The sphingosine-1-phosphate receptor 1 binding molecule FTY720 inhibits osteoclast formation in rats with ligature-induced periodontitis.

BACKGROUND AND OBJECTIVE: Osteoclast precursors (OPs) re-migrate from the bone surface into blood vessels through sphingosine-1-phosphate receptor 1 (S1PR1) expression. T cells also express S1PR1, mediating their migration from the lymph nodes into blood vessels. OP and T-cell migration are one of the sequential steps related to osteoclast formation. To characterize the role of S1PR1 in osteoclast formation induced by periodontitis, we investigated the effect of S1PR1-binding molecule FTY720 (FTY) on the number of OPs and T cells in periodontal tissue and peripheral blood of rats with ligature-induced periodontitis. MATERIAL AND METHODS: Rats were divided into four groups; control (Con), FTY, periodontitis (Peri), and periodontitis+FTY (Peri+FTY) groups. Ligatures were placed around the first molars in the left and right mandibles. The rats were intraperitoneally injected with vehicle or 3 mg/kg FTY daily until they were killed. The number of osteoclasts and cluster of differentiation (CD)11b, CD3 and receptor activator of NF-κB ligand (RANKL)-positive cells in first molar furcation were counted by tartrate-resistant acid phosphatase or immunohistochemistry staining. The number of CD11b- and CD3-positive cells in peripheral blood was estimated by flow cytometry. RESULTS: The number of osteoclasts in the Peri group was higher than Con, Peri+FTY and FTY groups (p < 0.05) and CD11b, CD3 and RANKL-positive cells were also higher in the Peri group than other groups in furcation (p < 0.05). While CD11b-positive cells in furcation of the Peri+FTY group were lower than the Peri group (p < 0.05), they were higher in peripheral blood (p < 0.05). Dissimilar to CD11b-positive cells, CD3-positive cells in the Peri+FTY group were lower in peripheral blood as well as furcation than the Peri group (p < 0.05). RANKL-positive cells in furcation of the Peri+FTY group were also lower than Peri group (p < 0.05). CONCLUSION: These results indicate that FTY may facilitate re-migration of OPs from the alveolar bone surface into blood vessels, blocking T-cell migration from the lymph nodes into blood vessels and subsequently reducing osteoclast formation induced by periodontitis. This suggests that S1PR1-S1P binding may play a role in osteoclast formation of periodontitis by modulating OP and T-cell migration.

Periodontitis mainly increases osteoclast formation via enhancing the differentiation of quiescent osteoclast precursors into osteoclasts.

BACKGROUND AND OBJECTIVES: Tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts are formed in sequential steps: proliferation and differentiation of hematopoietic progenitors into quiescent osteoclast precursors (QOPs), followed by fusion of QOPs. In this study, we investigated whether enhancement of osteoclast formation by periodontitis is derived from the stimulation of proliferation of hematopoietic progenitors or the differentiation of QOPs into osteoclasts. MATERIAL AND METHODS: Ligatures were placed around the first molars in the left mandibles of Fischer 344 inbred rats. The rats received drinking water containing bromodeoxyuridine (BrdU) (which can be incorporated into dividing nuclei) after ligation during the experimental period. The number of inflammatory cells in the distal area was counted. Alveolar bone loss was histologically estimated by measuring the distance from the cementoenamel junction to the alveolar bone crest in the distal area and determining the percentage of periodontal ligament area in the furcation. The number of osteoclasts and percentage of BrdU(+) nuclei in total osteoclasts nuclei were counted after TRAP and BrdU double labeling. RESULTS: The number of polymorphonuclear cells increased on day 1 and then rapidly decreased. The number of mononuclear cells increased in a time-dependent manner up to day 5 and remained the same until day 10. Alveolar bone loss of ligatured teeth increased in a time-dependent manner. The number of osteoclasts peaked on day 3 then gradually decreased. At peak, the percentage of BrdU(+) nuclei in total osteoclasts nuclei in the distal and furcation areas were 7.9% and 4.1%, respectively. CONCLUSION: These results indicate that most of the osteoclasts formed after periodontitis induction are derived from preformed QOPs, suggesting that enhancement of osteoclast formation by periodontitis might be mainly caused by stimulating the differentiation of QOPs into osteoclasts.

Diabetic characteristics and alveolar bone loss in streptozotocin- and streptozotocin-nicotinamide-treated rats with periodontitis.

