Body weight and dysautonomia in early Parkinson's disease.

OBJECTIVES: Patients with Parkinson's disease (PD) begin to lose weight several years before diagnosis, which suggests weight variation is associated with some factor(s) that precede the onset of motor symptoms. This study aimed to investigate the association of autonomic nervous system with body weight in patients with PD. MATERIALS AND METHODS: The subjects were 90 patients with early de novo PD. We examined the associations of body mass index (BMI) with sympathetic nervous activity reflected in orthostatic intolerance or cardiac uptake of 123 I-metaiodobenzylguanidine and parasympathetic nervous activity reflected in constipation or heart rate variability (HRV). RESULTS: Twelve patients (13.3%) were overweight (BMI>25 kg/m2 ), 62 patients (68.9%) were normal-weight (18.5≦BMI<25 kg/m2 ), and 16 patients (17.8%) were underweight (BMI<18.5 kg/m2 ). Underweight patients had greater disease severity and decrease in blood pressure on head-up tilt-table testing, higher cardiac washout ratio of 123 I-metaiodobenzylguanidine, and lower HRV and complained of constipation more often than those with normal-weight or overweight patients. On multiple regression analyses, the correlation of these variables with BMI maintained statistical significance after adjustment for age, sex, symptom duration, and motor subtype. CONCLUSIONS: Dysautonomia and disease severity are closely related to body weight independently of age, sex, symptom duration, and motor subtype. Dysautonomia may play a partial role on weight variation in the early stage of PD.

Effect of interfacial structure on bioinert properties of poly(2-methoxyethyl acrylate)/poly(methyl methacrylate) blend films in water.

In this study, we found that the surface made of a mixture of poly(2-methoxyethyl acrylate) (PMEA) and poly(methyl methacrylate) (PMMA) exhibited excellent blood compatibility by inhibiting platelet adhesion. To obtain a better understanding of this bioinertness, the polymer/water interface was characterized by neutron reflectivity measurements and sum frequency generation spectroscopy, in conjunction with bubble contact angle measurements. Based on the results, we can say that the outermost region of the blend film was reorganized in water. When the orientation of PMEA segments at the water interface became random with increasing immersion time, the fractional amount of lower-coordinated water molecules increased at the interface. Such an interfacial structure caused the suppression of platelet adhesion.

A synthetic peptide derived from staphylokinase enhances plasminogen activation by tissue-type plasminogen activator.

BACKGROUND: A synthetic nonadecapeptide (SP; GPYLMVNVTGVDGKGNELL) previously enhanced the activation of plasminogen by the SAK/plasmin complex. OBJECTIVES: To identify the binding site for SP on plasminogen and elucidate the effects of SP on plasminogen activation by the tissue-type plasminogen activator (t-PA). METHODS: The effects of SP on plasminogen activation were estimated using a chromogenic substrate and from the cleavage of plasmin on SDS-PAGE under reduced conditions. The binding to SP of various peptides derived from the amino acid sequence of plasminogen was analyzed with an IAsys biosensor. The SP-mediated structural change to plasminogen was analyzed by circular dichroism (CD) spectroscopy. The thrombolytic effects of SP were examined using a mouse model of thrombosis. RESULTS: SP enhanced the activation of plasminogen by t-PA. The catalytic efficiency (k(cat)/K(m)) of Glu-plasminogen activation by t-PA was 11.4-fold higher in the presence than absence of SP. The binding of SP to plasminogen was greatly inhibited by a synthetic peptide, FEKDKYILQGVTSWGLG, located close to the C-terminal of the plasminogen B region. Near-ultraviolet CD spectra of the complex between SP and Glu-plasminogen significantly differed from those of Glu-plasminogen. When SP was administered in a mouse model of thrombosis, early recanalization was observed in a dose-dependent manner. However, SP did not cause recanalization in t-PA gene-deficient mice. CONCLUSIONS: SP bound to the B region and promoted the activation of plasminogen by t-PA, and then induced effective thrombolysis.

Alpha2-antiplasmin is involved in the production of transforming growth factor beta1 and fibrosis.

