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T Mizuochi

Publications and source records attributed to T Mizuochi.

At least 37 records · Page 2Linked to original sources
PubMed2000

Autoantibody activity of IgG rheumatoid factor increases with decreasing levels of galactosylation and sialylation.

The occurrence of N-linked oligosaccharides lacking galactose is significantly higher than normal in serum IgG of patients with rheumatoid arthritis (RA) in whom rheumatoid factor (RF), an autoantibody against autologous IgG, has been detected. In the present study, IgGs with and without RF activity (IgGRF and non-RF IgG, respectively) were prepared from sera of RA patients, and their oligosaccharide structures were characterized in order to investigate the relationship between RF activity and glycosylation. Three IgGRF fractions and a non-RF IgG fraction were obtained based on their ability to bind to an IgG-Sepharose column. The specific RF activity, as measured by immunoassays, was highest in the IgGRF fraction, which bound most avidly to the IgG-Sepharose. When the oligosaccharides were released by hydrazinolysis, and analyzed by MALDI-TOF mass spectrometry and HPLC, in combination with sequential exoglycosidase treatment, all the IgG samples were found to contain a series of biantennary complex-type oligosaccharides. The incidence of galactose-free oligosaccharides was significantly higher in both IgGRFs and non-RF IgG from RA patients compared with IgG from healthy individuals. In all IgGRFs, the levels of sialylation and galactosylation were lower than those in non-RF IgG from RA patients; the sialylation of non-RF IgG was the same as that of IgG from healthy individuals. In addition, the decreases in galactosylation and sialylation of oligosaccharides in IgGRF correlated well with the increase in RF activity. These findings could contribute to our understanding of the mechanisms of IgG-IgG complex formation and the pathogenicity of these complexes in RA patients.

Arthritis, Rheumatoid↗
PubMed2000

Kinetic analysis of interaction of different types of rheumatoid factors with immobilized IgG using surface plasmon resonance.

Rheumatoid factors (RFs) are autoantibodies, which recognize antigens on a constant region of immunoglobulin G (IgG). Among various RF classes, RF of the IgG class (IgGRF) forms immune complexes in rheumatoid joints and is implicated in the pathogenesis of rheumatoid arthritis (RA). To characterize the formation of IgGRF immune complexes, in the present study, IgGRF was isolated from sera of RA patients, and its interaction with immobilized IgG was analyzed and compared to that of IgMRF or IgARF by means of surface plasmon resonance. On gel filtration, the IgGRF was eluted as a single peak corresponding to IgG, excluding the possible formation of self-associating IgGRF complexes in solution. Sensorgrams of the interaction of IgGRF with immobilized IgG revealed that it clearly bound to the IgG at 6 degrees C, but not at 30 degrees C. The degree of interaction decreased inversely with an increase in temperature, suggesting that IgGRF is much more reactive at lower temperatures. In contrast, the interaction of IgARF and IgMRF with IgG at 6 degrees C was similar to that at 30 degrees C. The association rate constant (k(a)) of IgGRF decreased with an increase in temperature, while those of IgARF and IgMRF were similar under various thermal conditions. The dissociation rate constant (k(d)) of IgGRF was greatly reduced at 25 degrees C, but those of IgARF and IgMRF slightly increased with an increase in temperature. These results suggested that the mode of interaction of IgGRF with IgG differed from in the cases of IgMRF and IgARF. The kinetic properties of the IgGRF-IgG interaction may facilitate elucidation of the IgGRF immune complex formation in rheumatoid joints.

Immunoglobulin G↗
PubMed2000

Genotyping of Pneumocystis carinii f. sp. hominis isolates in Japan based on nucleotide sequence variations in internal transcribed spacer regions of rRNA genes.

Genotyping of Pneumocystis carinii (Pc) isolated from 24 bronchoalveolar lavage (BAL) fluid specimens in Japan was examined based on nucleotide sequence variations in internal transcribed spacer regions 1 and 2 (ITS1 and ITS2, respectively) of rRNA genes. We found 11 ITS1 genotypes including 2 novel ones and 11 ITS2 genotypes including 3 new ones. Combining the ITS1 and ITS2 genotypes resulted in 30 ITS genotypes, of which 10 are newly described in this report. Two or more genotypes in ITS regions in a specimen were observed in 16 of 24 patients. Our results will be of help for the epidemiological investigation of Pc infection.

