Increased superoxide production by mononuclear cells of patients with hypertriglyceridemia and diabetes.

Diabetic patients with hypertriglyceridemia frequently develop atherosclerosis. Because superoxide (O2-) is suspected to play an important role in the initiation of atherosclerosis, we investigated whether an abnormal amount of O2- was produced by circulating mononuclear cells of patients with both diabetes mellitus and hypertriglyceridemia. The rate of production of superoxide dismutase-inhibitable O2- was measured when cells were stimulated by either 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) or by opsonized zymosan (OZ). In addition, the rates of O2- production by mononuclear cells drawn from three other groups (normal, solely diabetic, and solely hypertriglyceridemic) were determined. We found that the rate of O2- production by mononuclear cells from the diabetic hypertriglyceridemic group was significantly higher than that from normal, diabetic, and hypertriglyceridemic groups. When the rates of O2- 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 less than .01). However, the rate of O2- 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 O2- production in the diabetic hypertriglyceridemic mononuclear cells was a reflection of a hypertriglyceridemic condition and was not unique to the diabetic hypertriglyceridemic condition. Also, O2- may be involved in the pathogenesis of atherosclerosis in diabetic hypertriglyceridemic patients when atherogenic factors specific to diabetes are concomitantly present.

HLA-DPB1 allele associates with early-onset myasthenia gravis in Japan.

We investigated the HLA-DPB1 allele in 43 unrelated Japanese patients with myasthenia gravis (MG) using digestion of polymerase chain reaction (PCR)-amplified DNA with allele-specific restriction endonucleases (PCR-RFLP method). We found a higher frequency of the DPB1*0201 allele in female patients whose ages at onset were less than 30 years (83.3%) than in controls (35.6%). The study also included serologic typing of HLA-A, -B, -C, and -DR antigens in 72 patients with MG, and confirmed previous results demonstrating a strong association of HLA-DR53 with early onset of MG in females. These results indicate that both the DPB1*0201 allele and DR53 play key roles in the disease process of MG in early-onset females, and that the genetic background of Japanese females with early-onset MG is different from that of other patients with MG.

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.

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.

Molecular analysis of abnormal satellite I DNA from a BUF/Mna rat thymoma.

BUF/Mna rats develop spontaneous thymomas in an autosomal dominant manner. We constructed recombinant plasmid library of 90 and 185 base-paired (bp) satellite I DNA fragments isolated from BUF/Mna rat thymoma DNA. Four unusual clones containing 93, 95, 95, and 173 bp inserts were isolated by colony hybridization with Wistar rat satellite I DNA. Nucleotide sequence analysis of the inserts of the 4 clones revealed abnormal sequence organization and unusual subunit structure of the rat satellite I DNA. Sequence comparisons between normal and abnormal satellite I DNA suggested that the unusual subunit structure could be generated by the change of the Hinf I recognition sequence to an Eco RI cleavage site, in addition to random deletions, insertions and base substitutions. The heptanucleotide sequence TGGGAAC, which is strictly conserved in normal subunits, was completely lost in all these clones. Southern blot hybridization revealed the amplification of abnormal satellite I DNA in BUF/Mna rat thymomas.

Lymphocyte subsets of the peripheral blood in myasthenia gravis determined by two-color flow cytometry.

Lymphocyte subsets of the peripheral blood in 43 patients with myasthenia gravis (MG) were determined by two-color flow cytometry using a number of monoclonal antibodies. In the MG patients without thymectomy (Tx) and prednisolone (PSL) treatment, lymphocyte counts, B-cells, CD4+ cells and their subsets were normal, but numbers of T-cells, CD8+ cells and CD8+ CD 11-subsets were significantly decreased. Furthermore, proportions of activated cells in T-cells, CD 16+ Leu7- and CD16+ Leu7+ NK subsets were significantly high in the patients. The changes in T-cells, CD8+ cells and activated T-cells were less marked in the MG patients than Sjögren's syndrome (SS) used as a disease control. Contrary to MG patients, lymphocyte counts, CD4+ cells and their subsets were decreased, and the proportions of B-cells were high in SS patients. These results suggest altered immunologic conditions, immunologically active and deficient conditions, in both diseases, although the alterations were more prominent in SS than MG. PSL treatments and Tx significantly altered the lymphocyte profiles: PSL decreased lymphocytes, B-cells, T-cells, CD4+ cells and their subsets, while the proportions of CD8+ cells were increased. The changes were compatible with the known immunosuppressive effects of PSL. After Tx, lymphocytes and B-cells decreased, but the proportions of T-cells, CD8+ cells and their subsets, and NK cells subsets returned toward normal. CD4+ CD8+ cells were not increased in MG patients, and the cells did not decrease after Tx. Some of these observations might be relevant to clinical effects of Tx, although the mechanism responsible for these changes is still unknown.

