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S Aotsuka

Publications and source records attributed to S Aotsuka.

At least 19 recordsLinked to original sources
PubMed2005

Production and characterization of recombinant human anti-HBs Fab antibodies.

Recombinant human Fab antibodies were generated with different reactivities against the hepatitis B virus surface (HBs) antigen. To isolate the antibodies, a method was used that combined transformation of human B cells by Epstein-Barr virus (EBV) infection with a primer-vector system developed for isolating DNA fragments of human Ig Fab portions. With this method, monoclonal and oligoclonal cell lines producing anti-HBs antibodies were established and three anti-HBs Fab antibodies were isolated from two of these cell lines. From analysis of affinity characteristics, immunohistochemical activity, and cytolysis activity, these three Fab antibodies were classified into three different groups. The first group had high affinity for HBs, the second had the ability to kill HBV-infected cells, and the third was applicable to immunohistochemical staining with HBV-infected cells. The combined effect of these antibodies was also investigated by complement-dependent cytotoxicity assay.

Antibodies, Monoclonal↗
PubMed2005

Elevated immunoglobulin G antibodies to the proline-rich amino-terminal region of Epstein-Barr virus nuclear antigen-2 in sera from patients with systemic connective tissue diseases and from a subgroup of Sjögren's syndrome patients with pulmonary involvements.

Associations of Epstein-Barr virus (EBV) and autoimmune diseases have been hypothesized. We have analysed IgG antibodies to EBV nuclear antigen (EBNA)-2 in sera from Japanese patients with autoimmune systemic connective tissue diseases (CTD), exemplified by systemic lupus erythematosus (SLE), primary Sjogren's syndrome (SS), rheumatoid arthritis (RA), systemic sclerosis (SSc) and secondary SS (classical CTDs complicated with SS). An enzyme-linked immunosorbent assay (ELISA) which uses glutathione-S-transferase polypeptides fused to EBV nuclear antigen (EBNA)-2 and EBNA-1 was developed. Ratios of IgG antibody reactivity to whole IgG concentrations of sera were calculated to normalize EBNA-2 and EBNA-1 antibody levels to the hypergammaglobulinaemia that occurs in CTD. The ELISA optical density OD(450) readings of IgG antibodies to both the amino-terminal aa 1-116 of EBNA-2 and carboxyl-terminal aa 451-641 of EBNA-1 were elevated significantly in patients with SLE, primary SS, RA, SSc and secondary SS when compared to EBNA-1. The OD readings were divided by serum IgG concentrations to normalize for the hypergammaglobulinaemia. The specific levels of IgG antibodies to the amino-terminal region of EBNA-2 were elevated in patients with SLE, primary SS or RA, as well as those with secondary SS complicated with SLE or RA. The EBNA-2 amino-terminal region contains a polyproline tract and a proline-rich sequence and has considerable amino acid sequence homology with many cellular proline-rich proteins. High ratios of EBNA-2 aa 1-116 to EBNA-1 aa 451-641 IgG antibody levels which probably suggest reactivation of EBV latent infection were associated significantly with pulmonary involvement in SS patients. These results are consistent with the hypothesis that the sequence similarity between the amino-terminal region of EBNA-2 and proline-rich cellular proteins is associated with pathogenesis in a subpopulation of CTD patients, possibly by the molecular mimicry-epitope shift mechanism.

Adult↗
PubMed2002

Effects of intravenous anesthetics on interleukin (IL)-6 and IL-10 production by lipopolysaccharide-stimulated mononuclear cells from healthy volunteers.

BACKGROUND: Surgical trauma has been shown to augment the plasma concentrations of proinflammatory cytokines, which are important mediators of host defense mechanisms and the systemic inflammatory response syndrome (SIRS). Recently, it has been shown that certain kinds of surgery provoke not only a proinflammatory response (SIRS) but also a concurrent anti-inflammatory response. The aim of this study was therefore to examine the effects of intravenous anesthetics on the synthesis of interleukin (IL)-6 (a proinflammatory cytokine) and IL-10 (an anti-inflammatory cytokine) by lipopolysaccharide (LPS)-stimulated mononuclear cells from healthy volunteers. METHODS: Peripheral blood mononuclear cells (PBMCs) from 17 healthy volunteers, separated by centrifugation on a Ficoll-Hypaque gradient, were washed and suspended in RPMI containing 10% heat-inactivated fetal calf serum (FCS). After adding RPMI-FCS containing various concentrations of intravenous anesthetics (propofol, thiopental, ketamine and midazolam), the PBMCs were incubated overnight in the presence of a submaximal concentration of LPS. The supernatants were collected and their IL-6 and IL-10 contents were assayed using enzyme-linked immunosorbent assay kits. RESULTS: Propofol inhibited both IL-6 and IL-10 production at 0.5 microg/mL, 5 microg/mL and 50 microg/mL. Conversely, thiopental induced IL-10 production at 2 microg/mL and 20 microg/mL. CONCLUSION: Propofol appears to inhibit both IL-6 and IL-10 production by LPS-stimulated PBMCs in vitro. Further study is required to clarify the mechanism of the suppressive effect of propofol.

