Eiro Kubota

Synovial fluid cytokines and proteinases as markers of temporomandibular joint disease

PURPOSEnIn this article, biochemical markers in the synovial fluid (SF) for detecting intraarticular inflammation and early cartilage degradation of the temporomandibular joint (TMJ) disease were examined.nnnPATIENTS AND METHODSnSF was obtained from 25 TMJs in 22 patients with internal derangement (ID) or osteoarthritis (TMJ-OA), 15 asymptomatic TMJs in 11 normal volunteers, and 10 osteoarthritic knee joints (KNEE-OA). Cytokine levels were measured by enzyme-linked immunosorbent assay (ELISA), and the proteinase activities were detected by enzymography.nnnRESULTSnSF from TMJs with ID and OA showed higher (P < .05) levels (330.1 +/- 347.7 pg/100 microg SF protein) of IL-1beta than the asymptomatic control TMJs (76.7 +/- 95.3 pg/100 microg of SF protein). SF from TMJs with OA contained significantly (P < .05) higher levels of IL-1beta (531.8 +/- 379.6 pg/100 microg of SF protein) and IL-6 (979 +/- 552 pg/100 microg SF protein) than those with ID (IL-1beta: 216.7 +/- 280.1 pg, IL-6: 293 +/- 434 pg). Two matrix metalloproteinases (MMPs) with gelatinolytic activities at 92 kDa and 72 kDa were consistently detected in both the TMJ-SF (either normal or disease) and SF from KNEE-OA. Also detected were weak bands with molecular weight of 83 and 66 kDa. These bands were clearly shown, particularly in knee joints with advanced stages of OA. Western blot analysis delineated that these were active forms of MMP-9 (83 kDa) and MMP-2 (66 kDa). The same bands were also detected in TMJs with OA that showed high levels of IL-1beta and IL-6.nnnCONCLUSIONnThese findings suggest that concomitant increases in the levels of cytokines (IL-1 and IL-6) and active forms of MMPs could be potential catabolic markers for cartilage degradation in the TMJ.

Interleukin 1β and stromelysin (MMP3) activity of synovial fluid as possible markers of osteoarthritis in the temporomandibular joint

PURPOSEnThis study investigated the early signs of synovitis and cartilage degradation by means of synovial fluid analysis in temporomandibular joints (TMJs) with internal derangement (closed lock) or osteoarthritis (OA).nnnPATIENTS AND METHODSnSynovial fluid was obtained from 25 TMJs in 22 patients diagnosed with closed lock and from 15 asymptomatic TMJs of 12 normal controls. IL-1 beta concentrations were measured using enzyme-linked immunosorbent assay (ELISA), and proteinase activity was detected by means of gelatin enzymography.nnnRESULTSnNine of the 25 TMJs with closed lock (CL group) exhibited osteolytic changes on the surface of the condyle. TMJs in the normal control group did not show any bony changes. Mean IL-1 beta concentration in the synovial fluid (SF) protein in the CL group was 330.1 +/- 347.7 pg per 100 micrograms protein, which was significantly higher than in the normal control (76.7 +/- 95.3 pg/100 micrograms SF-protein). Synovial fluid from the TMJs with osteolytic changes contained higher levels of IL-1 beta (531.8 +/- 379.6 pg/100 micrograms SF-protein) than those without bony changes (216.7 +/- 280.1 pg/100 micrograms SF-protein). Matrix metalloproteinase (MMP) activity with a molecular weight of 50 kd (stromelysin or MMP3) was detected in a highly augmented form in two synovial fluid samples of seven closed lock patients.nnnCONCLUSIONnThe results suggest that IL-1 beta levels in synovial fluid of the TMJ have a positive correlation with OA change. The MMP3 activity detected was greatly increased in patients with cartilage degradation. These findings suggest that both changes may be important markers of early bone deterioration in TMJs that are undetectable by radiograph imaging.

Intra-articular levels of prostaglandin E2, hyaluronic acid, and chondroitin-4 and -6 sulfates in the temperomandibular joint synovial fluid of patients with internal derangement☆

