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.

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.

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.

Novel antigen expression on syngeneic somatic cell hybrids of murine spontaneous mammary tumor cells.

Spontaneous mammary carcinoma cells of C3H/He mice were fused with syngeneic L-cells, and two types of hybrid cell clones were obtained. In group A, hybrid cells showed contact inhibition, and parental H-2k and L-antigens were well expressed. Metacentric chromosomes of L-cell origin and estrogen dependency were also well preserved in this group. However, in group B, hybrid cells proliferated to pile up, and the expression of parental antigens, H-2 and L-cell antigens, was strongly suppressed. But, mouse mammary tumor antigens (MM-antigens), which were expressed on ascites mammary tumor cells with hypotetraploidy of C3H/He origin and which were not expressed on both parent cells, were newly expressed. The number of metacentric chromosomes was decreased, and estrogen dependency was lost in this group. The relationship between the expression of MM-antigens and that of H-2 antigens was reciprocal, and tumorigenicity was independent of cellular behavior in vitro and of MM-antigen expression. MM-antigen-positive hybrid cell clones were frequently obtained when tumor cells were fused with metaphase-rich L-cells.

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.

Mutagenicity of metabolites of carcinogenic aminoazo dyes.

The mutagenicity of 8 azo dyes and 6 p-phenylenediamine derivatives, which comprised the metabolites of carcinogenic 4-aminoazobenzene derivatives, was studied on Salmonella typhimurium TA98 and TA100. 4'-Hydroxy-N-methyl-4-aminoazobenzene and its O-sulfate and O-glucuronide, and 3-hydroxy-4-aminoazobenzene were mutagenic on TA98 in the presence of S-9 mix. p-Phenylenediamine and its o-methoxyl derivative were definitely mutagenic on TA98 with the addition of S-9 mix. All metabolites tested were non-mutagenic on TA100, although the mother azo dyes were mutagenic both on TA98 and TA100 in the presence of S-9 mix. These results rule out a possibility that the mutagenicity, at least on TA100 microbes, of carcinogenic 4-aminoazobenzene derivatives may be mediated by any of the ring-hydroxyl or azo reduction metabolites and their conjugates produced from the azo dyes by incubation with S-9 mix.

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.

Flunitrazepam for sleep disturbance in children with intractable epilepsy.

Add-on therapy with flunitrazepam (FNZ) was performed in 5 children with marked sleep disturbance and intractable seizures. Correction of the sleep disturbance was attained immediately after the start of FNZ administration in all patients. Furthermore, a significant decrease in the seizure frequency (3 patients) and improved quality of life (4 patients) were concomitantly observed. There was no adverse effect or interaction with conventional AEDs on long-term use.

Cephalosporins. II. 7-(O-Aminomethyl-phnylacetamido) cephalosporanic acids with six membered heterocycles in the C-3 side chain.

7-(o-Aminomethylphenylacetamido)cephalosporanic acids with six-membered heterocycles in the C-3 side chain were prepared by nucleophillic substitution of 7-ACA at the C-3 acetoxy group followed by N-acylation of the 7-amino group. The 7-side chain acid, o-aminomethylphenylacetic acid (5), was prepared by two new convenient routes, which involved Schmidt reaction of indanone (2) followed by cleavage of the lactam ring or reduction of o-cyanophenylacetic acid (10) starting from o-nitrotoluene. The antibacterial activity of the cephalosporins in this series depends on the heterocycle in the C-3 side chain. In general pyridazines gave cephalosporin derivatives possessing better activity than those with a pyridine or pyrimidine ring. The most active member of the new cephalosporins was 7-(o-aminomethylphenylacetamido)-3-(6-hydroxypyridazin-3-ylthilmethyl)-3-cephem-4-carboxylic acid (BB-S 150) (1g) which has in vitro antibacterial activity superior to cephalothin and cefazolin against both gram-negative and gram-positive organisms. The in vitro activity of BB-S 150 determined in mice was superior to cephalothin and comparable to cefazolin.

Cephalosporins. I. Cephaloglycin analogs with six-membered heterocycles in the C-3 side chain.

Cephaloglycin analogs with six-membered heterocycles in the C-3 side chain have been prepared by nucleophilic substitution of 7-aminocephalosporanic acid with appropriate azine thiols followed by 7-N-acylation with phenylglycine by the mixed anhydride method. Seventeen thiols of non-substituted or substituted pyridines, pyridazines, pyrimidines, pyrazines and triazines were used as the S-nucleophiles. In general, pyridazine thiols gave cephalosporins processing good antimicrobial activity against both gram-positive and gram-negative bacteria. Among them 6-hydroxypyridazine-3-thiol gave the most active compound of this series, BB-S 118 (1f), which was significantly more active than cephalexin and cephaloglycin in vitro against gram-positive and gram negative bacteria.

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.

[Analyses of T cell clonality in mice with autoimmune sialoadenitis].

T cells are believed to be deeply involved in the pathogenesis of organ-specific autoimmune diseases. However, it remains unknown whether antigen-specific immune responses occur at the inflammatory sites in vivo. As a model of human Sjögren's syndrome, we investigated the T cell clonality of mice with autoimmune sialoadenitis, i.e. IQI/Jcl-(Saegusa et al), thymus grafted (TG) nude-(Taguchi et al), and MRL/lpr-SCID (Hayashi et al) mice; using RT-PCR-SSCP method. As a result, accumulations of distinct T cell clones were demonstrated in the salivary and lacrimal glands in these mice, without V beta restriction. Moreover, a part of the accumulating clones were commonly detected among multiple glands, suggesting the existence of a common and specific immune response probably toward autoantigen(s) expressed on the affected glands.