Adult↗
PubMed2001

Endothelial cell-binding activity of anti-U1-ribonucleoprotein antibodies in patients with connective tissue diseases.

In order to elucidate the immunological properties of anti-U1-ribonucleoprotein (RNP) antibody, one of the autoantibodies detected in patients with connective tissue diseases (CTDs), we tested the endothelial cell-binding by anti-U1-RNP antibodies and epitopes on human pulmonary artery endothelial cells (HPAECs) to which the autoantibody bound. IgG fractions positive for anti-U1-RNP from patients with CTDs bound to the HPAECs. Furthermore, intact and F(ab')2 IgG anti-U1-RNP purified by affinity chromatography also bound to endothelial cells. The binding activity of IgG fractions positive for anti-U1-RNP to the endothelial cells could be effectively absorbed by U1-RNP-Sepharose. An immunoblotting assay of purified IgG anti-U1-RNP antibodies showed that these antibodies could bind to various membrane proteins of NP40-treated HPAECs such as 68, 48, 43, 38, 33, 29, 28 and 24 kDa. Some bands, 68, 33, 28 and 24 kDa, seemed to correspond to components of U1-RNP, i.e. 68 kDa, A, B' and C peptides, respectively. We confirmed that the anti-U1-RNP antibody from patients with CTDs can directly recognize a variety of antigens on the endothelial surface of the pulmonary artery, including the components of U1-RNP or other unknown polypeptides. These results suggest that binding to pulmonary artery endothelial cells of this autoantibody may be one of the triggers of endothelial cell inflammation in CTDs.

Animals↗
PubMed2001

Cloning and expression of human anti-tumor necrosis factor-alpha monoclonal antibodies from Epstein-Barr virus transformed oligoclonal libraries.

Peripheral blood was obtained from a healthy human volunteer and transformed with Epstein-Barr virus (EBV). This produced an oligoclonal cell library in culture medium that was screened by ELISA for anti-human tumor necrosis factor-alpha (TNFalpha) activity. RNA from two positive clones was applied to RT-PCR using antibody-specific primers, and the light (kappa and lambda) and heavy chain genes (gamma and mu) were cloned into the plasmid vector pFab1-His2. The antibodies produced in Escherichia coli as Fab fragments were assayed for anti-TNFalpha activity utilizing ELISA. Two IgG1/kappa anti-TNFalpha antibodies and two IgM/kappa anti-TNFalpha antibodies were isolated. DNA sequence analysis showed that the VL and VH gene families of IgM and IgG were the same. Both the antibodies showed almost the same activity on ELISA-testing. Ten clones randomly selected from light (kappa and lambda) and heavy (gamma and mu) chain genes in the oligoclonal cell library 1D5 were sequenced, and each gene (kappa, lambda, gamma, and mu) was found to be composed of one to three different genes. These data support the conclusion that the cell clone is oligoclonal at the molecular level.

Amino Acid Sequence↗
PubMed1999

Clinical and laboratory features of lupus patients with complicating pulmonary disease.

