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2.  Implication of interleukin 18 in production of matrix metalloproteinases in articular chondrocytes in arthritis: direct effect on chondrocytes may not be pivotal 
Annals of the Rheumatic Diseases  2005;64(5):735-742.
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, IL18Rß, 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 levels of signalling molecules were assayed by western blotting.
Results: IL18 mRNA was constitutively expressed in chondrocytes, and was enhanced by IL1ß stimulation. Flow cytometric analysis showed that IL1ß, tumour necrosis factor α, and IL18 up regulated IL18Rα expression levels. The level of IL18Rß mRNA was much lower than that of IL18Rα, and was slightly up regulated by IL1ß. 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.
PMCID: PMC1755478  PMID: 15834055
3.  Interleukin (IL) 18 stimulates osteoclast formation through synovial T cells in rheumatoid arthritis: comparison with IL1ß and tumour necrosis factor α 
Annals of the Rheumatic Diseases  2004;63(11):1379-1386.
Objective: To determine whether IL18 has any indirect effects on osteoclastogenesis mediated by T cells in RA synovium, and compare its effects with those of IL1ß and TNFα.
Methods: Resting T cells were isolated from peripheral blood of healthy donors, and stimulated with 2 µg/ml phytohaemagglutinin (PHA) and 0.5 ng/ml IL2 for 24 hours. Synovial T cells were isolated from RA synovial tissue. The levels of soluble receptor activator of the NF-κB ligand (RANKL), osteoprotegerin (OPG), IFNγ, M-CSF, and GM-CSF were determined by ELISA. Membrane bound RANKL expression was analysed by flow cytometry. Commercially available human osteoclast precursors were cocultured with T cells to induce osteoclast formation, which was determined with tartrate resistant acid phosphatase staining and pit formation assay.
Results: In PHA prestimulated T cells or RA synovial T cells, IL18, IL1ß, or TNFα increased soluble RANKL production and membrane bound RANKL expression in a dose dependent manner. IL18, IL1ß, and TNFα did not induce M-CSF, GM-CSF, IFNγ, or OPG production in PHA prestimulated T cells or RA synovial T cells. IL18 increased the number of osteoclasts and bone resorption area on dentine slices in the coculture of human osteoclast precursors with PHA prestimulated T cells or RA synovial T cells; its ability was equivalent to that of IL1ß, but less potent than that of TNFα. In the coculture system, OPG completely blocked osteoclast induction by IL18 or IL1ß, and greatly inhibited induction by TNFα.
Conclusion: IL18, IL1ß, or TNFα can indirectly stimulate osteoclast formation through up regulation of RANKL production from T cells in RA synovitis; IL18 is as effective as IL1ß, but less potent than TNFα.
PMCID: PMC1754791  PMID: 15479886
4.  Characterisation of cartilage intermediate layer protein (CILP)-induced arthropathy in mice 
Annals of the Rheumatic Diseases  2004;63(3):252-258.
Objectives: To characterise cartilage intermediate layer protein (CILP)-induced arthropathy in mice.
Methods: The first and second halves of the nucleotide triphosphate pyrophosphohydrolase (NTPPHase) non-homologous region of human CILP were prepared as recombinant proteins (C1 and C2, respectively), including three overlapping fragments of C2 (C2F1, C2F2, and C2F3). C57BL/6 mice were immunised with these proteins to induce arthritis. In addition, a separate group of mice were immunised repeatedly with the mixture of C1 and C2 to see the effect of chronic immunisation. Arthritis developed in the mice, and cellular and humoral immune responses against CILP were analysed.
Results: Immunisation with C2 and with the mixture C2F1/C2F2/C2F3 caused the severest arthritis to develop in mice. Immunisation with one of C1, C2F1, C2F2, or C2F3 caused milder arthritis, even though each of the fragments carried T cell epitopes. Immunisation either with C1 or C2 alone evoked cellular and humoral immune responses to both the C1 and C2 proteins. Further, the repeated immunisation with the C1/C2 mixture caused tendon calcification and bone irregularity, together with decreased NTPPH activity.
Conclusions: The results show that multiple T cell epitopes are needed for the development of CILP-induced arthritis, and present the characteristic new model of mild arthropathy accompanied by extra-articular calcifications. An immune response to putative murine CILP/NTPPH may be involved in the ectopic calcifications in the arthritic mice.
PMCID: PMC1754905  PMID: 14962958
7.  Impact of diabetes mellitus on long term survival after acute myocardial infarction in patients with single vessel disease 
Heart  2001;86(2):133-138.
OBJECTIVE—To assess the influence of diabetes on long term prognosis after reperfusion treatment and its interaction with multivessel disease.
DESIGN—A retrospective observational study.
SETTING—Hiroshima City Hospital.
PATIENTS—1660 consecutive patients with acute myocardial infarction who underwent coronary angiography within 24 hours after the onset of chest pain.
MAIN OUTCOME MEASURES—Influence of diabetes on 10 year survival after infarction was assessed using the generalised Wilcoxon test and Cox's proportional hazards regression. Follow up was completed in 1622 patients (98%).
RESULTS—Diabetic patients had more multivessel disease than non-diabetic patients (53% v 34%, p < 0.001). When only patients with single vessel disease were compared, diabetes was associated with a reduced 10 year survival after infarction (p = 0.002). On the other hand, in patients with multivessel disease there was no significant difference in survival between diabetic and non-diabetic patients (p = 0.70). Multivariate analysis also showed that diabetes was an independent risk factor related to 10 year mortality after infarction in patients with single vessel disease (odds ratio (OR) 1.81, 95% confidence interval (CI) 1.27 to 2.54; p = 0.001) and not in patients with multivessel disease (OR 1.17, 95% CI 0.85 to 1.60; p = 0.34).
CONCLUSIONS—Diabetes is an independent predictor of long term mortality after infarction in patients with single vessel disease. However, in the presence of multivessel disease, prognosis after infarction is impaired regardless of diabetes, and the influence of diabetes is less obvious.

