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To clarify roles of an endogenous pain modulatory system of the central nervous system (CNS) in hyperalgesia, we tried to identify qualitative and quantitative protein changes by a proteomic analysis using an animal model of hyperalgesia. Specifically, we first induced functional hyperalgesia on male Wistar rats by repeated cold stress (specific alternation of rhythm in temperature, SART). We then compared proteomes of multiple regions of CNS and the dorsal root ganglion between the hyperalgetic rats and non-treated ones by 2-D PAGE in the pI range of 4.0-7.0. We found that SART changed the proteomes prominently in the mesencephalon and cerebellum. We thus analyzed the two brain regions in more detail using gels with narrower pI ranges. As a result, 29 and 23 protein spots were significantly changed in the mesencephalon and the cerebellum, respectively. We successfully identified 12 protein spots by a MALDI-TOF/TOF MS and subsequent protein database searching. They included unc-18 protein homolog 67K, collapsin response mediator protein (CRMP)-2 and CRMP-4, which were reported to be involved in neurotransmitter release or axon elongation. Interestingly, mRNA expression levels of these three proteins were not changed significantly by the induction of hyperalgesia. Instead, we found that the detected changes in the protein spots are caused by the post-translational modification (PTM) of proteolysis or phosphorylation. Taken together, development of the hyperalgesia would be linked to PTM of these three CNS proteins. PTM regulation may be one of the useful ways to treat hyperalgesia.

Although adult human cartilage is physiologically avascular tissue, angiogenesis can be observed during the process of endochondral bone development. Inflammation in articular joints can also lead to neovascularization in cartilage. In such conditions, the expression of angiogenic factors, such as vascular endothelial growth factor (VEGF), has been shown to play a key role, controlling not only angiogenesis but also chondrocyte metabolism. Here we review recent research findings concerning the potential role of VEGF in cartilage, focusing in particular on its possible involvement in the pathogenesis of osteoarthritis.

Summary Objectives To evaluate osteoarthritis (OA) of the knee using positron emission tomography (PET) with 2- 18 F-fluoro-2-deoxy-d-glucose ( 18 F-FDG) as a tracer. Materials and methods Fifteen patients with medial-type knee OA and three healthy subjects were enrolled in the study. After clinical examination and conventional radiography, 18 F-FDG PET and magnetic resonance imaging (MRI) were performed. 18 F-FDG uptake was quantified as a standardized uptake value (SUV) and the localization of 18 F-FDG uptake was identified using fusion images created with MRI scans. Results 18 F-FDG generally accumulated in periarticular lesions and was absent in the articular cartilage. SUVs of the whole knee were higher in OA than in controls, and those in the medial condyle were higher than in the lateral condyle in OA. Prominent 18 F-FDG uptake was found in the intercondylar notch in OA and extended along the posterior cruciate ligament (PCL) in some cases. Periosteophytic accumulation was found in one-half of cases with definite osteophytes. Accumulation was also found in subchondral lesions and bone marrow, which corresponded with bone edema diagnosed by MRI. No significant correlation was found between SUV and clinical manifestations. Conclusions 18 F-FDG uptake was upregulated in OA and generally accumulated in periarticular lesions. Increased uptake was found in the intercondylar notch extending along the PCL, periosteophytic lesions, and bone marrow. These results provide in vivo pathognomonic insights into OA.

Objective To identify pathogenic and/or disease-specific short peptides in sera from patients with systemic sclerosis (SSc). Methods Serum samples from 40 patients with SSc, 30 patients with systemic lupus erythematosus, 21 patients with rheumatoid arthritis, 30 patients with osteoarthritis, and 26 healthy donors were tested. Short peptides with molecular weights of smaller than ∼3 kd, purified from the sera by magnetic bead–based hydrophobic interaction chromatography 18, were detected and their amino acid sequences determined using matrix-assisted laser desorption ionization–time-of-flight mass spectrometry. Effects of the identified peptides on fibroblasts and microvascular endothelial cells were tested using synthesized peptides and sera containing the peptides. Results A group of peptides with mass/charge (m/z) values of 1,865, 1,778, 1,691, 1,563, and 1,450 were detected predominantly in the SSc sera. These peptides were identified as family members of complement C3f-des-arginine (DRC3f) derived from C3b. The level of DRC3f (m/z 1,865) was related to vascular involvement in SSc and to SSc disease activity. The synthesized peptides of DRC3f and C3f, as well as the filtrated sera containing DRC3f, enhanced proliferation of microvascular endothelial cells, but not fibroblasts. Both DRC3f and C3f increased production of transforming growth factor β1 by dermal microvascular endothelial cells. Conclusion This comprehensive peptidomics analysis revealed the predominance of DRC3f in the sera of patients with SSc. Investigation of DRC3f may be a useful tool for the diagnosis and evaluation of disease activity in SSc. Moreover, its demonstrated effects on endothelial cells suggest a potential role for DRC3f in the pathophysiologic mechanisms of SSc.

Expression of CD69 on neutrophils and generation of anti-CD69 autoantibodies in patients with rheumatoid arthritis (RA) have been reported. Thus natural ligands for CD69 not yet identified and/or the anti-CD69 autoantibodies possibly affect neutrophils by evoking CD69 signaling, which may further affect joint-composing cells in RA. However, the effect of the CD69 signaling in neutrophils remains largely unclear. To elucidate the issue, we tried to identify proteins affected by the crosslinking of CD69 on neutrophils using a proteomic approach. Specifically, CD69 on granulocyte-macrophage colony stimulating factor (GM-CSF)-activated neutrophils was crosslinked by anti-CD69 monoclonal antibodies, and then intracellular proteins were detected using 2-dimensional electrophoresis and further identified by mass spectrometry and subsequent protein database searching. As a result, we successfully identified multiple proteins that increased their production by the CD69 signaling. Among the proteins, we focused on one of the up-regulated proteins, S100A9 calcium binding protein (S100A9), and investigated proteome changes brought by a recombinant S100A9 in a human synovial sarcoma cell line (SW982), a human chondrosarcoma cell line (OUMS-27), and a human T leukemia cell line (Jurkat). This revealed that the recombinant S100A9 altered proteomes of SW982 and OUMS-27, and to a lesser extent, that of the Jurkat cells. Further, S100A9 induced IL-1beta production from neutrophils and the SW982 cells. These data suggest that unidentified natural ligands for CD69 and/or the anti-CD69 autoantibodies possibly affect joint-composing cell types through the increased production of S100A9 in neutrophils, providing a new insight into functions of CD69 on neutrophils in RA.

Background Postoperative infection following total joint arthroplasty is thought to be one of the most serious and devastating complications. To develop an effective treatment for this complication, we tested a bioabsorbable antibacterial carrier that is made from novel cross-linked hyaluronic acid (HA) gel and gentamicin (GM).

