New method for isolation of human monocytes without adhesion process by using centrifugal elutriator has been recently established, and the effects of inflammatory cytokines such as TNF and MCAF on suspended monocytes were investigated. Both cytokines did not induce or only minimally induced superoxide release in human monocytes, but potently primed monocytes for enhanced release of superoxide upon stimulation with subsequent agonists, and these two cytokines primed monocytes in an additive manner. TNF induced minimal effects on intracellular signaling pathways, whereas MCAF rapidly induced intracellular signalings such as pH changes in a similar manner with FMLP.
To clarify interaction between monocytes and endothelial cells during inflammatory responses, double chamber in vitro migration assay system was established. Both monocytes and endothelium produced several inflammatory cytokines such as TNF, IL 1, MCAF, and GM-CSF into the apical but not basilar side of the endothelial cell monolayer during monocyte adhesion to and transmigration through endothelial cells. Thus, monocyte chemoattractant such as MCAF released into the apical side could inhibit monocyte transendothelial migration, suggesting possible in vivo selfregulatory system of inflammation.
In regard to the signal transduction in human monocytes, tyrosine kinases and their substrates are of particular importance, and several signaling molecules such as MAP kinase, STAT 5, and vav product have been identified. Although the signaling pathways utilized by soluble inflammatory stimuli such as cytokines and chemotactic factors have been substantially clarified, those utilized by adhesive stimuli are still largely unknown particularly in human phagocytes, and awaits much further investigation.

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CD44 molecules function as a receptor for various extracellular matrices. Recently, molecular structure of CD44 has been extensively analyzed and have been identified multiple isoforms produced by alternative splicing of mRNA.
In this study, we examined the expression of CD44 isoforms on different cell types isolated from periodontal tissue. In vitro cell lines of different cell types, human gingival fibroblasts (HGF), human periodontal ligament cells (HPDL) and human gingival epithelial cells (HGEC) were isolated and CD44 isoform expressions were analyzed. All of these in vitro cell lines expressed CD44 protein as determined by immunoprecipitation. Interestingly, the immunoprecipitated CD44 from HGEC showed higher molecular mass than those from HGF and HPDL.
To detect CD44 mRNA heterogeneity in details, reverse transcription polymerase chain reaction (RT-PCR) utilizing primer flanking the insertion site of alternatively spliced exons was introduced. All cells examined expressed one major band detected in the absence of alternatively spliced exons and additional bands. In particular, HGEC contained more abundant high molecular species in size than others. HGEC after confluent culture contained less amount of larger CD44 isoforms than the one before confluent culture.
These findings suggest that CD44 isoform expression in periodontal tissue is tissuespecifically regulated and its expression is modulated in epithelial cells.

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The present investigation was designed to clarify the possible relationship between the secretory leukocyte protease inhibitor (SLPI) in gingival crevicular fluid (GCF) and the periodontal clinical parameters or the presence of periodontopathic bacteria in subgingival plaque.
SLPI levels in GCF were determined with a commercially available ELISA system and the neutrophil elastase (NE) activity in GCF was measured with a low molecular weight substrate S-2484. Subgingival plaque samples were tested for the presence of eight periodontopathic bacteria by DNA probe analysis.
In thirty two examined sites from ten patients with periodontal maintenance care, the SLPI levels were positively associated with the GCF volume and the presence of gingival inflammation. Statistically significant negative correlations were found between SLPI levels and the presence of Porphyromonas gingivalis and Treponema denticola, whereas, the NE activity increased in the presence of the bacteria.
The results demonstrated that the bacteria with the capacity to produce proteolytic enzymes degraded or inactivated the protease inhibitor, suggesting that the proteolytic damage of periodontal tissue might be caused by the decrease of active protease inhibitor.

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Cyclodextrin (Cd) is a cyclic oligosaccharide and can include various kinds of compounds in its cavity. Cd applications improve the bioactivity of the compounds. Recently, it has been clarified that some oligosaccharides are immunostimulating agents, therefore, we studied the effects of Cds on IL-1 production from macrophages in mice. The addition of Cd increased IL-1 production in the cultured supernatants from LPS-stimulated macrophages of BALB/c mice.
Among three kinds of Cd, βCd was the most effective and γCd was less, but αCd did not show any effect at all. In C3H/HeJ mice, a similar enhancing effect using the 3 kinds of Cd was observed, however, it was much weaker. IL-1 production did not increase when the macrophages were precultured with βCd and later stimulated by LPS.
Using ELISA, we found that IL-1 molecules in the cultured supernatants was increasing. βCd induced more strongly the IL-1 mRNA expression of LPS-stimulated macrophages. The lymphocyte activating factor activity of recombinant IL-1 was not stimulated in the presence of βCd.
These results indicate that Cd enhances IL-1 production from LPS stimulated macrophages, although Cd itself does not induce the IL-1 syntheses, which is associated with the formation of the inclusion complex of Cd with LPS and the increase of LPS solubility in water.

