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Oral cancer and smoking are closely related, because the oral cavity, which is the route of ingestion of tobacco smoke, is in direct contact with the oral mucosa. Nicotine, one of the components of tobacco, can diffuse rapidly to the central nervous system and is responsible for tobacco addiction. Nicotine is present in high concentrations in the bloodstream of smokers; while the addictive effects of this alkaloid have extensively been studied, its effect on tumorigenesis is not clear yet. Therefore, in this study, we examined the effect of nicotine on cell proliferation and the signaling pathways it activates. The human oral squamous cell carcinoma cell line HSC-2 was used as a model system. We demonstrated the correlation between nicotine and epidermal growth factor receptor (EGFR) signaling. Nicotine treatment induced HSC-2 cell proliferation and migration and the phosphorylation of EGFR. Furthermore, nicotine treatment activated the EGFR downstream effectors phosphatidylinositol-3 kinase/AKT and p44/42 mitogen-activated protein kinases (ERK), which, in turn, promoted cell proliferation. Overall, our study suggests that nicotine promotes cell growth and migration through epidermal growth factor (EGF) signaling and plays an important role in oral cancer progression.

Loss of umami taste sensation affects quality of life and causes weight loss and health problems, particularly in the elderly. We recently expanded the use of the filter paper disc method to include assessment of umami taste sensitivity, using monosodium glutamate as the test solution. This test showed high diagnostic performance for discriminating between normal taste function and disorders in sensation of the umami taste, according to established cut-off values. The test also revealed: (1) some elderly patients suffered from specific loss of umami taste sensation with preservation of the other four taste sensations (sweet, salty, sour, and bitter); (2) umami taste disorder caused a loss of appetite and decline in weight, resulting in poor health; (3) appetite, weight and overall health improved after appropriate treatment for umami taste disorder. Because of the subjective nature of the test, however, it may not be useful for patients who cannot express which taste sensation is induced by a tastant, such as those with dementia. Most recently, using tissue samples collected from the tongue by scraping the foliate papillae, we showed that evaluation of umami taste receptor gene expression may be clinically useful for the objective genetic diagnosis of umami taste disorders.

The effects and mechanisms of tastes on labial minor salivary gland (LMSG) secretion were investigated in 59 healthy individuals. Stimulation with each of the five basic tastes (i.e., sweet, salty, sour, bitter, and umami) onto the tongue induced LMSG secretion in a dose-dependent manner. Umami and sour tastes evoked greater secretion than did the other tastes. A synergistic effect of umami on LMSG secretion was recognized: a much greater increase in secretion was observed by a mixed solution of monosodium glutamate and inosine 5′-monophosphate than by each separate stimulation. Blood flow (BF) in the nearby labial mucosa also increased following stimulation by each taste except bitter. The BF change and LMSG secretion in each participant showed a significant positive correlation with all tastes, including bitter. Administration of cevimeline hydrochloride hydrate to the labial mucosa evoked a significant increase in both LMSG secretion and BF, while adrenaline, atropine, and pirenzepine decreased LMSG secretion and BF. The change in LMSG secretion and BF induced by each autonomic agent was significantly correlated in each participant. These results indicate that basic tastes can induce the gustatory-salivary reflex in human LMSGs and that parasympathetic regulation is involved in this mechanism.

Immunohistochemistry for several neurochemical substances, the transient receptor potential cation channel subfamily V member 1 (TRPV1) and 2 (TRPV2), P2X3 receptor, and parvalbumin (PV), was performed on the nodose ganglion, pharynx, and epiglottis in human cadavers. The nodose ganglion was situated beneath the jugular foramen, and had a spindle shape with the long rostrocaudal axis. The pharyngeal branch (PB) issued from a rostral quarter of the nodose ganglion, whereas the superior laryngeal nerve (SLN) usually originated from a caudal half of the ganglion. In the nodose ganglion, sensory neurons were mostly immunoreactive for TRPV1 (89 %) or P2X3 (93.9 %). About 30 % of nodose neurons contained TRPV2 (35.7 %)-or PV (29.9 %)-immunoreactivity (-IR). These neurons mainly had small to medium-sized cell bodies, and were distributed throughout the ganglion. Neurodegenerative profiles such as shrinkage or pyknosis could not be detected in the examined ganglion. Occasionally, TRPV2-IR nerve fibers surrounded blood vessels in the epiglottis as well as in the nasal and oral parts of the pharynx. Isolated TRPV2-IR nerve fibers were also located beneath the epithelium. TRPV1-, P2X3-, or PV-IR nerve endings could not be detected in the pharynx or epiglottis. In the PB and SLN, however, numerous nerve fibers contained TRPV1-, TRPV2-, P2X3-, and PV-IR. The present study suggests that TRPV1-, TRPV2-, P2X3-, and PV-IR neurons in the human nodose ganglion innervate the pharynx and epiglottis through the PB and SLN. These neurons may respond to chemical, thermal, and mechanical stimuli during respiration and swallowing.