Keywords: myocardial infarction; diabetes; coronary angiography
PMCID: PMC1729851  PMID: 11454823
8.  Recognition of YKL-39, a human cartilage related protein, as a target antigen in patients with rheumatoid arthritis 
OBJECTIVE—To investigate whether autoimmunity to YKL-39, a recently cloned cartilage protein, occurs in patients with rheumatoid arthritis (RA).
METHODS—Autoantibody to YKL-39 was assayed by enzyme linked immunosorbent assay (ELISA) and western blotting in serum samples from patients with RA, systemic lupus erythematosus (SLE), and healthy donors, using recombinant YKL-39 protein. This reactivity was compared with that against a YKL-39 homologue, YKL-40 (human cartilage gp-39/chondrex), which has been reported to be an autoantigen in RA.
RESULTS—Autoantibody to YKL-39 was detected in seven of 87 patients with RA (8%), but not in serum samples from patients with SLE or healthy donors. YKL-40 reactivity was found in only one of 87 RA serum samples (1%), with no cross reactivity to YKL-39.
CONCLUSION—The existence of anti-YKL-39 antibody in a subset of patients with RA is reported here for the first time. Further, it was shown that the immune response to YKL-39 was independent of that to YKL-40. Clarification of the antibody and T cell responses to autoantigens derived from chondrocyte, cartilage, or other joint components may lead to a better understanding of the pathophysiology of joint destruction in patients with RA.

PMCID: PMC1753367  PMID: 11114282
19.  Effect of IL15 on T cell clonality in vitro and in the synovial fluid of patients with rheumatoid arthritis 
Annals of the Rheumatic Diseases  2000;59(9):688-694.
OBJECTIVE—Recent studies have suggested that interleukin (IL) 15 induces T cell accumulation in synovial lesions of rheumatoid arthritis (RA). This study aimed at determining whether this cytokine could explain in vivo T cell clonality in RA.
METHODS—Peripheral blood mononuclear cells (PBMC) from patients with RA were stimulated in vitro with IL15 or IL2. After isolation of mRNA from stimulated cells and synovial T cells, genes coding the V-D(N)-J (CDR3) region of T cell receptor β chains were amplified by a reverse transcriptase polymerase chain reaction. A single strand conformation polymorphism analysis was used to detect the clonotype(s) of accumulating T cells. Nucleotide and amino acid sequencing was also performed.
RESULTS—Stimulation of PBMC with IL15 resulted in oligoclonal expansion of T cells. However, IL15 induced clones from PBMC were mostly different from the dominantly expanding T cell clones in synovial fluid. Furthermore, IL15 and IL2 responding clones were only partially identical.
CONCLUSIONS—Although IL15 results in clonal accumulation of T cells, T cell clonality in rheumatoid joints could not be explained by the effect of IL15 alone. The results indicated the requirement of other factor(s), in addition to IL15, in the pathological process affecting RA joints. The results also suggested different responses by each T cell clone to IL15 or IL2.

