Combination use of immune complexes and a Ca2(+) channel blocker azelnidipine enhances interleukin-12 p40 secretion without T helper type 17 cytokine secretion in human monocyte-derived dendritic cells.

Immune complexes (ICs) improve the capacity of priming specific CD8(+) cytotoxic T cell responses of dendritic cells (DCs). ICs induce phosphorylation of mitogen-activated protein kinases (MAPK) and calcium influx, although the precise regulating mechanism still remains unclear. In the present study, we investigated the effect of a Ca2(+) channel blocker on the phosphorylation of p38 MAPK and extracellular signal-regulated kinase (ERK) in immature monocyte-derived DCs stimulated with lipopolysaccharide (LPS) or LPS-ICs, and the production of interleukin (IL)-12 family members (p40, p70, IL-23), T helper type 17 (Th17) cytokines (IL-6 and IL-23), tumour necrosis factor (TNF)-alpha and IL-10 were also investigated. In comparison with LPS stimulation, LPS-ICs stimulation enhanced p38 MAPK phosphorylation significantly, which was associated with an increase in IL-12 p40 monomer/homodimer secretion. LPS-ICs also enhanced TNF-alpha and IL-6 secretion, but suppressed IL-23 secretion. The use of azelnidipine (Aze), a long-acting L-type Ca2(+) channel blocker with a high lipid solubility, suppressed p38 MAPK phosphorylation stimulated with LPS or LPS-ICs, but surprisingly enhanced IL-12 p40 monomer/homodimer secretion stimulated with LPS-ICs. This IL-12 p40 secretion-enhancing effect was not accompanied by IL-10 or IL-23 production, but was associated with ERK phosphorylation. The use of Aze did not affect IL-12 p70 production. These results suggest that the use of Aze enhances ICs-mediated IL-12 p40 secretion without additional IL-23 secretion. Therefore, the use of Aze and ICs could be a new therapeutic approach to immunomolecular therapy, as it does not cause Th17 differentiation which induces autoimmunity or reduces anti-tumour immunity.

Induction of recipient cell-specific donor T-cell anergy by UV-C-irradiated recipient immature monocyte-derived dendritic cells.

The induction of donor T-cell anergy to recipient cells for reducing GVHD could be one way of expanding donor candidates for HLA-mismatched hematopoietic SCT. The present study was designed to clarify whether recipient cell-specific T-cell anergy could be induced by priming donor lymphocytes with recipient monocyte-derived DCs (mo-DCs) irradiated with ultraviolet-C (UV-C). By irradiation of mo-DCs with UV-C, the expression of DC-associated surface phenotypes such as CD83, CD80, CD86 and CD40 was reduced and the antigen-presenting ability of UV-C-irradiated mo-DCs was clearly decreased. By co-culturing normal donor 1 lymphocytes with UV-C-irradiated donor 2 immature mo-DCs, the response of the lymphocytes to donor 2 mature mo-DCs was markedly reduced as compared with that of the lymphocytes prestimulated with non-irradiated donor 2 immature mo-DCs or UV-C-irradiated mo-DCs derived from a different individual donor 3. The present study demonstrated that recipient cell-specific T-cell anergy could be induced by priming donor lymphocytes with UV-C-irradiated recipient immature mo-DCs in hematopoietic SCT. These data suggest the applicability of donor graft cells, which have been prestimulated with UV-C-irradiated recipient immature mo-DCs, for expanding donor candidates in HLA-mismatched hematopoietic SCT.

Arg60 to Leu mutation of the human thromboxane A2 receptor in a dominantly inherited bleeding disorder.