The beta-2 subunit of the mammalian brain voltage-gated sodium channel (SCN2B) was examined in the rat trigeminal ganglion (TG) and trigeminal sensory nuclei. In the TG, 42.6 % of sensory neurons were immunoreactive (IR) for SCN2B. These neurons had various cell body sizes. In facial skins and oral mucosae, corpuscular nerve endings contained SCN2B-immunoreactivity. SCN2B-IR nerve fibers formed nerve plexuses beneath taste buds in the tongue and incisive papilla. However, SCN2B-IR free nerve endings were rare in cutaneous and mucosal epithelia. Tooth pulps, muscle spindles and major salivary glands were also innervated by SCN2B-IR nerve fibers. A double immunofluorescence method revealed that about 40 % of SCN2B-IR neurons exhibited calcitonin gene-related peptide (CGRP)-immunoreactivity. However, distributions of SCN2B- and CGRP-IR nerve fibers were mostly different in facial, oral and cranial structures. By retrograde tracing method, 60.4 and 85.3 % of TG neurons innervating the facial skin and tooth pulp, respectively, showed SCN2B-immunoreactivity. CGRP-immunoreactivity was co-localized by about 40 % of SCN2B-IR cutaneous and tooth pulp TG neurons. In trigeminal sensory nuclei of the brainstem, SCN2B-IR neuronal cell bodies were common in deep laminae of the subnucleus caudalis, and the subnuclei interpolaris and oralis. In the mesencephalic trigeminal tract nucleus, primary sensory neurons also exhibited SCN2B-immunoreactivity. In other regions of trigeminal sensory nuclei, SCN2B-IR cells were very infrequent. SCN2B-IR neuropil was detected in deep laminae of the subnucleus caudalis as well as in the subnuclei interpolaris, oralis and principalis. These findings suggest that SCN2B is expressed by various types of sensory neurons in the TG. There appears to be SCN2B-containing pathway in the TG and trigeminal sensory nuclei.

Platelets are reportedly causal in hepatitis. We previously showed that in mice, lipopolysaccharide (LPS) induces a reversible and macrophage-dependent hepatic platelet accumulation (HPA), including translocation of platelets into Disse spaces and their entry into hepatocytes. Concanavalin A (ConA), which induces hepatitis in mice via both T cells and macrophages, also induces HPA. Here, we examined the relationship between HPA and ConA-hepatitis. ConA-hepatitis and HPA were evaluated by serum transaminases, hepatic 5-hydroxytryptamine, and/or electron microscopy. Unlike LPS-induced HPA, ConA-induced HPA was only moderately dependent on phagocytic macrophages. Against expectations, platelet-depletion significantly exacerbated ConA-hepatitis, and anti-P-selectin antibody and P-selectin receptor blockade reduced both ConA-induced HPA and hepatitis. Prior induction of HPA by pretreatment with low-dose LPS powerfully reduced ConA-hepatitis. Such protection by LPS-pretreatment was not effective in mice depleted of phagocytic macrophages. In platelet-depleted mice, LPS-pretreatment severely exacerbated ConA-hepatitis. In mice depleted of both macrophages and platelets, neither ConA nor LPS-pretreatment + ConA induced hepatitis. In mice deficient in IL-1α and IL-1β (but not in TNFα), ConA-induced hepatitis was mild, and a protective effect of LPS was not detected. These results suggest that (i) there are causal and protective types of HPA, (ii) the causal type involves hepatic aggregation of platelets, which may be induced by platelet stimulants leaked from injured hepatocytes, (iii) the protective type is inducible by administration of prior low-dose LPS in a manner dependent on phagocytic (or F4/80-positive) macrophages, and (iv) IL-1 is involved in both the causal and protective types.

Microbial infection is thought to modulate allergic disorders, and we previously demonstrated that not only mast cells (which release histamine), but also platelets are involved in the anaphylaxis induced in mice sensitised to ovalbumin (OVA). Here, we examined the effects of a lipopolysaccharide (LPS) from the oral bacterium Prevotella intermedia (Pi) on OVA-induced anaphylaxis. Upon intraperitoneal co-injection of Pi-LPS plus OVA into BALB/c mice, the Pi-LPS displayed a potent adjuvant effect comparable to that of alum (a standard adjuvant) in terms of its abilities to induce both anaphylactic shock and histamine-release following an antigen (OVA)-challenge. Moreover, an injection of Pi-LPS given to OVA+alum-sensitised mice shortly before an OVA-challenge augmented the shock-response. This LPS-pretreatment did not affect histamine-release, but did augment pulmonary platelet accumulation. Histamine was not by itself causal for shock-induction in sensitised mice. These results suggest that oral bacteria and/or their constituents (such as LPS) may help to sensitise the host to an antigen or exacerbate the host's allergic reactions ("aggravation effect"), probably by enhancing the platelet response to the antigen OVA.

