Fundamental study of osteoclast chemotaxis toward chemoattractants expressed in periodontitis.

BACKGROUND AND OBJECTIVE: Periodontitis is a chronic inflammatory disease that leads to bone resorption by osteoclasts (OCs). Several factors contribute to the differentiation of OCs from hematopoietic precursors. Cellular chemotactic factors are expressed in periodontitis tissue, but the effects of these chemoattractants on OCs are not well understood. Here we examined the effects of chemoattractants produced in inflamed periodontal tissue on OC chemotaxis. MATERIAL AND METHODS: Rat bone-marrow OCs were cultured in OC culture medium for 3 or 6 d. Using EZ-TAXIScan™, the chemotactic response of these OCs to several chemoattractants [monocyte chemotactic protein-1; macrophage inflammatory protein 1α; regulated on activation, normal T-cell expressed and secreted; stromal cell-derived factor-1α; and complement activation product 5a (C5a)] was measured. In addition, we measured the effect of C5a-specific inhibitors on chemotactic responses toward C5a. The recorded chemotactic responses were quantitatively analysed using ImageJ software. RESULTS: Chemoattractants associated with periodontal disease significantly increased the chemotactic activity of differentiated rat OCs in a concentration-dependent manner, with C5a inducing the highest chemotactic activity of OCs cultured for 3 or 6 d. The C5a-specific inhibitor significantly inhibited chemotaxis toward C5a in a concentration-dependent manner. CONCLUSION: We suggest that C5a plays an important role in pathologic bone resorption in periodontal disease by stimulating the chemotaxis of OCs. Therefore, C5a is a potential target for the treatment of periodontal disease.

Pharmacokinetic study of the oral administration of procaterol hydrochloride hydrate 50 µg in healthy adult Japanese men.

BACKGROUND: The pharmacokinetics of procaterol, a selective beta2-adrenergic agonist with a high intrinsic efficacy in man, could not be determined in humans when the drug was launched because of the low therapeutic dose and the low sensitivity of the analytical methods available at the time. However, a recently established analytical method using LC-MS/MS has been refined to enable the determination of the pharmacokinetic profile of procaterol and its metabolites in humans. METHODS: Procaterol hydrochloride hydrate 50 µg was administered orally to 8 healthy adult Japanese men. Plasma and urine samples collected from the subjects were analyzed by use of LC-MS/MS for procaterol and its metabolites. RESULTS: Following the oral administration of procaterol hydrochloride hydrate 50 µg, the plasma concentration of procaterol reached a Cmax of 136.4 pg/ml at ~1.44 h post-dose. The mean apparent terminal elimination half-life was ~3.83 h. DM-251 and DM-252, glucuronides of the optical isomers of procaterol, were the main metabolites and both were present in plasma at higher levels than procaterol in the plasma. The 24 h urinary excretion rates of unchanged procaterol, DM-251 and DM-252 were 15.7%, 12.4% and 11.2% of the procaterol administered, respectively. CONCLUSION: This study describes the pharmacokinetic profiles of procaterol and its metabolites following the oral administration of procaterol hydrochloride hydrate 50 µg. Procaterol and its glucuronides were found at high levels in the plasma and urine.

Pertussis toxin-sensitive G proteins as mediators of the signal transduction pathways activated by cytomegalovirus infection of smooth muscle cells.

We demonstrated recently that the arachidonic acid (AA) cascade is involved in cytomegalovirus (CMV)-induced generation of reactive oxygen species (ROS) and the activation of nuclear factor (NF)-kappaB in human smooth muscle cells (SMCs). Since AA release from neutrophils is mediated by pertussis toxin (PTx)-sensitive guanine nucleotide-binding (G) proteins, we hypothesized by analogy that CMV stimulates ROS generation in SMCs and ultimately activates NF-kappaB via a PTx-sensitive G protein-coupled pathway. Our first test of this hypothesis demonstrated that PTx blocked AA release induced by CMV infection of SMCs, as well as blocked the terminal products of this reaction, ROS generation and NF-kappaB activation. More proximal components of the pathway were then examined. CMV infection increased phosphorylation and activity of cytosolic phospholipase A2 (cPLA2), an enzyme causing AA release; these effects were inhibited by PTx. CMV infection activated mitogen-activated protein (MAP) kinase, a key enzyme for cPLA2 phosphorylation, an effect also inhibited by PTx. Finally, inhibition of MAP kinase kinase (MAPKK), which phosphorylates and thereby activates MAP kinase, inhibited CMV-induced ROS generation. These data demonstrate that a PTx-sensitive G protein-dependent signaling pathway mediates cellular effects of CMV infection of SMCs. The downstream events include phosphorylation and activation of MAP kinase by MAPKK and subsequent phosphorylation and activation of cPLA2 (with its translocation to cell membranes), followed by stimulation of the AA cascade, which generates intracellular ROS and thereby activates NF-kappaB.

