Shigeru Arimori

Increased Superoxide Production by Mononuclear Cells of Patients With Hypertriglyceridemia and Diabetes

Diabetic patients with hypertriglyceridemia frequently develop atherosclerosis. Because superoxide (O−2) is suspected to play an important role in the initiation of atherosclerosis, we investigated whether an abnormal amount of O−2 was produced by circulating mononuclear cells of patients with both diabetes mellitus and hypertriglyceridemia. The rate of production of superoxide dismutase–inhibitable O−2 was measured when cells were stimulated by either 4β-phorbol 12β-myristate 13α-acetate (PMA) or by opsonized zymosan (OZ). In addition, the rates of O−2 production by mononuclear cells drawn from three other groups (normal, solely diabetic, and solely hypertriglyceridemic) were determined. We found that the rate of O−2 production by mononuclear cells from the diabetic hypertriglyceridemic group was significantly higher than that from normal, diabetic, and hypertriglyceridemic groups. When the rates of O−2 production by mononuclear cells were plotted against the levels of plasma triglyceride for all individuals tested, they correlated positively (r = .73 in PMA stimulation and r = .79 in OZ stimulation, P < .01). However, the rate of O−2 production did not relate to other parameters, i.e., plasma cholesterol level, hemoglobin A1 level in erythrocytes, and the molar ratio of free cholesterol to phospholipid in mononuclear cells. Thus, we concluded that the observed elevated rate of O−2 production in the diabetic hypertriglyceridemic mononuclear cells was a reflection of a hypertriglyceridemic condition and was not unique to the diabetic hypertriglyceridemic condition. Also, O−2 may be involved in the pathogenesis of atherosclerosis in diabetic hypertriglyceridemic patients when atherogenic factors specific to diabetes are concomitantly present.

Reduction of platelet aggregation induced by euglycaemic insulin clamp

SummaryTo examine the effect of serum insulin independent of the level of blood glucose in vivo on platelet aggregation in healthy individuals, a euglycaemic insulin clamp was applied up to 4 h. During the clamp, blood glucose at 5.0 mmol/l and insulin levels at 100 μU/ml were maintained. Blood samples were drawn before, 2 and 4 h after the start of the insulin clamp. The platelet aggregation induced by 1 μmol/l and 2 μmol/l ADP, 1 μg/ml collagen and 2.7 μmol/l epinephrine was measured in the blood samples. Platelet aggregation induced by adenosine diphosphate, collagen and epinephrine in the 4 h sample was significantly reduced from the pre-clamp value of 8.4% to 3.9% (p<0.05), 26.2% to 7.0% (p<0.01) and 31.8% to 9.1% (p<0.01), respectively. On the other hand, when the same individuals were infused with physiological saline and blood glucose (4.4 mmol/l) and insulin level (10 mIU/l) were kept within normal values, there was no difference between the values of induced platelet aggregation in samples drawn before and during the insulin infusion. It was concluded that hyperinsulinaemia reduces platelet aggregation in vivo when euglycaemia was maintained.

Time course of superoxide generation by leukocytes-The MCLA chemiluminescence system

This study was performed to examine the pattern of Superoxide (O2−·) generation from leukocytes using the O2−· specific chemiluminescence (CL) method.Cypridina luciferin analog, 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-alpha]pyrazin-3-one (MCLA) was used as a CL probe. The appropriate conditions of the MCLA method was first determined for the evaluation of the time course of O2−· generation by leukocytes. The time course of O2−· generation obtained by the MCLA-CL system was compared with that by the luminol-dependent CL, electron spin resonance (ESR)/spin trapping, and cytochromec systems. Following stimulation by three different stimulants (PMA, OZ, FMLP), leukocytes continuously generated O2−· for up to 5 h in the MCLA-CL system, irrespective of the kind of stimulation. The curves obtained by generation ceased more rapidly in the luminol-CL, ESR/spin trapping, and cytochromec systems. A 50% activity of the initial value was observed at 70 min in the MCLA-CL system, but 30, 10 and 35 min in the other systems, respectively. The CL or O2−· generation value decreased to less than 1% (possible termination) at 300, 90, 120 and 180 min, respectively. With the exception of ESR studies with OZ, the cell viability was not significantly affected in any of the trials. These results indicate that leukocytes can generate O2−· much longer than previously estimated and that the MCLA-CL-system is the most suitable system for the measurement of the O2−· generation by leukocytes.