PURPOSEnThis study was conducted to measure the intra-articular levels of prostaglandin E2 (PGE2), hyaluronic acid, and chondroitin-4 and -6 sulfate in patients with temporomandibular joint (TMJ) internal derangement involving a closed lock, and to see if these levels correlate with the clinical or arthroscopic findings.nnnPATIENTS AND METHODSnFifteen female patients (16 joints) with a mean age of 36.7 years were diagnosed as having a closed lock by clinical examination and diagnostic MR imaging. The patients subjective pain was assessed by a visual analog scale (VAS) and a pain questionnaire (pain score), and the interincisal opening was measured. TMJ aspirates were obtained by washing of the joint with saline containing vitamin B12 as a marker for calibration of data. The samples were assayed for PGE2 with a radioimmunoassay, and the concentrations of unsaturated disaccaride isomers of hyaluronic acid (delta di-HA), chondroitin-4 sulfate (delta di-4S), and chondroitin-6 sulfate (delta di-6S) were measured by high-performance liquid chromatography. Immediately after collection of the synovial aspirates, diagnostic arthroscopy was performed on all but three joints to evaluate the severity of synovitis and cartilage degeneration. The degree of arthroscopic pathology was scored quantitatively. Intra-articular levels of PGE2, delta di-HA(HA), delta di-4S(C4S), and delta di-6S(C6S) were compared with patients age, mouth opening, VAS rating, pain scores, and arthroscopic scores for synovitis and cartilage degeneration.nnnRESULTSnThe PGE2 level did not correlate with the clinical or arthroscopic parameters. HA had a weak correlation with mouth opening (0.54). C4S and C6S were correlated with arthroscopic scores of TMJ degeneration (0.97, 0.89) and with age (0.75, 0.62). The ratio of C4S and C6S to HA was also correlated with the arthroscopic indices of degeneration (0.93, 0.8) and PGE2 level (0.74, 0.69), but not with age.nnnCONCLUSIONnThe PGE2 level in the TMJ synovial fluid does not specifically reflect the intensity of pain or synovitis, but the detection of high concentrations of C4S and C6S, compared with the amount of HA, is a possible marker of proteoglycan degradation in the TMJ.

Role of Gut Cryptopatches in Early Extrathymic Maturation of Intestinal Intraepithelial T Cells

Lympho-hemopoietic progenitors residing in murine gut cryptopatches (CP) have been shown to generate intestinal intraepithelial T cells (IEL). To investigate the role of CP in progenitor maturation, we analyzed IEL in male mice with a truncated mutation of common cytokine receptor γ-chain (CRγ−/Y) in which CP were undetectable. IEL-expressing TCR-γδ (γδ-IEL) were absent, and a drastically reduced number of Thy-1highCD4+ and Thy-1highCD8αβ+ αβ-IEL were present in CRγ−/Y mice, whereas these αβ-IEL disappeared from athymic CRγ−/Y littermate mice. Athymic CRγ−/Y mice possessed a small TCR- and αEβ7 integrin-negative IEL population, characterized by the disappearance of the extrathymic CD8αα+ subset, that expressed pre-Tα, RAG-2, and TCR-Cβ but not CD3ε transcripts. These TCR− IEL from athymic CRγ−/Y mice did not undergo Dβ-Jβ and Vδ-Jδ joinings, despite normal rearrangements at the TCR-β and -δ loci in thymocytes from euthymic CRγ−/Y mice. In contrast, athymic severe combined immunodeficient mice in which CP developed normally possessed two major TCR−αEβ7+ CD8αα+ and CD8− IEL populations that expressed pre-Tα, RAG-2, TCR-Cβ, and CD3ε transcripts. These findings underscore the role of gut CP in the early extrathymic maturation of CD8αα+ IEL, including cell-surface expression of αEβ7 integrin, CD3ε gene transcription, and TCR gene rearrangements.

Cationized gelatin delivery of a plasmid DNA expressing small interference RNA for VEGF inhibits murine squamous cell carcinoma

Double‐stranded RNA (dsRNA) plays a major role in RNA interference (RNAi), a process in which segments of dsRNA are initially cleaved by the Dicer into shorter segments (21–23 nt) called small interfering RNA (siRNA). These siRNA then specifically target homologous mRNA molecules causing them to be degraded by cellular ribonucleases. RNAi downregulates endogenous gene expression in mammalian cells. Vascular endothelial growth factor (VEGF) is a key molecule in vasculogenesis as well as in angiogenesis. Tumor growth is an angiogenesis‐dependent process, and therapeutic strategies aimed at inhibiting angiogenesis are theoretically attractive. To investigate the feasibility of using siRNA for VEGF in the specific knockdown of VEGF mRNA, thereby inhibiting angiogenesis, we have performed experiments with a DNA vector based on a siRNA system that targets VEGF (siVEGF). It almost completely inhibited the expression of three different isoforms (VEGF120, VEGF164 and VEGF188) of VEGF mRNA and the secretion of VEGF protein in mouse squamous cell carcinoma NRS‐1 cells. The siVEGF released from cationized gelatin microspheres suppressed tumor growth in vivo. A marked reduction in vascularity accompanied the inhibition of a siVEGF‐transfected tumor. Fluorescent microscopic study showed that the complex of siVEGF with cationized gelatin microspheres was still present around the tumor 10 days after injection, while free siVEGF had vanished by that time. siVEGF gene therapy increased the fraction of vessels covered by pericytes and induced expression of angiopoietin‐1 by pericytes. These data suggest that cationized‐gelatin microspheres containing siVEGF can be used to normalize tumor vasculature and inhibit tumor growth in a NRS‐1 squamous cell carcinoma xenograft model. (Cancer Sci 2006; 97: 313u2003–u2003321)