PMCID: PMC1753264  PMID: 10976081
20.  Characterisation of T cell clonotypes that accumulated in multiple joints of patients with rheumatoid arthritis 
Annals of the Rheumatic Diseases  1999;58(9):546-553.
OBJECTIVE—To investigate whether identical T cell clonotypes accumulate in multiple rheumatoid joints, the clonality of T cells that had infiltrated into synovial tissue (ST) samples simultaneously obtained from multiple joints of patients with rheumatoid arthritis (RA) was analysed.
METHODS—T cell receptor (TCR) β gene transcripts, amplified by reverse transcription-polymerase chain reaction from ST and peripheral blood lymphocytes of five RA patients, were subjected to single strand conformation polymorphism analysis and DNA sequencing.
RESULTS—Approximately 40% of accumulated T cell clonotypes found in one joint of a patient were found in multiple joints in the same patient. Furthermore, identical amino acid sequences were found in TCR β junctional regions of these clonotypes from different patients with at least one HLA molecule match.
CONCLUSIONS—The T cell clonotypes accumulating in multiple rheumatoid joints may be involved in the perpetuation of polyarthritis by reacting to antigens common to these multiple joints.

PMCID: PMC1752942  PMID: 10460187
21.  Clinical significance of CDC25A and CDC25B expression in squamous cell carcinomas of the oesophagus 
British Journal of Cancer  2001;85(3):412-421.
CDC25A, CDC25B and CDC25C belong to a family of protein phosphatases which activate the cyclin-dependent kinase at different points of the cell cycle. According to accumulating evidence, CDC25A and CDC25B seem to possess oncogenic properties. We have analysed these expressions by immunohistochemistry, western blot and RT-PCR in a series of 100 patients with squamous cell carcinoma of the oesophagus. When compared with non-cancerous cells, CDC25A and CDC25B were strongly expressed in the cytoplasm of cancer cells, with positive (+) classification in 46% (46 cases) and 48% (48 cases), respectively. There was no significant correlation between CDC25A and CDC25B expression, nor was there any association with the expression of other cell cycle-regulating molecules, including cyclin D1, Rb, p16INK4, p27KIP1 and PCNA (proliferating cell nuclear antigen). CDC25A (+), as well as CDC25B (+), was more frequently found in patients with deeper tumour invasion and lymph node metastasis, while tumour size was correlated only with CDC25A expression. Postoperative survival was significantly poorer for CDC25A (+) patients than CDC25A (–) patients, but was not affected by the CDC25B status. Nuclear localization of CDC25A was observed in 51 cases (51%), regardless of its cytoplasmic expression, and was not associated with clinico-pathological factors or prognosis. Multivariate analysis revealed only the CDC25A status to be an independent significant prognostic factor among these biological and clinico-pathological factors. CDC25A but not CDC25B may be a new prognostic factor for squamous cell carcinoma of the oesophagus. Thus, regulation of the G1 checkpoint in the cell cycle may be important in oesophageal carcinogenesis, which may also involve many other oncogenes. © 2001 Cancer Research Campaign
PMCID: PMC2364065  PMID: 11487274
squamous cell carcinoma of the oesophagus; CDC25A; CDC25B; prognosis
22.  Type II collagen is a target antigen of clonally expanded T cells in the synovium of patients with rheumatoid arthritis 
Annals of the Rheumatic Diseases  1999;58(7):446-450.
OBJECTIVE—To investigate whether type II collagen (CII) is recognised by oligoclonally expanded synovial T cells of patients with rheumatoid arthritis (RA).
METHODS—Peripheral blood mononuclear cells (PBMC) from 15 RA patients were stimulated with CII in vitro. T cell clones expanded by such stimulation were compared with the clonally expanded synovial T cells by using T cell receptor (TCR) B chain gene specific reverse transcription-polymerase chain reaction and subsequent single strand conformation polymorphism analyses.
RESULTS—Stimulation of the heterogeneous peripheral T cells with CII induced clonal expansion of T cells. In three of 15 patients, a proportion of these clones (approximately 17% to 25%) was found to be identical to expanded T cell clones in the synovium in vivo.
CONCLUSION—T cell clones that had TCR CDR3 sequences identical to those induced by purified CII were found in a proportion of RA patients. This finding suggests that CII is recognised by T cells that accumulate clonally in RA joints. Oligoclonal T cell expansion in RA joints is probably driven, at least in part, by intra-articular components such as CII.