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In order to investigate the dynamics of metalloproteinase-9 (MMP-9), we measured proMMP-9 and tissue inhibitor of metalloproteinase-1 (TIMP-1) in sera and joint fluids by sandwich enzyme linked immunosorbent assay (ELISA), and examined immunohistochemically the expression of the proteins in joint tissues from patients with rheumatoid arthritis (RA) . ProMMP-9 concentration in 86 sera (749.4+940.2 ng/ml) and 54 joints fluids (4539.9±7681.5 ng/ml) from patients with RA was significantly higher than those of patients with osteoarthritis (OA) and controls.
Immunohistochemistry showed that in rheumatoid synovium proMMP-9 localized in neutrophils and monocytes diffusely infiltrated the sublining layer. In addition, osteoclasts in rheumatoid subchondral bone were intensively stained.
Both the concentration of proMMP-9 and the molar ratio of proMMP-9/TIMP-1 positively correlated to the number proMMP-9 positive cells in RA synovia (r=0.607, r=0.507) and the score of Roony's diffuse infiltrated of lymphocytes (r=0.720, r=0.591), respectively, although they were not correlated to the stage and class of Steinbrocker's and to the clinical laboratorial data.
Our results suggest MMP-9 produced by neutrophils and monocytes plays a role in tissue destruction of RA joint, and may be regulated by lymphocytes.

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Intra-articular injections of MR-20S (ulinastatin, urinary trypsin inhibitor) were performed for 16 knee joints of 16 patients (6 males, 10 females, average age 65.1±8.2) with osteoarthritis (OA) .
Levels of 7 joint markers in the synovial fluids were measured before and after injection therapy. Levels of chondroitin 4-sulfate (C-4S), 6-sulfate (C-6S) and hyaluronic acid (HA) were measured using HPLC. Levels of type II procollagen C-peptide (pCOL II-C), MMP-3, TIMP-1 and PMN elastase were measured by ETA. Levels of pCOL II-C increased significantly after injections (p<0.01) . In the patients with C-4S levels of≥15 nmol/ml or C-6S levels of≥40 nmol/ml before injection, these chondroitin isomers decreased significantly (p<0.01) . No significant changes of HA, MMP-3, TIMP-1 and PMN elastase levels were observed after injection.
The present data suggested that intra-articular injection of MR-20S might affect the metabolism of cartilage and synovium in OA.

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Nonsteroidal antiinflammatory drugs (NSAIDs) are used extensively in the treatment of rheumatic diseases. However, the inhibition of PG synthesis is major factor accounting for damage to the gastroduodenal mucosa which has given rise to the term“NSAID gastropathy”. Recently, prodrug of NSAID have been developed. Since prodrug of NSAID pass through the gastrointestinal tract in the inactive form. GI disturbance by prodrug are expected to be weak.
A controlled double blind study was performed for 19 rheumatic patients in order to assess the damaging action on the gastroduodenal mucosa induced by a prodrug (inactive form) of piroxicam (ampiroxicam) in comparison with piroxicam.
Subjects were recieved either of the following two drugs combinations ; (1) piroxicam and ampiroxicam placebo, (2) piroxicam placebo and ampiroxicam. Endoscopic findings were evaluated before and 2 weeks after treatment.
The incidence of NSAID gastropathy was slightly lower in subjects administered ampiroxicam (50%) than in those administered piroxicam (78%) . In the ampiroxicam group with patients moderate indulgence of addictive habits, especially alcohol ingestion, were found to be significant factor promoting the development of GI damage by oral ampiroxicam. There was no correlation between the presence or absence of subjective symptomes and endoscopic findings.
The results of this study indicate that ampiroxicam (prodrug of piroxicam) was slightly able to avoid GI damage. However, the incidence of ampiroxicam induced GI damage was not significantly superior to piroxicam administered group. It would therefore appear still dangerous for clinicians.

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