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1.  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.
doi:10.1136/ard.2003.008045
PMCID: PMC1754905  PMID: 14962958
2.  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.


doi:10.1136/ard.60.1.49
PMCID: PMC1753367  PMID: 11114282
5.  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.


doi:10.1136/ard.59.9.688
PMCID: PMC1753264  PMID: 10976081
6.  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
7.  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
8.  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

Results 1-9 (9)