Combinations of HLA-DPB1 and HLA-DQB1 alleles determine susceptibility to early-onset myasthenia gravis in Japan

HLA class II alleles in the DQA1, DQB1, DRB1, and DPB1 genes were investigated in Japanese patients with myasthenia gravis (MG) by digestion of polymerase chain reaction amplified DNAs with allele specific restriction endonucleases (PCR-RFLP). A significantly higher frequency of DQB1*03, which includes *0301, *0302, *0303 and determines the serological DQ3 specificity, was observed in female patients less than 30 years in age at onset of disease compared with healthy controls (90.5 vs. 53.2%). This study also confirms the high incidence of DPB1*0201 in early-onset female patients compared to the controls (85.7 vs. 40.3%). Moreover, 81.0% of the early onset female patients were found to carry both DQB1*03 and DPB1*0201, compared to 17.7% of the controls. Since DQB1*03 and DPB1*0201 are not in linkage disequilibrium, both these alleles are supposed to be synergistically involved in disease development in early-onset female MG. In contrast, no obvious association of HLA-DQA1, DQB1, DRB1 and DPB1 alleles with either late-onset patients or patients with thymoma was observed. Clearly, the genetic background of Japanese females with early onset MG is different from that of other patients with MG.

Two-color flow cytometric analysis of thymic lymphocytes from patients with myasthenia gravis and/or thymoma

Phenotypic characteristics of lymphocyte components of the thymus were determined by two-color flow cytometry using monoclonal antibodies to T cell-differentiation antigens and activation antigens, and B cells in eight myasthenia gravis (MG) thymuses and eight thymomas including four associated with MG to clarify the roles of thymus in the diseases. Fifteen normal thymuses and four thymuses from non-MG patients were used as controls. Phenotypes of lymphoid components in thymoma resembled those in normal thymuses, in which the majority of lymphoid cells were immature (common) thymocytes (CD4+CD8+ or CD1+CD3-). The proportions of immature thymocytes, however, were relatively higher and those of mature thymocytes (CD4+CD8-, CD4-CD8+, CD1-CD3+ or CD2+CD3+) were lower in thymomas than normal thymuses. The results speculate that functions which support further differentiations of immature thymocytes into mature thymocytes are deficient in neoplastic epithelial cells of thymoma. In thymomas, therefore, occasional T cells might escape from the thymic surveillance system which eliminates auto-reactive T cells. Between thymomas associated with MG and those without, however, no significant difference was found in the proportions of thymocytes at various stages of maturation. In nonthymomatous thymuses from MG patients, an increase of both B cells (CD2-DR+ or CD2-CD21+) and activated T cells (CD2+DR+ or CD2+IL-2R+) was observed. Furthermore, immature thymocytes were significantly decreased and mature thymocytes were increased in the MG thymuses, as compared with normal thymuses, non-MG thymuses, and thymomas. These alterations were relevant to the histological findings observed in MG thymuses such as thymic involutions and germinal center formations: decreased immature thymocytes are considered to reflect thymic involutions, while germinal centers, in which lymphocytes from peripheral lymphoid organs are activated, are responsible for the increase of B cells, activated T cells, and mature T cells. In conclusions, our results suggest that neoplastic epithelial cells of thymomas are functionally deficient and that MG thymuses are immunologically active and resemble peripheral lymphoid organs.

Increased growth and collagen synthesis of bone marrow fibroblasts from patients with chronic myelocytic leukaemia

Summary. Growth and collagen synthesis were measured in human bone marrow fibroblasts derived from patients with chronic myelocytic leukaemia (CML) and normal individuals. The 3H‐thymidine uptake, growth rate and collagen synthesis of fibroblasts from patients with CML were significantly greater than those from normal subjects. Thus, there is increased proliferation and collagen synthesis of fibroblasts in patients with CML. These findings show that CML fibroblasts may display a greater sensitivity to stimulator contained in the fetal calf serum used for the cultures, and they are relevant to the myelofibrosis by bone marrow fibroblasts in this disease.