BACKGROUND: Fibrotic disease occurs in most tissues. Transforming growth factor (TGF)-beta is the major inducer of fibrosis. The fibrinolytic system is considered to play an important role in the degradation of extracellular matrices. However, the detailed mechanism of how this system affects fibrosis remains unclear. METHODS AND RESULTS: We examined experimental fibrosis in mice with a deficiency of alpha(2)-antiplasmin (alpha2AP), which is a potent and specific plasmin inhibitor. We found that the lack of alpha2AP attenuated bleomycin-induced TGF-beta(1) synthesis and fibrosis. In addition, the production of TGF-beta(1) from the explanted fibroblasts of alpha2AP(-/-) mice decreased dramatically as compared to that in wild-type mice. Moreover, we found that alpha2AP specifically induces the production of TGF-beta(1) in fibroblasts. CONCLUSION: The lack of alpha2AP attenuated TGF-beta(1) synthesis, thereby resulting in attenuated fibrosis. This is the first report to describe the crucial role that alpha2AP plays in TGF-beta(1) synthesis during the process of fibrosis. Our results provide new insights into the role of alpha2AP in fibrosis.

Lack of alpha2-antiplasmin improves cutaneous wound healing via over-released vascular endothelial growth factor-induced angiogenesis in wound lesions.

BACKGROUND: The fibrinolytic system is supposed to play an important role in the degradation of extracellular matrices for physiological and pathological tissue remodeling; however, the detailed mechanism regarding how this system affects cutaneous wound healing remains to be clarified. METHODS AND RESULTS: We performed experimental cutaneous wounding in mice with a deficiency of alpha(2)-antiplasmin (alpha(2)AP), which is a potent and specific plasmin inhibitor. We found that an accelerated wound closure was observed in alpha(2)AP-deficient (alpha(2)AP-/-) mice in comparison with wild type (WT) mice. Moreover, we observed that a greater increase of angiogenesis occurred in the process of wound healing in alpha(2)AP-/- mice than in the WT mice. Intriguingly, mRNA expression of vascular endothelial growth factor (VEGF), which is the best characterized positive regulator of angiogenesis, in wound lesions was found to show a greater increase in the early phase of the healing process in alpha(2)AP-/- mice than in WT mice. In addition, the amount of released-VEGF from the explanted fibroblasts of alpha(2)AP-/- mice increased dramatically more than in the WT mice. Finally, the intra-jugular administration of anti-VEGF antibody clearly suppressed the increased angiogenesis and accelerated wound closure in the wound lesion of alpha(2)AP-/- mice. CONCLUSION: The lack of alpha(2)AP markedly causes an over-release of VEGF from the fibroblasts in cutaneous wound lesions, thereby inducing angiogenesis around the area, and thus resulting in an accelerated-wound closure. CONCLUSIONS: This is the first report to describe the crucial role that alpha(2)AP plays following angiogenesis in the process of wound healing. Our results provide new insight into the role of alpha(2)AP on cutaneous wound healing.

Alpha2-antiplasmin plays a significant role in acute pulmonary embolism.

The importance of pulmonary embolism (PE) due to venous thrombosis is recognized in the treatment of vascular diseases. We have investigated the physiological effects of plasmin generation in experimental acute PE using mice deficient in plasminogen (Plg-/-) or alpha2-antiplasmin (alpha2-AP-/-). PE was induced by continuous induction of venous thrombus in the left jugular vein by endothelial injury due to photochemical reaction. The mortality of wild-type mice was 68.8% at 2 h after the initiation of venous thrombosis and it was significantly reduced in alpha2-AP-/- mice (41.7%). In contrast, Plg-/- mice did not survive. Histological evidence of thromboembolism in the lung was obtained in all mice. However, whereas a strict thromboembolism was observed in Plg-/- mice, only a few thrombi were detected in the lungs of alpha2-AP-/- mice. Plasma fibrinogen levels measured in mice were not different. When alpha2-AP was infused in alpha2-AP-/- mice, the mortality was indistinguishable from wild-type mice. Tissue-type plasminogen activator (tPA) did not reduce the mortality due to acute PE in wild-type mice. However, in alpha2-AP-/- mice, tPA (0.52 mg x kg-1) significantly decreased the mortality compared with that of alpha2-AP-/- mice without tPA. The bleeding time was not significantly prolonged in either type of mice treated with tPA. The lack of plasminogen increases the mortality due to acute PE while a lack of alpha2-AP decreases the mortality rate, which can be further reduced by tPA administration. Therefore, the combination of inhibition of alpha2-AP with thrombolytic therapy could be beneficial in the treatment of acute PE.

