Peroxiredoxin 2 is a novel autoantigen for anti-endothelial cell antibodies in systemic vasculitis.

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-1ß, 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.

Identification of autoantigens specific for systemic lupus erythematosus with central nervous system involvement.

Using proteomic analysis, we identified candidate autoantigens specific for central nervous system (CNS) involvement in systemic lupus erythematosus (SLE). Proteins, extracted from cultured human neuroblastoma cells, were separated both by SDS-PAGE (1-DE) and two-dimensional electrophoresis (2-DE), and transferred to membranes. Western blot analysis was performed using serum samples from 30 SLE patients with CNS involvement (CNS-Lupus) and from 30 SLE patients without CNS involvement (non-CNS-SLE). The detected autoantigens were identified using MALDI-TOF/TOF MS. On the 1-DE Western blot, we detected 32 antigenic bands in the serum samples from the CNS-Lupus patients. Among them, four bands were detected significantly more frequently in the CNS-Lupus patients than in the non-CNS-SLE patients. Three bands were detected in four or more of the CNS-Lupus patients but in only one or none of the non-CNS-SLE patients. We thus selected these seven bands for the next investigations. Next, we detected protein spots corresponding to the selected seven bands by 2-DE Western blot and identified four proteins. They are peroxiredoxin-4, ubiquitin carboxyl-terminal hydrolase isozyme L1, splicing factor arginine/serine-rich 3, and histone H2A type 1. These four candidate autoantigens for the anti-neuronal cell antibodies would be a useful marker for CNS-Lupus.

A metabolic aspect of osteoarthritis: lipid as a possible contributor to the pathogenesis of cartilage degradation.

Osteoarthritis (OA) is considered to be linked to obesity and body fat mass. Recent investigations, however, are aimed at clarifying the roles of adipose tissue-derived proteins and a wide variety of lipid mediators, including fatty acids, sphingolipids, and eicosanoids, in cartilage degradation in OA, in addition to the effects body weight itself. Here, we review recent progress in studies of OA, focusing on the potential role of lipid mediators in articular cartilage and introducing the concept that "OA is a metabolic disease" in which lipids essentially contribute to the pathophysiology of cartilage degradation.

Development and characteristics of pannus-like soft tissue in osteoarthritic articular surface in rat osteoarthritis model.

OBJECTIVES: Pannus is invasive granulation tissue found on the articular cartilage having rheumatoid arthritis (RA). However, pannus-like tissue has also been found in osteoarthritis (OA). Our previous study showed that pannus-like tissue in OA (OA pannus) was frequently found in human OA samples. The purpose of the study is to investigate the development and the characteristics of OA pannus in a rat OA model. DESIGN: Ligaments of the knee joint were transected in Wister rats to induce OA. The knee joints were removed at weeks 1, 2, 4 and 6, and subjected to histological study. Samples were stained with hematoxylin and eosin (HE), Safranin-O and immuno-stained for vimentin, CD34, type II collagen and MMP-3. The whole knee joint of OA rats was implanted in SCID mice and kept for a further 3 weeks. Then the histological findings were evaluated in HE sections. RESULT: OA pannus appeared at week 2 and extend over the articular surface. OA pannus cells were positive for vimentin and/or CD34. At week 6, a part of articular surface was restored with matrix. OA pannus cells expressed MMP-3 as well as type II collagen. Histological study of rat OA knees implanted in SCID mice showed that OA pannus cells filled the joint space and invaded articular cartilage. CONCLUSIONS: The presence of OA pannus was found in a rat OA model and its features were similar to those in human OA. OA pannus had both catabolic and reparative features, and the latter feature were speculated to be dominant in the later phase of the disease under a certain environmental condition.

The potential role of vascular endothelial growth factor (VEGF) in cartilage: how the angiogenic factor could be involved in the pathogenesis of osteoarthritis?

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.

Effects of celecoxib on human chondrocytes--enhanced production of chemokines.

OBJECTIVE: The purpose of this study was to examine the effects of a selective cyclooxigenase-2 (COX-2) inhibitor (celecoxib) comparing diclofenac. METHODS: Using chondrocytes derived from cartilage of non-arthritic (NA) subjects or patients with osteoarthritis (OA) or rheumatoid arthritis (RA), we examined the effects of celecoxib on incorporation of 3H-thymidine and 35S-sulfate, apoptosis, and production of matrix metalloproteinase (MMP)-1, MMP-3, MMP-13, and regulated upon activation, normal T cell expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1a and nitric oxide (NO). RESULTS: Celecoxib and diclofenac tended to reduce 3H-tymidine incorporation of chondrocytes. Celecoxib induced apoptosis in a dose-dependent manner, but to a lesser degree than diclofenac. Celecoxib inhibited proteoglycan synthesis (indicated by 35S-sulfate incorporation) in NA chondrocytes, but not in OA and RA chondrocytes. Celecoxib increased interleukin-1 (IL-1)-induced production of RANTES and MIP-1alpha by chondrocytes and decreased IL-1-induced NO production by chondrocytes, whereas it did not affect MMP production. CONCLUSION: Celecoxib had both beneficial and adverse effects on chondrocytes. RA, OA and NA chondrocytes showed different responses. Interestingly, celecoxib enhanced the production of chemokines.