Autoimmunity to chondrocyte-producing proteins has been reported in patients with osteoarthritis (OA) as well as in those with rheumatoid arthritis (RA). To answer whether or not OA-specific autoimmunity exist, we performed screening of chondrocyte-producing autoantigens by two-dimensional electrophoresis and Western blotting with each of 20 OA and 20 RA serum samples. We identified an apparently OA-specific autoantigen spot with a molecular mass of 52 kDa and a Isoelectric point of 4.1 as fibulin-4 by mass fingerprinting. By preparing recombinant proteins of fibulin-4, we determined prevalence of the autoantibodies to fibulin-4 in 92 patients with OA, 67 patients with RA, 40 patients with systemic lupus erythematosus, and 43 patients with systemic scleroderma. As a result, the IgG type anti-fibulin-4 autoantibodies were detected in 23.9% of sera from patients with OA, in 8.9% of sera from patients with RA, in 2.5% of sera from patients with systemic lupus erythematosus, and in 9.3% of sera from patients with systemic scleroderma. Furthermore, we immunized DBA/1J, ICR, BALB/c, and C57BL/6 mice with the recombinant fibulin-4 proteins to investigate arthritogenecity of fibulin-4. As a result, mild synovitis was detected in all of the four strains. In addition, we demonstrated expression of fibulin-4 in chondrocytes at both mRNA and protein levels in vivo and in vitro by RT-PCR, Western blotting, and immunohistochemistry. Taken together, fibulin-4, expressed in chondrocytes and recognized as an autoantigen mainly in OA rather than in RA, may play pathogenic roles in OA.

Relapsing polychondritis (RP) is a systemic inflammatory disease, in which autoimmunity to cartilage-related components is thought to be involved in its pathogenesis. However, the autoimmune profile in RP has not been studied fully. We therefore investigated autoantibodies/autoantigens in RP comprehensively, by 2-dimensional electrophoresis (2DE), subsequent western blotting (WB) and mass spectrometry, using cell-extracted proteins as the antigen source. As a result, we detected 15 autoantigens on 2DE-WB, and further identified five of them. On average, one RP serum recognized approximately 8 out of the 15 autoantigens. Frequencies of the autoantibodies to the 5 identified antigens of tubulin alpha ubiquitous/6, vimentin, alpha enolase, calreticulin, and colligin-1/-2 were 91%, 46%, 36%, 82%, and 36%, respectively. ELISA using recombinant proteins for them revealed that frequencies of the autoantibodies to tubulin alpha ubiquitous, vimentin, alpha enolase, calreticulin, and colligin-1 were 36%, 64%, 46%, 27%, and 18%, respectively. Our data demonstrated that the autoimmune reaction was not restricted to cartilage-related components, rather a variety of autoimmune responses occurred in patients with RP, which may be involved in the pathophysiology of RP. In addition, the proteomic approach using cell-extracted proteins would be a powerful way to investigate autoantigens.

We conducted a large scale epidemiological study in Japan and revealed a high mortality rate in RP patients with neurological involvement. Japanese RP patients developed encephalitis/meningitis (12 out of 239 cases, 5.0%), cerebral infarct/bleeding (5 cases, 2.1%) and cerebral vasculitis (4 cases, 1.7%). The mortality rate was 18%, in contrast to 8.1% of RP patients without neurological involvement. We suggested that neurological involvement appeared to be a major determinant of disease severity in patients with RP.

Objective: To clarify the effect of interleukin (IL) 18 on cartilage degeneration by studying the profile of IL18 receptor (IL18R) on chondrocytes and the direct effect of IL18 on production of matrix metalloproteinases (MMPs), aggrecanases, and tissue inhibitors of metalloproteinases (TIMPs) in articular chondrocytes. Methods: Monolayer cultured human articular chondrocytes were isolated from non-arthritic subjects and patients with rheumatoid arthritis or osteoarthritis. Gene expression of IL18, IL18Rα, IL18Rβ, MMPs, and aggrecanases was detected by RT-PCR. Protein levels of IL18Rα were analysed by flow cytometry. Protein levels of IL18, MMPs, and TIMPs were measured by ELISA. Aggrecanase-2 mRNA expression was quantitatively analysed by real time RT-PCR. Protein level s of signalling molecules were assayed by western blotting. Results: IL18 mRNA was constitutively expressed in chondrocytes, and was enhanced by IL1β stimulation. Flow cytometric analysis showed that IL1β, tumour necrosis factor α, and IL18 up regulated IL18Rα expression levels. The level of IL18Rβ mRNA was much lower than that of IL18Rα, and was slightly up regulated by IL1β. In chondrocytes responding to IL18, IL18 (1–100 ng/ml) slightly increased the production of MMP-1, MMP-3, and MMP-13, which was blocked by NF-κB inhibitor and p38 mitogen activated protein kinase inhibitor. IL18 up regulated mRNA expression of aggrecanase-2, but not aggrecanase-1. IL18 also slightly stimulated TIMP-1 production?through extracellular signal regulated kinase activation. Conclusion: IL18 induces production of MMPs from chondrocytes in inflammatory arthritis. Although the direct effect of IL18 on chondrocytes may not be pivotal for the induction of cartilage degeneration, IL18 seems to play some part in the degradation of articular cartilage in arthritis.

STUDY DESIGN Functional recovery and histopathological change after spinal cord injury in the Fas-deficient mice and the wild-type mice were investigated. OBJECTIVES To investigate the role of the Fas/Fas ligand (FasL) system as a signal transduction pathway leading to apoptosis after spinal cord injury. SUMMARY OF BACKGROUND DATA [corrected] Apoptosis observed after spinal cord injury has recently gained widespread interest as a cause of collateral damage after the initial injury. Apoptosis mediated by the Fas antigen in the postischemic brain or spinal cord was reported. Recently, the upregulation of Fas after spinal cord injury was also reported. However, the influence of Fas-mediated apoptosis on the extent of the secondary spinal cord injury has not yet been clarified. METHODS We investigated Fas-mediated apoptosis after spinal cord injury and examined the behavioral changes and the histopathological changes after spinal cord injury using MRL/Mp-lpr/lpr (MRL/lpr) mice, which were Fas-deficient mutant mice, and MRL/Mp-+/+ (MRL/+) mice, which were Fas-positive wild-type mice. RESULTS Locomotor recovery after spinal cord injury in MRL/lpr mice was superior to that observed in MRL/+ mice. In addition, the damaged area in MRL/lpr mice was significantly smaller than that seen in MRL/+ mice. Further, the frequency of apoptotic cells in the injured spinal cord of MRL/lpr mice was significantly less than that in MRL/+ mice. CONCLUSION We demonstrated the appearance of Fas-mediated apoptosis in the spinal cord after spinal cord injury. In addition, we elucidated for the first time that Fas-mediated apoptosis following spinal cord injury played an important role in the spinal cord damage and the ultimate neurologic injury.