IL-18, an important regulator of immune responses, is expressed in activated macrophages and also in nonimmune cells, such as keratinocytes and epithelial cells. Increased levels of serum IL-18 are reported in patients with a wide variety of diseases, but it is unclear which type of cell is the major source of serum IL-18. Here, we showed that the administration of liposomes encapsulating clodronate (Clo-lip) in mice selectively depleted F4/80+ phagocytic macrophages in the liver and spleen. Serum levels of mature IL-18 with 18 kDa were increased markedly in mice treated with Propionibacterium acnes and LPS, whereas administration of Clo-lip and gadolinium chloride, another widely used macrophage inactivator, showed no obvious effect on serum IL-18 levels, which were marginal in the liver, lung, and spleen and more pronounced in the intestines, especially in the duodenum. Treatment with P. acnes alone induced IL-18 more than twofold in each organ, and P. acnes and LPS induced a marked increase in IL-18 levels in the liver and spleen but decreased in the intestines. The administration of Clo-lip showed only a marginal effect on the IL-18 levels in these organs. Furthermore, serum levels of liver enzymes and TNF-α and liver injury (necrotic change and granuloma formation) induced by P. acnes and LPS were reduced moderately by Clo-lip. These results suggest that phagocytic macrophages do not actively contribute to the induction of serum IL-18 and liver injury in mice treated with P. acnes and LPS.

To examine the potential role of the histamine-forming enzyme, histidine decarboxylase (HDC), in oral inflammation and disease, we studied HDC activity in oral tissue after induction by bacterial agents. Following injection of E. coli-derived lipopolysaccharide (LPS) into mice, we measured the quantitative changes in HDC activity over time in dental pulp and gingiva. Oral tissue taken from individual mice was insufficient for detecting precise HDC activity, thus, we combined dental pulp or gingival tissues from four mice and assayed them over the course of 24 h. Our results indicate that LPS stimulated marked elevations of HDC activity in dental pulp and gingiva. This increase reached a maximum at 6 h after LPS injection and remained detectable at for least 24 h. Since mast cells are known to produce histamine through a difference mechanism than HDC induction, we compared LPS-induced HDC activity in dental pulp and gingiva to that in ear skin (a tissue rich in mast cells) and liver (a tissues lacking in mast cells). LPS also induced a marked increase in the HDC activity in liver and ear skin at 6 h after LPS injection. By contrast, saline injection had no effect on the HDC activity in any of the four tissues, although basal levels of HDC activity in ear skin was markedly higher than basal HDC activity in the other three kinds of tissues. Still, the relative increase in LPS-induced HDC activity in dental pulp and gingiva were much greater than that in ear skin. Since liver are devoid of mast cells and ear skin is considered the tissue richest in mast cells, the differences in HDC activity between tissues indicates that histamine induced by LPS may be produced by cells other than mast cells through another mechanism of action. These results also suggest that histamine produced in oral tissues in response to bacterial agents such as LPS could be involved in development of pulpitis or gingivitis (periodontitis), the most common diseases in the dental clinic, and that efforts to inhibit HDC activity, which elevates histamine levels in oral tissues, might offer the basis for novel treatment strategies.

Peptidoglycan recognition proteins (PGRPs), a novel family of pattern recognition molecules involved in innate immunity conserved from insects to mammals, recognize bacterial cell wall peptidoglycan (PGN) and have been suggested to act as anti-bacterial factors. In humans, four kinds of PGRPs (PGRP-L, -Iα, -Iβ and -S) have been cloned and all four of them bind PGN. Chemically synthesized pathogen-associated molecular patterns (PAMPs) in bacterial cell surface components (synthetic lipopeptide [TLR2 agonist], synthetic lipid A [TLR4 agonist], synthetic iE-DAP [NOD1 agonist], and muramyldipeptide [MDP, NOD2 agonist]) markedly upregulated the mRNA expressions of the four PGRPs and cell surface expressions of PGRP-Iα and -Iβ, but did not induce either mRNA expression or the secretion of inflammatory cytokines in oral epithelial cells. Suppression of the expressions of TLR2, TLR4, NOD1 and NOD2 by RNA interference specifically inhibited the upregulation of PGRP mRNA expression induced by lipopeptide, lipid A, iE-DAP and MDP, respectively. In addition, these PAMPs definitely activated nuclear factor (NF)-κB in the epithelial cells, and suppression of NF-κB activation clearly prevented the induction of PGRP mRNA expression induced by these PAMPs in the cells. These findings suggested that bacterial PAMPs induced the expression of PGRPs, but not proinflammatory cytokines. Although the function of human PGRPs has not been clarified so far, in contrast to insect PGRPs, human PGRPs might be also involved in host defence against bacterial invasion without accompanying inflammatory responses. Further studies are required to elucidate the function of human PGRPs.

