Effects of Sr2+, BO33-, and SiO32- on Differentiation of Human Dental Pulp Stem Cells into Odontoblast-Like Cells.

This study aimed to clarify the effects of strontium (Sr2+), borate (BO33-), and silicate (SiO32-) on cell proliferative capacity, the induction of differentiation into odontoblast-like cells (OLCs), and substrate formation of human dental pulp stem cells (hDPSCs). Sr2+, BO33-, and SiO32- solutions were added to the hDPSC culture medium at three different concentrations, totaling nine experimental groups. The effects of these ions on hDPSC proliferation, calcification, and collagen formation after 14, 21, and 28 days of culture were evaluated using a cell proliferation assay, a quantitative alkaline phosphatase (ALP) activity assay, and Alizarin Red S and Sirius Red staining, respectively. Furthermore, the effects of these ions on hDPSC differentiation into OLCs were assessed via quantitative polymerase chain reaction and immunocytochemistry. Sr2+ and SiO32- increased the expression of odontoblast markers; i.e., nestin, dentin matrix protein-1, dentin sialophosphoprotein, and ALP genes, compared with the control group. BO33- increased the ALP gene expression and activity. The results of this study suggested that Sr2+, BO33-, and SiO32- may induce hDPSC differentiation into OLCs.

Ganoin and acrodin formation on scales and teeth in spotted gar: A vital role of enamelin in the unique process of enamel mineralization.

Gars and bichirs develop scales and teeth with ancient actinopterygian characteristics. Their scale surface and tooth collar are covered with enamel, also known as ganoin, whereas the tooth cap is equipped with an enamel-like tissue, acrodin. Here, we investigated the formation and mineralization of the ganoin and acrodin matrices in spotted gar, and the evolution of the scpp5, ameloblastin (ambn), and enamelin (enam) genes, which encode matrix proteins of ganoin. Results suggest that, in bichirs and gars, all these genes retain structural characteristics of their orthologs in stem actinopterygians, presumably reflecting the presence of ganoin on scales and teeth. During scale formation, Scpp5 and Enam were initially found in the incipient ganoin matrix and the underlying collagen matrix, whereas Ambn was detected mostly in a surface region of the well-developed ganoin matrix. Although collagen is the principal acrodin matrix protein, Scpp5 was detected within the matrix. Similarities in timings of mineralization and the secretion of Scpp5 suggest that acrodin evolved by the loss of the matrix secretory stage of ganoin formation: dentin formation is immediately followed by the maturation stage. The late onset of Ambn secretion during ganoin formation implies that Ambn is not essential for mineral ribbon formation, the hallmark of the enamel matrix. Furthermore, Scpp5 resembles amelogenin that is not important for the initial formation of mineral ribbons in mammals. It is thus likely that the evolution of ENAM was vital to the origin of the unique mineralization process of the enamel matrix.

Effects of nicotine and lipopolysaccharide stimulation on adhesion molecules in human gingival endothelial cells.

Smoking is a risk factor for periodontitis, and the immune response of periodontal tissues in patients with periodontitis may be strongly affected by smoking. The purpose of this study was to elucidate the bioactivity and signal transduction of human gingival endothelial cells (HGECs) due to nicotinic stimulation using a cultured medium supplemented with lipopolysaccharide (LPS) as a model of periodontitis. HGECs were cultured in medium supplemented with LPS, nicotine, nicotine + LPS, and medium supplemented without nicotine or LPS (control). Cell proliferation was assessed using Alamar blue. Cytotoxicity was assessed by lactate dehydrogenase leakage. The expression of adhesion molecule-1 (ICAM-1, VCAM-1) was assessed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay. The expression of nicotinic acetylcholine receptor (nAChR) subunits (α3, α5, α7, ß2 and ß4) was evaluated by RT-PCR. The involvement of p38 mitogen-activated protein kinase (p38MAPK) and protein kinase C (PKC) cell signaling pathways in ICAM-1 and VCAM-1 expression was investigated by RT-qPCR with specific inhibitors. HGECs stimulated with LPS, nicotine and nicotine + LPS showed inhibition of cell proliferation, increase of cell death, and increase of gene and protein expression of ICAM-1. Moreover, HGECs showed the presence of α5 and α7 nAChR subunits. The expression of ICAM-1 in HGECs stimulated with LPS, nicotine, and nicotine + LPS was significantly suppressed by p38MAPK inhibitor, but not by a PKC inhibitor. The nAChR subunits of HGECs are α5 and α7, and that HGECs stimulated with nicotine and LPS express ICAM-1 via p38MAPK pathway.