Objective At sites of inflammation, T cells exert pathologic effects through direct contact with monocyte/macrophages, inducing massive up-regulation of interleukin-1 (IL-1) and tumor necrosis factor α (TNFα). We examined the regulatory effects of IL-18 on monocyte activation by direct contact with T lymphocytes in rheumatoid arthritis (RA). Methods Activated T cells were isolated from RA synovial fluid. Resting T cells and monocytes were isolated from peripheral blood mononuclear cells. RA synovial T cells or phytohemagglutinin (PHA)–stimulated T cells were fixed by paraformaldehyde and then cocultured with monocytes at a ratio of 4:1. Levels of TNFα, IL-1β, IL-10, and IL-18 were measured by enzyme-linked immunosorbent assay. Expression of adhesion molecules, IL-18 receptor, and TNF receptors was analyzed by flow cytometry. Expression of NF-κB p65, phosphorylated IκBα, and phosphatidylinositol 3-kinase (PI 3-kinase) p110 was analyzed by Western blotting. Results IL-18 dose-dependently enhanced the production of IL-1β and TNFα, but not IL-10, by monocytes following contact with RA synovial T cells or PHA-prestimulated T cells. NF-κB inhibitors N-acetyl-L-cysteine and Bay 11-7085 and PI 3-kinase inhibitor LY294002 inhibited the enhancing effects of IL-18, but MAPK p38 inhibitor SB203580, ERK inhibitor PD98059, and JNK inhibitor SP600125 did not. Increased levels of NF-κB in the nucleus, phosphorylated IκB, and PI 3-kinase were confirmed in monocytes cocultured with PHA-prestimulated T cells, and the levels were further increased by stimulation with IL-18. Neutralizing antibody to IL-18 inhibited monocyte activation induced by direct contact with PHA-prestimulated T cells. Via cell–cell contact, PHA-prestimulated T cells increased autocrine production of IL-18 by monocytes, which was mediated by activation of the NF-κB and PI 3-kinase pathways, and up-regulated the expression of the IL-18 receptor in monocytes. IL-18 up-regulated the expression of the TNF receptors vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) on monocytes. Blocking the binding of the TNF receptors VCAM-1 or ICAM-1 on monocytes to their ligands on stimulated T cells suppressed the IL-18–enhanced production of TNFα and IL-1β in monocytes induced by contact with PHA-prestimulated T cells. Conclusion IL-18 augments monocyte activation induced by contact with activated T cells in RA synovitis, which is dependent on activation of the NF-κB and PI 3-kinase pathways. IL-18 up-regulates the expression of the TNF receptors VCAM-1 and ICAM-1 on monocytes, which mediate the enhancing effects of IL-18 on T cell–monocyte contact.

We investigated the characteristics of morphology, DNA synthesis and argyrophilic nucleolar organizer regions (AgNORs) on murine Dunn osteosarcoma cells in response to heat (42 degrees C, 1 h) or dibutyryl cyclic adenosine 3',5'-monophosphate (Bt(2)cAMP). The cell morphology changes to a fibroblast-like appearance with long and thin protoplastic processes with the reduction of DNA synthesis by heat. It is closely similar to a response by Bt(2)cAMP. In the presence of 3 mM Bt(2)cAMP, the mean number of AgNORs was significantly decreased in 48 h compared with the untreated group. It was increased conversely by heat. Among these responsive cells, we can also find many cells stained without discrimination by the use of AgNORs staining. The present study provides a new clue to support differentiation of osteosarcoma cells from the viewpoint of hyperthermia in vitro.

The aim of the present study was to compare histological findings and clinical symptoms of patients with advanced stages of rheumatoid arthritis (RA). Synovial tissue specimens were obtained during reconstructive knee surgery from 93 RA patients (18 men; 75 women). The histological assessments of specimens were evaluated using two histological scoring systems reported by Rooney and Koizumi. Clinical symptoms (duration of morning stiffness, joint score, grip strength), laboratory data (erythrocyte sedimentation ratio, C-reactive protein (CRP), rheumatoid factor), X-ray findings (Larsen score) and drug usage were assessed before surgery. Significant statistical correlations between both histological scoring systems were observed; however, there was no significant correlation between the clinical findings and the histological scoring systems. A statistically significant correlation was found between the levels of CRP and Koizumi's scoring system. In addition, Koizumi score correlated significantly to X-ray findings. Rooney's scoring system had an inverse correlation to methotrexate history. Histological findings do not correlate to simultaneous clinical symptoms in advanced RA patients. However, our data indicate that observed histological changes reflect X-ray damage.

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 a (TNF-a) 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-a monoclonal antibody (mAb, 100 mgumouse) and histological changes were examined 4 weeks after the initial treatment. Secondly, a total of 100 mg of recombinant TNF-a or IL-6 (0.6 mguh) 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-a mAb treatment; conversely, the degree of synovial inflammation was significantly exacerbated by TNF-a administration. The levels of both IL-6 and TNF-a in sera were significantly increased by recombinant TNF-a administration, while TNF-a levels were unchanged by IL-6 administration. This suggests that TNF-a 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-a. Conclusion. This study provides strong evidence that TNF-a is a key molecule in the control of the inflammatory changes that occur in the RA synovium. In addition, TNF-a regulates IL-6 production. However, other inflammatory pathways independent of TNF-a may contribute to the bone and cartilage damage seen in RA. Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by hyperplasia of the synovium and an excess of inflammatory cells, leading to progressive destruction of the joints. In RA, several cytokines, e.g. interleukin (IL)-1, IL-6, IL-8, IL-12, IL-17, tumour necrosis factor-a (TNF-a), interferon-c (IFN-c) and granulocyte‐macrophage colony-stimulating factor (GM-CSF), are involved in almost all aspects of articular inflammation and destruction w1x. TNF-a has been considered a pivotal cytokine in the pathogenesis of RA, as significant clinical and laboratory evidence has been obtained by TNF-a blockade w1‐3x. Moreover, it has been verified that neutralized monoclonal antibodies (mAb) against TNF-a can reduce the production of other proinflammatory cytokines, such as IL-1 and GM-CSF, in cultured RA synovia w4x .I n the clinical trials performed to date, anti-TNF-a mAb has been effective in the majority of treated RA patients. Moreover, anti-TNF-a mAb may reduce tissue perfusion in the inflamed synovium, according to the results

Apurinic/apyrimidinic endonuclease 2 (Apex 2) plays a critical role in DNA repair caused by oxidative damage in a variety of human somatic cells. We speculated that chondrocyte Apex 2 may protect against the catabolic process of articular cartilage in osteoarthritis (OA). Higher levels of Apex 2 expression were histologically observed in severely compared with mildly degenerated OA cartilage from STR/OrtCrlj mice, an experimental model which spontaneously develops OA. The immunopositivity of Apex 2 was significantly correlated with the degree of cartilage degeneration. Moreover, the OA-related catabolic factor interleukin-1β induced the expression of Apex 2 in chondrocytes, while Apex 2 silencing using small interfering RNA reduced chondrocyte activity in vitro. The expression of Apex 2 in chondrocytes therefore appears to be associated with the degeneration of articular cartilage and could be induced by an OA-related catabolic factor to protect against the catabolic process of articular cartilage. Our findings suggest that Apex 2 may have the potential to prevent the catabolic stress-mediated down-regulation of chondrocyte activity in OA.

Sirtuin 2, which is mainly present in cytoplasm, plays an important role in mammalian development, caloric restriction, metabolic regulation cellular antioxidant potential and the regulation of aging. We found that the protein level of sirtuin 2 in human peripheral blood mononuclear cells (PBMCs) decreases with an advance in donor age in men and women. Our data suggest that sirtuin 2 level in PBMCs decreases with age in both men and women and may have a potential as a useful biomarker monitoring health conditions and aging.