Increase of IgA-bearing lymphocytes in peripheral blood from patients with IgA nephropathy.

Aberration of IgA-bearing B lymphocytes in patients with IgA nephropathy has been investigated. Twelve patients with IgA nephropathy demonstrated a marked increase of IgA-bearing lymphocytes in peripheral blood, while ten patients with chronic proliferative glomerulonephritis without mesangial deposition of IgA showed normal amounts of IgA-bearing lymphocytes. The increase of IgA-bearing lymphocytes reflected that of IgA-producing lymphocytes, since lymphocytes obtained from patients with IgA nephropathy restored a high percentage of IgA-bearing cells in vitro after treatment with trypsin. Quantitation of IgA-bearing lymphocytes in peripheral blood is a useful method for screening of patients with IgA nephropathy.

Increase of IgA-bearing peripheral blood lymphocytes in families of patients with IgA nephropathy.

IgA-bearing peripheral blood lymphocytes, serum IgA, urinary sediments and HLA types of patients with IgA nephropathy and members of their families were examined to elucidate whether some familial factors might be related to the development of IgA nephropathy. Ten patients with IgA nephropathy, 31 family members and 36 age-matched healthy persons were examined. All families included certain members with increased amounts of IgA-bearing peripheral blood lymphocytes. The pattern of the emergence of family members with increased IgA-bearing lymphocytes was vertical. Some family members who had increased IgA-bearing lymphocytes showed microhematuria at the time of the study. There was no significant correlation between the amounts of IgA-bearing peripheral blood lymphocytes and levels of serum IgA. HLA types of the ten patients did not show significant deviation from those in the general population. It is suggested that the measurement of IgA-bearing peripheral blood lymphocytes among family members is useful for the screening of patients with IgA nephropathy.

MDR1 (multidrug resistance) gene expression in adult acute leukemia: correlations with blast phenotype.

Resistance to multiple chemotherapeutic agents is related to the production of P-glycoprotein, a transmembrane drug efflux pump that is encoded by the multidrug resistance gene (MDR1). To detect low-level or heterogenous expression of the MDR1 gene in acute leukemia, we have developed sensitive, specific and semi-quantitative protocols for measuring levels of MDR1 mRNA, based on the polymerase chain reaction. Using this assay, we screened blasts from 20 patients with untreated adult acute leukemia for evidence of MDR1 gene expression. The level of MDR1 mRNA was normalized to beta 2-microglobulin mRNA and was defined by reference to the highly resistant trimetrexate-selected leukemia cells MOLT-3/TMQ200 (1.80). MDR1 mRNA was observed in 14 out of 20 patients. Higher MDR1 mRNAs were observed in three patients with phenotypes of undifferentiated or minimally differentiated nonlymphocytic acute leukemia, as compared with other types of acute leukemia (0.98 vs. 0.25). In contrast, lower MDR1 mRNAs were found in five patients with acute promyelocytic leukemia, as compared with other types of acute leukemia (0.08 vs. 0.45). These findings suggest that MDR1 gene expression is correlated with the leucocyte differentiation stage of leukemia. MDR1 gene expression may, in part, explain the responsiveness to chemotherapy in these distinct subtypes of acute leukemia.

Differential effects of IL-3, GM-CSF and G-CSF in an adult with congenital neutropenia.

Using a methylcellulose culture system, we studied the effects of recombinant human interleukin-3 (IL-3), recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), and recombinant human granulocyte colony-stimulating factor (G-CSF) on the growth of myeloid progenitor cells (CFU-C) from an adult patient with congenital neutropenia. The moderate clinical course and the maturation arrest at blast-promyelocyte stage in the marrow differentiated this patient from those described as having Kostmann-type congenital neutropenia. CFU-C growth in bone marrow cells from the patient responded to IL-3 normally in a dose-dependent manner. GM-CSF stimulated only macrophage colony formation in a dose-dependent manner comparable to that in normal subjects. Neither GM-CSF nor G-CSF stimulated any significant granulocyte colony formation. This evidence suggests that the hematopoietic progenitor cells in this patient had the potential for developing CFU-C with IL-3, and that the neutropenia in this patient could be a result of an intrinsic defect in myelopoiesis along a granulocytic pathway responsive to GM-CSF or G-CSF.