Convergent losses of SCPP genes and ganoid scales among non-teleost actinopterygians.

Various secretory calcium-binding phosphoprotein (SCPP) genes are expressed in the skin and jaw during the formation of bone, teeth, and scales in osteichthyans (bony vertebrates). Among these mineralized skeletal units is the ganoid scale, found in many fossil actinopterygians (ray-finned fish) but confirmed only in Polypteriformes (bichirs, reedfish) and Lepisosteiformes (gars) among extant clades. Here, we examined SCPP genes in the genome of seven non-teleost actinopterygian species that possess or do not possess ganoid scales. As a result, 39-43 SCPP genes were identified in Polypteriformes and Lepisosteiformes, whereas 22-24 SCPP genes were found in Acipenseriformes (sturgeons, paddlefish) and Amiiformes (bowfin). Most of these genes form two clusters in the genome of Polypteriformes, Lepisosteiformes, and Amiiformes, and these two clusters are duplicated in Acipenseriformes. Despite their distant phylogenetic relationship, Polypteriformes and Lepisosteiformes retain many orthologous SCPP genes. These results imply that common ancestors of extant actinopterygians possessed a large repertoire of SCPP genes, and that many SCPP genes were lost independently in Acipenseriformes and Amiiformes. Notably, most SCPP genes originally located in one of the two SCPP gene clusters are retained in Polypteriformes and Lepisosteiformes but were secondarily lost in Acipenseriformes and Amiiformes. In Lepisosteiformes, orthologs of these lost genes show high or detectable expression levels in the skin but not in the jaw. We thus hypothesize that many SCPP genes located in this cluster are involved in the formation of ganoid scales in Polypteriformes and Lepisosteiformes, and that their orthologs and ganoid scales were convergently lost in Acipenseriformes and Amiiformes.

Coevolution of enamel, ganoin, enameloid, and their matrix SCPP genes in osteichthyans.

We resolve debate over the evolution of vertebrate hypermineralized tissues through analyses of matrix protein-encoding secretory calcium-binding phosphoprotein (SCPP) genes and phylogenetic inference of hypermineralized tissues. Among these genes, AMBN and ENAM are found in both sarcopterygians and actinopterygians, whereas AMEL and SCPP5 are found only in sarcopterygians and actinopterygians, respectively. Actinopterygian AMBN, ENAM, and SCPP5 are expressed during the formation of hypermineralized tissues on scales and teeth: ganoin, acrodin, and collar enamel in gar, and acrodin and collar enameloid in zebrafish. Our phylogenetic analyses indicate the emergence of an ancestral enamel in stem-osteichthyans, whereas ganoin emerged in stem-actinopterygians and true enamel in stem-sarcopterygians. Thus, AMBN and ENAM originated in concert with ancestral enamel, SCPP5 evolved in association with ganoin, and AMEL evolved with true enamel. Shifts in gene expression domain and timing explain the evolution of different hypermineralized tissues. We propose that hypermineralized tissues in osteichthyans coevolved with matrix SCPP genes.

Properties of fucoidans beneficial to oral healthcare.

