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In recent years there have been rapid changes in the social environment of preschool children with parents becoming the only center of family - in particular as a result of an increase in the number of working mothers and changes in lifestyles. Accordingly, preschool children are unable to maintain regular habits; causing concern about the effect on their physical and mental development. Additionally parents are at a loss with contemporary child care which increasingly gives priority to facilities and rationalization. A questionnaire and a new K-system development examination were used to study the habits and developmental stages of three-year-olds. Results indicate a positive correlation between family mealtimes, greetings and conversation, family experiences, a positive sense of family; and the development of preschoolers. It is necessary for child care support to help create positive surroundings in the home.

In this work we report a novel method to efficiently induce a murine model of Graves’ hyperthyroidism. Inbred mice of different strains were immunized by i.m. injection with adenovirus expressing thyrotropin receptor (TSHR) or β-galactosidase (1 × 1011 particles/mouse, three times at 3-wk intervals) and followed up to 8 wk after the third immunization. Fifty-five percent of female and 33% of male BALB/c (H-2d) and 25% of female C57BL/6 (H-2b) mice developed Graves’-like hyperthyroidism with elevated serum thyroxine (T4) levels and positive anti-TSHR autoantibodies with thyroid-stimulating Ig (TSI) and TSH-binding inhibiting Ig (TBII) activities. In contrast, none of female CBA/J (H-2k), DBA/1J (H-2q), or SJL/J (H-2s) mice developed Graves’ hyperthyroidism or anti-TSHR autoantibodies except SJL/J, which showed strong TBII activities. There was a significant positive correlation between TSI values and T4 levels, but the correlations between T4 and TBII and between TSI and TBII were very weak. TSI activities in sera from hyperthyroid mice measured with some chimeric TSH/lutropin receptors suggested that their epitope(s) on TSHR appeared similar to those in patients with Graves’ disease. The thyroid glands from hyperthyroid mice displayed diffuse enlargement with hypertrophy and hypercellularity of follicular epithelia with occasional protrusion into the follicular lumen, characteristics of Graves’ hyperthyroidism. Decreased amounts of colloid were also observed. However, there was no inflammatory cell infiltration. Furthermore, extraocular muscles from hyperthyroid mice were normal. Thus, the highly efficient means that we now report to induce Graves’ hyperthyroidism in mice will be very useful for studying the pathogenesis of autoimmunity in Graves’ disease.

Nitric oxide (NO) is a multifunctional messenger molecule generated from L-arginine by a family of enzymes, including nitric oxide synthase (NOS). This study was performed to examine whether NO modulates the production of matrix metalloproteinases (MMPs), which degrade all components of extracellular matrix (ECM), in rheumatoid synovial cells. We investigated the effects of exogenously generated NO by a NO donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP), on the MMPs production by rheumatoid synovial cells. Culture media conditioned by SNAP-treated synovial cells were examined by gelatin zymography and immunoblot analysis. Incubation of synovial cells with SNAP resulted in gelatinase A production in a dose-dependent fashion. Furthermore, RT-PCR analysis demonstrated that MMP-2 mRNA expression was induced in SNAP-treated synovial cells. In contrast, SNAP did not influence the production of TIMP-1 and TIMP-2, which preferentially inhibit MMP-2, by rheumatoid synovial cells. Our data indicate that NO could modulate MMP production by rheumatoid synovial cells and therefore contribute to ECM degradation of articular components in RA.

Overgrowth of rheumatoid synoviocytes, which results in joint destruction, is due to impaired balance between cell proliferation and cell death (apoptosis). Ceramide is an important lipid messenger involved in mediating a variety of cell functions including apoptosis. We investigated the effects of ceramide on growth-promoting anti-apoptotic signals in rheumatoid synovial cells. Human synovial cells isolated from patients with rheumatoid arthritis (RA) were stimulated with platelet-derived growth factor (PDGF) in the presence or absence of C2-ceramide. The kinase activity of Akt, MEK, and ERK1/2 was analyzed in PDGF-stimulated synovial cells by Western blot analysis. Pretreatment with C2-ceramide completely inhibited PDGF-induced cell cycle progression of rheumatoid synovial cells. PDGF stimulation induced phosphorylation and activation of Akt, MEK, and ERK1/2 in rheumatoid synovial cells. C2-ceramide inhibited the activation of Akt, MEK and ERK1/2 in PDGF-stimulated synovial cells. Our data demonstrated that inhibition of anti-apoptotic kinases, such as Akt and ERK1/2, may play an important role in ceramide-mediated apoptosis of rheumatoid synovial cells.