Adult↗
PubMed1999

Role of macrophage lysosomal enzymes in the degradation of nucleosomes of apoptotic cells.

Although apoptotic cells are recognized and engulfed by macrophages via a number of membrane receptors, little is known about the fate of apoptotic cells after the engulfment. We observed in this study that nucleosomal DNA fragments of apoptotic cells disappeared when they were engulfed by the macrophage cell line J774.1 at 37 degrees C. Pretreatment of J774.1 cells with chloroquine inhibited intensive DNA degradation, indicating that the cleavage of nucleosomal DNA fragments of apoptotic cells may take place in the lysosomes of J774. 1. When apoptotic cells were exposed to a lysosome-rich fraction derived from J774.1 cells under an acidic condition, nucleosomal DNA fragments of apoptotic cells were no longer detectable by agarose gel electrophoresis. Additionally, we found that the lysosome-rich fraction of J774.1 cells contained an acid DNase that is similar to DNase II with respect to its m.w., optimal pH, and sensitivity to the inhibitors of DNase II. By exposure of apoptotic cells to the lysosomal-rich fraction, nucleosomal core histones of apoptotic cells were hydrolyzed along with degradation of nucleosomal DNA fragments. Addition of pepstatin A to the reaction buffer resulted in accumulation of approximately 180-bp DNA fragments and inhibition of hydrolysis of nucleosomal core histones. Leupeptin or CA-074 partially inhibited the degradation of nucleosomal DNA fragments and core histones. These findings suggest that lysosomal enzymes of macrophages, e.g., DNase II-like acid DNase and cathepsins, are responsible for the degradation of nucleosomes of apoptotic cells.

Animals↗
PubMed1998

Liposome oligomannose-coated with neoglycolipid, a new candidate for a safe adjuvant for induction of CD8+ cytotoxic T lymphocytes.

The cytotoxic T lymphocyte (CTL) response has recently been shown to play a role in protection against human immunodeficiency virus (HIV) and it is therefore thought that a vaccine against HIV must be able to elicit a CTL response. The development of a safe, effective adjuvant is very important because alum, the only adjuvant available for use in humans at present, can barely induce a response of this type. We demonstrate here that liposomes that contain an immunodominant peptide (15 amino acids) of the envelope glycoprotein gp120 of HIV-1 and that are coated with mannopentaose-dipalmitoylphosphatidylethanolamine conjugate induce a major histocompatibility complex class I-restricted CD8+ CTL response in mice with a single subcutaneous immunization, whereas non-coated liposomes do not. Since no damage to the skin at the injection site was caused by the liposomes, and since the oligomannose-coated liposomes consist of innocuous materials ubiquitously distributed throughout the human body, they may be highly suitable for use as a safe adjuvant in vaccines inducing a CTL response against HIV.

Adjuvants, Immunologic↗
PubMed1998

The antigen presentation pathway in medullary thymic epithelial cells, but not that in cortical thymic epithelial cells, conforms to the endocytic pathway.

Murine medullary thymic epithelial cells (mTEC), but not cortical thymic epithelial cells (cTEC), are able to present a soluble antigen, ovalbumin, to helper T cells (Mizuochi, T. et al., J. Exp. Med. 1992. 175: 1601-1605). This functional difference between the mTEC and the cTEC is particularly important when we consider the thymic selection of the T cell repertoire. In the previous report, we proposed that mTEC and cTEC utilize two distinct antigen processing/presenting pathways (Kasai, M. et al., Eur. J. Immunol. 1996. 26: 2101-2107). In this report, we further confirmed this difference by analyzing (a) localization of MHC class II, H2-DM, and invariant chain (li) molecules, (b) the biochemical nature of MHC class II molecules, (c) the sensitivity of MHC class II alphabeta heterodimer formation to concanamycin A, a vacuolar H+-ATPase inhibitor, and (d) the subcellular distribution of MHC class II, H2-DM, and li molecules, in both TEC. Our results demonstrated that, in the mTEC, MHC class II, H2-DM and li molecules gain access to the endocytic pathway, where the luminal condition is acidic and thus li molecules are efficiently degraded and H2-DM molecules function well. In the cTEC, however, such molecules seemed to gain access to an alternative transport pathway, e.g. a secretory pathway, where the luminal condition is not fully acidic. These two distinct antigen processing pathways may account for the functional difference between mTEC and cTEC.