Fucoidans are sulfated polysaccharides that are found in marine algae and have many useful activities, including antitumor effects, promotion of apoptosis of cancer cells, and antiviral, anti-inflammatory, and antiallergic actions. In oral medicine, several case reports have shown that fucoidan-containing creams and tablets markedly improved recurrent aphthous stomatitis, symptomatic inflamed tongue, and recurrent oral herpes labialis. The aim of this study was to examine the properties of fucoidans for use in oral healthcare. The antimicrobial, anti-adhesion, endotoxin-neutralizing, and cyclooxygenase (COX)-1 and COX-2 inhibitory activities of fucoidans were examined. Four key results were obtained: fucoidans showed strong antimicrobial activity against Candida albicans, Streptococcus mutans, and Porphyromonas gingivalis; significantly inhibited the adhesion of S. mutans to bovine teeth and porcelain; were suggested to bind to and neutralize endotoxin (lipopolysaccharide) in an LAL assay; and showed COX-1 and/or COX-2 inhibitory activity. These results suggested that fucoidans may be useful in the field of oral healthcare.

The Evolution of Unusually Small Amelogenin Genes in Cetaceans; Pseudogenization, X-Y Gene Conversion, and Feeding Strategy.

Among extant cetaceans, mysticetes are filter feeders that do not possess teeth and use their baleen for feeding, while most odontocetes are considered suction feeders, which capture prey by suction without biting or chewing with teeth. In the present study, we address the functionality of amelogenin (AMEL) genes in cetaceans. AMEL encodes a protein that is specifically involved in dental enamel formation and is located on the sex chromosomes in eutherians. The X-copy AMELX is functional in enamel-bearing eutherians, whereas the Y-copy AMELY appears to have undergone decay and was completely lost in some species. Consistent with these premises, we detected various deleterious mutations and/or non-canonical splice junctions in AMELX of mysticetes and four suction feeding odontocetes, Delphinapterus leucas, Monodon monoceros, Kogia breviceps, and Physeter macrocephalus, and in AMELY of mysticetes and odontocetes. Regardless of the functionality, both AMELX and AMELY are equally and unusually small in cetaceans, and even their functional AMELX genes presumably encode a degenerate core region, which is thought to be essential for enamel matrix assembly and enamel crystal growth. Furthermore, our results suggest that the most recent common ancestors of extant cetaceans had functional AMELX and AMELY, both of which are similar to AMELX of Platanista minor. Similar small AMELX and AMELY in archaic cetaceans can be explained by gene conversion between AMELX and AMELY. We speculate that common ancestors of modern cetaceans employed a degenerate AMELX, transferred from a decaying AMELY by gene conversion, at an early stage of their transition to suction feeders.

Immunolocalization of enamel matrix protein-like proteins in the tooth enameloid of spotted gar, Lepisosteus oculatus, an actinopterygian bony fish.

Enameloid is a well-mineralized tissue covering the tooth surface in fish and it corresponds to the outer-most layer of dentin. It was reported that both dental epithelial cells and odontoblasts are involved in the formation of enameloid. Nevertheless, the localization and timing of secretion of ectodermal enamel matrix proteins in enameloid are unclear. In the present study, the enameloid matrix during the stages of enameloid formation in spotted gar, Lepisosteus oculatus, an actinopterygian, was examined mainly by transmission electron microscopy-based immunohistochemistry using an anti-mammalian amelogenin antibody and antiserum. Positive immunoreactivity with the antibody and antiserum was found in enameloid from the surface to the dentin-enameloid junction just before the formation of crystallites. This immunoreactivity disappeared rapidly before the full appearance of crystallites in the enameloid during the stage of mineralization. Immunolabelling was usually found along the collagen fibrils but was not seen on the electron-dense fibrous structures, which were probably derived from matrix vesicles in the previous stage. In inner dental epithelial cells, the granules in the distal cytoplasm often showed positive immunoreactivity, suggesting that the enamel matrix protein-like proteins originated from inner dental epithelial cells. Enamel matrix protein-like proteins in the enameloid matrix might be common to the enamel matrix protein-like proteins previously reported in the collar enamel of teeth and ganoine of ganoid scales, because they exhibited marked immunoreactivity with the same anti-mammalian amelogenin antibodies. It is likely that enamel matrix protein-like proteins are involved in the formation of crystallites along collagen fibrils in enameloid.