The first fluorescent sensor for boronic and boric acids with sensitivity at sub-micromolar concentrations--a cautionary tale.

The first reported fluorescent sensor for boronic and boric acids is actually not a sensor for boronic and boric acids but rather is a sensor for protons; the system is also not the first fluorescent sensor since Alizarin has been used as a fluorescent sensor for boric acids since 1936.

Modular fluorescence sensors for saccharides.

Modular and modular polymer supported fluorescence photoinduced electron transfer (PET) sensors 2 and 3 with two boronic acid receptor units, a pyren-1-yl fluorophore, and hexamethylene linker show selective saccharide binding in aqueous methanolic solution at pH 8.21.

Enhanced superoxide generation and the decreased superoxide scavenging activity of peripheral blood leukocytes in Behçet's disease--effects of colchicine.

Enhanced superoxide radical (O2-) generation by polymorphonuclear cells (PMN) has been suggested to mediate tissue damage associated with Behçet's disease (BD). Because superoxide dismutase (SOD) provides protection from O2- we investigated whether the superoxide scavenging activity (SSA) of PMN, mononuclear cells (MNC) or plasma has a correlation to the amount of O2- generated by PMN in BD. We also studied the effects of colchicine, a potent inhibitor of phagocytosis, on the SSA of PMN. In BD, O2- release by both non-stimulated PMN and those stimulated with opsonized zymosan (OZ), or 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA), was enhanced. The SSA of PMN, but not MNC and plasma, was significantly lower in BD patients as compared with healthy controls. The SSA of PMN showed a strong negative correlation with their O2- release. The SSA of PMN was lower in the BD patients with elevated erythrocyte sedimentation rates and C-reactive protein levels than in those with normal test results. In addition, we found that colchicine treatment increased the SSA of PMN toward normal levels in BD patients. In vitro studies demonstrated that the SSA in PMN obtained from healthy adults was decreased after stimulation with either OZ or PMA. Colchicine could prevent the decrease in the SSA when PMN were stimulated with OZ, but not when stimulated with PMA. Our results suggest that the enhanced O2- release by PMN in vivo may be responsible for the decreased SSA of PMN in BD and the PMN might be able to release more O2- in tissues. Colchicine may increase the SSA of PMN toward normal levels by blocking phagocytosis.

Activation antigens expressed on T-cells of the peripheral blood in Sjögren's syndrome and rheumatoid arthritis.

We analyzed activated T-cells in the peripheral blood (PB) of 29 patients with Sjögren's syndrome (SS: 13 I degrees-SS and 16 II degrees-SS patients) and 11 patients with rheumatoid arthritis (RA) by two-color flowcytometry using antibodies to antigens serially expressed on the cell surface after activation--interleukin-2 receptor (IL-2R), HLA-DR, T-lineage specific antigen (TLiSA-1) and very late antigen (VLA-1). The early and intermediate activation antigens, DR and TLiSA-1, were significantly increased in the PB of SS and/or RA patients. The proportions of activated T-cells were higher in CD8+ cells than those in CD4+ cells. T-cells expressing IL-2R or VLA-1, which appear in the very early or late stages of activation, were also increased in SS and/or RA patients. The results suggest that activated T-cells in the PB of both diseases might be recruited from continuously activated lymphoid organs, and that the activated cells migrate from PB into target tissues. In addition, the increase of activated T-cells in PB might be one of the causes of the deficient cell-mediated immunity reported in these patients.

Accessory molecules expressed on the peripheral blood or synovial fluid T lymphocytes from patients with Sjögren's syndrome or rheumatoid arthritis.