To investigate the role of human gingival fibroblasts (HGF) in periodontitis, we examined the expression of interleukin-2 receptor (IL-2R) and IL-15R subunits in HGF from normal and inflamed regions and the role of endogenous IL-15 in IL-2-mediated signaling. Normal HGF expressed IL-2Rβ and common γ-chain (γc) but not IL-2Rα or IL-15Rα, whereas inflamed HGF expressed IL-2Rα, IL-15Rα, IL-2Rβ and γc. Exogenous IL-2 and IL-15 induced production of monocyte chemoattractant protein-1 (MCP-1) but not IL-8 in normal HGF, and induced the production of both chemokines in inflamed HGF. Both HGF constitutively transcribed 48aa-IL-15 isoform, and the isoform existed as a membrane-bound form. Pretreatment with anti-IL-15 neutralizing mAb completely inhibited the production of MCP-1 induced by IL-2 and IL-15 and IL-2-induced phosphorylation of Janus tyrosine kinase 1 and 3 in HGF. The pretreatment and RNA interference targeted to IL-15 mRNA resulted in total inhibition of the IL-2Rβ and γc expression at mRNA and protein levels. Furthermore, inhibition of nuclear factor (NF)-κB abrogated the expression of IL-2Rβ and γc, and IL-2-induced-nuclear translocation of NF-κB was completely inhibited by the RNA interference in HGF. These results suggest that endogenous membrane-bound IL-15 sustains recruitment of IL-2Rβ and γc through activation of NF-κB in HGF.

Peptidoglycan recognition proteins (PGRPs), a novel family of pattern recognition molecules (PRMs) in innate immunity conserved from insects to mammals, recognize bacterial cell wall peptidoglycan (PGN) and are suggested to act as anti-bacterial factors. In humans, four kinds of PGRPs (PGRP-L, -Ialpha, -Ibeta and -S) have been cloned and all four human PGRPs bind PGN. In this study, we examined the possible regulation of the expression of PGRPs in oral epithelial cells upon stimulation with chemically synthesized pathogen-associated molecular patterns (PAMPs) in bacterial cell surface components: Escherichia coli-type tryacyl lipopeptide (Pam3CSSNA), E. coli-type lipid A (LA-15-PP), diaminopimelic acid containing desmuramyl peptide (gamma-D-glutamyl-meso-DAP; iE-DAP), and muramyldipeptide (MDP). These synthetic PAMPs markedly upregulated the mRNA expression of the four PGRPs and cell surface expression of PGRP-Ialpha and -Ibeta, but did not induce either mRNA expression or secretion of inflammatory cytokines, in oral epithelial cells. Suppression of the expression of Toll-like receptor (TLR)2, TLR4, nucleotide-binding oligomerization domain (NOD)1 and NOD2 by RNA interference specifically inhibited the upregulation of PGRP mRNA expression induced by Pam3CSSNA, LA-15-PP, iE-DAP and MDP respectively. These PAMPs definitely activated nuclear factor (NF)-kappaB in the epithelial cells, and suppression of NF-kappaB activation clearly prevented the induction of PGRP mRNA expression induced by these PAMPs in the cells. These findings suggested that bacterial PAMPs induced the expression of PGRPs, but not proinflammatory cytokines, in oral epithelial cells, and the PGRPs might be involved in host defence against bacterial invasion without accompanying inflammatory responses.

Within a few minutes of an intravenous injection of a lipopolysaccharide (LPS) into mice, platelets accumulate, largely in the lung. At higher doses, LPS induces rapid shock (within 10 min), leading to death within 1 h. This type of shock differs from so-called endotoxin shock, in which shock signs and death occur several hours or more later. Here, we found that platelet depletion (by a monoclonal anti-platelet antibody) prevented LPS-induced rapid shock, but increased delayed lethality. In Japan, glycyrrhizin (GL), a compound isolated from licorice, is daily and slowly infused intravenously into chronic hepatitis C patients. A single bolus intravenous injection into mice of GL (200 mg/kg or less) shortly before (or simultaneously with) LPS injection reduced the pulmonary platelet accumulation and the severity of the rapid shock, and prevented death in both the early and later periods. GL itself, at 400 mg/kg, produced no detectable abnormalities in the appearance or activity of mice. Intraperitoneal injection of aspirin or dexamethasone had only marginal effects on LPS-induced platelet responses and lethality. These results suggest that platelets play important roles in the development of both the rapid and delayed types of shock induced by LPS. Although the mechanism by which GL suppresses platelet responses and delayed lethality remains to be clarified, GL might provide a strategy for alleviating the acute respiratory distress syndrome seen in sepsis. Our results may also support the proposal by Cinatl et al. [Cinatl J, Morgenstern B, Bauer G, Chandra P, Ravenau H, Doerr HW. Glycyrrhizin, an active component of liquorice roots, and replication of SARS-associated coronavirus. Lancet 2003; 361: 2045-6.] that GL may be an effective drug against severe acute respiratory syndrome.