Layilin (LAYN) is thought to be involved in reorganization of cytoskeleton structures, interacting with merlin, radixin, and talin. Also, LAYN is known to be one of the receptors for hyaluronic acid (HA). In rheumatoid arthritis (RA), inflammatory cytokines like tumor necrosis factor α (TNF-α) have been known to play pathological roles. HA with low molecular weight is speculated to exacerbate inflammation in RA. In this context, differences of quantity and functions of HA receptors would affect the severity of inflammation in RA. Chondrocytes, which play critical roles in maintaining articular cartilage and are affected in RA, express at least kinds of HA receptors like CD44 and LAYN. However, roles and regulation of LAYN in articular chondrocytes have been poorly understood. To clarify regulation of LAYN in chondrocytes, we here investigated whether TNF-α affected expression levels of LAYN in human articular chondrocytes. Next, to clarify LAYN-specific roles in chondrocytes, we investigated whether binding of antibodies to the extracellular domain of LAYN affected secretion of inflammatory cytokines using a chondrosarcoma cell line. As a result, we found that TNF-α up-regulated expression levels of LAYN in the chondrocytes. Further, the LAYN signaling was found to enhance secretion of inflammatory factors, IL-8 and complement5 (C5)/C5a, from the cells. Our results indicate that LAYN would be involved in the enhancement of inflammation and degradation of cartilage in joint diseases such as RA and OA.

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.

Objective Periarticular osteopenia is frequently observed in rheumatoid arthritis (RA). Bone loss has been considered to be at least partly due to inadequate bone formation, which in turn, is largely dependent on the number of osteoblasts and the osteoblastic activity. Normal human somatic cells undergo a finite number of cell divisions and ultimately enter a nondividing state called replicative senescence. It has been proposed that the telomere, the terminal sequence of chromosomes, is the mitotic clock that triggers senescence. In the present study, we sought to clarify the relationship between periarticular osteopenia and osteoblast replicative senescence in RA. Methods We examined age-related changes in cellular activity (alkaline phosphatase activity, osteocalcin and C-terminal type I procollagen secretion, and cAMP response to parathyroid hormone), replicative capacity, and senescent cell expression in osteoblasts from periarticular bone samples obtained from 15 patients with RA and 15 age-matched patients with osteoarthritis (OA). Cellular replicative capacity was analyzed by the mean telomere length and in vitro remaining replicative lifespan of the cells. Results In both OA and RA groups, the cell proliferation rate, the levels of osteoblastic markers, mean telomere length, and replicative lifespan in osteoblastic cells gradually decreased with the increasing age of the donor. The percentage of senescent osteoblastic cells in the periarticular bone increased with age in both groups, and the rate of expression of senescent cells was higher in RA patients than in age-matched OA patients. The osteoblastic activities and replicative capacity of osteoblastic cells from RA patients were lower than those from OA patients at any donor age. The age-related decreases in the osteoblastic activity and replicative capacity of osteoblastic cells from periarticular bone were greater in RA patients than in OA patients. Conclusion Our results suggest that osteoblast replicative senescence in periarticular bones occurs more rapidly with aging in RA than in OA patients and contributes to periarticular osteopenia in RA.

Objective To elucidate the involvement of telomerase activity in the pathogenesis of rheumatoid arthritis (RA) and pigmented villonodular synovitis (PVS). Methods Peripheral blood lymphocytes (PBL), synovial infiltrating lymphocytes, and synoviocytes were isolated from peripheral blood samples and synovial tissue obtained from 18 patients with RA, 9 with PVS, 12 with osteoarthritis (OA), and 10 with knee joint trauma. Cellular telomerase activity was measured by the telomeric repeat amplification protocol assay. In RA patients, the telomerase activity level in synovial infiltrating lymphocytes was assessed for correlations with histologic features in rheumatoid synovium. Results A high level of telomerase activity was detected in the PBL and synovial infiltrating lymphocytes from RA patients and in the synoviocytes from PVS patients, whereas the enzyme activity was expressed at a low-to-borderline level in the PBL and synovial lymphocytes from OA, PVS, and trauma patients and was absent in the synoviocytes from RA as well as OA and trauma patients. In RA patients, the telomerase activity level in synovial infiltrating lymphocytes was significantly correlated with the intensity of synovial lining hyperplasia, microvessel proliferation, lymphocyte infiltration, and percentage of synovial cells positive for proliferating cell nuclear antigen in rheumatoid synovium. Conclusion Telomerase activation in lymphocytes may provide insights into the progression of synovitis and synovial proliferation in RA. Moreover, the enzyme may be implicated in the proliferation of synoviocytes in PVS.

Objective: The purpose of this study was to analyze the biochemical characteristics of synovial fluids after treatment of patients with rheumatoid arthritis (RA) with intra-articularly injected hyaluronate (HA).¶Treatment: In a double-blind study, 13 patients received intra-articular injections of 1% HA in phosphate buffered saline (PBS) while 13 other patients were injected 0.01% HA in PBS, as the control group. Injections were administered once a week for five weeks.¶Methods: Clinical efficacy and characteristics of synovial fluid were compared between groups.¶Results: Significant clinical improvement was observed in the HA-treated group compared to the control group. The viscosity of retained synovial fluid increased. Stringing and HA concentration increased significantly whereas the concentrations of protein and chondroitin sulfate 4 and 6 decreased significantly. The HA molecular weight remained the same in both groups.¶Conclusion: Intra-articular HA injection altered the properties of synovial fluid and proved efficacious for patients with RA.

We investigated the role of 1α,25-dihydroxyvitamin D3 (1α,25[OH]2 D3 ) in modulating tumor cell invasiveness through the extracellular matrix (ECM) and pulmonary metastasis in B16 mouse melanoma. The pretreatment of B16 cells for 48 hours with 1α,25(OH)2 D3 significantly inhibited in vitro invasiveness through the ECM by a mechanism that is not directly correlated with the inhibition of cell proliferation. When cells were treated with 1α,25(OH)2 D3 for only 8 hours during the assay, no inhibitory effect was observed, suggesting that pretreatment with the hormone for more than 8 hours is necessary to inhibit the invasive potential of B16 cells. The activity of B16 cells to adhere to reconstituted basement membrane (Matrigel) and type IV collagenolysis was inhibited by pretreatment of the cells with 1α,25(OH)2 D3 for 48 hours. Cell motility was not influenced by the hormone. Mice were inoculated subcutaneously with 3 × 106 B16 cells and were given 1α,25(OH)2 D3 (0.5 μg/kg) or vehicle daily for 28 days, beginning 1 day after tumor inoculation. In the 1α,25(OH)2 D3 -treated group, no significant inhibition in exponential tumor growth, body weight, and serum level of calcium was observed until the twenty-eighth day. The mean serum concentration of the hormone was about 50 ng/mL, and there were no significant changes in its concentration during the treatment period. In both spontaneous and experimental metastasis models of tumor-bearing mice, treatment with 1α,25(OH)2 D3 inhibited pulmonary metastasis. These findings suggest that 1α,25(OH)2 D3 acts on B16 cells, inhibiting invasiveness through the ECM that is caused by the inhibition of cell adhesion to the ECM and the degradation of the ECM by the cells. 1α,25(OH)2 D3 may have the potential to inhibit metastasis by a mechanism that is not exclusively based on its anti-cell proliferative effect. (J Lab Clin Med 1999;133:120-8)

Interleukin 1 alpha (IL-1alpha) production and invasiveness through mouse lung endothelial cells (MLE) were investigated in high-metastatic RCT+ and low-metastatic RCT- clones established from poorly differentiated murine sarcoma. Apparently, a higher level of IL-1alpha was derived from RCT+ cells than from RCT- cells. In an invasion assay, the number of cells which penetrated the MLE monolayer in RCT+ was significantly greater than that in RCT-. The invasiveness of RCT+ and RCT- cells was stimulated by additional recombinant mouse IL-1alpha (rIL-1alpha) in a dose-dependent manner. Anti-mouse IL-1alpha monoclonal antibody (anti-IL-1alpha mAb) significantly inhibited the invasiveness of RCT+ and RCT- cells through the MLE monolayer. However, in RCT+ cells these effects were higher than in RCT- cells. In an attachment assay, the ability of RCT+ cells to attach to the MLE monolayer was significantly higher than that of RCT- cells. The attachment ability of RCT+ and RCT- cells to the MLE monolayer was significantly increased by the pretreatment with rIL-1alpha in a dose-dependent manner. In a retraction assay, conditioned medium of RCT+ stimulated the retraction of the MLE monolayer more markedly in comparison with conditioned medium of RCT-. The retraction of the MLE monolayer was stimulated by additional rIL-1alpha in a dose-dependent manner. The increased retraction of the MLE monolayer was closely associated with the enhancement in tumor cell invasiveness. These findings suggest that IL-1alpha derived from RCT+ and RCT- cells might contribute to the enhancement of tumor cell invasion by stimulating the attachment to the MLE monolayer and retraction of the MLE monolayer.