Positron emission tomography with 18F-FDG in osteoarthritic knee.

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.

Tryptase enhances release of vascular endothelial growth factor from human osteoarthritic chondrocytes.

OBJECTIVE: A contribution of mast cells and its mediators in the pathogenesis of arthritis has been postulated. We aimed to clarify the role of mast cell-derived serine protease tryptase and proteinase activated receptor (PAR)-2-mediated signaling in chondrocytes. METHODS: Human articular cartilage specimens were obtained from patients with osteoarthritis (OA), rheumatoid arthritis (RA) and with traumatic fracture without arthritis (PT; as controls) who underwent joint surgery. Isolated chondrocytes were cultured in vitro by monolayer, and confluent cells were incubated with recombinant human lung Beta tryptase or with a PAR-2 agonist peptide. The secreted level of vascular endothelial growth factor (VEGF) in culture supernatant was measured using commercially available ELISA kits, and expression of VEGF mRNA was analyzed using real-time PCR. RESULTS: The tryptase-stimulated chondrocytes from OA or RA, but not from PT patients, produced significantly higher amount of VEGF in their supernatants. The response was blocked by a G-protein receptor inhibitor pertussis toxin, however, was not reproduced by incubation of cells with the PAR-2 agonist, suggesting a presence of non-PAR-2 dependent signals for the VEGF induction. In addition, actinomycin D and cycloheximide did not exert significant inhibition, indicating a regulation of VEGF release by tryptase. CONCLUSION: The inflammatory mediator, mast cell-derived protease tryptase may modulate chondrocyte metabolism through induction of VEGF release.

Treatment of solitary small hepatocellular carcinoma: consideration of hepatic functional reserve and mode of recurrence.

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) recurs frequently after initial treatment. The subsequent prognosis varies with the mode of recurrence. Some patients die of hepatic failure even though the HCC is controlled. We consider the clinical stage (CS), using the modified Child-Pugh classification, to be an important factor influencing the prognosis of these patients. METHODOLOGY: To determine the most effective treatment for HCC, we examined 105 patients with solitary small HCC who were followed-up for more than 1 year after initial treatment. All of them were judged to be cured according to imaging or histological studies. The initial treatments were hepatic resection (n = 43), percutaneous ethanol injection therapy (PEIT, n = 33), and percutaneous microwave coagulation therapy (PMCT, n = 29). The modes of recurrence were divided into intrahepatic metastasis (IM) and multicentric occurrence (MO). RESULTS: Prognosis of MO was superior to that of IM in CS I patients, but there was no difference in prognosis between these modes in CS II. The hepatic resection group had more MO recurrences in CS I patients and more IM recurrences in CS II patients. IM developed frequently after PEIT and PMCT, regardless of the CS. Prognosis with hepatic resection was superior to that of the other treatments in CS I patients, but there was no difference in prognosis among the 3 treatment modalities in CS II patients. CONCLUSIONS: These data indicate that hepatic resection is the first choice for treating HCC in CS I patients, and that PEIT or PMCT is preferable for CS II patients.

Management of acute chylothorax with hydrops fetalis diagnosed in the third trimester of pregnancy.

A fetus with large pleural effusion and hydrops fetalis diagnosed in the third trimester was successfully treated with prompt vaginal delivery followed by drainage of the pleural cavity, after confirmation of congenital chylothorax and re-expansion of the lung with prenatal thoracentesis.

Germline-specific expression of the Oct-4/green fluorescent protein (GFP) transgene in mice.

The Pic-1, Oct-1,2, Unc-86 (POU) transcription factor Oct-4 is specifically expressed in the germ cell line, and a previous study has indicated that the expression of the lacZ gene inserted into an 18 kb genomic fragment encompassing the Oct-4 gene can come close to mimicking the endogenous embryonic expression pattern of Oct-4 in transgenic mice. In the present study transgenic mice expressing green fluorescent protein (GFP) in the germ cell line were generated using the same Oct-4 genomic fragments and the expression pattern was analyzed in detail through all stages of germ cell development. The GFP expressing primordial germ cells were first detected as early as 8.0 days post-coitum (d.p.c.; early head fold stage) at the base of the allantois in living embryos. The GFP expression was thereafter found in both male and female germ cells at all developmental stages except in male germ cells after differentiating into type A spermatogonia in the postnatal testis. There was also a lower level of expression in female germ cells in the prophase of the first meiotic division. These transgenic mice therefore proved to be powerful tools for isolating living germ cells at various developmental stages to study their nature and to isolate new genes.