Amino Acid Sequence↗
PubMed1998

Delayed progression of murine AIDS in C57BL/6 mice pre-immunized with a highly antigenic 10-mer peptide encoded by the murine AIDS defective virus gag p12 gene.

C57BL/6 (B6) mice were immunized with a highly antigenic 10-mer peptide (P12-10), which is encoded by the murine AIDS (MAIDS) defective virus gag p12 gene, emulsified in incomplete Freund's adjuvant (ICFA). One week later, the mice were inoculated with the MAIDS virus to see if the immunization affects progression of MAIDS. It was demonstrated that the immunization significantly delayed progression of MAIDS, although it failed to induce appreciable cytotoxic T lymphocyte (CTL) responses against the P12-10 antigen. In contrast, immunization of B6 mice with the P12-10 coupled with liposome induced substantial CTL responses but failed to protect the mice against MAIDS development. This segregation between CTL activity and in vivo protection efficacy might be worth considering when we exploit vaccines for augmenting cellular immunity mediated by CD8+ T cells.

Animals↗
PubMed1998

Structural changes in the oligosaccharide moiety of human IgG with aging.

In order to elucidate the relationship between glycosylation of IgG and aging, oligosaccharide structures of human IgG purified from sera of men and women aged 18 to 73 years were investigated. Oligosaccharides were liberated quantitatively from IgG by hydrazinolysis followed by N-acetylation and were tagged with p-aminobenzoic acid ethyl ester. The oligosaccharide structures were then analyzed by HPLC in conjunction with sequential exoglycosidase digestion. All IgG samples were shown to contain a series of biantennary complex type oligosaccharides which consisted of +/-Galbeta1-4GlcNAcbeta1-2Manalpha1-6(+/-GlcNAcbeta 1-4)(+/-Galbeta1-4GlcNAcbeta1-2Man(alpha)1-3)Man(beta)1-+ ++4GlcNAcbeta1-4(+/- Fucalpha1-6)GlcNAc and their mono- and disialo glycoforms in different ratios. In female IgG samples only, the incidence of non-galactosylated oligosaccharides with non-reducing terminal GlcNAc residues increased with aging (r>0.8), whereas that of digalactosylated oligosaccharides decreased (r<-0.8). A weaker correlation was observed between aging and the incidence of neutral and monosialo oligosaccharides in female IgG (r=0.461 and r= -0.538, respectively) and between aging and the incidence of oligosaccharides with a bisecting GlcNAc in both male and female IgG samples (r=0.566 and r=0.440, respectively). In addition, a significant change with aging in the galactosylation of IgG oligosaccharides was observed in females in their thirties, fifties, and sixties (p<0.02, p<0.01, and p<0.04, respectively). These findings may contribute to our understanding of autoimmune diseases such as rheumatoid arthritis in which glycosylation is involved.

Adolescent↗
PubMed1998

Detection of disease-specific augmentation of abnormal immunoglobulin G in sera of patients with rheumatoid arthritis.