Many animal models have been developed for a study of rheumatoid arthritis (RA). However, RA animal models are not always similar to RA patients in their response to antirheumatic drugs. Recently, humanized monoclonal antibody (mAb) has been developed for the treatment of RA, but at present there is no animal model on which to screen this mAb therapy because of problems with cross-reactivity. We therefore considered the development of a novel animal model for the screening of antirheumatic drugs using the severe combined immunodeficiency (SCID) mouse in order to prevent the rejection of human transplant cells. Following subcutaneous implantation of synovial tissue in the SCID mouse, all target cells within the SCID-HuRAg mouse were of human origin, having migrated from the implanted tissue. Moreover, human interlukin-6 and rheumatoid factor were detected in this mouse serum. We therefore propose that this SCID-HuRAg mouse is a novel, useful animal model for the study and development of new drugs for RA patients. This novel RA animal model is reviewed in this chapter.

Human fibrosarcoma HT1080 cell surface phenotype analysis revealed the expression of "cluster of differentiation 15" (CD15) antigen and to a lesser extent, of "very late antigen-4" (VLA-4). Expression of "endothelial-leukocyte adhesion molecule-1" (ELAM-1) was negligible on resting human umbilical vascular endothelial cells (HUVECs), but its expression could be induced by HT1080 conditioned medium. HT1080 cell adhesion to HUVECs was partially dependent on CD15/ELAM-1 adhesion molecules. HT1080 cell adhesion to HUVECs induced the enhancement of nitric oxide (NO) production from HUVECs. Exogenous NO and NO from HUVECs enhanced ELAM-1 expression on HUVECs, HT1080 cell adhesion to HUVECs, permeability of the HUVEC monolayer, and HT1080 cell invasion through the HUVEC monolayer. These enhancements were not induced by NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME). These results suggest that NO expression induced by tumor cells via the CD15/ELAM-1 adhesion system may contribute to enhancement of tumor cell adhesion to endothelial cells and hyperpermeability of the endothelium, facilitating tumor cell invasion.

We investigated the clinical relevance of doubling time (DT) of serum laboratory data obtained in routine clinical examination of patients with primary bone and soft tissue tumors, in comparison with major clinical and pathological parameters (age at presentation, sex, tumor size, location, clinical stage and histologic grade) by uni‐ and multivariate analyses. In 64 patients with primary bone and soft tissue tumors (primary bone tumors: 39, primary soft tissue tumors: 25) and 68 cancer patients, the pretreatment DT values of serum C‐reactive protein (CRP), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), calcium (Ca), phosphate (P) levels were measured, as well as the erythrocyte sedimentation rate (ESR: mm/60 min); these values were then compared with overall survival, local recurrence‐free survival and metastasis‐free survival. Only DT of CRP and ALP (CRP‐DT, ALP‐DT) were found to be correlated with disease outcome in patients with primary bone and soft tissue tumors. In cancer patients, only CRP‐DT showed a relation with clinical stage and histologic grade, but the ALP‐DT in patients with bone metastasis was significantly shorter than that in patients with metastases at other sites or in those with no metastasis. Among all tumor patients, those with bone metastasis showed the shortest ALP‐DT compared with those with lung, liver and brain metastasis. Univariate analysis showed that shorter CRP‐DT and ALP‐DT are associated with poor overall survival, and the development of local recurrence and metastasis. These findings suggest that pretreatment CRP‐ and ALP‐DT could be additional prognostic parameters for disease outcome in patients with primary malignant bone and soft tissue tumors. However, in multivariate analysis, only ALP‐DT, but not CRP‐DT, was an independent prognostic parameter for these disease outcomes.

We investigated the invasiveness through the extracellular matrix and pulmonary metastatic potential in high laminin-adherent [LN(+)] and low laminin-adherent [LN(-)] Dunn oseosarcoma cells selected for adhesiveness to laminin. In the invasion assay using a reconstituted basement membrane (matrigel) in a Boyden chamber, LN(+) cells proved to be more invasive than LN(-) and the parental Dunn cells. Pulmonary metastatic potential was correlated with invasiveness through the matrigel in three cell types. The ability of LN(+) cells to attach to laminin and the matrigel was significantly higher than that of LN(-) or the parental Dunn cells. LN(-) cells showed much lower attachment ability compared to the other cells. There were no significant differences in type IV collagenolysis and cell migration among the three cell types. In LN(+) and the parental Dunn cells, laminin significantly stimulated type IV collagenolytic and migration activities. LN(-) cells showed no significant differences of type IV collagenolysis and cell migration in response to laminin. These findings suggested that the different invasiveness of these cells was associated with the difference in the abilities of cell attachment to laminin and type IV collagenolysis and cell migration activities stimulated by laminin. YIGSR inhibited not only the laminin-mediated cell attachment but also the type IV collagenolysis and cell migration induced by the cell attachment to laminin. The amount of laminin receptor of LN(+) cells was about fourfold that of LN(-) and twofold that of the parental Dunn cells, suggesting the stimulatory effect of laminin on the invasiveness through the matrix is associated with the level of laminin receptor expression in these cells. The present study provides several findings suggesting that laminin has important roles in invasiveness through the extracellular matrix and metastasis formation in Dunn osteosarcoma cells.

To investigate the pathophysiology of Behçet's disease (BD) and find biomarkers for the disease, we analysed protein profiles of peripheral blood mononuclear cells (PBMCs).Proteins, extracted from PBMCs, were comprehensively analysed in 16 patients with BD, 16 patients with rheumatoid arthritis (RA), 12 patients with Crohn's disease (CD), and 16 healthy control subjects (HC) by 2-dimensional differential gel electrophoResis (2D-DIGE). Differently expressed proteins were identified by mass spectrometry.563 protein spots were detected. We completely discriminated between the BD and HC groups, between the BD and RA groups, and between the BD and CD groups by multivariate analysis of intensity of 23, 35, and 1 spots, respectively. The spots contributing to the differences included proteins related to cytoskeleton, transcription/translation, T cell activation, bone turnover, regulating apoptosis, and microbial infection. Intensity of 3 spots (tyrosine-protein phosphatase non-receptor type 4, threonine synthase-like 2, and β-actin) provided area under the receiver operating characteristic curves (AUROC) of 0.889 for discrimination between the BD group and the non-BD groups. Informatively, intensity of the above 1 spot completely discriminated the CD group from the other groups (AUROC 1.000). This spot, identified as β-actin, had different pI from the above β-actin-spot probably due to different post-translational modification.PBMC protein profiles, especially the profile of the 3 spots, would be candidate biomarkers for BD. The latter β-actin subtype would be useful for discriminating inflammatory bowel diseases from BD and other diseases. The identified proteins may play important roles in the pathophysiology of BD.