Recent advances in molecular genetics have revealed the mechanisms underlying a variety of inherited human disorders. Among them, mutations in G protein-coupled receptors have clearly demonstrated two types of abnormalities, namely loss of function and constitutive activation of the receptors. Thromboxane A2 (TXA2) receptor is a member of the family of G protein-coupled receptors and performs an essential role in hemostasis by interacting with TXA2 to induce platelet aggregation. Here we identify a single amino acid substitution (Arg60-->Leu) in the first cytoplasmic loop of the TXA2 receptor in a dominantly inherited bleeding disorder characterized by defective platelet response to TXA2. This mutation was found exclusively in affected members of two unrelated families with the disorder. The mutant receptor expressed in Chinese hamster ovary cells showed decreased agonist-induced second messenger formation despite its normal ligand binding affinities. These results suggest that the Arg60 to Leu mutation is responsible for the disorder. Moreover, dominant inheritance of the disorder suggests the possibility that the mutation produces a dominant negative TXA2 receptor.

Does protein kinase C activation mediate thrombin-induced arachidonate release in human platelets?

Thrombin stimulated rapid formation of diacylglycerol, inositol 1,4,5-trisphosphate (IP3) and thromboxane B2 (TXB2) in human platelets. Formation of diacylglycerol and IP3 appeared to precede that of TXB2. Activation of protein kinase C by diacylglycerol combining with Ca+2 mobilization by IP3 has been implicated in mediating arachidonate release. However, addition of the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) to platelet suspension did not inhibit thrombin-stimulated arachidonate release and TXB2 synthesis, whereas addition of the Ca+2 antagonist, 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester (TMB-8) or the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) abolished arachidonate release. The correlation of IP3 production with arachidonate release on increasing the concentrations of thrombin was further examined. IP3 production reached near maximum at 0.2 U/ml, whereas TXB2 synthesis continued to increase at 1 U/ml. These results suggest that protein kinase C activation may not mediate arachidonate release and that Ca+2 mobilization by IP3 may only partially account for arachidonate release in platelets stimulated with relatively high concentrations of thrombin.

Erythroid involvement in CD36 deficiency.

OBJECTIVE: The CD36 molecule is expressed in platelets, monocytes, erythroblasts, and other different tissues. The two types of platelet CD36 deficiency, types I and II, are associated with the absence and presence of CD36 on monocytes, respectively. To clarify the involvement of the erythroid lineage in CD36 deficiency, we investigated the phenotype and RNA expression of CD36. MATERIALS AND METHODS: CD36 expression was examined in 296 patients with several cardiovascular diseases in our outpatient clinic. There were 12 patients with type I deficiency and 16 with type II CD36 deficiency. A bone marrow sample was examined in five type I and four type II patients. Expression of CD36 mRNA was examined in burst-forming unit-erythroid (BFU-E). The sequences of reverse transcriptase polymerase chain reaction (RT-PCR) products of the CD36 mRNA from monocytes were examined. RESULTS: As expected, CD36 was deficient in erythroblasts from all five patients with type I deficiency. CD36 was present in erythroblasts from three of the four with type II deficiency, suggesting that their abnormality is restricted to platelets (type IIa). CD36 was unexpectedly absent from erythroblasts of a single type II patient (type IIb). CD36-specific mRNA was identified in BFU-E from each of two normals, six type I, and six type II patients, including type IIb. The sequences of RT-PCR products of the CD36 mRNA in a patient with type IIa and another with type IIb showed homozygous wild alleles. CONCLUSION: The findings provide evidence for further heterogeneity among CD36-deficient individuals and the existence of a basic principle mechanism of type II, such as glycosylation abnormality.

Disorders of platelet function.

Qualitative platelet disorders are described and reviewed above. The acquired platelet function defects are very common, and sometimes result in hemorrhage, especially in association with trauma or surgery. However, the specific biochemical defect is absent, and no characterized platelet abnormalities have been recognized. On the other hand, the hereditary qualitative platelet defects are rare, but the platelet abnormalities are characteristic. The study of these patients had led to an increased understanding of the normal primary hemostatic mechanism. Recently, the molecular basis analysis of the platelet defects has been developed. This will help us understand the molecular events involved in platelet adhesion and aggregation.

Mutations of the platelet thromboxane A2 (TXA2) receptor in patients characterized by the absence of TXA2-induced platelet aggregation despite normal TXA2 binding activity.

