Cathelicidin antimicrobial peptide inhibits fibroblast migration via P2X7 receptor signaling.

Fibrosis is one of the most common pathological alterations in heart failure, and fibroblast migration is an essential process in the development of cardiac fibrosis. Experimental autoimmune myocarditis (EAM) is a model of inflammatory heart disease characterized by inflammatory cell infiltration followed by healing without residual fibrosis. However, the precise mechanisms mediating termination of inflammation and nonfibrotic healing remain to be elucidated. Microarray analysis of hearts from model mice at multiple time points after EAM induction identified several secreted proteins upregulated during nonfibrotic healing, including the anti-inflammatory cathelicidin antimicrobial peptide (CAMP). Treatment with LL-37, a human homolog of CAMP, activated MAP kinases in fibroblasts but not in cardiomyocytes, indicating that fibroblasts were the target of CAMP activity. In addition, LL-37 decreased fibroblast migration in the in vitro scratch assay. P2X7 receptor (P2X7R), a well-known receptor for LL-37, was involved in LL-37 mediated biological effect on cardiac fibroblasts. Stimulation of BzATP, a P2X7R agonist, activated MAPK in fibroblasts, whereas the P2X7R antagonist, BBG, as well as P2X7R deletion abolished both LL-37-mediated MAPK activation and LL-37-induced reduction in fibroblast migration. These results strongly suggest that CAMP upregulation during myocarditis prevents myocardial fibrosis by restricting fibroblast migration via activation of the P2X7R-MAPK signaling pathway.

Therapeutic administration of IL-11 exhibits the postconditioning effects against ischemia-reperfusion injury via STAT3 in the heart.

Activation of cardiac STAT3 by IL-6 cytokine family contributes to cardioprotection. Previously, we demonstrated that IL-11, an IL-6 cytokine family, has the therapeutic potential to prevent adverse cardiac remodeling after myocardial infarction; however, it remains to be elucidated whether IL-11 exhibits postconditioning effects. To address the possibility that IL-11 treatment improves clinical outcome of recanalization therapy against acute myocardial infarction, we examined its postconditioning effects on ischemia/reperfusion (I/R) injury. C57BL/6 mice were exposed to ischemia (30 min) and reperfusion (24 h), and IL-11 was intravenously administered at the start of reperfusion. I/R injury mediated the activation of STAT3, which was enhanced by IL-11 administration. IL-11 treatment reduced I/R injury, analyzed by triphenyl tetrazolium chloride staining [PBS, 46.7 ± 14.4%; IL-11 (20 µg/kg), 28.6 ± 7.5% in the ratio of infarct to risk area]. Moreover, echocardiographic and hemodynamic analyses clarified that IL-11 treatment preserved cardiac function after I/R. Terminal deoxynucleotide transferase-mediated dUTP nick-end labeling staining revealed that IL-11 reduced the frequency of apoptotic cardiomyocytes after I/R. Interestingly, IL-11 reduced superoxide production assessed by in situ dihydroethidium fluorescence analysis, accompanied by the increased expression of metallothionein 1 and 2, reactive oxygen species (ROS) scavengers. Importantly, with the use of cardiac-specific STAT3 conditional knockout (STAT3 CKO) mice, it was revealed that cardiac-specific ablation of STAT3 abrogated IL-11-mediated attenuation of I/R injury. Finally, IL-11 failed to suppress the ROS production after I/R in STAT3 CKO mice. IL-11 administration exhibits the postconditioning effects through cardiac STAT3 activation, suggesting that IL-11 has the clinical therapeutic potential to prevent I/R injury in heart.

STAT3/Pim-1 signaling pathway plays a crucial role in endothelial differentiation of cardiac resident Sca-1+ cells both in vitro and in vivo.