BACKGROUND AND OBJECTIVE: Experimental models showing variable diabetic status are necessary to understand the relationship between diabetes and periodontitis. The streptozotocin (STZ)-induced diabetes model allows control of diabetic status by nicotinamide (NA), which protects against STZ-induced ß-cell necrosis. Therefore, we compared diabetic characteristics and alveolar bone loss in STZ- and STZ-NA-treated rats with periodontitis. MATERIAL AND METHODS: STZ-treated rats were generated by intravenous (IV) administration of STZ (50 mg/kg). STZ-NA-treated rats were induced by intraperitoneal administration of NA (270 mg/kg) 15 min before IV administration of STZ (65 mg/kg). Periodontitis was induced by ligature around the left mandibular first molar 1 wk after injection. Blood glucose level, glucose tolerance and serum insulin levels were determined at day 0 and/or 20 after ligature. At day 20, tibia bone loss was assessed using micro-computed tomography and hematoxylin and eosin staining. Alveolar bone loss was histologically measured as the distance of the cementoenamel junction to the alveolar bone crest in distal and the percentage of periodontal ligament area in the first molar furcation, respectively. The number of inflammatory cells, receptor activator of nuclear factor kappa-B ligand (RANKL)-positive cells and the area of osteoid were determined. RESULTS: In STZ-treated rats, obvious hyperglycemia over 300 mg/dL and severe body weight loss were observed. The insulin level was approximately 14% compared to that of control rats. STZ-NA-treated rats were impaired in glucose tolerance compared to control rats; however, body weight and insulin levels were not significantly different. Tibia bone loss was increased in STZ-treated rats, but significant change was not observed in STZ-NA-treated rats compared to control rats. In ligatured teeth, alveolar bone loss was increased in both STZ- and STZ-NA-treated rats compared to control rats. Alveolar bone loss, the number of inflammatory cells and RANKL-positive cells in STZ-treated rats were greater than in STZ-NA-treated rats. The area of osteoid decreased in STZ-treated rats compared to control, but not STZ-NA-treated rats. CONCLUSION: These results indicate that STZ- and STZ-NA-treated rats exhibit diabetic characteristics similar to type 1 diabetes mellitus and a pre-diabetic state, respectively. In addition, alveolar bone loss in response to periodontitis and tibia loss depend on diabetic status. Diabetic status-dependent bone remodeling imbalance and inflammation could affect the alveolar bone loss in the two models. Both STZ- and STZ-NA-treated rats may be useful to investigate differences in periodontitis sensitivity associated with diabetic status and to develop therapeutic agents for periodontitis in patients with diabetes.

The effect of orally administered epigallocatechin-3-gallate on ligature-induced periodontitis in rats.

BACKGROUND AND OBJECTIVE: Epigallocatechin-3-gallate (EGCG) is known for its beneficial properties, including anti-inflammatory and anti-oxidative activities. Recently, reports have suggested that EGCG plays a pivotal role in regulating cytokine expression and osteoclastic activity. In the present study, we investigated whether orally administered EGCG has a therapeutic effect on ligature-induced periodontitis. MATERIALS AND METHODS: Forty-eight Sprague-Dawley rats were treated with EGCG or phosphate-buffered saline. Periodontitis was induced by tying a ligature for 7 d. After removing ligation, EGCG (200 mg/kg) or phosphate-buffered saline was administered via oral gavage on a daily basis. Rats were killed after 1, 2 and 4 wk of administration. Histologic and histomorphometric analyses, tartrate resistant acid phosphatase staining and immunohistochemistry were carried out. RESULTS: In the control group, bone loss did not recover even after the causative factor of periodontitis was eliminated. On the other hand, distance from cemento-enamel junction to alveolar bone crest, long junctional epithelium and collagen destruction were reduced in the EGCG group. Decreased interleukin (IL)-6 expression was shown from the early stage of EGCG administration, followed by reduced tumor necrosis factor (TNF) expression at week 4 EGCG group. The CT area showed a higher decrease of IL-6 expression between the control and EGCG group than alveolar bone area. Downregulation of TNF and IL-6 expression led to a decrease in osteoclast number and activity, which resulted in reduced bone loss. CONCLUSIONS: Systemic administration of EGCG could have a therapeutic effect on damaged periodontal tissue. Inhibited cytokine expression, including TNF and IL-6 is responsible for the reduction in osteoclast formation, osteoclastic activity and collagen destruction.

Effects of HB-EGF and epiregulin on wound healing of gingival cells in vitro.

OBJECTIVE: Gingival wound healing is important to periodontal disease and surgery. This in vitro study was conducted to assess the manner in which heparin-binding epidermal growth factor-like growth factor (HB-EGF) and epiregulin cooperatively participate in the wound-healing process in the gingival epithelial and fibroblast cells of the oral mucosa. MATERIAL AND METHODS: Gingival epithelium and fibroblast were separated from gingival tissue biopsies and prepared to primary cultures. The changes in the mRNA expression were evaluated via real-time PCR. The effects on cell proliferation, migration, and repopulation were evaluated in vitro. RESULTS: The different regulation of expressions of HB-EGF, epiregulin, and epidermal growth factor receptors was observed over time and with different gingival cell types. HB-EGF exerted a cell migration-inducing effect on both epithelial and fibroblast cells, whereas epiregulin did not. Both growth factors functioned as mitogens for epithelial cell proliferation, but not for fibroblast proliferation. HB-EGF strongly promoted epithelial cell repopulation and mildly promoted fibroblast repopulation, whereas epiregulin promoted only fibroblast repopulation. CONCLUSION: These results indicated that both growth factors might function importantly in the wound-healing process of human gingival tissue via the different regulation of the expression, cell migration, proliferation, and repopulation.