The objective of this study was to determine the incidence of pulmonary involvement in patients with systemic lupus erythematosus (SLE) and to clarify the clinical and laboratory characteristics in SLE patients with various pulmonary involvements. A retrospective study (n = 137) revealed that the types of pulmonary involvement found in SLE patients were: pleuritis (9%), interstitial pneumonia (8%), pulmonary infarction (7%), pulmonary infection (4%), pulmonary hypertension (2%), restrictive dysfunction (28%) and decreased diffusion capacity (43%). The incidences of pericarditis (P < 0.01), arthralgia (P < 0.05) and hypoalbuminemia (P < 0.05) were significantly greater in patients with pleuritis than in those without, while in patients with interstitial pneumonia, the incidence of anti-SS-A antibody (P < 0.05) and sicca syndrome (P < 0.05) were significantly greater than in those without. A longitudinal follow-up study of patient groups with various pulmonary involvements revealed: 1. significant changes of erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), lactate dehydrogenase (LDH) and thrombomodulin (TM) in patients with pleuritis, and 2. significant changes of WBC and LDH in patients with interstitial pneumonia. The increased ESR, CRP and TM levels during disease episodes suggest that the involvement of inflammatory processes is related to vasculitic events in the pathogenesis of lupus pleuritis. A higher incidence of anti-SS-A antibody in lupus patients with interstitial pneumonia suggests a potential role for this autoantibody in the pathogenesis of this complication.

Adolescent↗
PubMed1999

Up-regulation of intercellular adhesion molecule-1 (ICAM-1), endothelial leucocyte adhesion molecule-1 (ELAM-1) and class II MHC molecules on pulmonary artery endothelial cells by antibodies against U1-ribonucleoprotein.

In order to elucidate the pathogenic role(s) of autoantibodies in connective tissue disease (CTD), we examined whether autoantibodies against U1-ribonucleoprotein (RNP) and double-stranded (ds) DNA can up-regulate ICAM-1, ELAM-1 and class I and II MHC molecule expression on pulmonary artery endothelial cells (HPAEC). ICAM-1, ELAM-1 and class II MHC molecule expression on HPAEC cultured in the presence of anti-U1-RNP-containing and anti-dsDNA-containing IgG from CTD patients was up-regulated significantly in comparison with that on HPAEC cultured with IgG from normal healthy volunteers. Affinity chromatographic enrichment and depletion of the anti-U1-RNP antibody content of anti-U1-RNP-containing IgG confirmed that the anti-U1-RNP antibody did up-regulate ICAM-1, ELAM-1 and class II MHC molecule expression. The finding that an IgG F(ab')2-purified anti-U1-RNP antibody also up-regulated expression of these molecules may indicate that mechanisms other than Fc receptor-mediated stimulation are involved. These in vitro findings suggest that autoantibodies against U1-RNP and dsDNA play important roles in the immunopathological processes leading to the proliferative pulmonary arterial vasculopathy observed in CTD patients with pulmonary hypertension by up-regulating adhesion and class II MHC molecule expression on endothelial cells.

Adult↗
PubMed1999

Preparation of recombinant human monoclonal antibody Fab fragments specific for Entamoeba histolytica.

Genes coding for human antibody Fab fragments specific for Entamoeba histolytica were cloned and expressed in Escherichia coli. Lymphocytes were separated from the peripheral blood of a patient with an amebic liver abscess. Poly(A)+ RNA was isolated from the lymphocytes, and then genes coding for the light chain and Fd region of the heavy chain were amplified by a reverse transcriptase PCR. The amplified DNA fragments were ligated with a plasmid vector and were introduced into Escherichia coli. Three thousand colonies were screened for the production of antibodies to E. histolytica HM-1:IMSS by an indirect fluorescence-antibody (IFA) test. Lysates from five Escherichia coli clones were positive. Analysis of the DNA sequences of the five clones showed that three of the five heavy-chain sequences and four of the five light-chain sequences differed from each other. When the reactivities of the Escherichia coli lysates to nine reference strains of E. histolytica were examined by the IFA test, three Fab fragments with different DNA sequences were found to react with all nine strains and another Fab fragment was found to react with seven strains. None of the four human monoclonal antibody Fab fragments reacted with Entamoeba dispar reference strains or with other enteric protozoan parasites. These results indicate that the bacterial expression system reported here is effective for the production of human monoclonal antibodies specific for E. histolytica. The recombinant human monoclonal antibody Fab fragments may be applicable for distinguishing E. histolytica from E. dispar and for use in the serodiagnosis of amebiasis.

Amino Acid Sequence↗
PubMed1999

Bacterial expression of a neutralizing mouse monoclonal antibody Fab fragment to a 150-kilodalton surface antigen of Entamoeba histolytica.