Mitochondrial regulation of cell death: mitochondria are essential for procaspase 3-p21 complex formation to resist Fas-mediated cell death.

Death receptor Fas transduces cell death signaling upon stimulation by Fas ligand, and this death signaling is mediated by caspase. Recently, we reported that the cell cycle regulator p21 interacts with procaspase 3 to resist Fas-mediated cell death. In the present study, the molecular characterization and functional region of the procaspase 3-p21 complex was further investigated. We observed the p21 expression in the mitochondrial fraction of HepG2 cells and detected Fas-mediated cell death only in the presence of actinomycin D. However, mitochondrial-DNA-lacking HepG2 (MDLH) cells showed this effect even in the absence of actinomycin D. Both p21 and procaspase 3 were expressed in MDLH cells, but the procaspase 3-p21 complex formation was not observed. Interestingly, the resistance to Fas-mediated cell death in the MDLH cells without actinomycin D was recovered after microinjection of HepG2-derived mitochondria into the MDLH cells. We conclude that mitochondria are necessary for procaspase 3-p21 complex formation and propose that the mitochondrial role during cell death is not only death induction but also death suppression.

Combined effect of the 5-HT2 receptor antagonist DV-7028 and aspirin or heparin on coronary cyclic flow reductions in dogs.

OBJECTIVE: The aim was to study the combined effect of DV-7028, a selective 5-hydroxytryptamine2 receptor antagonist, and aspirin or heparin on cyclic flow reductions in the canine coronary artery. METHODS: Anaesthetised open chest beagle dogs under artificial respiration were used. Cyclic flow reductions were induced by partial occlusion of the left anterior descending coronary artery at the site of endothelial injury. After induction of cyclic flow reductions, test drugs were given to the animals intravenously. RESULTS: DV-7028 (0.1 mg.kg-1) reduced the frequency of cyclic flow reductions by 77% and improved the nadir of coronary blood flow velocity that indicated the severity of cyclic flow reductions. Also, aspirin (1 or 3 mg.kg-1) or heparin (200 U.kg-1) attenuated the cyclic flow reductions. In experiments with drug combinations, DV-7028 was given to animals that had already received aspirin (1 mg.kg-1) or heparin (200 U.kg-1). DV-7028 (0.1 mg.kg-1) completely abolished the cyclic flow reductions remaining after aspirin treatment in three of four animals. Heparin inhibited the cyclic flow reductions in one of five animals and the addition of DV-7028 abolished the remaining cyclic flow reductions in the other four animals. After combined injection of DV-7028 with aspirin or heparin, the coronary blood flow with cyclical reductions returned to the baseline. CONCLUSIONS: The 5-HT2 receptor antagonist DV-7028 can inhibit the cyclic flow reductions that are resistant to aspirin or heparin. The combined regimen of DV-7028 and aspirin or heparin in treatment of acute coronary stenosis is more effective than that of aspirin or heparin alone.

Effect of medium pH on osteoclast activity and osteoclast formation in cultures of dispersed rabbit osteoclasts.

We investigated the effect of medium pH on activity of isolated osteoclasts and have also looked at the possibility that medium pH affects osteoclast numbers during culture. Osteoclast-containing cell suspensions prepared from neonatal rabbits were cultured on bovine bone slices at pH 6.5, 7.0, or 7.5. After 24 or 48 h of culture, the cells and bone slices were fixed and stained for tartrate-resistant acid phosphatase (TRAP). After counting the osteoclasts, the cells were removed and the resorption lacunae stained by immunostaining using anticollagen type I antibody and then quantitated. We found that the resorptive activity of isolated rabbit osteoclasts was sharply increased at pH 6.5-7. Osteoclast differentiation and proliferation, on the other hand, were optimal at pH 7.0-7.5 but decreased at pH 6.5. The results thus imply that pH regulation of the bone surface environment can dramatically alter both the number of osteoclasts and their resorptive activity.