Galactose-free immunoglobulin G (IgG), which is known to be higher in the sera of patients with rheumatoid arthritis, was prepared from IgG of healthy volunteers using enzymes. Its reactivity to lectins was analyzed. The galactose-free IgG showed no reactivity to Ricinus communis agglutinin 120 but displayed greater reactivity to concanavalin A and Lens culinaris lectin than did intact human IgG. Then, IgG in serum samples was bound to protein A immobilized on a nitrocellulose membrane, and its reactivity to biotinylated concanavalin A was measured with streptavidin-conjugated horseradish peroxidase. When the reactivity to concanavalin A of IgG in sera from healthy individuals and patients with rheumatoid arthritis (RA), osteoarthritis, systemic lupus erythematosus, or hepatic disease was compared, higher levels were shown in patients with RA, notably in 60% of the seronegative patients and 80% of the early phase patients. Therefore, it was suggested that augmentation of the abnormal IgG in sera was highly specific to patients with RA and that this novel serum test could be very useful for an accurate diagnosis of this disease.

Arthritis, Rheumatoid↗
PubMed1998

Heterosexual transmission of a murine AIDS virus.

Heterosexual transmission of a murine leukemia virus mixture named LP-BM5 MuLV, which is known as the murine AIDS virus, was investigated. Our results indicated that the heterosexual transmission of LP-BM5 MuLV occurs in both directions with high frequency and that the frequencies of virus transmission in the cervix and penis are higher than those in other genital organs. The results suggested that infection by LP-BM5 MuLV via heterosexual transmission may initially take place at particular retrovirus-sensitive sites (cells) in the genital organs.

Animals↗
PubMed1998

Binding specificities of lectins to immobilized glycoproteins and oligosaccharides differ from those of immobilized lectins to oligosaccharides.

The carbohydrate-binding specificities of lectins in solution to glycoproteins and neoglycolipids immobilized on a solid phase were analyzed in order to establish a simple, rapid method for structural analysis of the carbohydrate moieties of small amounts of individual glycoproteins blotted on membrane. Eight glycoproteins containing typical O-linked tetrasaccharides or a series of typical N-linked oligosaccharides of the high-man-nose type, hybrid type, and complex type and 6 neoglycoproteins containing mono- or di-saccharides were dot blotted on membranes and the membranes were then reacted with 8 kinds of horseradish peroxidase-conjugated lectins before and after heat treatment. Neoglycolipids containing the glycoprotein-derived oligosaccharides immobilized on a thin layer chromatography plate were also reacted with lectins. The heat treatment of the membrane increased lectin reactivity toward the glycoproteins. The carbohydrate-binding behavior of lectins, Phaseolus vulgaris erythroagglutinin, wheat germ agglutinin, and concanavalin A in solution toward glycoproteins and neoglycolipids immobilized on a solid phase differed from that of immobilized lectins toward oligosaccharides in solution. This difference should be noted in lectin detection of specific carbohydrates of individual glycoproteins on membrane.

Glycoproteins↗
PubMed1997

Accumulation of somatic hypermutation and antigen-driven selection in rapidly cycling surface Ig+ germinal center (GC) B cells which occupy GC at a high frequency during the primary anti-hapten response in mice.

Well-developed germinal centers (GC) contain rapidly dividing surface immunoglobulin-negative (sIg-) B cells (centroblasts), and most of their progeny are sIg+ B cells (centrocytes) in a resting state. It has been predicted that somatic hypermutation occurs in centroblasts, whereas antigen-driven selection takes place in centrocytes. The present analysis indicates that murine GC B cells bearing sIg with specificity for an immunizing antigen are in a rapidly cycling state and increase exponentially in number to occupy spleen GC at high frequency during the 1st week after primary immunization; however, the number of these cells is significantly reduced in the 2nd week of immunization. During that period, these proliferating sIg+ GC B cells accumulate somatic hypermutations with nucleotide exchanges indicative of affinity maturation. These sIg+ GC B cells co-express B7-2, ICAM-1, and LFA-1, and have potent antigen-presenting activity which results in T cell activation in vitro. These observations indicate that the sIg+ GC B cells accumulate somatic hypermutations and undergo antigen-driven selection through proliferation, probably upon activation by T cells. This sIg+ GC B cell population may represent cell cycling centrocytes; however, the possibility that these may represent centroblasts undergoing re-expression of sIg could not be excluded.

Amino Acid Sequence↗
PubMed1997

Specific immunosuppression of corneal allograft rejection by combination of anti-VLA-4 and anti-LFA-1 monoclonal antibodies in mice.

