophages with 25 microg/mL OxLDL for 24 hours increased the level of CD36 protein by 56% and that of CD36 mRNA by 58%. Lysophosphatidylcholine did not affect the expression of CD36. The effects of OxLDL were demonstrated in macrophages that had already differentiated to the point where CD36 expression was almost maximal. Interferon-gamma (IFN-gamma) reduced the expression of CD36 in a dose-dependent manner. A concentration of 1000 U/mL IFN-gamma significantly reduced the expression of CD36 protein by 57% and that of CD36 mRNA by 30%. In conclusion, CD36 may be important in the formation of foam cells by induction through its ligand (OxLDL). Moreover, some local factors, such as IFN-gamma, may suppress CD36 expression on macrophages in human atherosclerotic lesions.

CD36 Antigens↗
PubMed1998

Difference of (Ca2+)i movements in platelets stimulated by thrombin and TRAP: the involvement of alpha(IIb)beta3-mediated TXA2 synthesis.

This study investigated the difference of [Ca2+]i movement in platelets in response to thrombin and TRAP. The involvement of alpha(IIb)beta3 in this signaling was also studied. Stimulation of platelets with thrombin at 0.03 U/ml caused platelet aggregation and a two-peak increase in [Ca2+]i. The second peak of [Ca2+]i, but not the first peak was abolished by the inhibition of platelet aggregation with alpha(IIb)beta3 antagonists or by scavenging endogenous ADP with apyrase. A cyclooxygenase inhibitor, aspirin, and a TXA2 receptor antagonist, BM 13505, also abolished the second peak of [Ca2+]i but not the first peak, although these regents did not inhibit aggregation. Under the same assay conditions, measurement of TXB2 demonstrated that alpha(IIb)beta3 antagonists and aspirin almost completely inhibited the production of TXB2. In contrast to thrombin-stimulation, TRAP caused only a single peak of [Ca2+]i even in the presence of platelet aggregation, and a high level of [Ca2+]i increase was needed for the induction of platelet aggregation. The inhibition of aggregation with alpha(IIb)beta3 antagonists had no effect on [Ca2+]i change and TXB2 production induced by TRAP. Inhibition studies using anti-GPIb antibodies suggested that GPIb may be involved in the thrombin response, but not in the TRAP. Our findings suggest that low dose thrombin causes a different [Ca2+]i response and TXA2 producing signal from TRAP. Endogenous ADP release and fibrinogen binding to alpha(IIb)beta3 are responsible for the synthesis of TXA2 which results in the induction of the second peak of [Ca2+]i in low thrombin- but not TRAP-stimulated platelets.

Adenosine Diphosphate↗
PubMed1998

A Glanzmann thrombasthenia-like phenotype caused by a defect in inside-out signaling through the integrin alpha(IIb)beta3.

Activation of the platelet integrin alpha(IIb)beta3, an essential step in platelet aggregation, is regulated by intracellular signal pathways (inside-out signaling). In this study, we characterize a 35-year-old Japanese female, HM, with a life-long history of mucocutaneous bleeding. HM showed a Glanzmann thrombasthenia-like phenotype with normal expression of alpha(IIb)beta3, and failure of platelet aggregation induced by various agonists. An activation-independent ligand mimic monoclonal antibody (mAb), OP-G2, and RGDS peptides bound normally to the patient's alpha(IIb)beta3, while an activating anti-beta3 mAb, AP5, induced normal aggregation of HM platelets. The nucleotide sequence of the entire coding region of the patient's alphaIIb and beta3, including the cytoplasmic domains of each subunit, revealed no abnormalities. Agonist-induced phosphorylation of platelet pleckstrin and myosin light chain was not impaired. Recently, we proposed that a Na+/Ca2+ exchanger is involved in inside-out signaling, especially in the case of chymotrypsin-induced alpha(IIb)beta3 activation (Blood 88: 2594, 1996). However, chymotrypsin-induced platelet aggregation occurred normally in patient HM. Measurement of changes in cytosolic free calcium concentration ([Ca2+]i) revealed that the plateau level of [Ca2+]i after thrombin stimulation was significantly inhibited in patient HM. Our data suggest that patient HM exhibits a Glanzmann thrombasthenia-like phenotype associated with an abnormality in inside-out signaling which would otherwise activate alpha(IIb)beta3.

Adult↗
PubMed1998

[Spontaneous resolution of pulmonary cryptococcosis--report of 2 cases].

We encountered two patients with pulmonary cryptococcosis that resolved spontaneously without antifungal treatment. A 59-year-old man (patient 1) and 37-year-old man (patient 2) were admitted to our hospital for the examination of abnormal shadows on their chest x-ray films. Transbronchial lung biopsies were performed, and cryptococci were detected in both patient's lung tissues. Latex aggultination tests for cryptococcal antigen were positive (1:16 and 1:4, respectively). The size of the shadows reduced spontaneously and the titers of the cryptococcal antigen in sera decreased within three months although no antifungal therapy was performed. Bronchoalveolar lavage fluid (BALF) was obtained from each patient and examined. In patient 1, the lymphocyte to CD 4/8 ratio was high in the BALF obtained on admission and the lymphocyte count was low; however, the CD 4/8 ratio was still high in a BALF sample obtained 3 months after discharge. In patient 2, the findings of the BALF analysis on admission were almost normal. The analysis of BALF may contribute to the immunological conditions to in the patients with pulmonary cryptococcosis.

Adult↗
PubMed1997

Markedly reduced expression of platelet c-mpl receptor in essential thrombocythemia.

Thrombopoietin (TPO) is implicated as a primary regulator of megakaryopoiesis and thrombopoiesis through binding to the cytokine receptor c-Mpl (the product of the c-mpl proto-oncogene). In an effort to determine the pathophysiological role of TPO-c-Mpl system in essential thrombocythemia (ET), we have examined the levels of serum TPO and the expression and function of platelet c-Mpl in 17 patients with ET. In spite of extreme thrombocytosis, serum TPO levels were slightly elevated or within normal range in most, if not all, patients with ET (mean +/- SD, 1.31 +/- 1.64 fmol/mL), as compared with normal subjects (0.76 +/- 0.21 fmol/mL). Flow cytometric and Western blot analyses revealed that the expression of platelet c-Mpl was strikingly reduced in all patients with ET. Furthermore, the expression of platelet c-mpl mRNA was found to be significantly decreased in the ET patients tested. In contrast, almost identical levels of GPIIb/IIIa protein and mRNA were expressed in platelets from ET patients and normal controls. In addition to expression level, activation state of platelet c-Mpl was investigated in ET patients. Immunoblotting with anti-phosphotyrosine antibody showed that no aberrant protein-tyrosine phosphorylation was observed in platelets of ET patients before treatment with TPO, and the levels of TPO-induced protein-tyrosine phosphorylation, including c-Mpl-tyrosyl phosphorylation, roughly paralleled those of c-Mpl expression, suggesting that c-Mpl-mediated signaling pathway was not constitutively activated in platelets of ET patients. These results suggested that the TPO-c-Mpl system may not be directly linked to pathogenesis of ET, and that gene(s) mutated in ET may be important in regulating the levels of c-mpl gene expression in addition to the growth and differentiation of multipotential hematopoietic stem cells.

Adolescent↗