Inhibitory effects of an anti-rheumatic agent T-614 on immunoglobulin production by cultured B cells and rheumatoid synovial tissues engrafted into SCID mice.

OBJECTIVE: To clarify the pharmacological action of an anti-rheumatic agent T-614, we investigated its effects on immunoglobulin (Ig) production by cultured B cells and Ig secretion from synovial tissues of patients with rheumatoid arthritis (RA) using SCID mice engrafted with human RA tissue (SCID-HuRAg). METHODS: Murine B cells were prepared from mouse spleen by a T-cell depletion method. The cells were cultured with lipopolysaccharide (LPS) and/or interleukin 4 (IL-4) in the absence or presence of T-614. Human B cells were isolated from peripheral blood of healthy donors and the Ig production was induced by co-culture with autologous T cells and anti-CD3 antibody. SCID-HuRAg was prepared according to our previous method. T-614 was orally administered to the mice once daily for 4 weeks starting on the fourth week after the implantation. Then, peripheral blood was obtained and the implanted tissues were removed. Igs in the culture media or the sera were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: In murine B-cell cultures, T-614 significantly decreased not only the IgM production stimulated with LPS but IgG1 production induced by LPS and IL-4. Regarding human B cells stimulated with T cells, it also inhibited IgM and IgG production. In SCID-HuRAg mice, high concentrations of polyclonal human IgG were detectable in the sera of all mice. A significant decrease in the IgG level was observed in the T-614-treated group compared with the control group. CONCLUSIONS: We showed that T-614 inhibited Ig production by the cultured B cells and also decreased the high level of human IgG observed in SCID-HuRAg mice. These results may support its effect on plasma Ig in RA patients and provide insights into the mechanisms of its anti-rheumatic effect.

A peptide isolated from alpha B-crystallin is a novel and potent inhibitor of platelet aggregation via dual prevention of PAR-1 and GPIb/V/IX.

BACKGROUND: The ability of low-molecular-weight heat shock protein (HSP) to modulate thrombin-induced platelet aggregation has been investigated. OBJECTIVES: We examined the inhibitory effects on platelet aggregation of nine amino acid sequences isolated from HSP20 or alpha B-crystallin and their various derivatives. METHODS AND RESULTS: Platelet aggregation induced by various agonists was performed. These findings indicated that a peptide (Trp-Ile-Arg-Arg-Pro-Phe-Phe-Pro-Phe) from alpha B-crystallin significantly inhibits platelet aggregation induced by thrombin, TRAP (a protease activated receptor-1 agonist) and botrocetin, ristocetin (a stimulator of the platelet glycoprotein Ib/V/IX-von Willebrand factor axis), but not a protease-activated receptor-4 agonist, collagen and ADP. The inhibitory activity against thrombin or botrocetin is mainly linked to Arg-Arg-Pro-Phe or Trp-Ile-Arg-Arg-Pro, respectively, among nine amino acids. Additionally, during in vivo experiments, Trp-Ile-Arg-Arg-Pro-Phe-Phe-Pro-Phe shows a significant antithrombotic effect without marked bleeding. CONCLUSION: Our results provide the basis for a potential new aspect of antiplatelet compound for the therapy of thrombosis and cardiovascular disease.

The role of TNF-alpha in the pathogenesis of inflammation and joint destruction in rheumatoid arthritis (RA): a study using a human RA/SCID mouse chimera.