T-cell clonal change after allo-kidney transplantation in humans.

Whether T cells circulating peripherally express changes at a clonal level after renal transplantation is uncertain. To clarify this issue, we analyzed T-cell clonality of peripheral blood lymphocytes (PBLs) in 12 renal transplant recipients by a novel polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) method that can discriminate T-cell clones with different T-cell receptor (TCR) Vbeta motifs. The PCR-SSCP study showed that after transplantation, only a few distinct T-cell clonotypes accumulated in the absence of clinical episodes, irrespective of the compatibility of HLA antigens. In contrast, various T-cell clones appeared in cases of acute rejection (AR) and infection. These subsided immediately after the AR was resolved; however, they remained long after the resolution of the infection. In a case of AR followed by an infectious episode, distinct T-cell clones appeared concomitantly with each episode. Several of them disappeared or remained thereafter. In one case, significant numbers of accumulating bands were observed by in-vitro stimulation by mixed lymphocyte reaction (MLR); several were identical to those found in vivo. However, some of those that did not appear in vitro were apparent in vivo. In conclusion, the appearance of T-cell clonotypes at a peripheral level indicates the existence of immunologically activated T-cell clones, which were significantly affected by immunosuppressive therapy. It was also determined that the T-cell immune system is much more complicated in vivo than in vitro.

Polygyny and Monoandry in the Ant Formica japonica (Hymenoptera: Formicidae).

Queen number, mating frequency and nest kin-structure of the ant Formica japonica were studied in the field and the laboratory. Nest excavation in the study site, the east slope of Mt. Fuji, Gotenba, Japan, revealed that F. japonica is weakly polygynous all year round and the queen number increases after the nuptial flight season, suggesting the adoption of newly mated queens by established nests. Dissection and laboratory rearing demonstrated that nearly all queens in polygynous nests had mated and were fertile with mature oocytes in their ovaries. Multilocus DNA fingerprinting was used to examine kin relationships among ants found in the same nests. The fingerprint band patterns were apparently governed by a simple genetic rule and suggested monoandry (single mating per queen). The mean band sharing score of DNA fingerprints among full sisters was 0.90, and the mean value between queens and their daughters was 0.75. Comparison of DNA fingerprints of adult and pupal workers with pupal gynes suggested that multiple queens in a nest may contribute unequally to gyne (new queen) production.

Mutation rate of GB virus C/hepatitis G virus over the entire genome and in subgenomic regions.

A patient on maintenance hemodialysis was infected with a recently discovered putative non-A to -E hepatitis virus designated GB virus C (GBV-C) or hepatitis G virus (HGV) by transfusion. The viral isolate was recovered from the patient soon after she turned positive for GBV-C/HGV RNA in serum (GS185) and 8.4 years thereafter (GS193), and the entire nucleotide sequences were determined. They both had a genomic length of 9391 nucleotides with a defective C gene made of only 42 nucleotides. Between GS185 and GS193, 31 (0.33%) nucleotides were different, which changed 5 (0.18%) of the encoded 2842 amino acids. Thus, GBV-C/HGV was estimated to have a mutation rate of 3.9 x 10(-4) base substitutions per site per year. Nucleotide conversions were distributed over subgenomic regions, except in the 5' untranslated region of 552 nucleotides and a defective short C gene, which were conserved in sequence. The change in the putative envelope genes (E1 and E2) was no different from that in the entire genome with only 6 (0.35%) nucleotide substitutions among the 1730, just 1 of which induced an amino acid conversion. Taken along with the comparison of the two isolates with the reported five GBV-C or HGV isolates, these results indicate that GBV-C/HGV would not have hypervariable regions and would use a strategy for viral persistence that is different from immune escape.

Correlation of clonal T cell expansion with disease activity in systemic lupus erythematosus.

