Biomedical subjects
Publications and source records attributed to M Harigai.
Explore the source record for details and available documents.
Vascular damage in systemic sclerosis (SSc) may be a factor in the abnormal regulation of several vasoactive agents. It has been well confirmed that plasma endothelin-1, as a vasoconstrictive factor, is elevated in patients with SSc. However, it is still controversial whether the levels of serum nitric oxide (NO), a strong vasodilator, are increased or decreased in SSc patients compared to healthy donors. In this study, we measured the levels of serum NO metabolites in SSc patients and determined the contribution of the excessive production of NO synthase (NOS)-2 by skin fibroblasts to NO synthesis. Serum NO levels of 45 patients with SSc were significantly higher than those of 20 healthy volunteers. In addition, some clinical features of SSc (the extent of skin fibrosis, short disease duration, and the complication of active fibrosing alveolitis) were all correlated positively with the levels of NO metabolites in SSc patients. To evaluate the levels of NOS-2 produced by skin fibroblasts, skin fibroblast cultures were established from SSc patients and healthy volunteers. Reverse transcription-polymerase chain reaction indicated that NOS-2 mRNA was spontaneously expressed in cultured fibroblasts derived from SSc patients, but not in those derived from healthy normal controls. Immunohistochemical staining also showed that NOS-2 proteins were detected in SSc fibroblasts but not in normal fibroblasts. The production of NO by cultured fibroblasts was visualized directly by a reagent (DAF-2 DA) used for the fluorescent detection of NO. Cultured SSc fibroblasts were capable of NO synthesis in culture media containing L-arginine, whereas normal fibroblasts (with no expression of NOS-2) did not synthesize detectable NO. These observations indicate that NO production is increased markedly in early-stage diffuse cutaneous SSc patients with active fibrosing alveolitis, and that constitutive NOS-2 expression in SSc fibroblasts may contribute to increased NO production.
Recently, we reported that serum concentration of IL-18 is strikingly high in patients with adult-onset Still's disease (AOSD). The aim of the present study was to screen for genetic polymorphisms in the human IL-18 (hIL-18) gene and to determine the association of polymorphisms with susceptibility to AOSD. We investigated the 6.7 kb region upstream of exon 2 of hIL-18 gene, in which a promoter activity had been reported. Sixteen AOSD patients, 144 rheumatoid arthritis (RA) patients and 92 healthy control individuals were studied. We found seven single nucleotide polymorphisms and a single 9 bp insertion which were frequently present in the AOSD patients. Three haplotypes including a unique combination of these polymorphisms were also determined. Of them, haplotype S01 contained all eight of these polymorphisms. The frequency of individuals carrying a diplotype configuration, ie a combination of two haplotypes, of S01/S01 was significantly higher in the AOSD patients than in the healthy controls (P=0.00059, Fischer's exact probability test, odds ratio [OR]=7.81, 95% confidence interval [95% CI]=2.48-24.65) and the RA patients (P=0.015, Fischer's exact probability test, OR=4.0, 95% CI=1.39-11.54). We therefore conclude that possession of the diplotype configuration of S01/S01 is a major genetic risk factor for susceptibility to AOSD.
To specify when and where Ig class switch recombination (CSR) takes place, a good molecular marker closely associated with active CSR is required. CSR is accompanied by deletion of circular DNA from the Ig heavy chain locus. The circular DNA contains a DNA segment between Smu and a target S region including its I promoter, which is driven by specific cytokine stimulation before CSR. We found that the specific I promoter is still active in looped-out circular DNA and directs production of I-Cmu transcripts termed "circle transcripts." Reverse transcription-PCR demonstrated transient induction of specific circle transcripts upon CSR in a murine lymphoma cell line, CH12F3-2A, as well as spleen B cells. Production of the circle transcripts appeared to depend on expression of activation-induced cytidine deaminase (AID), an essential factor for CSR. A comparison of kinetics between circle transcripts and circular DNA showed more rapid disappearance of circle transcripts. Thus, circle transcripts may serve as a hallmark for active CSR in vitro and in vivo.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
The spectroscopic properties of photoactive yellow protein (PYP) partially digested by chymotrypsin were studied. Chymotrypsin yielded three major products that were yellow but distinguishable by SDS-PAGE. They were readily separated by DEAE-Sepharose column chromatography. Protein sequencing and mass spectrometry demonstrated that chymotrypsin cleaved the N-terminal 6, 15, or 23 amino acids (T6, T15, and T23). The blue-shifts of the absorption maxima and the increases in the apparent pK(a) of the chromophores relative to those of intact PYP were less than 4 nm and 0.2, respectively. The absorption spectra of the near-UV intermediates produced from T6, T15, and T23 were identical to that of intact PYP, but with lifetimes that were 140, 2,300, and 4,500 times longer, respectively. These observations suggest that the recovery of the dark state of PYP from the near-UV intermediate is accelerated by the N-terminal region, and that this region acts as a regulatory factor for the photocycle of PYP.