Inhibitory effect of evodiamine alone and in combination with rosiglitazone on in vitro adipocyte differentiation and in vivo obesity related to diabetes.

OBJECTIVE: Evodiamine (evo) has been shown to exert anti-inflammatory, antinociceptive and anticancer effects. In this study, we investigated the effects of evo alone and in combination with rosiglitazone (rosi) on in vitro adipocyte differentiation and in vivo obesity related to diabetes. METHODS: Adipocyte differentiation was investigated in vitro using 3T3-L1 and C3H10T1/2 cells. To determine the degree of differentiation, Oil Red O staining and reverse transcription-PCR were carried out. Four groups of db/db mice were treated intraperitoneally once per day with vehicle, evo, rosi and evo+rosi. The mice were killed after 14 days and the blood, liver and adipose tissue were analyzed. RESULTS: The presence of evo or evo combined with rosi during adipogenic induction has been shown to inhibit adipocyte differentiation to a significant degree, particularly at the commitment and early induction stages. The evo and evo+rosi groups of db/db mice evidenced significant reductions in body weight gain. The ratio of epididymal white adipocyte tissue weight to body weight of the evo group was also significantly reduced. It is important to note that in the evo+rosi treatment, blood glucose levels were reduced to a degree similar to that of the rosi group, and plasma insulin levels were reduced significantly better than that of rosi group. Furthermore, hepatic lesions associated with fat and glycogen deposition were morphologically improved in the evo and evo+rosi groups. CONCLUSION: The results of this study showed that evo exerts an inhibitory effect on in vitro adipocyte differentiation and in vivo obesity, and also an improvement effect on insulin resistance. These desirable effects of evo were noted even in the presence of rosi. These results indicate that evo improves the undesirable effects of rosi, including adipogenesis, body weight gain and hepatotoxicity, while preserving its desirable blood-glucose-lowering effect.

Cervical chondroid chordoma in a Shetland sheep dog.

A spayed female Shetland sheep dog aged 12 years was presented for examination with ataxia and hindlimb paralysis. Extradural spinal cord compression was found at the level of vertebrae C6-C7 by radiography and myelocomputed tomography. A jelly-like mass (0.6 x 1.3 cm) was removed surgically. Histopathological findings were characterized by proliferation of vacuolated polygonal cells (physaliphorous cells) in a mucinous matrix and the presence of chondroid tissue shown immunohistochemically to express S-100. The physaliphorous cells were immunolabelled strongly for vimentin and S-100, and weakly for cytokeratin. A diagnosis of canine cervical chondroid chordoma was made. This is considered to be the first report of a chondroid chordoma originating from the cervical region of the spine in the dog.

Molecular mechanisms of hydroxyurea(HU)-induced apoptosis in the mouse fetal brain.

Hydroxyurea (HU), a potent mammalian teratogen, affects proliferating embryonic cells and inhibits DNA synthesis. The teratogenic potential of HU has been well known in experimental animals for several decades. In this study, we investigated molecular mechanisms of HU-induced apoptosis in the telencephalon of the fetal brain by exposing pregnant mice to HU on day 13 of gestation. The number of TUNEL-positive cells began to increase at 3 h, peaked at 12 h, and rapidly decreased at 24 h. Although changes of p53 mRNA expression were not observed by RT-PCR, a p53-positive reaction was detected immunohistochemically in the nuclei of neuroepithelial cells from 1 h to 6 h, and p53-protein expression was simultaneously identified by Western blot analysis. The expression of p53-target genes was detected at both the mRNA and protein. The mRNA levels of apotosis-related genes (fas, fasL, and bax) and cell cycle-related genes (mdm2 and p21) were significantly elevated, and the degree to and sequence in which these target genes expressed was similar to those for fas, fasL, mdm2 and p21. Flow-cytometric and Western blot analyses of cell cycle-related proteins suggested that neuroepithelial cells are arrested at the S checkpoint from 3 to 6 h and at the G2/M checkpoint at 12 h, respectively. HU-induced apoptosis is considered to be mediated by p53 in the fetal brain.

Hydroxyurea (HU)-induced apoptosis in the mouse fetal tissues.