A mouse monoclonal antibody (MAb) (EH3015, IgG1 with a K light chain) prepared by hybridoma technology recognizes a 150-kD surface antigen of Entamoeba histolytica and inhibits adherence and cytotoxicity of the ameba to mammalian cells. The genes encoding the light chain and the Fd region of the heavy chain of the MAb were cloned and expressed in Escherichia coli. The plasmid used was designed for the expression of Fab with a hexa-histidine tag in the periplasmic space. Recombinant Fab fragments were purified and analyzed by an indirect immunofluorescence antibody test and Western immunoblot. The specificity of the recombinant Fab fragment was comparable with the parent whole IgG. In addition, the Fab fragments significantly inhibited the adherence of E. histolytica to erythrocytes. These results suggest that the production of a neutralizing MAb in Escherichia coli is practical and efficient with this expression system.

Amino Acid Sequence↗
PubMed1999

Bacterial expression of a human recombinant monoclonal antibody fab fragment against hepatitis B surface antigen.

The Fab fragment was cloned from the monoclonal cell line TAPC301-CL4, which was produced using the Epstein-Barr virus (EBV) transformation method. This cell line produces a human monoclonal antibody (CL4MAb) against the hepatitis B surface antigen (HBsAg). This MAb was shown to have hepatitis B virus (HBV) neutralizing activity in chimpanzees. The Fab fragment was produced by subjecting the heavy and light chain antibody genes of the TAPC301-CL4 cell line to reverse transcription-polymerase chain reaction, cloning the products in the plasmid vector pFab1-His2 and introducing the plasmid into bacteria. Sequence analyses of the CL4Fab fragment revealed that the light and heavy chains belong to the Vk3a and VH3 groups of the immunoglobulin (Ig) family, respectively. An enzyme-linked immunosorbent assay confirmed that specificity of the recombinant CL4Fab antibody against HBsAg was the same as that of the parental MAb. Flow cytometric analysis using PLC/PRF/5 (Alexander) cells, which express HBsAg, showed the reactivities of the CL4MAb and CL4Fab antibody were the same. These results suggest that the recombinant CL4Fab antibody produced by Escherichia coli using the new vector-primer system developed for human IgG Fab fragments has a very high affinity for the HBsAg and may be useful clinically. A source for generation of human MAb for human therapy with very stable and specific expression was thus produced by isolating antibodies from EBV-transformed cell lines.

Amino Acid Sequence↗
PubMed1999

Immunological analysis of pulmonary hypertension in connective tissue diseases.

OBJECTIVE: To analyze the immunological characteristics of the sera of patients with inflammatory connective tissue diseases complicated by pulmonary hypertension (PH). METHODS: Sera of 24 patients with mixed connective tissue disease complicated by PH (MCTD-PH), sera of 11 patients with other connective tissue diseases complicated by PH (Other-PH; 6 systemic sclerosis, 3 systemic lupus erythematosus, 2 rheumatoid arthritis), and sera of 15 patients with MCTD not complicated by PH (MCTD-non-PH) were tested for IgG antibodies against U1RNP proteins, U1RNP-70K protein, U1RNP-A protein, and U1RNP-C protein, and for IgG and IgM antibodies to beta2-glycoprotein I dependent cardiolipin (CL) and human umbilical vein endothelial cells. We also measured the serum levels of von Willebrand factor related antigens and interleukin 6 (IL-6). RESULTS: (1) The titers of the anti-U1RNP, anti-U1RNP-70K, anti-U1RNP-A, and anti-U1RNP-C antibodies were significantly higher in the MCTD-PH and MCTD-non-PH groups than in the Other-PH group. However, there were no statistically significant differences in the titers of these 4 antibodies between the MCTD-PH and MCTD-non-PH groups. (2) The titers of the IgG aCL and the serum IL-6 levels were significantly higher in the MCTD-PH group than in the MCTD-non-PH group. (3) Statistically significant correlations between the anti-U1RNP and IgG anti-CL antibody titers, and between the IgG anti-endothelial cell and IgG anti-CL antibody titers were observed within the MCTD-PH and Other-PH groups, but not within the MCTD-non-PH group. CONCLUSION: The occurrence of anti-U1RNP, anti-endothelial cell, and anti-CL antibodies is associated with PH in certain patients with connective tissue disease.

Autoantibodies↗
PubMed1997

Changes in levels of mRNAs for cell wall-related enzymes in growing cotton fiber cells.

mRNAs for cell wall-related enzymes in developing cotton fiber cells were measured by reverse transcription-PCR analysis. Both endo-1,4-beta-glucanase and expansin mRNA levels were high during cell elongation but decreased when cell elongation ceased, and xyloglucan decreased. The endo-1,3-beta-glucanase mRNA level was very low in the elongating cells but increased gradually at the onset of secondary wall synthesis, accompanying the massive deposition of cellulose. Endoxyloglucan transferase and sucrose synthase mRNA levels were constant during all stages of growth.