We investigated the roles of extracellular matrix proteins, laminin and fibronectin, in promoting invasiveness through the extracellular matrix in high-metastatic [RCT(+)] clone cells established from poorly differentiated murine RCT sarcoma in C3H/He mice. Laminin stimulated the type IV collagenolytic activity of RCT(+) cells. After more than 6 h of incubation, the type IV collagenolysis of the cell-conditioned medium was significantly higher in laminin-treated groups compared with the control. The migration activity of RCT(+) cells was stimulated by laminin. However, fibronectin did not influence the type IV collagenolysis or cell migration in this clone cell. The amino acid sequence YIGSR, which is derived from laminin, inhibited the laminin-mediated cell attachment and the laminin-promoted type IV collagenolysis, as well as cell migration of RCT(+) cells. RGD derived from fibronectin did not influence the cell attachment to laminin or Matrigel in this clone. In the invasion assay employing a Matrigel coated filter in a Boyden chamber, YIGSR showed greater inhibition of invasion through the Matrigel than did RGD with RCT(+) cells. YIGSR might inhibit the promoted-matrix degradation and cell migration in response to the cell attachment to laminin by competing with laminin for binding to cell surface laminin receptor. We suggest that laminin-mediated cell attachment to the extracellular matrix may play a role in promoting the matrix degradation and cell migration during metastatic cascades.

Background. Besides its involvement in the cardiovascular system, the renin-angiotensin-aldosterone (RAS) system has also been suggested to play an important role in inflammation. To explore the role of this system in cartilage damage in arthritis, we investigated the expression of angiotensin II receptors in chondrocytes. Methods. Articular cartilage was obtained from patients with osteoarthritis, rheumatoid arthritis, and traumatic fractures who were undergoing arthroplasty. Chondrocytes were isolated and cultured in vitro with or without interleukin (IL-1). The expression of angiotensin II receptor types 1 (AT1R) and 2 (AT2R) mRNA by the chondrocytes was analyzed using reverse transcription-polymerase chain reaction (RT-PCR). AT1R expression in cartilage tissue was analyzed using immunohistochemistry. The effect of IL-1 on AT1R/AT2R expression in the chondrocytes was analyzed by quantitative PCR and flow cytometry. Results. Chondrocytes from all patient types expressed AT1R/AT2R mRNA, though considerable variation was found between samples. Immunohistochemical analysis confirmed AT1R expression at the protein level. Stimulation with IL-1 enhanced the expression of AT1R/AT2R mRNA in OA and RA chondrocytes. Conclusions. Human articular chondrocytes, at least partially, express angiotensin II receptors, and IL-1 stimulation induced AT1R/AT2R mRNA expression significantly.

Aim Although sphingosine-1-phosphate (S1P) is suggested to have an important role in arthritis, its function in chondrocytes remains unknown. In contrast, vascular endothelial growth factor (VEGF) has been speculated to contribute to the pathogenesis of osteoarthritis (OA), most likely by regulating angiogenesis. We here investigated the in vitro effect of S1P on VEGF expression in human articular chondrocytes from OA patients. Methods Human articular cartilage samples were obtained from patients with OA under informed consent. Chondrocytes were isolated by an enzymatic procedure, grown in monolayer culture, and then stimulated with S1P in the presence or absence of mitogen-activated protein kinase (MAPK) inhibitors or the Gi protein inhibitor pertussis toxin (PTX). VEGF expression and secretion in culture supernatants were analyzed using real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Results Although S1P did not enhance basal secretion of matrix metalloproteinase (MMP)-1 and MMP-13, it stimulated VEGF expression in human articular chondrocytes, both at the messenger RNA and protein levels. MAPK inhibitors SB203580 and PD98059 were not effective at suppressing VEGF induction; rather, blocking extracellular signal-regulated kinase (ERK) MAPK enhanced VEGF expression. The Gi protein inhibitor PTX partially attenuated S1P-induced VEGF secretion. Conclusion Our results suggest that S1P may contribute to the regulation of VEGF expression in human chondrocytes. S1P may therefore play a unique role in the pathophysiology of OA by regulating VEGF expression in chondrocytes.

Saturable, specific, high-affinity calcitonin receptors were demonstrated in cultured neoplastic mononuclear spindle cells from a giant-cell tumor of the sacrum of a 38-year-old woman. The receptor was analyzed by autoradiography and125I-calcitonin binding assay. Binding reversibility of125I-calcitonin to the cells was not complete and the structural specificity was indicated by the inability of unrelated hormones to compete with calcitonin. The 24 000 receptors/cell and dissociation constant (K d ) of 8.0×10−10 M, calculated from linear Scatchard plots, suggested the existence of a single class of calcitonin binding sites in the neoplastic mononuclear cells. Flow-cytometric analysis in the primary culture showed that mononuclear cells consisted of mononuclear round cells of monocyte/macrophage lineage, which express more calcitonin receptors than neoplastic mononuclear spindle cells. Administration of calcitonin caused morphological and physiological alterations, resulting in involutional or irregular cytoplasmic shapes and inhibition of DNA synthesis in neoplastic mononuclear cells accompanied by the escape phenomenon. Cells preincubated with caleitonin showed a decrease in125I-calcitonin binding activity, which could account for the escape phenomenon. The decrease in125I-calcitonin binding was rapid, but the recovery was not observed for 24 h after elimination of calcitonin. This decrease may be caused by the disappearance of residual receptors or by a decrease in calcitonin affinity. The calcitonin-induced morphological changes and the inhibition of DNA synthesis of cells were revealed to be mediated by caleitonin receptors.

We investigated the invasiveness of tumor cells through the extracellular matrix and the influence of epidermal growth factor (EGF) on tumor cell invasion using in vitro systems in high‐[RCT(+)] and low‐metastatic [RCT(–)] clones established from poorly differentiated murine RCT sarcoma in C3H/He mice. In the invasion assay using a filter coated with reconstituted basement membrane (Matrigel) in a Boyden chamber, RCT(+) cells were more invasive than RCT(‐) cells. The attachment of RCT(+) cells to extracellular matrix components and the degradation of type IV collagen by the cells were significantly greater than with RCT(–) cells. However, there was no significant difference in the migration of cells to the extracellular matrix components between cultured RCT(+) and RCT(‐) cells. These findings suggested that the different invasiveness of these clone cells was associated with the difference in the ability of attachment to and degradation of the matrix. The level of laminin receptor expression in RCT(+) cells was about four‐fold that in RCT(–) cells and laminin stimulated the type IV collagenolytic activity of RCT(+) cells, suggesting that RCT(+) cell attachment to laminin via laminin receptor on the cell surface induced the production of type IV collagenase by the tumor cells. EGF did not affect the invasiveness of RCT(–) cells. In RCT(+) cells, EGF stimulated the invasiveness through Matrigel, the attachment to extracellular matrix components and the degradation of type IV collagen through high‐affinity EGF receptors (EGFR), with Kt of pM order, while the migration to the matrix was not influenced by EGF. These findings suggest that the stimulatory effect of EGF on invasion is related to the acceleration of cell adhesion, and the degradative cascade of the extracellular matrix and high‐affinity EGFRs play an important role in the effect of EGF on in vitro invasiveness in this tumor.