Cardiac stem cells potentially differentiate into cardiac cells, including cardiomyocytes and endothelial cells (ECs). Previously we demonstrated that STAT3 activation by IL-6 family cytokines, such as leukemia inhibitory factor (LIF), induces the endothelial differentiation of cardiac Sca-1+ cells. In this study, we addressed molecular mechanisms for EC differentiation of Sca-1+ cells. First, DNA array experiments were performed to search for the molecules induced by LIF. Among 134 genes that LIF upregulated by more than 4 fold, we focused on Pim-1 gene transcript, because Pim-1 is associated with the differentiation of some cell lineages. Real time RT-PCR analyses confirmed that LIF stimulation upregulated Pim-1 expression. Adenoviral transfection of dominant negative (dn) STAT3 inhibited LIF-mediated induction of Pim-1, while the overexpression of constitutively active STAT3 upregulated Pim-1 expression, suggesting that STAT3 activation is necessary and sufficient for Pim-1 induction. Moreover, in STAT3-deficient Sca-1+ cells, LIF failed to induce Pim-1 expression and EC differentiation. Importantly, the overexpression of dnPim-1 abrogated the induction of EC markers, indicating Pim kinase activity is indispensable for STAT3-mediated EC differentiation in vitro. Finally, Sca-1+ cells labeled with LacZ were transplanted into post-infarct myocardium and the transdifferentiation was estimated. The overexpression of wild-type STAT3 by adenovirus vector significantly promoted EC differentiation, while STAT3 gene ablation reduced the frequency of differentiating cells in post-infarct myocardium. Furthermore, transplanted Sca-1+ cells overexpressing dnPim-1 showed the reduced frequency of EC differentiation and capillary density. Collectively, Pim-1 kinase is upregulated by STAT3 activation in cardiac Sca-1+ cells and plays a pivotal role in EC differentiation both in vitro and in vivo.

Therapeutic activation of signal transducer and activator of transcription 3 by interleukin-11 ameliorates cardiac fibrosis after myocardial infarction.

BACKGROUND: Glycoprotein 130 is the common receptor subunit for the interleukin (IL)-6 cytokine family. Previously, we reported that pretreatment of IL-11, an IL-6 family cytokine, activates the glycoprotein 130 signaling pathway in cardiomyocytes and prevents ischemia/reperfusion injury in vivo; however, its long-term effects on cardiac remodeling after myocardial infarction (MI) remain to be elucidated. METHODS AND RESULTS: MI was generated by ligating the left coronary artery in C57BL/6 mice. Real-time reverse transcription polymerase chain reaction analyses showed that IL-11 mRNA was remarkably upregulated in the hearts exposed to MI. Intravenous injection of IL-11 activated signal transducer and activator of transcription 3 (STAT3), a downstream signaling molecule of glycoprotein 130, in cardiomyocytes in vivo, suggesting that cardiac myocytes are target cells of IL-11 in the hearts. Twenty-four hours after coronary ligation, IL-11 was administered intravenously, followed by consecutive administration every 24 hours for 4 days. IL-11 treatment reduced fibrosis area 14 days after MI, attenuating cardiac dysfunction. Consistent with a previous report that STAT3 exhibits antiapoptotic and angiogenic activity in the heart, IL-11 treatment prevented apoptotic cell death of the bordering myocardium adjacent to the infarct zone and increased capillary density at the border zone. Importantly, cardiac-specific ablation of STAT3 abrogated IL-11-mediated attenuation of fibrosis and was associated with left ventricular enlargement. Moreover, with the use of cardiac-specific transgenic mice expressing constitutively active STAT3, cardiac STAT3 activation was shown to be sufficient to prevent adverse cardiac remodeling. CONCLUSIONS: IL-11 attenuated cardiac fibrosis after MI through STAT3. Activation of the IL-11/glycoprotein 130/STAT3 axis may be a novel therapeutic strategy against cardiovascular diseases.

Glycoprotein 130 cytokine signal as a therapeutic target against cardiovascular diseases.