It has been proposed that T cell activation may play an important role in the pathogenesis of systemic lupus erythematosus (SLE). In order to examine this hypothesis, we assessed the whole degree of clonal accumulation of T cells using RT-PCR and subsequent single-strand conformation polymorphism (SSCP) analysis. The analysis of the beta chain of the TCR revealed little clonotypic T cell expansion in the peripheral blood of lupus patients in remission, whereas in patients with active disease many dominant T cell clonal expansions without any distinct V beta bias were observed. The alteration in the number of T cell clones correlated well with disease activities, since these T cell expansions decreased as patients had improved. Furthermore, similar but more intense accumulations of T cell clones were found in pleural and pericardial effusions of patients with lupus serositis. Some of these identical expanded clonotypes were observed irrespective of the lesions and the times of sampling, and some of them were identical to those observed in the peripheral blood. These results suggest that the T cell clonal expansions correlate with the disease activities and that the expansion might contribute to the pathogenic lesion formation in SLE.

GB virus C and hepatitis C virus infections in hemodialysis patients in eight Japanese centers.

RNA of a putative non-A to E hepatitis virus, designated GB virus C (GBV-C), was detected in 40 (6.2%) of 645 hemodialysis patients, at a frequency significantly higher than in 3 (0.9%) of 336 blood donors in Japan (p < 0.001). A history of transfusion was more frequent (88 vs. 58%, p < 0.001), the duration of dialysis was longer (13.2 +/- 7.9 vs. 7.9 +/- 6.5 years, p < 0.001), and the detection of hepatitis C virus RNA was more often (38 vs. 18%, p < 0.01) in the 40 patients with GBV-C RNA than in the 605 patients without it. The prevalence of GBV-C RNA varied widely from 0 to 10% among the 8 dialysis centers. These results indicate that hemodialysis patients would be at increased risk of GBV-C transmitted by transfusions. The detection of GBV-C RNA in the 5 patients without a history of transfusion and a high prevalence restricted to certain dialysis centers would reflect nosocomial infection.

Novel mechanisms of selective apoptosis in synovial T cells of patients with rheumatoid arthritis.

OBJECTIVE: To analyze whether T cells infiltrating the synovium of patients with rheumatoid arthritis (RA) express functional Fas antigen. METHODS: Mononuclear T cells, mainly from synovial tissues, synovial fluids (SF), and peripheral blood mononuclear cells (PBMC) from 14 patients with RA and 5 with osteoarthritis (OA), were treated with anti-Fas monoclonal antibody (Mab) (CH11) for 24 h in vitro. Cell viability and DNA fragmentation were examined. The expression of Fas antigen, Fas ligand, and T cell subpopulations was examined using flow cytometry and reverse transcription polymerase chain reaction. RESULTS: More than 50% of T cells from synovial tissue and SF of patients with RA underwent apoptosis, whereas no effect was observed in PBMC from RA or PBMC and synovial T cells from OA, suggesting that functional Fas antigens are specifically expressed on synovial T cells. Flow cytometric analysis demonstrated higher expression of Fas antigen in T cells from RA synovium than from PBMC. The T cell subpopulations susceptible to anti-Fas Mab were mainly CD45RO+ and CD4 or CD8 single positive T cells, indicating that activated mature T cells express functional Fas antigen. Fas ligand was overexpressed only in synovial nonadherent cells in RA at the level of mRNA, whereas T cells account for more than 60% of the total, but not in OA. CONCLUSION: These findings suggest the majority of activated T cells infiltrating the synovium express functional Fas antigen and Fas ligand specifically in patients with RA but not OA. This phenomenon may provide a clue to understanding the pathogenesis of RA.

Characterization of T cell receptor beta chains of accumulating T cells in skin allografts in mice.

The study of T cells involved in the immune reaction that occurs in engrafted organs should provide information that would be helpful in the regulation of allograft rejection in organ transplantation. Toward this end, we focused on detection and characterization of accumulating T cells in mouse skin allografts from B10.A(4R) to C57BL/6 mice in vivo. T cell receptor beta genes were amplified by reverse transcriptase-PCR from mRNA of the skin grafts, and accumulating T cell receptor beta gene clonotypes were identified by their single strand conformation polymorphism. Their joining region usage and the amino acid sequences of the complementarity-determining region-3 were then determined. The results were as follows: (1) Distinct oligoclonal accumulation of T cells was more prevalent in the skin allografts than in the syngenic skin grafts. (2) Although the accumulating T cell clonotypes appeared to use many different variable-region gene families, preferential combinations of variable region-joining region were found. (3) Several homologous amino acid sequences were found in these accumulating TCR beta genes in allografts, suggesting that these T cells are driven by the same or similar antigens. (4) In addition, little T cell accumulation was found in spleens from the mice with allografts or syngenic skin grafts. Taken together, accumulating T cells in the skin allografts were detected in vivo, and some appeared to have characteristics in common. This may lead to T cell clonotype-specific therapy in organ transplantation.