We previously demonstrated that the cells expressed by activation antigens were increased in several T cell subsets from the peripheral blood (PB) and joint fluid (JF) of patients with Sjögren's syndrome (SS) or rheumatoid arthritis (RA). In the present report, we further determined by three-color flow cytometry the homing receptors (Leu8) for peripheral lymph nodes expressed on naive (CD45RA+) or memory (CD45RA-) CD4+ cells and on the two subsets of CD8+ cells (CD11b+ and CD11b-) in the PB and JF from SS and RA patients. In addition, the activation antigens (HLA-DR) and two adhesion molecules, including the alpha-chain of the leukocyte function associated antigen-1 (LFA 1 alpha: CD11a) and its ligand (intercellular adhesion molecule-1: ICAM-1: CD54) expressed on T cells, were compared. We found that CD45RA-CD4+ cells were markedly increased in JF, while CD45RA+CD4+ cells were almost absent. Leu8+ cells were decreased in both CD45RA-CD4+ and CD8+CD11b-cells (cytotoxic T cells) in the JF, and were also decreased in the CD8+CD11b-subset in the patients' PB. Furthermore, activated (DR+) T cells were markedly increased in JF, and the cells expressed more adhesion molecules in both the PB and JF from patients, compared with the DR-T cells. The DR+ T cells therefore are considered to be memory T cells, which are more efficient for cell-to-cell interactions. These observations also suggest that the Leu8- and DR+ T cells with increased adhesion molecules might preferentially migrate into inflammatory tissues, and that naive T cells are being further converted to to memory T cells by in vivo stimulation within the tissues.

Association of enhanced superoxide generation by neutrophils with low superoxide scavenging activity of the peripheral blood, joint fluid, and their leukocyte components in rheumatoid arthritis: effects of slow-acting anti-rheumatic drugs and disease activity.

To study the role of the superoxide radical (O2-) in the pathogenesis of rheumatoid arthritis (RA), both the O2- generation of peripheral blood (PB) polymorphonuclear cells (PMN) and the superoxide scavenging activity (SSA) of PB-PMN, plasma, joint fluid (JF) and PB- or JF-mononuclear cells (MNC) were measured in forty-five patients with RA using the highly sensitive and specific 2-methyl-6-methoxyphenyl-3,7-dihydroimidazo[1,2-alpha] pyrazin-3-one dependent chemiluminescence and the electron paramagnetic resonance/spin trapping methods, respectively. Since many drugs, particularly the slow-acting anti-rheumatic drugs (SARDs) used in RA, may alter O2- metabolism, the effects of SARDs on SSA were also studied. The basal O2- release and opsonized zymosan- or phorbol myristate acetate-stimulated O2- generation by PB-PMN from RA patients were significantly increased, while the SSA of PMN was decreased as compared to those from healthy controls. In addition, the SSA of PMN showed a negative correlation with their O2- generation rates. The JF-PMN showed lower SSA levels than PB-PMN. A negative correlation was also found between the SSA of the plasma and the erythrocyte sedimentation rates. The SSA of the plasma, PB-PMN, JF and JF-PMN were significantly higher in patients treated with SARDs than those without. In cell-free systems, sulfasalazine (SAP) and its metabolite, 5-amino-salicylic acid, had a direct SSA within a millimolar range. The other metabolites of SASP and D-penicillamine had an indirect SSA, since they affected the O2- generating system. Auranofin and bucillamine had no SSA. However, none of the SARDs examined could scavenge O2- at concentrations reported in patients' plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

T cells bearing gamma/delta T cell receptor and their expression of activation antigen in peripheral blood from patients with Sjögren's syndrome.

T cells bearing gamma/delta T cell receptor (gamma/delta + T cells) and their expression of activation antigen (HLA-DR) or the marker of natural killer (NK) cells (CD56), were examined in the peripheral blood lymphocytes (PBL) from twenty-two patients with Sjögren's syndrome (SS) by three-color flowcytometry to elucidate possible pathological roles of the T cell subset in SS. The frequency of gamma/delta + T cells in PBL was not elevated in SS patients, while that of gamma/delta - T cells, which are T cells bearing the alpha/beta T cell receptor (alpha/beta + T cells), was significantly low in the patients, as compared with 22 healthy controls. We found that the proportions of activated cells (HLA-DR+) in both the gamma/delta + and alpha/beta+T cell subsets were significantly higher in the patients than in the controls. The proportions of HLA-DR+ cells in cells in both patients and controls. Furthermore, the frequency of activated cells in both T cell subsets correlated with the duration of disease in SS patients. However, no difference was found in the percentages of total CD56+ cells, CD56+CD3- cells (true NK cells), CD56+CD3+T cells, CD56+gamma/delta+T cells, or CD56-gamma/delta+T cells between the patients and controls. The above results indicate that immunologic activation in SS patients is progressive and involves both alpha/beta+ and gamma/delta+ T cell subsets.