Acidic extracellular pH increases calcium influx‐triggered phospholipase D activity along with acidic sphingomyelinase activation to induce matrix metalloproteinase‐9 expression in mouse metastatic melanoma

Acidic extracellular pH is a common feature of tumor tissues. We have reported that culturing cells at acidic pH (5.4–6.5) induced matrix metalloproteinase‐9 expression through phospholipaseu2003D, extracellular signal regulated kinaseu20031/2 and p38 mitogen‐activated protein kinases and nuclear factor‐κB. Here, we show that acidic extracellular pH signaling involves both pathways of phospholipaseu2003D triggered by Ca2+ influx and acidic sphingomyelinase in mouse B16 melanoma cells. We found that BAPTA‐AM [1,2‐bis(2‐aminophenoxy)‐ethane‐N,N,N′,N′‐tetraacetic acid tetrakis (acetoxymethyl) ester], a chelator of intracellular free calcium, and the voltage dependent Ca2+ channel blockers, mibefradil (for T‐type) and nimodipine (for L‐type), dose‐dependently inhibited acidic extracellular pH‐induced matrix metalloproteinase‐9 expression. Intracellular free calcium concentration ([Ca2+]i) was transiently elevated by acidic extracellular pH, and this [Ca2+]i elevation was repressed by EGTA and the voltage dependent Ca2+ channel blockers but not by phospholipaseu2003C inhibitor, suggesting that acidic extracellular pH increased [Ca2+]i through voltage dependent Ca2+ channel. In contrast, SR33557, an L‐type voltage dependent Ca2+ channel blocker and acidic sphingomyelinase inhibitor, attenuated matrix metalloproteinase‐9 induction but did not affect calcium influx. We found that acidic sphingomyelinase activity was induced by acidic extracellular pH and that the specific acidic sphingomyelinase inhibitors (perhexiline and desipramine) and siRNA targeting aSMase/smpd1 could inhibit acidic extracellular pH‐induced matrix metalloproteinase‐9 expression. BAPTA‐AM reduced acidic extracellular pH‐induced phospholipaseu2003D but not acidic sphingomyelinase acitivity. The acidic sphingomyelinase inhibitors did not affect the phosphorylation of extracellular signal regulated kinase 1/2 and p38, but they suppressed nuclear factor‐κB activity. These data suggest that the calcium influx‐triggered phospholipaseu2003D and acidic sphingomyelinase pathways of acidic extracellular pH induced matrix metalloproteinase‐9 expression, at least in part, through nuclear factor‐κB activation.

Glycosaminoglycan components in temporomandibular joint synovial fluid as markers of joint pathology

PURPOSEnThis study investigated the correlation between temporomandibular joint (TMJ) disease and the composition of glycosaminoglycans (GAGs) components in the synovial fluid (SF).nnnMATERIALS AND METHODSnSynovial fluid (SF) was obtained from 30 TMJs of 28 female patients diagnosed as having a displaced disc with reduction (WR) (seven joints), a displaced disc without reduction (WOR) (13 joints), osteoarthritis (OA) (five joints), or rheumatoid arthritis (RA) (five joints) by MR imaging and clinical examination. After the SF was directly aspirated, It was digested with chondroitinase ABC and hyaluronidase, and the concentration of unsaturated disaccharide isomers of chondroitin 6-sulfate (delta di-6S), chondroitin 4-sulfate (delta di-4S) and hyaluronic acid (delta di-HA) were measured by high-performance liquid chromatography (HPLC) combined with fluorometry. The ratio of delta di-6S or delta di-4S to delta di-HA, and delta di-6S to delta di-4S, were calculated.nnnRESULTSnThere were no significant differences in concentrations of delta di-6S, delta di-4S, or delta di-HA among the groups. The ratio of delta di-6S to delta di-4S was 2.7 +/- 1.4 in OA, 2.6 +/- 0.9 in joints with WOR, 2.9 +/- 1.2 in joints with WR, and 1.3 +/- 0.4 in RA synovial fluid. Differences in the delta di-6S: delta di-4S ratio between RA and the other conditions were statistically significant (P < .05).nnnCONCLUSIONnThese results suggest that the delta di-6S:delta di-4S ratio in the synovial fluid of the TMJ reflects the proteoglycan metabolism of the joint tissues, particularly of the articular cartilage and synovial tissue. This ratio could be used to diagnose joint diseases and to predict articular cartilage destruction or synovial proliferation caused by these diseases.