The presence of a putative autoantigen of autoimmune disorder in a target organ may cause accumulation of specific T cells in the inflammatory region. One of the mechanisms of such accumulation involves the migration of specific-circulating T cells through the endothelial cells into the target lesion. The presence of only a few specific T cells responsive to a putative autoantigen has hampered the investigation of specific migration of circulating T cells to the target organ. We used a superantigen to investigate specific T-cell adhesion to endothelial cells, because it stimulates a large proportion of T cells with particular V beta elements and adhesion of T cells to the endothelium is a vital step in the migration process. Adhesion of murine T cells to the human endothelial cell line, EA.hy926, was specifically increased in the presence of staphylococcal enterotoxin B (SEB). The increase was interferon-gamma (IFN-gamma)-dependent, and consisted mainly of CD4+ T cells. V beta 8.1,2+ T cells preferentially adhered to endothelial cells in the presence of SEB compared with V beta 6+ T cells. Pretreatment of endothelial cells with SEB increased the adherence of V beta 8.1,2+ T cells, while anti-human leucocyte antigen (HLA)-DR and -DQ antibodies inhibited the increased adherence of V beta 8.1,2+ T cells. Our results demonstrate that increased T-cell adhesion to endothelial cells is SEB specific, and that the specificity is dependent on major histocompatibility complex (MHC) class II molecules expressed on endothelial cells and on the recognition of the SEB-MHC class II complex by V beta 8.1,2+ T cells.

A 46-year-old woman with rheumatoid arthritis had been on non-steroidal antiinflammatory agents for eighteen years until she developed cushingoid features and hypertension resistant to antihypertensive drugs. She had high plasma cortisol and 24 h urinary 17-hydroxycorticosteroids (17HCS) which were not suppressed by 8 mg dexamethasone per day for two days. The circadian rhythm of plasma cortisol was absent and plasma ACTH concentrations were suppressed before and after intravenous administration of CRH. Abdominal computed tomography demonstrated a tumor (3.0 x 3.0 x 2.3 cm) in the right adrenal gland and a 131I-6 beta-19-nor-methylcholesterol scan revealed marked uptake on the same side. The patient underwent a right adrenalectomy and the diagnosis of a cortisol secreting benign adenoma was histologically confirmed. Blood pressure declined and cushingoid features regressed, but three months after the operation and while the patient was on replacement, she complained of pain on motion, marked tenderness and swelling of fingers, wrists, elbows, knees and foot joints, and had very high rheumatoid factors. Treatment with immunosuppressive drugs and oral and intraarticular administration of glucocorticoids were necessary to relieve the clinical symptoms of rheumatoid arthritis. In summary, we report a patient with rheumatoid arthritis and Cushing's syndrome due to an adrenal adenoma, in whom rheumatoid arthritis was exacerbated after curing the Cushing's syndrome. This suggests that it is imperative to follow the development and/or course of autoimmune diseases after the treatment of Cushing's syndrome.

高γグロブリン血症と白血球数減少症を検出して以来, 14年後にSjogren症候群を, 20年後に全身性エリテマトーデスを合併した症例を経験した.症例は37歳家婦で, 17歳時(1973年)に学校検診で高γグロブリン血症を指摘され精査のために当科に第1回目入院する.各種検査を施行するも高γグロブリン血症と白血球減少症を認める以外に確定診断は得られなかった. 29歳(1985年)易感染症と全身倦怠感を自覚し当科を受診する.頚部および腋窩部にリンパ節腫脹を認め,抗核抗体,抗SS-A抗体,抗SS-B抗体が陽性であった. 33歳(1989年)眼・口腔内乾燥症状とRaynaud現象が出現した. 1992年8月抗dsDNA抗体が陽性となり1993年1月には発熱,関節痛,尿蛋白が出現し抗Sm抗体も陽性となり同年10月入院した.精査のうえ, Sjogren症候群と全身性エリテマトーデスの合併と診断した.プレドニゾロン投与,ステロイドパルス療法にて,臨床症状,所見はすみやかに改善した. 本症例は両疾患の発症,進展および両疾患の関連を追求するに示唆に富む症例と考え報告した.

The patient was a 63-year-old woman. She was admitted to our hospital with acute renal failure and multiple mononeuritis in 2002. She was diagnosed as microscopic polyangiitis based on positive for MPO-ANCA. Remission was induced by combination therapy with methylprednisolone pulse therapy and plasma exchange. Because condition of the disease was stable, prednisolone was discontinued from August 2006. Elevation of serum creatinine and microscopic hematuria was detected in November 2007. Fever and dyspnea occurred in January 24 2008. Elevation of CRP and serum creatinine was found, and infiltration in bilateral lung was noted on chest X-ray. She was admitted on the same day. After admission, she presented with hemosputum and exacerbation of dyspnea. Chest CT revealed diffuse consolidation and ground glass opacity, and MPO-ANCA converted to be positive. Diagnosis of diffuse alveolar hemorrhage and rapidly progressive glomerulonephritis with microscopic polyangiitis was made, and she was managed by artificial respirator and CHDF in ICU. Combination therapy with steroid pulse therapy and plasma exchange re-induced remission. Mizoribine was administrated as maintenance therapy with oral prednisolone.