OBJECTIVE: In order to elucidate which cytokine preferentially stimulates the synovium in patients with rheumatoid arthritis (RA), we investigated the roles of tumour necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) using SCID mice engrafted with human RA tissue (SCID-HuRAg). METHODS: The SCID-HuRAg mice were prepared according to our previously described method. First, SCID-HuRAg mice were treated with chimeric anti-TNF-alpha monoclonal antibody (mAb, 100 microg/mouse) and histological changes were examined 4 weeks after the initial treatment. Secondly, a total of 100 microg of recombinant TNF-alpha or IL-6 (0.6 microg/h) was administered daily to mice using an osmium pump. The histological changes and serum cytokine levels were examined 4 weeks after the initial administration. Human immunoglobulin G (IgG) was administered to mice as a control. RESULTS: Synovial inflammatory cells were significantly decreased after the anti-TNF-alpha mAb treatment; conversely, the degree of synovial inflammation was significantly exacerbated by TNF-alpha administration. The levels of both IL-6 and TNF-alpha in sera were significantly increased by recombinant TNF-alpha administration, while TNF-alpha levels were unchanged by IL-6 administration. This suggests that TNF-alpha controls IL-6 production. Despite the profound changes in inflammation, we found no effects on bone and no articular cartilage damage was produced by TNF-alpha. CONCLUSION: This study provides strong evidence that TNF-alpha is a key molecule in the control of the inflammatory changes that occur in the RA synovium. In addition, TNF-alpha regulates IL-6 production. However, other inflammatory pathways independent of TNF-alpha may contribute to the bone and cartilage damage seen in RA.

Mechanism of prostaglandin E2-stimulated heat shock protein 27 induction in osteoblast-like MC3T3-E1 cells.

We investigated the effect of prostaglandin E2 (PGE2) on the induction of heat shock protein 27 (HSP27) and HSP70, and the mechanism behind the induction in osteoblast-like MC3T3-E1 cells. PGE2 time-dependently increased the level of HSP27 without affecting the level of HSP70. PGE2 stimulated the accumulation of HSP27 dose-dependently in the range between 10 nM and 10 microM. PGE2 stimulated the increase in the level of the mRNA for HSP27. Staurosporine and calphostin C, inhibitors of protein kinase C (PKC), suppressed the PGE2-induced HSP27 accumulation. The effect of PGE2 on HSP27 accumulation was reduced in the PKC down-regulated cells. BAPTA/AM, a chelator of intracellular Ca2+, or TMB-8, an inhibitor of intracellular Ca2+ mobilization, reduced the accumulation of HSP27 induced by PGE2. Dibutyryl cAMP had little effect on the basal level of HSP27. PGE2 induced the phosphorylation of both p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase. PD98059 and U-0126, inhibitors of the upstream kinase of p44/p42 MAP kinase, reduced the accumulation of HSP27 induced by PGE2. SB203580, a specific inhibitor of p38 MAP kinase, suppressed the HSP27 accumulation induced by PGE2. U-73122, an inhibitor of phospholipase C, and calphostin C reduced the PGE2-induced phosphorylation of both p44/p42 MAP kinase and p38 MAP kinase. These results indicate that PGE2 stimulates the induction of HSP27 through PKC-dependent activations of both p44/p42 MAP kinase and p38 MAP kinase in osteoblasts.

Corticosteroid treatment induces chondrocyte apoptosis in an experimental arthritis model and in chondrocyte cultures.

OBJECT: In order to examine the mechanisms involved in steroid-induced arthropathy after intra-articular corticosteroid injection, a histological examination was performed in vivo using severe combined immunodeficiency (SCID) mice that were implanted with human articular cartilage into the back (SCID/hu model). In addition, the effect of corticosteroids on chondrocyte apoptosis was evaluated in vitro using cultured human chondrocytes. METHOD: Human articular cartilage was obtained during knee surgery and implanted subcutaneously into the backs of SCID mice. One month later, weekly injections of corticosteroid (hydrocortisone acatate: 1 mg/0.2 ml, triamcinolone acetonide: 0.2 mg/0.2 ml, dexamethasone acetate: 0.1 mg/0.2 ml) in the subcutaneous cavity around the grafted cartilage in SCID mice were initiated. After six weeks of treatment, the grafted cartilage pieces were removed from the SCID mice and examined histologically. Chondrocyte apoptosis after corticosteroid treatment was also investigated using cultured human chondrocytes. RESULT: In the corticosteroid treated, grafted articular cartilage, apoptotic chondrocytes were apparent in the superficial and middle layers of cartilage. But a reduced intensity of Safranin O staining was not remarkable. In the cultured chondrocytes, apoptotic changes were also observed after corticosteroid treatment. CONCLUSION: Corticosteroid treatment induces chondrocyte apoptosis and it may be important to understand the steroid-induced arthropathy.