Histamine modulates immune responses. There are at least two ways histamine might be supplied: one is its release from cells that pool pre-formed histamine and the other is its de novo formation via induction of histidine decarboxylase (HDC). Lipopolysaccharide (LPS) and the proinflammatory cytokine interleukin (IL)-1 induce a marked elevation of HDC activity in various tissues or organs. To examine the contribution of mast cells to HDC induction in mice given LPS or IL-1, we examined the effects of LPS and IL-1 on HDC activity and/or histamine content in various organs (liver, lung, spleen or bone marrow) in mast cell-deficient mice (W/Wv), their normal littermates (+/+) and BALB/c mice deficient in IL-1alpha, IL-1beta and tumor necrosis factor (TNF)-alpha (IL-1alpha beta/TNFalphaKO mice). In non-stimulated mice, the histamine in the lung and spleen was contained largely within mast cells. The LPS-stimulated increase in HDC activity in a given organ was similar between +/+ and W/W(v) mice, and between IL-1alpha beta/TNFalphaKO BALB/c and control BALB/c mice, and led to increases in histamine. In W/Wv and +/+ mice, IL-1alpha also elevated HDC activity. These results suggest that (i) in liver, lung and spleen, either the major cells supplying histamine via HDC induction in response to LPS and IL-1 are not mast cells, or mast cells are not a prerequisite for the induction of HDC; (ii) the cells in which HDC is induced by LPS and IL-1 are similar or identical in a given organ; and (iii) neither IL-1 nor TNF-alpha is a prerequisite for the induction of HDC by LPS.

The lack of published information about the minor salivary glands is due in part to the difficulties experienced in collecting and quantifying their secretions. In fact, no method exists for measuring their secretions that is both simple and accurate. This investigation examined the accuracy of our newly developed method (which simply employs the iodine-starch reaction) in 10 healthy non-medicated adults. A strip painted with a solution of iodine in absolute alcohol then with a fine starch powder mixed with castor oil was placed at a designated location on the lower-lip mucosa for 2 min to collect saliva. Black-stained spots of various sizes corresponding to the individual glands could be accurately visualized. After removal of the strip, the total stained area (mm2) was calculated by digitizing the spot areas using a computer system. The correlation coefficient (r) between known volumes of saliva and stain size was 0.995, indicating a close correlation. The correlation coefficient (r) between area values obtained in the first trial in each subject (Y) and the second (X; 10 min later) was 0.963, and the simple regression equation was close to Y=X, indicating good reproducibility. The mean flow rate microl/cm2 per min) obtained by converting mean total area to volume and thence to flow rate was 0.49+/-0.26, in good agreement with published values obtained by others. These results suggest that our newly developed method allows both the distribution and secretion rate of the minor salivary glands to be observed, and that it should be of practical value due to its simplicity, accuracy, and reproducibility.

Mast cells and basophils are believed to trigger allergic reactions and anaphylaxis. They rapidly release histamine (H), a typical mediator of inflammation, in response to antigens. In the mouse, platelets contain much 5-hydroxytryptamine (5HT), an additional inflammatory mediator, while human platelets contain both H and 5HT. Here, we examined the response of platelets in sensitized mice to antigen challenge. Platelets accumulated in the lung and liver almost immediately after intravenous injection of ovalbumin (OVA), in mice sensitized to it, and platelet degranulation occurred during these reactions. These responses of platelets preceded H release from mast cells and/or basophils, occurred at doses of OVA lower than those inducing H release, and contributed to the signs of shock. We reported previously that intravenous injection into mice of LPS (a membrane constituent of gram-negative bacteria) induces a similar platelet response (accumulation of platelets in the lung and liver) and shock. Blood that has passed through the body (other than the digestive tract) passes first to the lungs before being recirculated by the heart, and blood that has passed through the digestive tract passes next to the liver. Thus, our findings suggest that in addition to their role in haemostasis, platelets, tiny anuclear cytoplasts, may be important in both innate and acquired immunity, and that the lung and liver may be the fronts at which platelets wage war on pathogens. British Journal of Pharmacology (2002) 137, 146–152. doi:10.1038/sj.bjp.0704852

Thymine DNA glycosylase (TDG) is a base excision repair enzyme that interacts with the small ubiquitin-related modifier (SUMO)-targeted ubiquitin E3 ligase RNF4 and functions in the active DNA demethylation pathway. Here we showed that both SUMOylated and non-modified forms of endogenous TDG fluctuated during the cell cycle and in response to drugs that perturbed cell cycle progression, including hydroxyurea and nocodazole. Additionally, we detected a SUMOylation-independent association between TDG and RNF4 in vitro as well as in vivo , and observed that both forms of TDG were efficiently degraded in RNF4-depleted cells when arrested at S phase. Our findings provide insights into the in vivo dynamics of TDG SUMOylation and further clarify the TDG–RNF4 interaction.

There is a close relationship between perception of umami, which has become recognized as the fifth taste, and the human physical condition. We have developed a clinical test for umami taste sensitivity using a filter paper disc with a range of six monosodium glutamate (MSG) concentrations. We recruited 28 patients with taste disorders (45–78 years) and 184 controls with no taste disorders (102 young [18–25 years] and 82 older [65–89 years] participants). Filter paper discs (5 mm dia.) were soaked in aqueous MSG solutions (1, 5, 10, 50, 100 and 200 mM), then placed on three oral sites innervated by different taste nerves. The lowest concentration participants correctly identified was defined as the recognition threshold (RT) for MSG. This test showed good reproducibility for inter- and intra-observer variability. We concluded that: (1) The RT of healthy controls differed at measurement sites innervated by different taste nerves; that is, the RT of the anterior tongue was higher than that of either the posterior tongue or the soft palate in both young and older individuals. (2) No significant difference in RT was found between young adults and older individuals at any measurement site. (3) The RT of patients with taste disorders was higher before treatment than that of the healthy controls at any measurement site. (4) The RT after treatment in these patients improved to the same level as that of the healthy controls. (5) The cutoff values of RT, showing the highest diagnostic accuracy (true positives + true negatives), were 200 mM MSG for AT and 50 mM MSG for PT and SP. The diagnostic accuracy at these cutoff values was 0.92, 0.87 and 0.86 for AT, PT and SP, respectively. Consequently, this umami taste sensitivity test is useful for discriminating between normal and abnormal umami taste sensations.