Previously, we reported five cases of platelet dysfunction characterized by the absence of thromboxane A2 (TXA2) - induced platelet aggregation despite normal TXA2 binding activity. In this platelet disorder, patients were divided into two groups; i.e. those whose platelets lacked or did not lack phospholipase C (PLC) activation (Group A and Group B, respectively) (Thromb Haemost 1996; 76: 1080). Furthermore, in one of the patients, we showed that a single amino acid substitution (Arg60 to Leu) in the first cytoplasmic loop of the TXA2 receptor (TXR) was responsible for this platelet disorder. However, mutational analysis of the TXR in the remaining patients has not been performed. Based on this background, we investigated the mutations of the TXR in these patients, and found that all of the patients have the same abnormality of the TXR (Arg60-->Leu), although the Group A patients were homozygous and the Group B patients were heterozygous for this mutation. This mutation is the only abnormality which has been found in this platelet disorder, and in patients heterozygous for this mutation, the mutant type TXR suppresses wild-type receptor-mediated platelet aggregation by a mechanism independent of PLC activation.

Pathogenetic analysis of five cases with a platelet disorder characterized by the absence of thromboxane A2 (TXA2)-induced platelet aggregation in spite of normal TXA2 binding activity.

Five patients with mild bleeding tendencies characterized by defective thromboxane A2 (TXA2)-induced platelet aggregation are reported. The platelets of all the patients had the ability to bind exogenous TXA2. Bleeding time was markedly prolonged in one patient. In three of the five patients, synthetic TXA2 mimetic (STA2)-induced platelet responses, including IP3 formation, Ca2+ mobilization, phosphatidic acid formation and GTPase activities were selectively defective, suggesting impaired coupling between the TXA2 receptor and phospholipase C activation. However, in the remaining two patients, these responses were all within normal limits. This suggests that the defective site of this type of platelet disorder is heterogenous and that signaling mechanisms other than the TXA2 receptor-phospholipase C pathway are also involved in TXA2-induced platelet aggregation.

Expression of eosinophil peroxidase in the immature basophil cell line KU812-F.

Although peroxidase activity in basophils can be detected by optical and ultrastructural cytochemistry, its characteristics remain to be determined. We have demonstrated the characteristics of peroxidase activity induced in the immature basophil cell line, KU812-F. Ultrastructurally, peroxidase activity was detected in granules as well as in the perinuclear space and endoplasmic reticulum. Immunocytochemistry revealed that KU812-F cells were stained by anti-eosinophil peroxidase antibodies, and eosinophil peroxidase mRNA, not myeloperoxidase, was detected in the cells using Northern hybridization and reverse transcription-polymerase chain reaction. Eosinophil peroxidase can be one of the molecules shared with eosinophils and basophils. The biological function of eosinophil peroxidase detected in basophils remains uncertain.

High-dose cytosine arabinoside and etoposide with total body irradiation as a preparatory regimen for allogeneic hematopoietic stem-cell transplantation in patients with acute lymphoblastic leukemia.

One approach to improving the outcome of allogeneic hematopoietic stem-cell transplantation for acute lymphoblastic leukemia (ALL) is to intensify the pretransplant conditioning regimen without increasing toxicity. We used an intensified conditioning regimen consisting of high-dose cytosine arabinoside (3 g/m(2) twice daily i.v. for 3 consecutive days, total six doses), high-dose etoposide (1 g/m(2) once daily i.v. during the first 2 days) and total body irradiation (TBI) (HDACE-TBI) in ALL patients. We retrospectively analyzed 21 patients treated with HDACE-TBI, of whom 18 were in complete remission (CR) and three were in non-CR at transplantation. Although gastrointestinal toxicities were common, critical regimen-related toxicities were not seen in any patients. One patient demonstrated veno-occlusive disease, which could be controlled conservatively. The disease-free survival rate of 18 patients in CR at transplantation was 61%. These results demonstrate that the HDACE-TBI combination regimen is a feasible alternative to other preparatory regimens and does not increase the regimen-related toxicity.

Vascular thromboxane formation in hemostasis mechanism: correlation between bleeding time and vascular TXB2 in a patient with congenital platelet cyclo-oxygenase deficiency.