SCPP Genes and Their Relatives in Gar: Rapid Expansion of Mineralization Genes in Osteichthyans.

Gar is an actinopterygian that has bone, dentin, enameloid, and ganoin (enamel) in teeth and/or scales. Mineralization of these tissues involves genes encoding various secretory calcium-binding phosphoproteins (SCPPs) in osteichthyans, but no SCPP genes have been identified in chondrichthyans to date. In the gar genome, we identified 38 SCPP genes, seven of which encode "acidic-residue-rich" proteins and 31 encode "Pro/Gln (P/Q) rich" proteins. These gar SCPP genes constitute the largest known repertoire, including many newly identified P/Q-rich genes expressed in teeth and/or scales. Among gar SCPP genes, six acidic and three P/Q-rich genes were identified as orthologs of sarcopterygian genes. The sarcopterygian orthologs of most of these acidic genes are involved in bone and/or dentin formation, and sarcopterygian orthologs of all three P/Q-rich genes participate in enamel formation. The finding of these genes in gar suggests that an elaborate SCPP gene-based genetic system for tissue mineralization was already present in stem osteichthyans. While SCPP genes have been thought to originate from ancient SPARCL1, SPARCL1L1 appears to be more closely related to these genes, because it established a structure similar to acidic SCPP genes probably in stem gnathostomes, perhaps at about the same time with the origin of tissue mineralization. Assuming enamel evolved in stem osteichthyans, all P/Q-rich SCPP genes likely arose within the osteichthyan lineage. Furthermore, the absence of acidic SCPP genes in chondrichthyans might be explained by the secondary loss of earliest acidic genes. It appears that many SCPP genes expanded rapidly in stem osteichthyans and in basal actinopterygians.

Immunohistochemical and Western Blotting Analyses of Ganoine in the Ganoid Scales of Lepisosteus oculatus: an Actinopterygian Fish.

In order to compare its characteristics with those of jaw tooth collar enamel, normally developing and experimentally regenerating ganoine from ganoid scales of Lepisosteus oculatus (spotted gar), an actinopterygian fish species, was examined by Western blotting and immunohistochemistry. Amelogenin, a major enamel matrix protein (EMP), is widely found from sarcopterygian fish to mammals. Therefore, we used antimammalian amelogenin antibodies and antisera: an antibody against bovine amelogenin; antiserum against porcine amelogenin; and region-specific antibodies or antiserum against the C-terminus, middle region, or N-terminus of porcine amelogenin in this study. Positive immunoreactivity with the antibody against bovine amelogenin, antiserum against porcine amelogenin, and the middle and C-terminal region-specific antibodies was detected in both normally developing and regenerating ganoine matrix, as well as in granules found within inner ganoine epithelial cells. These immunohistochemical analyses indicated that the Lepisosteus ganoine matrix contains EMP-like proteins with epitopes similar to mammalian amelogenins. In Western blotting analyses of regenerating ganoid scales with the antibovine amelogenin antibody, two protein bands with molecular weights of approximately 78 and 65 kDa were detected, which were similar to those found in Lepisosteus tooth enamel. Our study suggests that in Lepisosteus, EMP-like proteins in the ganoine matrix corresponded to those in tooth enamel. However, it was revealed that the 78 and 65 kDa EMP-like proteins were different from 27 kDa bovine amelogenin.

The spotted gar genome illuminates vertebrate evolution and facilitates human-teleost comparisons.