Patients with gustatory disorders often complain of persistent impairment of umami taste, a synonym for savory or broth-like flavor, even after the recovery of the other four basic taste sensations (sweet, salty, sour, and bitter). Umami taste is important for maintaining not only the quality of life but also good health, and therefore, it seems important to assess and treat the impairment of umami taste perception. At present, however, there is no clinical test for umami taste, even though tests for the other four basic tastes have been widely used in quantitative gustometry. We have developed a new method to clinically assess the sensitivity of umami taste using a filter paper disc (FPD) with monosodium glutamate (MSG) as the prototype umami substance. We found that some elderly patients complained only of the loss of umami taste sensation, whereas the sensation of the other four basic tastes (sweet, salty, sour, and bitter) was normal. Those patients without normal umami taste sensitivity were all over 65 years old, and complained of appetite and weight loss, resulting in poor general health. After treatment, their umami taste threshold recovered, and they regained appetite and weight.

A multifunctional calcium-binding protein, centrin-1, is specifically expressed in male germ cells, certain neurons and ciliated cells. We identified centrin-1 as a protein interacting with SUMO-2/3 using yeast two-hybrid screening of a mouse testicular cDNA library. In bead halo assays, the interaction between centrin-1 and SUMO-2/3 was reduced in the presence of EGTA and facilitated by the addition of CaCl2. immunostaining of seminiferous tubules in 35-day-old mouse testes revealed that cells in the layer containing spermatogonia showed colocalization of SUMO-2/3 with centrin-1 in cytoplasmic spots. Identification of centrin-1 as the EGTA-sensitive SUMO-2/3-interacting protein indicates the possible role of calcium in modulating the centrin-1–SUMO-2/3 interaction and suggests the importance of this interaction in mouse testis. Copyright © 2010 John Wiley & Sons, Ltd.

This study was designed to assess the functional role of interleukin-18 (IL-18) in periodontal tissues. IL-18 transgenic (IL-18Tg) mice and wild-type (WT) mice were inoculated intraorally with Porphyromonas gingivalis. Alveolar bone loss, gingival cytokine levels, and gingival gene expression were assessed using morphometric analysis, enzyme-linked immunosorbent assay (ELISA), and semiquantitative reverse transcription polymerase chain reaction, respectively. P. gingivalis-infection induced periodontal bone loss in IL-18Tg mice, whereas bone loss did not occur in WT mice. Interferon gamma was downregulated only in IL-18Tg mice by P. gingivalis-infection. P. gingivalis-infection upregulated the expression of receptor activator of nuclear factor kappa B ligand (RANKL) in IL-18Tg mice vs. WT-type mice.These results suggest that IL-18Tg mice are highly susceptible to bone loss induced by the periodontal pathogen P. gingivalis, which may be mediated via RANKL-dependent pathway.

This study was designed to determine if sympathetic nerve fibers exist in dentinal tubules in rat normal dental pulp, and if they sprout into the tubules in the inflamed condition. Sympathetic nerve fibers in rat molar dental pulp were labeled using an anterograde axonal transport technique involving injection of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) into the superior cervical ganglion. In the normal dental pulp, scattered WGA-HRP reaction products were observed in unmyelinated nerve endings in odontoblast layer and subodontoblastic region, mostly close to odontoblast cell bodies. More reaction products were detected in the above areas in the inflamed dental pulp than that in the normal pulp. However, no labeled nerve fibers were observed in dentinal tubules in either normal or inflamed dental pulp. These results indicate that although sympathetic nerve fibers do indeed sprout in inflamed dental pulp, they do not penetrate into the dentinal tubules.

Interleukin (IL)-18 is one of the inflammatory cytokine which is expressed not only in activated macrophages but also in non-immune cells, such as keratinocytes and epithelial cells. It is unclear which type of cell is the major source of serum IL-18. We showed that serum levels of IL-18 were increased in mice treated with Propionibacterium acnes and lipopolysaccharide (LPS), whereas, administration of clodronate-liposomes (Clo-lip) to induce depletion of macrophages showed no obvious effect on IL-18 levels. IL-18 levels were marginal in the liver, lung and spleen. Treatment with P. acnes alone induced IL-18 in each organ, and P. acnes and LPS induced increase in IL-18 levels in the liver and spleen, but decrease in the intestines. The administration of Clo-lip in mice showed only a marginal effect on the IL-18 levels in the organs. These results suggest that IL-18 expressed in keratinocytes/epithelial cells contributes to serum IL-18 levels.