PMCID: PMC1752913  PMID: 10381490
23.  Long term persistent accumulation of CD8+ T cells in synovial fluid of rheumatoid arthritis 
Annals of the Rheumatic Diseases  1997;56(10):613-621.
OBJECTIVE—To characterise the type and kinetics of T cell clones in synovial lesions of patients with rheumatoid arthritis (RA).
METHODS—Mononuclear cells from serial samples of synovial fluid (SF) and peripheral blood from nine RA patients were separated phenotypically using antibody coated magnetic beads. After mRNA preparation, reverse transcription-polymerase chain reaction (RT-PCR) was performed to amplify V-D(N)-J (that is, the third complementarity determining, CDR3) regions of their T cell receptor beta chain genes. This was followed by single strand conformation polymorphism (SSCP) analysis to detect the clonotypes of accumulating T cells. Amino acid sequences of the dominant clones were also determined.
RESULTS—Although peripheral T cells were heterogeneous, accumulation of oligoclonal T cells was detected in SF. The predominant accumulating clone was the CD8 subset, which was persistently present in serial samples obtained over almost one year of follow up. A proportion of these cells expressed CD25 or CD45RO, or both, suggesting they are `memory' clones.
CONCLUSION—The persistent presence of CD8+ T cell clones in RA joints indicates that they may be involved in the perpetuation of the chronic inflammatory process in RA joints.

PMCID: PMC1752266  PMID: 9389223
24.  Characterisation of fibroblast-like cells in pannus lesions of patients with rheumatoid arthritis sharing properties of fibroblasts and chondrocytes 
Annals of the Rheumatic Diseases  1997;56(4):262-267.
OBJECTIVE—To better understand the characteristics of synoviocytes located in the rheumatoid arthritis (RA) pannus.
METHODS—One cell line, termed PSC, was cloned from RA pannus lesions. Phenotypic analysis was done by contrast microscopy, indirect immunostaining, and safranin O staining. Transcription of several protooncogenes and matrix degrading enzymes was evaluated. The expression of mRNA for collagen II was detected by in situ hybridisation. The ability of anchorage independent growth was assessed by soft agarose culture.
RESULTS—PSCs showed a high transcription of protooncogenes c-fos, c-myc and c-jun. They also expressed mRNA for matrix degrading enzymes, such as collagenase, cathepsin B, and cathepsin L. Anchorage independent growth assay demonstrated that PSCs formed colonies in soft agar culture. Phenotypic analysis showed that this fibroblast-like PSC was stained intensely with anti-vimentin and anti-fibroblast antibody. In situ reverse transcriptase assay showed that the cell line expressed type II collagen mRNA.
CONCLUSION—Alternative fibroblast-like cells were identified in the pannus lesion of RA sharing properties of fibroblasts and chondrocytes. These findings suggest that this fibroblast-like cell derived from pannus lesions may contribute to the destruction of the cartilage in RA.

PMCID: PMC1752353  PMID: 9166000
25.  Multi-forms of human MTH1 polypeptides produced by alternative translation initiation and single nucleotide polymorphism. 
Nucleic Acids Research  1999;27(22):4335-4343.
The human MTH1 gene for 8-oxo-7,8-dihydrodeoxyguanosine triphosphatase, produces seven types (types 1, 2A, 2B, 3A, 3B, 4A and 4B) of mRNAs. The B-type mRNAs with exon 2b-2c segments have three additional in-frame AUGs in their 5' regions. We report here that these transcripts produce three forms of MTH1 polypeptides (p22, p21 and p18) in in vitro translation reactions. Three polypeptides were also detected in extracts of human cells, using western blotting. B-type mRNAs with a polymorphic alteration (GU-->GC) at the beginning of exon 2c that converts an in-frame UGA to CGA yielding another in-frame AUG further upstream, produced an additional polypeptide (p26) in vitro. Substitution of each AUG abolished the production of each corresponding polypeptide. Cell lines from individuals with the GC allele contain more B-type mRNAs than do those of GT homozygotes, and the former produce all of four polypeptides but the latter lack p26. Amounts of each polypeptide reflected copy number of the GC allele in each cell line. There is an apparent linkage dis-equilibrium between the two polymorphic sites, GT/GC at exon 2c and Val83/Met83 at codon 83 for p18.
PMCID: PMC148714  PMID: 10536140

Results 1-25 (47)