Restoration of BRAK / CXCL14 gene expression by gefitinib is associated with antitumor efficacy of the drug in head and neck squamous cell carcinoma.

Clinical efficacy of gefitinib (ZD1839, Iressa), which is an inhibitor specific for epidermal growth factor (EGF) receptor tyrosine kinase, has been shown in non‐small‐cell lung carcinoma patients with EGF receptor mutations, so these mutations are useful marker(s) to find a responder for the drug. Recent studies have shown that the EGF receptor gene mutation is rare in squamous cell carcinoma in the esophageal and head and neck regions. We previously reported that the expression of the chemokine BRAK/CXCL14 in head and neck squamous cell carcinoma (HNSCC) cells was down‐regulated by EGF treatment, and that forced expression of BRAK in tumor cells decreased the tumorigenicity of the cells in xenografts. Thus, we investigated the relationship between restoration of BRAK expression by gefitinib and the efficacy of the drug for tumor suppression. We found that EGF down‐regulated BRAK expression through the MEK–extracellular signal regulated kinase pathway and that this down‐regulated expression was restored by gefitinib in vitro. Oral administration of gefitinib significantly (Pu2003<u20030.001) reduced tumor growth of xenografts of three HNSCC cell lines (HSC‐2, HSC‐3, and HSC‐4), in female athymic nude mice, accompanied by an increase in BRAK expression specifically in tumor tissue. This tumor‐suppressing effect of the drug was not observed in the case of BRAK non‐expressing cells. Furthermore introduction of BRAK shRNA vector reduced both the expression levels of BRAK in HSC‐3 cells and the antitumor efficacy of gefitinib in vivo. Our data showing an inverse relationship between BRAK expression levels in tumor cells and the tumor growth rate indicate that the gefitinib‐induced increase in BRAK expression is beneficial for tumor suppression in vivo. (Cancer Sci 2009)

Evidence of reactive oxygen species generation in synovial fluid from patients with temporomandibular disease by electron spin resonance spectroscopy

Abstract Reactive oxygen species (ROS) have been implicated in the pathogenesis of temporomandibular disorders. In the present study, we provide the first evidence of ROS generation in the synovial fluid from human temporomandibular disorder patients, as shown by electron spin resonance (ESR) and spin trapping. Three distinct ESR spectra of DMPO spin adducts were observed in the synovial fluid. They corresponded to three free radical species: hydroxyl radical (HO•), hydrogen radical (H•), and carbon-center radical (R•). Among them, the 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)-OH spectrum was the most prominent, suggesting that HO• was dominantly generated in the synovial fluid from temporomandibular disorder patients. Desferrioxamine (DFO), an iron chelator, strongly depressed the DMPO-OH signal intensity in the synovial fluid from patients with temporomandibular disorders. We successfully demonstrated ROS-induced oxidative stress in the synovial fluid from temporomandibular disorder patients. ROS generation in the temporomandibular joint could lead to exacerbation of inflammation and activation of cartilage matrix degrading enzymes that proceed to degenerative change of the temporomandibular joint. Thus, iron-dependent generation of HO • might have a crucial role in the pathogenesis of temporomandibular disorders.

Essential Role of LFA-1 in Activating Th2-Like Responses by α-Galactosylceramide-Activated NKT Cells

NKT cells produce large amounts of cytokines associated with both the Th1 (IFN-γ) and Th2 (IL-4) responses following stimulation of their invariant Vα14 Ag receptor. The role of adhesion molecules in the activation of NKT cells by the Vα14 ligand α-galactosylceramide (α-GalCer) remains unclear. To address this issue, LFA-1−/− (CD11a−/−) mice were used to investigate IL-4 and IFN-γ production by NKT cells following α-GalCer stimulation. Intriguingly, LFA-1−/− mice showed increased IL-4, IL-5, and IL-13 production and polarized Th2-type responses in response to α-GalCer in vitro and in vivo. Furthermore, the Th2-specific transcription factor GATA-3 was up-regulated in α-GalCer-activated NKT cells from LFA-1−/− mice. These results provide the first genetic evidence that the adhesion receptor LFA-1 has a crucial role in Th2-polarizing functions of NKT cells.