We encountered a patient with congenital platelet cyclo-oxygenase deficiency with normal ability to synthesize vascular prostaglandin I2 (PGI2) and thromboxane A2 (TXA2). The patient's peripheral blood monocytes did not show cyclo-oxygenase (COX) activity, but cultured bone marrow fibroblasts showed COX activity. To determine the mechanism of primary hemostasis in this patient, we examined the effect of oral administration of aspirin (1 g) on bleeding time and thromboxane B2 (TXB2), 6-keto prostaglandin F1 alpha (6-keto-PGF1 alpha) production in the blood emerging from the incision in this patient. The bleeding time was markedly prolonged by the administration of aspirin, and this prolongation was associated with the inhibition of TXB2 in the effluent blood, which seemed to be derived from the vessel wall. These findings suggest that vascular TXA2 production plays an important role in the maintenance of hemostasis.

Primary Thrombocythemia in Japan: A Survey of 225 Patients.

Data on 225 Japanese patients with primary or essential thrombocythemia (ET) were analyzed in an attempt to characterize the clinical and laboratory features in subgroups with thrombosis (T), hemorrhages (H), thrombohemorrhagic events (TH) or a non-thrombohemorrhagic (O) group, and in order to examine survival and the incidence of blastic transformation in the entire group and in the different subgroups. Higher platelet and leukocyte counts were related to hemorrhage (H and TH), prolonged activated partial thromboplastin times and high LDH levels to H while elevated FDP levels were more frequently linked to T. Increased spontaneous platelet aggregation (SPA) was noted in 80.3% of the entire group, independent of whether there was a tendency for thrombohemorrhagic events or not. Bleeding time, as measured by the Duke method, and hemoglobin levels were not different in the various subgroups. Transformation occurred in 11 patients (1.9% per year); seven developed acute leukemia (myeloblastic 4, lymphoblastic 2, megakaryoblastic 1) at a rate of 1.2% per year; and 4 developed other types of chronic myeloproliferative disorders. Nineteen patients died (3.3% per year), six from leukemia (32%), 4 from bleeding (21%) and 9 from unrelated diseases (47%). Survival was estimated to be 65% at ten years, and was significantly longer in females, younger individuals, and the groups with lower leukocyte counts, but did not differ between the subgroups when platelet count and hemoglobin level were considered. Survival was similar in patients with platelet counts between 700-1000 × 10(9)/L and in those with an even higher platelet count. These findings suggest that (1) young female patients with low leukocyte counts may survive longer, (2) SPA is not indicative of either a thrombotic or an hemorrhagic tendency and (3) the limit of the platelet count for establishing the diagnosis of this disorder could perhaps be lowered to 700 × 10(9)/L.

Paroxysmal nocturnal hemoglobinuria with myelofibrosis: progression to acute myeloblastic leukemia.

A 58-year-old male was diagnosed as having paroxysmal nocturnal hemoglobinuria (PNH) with myelofibrosis in 1984. The administration of hydroxyurea and low dose splenic irradiation were initiated for abdominal distention due to splenomegaly in 1987. In May 1990 the patient developed smouldering acute myeloblastic leukemia (AML); and the blasts proliferated in response to G-CSF administered for refractory pneumonia. The patient died of pneumonia and pleural involvement of leukemia in September 1990. FACS analysis of the blasts using anti-decay accelerating factor (DAF) (CD55) and CD59 (membrane attack complex inhibition factor: MACIF) monoclonal antibodies demonstrated that 25.5% and/or 87.3% of the blasts were negative for DAF or CD59 respectively. There is the earlier evidence that about 90% leukemic myeloblasts from non-PNH AML patients are positive for DAF, and nearly 100% of non-PNH neutrophils have been shown to be positive for both DAF and CD59. Our data suggest that the leukemic blasts from this patient may have derived from the PNH clone.

Different effects of three kinds of somatostatin (15-28, 1-14, 1-28) on rabbit's platelet aggregation.