Hemophagocytic syndrome (HPS) is characterized by the activation of the mononuclear phagocytic system with prominent hemophagocytosis in the bone marrow and reticuloendothelial systems, and its occurrence is usually associated with variable disorders such as viral infections and malignant lymphoma. Recently, it was reported that HPS also occurred in association with underlying connective tissue disease, especially systemic lupus erythematosus. We report here a case of recurrent HPS complicated with systemic sclerosis. A 32-year-old woman had been diagnosed as systemic sclerosis since 1994. She was admitted due to unknown high fever and severe pancytopenia in 1997, and the diagnosis of HPS was determined because of hemophagocytosis in bone marrow and hyperferritinemia. Her symptoms were improved by immunosuppressive therapies including steroid pulse therapy and oral prednisolone (60 mg/day). She was followed by the treatment of oral prednisolone which was gradually tapered in our out-patient clinic. In August of 1999 high fever and severe anemia were recurred, and she was admitted again to our hospital because of the diagnosis as recurrent HPS. She had been treated with 40 mg/day of oral prednisolone and fever was immediately disappeared and hemoglobin was gradually increased. HPS is considered to be a rare complication with systemic sclerosis, and the etiology has been unknown. IL-18 is a novel cytokine which is a potent inducer of interferon-gamma, and its properties may be a proinflammatory regulation and activation of monocyte/macrophage and histiocyte through the expression of interferon-gamma. Therefore, the significance of IL-18 in the pathophysiology of HPS was recently reported. In this case, we investigated the significance of IL-18 and revealed the levels of serum IL-18 were well correlated with disease activity of HPS.
OBJECTIVE: Expression and function of tumor necrosis factor-alpha (TNF-alpha) converting enzyme (TACE) in synovia of patients with rheumatoid arthritis (RA) were examined to investigate posttranslational regulation of TNF-alpha production by TACE in RA. METHODS: Expression of TACE protein was evaluated by immunohistochemistry. Cytokines and soluble cytokine receptors were measured by ELISA. TACE mRNA was detected by RT-PCR. The enzymatic activity of TACE was measured using TACE-specific fluorogenic substrate. RESULTS: Expression of TACE at protein level in synovial tissue (ST) of patients with RA was significantly stronger than that of patients with osteoarthritis (OA). In RA, TACE was mainly expressed in CD68+ macrophage-like synovial cells. ST from 9 of 9 RA and 3 of 8 OA patients expressed TACE mRNA. RA ST cells possessed significantly higher TACE-like enzymatic activity than OA ST. A synthetic TACE inhibitor significantly reduced the release of TNF-alpha and p75 TNF receptor from RA ST cells. CONCLUSION: TACE is an important regulator of the secretion of TNF-alpha from synovia of patients with RA.
OBJECTIVE: To investigate the expression and function of interleukin 18 (IL-18) in synovial tissue (ST) of patients with rheumatoid arthritis (RA). METHODS: The localization of IL-18 in ST was analyzed by immunohistochemistry. IL-18 and IL-18 receptor (IL-18R) mRNA were detected by RT-PCR. Expression of IL-18 at the protein level was analyzed by Western blotting. Cytokines in culture supernatants were measured by ELISA. RESULTS: From immunohistochemical analysis, IL-18-producing cells were localized in the lining layer and sublining region of RA ST. Most of them coexpressed CD68 antigen. In ST from patients with osteoarthritis (OA), IL-18-producing cells were localized only in the sublining region and the numbers of these cells were small. From RT-PCR, RA ST expressed mRNA of IL-18, as well as alpha- and beta-chains of IL-18R. OA ST did not express or very weakly expressed mRNA of alpha- and beta-chains of IL-18R. ST from RA patients produced significantly larger amounts of IL-18 in vitro than OA ST. Western blotting revealed that RA ST expressed mature IL-18 more abundantly than OA ST. IL-12 alone stimulates interferon-gamma (IFN-gamma) production by RA synovial tissue cells, but IL-18 alone could not. In the presence of IL-12, however, IL-18 could synergistically stimulate IFN-gamma production by RA synovial tissue cells. OA synovial tissue cells responded to neither IL-12 nor IL-12 + IL-18. IL-18 showed synergistic effects with IL-12 on promoting the ability of synovial T cells from RA patients to produce IFN-gamma. CONCLUSION: These findings suggest that mature IL-18 is expressed in RA synovia and contributes to the production of IFN-gamma by infiltrating T cells.