To connect human biology to fish biomedical models, we sequenced the genome of spotted gar (Lepisosteus oculatus), whose lineage diverged from teleosts before teleost genome duplication (TGD). The slowly evolving gar genome has conserved in content and size many entire chromosomes from bony vertebrate ancestors. Gar bridges teleosts to tetrapods by illuminating the evolution of immunity, mineralization and development (mediated, for example, by Hox, ParaHox and microRNA genes). Numerous conserved noncoding elements (CNEs; often cis regulatory) undetectable in direct human-teleost comparisons become apparent using gar: functional studies uncovered conserved roles for such cryptic CNEs, facilitating annotation of sequences identified in human genome-wide association studies. Transcriptomic analyses showed that the sums of expression domains and expression levels for duplicated teleost genes often approximate the patterns and levels of expression for gar genes, consistent with subfunctionalization. The gar genome provides a resource for understanding evolution after genome duplication, the origin of vertebrate genomes and the function of human regulatory sequences.

Immunohistochemical and Western blot analyses of collar enamel in the jaw teeth of gars, Lepisosteus oculatus, an actinopterygian fish.

Although most fish have no enamel layer in their teeth, those belonging to Lepisosteus (gars), an extant actinopterygian fish genus, do and so can be used to study amelogenesis. In order to examine the collar enamel matrix in gar teeth, we subjected gar teeth to light and electron microscopic immunohistochemical examinations using an antibody against bovine amelogenin (27 kDa) and antiserum against porcine amelogenin (25 kDa), as well as region-specific antibodies and antiserum against the C-terminus and middle region, and N-terminus of porcine amelogenin, respectively. The enamel matrix exhibited intense immunoreactivity to the anti-bovine amelogenin antibody and the anti-porcine amelogenin antiserum in addition to the C-terminal and middle region-specific antibodies, but not to the N-terminal-specific antiserum. These results suggest that the collar enamel matrix of gar teeth contains amelogenin-like proteins and that these proteins possess domains that closely resemble the C-terminal and middle regions of porcine amelogenin. Western blot analyses of the tooth germs of Lepisosteus were also performed. As a result, protein bands with molecular weights of 78 kDa and 65 kDa were clearly stained by the anti-bovine amelogenin antibody as well as the antiserum against porcine amelogenin and the middle-region-specific antibody. It is likely that the amelogenin-like proteins present in Lepisosteus do not correspond to the amelogenins found in mammals, although they do possess domains that are shared with mammalian amelogenins.

Comparative studies on microvascular endothelial cells isolated from periodontal tissue.

BACKGROUND: Most available periodontal studies regarding the endothelial cell (EC) were investigated by using human umbilical vein endothelial cells (HUVECs); however, ECs can display remarkable heterogeneity in vascular beds of different origins. The aim of the present study, therefore, is to characterize microvascular ECs isolated from periodontal tissue and investigate their growth and gene expression compared to HUVECs (macrovascular). METHODS: Periodontal ligament ECs (PDL-ECs) and gingiva ECs (G-ECs) were isolated by coupling to monoclonal anti-CD31 antibody magnetic beads. Both PDL-ECs and G-ECs were characterized to definitively demonstrate that the culture represented pure ECs. Their growth was determined by resazurin reduction assay. Interleukin (IL)-8, intercellular adhesion molecule 1 (ICAM-1), and E-selectin gene expression were determined by real-time quantitative reverse-transcription polymerase chain reaction after treatment with Escherichia coli lipopolysaccharide (LPS). RESULTS: PDL-ECs and G-ECs revealed specific EC characteristics but formed tube-like structures and had slower growth rates than HUVECs. After E. coli LPS treatment, PDL-ECs and G-ECs showed similar dose-dependently increased levels of IL-8, ICAM-1, and E-selectin mRNA expression; however, their expressions were in contrast to HUVECs. PDL-ECs and G-ECs showed obviously increased ICAM-1 mRNA expression, whereas HUVECs showed markedly increased E-selectin mRNA expression after treatment with 0.1 µg/mL E. coli LPS. CONCLUSIONS: ECs isolated from periodontal tissue show different growth and gene expression from those of HUVECs. Thus, these microvascular ECs appear to be a more valuable in vitro model system than HUVECs (macrovascular) to further study pathogenesis and angiogenesis of periodontal disease.