Serotonin inhibits release of substance P evoked by tooth pulp stimulation in trigeminal nucleus caudalis in rabbits.

The influence of the serotonin (5-HT) system on the release of immunoreactive substance P after electrical stimulation of the lower incisor pulp was examined by perfusion of the superficial layers of the subnucleus caudalis of the brain stem trigeminal sensory nuclear complex of rabbits in situ. Increased release of immunoreactive substance P was observed after electrical stimulation of the pulp at 40 V. Stimulation of the nucleus raphe magnus significantly increased the release of 5-HT and completely inhibited the release of immunoreactive substance P, evoked by stimulation of the tooth pulp. Local application of 5-HT (10(-6) M) inhibited the release of immunoreactive substance P induced by stimulation and this inhibition was antagonized by methysergide (10(-4) M) applied concomitantly to the superficial layers of the trigeminal nucleus. These results suggest a functional interaction between substance P and 5-HT in the superficial layers of the trigeminal nucleus for regulation of transmission of dental pain.

DX-9065a, an orally active factor Xa inhibitor, does not facilitate haemorrhage induced by tail transection or gastric ulcer at the effective doses in rat thrombosis model.

DX-9065a is an antithrombin III (AT III)-independent and selective inhibitor of activated blood coagulation factor X (FXa). We evaluated the effects of DX-9065a and warfarin on bleeding time and blood loss in rat tail transection model and on blood loss in hydrochloride (HCl)-induced rat gastrointestinal haemorrhage model. The blood loss was determined by measuring the haemoglobin content in saline immersed with transected tail or hematin chloride content in the gaster after HCl administration. DX-9065a or warfarin was administered orally at 1 h or 15-21 h before the haemorrhagic stimuli, respectively. The dose required for 50% inhibition of thrombus formation (ID50) was 21 mg/kg for DX-9065a and 0.75 mg/kg for warfarin in a copper wire-inserted arteriovenous (AV) shunt model. In contrast to DX-9065a (10 or 30 mg/kg), warfarin (0.75 mg/kg) significantly prolonged the bleeding time. In rat tail transection model, the blood loss for the control group was 102+/-41 microl at 20 min after the transection. While warfarin (0.75 mg/kg) facilitated the blood loss about 5 times as much as the control, DX-9065a (10 or 30 mg/kg) did not. In rat gastrointestinal model, the blood loss for the control group was 15.9+/-5.6 microl at 15 min after HCl administration. In contrast to DX-9065a (10 or 30 mg/kg), warfarin (0.75 mg/kg) increased the blood loss about twice as much as the control. Thus, compared with warfarin, DX-9065a only increased bleeding time or blood loss to a minor extent in the doses tested. These observations suggest that direct inhibition of FXa could be preferable to warfarin in the suppression of thrombosis without haemorrhagic complications.

Development of a new calcium phosphate cement that contains sodium calcium phosphate.

A cement powder consisting of sodium calcium phosphate, Na3Ca6(PO4)5, in addition to tetracalcium phosphate and beta-tricalcium phosphate was prepared by pulverizing blocks of 4 wt% sodium-, 11 wt% carbonate-containing apatite samples that were heated at 1700 degrees C for 5 h. When mixed with 30 wt% malic acid or citric acid at a powder liquid ratio of 3:1, the cement set in 3 or 7 min at room temperature with compressive strength being around 52 or 27 MPa. In HeLa-cell cultures, the cement mixed with malic acid was less cytotoxic than the cement mixed with citric acid, which was far less cytotoxic than a commercial carboxylate cement used as a negative control, suggesting malic acid to be superior to citric acid as a liquid in this regard. Similar findings were also obtained with osteoclasts, of which culture experiments clearly suggested that the number of osteoclasts on the cement mixed with malic acid was significantly greater than that on the cement mixed with citric acid. Since osteoclastic response to substrates could be used as a maker in evaluating their bioresorbability associated with osteoclasts, the above finding may suggest that the cement that is to be mixed with malic acid would be more useful as bone substitutes.