In polymyositis (PM)/dermatomyositis (DM), T cells infiltrate the muscle tissues and interact with muscle cells via cell surface molecules. Recently, myoblasts have been reported to express CD40, but little is known about the role of CD40 in myoblasts. In the present study we examined the expression and involvement of CD40 and CD40 ligand (CD40L) in the interaction between muscle cells and T cells in PM/DM. Immunohistochemical staining revealed that CD40 was expressed on muscle cells in five of five PM and four of five DM patients, and that infiltrating mononuclear cells (MNCs) expressed CD40L in all cases of PM/DM. These CD40L-expressing MNCs were primarily CD4+ T cells. IFN-gamma, which is known to induce CD40 expression on various types of cells, was also expressed on the MNCs in four of the PM and four of the DM patients. Although cultured human myoblasts (SkMC 2859) did not express CD40 constitutively, IFN-gamma induced CD40 expression in a dose-dependent manner. To clarify the functional roles of CD40-mediated signals, the effects of a trimeric form of recombinant human CD40L on cytokine production were studied in SkMC 2859 that were prestimulated with IFN-gamma to express CD40. Recombinant human CD40L markedly increased the production of IL-6, IL-8, IL-15, and monocyte chemoattractant protein-1 of SkMC 2859. The expression of these humoral factors in muscle cells of PM and DM was demonstrated by immunohistochemistry. These results suggest that interaction between T cells and muscle cells via the CD40-CD40L system contributes to the immunopathogenesis of PM/DM by augmenting inflammation via cytokine production by the muscle cells.
A 60-year-old woman was admitted to our hospital with complaints of muscle weakness and erythema on her extremities. Gottron's sign, heliotrope rash, elevation of serum myogenic enzymes, electromyography and magnetic resonance imaging findings established a diagnosis of dermatomyositis (DM). She was treated with 60 mg of daily prednisolone. One week later, she suddenly developed splenic and renal infarctions, which were considered to have resulted from vasculopathy associated with DM. Cyclophosphamide and anticoagulants along with increasing the dosage of corticosteroid were effective. This is the first report describing splenic and renal infarctions in a patient with adult-onset DM.
OBJECTIVE: To investigate the diagnostic value of the serum concentrations of KL-6, a mucinous glycoprotein expressed on type II pneumocytes, for interstitial pneumonia (IP) in various collagen diseases. METHODS: Serum KL-6 levels were measured by ELISA. RESULTS: The mean values and the positive rates of serum KL-6 levels for patients with rheumatoid arthritis, systemic sclerosis, or polymyositis/dermatomyositis with IP were significantly higher than those without IP. Sensitivity, specificity, positive and negative predictive values of serum KL-6 level for IP associated with collagen diseases were 60.7, 98.9, 97.4, and 77.1%, respectively. The mean serum KL-6 level of patients with active IP was significantly (p = 0.0001) higher than that of patients with inactive IP. Serum KL-6 levels increased with the deterioration of IP, while the successful treatment of IP resulted in a significant decrease of these levels. CONCLUSION: Serum KL-6 concentration levels are a useful marker for diagnosis and evaluation of the disease activity of IP associated with collagen diseases.