Decreased plasma superoxide scavenging activity in immunological disorders — carboxyethylgermanium sesquioxide (Ge-132) as a promoter of prednisolone

SummaryWe investigated so-called superoxide scavenging activity (SSA) of plasma in patients with several immunological disorders, such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), polymyo-dermatomyositis (PM), progressive systemic sclerosis (PSS), myasthenia gravis (MG) and autoimmune thyroid disease (AT), using the electron paramagnetic resonance/spin trapping technique. Since carboxyethylgermanium sesquioxide, Ge-132, has been reported to modulate both the immune response and leukocyte functions, we have studiedin vivo effect of Ge-132 on plasma SSA and other laboratory parameters in these disorders. The plasma SSA was significantly lower in RA, SLE, PM and PSS, but not in MG and AT, as compared with that in healthy controls. An inverse correlation was observed between plasma SSA and parameters such as erythrocytes sedimentation rates, absolute number of leukocytes, C-reactive protein and serum globulin levels. Furthermore, plasma SSA was significantly decreased in rheumatoid factor-positive patients as compared to negative patients. No correlation was observed between plasma SSA and factors such as ages, sex of patients or the other laboratory parameters, such as serum albumin, triglyceride, cholesterol, hemoglobin and serum iron levels. Patients treated with prednisolone, especially ones with RA, showed an increase of plasma SSA. It appears that Ge-132 promotes prednisolone effects. Our results indicate that a decrease in plasma SSA is not disease specific, but inversely correlates with the severity and activity of inflammation. The methodology to measure plasma SSA presented in this work provides a helpful tool for determining the actual activity of the diseases as well asin vivo studies of antiinflammatory agents.

Flow cytometric analysis of cell-surface antigen expressions on acute myeloid leukemia cell populations according to their cell-size

Acute myeloid leukemia (AML) cells which expanded from a single leukemic cell show certain degrees of morphological and biological heterogeneity. In the present study, we determined cell-surface antigen expressions (CD13, 33, 34 and 38, and HLA-DR) on AML cells based on their cell-size (large vs small cells) by flow cytometry. We found that the cell-surface antigens were more strongly expressed on the large leukemic cells than the small cells, regardless of FAB subtypes. Furthermore, our preliminary study demonstrated that AML patients who showed a relatively small difference in antigen expression between large and small leukemic cells had longer remission durations and survival periods, compared with those with a more prominent difference in antigen expression. Thus, the heterogeneity of AML cells determined by the combination of cell-surface antigen expressions and cell-size may be associated with clinically important biological behaviors.

Serum Apolipoprotein H Levels in Systemic Lupus Erythematosus Are Not Influenced by Antiphospholipid Antibodies

Anticardiolipin antibodies (aCL) were recently discovered to recognize a complex consisting of phospholipids and apolipoprotein H (apo H). In this study, we determined the serum apo H levels in 36 systemic lupus erythematosus (SLE) patients with or without antiphospholipid antibodies (aPL), including aCL and lupus anticoagulants, to clarify the possible effects of aPL on apo H levels in vivo. The apo H levels were low in SLE patients as compared with 22 healthy controls. However, no associations were found between apo H levels and circulating aPL or clinical features of the antiphospholipid antibody syndrome. A secondary hyperlipidemic state, which probably related to lupus nephritis (proteinuria) and/or prednisolone treatment, increased apo H levels in SLE patients.

BG-104 enhances the decreased plasma superoxide scavenging activity in patients with Behçet's disease, Sjögren's syndrome or hematological malignancy

SummaryBG-104, a compound of Chinese herbs, has been reported to exert superoxide scavenging activity (SSA) in cell free systems. This report addressesin vivo effects of BG-104 in various disorders. The plasma SSA and laboratory parameters were determined in patients Behçets disease (BD), Sjögrens syndrome (SjS) or hematological malignancy (M), and the effects of BG-104 treatment on these parameters were studied and compared with those of another antioxidant, vitamin E (alpha-tocopherol).The plasma SSA was significantly lower both in patients with BD and M, and in patients with SjS without antioxidant treatment as compared to that in healthy controls, and it showed an inverse correlation with disease activities. The treatment with BG-104 and/or vitamin E significantly enhanced the plasma SSA in all disorders studied. Both the erythrocyte sedimentation rates, the absolute number of neutrophils, as well as C-reactive protein levels were significantly lower in patients treated with BG-104 and/or vitamin E than those without these drugs. These results indicate that the BG-104 has an anti-inflammatory effect through enhancing plasma SSA in patients with BD, SjS or M.