Antibodies to the anti-oxidative peroxiredoxin (Prx) enzymes occur in both systemic autoimmune disease and vasculitis in adulthood. Because increased oxidative stress induces vasculitis in Kawasaki disease (KD), autoimmunity to Prxs in patients with KD was investigated. The presence of antibodies to Prx 1, 2 and 4 was analyzed by ELISA and Western blot. Of 30 patients with KD, 13 (43.3%) possessed antibodies to Prx 2, whereas these antibodies were present in only 1 of 10 patients (10.0%) with sepsis (4 with purulent meningitis and 6 with septicemia). In contrast, antibodies to Prx 1 and 4 were not detected in either group. There was no significant correlation among the titers of the three antibodies. Clinical parameters were compared between anti-Prx 2-positive and -negative patients. The presence of anti-Prx 2 antibodies correlated with a longer period of fever and poor response to high-dose γ-globulin therapy in patients with KD. Anti-Prx 2-positive patients had significantly greater excretion of urinary 8-isoprostaglandin than did anti-Prx 2-negative patients. These results provide the first evidence for an antibody to Prx 2 in patients with KD. They also suggest that this antibody might serve as a marker of disease severity and be involved in the pathophysiology of vasculitis in some patients with KD.

In our previous proteomic surveillance, we found that at least 11 proteins in neutrophils were increased more than 2.5-fold by the stimulation of GM-CSF. In this paper, focusing on one of the 11 proteins, S100 calcium binding protein A8 (S100A8), we tried to elucidate the effect of S100A8 and the cooperative effect of S100A8 and GM-CSF on production and secretion of cytokines of neutrophils.S100A8 in neutrophil was detected by western blotting, and concentrations of S100A8 in synovial fluid (SF) from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) were measured by ELISA. Cytokine levels in the culture medium of neutrophils incubated with and without S100A8 were measured by an antibody array. IL-8 and IL-16 levels in the culture medium of neutrophils stimulated with S100A8, GM-CSF, and the combination of S100A8 and GM-CSF were measured by ELISA. The mRNA levels of IL-8 and IL-16 in the stimulated neutrophils were analysed by real-time PCR.The western blotting analysis confirmed that S100A8 is up-regulated in neutrophil by the stimulation of GM-CSF. Furthermore, the ELISA analysis confirmed that S100A8 was significantly elevated in SF of patients with RA compared to SF of patients with OA. S100A8 induced mRNA expression and secretion of IL-8 and IL-16. S100A8 further enhanced production of IL-8 by GM-CSF but not that of IL-16.These data suggest that S100A8 may be involved in the exacerbation of RA, and that S100A8 may be a therapeutic target of RA.

Objectives Matrix metalloproteinase (MMP) 13 is a pathogenic collagenase that causes cartilage destruction and plays a leading role in causing osteoarthritis. This study focused on 114 genes that are differentially expressed between intact and damaged osteoarthritis cartilage, in order to determine which molecules are involved in suppressing MMP-13 expression. Methods MMP-13 concentrations were measured in the supernatant of human osteoarthritis chondrocyte cultures transfected with small interfering RNA (siRNA) against the 114 genes. MMP-13 levels changed most dramatically in response to siRNA against prostaglandin EP2 receptor. The authors performed further measurements of MMP-13 production in osteoarthritis chondrocytes stimulated by the EP2 agonist butaprost in the presence or absence of interleukin-1β (IL-1β) and/or cyclooxygenase-2 (COX-2) inhibitor. They also assessed the effect of butaprost on chondrocyte viability, and investigated the involvement of the cAMP–protein kinase A (PKA) pathway on EP2 signalling using inhibitors. Cartilage-related gene expression was examined in chondrocytes treated with butaprost. The authors also investigated which E series of prostaglandin (EP) receptors are expressed in osteoarthritis cartilage. Results MMP-13 messenger RNA expression was significantly affected by two molecules, EP2 receptor and SLC14A1, a urea transporter. In IL-1β-treated osteoarthritis chondrocytes, butaprost suppressed MMP-13 production, which was further decreased by COX-2 inhibitor. EP2 signalling downregulated MMP-13 mRNA expression via the cAMP–PKA pathway without affecting cell viability. Although EP2 signalling enhanced IL-6 expression, the expressions of several catabolic factors (MMP-1, MMP-3, MMP-13, ADAMTS5, IL-1β and tumour necrosis factor alpha) were inhibited. EP2 receptor was the major EP receptor in osteoarthritis cartilage. Conclusion The results suggest that EP2 signalling has ‘anti-catabolic’ effects in osteoarthritis chondrocytes.

Anti-endothelial cell antibodies (AECA) were antibodies targeting the antigens expressed on the endothelial cell surface. It has been reported that AECA were detected frequently in patients with vasculitis and were associated with disease activity and vasculitis symptoms. Consequently, AECA are thought to be involved in pathophysiology of vasculitis, including Kawasaki disease (KD); however, the role of AECA is not clear yet. One of the causes is that target proteins of AECA have been poorly identified. Therefore, we try to detect new target proteins of AECA in patients with vasculitis using proteomics. We have identified 63 proteins out of about 150 endothelial cell-specific candidate target proteins of AECA in patients with vasculitis so far. One of the identified proteins was peroxiredoxin2 (Prx2), an antioxidant enzyme. Our research suggests that the anti-Prx2 antibodies are detected frequently in patients with vasculitis and may have pathogenic roles in vasculitis via inflammatory cytokines/chemokines production and inhibition of anti-oxidative activity of Prx2. In this paper, we overview our study of the autoantigens detected by AECA in patients with vasculitis, and will provide some data on clinical significance of autoantibodies to Prx2, a target protein of AECA, in patients with KD.

Summary Anti-endothelial cell antibodies (AECA) have been frequently detected in systemic vasculitis, which affects blood vessels of various sizes. To understand the pathogenic roles of AECA in systemic vasculitis, we attempted to identify target antigens for AECA comprehensively by a proteomic approach. Proteins extracted from human umbilical vein endothelial cells (HUVEC) were separated by two-dimensional electrophoresis, and Western blotting was subsequently conducted using sera from patients with systemic vasculitis. As a result, 53 autoantigenic protein spots for AECA were detected, nine of which were identified by mass spectrometry. One of the identified proteins was peroxiredoxin 2 (Prx2), an anti-oxidant enzyme. Frequency of anti-Prx2 autoantibodies, measured by enzyme-linked immunosorbent assay (ELISA), was significantly higher in systemic vasculitis (60%) compared to those in collagen diseases without clinical vasculitis (7%, P < 0·01) and healthy individuals (0%, P < 0·01). Further, the titres changed in parallel with the disease activity during time–courses. The presence of anti-Prx2 autoantibodies correlated significantly with elevation of serum d-dimers and thrombin–antithrombin complex (P < 0·05). Immunocytochemical analysis revealed that live endothelial cells expressed Prx2 on their surface. Interestingly, stimulation of HUVEC with rabbit anti-Prx2 antibodies increased secretion of interleukin (IL)-6, IL-1β, IL-1ra, growth regulated oncogene (GRO)-α, granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM–CSF), IL-8 and monocyte chemoattractant protein (MCP)-1 more than twofold compared to that of with rabbit immunoglobulin (Ig)G. Taken together, our data suggest that anti-Prx2 autoantibodies would be a useful marker for systemic vasculitis and would be involved in the inflammatory processes of systemic vasculitis.