Involvement of descending monoaminergic systems in the transmission of dental pain in the trigeminal nucleus caudalis of the rabbit.

Perfusates were taken from the superficial layers of the subnucleus caudalis of the trigeminal sensory nuclear complex (SpVc), the first relay station of dental pain, with a push-pull cannula system and were assayed for endogenous serotonin (5-HT) and catecholamines by high-pressure liquid chromatography with an electrochemical detection. Spontaneous release of 5-HT and epinephrine was observed, while that of norepinephrine was not. Tooth pulp stimulation (ST) tended to increase the level of 5-HT in the perfusates. Pretreatment with morphine at a dose of 10 mg/kg (i.v.) significantly enhanced the release of 5-HT. However, there was no significant difference in morphine effect on the 5-HT level between stimulated and non-stimulated animals. Systemic administration of morphine (10 mg/kg i.v.) completely inhibited the release of immunoreactive substance P from the superficial layers of SpVc evoked by ST, and this inhibition was antagonized by local application of methysergide (10(-4) M). These results suggest that in the superficial layers of SpVc, morphine may primarily activate the descending 5-HT pathway which serves to modulate dental pain transmission.

Effects of opioids and opioid peptide on the release of substance P-like material induced by tooth pulp stimulation in the trigeminal nucleus caudalis of the rabbit.

The superficial layer in subnucleus caudalis of the brain-stem trigeminal sensory nuclear complex (SpVc) in the rabbit was perfused with artificial cerebrospinal fluid using a push-pull perfusion cannula system. Immunoreactive substance P (iSP) and [Met5]enkephalin (iME) released into the perfusates following electrical stimulation of the lower incisor pulp were measured. An increase in the release of iSP and iME lasting for 1 h or more was observed following electrical stimulation with 40 V. The increase in iSP release depended on the intensity of stimulation. Systemic morphine (10 mg/kg i.v.) completely inhibited the stimulus-evoked iSP release and this inhibition was antagonized by pretreatment with naloxone (5 mg/kg i.v.). The stimulus-evoked iSP release was also inhibited by local application of morphine (10(-6) M) or the opioid peptide [D-Ala2,Met5]enkephalinamide (10(-4) M). However, the local application of naloxone (5 X 10(-7) M) only partially antagonized the inhibitory effects of locally applied morphine and the opioid peptide. These results suggest that there is a functional interaction between SP and enkephalin systems in the superficial layer of SpVc for the regulation of dental pain transmission.

Membrane potential of mesenteric artery from carvedilol-treated spontaneously hypertensive rats.

The effects of chronic treatment of stroke-prone spontaneously hypertensive rats (SHRSP) with carvedilol, an antihypertensive agent which has both alpha- and beta-adrenoceptor-blocking actions, on membrane potential and relaxation of mesenteric resistant artery were studied. Five-week old SHRSP were treated with carvedilol for three months. At 16 weeks, the resting membrane potential of arteries from carvedilol-treated SHRSP was more negative than that of arteries from untreated SHRSP. The magnitude of acetylcholine-induced hyperpolarization in arteries from carvedilol-treated SHRSP was not different from that of arteries from untreated SHRSP. In the presence of noradrenaline, the membrane potential of arteries from carvedilol-treated SHRSP was more negative than that of arteries from untreated SHRSP. The membrane potential of arteries from carvedilol-treated SHRSP in the presence of noradrenaline and acetylcholine was more negative than that of arteries from untreated SHRSP. The acetylcholine-induced relaxation in noradrenaline-precontracted preparations from carvedilol-treated SHRSP was greater than that in preparations from untreated SHRSP and was smaller than that in preparations from Wistar Kyoto rats. Scanning electronmicroscopy showed that carvedilol-treatment decreased the structural abnormalities of the endothelium of arteries from SHRSP. These results indicate that chronic carvedilol treatment made the membrane potential of smooth muscle more negative and improved endothelial function in the mesenteric artery of SHRSP, which may contribute to the antihypertensive effect of carvedilol.

A specific inhibitor of factor Xa, DX-9065a, exerts effective protection against experimental tumor induced disseminated intravascular coagulation in rats.