Oral epithelium is endowed with innate immune receptors for bacterial components, which play important roles in host defense against bacterial infection. We examined the expression of various Toll-like receptors (TLRs), NOD1 and NOD2 in oral epithelial cells, and the production of β-defensin 2 and peptidoglycan recognition proteins (PGRPs) upon stimulation with their respective chemically synthesized ligands. We found a clear expression of TLR4 as well as TLR2, and a strong expression of NOD1 and NOD2 in normal oral epithelial tissues by immunohistochemical analysis. In the inflamed oral epithelium, cell-surface localizations of TLR2 and TLR4 were more clearly observed than in the healthy tissue. We also showed that oral epithelial cells in culture constitutively expressed TLR3 and TLR7 in addition to TLR2, TLR4, NOD1, and NOD2, and stimulation with synthetic ligands for these receptors (TLR2 agonistic lipopeptide, TLR3 agonistic poly I:C, TLR4 agonistic lipid A, TLR7 agonistic single-stranded RNA, NOD1 agonistic iE-DAP and NOD2 agonistic muramyldipeptide) markedly up-regulated the expression of antibacterial factors, such as β-defensin 2 and PGRPs, but not the proinflammatory cytokines. These findings indicate that these molecules in oral epithelial cells are functional receptors that induce antibacterial responses without excessive inflammatory responses.

This study was designed to determine if sympathetic nerve fibers exist in dentinal tubules in rat normal dental pulp, and if they sprout into the dentinal tubules in response to artificial cavity preparation in dentin. Sympathetic nerve fibers in rat molar dental pulp were labeled using an anterograde axonal transport technique involving injection of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) into the superior cervical ganglion (SCG). They were then observed using light and electron microscopes. In normal dental pulp (control), scattered WGA-HRP reaction products were observed in unmyelinated nerve endings in the odontoblast layer and subodontoblastic region. In injured pulp 3 weeks after cavity preparation, reaction products were about 1.8-times more plentiful in the above areas (versus control pulp). However, no labeled nerve fibers were observed in the dentinal tubules in either control or injured dental pulp. These results indicate that although sympathetic nerve fibers do indeed sprout in rat dental pulp in response to cavity preparation, they do not penetrate into the dentinal tubules in which postganglionic nerve endings derived from the SCG were not originally present.

Background: Previous findings suggest that antigen challenge (AC) may induce histidine decarboxylase (HDC) in cells other than mast cells (MCs) via MC-derived IL-1. We examined this hypothesis. Methods: Mice were sensitized to ovalbumin. After the sensitization, an AC was delivered intravenously. Results: In control mice, AC markedly induced HDC at a postanaphylactic time in the liver, lung, spleen, and ears. In MC-deficient W/Wv mice, AC also induced HDC, although the effect was weaker than in control mice. AC increased IL-1 in the tissues, the pattern being similar in W/Wv and control mice. AC induced HDC similarly in IL-1-deficient and control mice. In control mice, AC decreased histamine in the tissues (except the liver) for several hours. Conclusion: (1) AC induces HDC in both MC-dependent and MC-independent ways. (2) AC induces IL-1 mostly in non-MCs, but this IL-1 is not a prerequisite for the induction of HDC by AC. (3) HDC induction may contribute to the replenishment of the reduced pool of MC histamine in the anaphylactic period. (4) In the case of MC-dependent HDC induction, AC may stimulate MCs in such a way as to induce HDC within the MCs themselves, and/or AC-stimulated MCs may stimulate HDC induction in other cells, which will need to be directly identified in future studies.

Among the bisphosphonates, the nitrogen-containing bisphosphonates have much stronger anti-bone-resorptive activities than bisphosphonates containing no nitrogen, but nitrogen-containing bisphosphonates mostly have inflammatory side effects. Our previous murine-model experiments with a single intraperitoneal bisphosphonate injection demonstrated that (i) nitrogen-containing bisphosphonates induce various inflammatory reactions via an IL-1-dependent mechanism, (ii) alendronate (an nitrogen-containing bisphosphonate) produces a clear sclerotic line in the tibia that is easily detectable by radiography a few weeks later (tentatively called the bisphosphonate line, a useful marker for the anti-bone-resorptive activities of bisphosphonates), and (iii) clodronate (a non-nitrogen-containing bisphosphonate) reduces the inflammatory reactions induced by alendronate but does not reduce the bisphosphonate line formation induced by alendronate. We compared the effects of clodronate, aspirin and dexamethasone on the inflammatory reactions induced by alendronate (40 micromol/kg) (induction of the histamine-forming enzyme, accumulation of pleural exudate and splenomegaly) and on the bisphosphonate line formation induced by alendronate (0.1 micromol/kg). The effects of aspirin (833 micromol/kg) were weak. However, like clodronate, dexamethasone (10 micromol/kg, injected 5 min. after alendronate), strongly inhibited the alendronate-induced inflammatory reactions but did not reduce the alendronate-induced bisphosphonate line formation. Alendronate produced normal bisphosphonate lines in IL-1-deficient mice, too. These results suggest that clodronate and/or dexamethasone may be suitable for preventing or reducing the inflammatory side effects of nitrogen-containing bisphosphonates while preserving their powerful anti-bone-resorptive activities (although in practice the known side effects of dexamethasone may limit its use), and that the anti-bone resorptive activities of nitrogen-containing bisphosphonates are not influenced by IL-1.