Cell Wall↗
PubMed1997

Histological evaluation of destructive monoarthropathy in mixed connective tissue disease.

The synovium from a patient with mixed connective tissue disease and destructive ankle monoarthritis was studied in detail to determine its immunohistological characteristics. Fibrinoid necrotic tissue on the surface of the synovium, multi-layered lining cells, increased numbers of capillaries, interstitial edema, infiltration of macrophages, relatively small numbers of lympho-plasma cells and polymorphonuclear leukocytes, scattered bone fragments, and multinucleated giant cells were observed. Many cells in the lining and sublining area were positive for CD68 and MAC387. Lower layers of increased lining cells which had a spindle shape were positively stained with anti-HLA-DR antibody. The small arteries in the deeper part of the synovium revealed obstruction or highly stenotic change. These findings suggest that obstructive circulatory disturbance due to endothelial injury might influence the progression of arthritis.

Adult↗
PubMed1997

[Antinuclear antibody screening by an enzyme-linked immunosorbent assay using multiple nuclear antigens].

We assessed the clinical value of an enzyme-linked immunosorbent assay (ELISA) kit (MESACUP ANA, MANA) which uses multiple nuclear antigens for screening antinuclear antibody (ANA). Since the screening procedure for ANA by the indirect fluorescence method (FANA) requires trained personnel and gives rather subjective results, development of a suitable screening test for ANA by ELISA which uses specific nuclear antigens has been long awaited. Serum samples from 350 patients with connective tissue diseases (CTD), and from 300 normal subjects were assayed for ANA by ELISA and the results were compared with those of FANA and of single-antigen ELISAs for RNP, Sm, SS-A, SS-B, Scl-70, Jo-1, dsDNA, and ssDNA. For discrimination between patients with systemic lupus erythematosus and normal subjects, the sensitivity, specificity and accuracy for MANA were 85.7%, 97.5%, and 91.6%, and those for FANA were 95.8%, 77.0%, and 86.4%, respectively. Analysis of CTD sera showing discordant results between FANA and MANA using single-antigen ELISAs revealed that FANA - MANA + sera (n = 8) contained antibodies against SS-A (n = 1) or dsDNA (n = 3) or ssDNA (n = 3), and that some FANA + MANA - sera (n = 82), which were frequently found in RA patients (43/98, 43.9%), also contained antibodies against RNP (n = 3) or ssDNA (n = 13). MANA + normal sera (n = 9) were found to contain antibodies against SS-A (n = 1) or dsDNA (n = 1) or ssDNA (n = 2). In conclusion, MANA was found to be objective, much less labor intensive and suitable for screening large numbers of CTD sera for disease-specific ANAs.

Antibodies, Antinuclear↗
PubMed1996

Prospective study of the clinical value of determining circulating IgA-alpha 1-antitrypsin complex using a prototype ELISA kit in patients with rheumatoid arthritis.

OBJECTIVE: To evaluate the clinical value of determining circulating IgA-alpha 1-antitrypsin (IgA-AT) complex in rheumatoid arthritis. METHODS: The IgA-AT complex was assayed by a prototype ELISA kit using a specific monoclonal antibody against the complex. RESULTS: The median level of serum IgA-AT complex in rheumatoid patients (2.26 AU ml-1) was significantly higher than in osteoarthritis patients (1.37 AU ml-1, P < 0.05) and healthy volunteers (1.03 AU ml-1, P < 0.001). The concentration of IgA-AT complex in rheumatoid arthritis patients at baseline was correlated with the number of painful joints (P < 0.05), number of swollen joints (P < 0.01), erythrocyte sedimentation rate (P < 0.05), and modified Lansbury index (P < 0.01). The median serum level of IgA-AT complex in rheumatoid patients at baseline was higher than that at three months (P < 0.01), six months (P < 0.01), and 12 months (P < 0.01) after the start of treatment. The difference and ratio of IgA-AT complex levels before and after treatment were significantly associated with radiographic progression. CONCLUSIONS: The findings validate the usefulness of determining IgA-AT complex using ELISA in the management of rheumatoid arthritis.

Adult↗