Contrasting effects of midazolam on induction of heat shock protein 27 by vasopressin and heat in aortic smooth muscle cells.

We previously showed that vasopressin stimulates the induction of heat shock protein (HSP) 27, a low molecular-weight HSP, through protein kinase C activation in aortic smooth muscle A10 cells. In the present study, we examined the effects of midazolam, an intravenous anesthetic, on the HSP27 induction stimulated by vasopressin, heat, or sodium arsenite (arsenite) in A10 cells. Midazolam inhibited the accumulation of HSP27 induced by vasopressin or 12-O-tetradecanoylphorbol 13-acetate (TPA), a direct activator of protein kinase C. Midazolam also reduced the vasopressin-induced level of the mRNA for HSP27. In contrast, midazolam enhanced the HSP27-accumulation induced by heat or arsenite. Midazolam also enhanced the heat-increased level of the mRNA for HSP27. However, midazolam had no effect on the dissociation of the aggregated form of HSP27 following stimulation by vasopressin, heat, or arsenite. These results suggest that midazolam suppresses vasopressin-stimulated HSP27 induction in vascular smooth muscle cells, and that this inhibitory effect is exerted at a point downstream from protein kinase C. In contrast, midazolam enhanced heat- or arsenite-stimulated HSP27 induction. Thus, midazolam has dual effects on the HSP27 induction stimulated by various stresses in vascular smooth muscle cells.

Inhibition by adenylyl cyclase-cAMP system of ET-1-induced HSP27 in osteoblasts.

We have previously reported that endothelin-1 (ET-1) stimulates heat shock protein (HSP) 27 induction in osteoblast-like MC3T3-E1 cells and that p38 mitogen-activated protein (MAP) kinase acts at a point downstream from protein kinase C (PKC) in HSP27 induction. In the present study, we investigated the effect of the adenylyl cyclase-cAMP system on ET-1-stimulated induction of HSP27 in MC3T3-E1 cells. Dibutyryl-cAMP (DBcAMP) dose dependently inhibited the HSP27 accumulation stimulated by ET-1. Forskolin and cholera toxin significantly suppressed the ET-1-stimulated accumulation of HSP27. However, dideoxyforskolin, a forskolin derivative that does not activate cAMP, failed to suppress the ET-1-induced HSP27 accumulation. Forskolin reduced the p38 MAP kinase phosphorylation induced by ET-1 or 12-O-tetradecanoylphorbol-13-acetate (TPA). PGE(1), an extracellular agonist that activates cAMP production, reduced the ET-1-induced HSP27 accumulation. In addition, the phosphorylation of p38 MAP kinase induced by ET-1 or TPA was suppressed by PGE(1). Forskolin, DBcAMP, and PGE(1) suppressed the ET-1-stimulated increase in the mRNA level for HSP27. These results indicate that the adenylyl cyclase-cAMP system has an inhibitory role in ET-1-stimulated HSP27 induction in osteoblasts and that the effect is exerted at the point between PKC and p38 MAP kinase in osteoblasts.

Direct monitoring kinetic studies of DNA polymerase reactions on a DNA-immobilized quartz-crystal microbalance.

Catalytic reactions of DNA polymerase I from E. coli (Klenow fragment, KF) were monitored directly with a template/primer (40/25- or 75/25-mer)-immobilized 27-MHz quartz-crystal microbalance (QCM). The 27-MHz QCM is a very sensitive mass-measuring device in aqueous solution, as the frequency decreases linearly with increasing mass on the QCM electrode at the nanogram level. Three steps in polymerase reactions which include 1) binding of DNA polymerase to the primer on the QCM (mass increase); 2) elongation of complementary nucleotides along the template (mass increase); and 3) release of the enzyme from the completely polymerized DNA (mass decrease), could be monitored continuously from the time dependencies of QCM frequency changes. The binding constant (Ka) of KF to the template/primer DNA was 10(8)M(-1) (k(on) = 10(5)M(-1)s(-1) and k(off)= 10(-3)s(-1)), and decreased to 10(6)M(-1) (k'on = 10(4)M(-1)s(-1) and k'off = 10(-2)s(-1)) for completely polymerized DNA. This is due to the 10-fold decrease in binding rate constant (k(on)) and 10-fold increase in dissociation rate constant (k(off)) for completed DNA strands. Ka values depended slightly on the template and primer sequences. The kinetic parameters in the elongation process (k(cat) and Km) depended only slightly on the DNA sequences. The repair process during the elongation catalyzed by KF could also be monitored in real time as QCM frequency changes.