Familial Sjögren's syndrome in the Japanese: immunogenetic and serological studies.

Serological abnormalities and HLA haplotypes were studied in a Japanese family of two patients with Sjögren's syndrome (SS) associated with other autoimmune diseases. In contrast to the reports in the U.S.A. and Europe, we found a significant excess of HLA-DRw53 antigen in the family members. Two family members, mother and niece of the probands, had suffered from other connective tissue diseases. Although none of the siblings of probands had manifestations of connective tissue diseases, two siblings had several autoantibodies. No consistent segregation, however, was found between the HLA haplotypes and the serological abnormalities in the relatives. Therefore, HLA alone can not explain the familial clustering of autoimmune diseases including SS and of autoantibodies.

Reactivities of antiphospholipid antibodies to blood cells and their effects on platelet aggregations in vitro.

To clarify the pathogenesis of antiphospholipid antibody (aPL) syndrome, the reactivities of anticardiolipin antibodies (aCL) in sera of patients with systemic lupus erythematosus (SLE) or other diseases to fresh, activated or destroyed blood cells were examined by the inhibition assay using an enzyme-linked immunosorbent assay. In addition, the effects of lupus anticoagulants (LA) in the patients' plasma and of immune complexes formed between LA and PL antigens on platelet aggregations were also determined. The IgG-aCL activity of patients' sera was markedly inhibited by pre-incubation with freeze-thawed blood cells, including erythrocytes (RBC), mononuclear cells (MNC) and platelets, but not fresh platelets or RBC. The aCL activity was slightly inhibited by fresh MNC, and was definitely inhibited by thrombin-activated platelets and polymorphonuclear cells (PMN) stimulated with phorbol 12-myristate 13-acetate (PMA). However, the activity was not inhibited by platelets stimulated with adenosine 5'-diphosphate (ADP; 10 microM). Twenty-two LA positive plasma and 17 LA negative plasma from patients similarly enhanced the aggregation of platelets which were obtained from healthy adults and stimulated with low concentrations of ADP (1 or 2 microM). However, such enhancement of platelet aggregation was not observed when high concentrations of ADP (5 microM) or collagen (2 micrograms/ml) were used as stimulators. In four of the 16 LA positive plasma examined, the mixture of plasma and phospholipid reagent for activated partial thromboplastin time induced platelet aggregations without the other stimulations, but the plasmas themselves did not induce such a reaction. The above results indicate that the aPL from patients do not react with intact blood cells in vitro, but they can react with activated or destroyed blood cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Lymphocyte subsets of the peripheral blood in Sjögren's syndrome and rheumatoid arthritis.

Lymphocyte subsets were determined in the peripheral blood from twenty-three patients with primary Sjögren's syndrome (SS) and sixteen patients with clinically active rheumatoid arthritis (RA) by two-color flowcytometry using various monoclonal antibodies. In both diseases, T-cells (CD3+), suppressor/cytotoxic cells (CD8+) and their cytotoxic subset (CD8+CD11-) were decreased, as compared with thirty-one healthy controls. B-cells (CD 21+ and CD 3-DR+) and activated T-cells (CD 3+DR+) were increased in SS patients. Helper T-cells (CD 4+Leu8-), suppressor-inducer T-cells (CD4+Leu8+), suppressor T-cells (CD8+Leu 15+) and three natural killer (NK) cell subsets determined by both CD16 and Leu7 antibodies did not differ between controls and SS or RA, although Leu7+NK cells were significantly increased in SS patients. In addition, we found that the treatment with low-dose prednisolone decreased B-cells and suppressor-inducer T cells, and increased suppressor T-cells, cytotoxic T-cells and Leu7+NK cells. The results indicate similar changes in the proportion of lymphocyte subsets and suggest immunologically activated and deficient conditions in both diseases. Immunomodulating effects of the treatment with low-dose prednisolone on some of the lymphocyte subsets in patients with these diseases were also supported by the study.