Objective: Recently, attention has been attracted by the finding that overexpression of caveolin-1 induces cellular senescence in age-related diseases. We aimed to ascertain whether angiogenic growth factors (AGFs) can inhibit interleukin (IL)-1β-induced senescence in human chondrocytes by downregulation of caveolin-1. Methods: We investigated the intracellular signalling pathways involved in chondrocyte ageing. Human chondrocytes were isolated from the articular cartilage of patients undergoing arthroplastic knee surgery in osteoarthritis (OA). Chondrocytes were stimulated with or without IL-1β (10 ng/mL) in the presence or absence of vascular endothelial growth factor, basic fibroblast growth factor or hepatocyte growth factor (20 ng/mL). After 72-h incubation, we observed the expression of caveolin-1 in human chondrocytes by immunohistochemistry, and analysed the protein levels of caveolin-1 by Western blot. We examined the time-course of phosphorylation patterns of mammalian mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3-K) by Western blot, and used several specific protein kinase inhibitors to evaluate the involvement of the intracellular signalling pathways. Also, chondrocyte replicative lifespan was analyzed in the presence or absence of AGFs. Results: Treatment with AGFs inhibited IL-1β-induced overexpression of caveolin-1 in human OA chondrocytes. Treatment with AGFs all down-regulated protein levels of IL-1β-accelarated expression of caveolin-1 in chondrocytes. IL-1β significantly decreased the cellular replicative lifespan in chondrocytes. Treatment with AGFs prevented the IL-1β-induced shortening of chondrocyte replicative lifespan. The specific inhibitors for MAPK/extracellular signal-regulated kinase and PI3-K cancelled the AGF-induced downregulation of overexpression of caveolin-1. Conclusion: Our results suggest that AGFs downregulated IL-1β-induced chondrocyte ageing and overexpression of caveolin-1 in human chondrocytes, which is mediated by kinase cascades involving the p42/44 MAP kinase and PI3-K/Akt signalling pathways.

We analyzed serum short peptides comprehensively to know whether they were useful to characterize IgA nephropathy (IgAN). Serum samples from 26 patients with untreated IgAN and 25 healthy donors were tested. Short peptides with molecular weights of approximately 7 kDa, purified from the serum samples by magnetic-beads-based weak cation exchange, were detected by mass spectrometry. Then the peptide peaks detected were subjected to the multivariate data analysis by SIMCA-P+ containing principal component analysis (PCA) and orthogonal partial-least-squares-discriminate analysis (OPLS-DA). A total of 92 peptide peaks were detected in the tested serum samples. The OPLS-DA analysis revealed that the profile of all the peptide peak intensities discriminated the IgAN group and the healthy group completely with a high R2 value (0.919) and a high Q2 value (0.861). Further, the profile of only five peptide peaks was found to discriminate the two groups. By tandem mass spectrometry and database searching, three of the five peptides which increased in the IgAN group were identified as fragments of fibrinogen alpha chain, and the two peptides which increased in the healthy group were identified as fragments of complement C3f and kininogen-1 light chain. Taken together, the profile of the serum short peptides would be useful to discriminate IgAN and healthy conditions. Further, the five peptides may be candidate serum markers for IgAN and may be related to pathogenesis of IgA.

Prostaglandin E2 (PGE2) is expressed in articular joints with inflammatory arthropathy and may exert catabolic effects leading to cartilage degradation. As we observed in a preliminary experiment that PGE2 suppressed the expression of SERPINE1/plasminogen activator inhibitor (PAI)-1 mRNA in chondrocytes, we focused on the effect of PGE2 on PAI-1 in a panel of cultured chondrocytes obtained from osteoarthritic patients. Specifically, articular cartilage specimens were obtained from patients with osteoarthritis who underwent joint surgery. Isolated chondrocytes were cultured in vitro as a monolayer and stimulated with PGE2. Stimulated cells and culture supernatants were analyzed using Western blotting and enzyme-linked immunosorbent assay. The results confirmed that the in vitro PGE2 stimulation suppressed the expression of PAI-1 in the tested chondrocyte samples. The inhibitory effect was partly abrogated by an antagonist of EP4 receptor of PGE2, but not by an EP2 antagonist. Although PGE2 induced activations of mitogen-activated protein kinases (MAPK), blocking of the MAPK did not abrogate the suppressive effect of PGE2, implying a distinct signaling pathway. In summary, prostaglandin is suggested to modulate the plasminogen system in chondrocytes. Further elucidation of the interaction might open a new avenue to understand the degradative process of cartilage.

Kayo Masuko1, Minako Murata2, Naoya Suematsu1, Kazuki Okamoto1, Kazuo Yudoh2, Hiroyuki Shimizu3, Moroe Beppu3, Hiroshi Nakamura4, Tomohiro Kato11Department of Biochemistry; 2Department of Frontier Medicine, Institute of Medical Science; 3Department of Orthopedic Surgery, St. Marianna University School of Medicine, Kawasaki-shi, Kanagawa, Japan; 4Department of Joint Disease and Rheumatism, Nippon Medical School, Bunkyo-ku, Tokyo, JapanAbstract: Prostaglandin E2 (PGE2) is expressed in articular joints with inflammatory arthropathy and may exert catabolic effects leading to cartilage degradation. As we observed in a preliminary experiment that PGE2 suppressed the expression of SERPINE1/plasminogen activator inhibitor (PAI)-1 mRNA in chondrocytes, we focused on the effect of PGE2 on PAI-1 in a panel of cultured chondrocytes obtained from osteoarthritic patients. Specifically, articular cartilage specimens were obtained from patients with osteoarthritis who underwent joint surgery. Isolated chondrocytes were cultured in vitro as a monolayer and stimulated with PGE2. Stimulated cells and culture supernatants were analyzed using Western blotting and enzyme-linked immunosorbent assay. The results confirmed that the in vitro PGE2 stimulation suppressed the expression of PAI-1 in the tested chondrocyte samples. The inhibitory effect was partly abrogated by an antagonist of EP4 receptor of PGE2, but not by an EP2 antagonist. Although PGE2 induced activations of mitogen-activated protein kinases (MAPK), blocking of the MAPK did not abrogate the suppressive effect of PGE2, implying a distinct signaling pathway. In summary, prostaglandin is suggested to modulate the plasminogen system in chondrocytes. Further elucidation of the interaction might open a new avenue to understand the degradative process of cartilage.Keywords: chondrocyte, prostaglandin, PGE2, PAI-1

The effects of an orally administered combination of a glucosamine-chondroitin-quercetin glucoside (GCQG) supplement on the synovial fluid properties of patients with osteoarthritis (OA) and rheumatoid arthritis (RA) were investigated from the clinical nutrition view point. In this study, forty-six OA and twenty-two RA patients were administered with the GCQG supplement orally for 3 months. Several parameters of the knee joints were monitored before and after supplementation. The OA patients showed a significant improvement in pain symptoms, daily activities (walking and climbing up and down stairs), and visual analogue scale, and changes in the synovial fluid properties with respect to the protein concentration, molecular size of hyaluronic acid, and chondroitin 6-sulphate concentration were also observed. However, no such effects were observed in the RA patients. These results suggest that the GCQG supplement exerted a special effect on improving the synovial fluid properties in OA patients.