Mechanism of prostaglandin D(2)-stimulated heat shock protein 27 induction in osteoblasts.

We previously showed that prostaglandin D(2) (PGD(2)) stimulates activation of protein kinase C (PKC). We investigated whether PGD(2) stimulates the induction of heat shock protein (HSP) 27 and HSP70 in osteoblast-like MC3T3-E1 cells and the mechanism underlying the induction. PGD(2) increased the levels of HSP27 while having little effect on HSP70 levels. PGD(2) stimulated the accumulation of HSP27 dose dependently in the range between 10 nM and 10 microM. PGD(2) induced an increase in the levels of mRNA for HSP27. The PGD(2)-stimulated accumulation of HSP27 was reduced by staurosporine or calphostin C, inhibitors of PKC. PGD(2) induced the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase. The HSP27 accumulation induced by PGD(2) was significantly suppressed by PD98059, an inhibitor of the upstream kinase of p44/p42 MAP kinase, or SB203580, an inhibitor of p38 MAP kinase. Calphostin C suppressed the PGD(2)-induced phosphorylation of p44/p42 MAP kinase and p38 MAP kinase. PD98059 or SB203580 suppressed the PGD(2)-increased levels of mRNA for HSP27. These results strongly suggest that PGD(2) stimulates HSP27 induction through p44/p42 MAP kinase activation and p38 MAP kinase activation in osteoblasts and that PKC acts at a point upstream from both the MAP kinases.

Reconstituting telomerase activity using the telomerase catalytic subunit prevents the telomere shorting and replicative senescence in human osteoblasts.

The rate of bone formation is largely determined by the number of osteoblasts, which in turn is determined by the rate of replication of progenitors and the life span of mature cells, reflecting the timing of death by apoptosis. However, the exact age-dependent changes of the cellular activity, replicative potential, and life span of osteoblasts have not been investigated to date. Here, we present evidence that the cellular activity, telomere lengths, and replicative life span of osteoblastic cells obtained from juxta-articular bone marrow gradually decrease with the advance of donor age. Recently, telomerase reverse transcriptase (hTERT) has been identified as a human telomerase catalytic subunit. We transfected the gene encoding hTERT into telomerase-negative human osteoblastic cells from donors and osteoblastic cell strain NHOst 54881 cells and showed that expression of hTERT induces telomerase activity in these osteoblastic cells. In contrast to telomerase-negative control cells, which exhibited telomere shortening and senescence after 10-15 population doublings, telomerase-expressing osteoblastic cells had elongated telomere lengths and showed continued alkaline phosphatase activity and procollagen I C-terminal propeptide (PICP) secretion for more than 30 population doublings. These results indicate that osteoblasts with forced expression of hTERT may be used in cell-based therapies such as ex vivo gene therapy, tissue engineering, and transplantation of osteoblasts to correct bone loss or osteopenia in age-related osteoporotic diseases.

Intra-articular injection of hyaluronate (SI-6601D) improves joint pain and synovial fluid prostaglandin E2 levels in rheumatoid arthritis: a multicenter clinical trial.