(+)-2S-2-[4-[[(35S)-1-acetimidoyl-3-pyrrolidinyl]oxy]phenyl]-3-[7- amidino-2-napthyl]propanoic acid hydrochloride pentahydrate (DX-9065a) is an antithrombin III-independent, selective inhibitor of activated blood coagulation factor X (FXa). We investigated the protective effects of DX-9065a against tumor-bearing experimental disseminated intravascular coagulation (DIC) induced by the inoculation of AH-109A cells into rats. DX-9065a was subcutaneously administered at doses of 0.03 and 0.1 mg/kg/hour through an osmotic pump transplanted immediately after the inoculation of the tumor cells during the observation period. Platelet count decreased 12 days after the inoculation, concomitant with an increase in the thrombin-antithrombin III complex and fibrin and fibrinogen degradation products. Doses of 0.03 and 0.1 mg/kg/hour of DX-9065a significantly inhibited the decrease in plasma fibrinogen concentration and platelet count 13 days after the inoculation, respectively. These findings suggest that direct, selective inhibition of FXa by DX-9065a improves the hypercoagulable state induced by the progress of solid tumor.

Effects of chronic treatment with carvedilol on smooth muscle tone and endothelium-dependent relaxation of aorta in stroke-prone spontaneously hypertensive rats.

Effects of chronic treatment of stroke-prone spontaneously hypertensive rats (SHRSP) with carvedilol, an agent which has both alpha and beta-adrenoceptor blocking actions, on spontaneous muscle tone and on structural and functional abnormalities of endothelium were studied. The treatment of SHRSP with the drug of the dose of 30 to 200 mg/kg/day lowered the blood pressure significantly. Spontaneous muscle tone in endothelium-removed preparation disappeared by the treatment. Noradrenaline-induced contraction was depressed by the treatment in endothelium intact preparation but not in endothelium removed preparation. The treatment prevented the structural and functional abnormalities of endothelium. Similar results were obtained by the treatment with propranolol. These results indicate that carvedilol prevented abnormal contraction of SHRSP aorta through protective effects on smooth muscle and endothelium. These effects may play roles in blood pressure lowering effect of carvedilol.

Immunohistochemical localization of glycosaminoglycans with the use of monoclonal antibodies in salivary pleomorphic adenomas.

Immunohistochemical identification of glycosaminoglycans (GG) in salivary pleomorphic adenomas (63 cases) was made to evaluate chondrogenesis in modified myoepithelial cell regions. Monoclonal antibodies (MoAbs) to GGs were used in conjunction with specific enzyme digestion, and chondroitin 4S proteoglycan (C4SPG), chondroitin 6S PG(C6SPG), dermatan sulfate PG(DSPG), heparan sulfate PG(HSPG) and keratan sulfate PG(KSPC) were identified. Modified myoepithelial cells showing fibrillar and plasmatoid shapes contained KSPG (68%), DSPG (32%) and CSPG(C6SPG:22%, C4SPG:33%). Foci of chondroidally changed cells stained intensely for KSPG (53%), and less so for CSPG(C6SPG:22%, C4SPG:33%), and DSPG (19%). Perinuclear matrix in chondroidal tissue reacted most strongly. Almost all types of modified myoepithelial cells, or outer layer tumor cells of tubulo-ductal structures, produced or synthesized CSPG, DSPG, and HSPG. Certain cells located in hyalinous and myxomatous tissues may undergo chondroidal metaplasia, and clusters of such cells may produce GGs and PGs in the perinuclear zone similar to those GGs in matrix synthesized in chondroidal tissue. GG-synthesizing cells might continuously produce KSPG until the cartilage matrix is completed.

Experimentally induced periodontitis in beagle dogs causes rapid increases in osteoclastic resorption of alveolar bone.