Hydroxyurea (HU), a ribonucleotide reductase inhibitor, induces morphological anomalies in the central nervous system (CNS), craniofacial tissues and limb buds in animals, and neonatal respiratory distress in humans. In the present study, pregnant mice were treated with 400 mg/kg of HU at day 13 of gestation, and their fetuses were examined from 1 to 48 hours after treatment (HAT) to find a clue to clarify the mechanisms of HU-induced fetotoxicity and teratogenecity. At 6 and 12 HAT, a moderate to marked increase in the number of pyknotic cells was detected in the CNS and lung. A mild increase in the number of pyknotic cells was also found in the craniofacial mesenchymal tissues, limb buds and so on. These pyknotic cells had nuclei positively stained by the TUNEL method, which is widely used for the detection of apoptotic nuclei, and they also showed electron microscopic characteristics identical to those of apoptotic cells. The present results suggest that the HU-induced fetotoxicity is characterized by excess apoptotic cell death in the fetal tissues, and that such excess cell death in the fetal CNS, lung, craniofacial tissue and limb bud may have a certain relation to the later occurrence of morphological or functional anomalies reported in these tissues following HU-administration.

Comparative histopathological characteristics of highly pathogenic avian influenza (HPAI) in chickens and domestic ducks in 2008 Korea.

We compared characteristic lesions occurring in chickens and domestic ducks naturally infected with H5N1 HPAI virus in April and May 2008. Infected chickens generally exhibited pale-green, watery diarrhoea, depression, neurological signs and cyanosis of wattles and combs, and infected ducks generally exhibited neurological signs and watery diarrhoea. Gross petechial or ecchymotic haemorrhage affected the heart, proventriculus, liver, muscle, fat, and pancreas in chickens, and muscle in ducks. Necrotic foci were primarily present in the pancreas of both species and in the heart of domestic ducks. Histopathologically, chickens exhibited multifocal encephalomalacia, multifocal lymphohistiocytic myocarditis, multifocal necrotic pancreatitis and haemorrhage of several organs and tissues; ducks exhibited lymphohistiocytic meningoencephalitis with multifocal haemorrhages, multifocal necrotic pancreatitis, and severe necrotic myocarditis with mineralisation. The characteristic histopathologic findings of 2008 HPAI were multifocal encephalomalacia and necrotic pancreatitis accompanied by lymphohistiocytic myocarditis, and haemorrhage in various organs and tissues in chickens, whereas in ducks, they were severe necrotic myocarditis with mineralisation and necrotic pancreatitis, accompanied with lymphohistiocytic meningoencephalitis. The high mortality of domestic ducks may be intimately associated with heart failure resulting from increased H5N1 HPAI viral cardiotropism.

Identification and analysis of MHC class II DRB1 (Patr-DRB1) alleles in chimpanzees.

The MHC-DRB1 gene is known to display the most extensive allelic polymorphisms among MHC class II genes. We attempted the selective identification of chimpanzee (Pan troglodytes) DRB1 (Patr-DRB1) alleles using the polymerase chain reaction (PCR) technique in three steps: first, we performed Patr-DRB1*02 lineage-specific 8-kb PCR for *02 lineage detection in each chimpanzee; second, we performed 620-bp PCR for amplification of full-length exon 2; and finally, we carried out an insert check using the pattern of microsatellite repeat length variability. In the genomic DNA of 23 chimpanzees, nine Patr-DRB1 alleles containing two new alleles were detected. Our approach provides a relatively effective method of identifying Patr-DRB1 alleles in individual chimpanzees and should also contribute to our understanding of the features of MHC molecules in non-human primates.

Sequence analysis of the MHC class II DPB1 gene in chimpanzees (Pan troglodytes).

The diversity of the MHC class II region in non-human primates is a focus of biomedical research because this region plays a crucial role in the recognition of antigens in the immune system. In particular, the chimpanzee [Pan troglodytes (Patr)], which belongs to the superfamily Hominoidea, has been used as a human model for the study of diseases such as human hepatitis C virus (HCV), human hepatitis B virus (HBV) and human immunodeficiency virus (HIV) infections, to which only humans and chimpanzees are susceptible. In the present study, polymorphisms of the MHC-DPB1 gene (Patr-DPB1) in a chimpanzee colony in Japan were examined using a stepwise polymerase chain reaction (PCR) technique. In order to design a suitable primer pair which would amplify exon 2 of the Patr-DPB1 gene, a fragment of approximately 8 kb from exon 1 to exon 3 was amplified from chimpanzee genomic DNA. After designing a 500-bp primer pair at the 3' region of intron 1 and the 5' region of intron 2, analysis of DPB1 exon 2 alleles of each chimpanzee was carried out. Twenty-two chimpanzees were used in our study, and we identified seven alleles by sequence analysis on the Patr-DPB1 gene, including one new allele. The obtained nucleotide sequence patterns suggest that Patr-DPB1 alleles emerge by genetic variations such as the exchange of sequence motifs and the accumulation of point mutations.