Specificity of antimicrobial peptide LL-37 to neutralize periodontopathogenic lipopolysaccharide activity in human oral fibroblasts.

BACKGROUND: The antimicrobial peptide LL-37 is known to have a potent lipopolysaccharide (LPS)-neutralizing activity in various cell types. Because of observed heterogeneity within periodontopathogenic LPS, the authors hypothesized that LL-37 had specificity to neutralize such LPS activity. The present study, therefore, aims to investigate the LPS-neutralizing activity of LL-37 to various periodontopathogenic LPS in interleukin-8 (IL-8) production after challenging them in human oral fibroblasts. METHODS: Human periodontal ligament fibroblasts (PDLF) and gingival fibroblasts (GF) were cultured from biopsies of periodontal ligament and gingival tissues. After cell confluence in 24-well plates, LPS (10 µg/mL) from Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans were added with or without LL-37 (10 µg/mL). After 18 hours, the supernatant was collected and analyzed in IL-8 production by enzyme-linked immunosorbent assay. RESULTS: All periodontopathogenic LPS statistically significantly induced IL-8 production in both PDLF and GF (P <0.01). After neutralization with LL-37, both PDLF and GF showed a statistically significant reduction in IL-8 production compared with LPS-treated groups without LL-37 (P <0.01), and the percentage of reduction in IL-8 production in PDLF appeared to be higher than in GF. In addition, the percentage of reduction in IL-8 production varied considerably according to each periodontopathogenic LPS. CONCLUSIONS: The antimicrobial peptide LL-37 had an ability to suppress periodontopathogenic LPS-induced IL-8 production in both PDLF and GF. Its LPS-neutralizing activity revealed specificity to periodontopathogenic LPS and seemed to be dependent on the heterogeneity within LPS between different genera.

Effect of aging on BCG immunostimulation of Porphyromonas gingivalis infection in mice.

The need for population care increases with the age of the population. Pneumonia is the fourth leading cause of death in Japan, yet the risk of pneumonia could be reduced by eliminating opportunistic infection sources such as oral bacteria (e.g. Porphyromonas gingivalis). Previously, we reported removal of P. gingivalis by macrophages during the early stages of cellular immunity, although neither neutrophils nor antibodies participated in the antimicrobial activity. BCG is a live vaccine against tuberculosis, and is thought to maintain cellular immunity as the antigen remains in vivo for longer periods. In this experiment, we attempted to clarify the relationship between aging and the elimination of P. gingivalis by examining the protective capacity of BCG against P. gingivalis infection in mice of various ages. In young mice, the reduction in numbers of P. gingivalis was accompanied by increased IFN-γ and IL-12 levels, and nitric oxide was continuously produced. The augmentation of bactericidal activity, namely the effects of the vaccine, was clear in young mice, but weaker in older mice. Activation of cellular immunity was not observed in older mice, even when boosters were administered.

Chick tooth induction revisited.

Teeth have been missing from Aves for almost 100 million years. However, it is believed that the avian oral epithelium retains the molecular signaling required to induce odontogenesis, and this has been widely examined using heterospecific recombinations with mouse dental mesenchyme. It has also been argued that teeth can form from the avian oral epithelium owing to contamination of the mouse mesenchyme with mouse dental epithelial cells. To investigate the possibility of tooth formation from chick oral epithelium and the characteristics of possible chick enamel, we applied LacZ transgenic mice during heterospecific recombination and examined the further tooth formation. Transmission electron microscopy was used to identify the two tissues during development after heterospecific recombination. No mixing was detected between chick oral epithelium and mouse dental mesenchyme after 2 days, and secretory ameloblasts with Tomes' processes were observed after 1 week. Teeth were formed after 3 weeks with a single cusp pattern, possibly determined by epithelial factors, which is similar to that of the avian tooth in the late Jurassic period. These recombinant teeth were smaller than mouse molars, whereas perfect structures of both ameloblasts and enamel showed histological characteristics similar to those of mice. Together these observations consistent with previous report that odontogenesis is initially directed by species-specific mesenchymal signals interplaying with common epithelial signals.