We report a case of Sjögren's syndrome (SS) and systemic sclerosis (SSc) complicated with acute pancreatitis. A 51-year-old woman had been diagnosed as SS in 1973. She noticed Raynaud's phenomenon in 1977. In 1988, interstitial pneumonia (IP) was pointed out and she was treated with methylprednisolone (mPSL) pulse therapy. Prednisolone (PSL) was gradually tapered to 3-5 mg daily and she visited our outpatient clinic in 1995. On her first admission to our hospital in 1996, she showed xerostomia, keratoconjunctivitis sicca, sclerotic skin changes of her distal extremities and face, thickening of her sublinguinal frenulum, and regurgitative esophagitis. She was positive with anti-SS-A and SS-B antibodies. She was diagnosed as SS and SSc. Radiographic and laboratory data also established the diagnosis of inactive IP, renal tubular acidosis (RTA) and chronic renal failure (CRF). In April 30th 1997, she was admitted to our hospital again with complaints of dyspnea, dysesthesia, epigastralgia and petechia. Active IP and mononeuritis multiplex were diagnosed, and petechia was considered to be associated with vasculopathy. Her serum amylase level was 891 mU/ml on admission and spontaneously increased to 2440 mU/ml on May 12th along with increase of fibrinogen degradation product, D-dimer and alpha 2 plasmin-plasmin inhibitor complex levels. Ultrasonography depicted swelling of her pancreatic head and the diagnosis of acute pancreatitis was made. She was treated with protease inhibitors and intravenous hyperalimentation for acute pancreatitis. mPSL pulse therapy (500 mg/day for 3 days) was instituted for IP and mononeuritis multiplex on May 22, followed by 50 mg of daily PSL. While IP and mononeuritis multiplex gradually improved by the high-dose steroid therapy, serum amylase level raised to more than 4293 mU/ml, suggesting the modification of pancreatitis by the treatment with steroid. Since she did not respond to the conservative therapy for acute pancreatitis, she was treated with plasmapheresis, which turned out to be very effective. However, she was suffered from fungal pneumonia and died of respiratory failure. As far as we know, only three cases of SS with acute pancreatitis have been reported so far. The immunopathological mechanisms of development of acute pancreatitis in our case, especially focusing on the significance of microvasculopathy and hypercoagulability, were discussed.
bcl-2 gene expression is induced by 17beta-estradiol (E2) in T47D and MCF-7 human breast cancer cells, and the mechanism of E2 responsiveness was further investigated by analysis of the bcl-2 gene promoter. The -1602 to -1534 distal region (bcl-2j) of the promoter was E2-responsive; however, in gel mobility shift assays, the estrogen receptor alpha (ER(alpha)) did not bind [(32)P]bcl-2j, whereas Sp1 protein formed a retarded band complex. Further analysis demonstrated that the upstream region (-1603 to -1579) of the bcl-2 gene promoter contained two GC/GA-rich sites at -1601 (5'-GGGCTGG-3') and -1588 (3'-GGAGGG-5') that bound Sp1 protein. Subsequent studies confirmed that transactivation by E2 was dependent on ER(alpha)/Sp1 interactions with both GC-rich sites, and this was confirmed by in vitro footprinting. In contrast, a 21-base pair E2-responsive downstream region (-1578 to -1534) did not bind Sp1 or ER(alpha) protein; however, analysis of a complex binding pattern with nuclear extracts showed that ATF-1 and CREB-1 bound to this motif. These data coupled with results of transient transfection studies demonstrated that transcriptional activation by E2 of the -1578 to -1534 region of the bcl-2 gene promoter was dependent on induction of cAMP and subsequent activation through a cAMP response element. Thus, hormone regulation of bcl-2 gene expression in breast cancer cells involves multiple enhancer elements and E2-mediated transactivation does not require direct binding of the estrogen receptor with promoter DNA.
OBJECTIVE: To investigate the immunopathological significance of CD40/CD40 ligand system for B cell hyperactivation in SLE patients, the expression and the function of CD40 on B cells were compared with those of normal controls. METHODS: Expression of CD40 was evaluated by flow cytometry. DNA synthesis of B cells were measured by 3H - TdR incorporation. Antibody production was assessed by ELISA. RESULTS: There was no significant difference between SLE and normal controls in CD40 expression on peripheral blood B cells. Recombinant CD40 ligand-leucine zipper fusion protein (CD40L-LZ) significantly enhanced 3H - TdR incorporation by both SLE and normal B cells (P<0.01). 3H - TdR incorporation of SLE B cells without stimuli (P<0.001) and with CD40L-LZ stimulation (P<0.05) were significantly lower in SLE patients compared with normal controls. Active SLE B cells spontaneously produced significantly larger amounts of total IgG than normal B cells (P<0.05). CD40L-LZ significantly increased the production of total IgG by SLE B cells (P<0.05), but not by normal B cells. Active SLE B cells spontaneously produced IgG anti-dsDNA and IgG anti-ssDNA antibodies. CD40L-LZ significantly increased the production of these autoantibodies by SLE B cells (P<0.05). B cells from normal controls do not produce these autoantibodies spontaneously nor in response to CD40L-LZ. CONCLUSION: These findings indicate that signalling via CD40 plays an important role in B cell proliferation and autoantibody production in human SLE.
Explore the source record for details and available documents.
Explore the source record for details and available documents.