We studied the different effects of three kinds of somatostatin (somatostatin 1-14, somatostatin 15-28, somatostatin 1-28) on the aggregation of rabbit's platelets. It was clarified that somatostatin 15-28 had inhibitory effects on rabbit's platelet aggregation stronger than somatostatin 1-14 did, and that somatostatin 1-28 did not have any such effects. These anti-aggregatory effects of somatostatin were stronger when induced by collagen than induced by ADP.

Inhibition of ATRA-induced myeloid differentiation in acute promyelocytic leukemia by a new protein tyrosine phosphatase inhibitor, 3,4-dephostatin.

We previously reported that the expression and the activity of SHP-1, a non-transmembrane protein tyrosine phosphatase (PTPase) increased during myeloid differentiation of an acute promyelocytic leukemia cell line (HT93) induced by all-trans retinoic acid (ATRA). To examine whether inhibition of SHP-1 activity attenuates myeloid differentiation, we used a new PTPase inhibitor, 3,4-dephostatin, and studied its effect on myeloid differentiation. Suppressive effects on immunoprecipitated SHP-1 phosphatase activity and myeloid cell differentiation were detected. These results suggest that SHP-1 is a substrate for 3,4-dephostatin, and that SHP-1 PTPase activity is closely related to myeloid differentiation.

Severe cryoglobulinemia in a patient with asymptomatic hepatitis C virus infection.

A patient with severe type II cryoglobulinemia after ten years of asymptomatic hepatitis C virus (HCV) infection is reported. Laboratory data showed hypergammaglobulinemia with polyclonal IgG and monoclonal IgM, blood hyperviscosity, high level of cryocrit (60%), HCV viremia, and normal levels of serum transaminases. Due to cold exposure, acrocyanosis and cardiac ischemic attack occurred; he recovered after being artificially warmed. Administration of prednisolone and natural interferon-alpha for three months resulted in significant decreases in the levels of serum IgM and cryocrit.

Myocardial CD36 expression and fatty acid accumulation in patients with type I and II CD36 deficiency.

Long-chain fatty acids (LCFA) are one of the major cardiac energy substrates, so understanding LCFA metabolism may help in elucidating the mechanisms of various heart diseases. CD36 is a multifunctional membrane glycoprotein that acts not only as a receptor for thrombospondin, collagen and oxidized low density lipoprotein but also as a receptor for LCFA. We investigated the relationship between CD36 expression in myocardial capillary endothelial cells and myocardial LCFA uptake in patients with CD36 deficiency. We analyzed CD36 expression in blood cells from 250 patients with heart diseases by means of a flow cytometer. In 218 patients, myocardial LCFA scintigraphy was performed with 123I-beta-methyl-p-iodophenyl pentadecanoic acid (BMIPP). In 5 patients, myocardial capillary endothelial cells were examined immunohistochemically for CD36 expression. Eleven patients (4%) showed signs of type I CD36 deficiency (neither platelets nor monocytes expressed CD36). Twenty patients (8%) had type II CD36 deficiency (monocytes expressed CD36 but platelets did not). In all 11 patients with type I CD36 deficiency, no BMIPP accumulation was observed in the heart, but in 13 patients with type II CD36 deficiency, BMIPP accumulation in the heart was focally reduced, but there were no patients without BMIPP accumulation in the heart. Although the myocardial capillary endothelial cells from two CD36-positive patients expressed CD36, those from two patients with type I CD36 deficiency did not. In a patient with type II CD36 deficiency, some capillary endothelial cells displayed patchy CD36 expression. CD36 deficiency was documented in 31 (12%) patients with heart diseases. Because CD36 was not expressed in the myocardial capillary endothelial cells in patients with type I CD36 deficiency, type I CD36 deficiency is closely related to lack of myocardial LCFA accumulation and metabolism in the myocardium.

[Infections during induction chemotherapy of acute leukemia and their control V. Clinical evaluation of a large dose of amikacin injected intravenously].