The participation of nitric oxide in peritoneal exudate cell cytotoxicity of mice by Fusobacterium nucleatum.

Previously we reported that mice infected recurrently with live Fusobacterium nucleatum(Fn) synthesize a significant amount of NO between 12 hr and 24 hr after Fn injection. Fn is a gram-negative rod periodontal pathogen. NO could not be induced by heat-killed Fn or in untreated mice. This NO, derived from the iNOS after infection of live Fn, was not involved in the Fn reduction because Fn clearance occurs within 6 hr. We investigated in this study whether this NO was involved in cytotoxicity in peritoneal exudate cells (PEC) in vivo. The mice were divided into two groups: those treated with live Fn (immune) and those left untreated (normal). PEC number, NO production, detection of apoptosis or death cells, and lactate dehydrogenase (LDH) release activity after injection of live Fn were compared in these groups. In the immune group, the increase of the total cell numbers caused by an increase in neutrophils, a significant NO production only after injection of live Fn at 24 hr and identification of iNOS positive macrophages were confirmed. The apoptotic rate was very low and did not increase at 24 hr in vivo. Therefore, apoptosis was seldom relevant to the NO. In the immune group, LDH activity was remarkable high at 24 hr, and dead cells and macrophages phagocytizing cell fragments increased at the same time. Pretreatment of L NMMA, an inhibitor of iNOS, suppressed LDH activity and cell death. Therefore, the NO derived from the iNOS is involved in the cytotoxicity. These results suggest that NO may contribute to the inflammatory response during Fn infection in periodontitis.

Usefulness of measuring plasma cytokines in oral and maxillofacial surgery.

We determined the intra- and postoperative plasma concentrations of cytokines (tumor necrosis factor [TNF]-Alpha, interleukin [IL]-1Beta, IL-6, and IL-8) in oral surgery patients with procedures ranging in duration from 20 to 375 min and investigated their relationship to the intensity of the surgical stress over time. No significant differences from baseline levels were observed in the levels of TNF-Alpha, IL-1Beta, and IL-8. By contrast, increased IL-6 levels were noted only on the first postoperative day, when they reached 1500% of the baseline level, after which they decreased to preoperative levels by the third postoperative day and tended to reflect the intensity of surgical stress.

The reduction of Fusobacterium nucleatum in mice is irrelevant to the nitric oxide induced by iNOS.

Previously we reported that mice infected recurrently with live Fusobacterim nucleatum (Fn) synthesize a significant amount of NO between 12 hr and 24 hr after the Fn injection. We now investigated whether the NO has the capability of killing Fn, a gram-negative rod periodontal pathogen. The mice were divided into three groups: treated with live bacteria (LB), treated with heat-killed bacteria (HKB) and untreated: normal (N). The Fn reduction, NO production and cell number after Fn injection were then compared in these mice. In the LB group, no Fn was detected at 6 hr, whereas it was still detected in the HKB and N groups at 24 hr as assessed by both colony counts and PCR assays. A significant amount of NO was synthesized in the LB group at 24 hr after the Fn injection. Fn is not killed by SNAP-generated NO in vitro. An increase in the total cell number was accompanied by an increase of the neutrophil numbers in the LB group. Intracellular O2(-) generation (including ONOO(-)) was visualized using dihydrorhodamine (DHR)-123. The peak of O2(-) generation by PEC was shown to be at 3 hr in all 3 groups. The number of O2(-) positive cells in the LB group at 3 hr was remarkably high, and most of them were likely to be neutrophils. The Fn reduction would be performed cooperatively via oxygen dependent and oxygen independent mechanisms. Thus reactive oxygen species (ROS) included in the oxygen dependent mechanism appear to be important for Fn reduction. However the significant amounts of NO derived from the iNOS synthesized in the LB group between 12 hr and 24 hr after injection of LFn were not involved in the Fn reduction.