It has been reported that allograft rejection is mediated by a variety of adhesion molecules. Using a corneal allograft model in mice, we studied the role of very late antigen (VLA)-4 and leukocyte function-associated antigen (LFA)-1 adhesion molecules in corneal allograft rejection and the effects of monoclonal antibodies (mAbs) to them in suppressing corneal rejection. C3H/He donor corneas were transplanted into BALB/c corneal beds. The allografted mice were treated with a control mAb (M18/2), mAbs to VLA-4, or LFA-1 or their combination by i.p. injection until day 7. The expression of VLA-4, LFA-1, major histocompatibility complex (MHC) class II antigens, interleukin (IL)-2, IL-2 receptor and interferon gamma (IFNgamma) in the grafted cornea were studied immunohistochemically. Cytotoxic T lymphocyte (CTL) responses to donor alloantigens were assessed. The skins from a syngeneic donor or a third-part strain were transplanted 8 weeks after the initial keratoplasty onto the mice treated with anti-LFA-1 plus anti-VLA-4 mAbs. Fourteen of 16 allografts in non-treated mice and control mAb-treated mice became opaque by 2 weeks after transplantation. At 2 weeks, non-treated allografts showed expression of MHC class II antigens on keratocytes and mononuclear cells at the host-graft junction. Also, mononuclear cells expressing VLA-4, LFA-1, IL-2, IL-2 receptor and IFNgammawere present in the stroma at the host-graft junction. The allografts treated with either anti-VLA-4 or anti-LFA-1 alone, or anti-VLA-4 plus anti-LFA-1 remained transparent for more than 2 weeks, and the survival rates at 14 weeks was 0%, 16.7%, and 75.0%, respectively. The combined use of anti-VLA-4 and anti-LFA-1 mAbs prolonged graft survival significantly (P<0.05) at 14 weeks as compared with anti-LFA-1 mAb alone. At 3 weeks, CTL responses to donor alloantigens were depressed in mice treated with either anti-LFA-1 alone or anti-LFA-1 plus anti-VLA-4. Specific prolongation of donor-syngeneic skin was observed after treatment with the combination of these two mAbs. These results indicate that VLA-4 and LFA-1 have important roles in rejection process of corneal allografts, and that the combined use of mAbs to these molecules has remarkable effects on inducing alloantigen-specific immunosuppression in corneal transplantation.

Animals↗
PubMed1997

A polypeptide encoded within the murine AIDS defective virus stimulates primary proliferation of CD8+ T-cells.

The murine AIDS (MAIDS) is a retrovirus-induced disease that shows severe immunodeficiency with abnormal lymphoproliferation in susceptible strains of mice. To clarify the antigenicity of gag gene products of the LP-BM5 defective virus, which is known as the causative virus of MAIDS, we expressed and purified the gag p12 gene product (P12) by using a baculovirus expression vector system. The P12 protein strongly stimulated the proliferation of normal C57BL/6 (B6) lymph node T-cells in vitro. Furthermore, a 25-mer synthetic polypeptide within the P12 sequence gave rise to the similar or even higher activation of T-cells. The phenotype of responding T-cells was found to be CD8+ CD44low, indicating that naive CD8+ T-cells respond against a peptide encoded within a MAIDS defective virus gag p12 gene. Finally, the expression of T-cell receptor (TcR) V beta on the responding CD8+ T-cells was analyzed. Although CD8+ T-cells with the particular V beta chains were expanded in response to the 25-mer peptide, this polypeptide does not seem to be a superantigen, since this response is MHC class I-restricted and the V beta preference is not striking. The presentation pathway of this highly antigenic polypeptide will be discussed.

Amino Acid Sequence↗
PubMed1997

Alteration of N-linked oligosaccharide structures of human chorionic gonadotropin beta-subunit by disruption of disulfide bonds.