In this study, we treated severe infections (21 cases) accompanied with induction chemotherapy in 20 patients with acute leukemia by the combination of a large dose (600 approximately 1,200 mg/day) of amikacin with other antibiotics. Infections during induction chemotherapy of acute leukemia consisted of sepsis (8 cases), pneumonia (7) and others (6), and most of causative organisms were Gram-negative bacteria, such as Ps. aeruginosa (7 strains), Flavobacterium (5), Serratia (3), Ps. cepacia (2), E. coli (2) and others. The combination chemotherapy of a large dose of amikacin with other antibiotics was found to be effective (71.4%) for such infections. Side effects were negligible except for drug eruption. Therefore, a large dose of amikacin should be given for the treatment of severe infections accompanied with induction chemotherapy of acute leukemia.

[Analysis of platelet shape change, inositol metabolism, and Ca mobilization in patients with platelet dysfunction].

Agonists-induced platelet shape change, inositol metabolism, and Ca mobilization were investigated in patients with various platelet dysfunctions. The platelet shape change determined by our method revealed that arachidonate-induced platelet shape change was completely defective in patients with cyclo-oxygenase (CO) deficiency (A). STA2-induced platelet shape change was also defective in one of five patients with impaired aggregation to STA2 (B). Thrombin-induced platelet shape change was weak in patients with Bernard-Soulier syndrome. In patient with Hermansky-Pudlak syndrome, the platelets did not respond normally to STA2, arachidonate or PMA. These findings suggested that the determinations of platelet shape change by our method was useful in diagnosing platelet dysfunctions. Inositol metabolism and Ca mobilization in response to thrombin, STA2, or NaF were also investigated in patient A,B, and impaired aggregation to A23187 in patient C. The responses were normal in patient A, suggested that CO activity did not affect them. Inositol metabolism was also normal in patient C, although Ca mobilization in response to A23187 was delayed, and that in response to thrombin was defective in the absence of extracellular Ca2+. This suggests that the patient's platelets have a defective IP3-induced Ca mobilization pathway. STA2 selectively failed to induce IP3 formation and Ca mobilization in patient B, although 3H-labelled thromboxane ligand (3H-U46619) bound to the patient's platelets, normally. These findings suggested that the patient's platelets have a defect in postreceptor signal transduction, especially thromboxane receptor-mediated phospholipase C activation pathway.

[Essential thrombocythemia in transformation to smouldering megakaryoblastic leukemia with myelofibrosis].

Leukemic transformation in essential thrombocythemia (ET) is rare. We describe a patient with ET which transformed to megakaryoblastic leukemia with myelofibrosis after treatment with melphalan for 8 years. His course after transformation smouldered for 20 months without antileukemic chemotherapy. A 61-year-old man was referred by a local doctor to Niigata University Hospital due to nasal bleeding in June 1984. Complete blood count (CBC) was as follows; hemoglobin 12.4 g/dl, platelets 268.8 x 10(4)/microliters, and white blood cells 11,900/microliters, with differentials of 39% PMN, 1% basophils, 2% eosinophils, 4% monocytes, and 13% lymphocytes. Bone marrow examination revealed hyperplasia of megakaryocytes without increase of reticulin fibers. Neutrophil alkaline phosphatase activity and karyotype of marrow cells were normal. ET was diagnosed. He was followed up by local doctor. The platelet count was controlled at a level of approximately 40 x 10(4)/microliters with melphalan for eight years. In January 1992 he developed pain in his lower extremities. He was admitted to our hospital on May 29, 1992. CBC was as follows; hemoglobin 8.9 g/dl, platelets 14.3 x 10(4)/microliters, and white blood cells 3,500/microliters, with differentials of 25% PMN, 5% monocytes, 28% lymphocytes, and 24% blasts. Bone marrow aspiration was unsuccessful and bone marrow biopsy revealed increases in fibroblasts and collagen fibers. Circulating blasts were positive for CD4, CD7, CD25, CD13, CD33, CD34, and HLA-DR and partly positive for CD41 and CD36. In ultrastructural cytochemistry blasts were positive for platelet peroxidase but negative for myeloperoxidase. Cytogenetic study revealed 46, XY, +der (1) t(1:7) (p11;q11) in all of five metaphases. He was diagnosed with megakaryoblastic leukemia accompanied by myelofibrosis.(ABSTRACT TRUNCATED AT 250 WORDS)