OBJECTIVE: The relationship between clinicalfeatures and biochemical parameters of synovialfluid after serial intra-articular injections of sodium hyaluronate (SI-6601D) was investigated. METHODS: SI-6601D (sodium hyaluronate with an average molecular weight of 8.4 x 10(5); 25mg/2.5ml/syringe) was injected intra-articularly into the knees of 25 patients with rheumatoid arthritis (RA) every week for 5 consecutive weeks. Clinical and biochemical parameters were monitored before and after injection. Clinicalfindings included pain, as a summation of 3 categories (pain at rest, pain in motion and pain in passive motion, each assessed on a 4-step rating scale), and inflammation, also as a summation of 3 categories (swelling, patellar ballotement and local warmth, each assessed on a 4-step rating scale). Pain on walking of patient was qualitatively assessed by visual analogue scale (VAS). The aspirated volume of synovialfluid (SFV) was recorded and levels of prostaglandin (PG) E2, transforming growth factor beta-1, tumor necrosis factor alpha, interleukin I receptor antagonist, chondroitin 4-sulfate (C4S) and chondroitin 6-sulfate were measured. RESULTS: Significant improvement in pain symptoms (p < 0.0001), inflammation (p < 0.0001), VAS pain (p < 0.001) and SFV (p < 0.05) were observed after the 5 injections. Levels of PGE2 (p < 0.05) and C4S (p < 0.05) in the synovialfluid were significantly decreased. DISCUSSION: SI-6601D improved local clinical symptoms in RA patients by suppressing PGE2 and, therefore, may be a useful treatment for local inflammation in RA.

Methotrexate inhibits rheumatoid synovitis by inducing apoptosis.

OBJECTIVE: To clarify the pharmacological action of methotrexate (MTX) on the synovium of patients with rheumatoid arthritis (RA) using severe combined immunodeficient (SCID) mice in which human RA synovial tissue had been grafted (SCID-HuRAg). METHODS: One month after engraftment of human RA tissue into SCID mice, MTX (0.3 mg/kg) was administered orally, then the appearance of apoptosis in the grafted tissue was examined by TdT mediated dUTP nick end labeling (TUNEL) staining and electron microscopy at various time points after MTX administration. In cultured synovial cells, synovial apoptotic changes after MTX treatment were studied by agarose gel electrophoresis and flow cytometric analysis. To compare the histological changes induced by MTX with those induced by other disease modifying antirheumatic drugs (DMARD) and a nonsteroidal antiinflammatory drug, histological examination of the grafted synovial tissues from SCID-HuRAg mice was conducted after 4 weeks of oral administration of MTX (0.3 mg/kg/week), salazosulfapyridine (30 mg/kg/day), auranofin (0.2 mg/kg/day), bucillamine (10 mg/kg/day), or indomethacin (2 mg/kg/day). RESULTS: A significant decrease in the number of inflammatory cells was observed in the grafted synovial tissue of MTX treated SCID-HuRAg. A similar antiinflammatory effect was not observed with the other DMARD. Induction of apoptosis was noted with MTX treatment but not with the others. The pro-apoptotic effect of MTX was also observed in synovial cell cultures. CONCLUSION: MTX induces apoptosis in RA synovium that, in turn, may contribute to its antiinflammatory effect on RA synovitis.

Involvement of p38 MAP kinase in TGF-beta-stimulated VEGF synthesis in aortic smooth muscle cells.

Although it is known that transforming growth factor (TGF)-beta induces vascular endothelial growth factor (VEGF) synthesis in vascular smooth muscle cells, the underlying mechanisms are still poorly understood. In the present study, we examined whether the mitogen-activated protein (MAP) kinase superfamily is involved in TGF-beta-stimulated VEGF synthesis in aortic smooth muscle A10 cells. TGF-beta stimulated the phosphorylation of p42/p44 MAP kinase and p38 MAP kinase, but not that of SAPK (stress-activated protein kinase)/JNK (c-Jun N-terminal kinase). The VEGF synthesis induced by TGF-beta was not affected by PD98059 or U0126, specific inhibitors of the upstream kinase that activates p42/p44 MAP kinase. We confirmed that PD98059 or U0126 did actually suppress the phosphorylation of p42/p44 MAP kinase by TGF-beta in our preparations. PD169316 and SB203580, specific inhibitors of p38 MAP kinase, significantly reduced the TGF-beta-stimulated synthesis of VEGF (each in a dose-dependent manner). PD169316 or SB203580 attenuated the TGF-beta-induced phosphorylation of p38 MAP kinase. These results strongly suggest that p38 MAP kinase plays a part in the pathway by which TGF-beta stimulates the synthesis of VEGF in aortic smooth muscle cells.