Nitrogen-containing bisphosphonates (N-BPs), powerful anti-bone-resorptive drugs, have inflammatory side effects, while histamine is not only an inflammatory mediator, but also an immuno-modifier. In murine models, a single intraperitoneal injection of an N-BP induces various inflammatory reactions, including the induction of the histamine-forming enzyme histidine decarboxylase (HDC) in tissues important in immune responses (such as liver, lungs, spleen, and bone marrow). Lipopolysaccharide (LPS) and the proinflammatory cytokines IL-1 and TNF are also capable of inducing HDC. We reported previously that in mice, (i) the inflammatory actions of N-BPs depend on IL-1, (ii) N-BP pretreatment augments both LPS-stimulated IL-1 production and HDC induction, and (iii) the co-administration of clodronate (a non-N-BP) with an N-BP inhibits the latter's inflammatory actions (including HDC induction). Here, we add the new findings that (a) pretreatment with alendronate (a typical N-BP) augments both IL-1- and TNF-induced HDC elevations, (b) LPS pretreatment augments the alendronate-induced HDC elevation, (c) co-administration of clodronate with alendronate abolishes these augmentations, (d) alendronate does not induce HDC in IL-1-deficient mice even if they are pretreated with LPS, and (e) alendronate increases IL-1beta in all tissues tested, but not in the serum. These results suggest that (1) there are mutual augmentations between alendronate and immuno-stimulants (IL-1, TNF, and LPS) in HDC induction, (2) tissue IL-1beta is important in alendronate-stimulated HDC induction, and (3) combination use of clodronate may have the potential to reduce the inflammatory effects of alendronate (we previously found that clodronate, conveniently, does not inhibit the anti-bone-resorptive activity of alendronate).

Using laser-Doppler flowmetry we investigated whether reflex vasodilator responses would be evoked by the painful stimulation of the trigeminal region in human gingiva. GBF increases were elicited by the painful electrical stimulation to the tooth-pulp or gingiva, in addition to application by capsaicin onto the tongue, without an increase in systemic blood pressure. Thus, the pain of various oral tissues would cause the reflex vasodilatation in human gingiva.

Sympathetic regulation of periodontal ligament blood flow (PLBF) is well-attested; however, vasodilator responses mediated by parasympathetic nerve fibers have yet to be conclusively demonstrated in the periodontal ligament (PL). The present study was designed to determine whether parasympathetic vasodilator mechanisms do or do not exist in the cat PL. In our cats, the cervical sympathetic trunks were sectioned bilaterally prior to any stimulation in order to eliminate sympathetic effects on the vascular beds under study. Dynamic changes in PLBF, with mandibular lip blood flow (LBF) recorded for comparison, were investigated in cat mandibular canine teeth using laser Doppler flowmetry. The peripheral cut ends of the facial and glossopharyngeal nerve roots, which have been reported to contain parasympathetic nerve fibers to the oral tissues, were electrically stimulated intracranially. Such stimulation caused blood flow to increase in the ipsilateral PL and lip, without an increase in systemic blood pressure. These vasodilator responses in the PL and lip were sensitive to ganglion blockade (with hexamethonium), indicating vasodilation via activation of parasympathetic vasodilator fibers. In contrast, although intracranial stimulation of the trigeminal nerve root also induced increases in both PLBF and LBF, these were unaffected by hexamethonium, but reduced by tripelennamine, indicating antidromic vasodilatation via the trigeminal sensory nerve. These results suggest that parasympathetic vasodilator mechanisms do exist in feline PL.

A transistor pH electrode (pH-ISFET electrode) was placed in each of four human mouths and bacteria were allowed to accumulate on it for 1, 2 or 3 days. Changes of plaque pH were measured in situ after application of sucrose solution (0.1, 1 or 3%) and, then, quick pH recovery by mouth-rinses with water was estimated. Although the acidic pH after application of 0.1% sucrose was easily and quickly recovered from by a few mouth-rinses with water, rather frequent mouth-rinses with water were required to recover from the acidic pH of 2- or 3-day-old plaque after the application of a 1 or 3% sucrose solution. For example, 15 (mean, n = 4) mouth-rinses with water were required for the 3 day-old plaque pH to recover after the application of 3% sucrose solution.

Dynamic changes of pulpal blood flow (PBF) and gingival blood flow (GBF) induced by intra-venous injection of two kinds of vasoactive drugs were observed in dogs by means of Laser Doppler Velocimetry. Intra-venous injection of norepinephrine caused PBF to increase, corresponding to the blood pressure (BP) increase, while GBF decreased. Orciprenaline sulfate caused PBF to decrease parallel to the BP decrease, as compared with a GBF increase. The effects of these vasoactive drugs lasted longer on GBF than on PBF and BP. These results indicate that the regulation of blood flow in the dental pulp is more dependent on systemic blood pressure than on local vasoconstriction or vasodilation.