This study was undertaken to observe osteoclast differentiation related to inflammatory progression in aggressive periodontitis induced in beagle dogs by ligature of the gingival sulcus. To monitor osteoclastic activity, we used histochemical methods (staining for tartrate-resistant acid phosphatase [TRAP]) to visualize osteoclasts and their TRAP-positive precursors and biochemical methods (ELISA assay of pyridinium crosslinks) to detect bone matrix degradation products in gingival crevicular fluid (GCF), serum, and urine. For histochemical study, tissue specimens were prepared from 3 adult female beagle dogs induced with experimental periodontitis by silk ligature placement below the gingival margin of mandibular molars ligated for 3, 7, and 21 days. For biochemical study for pyridinoline measurement, the 24 mandibular molars of 4 male beagle dogs were ligated. GCF, urine, and serum were collected at day 0 and at 3, 7, 14, and 21 days after ligation. In the early inflammatory phase of ligature-induced periodontitis (day 3), TRAP+ mononuclear and TRAP+ multinucleated cells were present in the gingival connective tissue, and active bone-resorbing cells were found in excavated lacunae at the alveolar crest, but osteoclasts were not infiltrating the periodontal ligament during this early phase. During later stages of the inflammatory process (7 and 21 days), osteoclasts appeared at both the gingival and ligament side of the alveolar bone. Osteoclastic bone resorption appeared to be more severe on the bone surface at the gingival side than on the bone surface of the periodontal ligament side. Measurement of pyridinoline significantly increased in GCF and urine 3 days after ligation. The results suggested that bone at the crest of the alveolar bone is rapidly resorbed within 3 days of inducing experimental periodontitis.

Long-term follow-up of periodontitis in a patient with Chédiak-Higashi syndrome. A case report.

Chediak-Higashi syndrome (CHS) is an extremely rare hereditary disease characterized by leukocyte dysfunction. We report on a 21-year-old woman who presented at the age 9 years with CHS and serious periodontal tissue destruction around erupted teeth. The patient had received systemic, radiographic, immunological, microbial, and clinical periodontal examinations since childhood. The chemotactic activity of neutrophils in the Boyden chamber assay was 22% of the control, and leukocyte bactericidal activity was one-third of the control. Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia were isolated from periodontal pockets. Periodontal treatment including oral hygiene was provided, followed by professional tooth cleaning from the age of 12 to 21 years. However, the mobility of teeth and the inflammation of periodontal tissue progressed. This CHS patient presented with periodontal disease of extremely early onset, which was resistant to periodontal treatment.

Lectin-binding glycoconjugates and immunoreactive proteoglycans in resorption pits freshly excavated by isolated rabbit osteoclasts.

Staining with FITC-conjugated concanavalin A, wheatgerm agglutinin and peanut agglutinin demonstrated that abundant sugar residues are present in resorption pits produced in vitro and over the cell surface of rabbit osteoclasts resorbing bovine femoral bone slices. After chondroitinase ABC digestion the stubs of chondroitin 4-sulphate and dermatan sulphate could be detected in the resorption pits by monoclonal antibodies.

Failure of aspirin and diltiazem to prevent the formation of acute coronary thrombi in dogs.

The aim of the present study was to determine the efficacy of aspirin and diltiazem in preventing the formation of coronary thrombi in dogs. Canine coronary thrombi were produced by inserting a small catheter filled with collagen powder into the endothelial-injured, partially occluded left anterior descending coronary artery. Neither aspirin (bolus of 30 mg/kg, followed by 100 mg/kg/h by infusion), nor diltiazem (0.1 mg/kg, followed by 0.3 mg/kg/h by infusion) prevented the formation of coronary thrombi. The mortality in aspirin group was significantly higher than that in control and diltiazem groups. These results indicate that aspirin and diltiazem do not inhibit thrombus formation in the canine model of coronary thrombosis.

Electroretinogram in cone dystrophy.

Electroretinograms (ERGs) in 10 cases of cone dystrophy were studied with special respect to log(bp/bs) that represents the log of photopic ERG amplitude divided by scotopic ERG amplitude. Photopic ERGs were either greatly diminished or nonrecordable, and scotopic ERGs showed either normal or reduced amplitudes. All cases had a low value of log(bp/bs), less than the lower normal limit, indicating significantly greater impairment in cone function than in rod function. These results have proved log(bp/bs) to be helpful in confirming the diagnosis of cone dystrophy especially in cases with recordable photopic ERG and reduced scotopic ERG. Among other ERG parameters, the photopic ERG b-wave implicit time that was determined with averaging and digital amplification of the responses in 4 cases showed prolongation in 3 cases while normal in one case. Oscillatory potentials were nonrecordable or barely recordable in all cases.