The human chorionic gonadotropin beta-subunit (hCGbeta) is a glycoprotein in which 12 cysteine residues pair to form six intramolecular disulfide bonds. In order to elucidate the effect of each disulfide bond on glycosylation of the molecule, we analysed structures of asparagine-linked oligosaccharides of various recombinant hCGbeta produced in Chinese hamster ovary (CHO) cells: wild-type hCGbeta (betaWT) and mutants in which any one of the six intramolecular disulfide bonds had been disrupted by site-directed mutagenesis. SDS-PAGE analysis of betaWT and these mutants before and after digestion with endoglycosidase F and H revealed structural changes in the oligosaccharide moieties of some mutants. In addition, structural analysis of oligosaccharides obtained from metabolically labeled betaWT and a mutant showed that the mutant contained additional high mannose type oligosaccharides. These results suggest that elimination of a specific disulfide bond, resulting in a change in the protein conformation, disturbs the normal assembly of the mature complex type oligosaccharides in the hCGbeta molecule.

Acetylglucosamine↗
PubMed1997

MHC class I presentation of an exogenous polypeptide antigen encoded by the murine AIDS defective virus.

Peptides derived from endogenous proteins are presented by MHC class I molecules, whereas those derived from exogenous proteins are presented by MHC class II molecules. This strict segregation has been reconsidered in recent reports in which exogenous antigens are shown to be presented by MHC class I molecules in the phagocytic pathway. In this report, the presentation pathway of an exogenously added highly antigenic polypeptide encoded by the murine AIDS (MAIDS) defective virus gag p12 gene is investigated. A 25-mer polypeptide (P12-25) encoded within the gag p12 region of the MAIDS defective virus was found to be effective in stimulating unprimed B6 (H-2b) CD8+ T cells in vitro. The presentation of P12-25 is sensitive to cytochalasin B and D, brefeldin A and gelonin, a ribosome-inactivating protein synthesis inhibitor, but less sensitive or resistant to lactacystin, a highly specific inhibitor of the proteasome. Interestingly, CA-074, a selective inhibitor of cathepsin B, inhibited presentation of the polypeptide, indicating its involvement in the degradation of the P12-25 polypeptide. In fact, when P12-25 was digested with purified cathepsin B in vitro, a highly antigenic 11-mer peptide containing the class I (H-2Db)-binding motif was obtained. Our results favor the phagosome/macropinosome-to-cytosol-to-endoplasmic reticulum (ER)-to-cell surface pathway for exogenous antigens presented by MHC class I molecules. These findings may be relevant to exploiting peptide vaccines that specifically elicit CD8+ T cell immunity in vivo.

Acetylcysteine↗
PubMed1997

Induction of intestinal lesions in nu/nu mice induced by transfer of lymphocytes from syngeneic mice infected with murine retrovirus.

BACKGROUND: Murine leukemia virus, LP-BM5, induces severe immunodeficiency with abnormal lymphoproliferation in susceptible C57BL/6 mice. In a previous study, it was shown that a Sjögren's syndrome-like systemic exocrinopathy is induced in the virus infected mice. AIMS: To examine lymphocyte functions of the virus infected mice. METHODS: Four-week old mice were inoculated with the virus and their spleen cells were transferred into syngeneic nu/nu mice. Their organs were examined by light and electron microscopy. Phenotypes of the colon infiltrating cells were examined by flow cytometry. RESULTS: All nu/nu recipients had died by six weeks after cell transfer, showing runting disease like cachexia with diarrhoea and anal bleeding. Histopathological examination revealed that systemic exocrinopathy was adoptively transferable and that the colon became thickened due to mononuclear cell infiltration into the mucosal and submucosal layer with hyperplasia of intestinal epithelial cells. No virus particles were found in the colon. Flow cytometric analyses revealed that most of the infiltrating CD4+ T cells showed CD45RBlow. No intestinal lesions were observed in the virus infected mice nor in nu/nu mice inoculated with normal lymphocytes. CONCLUSION: Lymphocytes of the virus infected mice induced colitis and hyperplasia of intestinal epithelial cells as well as systemic exocrinopathy in nu/nu mice. Our experimental system may give some insight into intestinal lesions associated with virus infection.

Animals↗