Summary Membrane-type 1 matrix metalloproteinase (MT1-MMP) is thought to be a putative regulator of pro-gelatinase A (MMP-2) in the rheumatoid synovium. In this study, we examined the effects of IL-1β, one of the inflammatory cytokines, on the expression of MT1-MMP and the activation of pro-MMP-2 using rheumatoid synovial cells. We also studied the effects of KE-298 (2-acetylthiomethyl-4-(4-methylphenyl)-4-oxobutanoic acid), a new disease-modifying anti-rheumatic drug (DMARD), on MT1-MMP expression of rheumatoid synovial cells. Type B synovial cells (fibroblast-like synovial cells) were cultured with KE-298 (25–100 µg/ml) in the presence of IL-1β for 48 h. Activation of pro-MMP-2 secreted from synovial cells was analysed by gelatin zymography. Reverse transcription–polymerase chain reaction (RT–PCR) methods were used to detect MT1-MMP mRNA. MT1-MMP protein expression on synovial cells was examined by anti-MT1-MMP immunoblot. An active form of MMP-2 was demonstrated in the culture media conditioned by IL-1β-stimulated synovial cells. In addition, MT1-MMP mRNA and protein expression of rheumatoid synovial cells were increased by IL-1β treatment. KE-298 blocked this IL-1β-induced pro-MMP-2 activation and MT1-MMP expression, but did not affect IL-1β-induced tissue inhibitor of metalloproteinase-2 (TIMP-2) secretion from rheumatoid synovial cells. These findings indicate that activation of rheumatoid synovial cells by IL-1β results in the induction of MT1-MMP expression. Given that MT1-MMP promotes matrix degradation by activating pro-MMP-2, these results suggest a novel mechanism whereby cytokine may contribute to articular destruction in rheumatoid arthritis (RA). KE-298 may prevent this process by down-regulating MT1-MMP expression.

Nitric oxide (NO) is elevated in the synovial fluids and sera of patients with rheumatoid arthritis (RA) and is thought to be an important proinflammatory mediator in the rheumatoid synovium. To test the hypothesis that NO might modulate the apoptosis-inducing signal pathway, we investigated the effects of NO on rheumatoid synovial-cell apoptosis induced by Fas ligation with anti-Fas antibody. Pretreatment of synovial cells with the NO donor S-nitro-N-acetylpenicillamine (SNAP) prevented the Fas-mediated induction of apoptosis. The activation of caspase-3 was required to mediate Fas-induced synovial cell apoptosis. The NO donor SNAP inhibited Fas-induced caspase-3 activation in rheumatoid synovial cells. However, NO did not interrupt Fas-induced caspase-8 cleavage or subsequent cytochrome c release into the cytosol in rheumatoid synovial cells. These data indicate that NO prevents apoptosis in rheumatoid synovial cells by directly inhibiting caspase-3 activation. Thus, we propose that NO interferes with cell death signal transduction and may contribute to rheumatoid synovial cell proliferation by inhibiting induction of apoptosis.

SummarySecondary amyloidosis (AA amyloidosis) is a systemic disease characterized by the extracellular tissue deposition of insoluble amyloid A (AA) protein. Aberrant metabolism of serum amyloid A (SAA) by macrophages is only one of many putative mechanisms which may be important in AA amyloidogenesis. In this study, we investigated the effects of cytokines on human monocyte-mediated SAA proteolysis. Human peripheral blood mononuclear cells (PBMC) or CD14+ monocytes were cultured with SAA, and the culture supernatants were then subjected to anti-SAA immunoblot. CD14+ monocytes degraded SAA completely. Whereas, when CD14+ monocytes were pretreated with IL-1β or IFN-γ, increasing amounts of SAA-related derivatives were detected in culture supernatants. These findings suggest that activation of monocytes by IL-1β or IFN-γ hampers the proteolysis of a precursor protein and leads to a partial degradation of SAA. This down-regulated proteolysis of SAA protein by cytokine-stimulated monocytes may play a role in the mechanism of AA amyloid formation as well as its removal.

Prostaglandins formed by cyclooxygenase (COX) enzymes are important mediators of inflammation. The contribution of inducible COX-2 in the rheumatoid synovium is well documented. In this study, we evaluated the contribution of nitric oxide (NO) to COX-2 expression in rheumatoid synovial cells. Exposure of rheumatoid synovial cells to a NO donor, SNAP, induced COX-2 protein expression in a dose-dependent manner. RT-PCR analysis also demonstrated that COX-2 mRNA was induced in SNAP-treated synovial cells. Dexamethasone at therapeutic concentrations markedly inhibited this NO-mediated COX-2 expression in synovial cells. In contrast to its effect on COX-2 expression, SNAP did not affect the constitutive expression of COX-1 in rheumatoid synovial cells. Our findings suggest that NO is an important modulator of COX-2 expression and that glucocorticoids exert their anti-inflammatory action in rheumatoid synovium, at least in part, by suppression of COX-2 induction.

There is accumulating evidence that human T-lymphotropic virus-1 (HTLV-1) infection contributes to the development of various inflammatory disorders. To elucidate the relation between the infection and Sjogren's syndrome, seroepidemiological and virological studies were conducted on patients with this syndrome in Nagasaki Prefecture, Japan, an area heavily endemic for HTLV-1. The HTLV-1 seroprevalence rate among the patients with Sjogren's syndrome (17/74, 23%) was significantly higher than that among blood donors (916/27,284, 3%), whereas the difference between patients with systemic lupus erythematosus and blood donors was insignificant. Moreover, among Sjogren's syndrome patients the seroprevalence was high irrespective of age, unlike that among blood donors, which rose with age. Titres of serum antibodies in the HTLV-1 seropositive patients with Sjogren's syndrome were similar to those among patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and significantly higher than those among healthy carriers. IgM class antibodies were commonly detected in the serum of patients with Sjogren's syndrome. However, unlike that in HAM/TSP patients, the viral load in peripheral-blood mononuclear cells was not necessarily high in the seropositive Sjogren syndrome group. Salivary IgA antibodies to HTLV-1 were common among seropositive patients with Sjogren's syndrome (5/7), which might be due to increased viral activity in the salivary glands. These antibodies were barely detectable in HAM/TSP patients (prevalence 1/10) or in healthy carriers (0/11). The findings strongly suggest that HTLV-1 is involved in the pathogenesis of the disease in a subset of patients with Sjogren's syndrome in endemic areas.

Optical rotatory dispersion (ORD) and circular dichroism (CD) of sulfur-containing nucleosides (4-thiouridine, 6-thioguanosine and 6-thioinosine) and a nucleotide (4-thiouridine 2′(3′)-monophosphate) as well as a complex between ribonuclease (EC 2.7.7.16) and the nucleotide were measured in the near-ultraviolet and ultraviolet regions. All thionucleosides and the nucleotide displayed multiple Cotton effects which were correlated with their absorption maxima. Their CD spectra corresponded to the multiple Cotton effects except for 6-thioguanosine and 6-thioinosine which exhibited such low intensities that their bands could not be detected. The Cotton effects and CD bands were displaced toward shorter wavelengths upon alkaline treatment. The ribonuclease-thionucleotide complex showed ORD and CD near the absorption maximum at 331 mμ of the nucleotide quite different from the curves computed on the basis of the constituents. This suggests that the change in dissymmetry in the nucleotide molecule occurred on the formation of the complex. The ultraviolet ORD and CD were almost the same as those of the constituents, indicating the absence of any drastic conformational change in the protein molecule accompanying complex formation.

Circular dichroism and optical rotatory dispersion of bovine liver catalase have been measured over wide wavelength range of 200–650 mμ. Native catalase showed a small negative CD band in the Soret absorption region, which corresponded to a small negative Cotton effect. The denaturation with acid (pH 3.0), alkali (pH 12.0) and 8 M urea caused almost complete loss of this Soret CD band and the Cotton effect. Addition of the ligand molecules such as KCN and NaN3 to native catalase caused an appearance of new small CD band near 450 mμ. In the near-ultraviolet region native molecule exhibited a CD band at 285 mμ, while the denaturations caused blue shift of the band with a large enhancement in intensity at pH 12.0 as well as diminutions at pH 3.0 and by 8 M urea. The liganded catalase molecules showed a new band near 340 mμ in addition to the CD band at 280–290 mμ. These CD bands may be related to aromatic amino acids and also their interaction with the heme groups in the molecules. In the ultraviolet region all proteins except for the denatured one with 8 M urea exhibited two intense negative CD bands and a deep trough of Cotton effect at 233 mμ, which indicated the presence of α-helical structure in their molecules. It was concluded from these data that the helix content of the native molecule was about 50%, whereas the acid- and alkali-denatured molecules showed the less helical content of 20–25% and 15–20%, respectively. Treatment with 8 M urea seemed to cause complete loss of helical conformation in the molecule. However, addition of ligands (KCN and NaN3) to the heme groups of catalase caused reduction of helical content in the molecule whose enzymic activity was completely lost. The estimated helical content was approx. 30%.