Postnatal cardiomyocytes have only limited capacity of proliferation. Therefore, the myocardium is intrinsically equipped with cardioprotective machineries and protects itself from pathological stresses. One of the most important cardioprotective systems is the signal network of autocrine/paracrine factors, including neurohumoral factors, growth factors, and cytokines. In this review, we focus on the roles of interleukin-6 (IL-6) family cytokines, also known as glycoprotein 130 (gp130) cytokines, in cardioprotection. These cytokines make a complex with their specific cytokine receptor α-subunits. The cytokine-receptor α-subunit complex binds to gp130, a common receptor of the IL-6 family, followed by the activation of JAK/STAT, ERK, and PI3 kinase/Akt pathways. In cardiomyocytes, signals through gp130 promote cell survival and angiogenesis through the JAK/STAT pathway. Activation of gp130 in cardiac stem cells induces their endothelial transdifferentiation, leading to neovascularization. Recently, accumulating evidence has revealed that altered JAK/STAT activity is associated with heart failure, suggesting that the JAK/STAT pathway is a therapeutic target against cardiovascular diseases. Interestingly, activation of the JAK/STAT pathway with interleukin-11 (IL-11) exhibits preconditioning effects in ischemia/reperfusion model. Moreover, IL-11 treatment after coronary ligation prevents cardiac remodeling through the JAK/STAT pathway. Since IL-11 is used for patients with thrombocytopenia, we propose that IL-11 is a candidate cytokine clinically available for cardioprotection therapy.

Oral complaints and stimulated salivary flow rate in 1188 adults.

BACKGROUND: Recently, oral sensory complaints (OSC) were proposed as a disease entity to represent idiopathic sensory disturbances of dry mouth, burning mouth, and taste disturbance, even though neither the status of OSC in the general population nor its underlying mechanism has yet been elucidated. Moreover, these three OSC-related complaints have not been assessed in combination by means of a visual analog scale (VAS) in a large-scale, community-dwelling population of a broad age range. METHODS: In a 1188-member community-dwelling adult population, comprised of 373 males and 815 females, aged 20-90 years, the three OSC-related complaints and stimulated salivary flow rate (SSFR) were assessed by means of a VAS and modified Saxon test, respectively. Association of each complaint with age, gender, SSFR, and other complaints was analyzed. RESULTS: Increases in both prevalence and intensity of subjective dry mouth and burning mouth were associated closely with decreasing SSFR. Even for taste disturbance, which may be affected less significantly by salivation status than the other two complaints, a significant association was suggested between decreasing SSFR and especially severe taste disturbance. However, these oral complaints were found in considerable prevalence even in the individuals with high SSFR. Often overlapping presentation of these complaints and a close association in intensity between the complaints to each other were also found. CONCLUSIONS: Hyposalivation may be a significant and common etiology for the three oral complaints, although the considerable prevalence of complaints without hyposalivation suggests other etiologies, including those related to the OSC.

Head and neck squamous cell carcinoma: usefulness of diffusion-weighted MR imaging in the prediction of a neoadjuvant therapeutic effect.

The purpose of our study was to evaluate the usefulness of diffusion-weighted imaging in predicting the responses to neoadjuvant therapy for head and neck squamous cell carcinomas. Diffusion-weighted, T2-weighted, and gadolinium-enhanced T1-weighted images were obtained from 28 patients with untreated head and neck squamous cell carcinomas with histological proof. A blinded radiologist evaluated the quantitative and qualitative signal intensities and apparent diffusion coefficients (ADCs) in the lesions on each sequence. All patients were treated by neoadjuvant therapies, and the post-therapeutic tumor regression rate was determined. Both the quantitative and qualitative signal intensities on diffusion-weighted images showed positive correlations (r = 0.367 and 0.412, p < .05), and the ADCs showed a weak, inversed correlation (r = -0.384, p < .05) with the tumor regression rates. Diffusion-weighted imaging including an assessment by ADCs may be able to predict tumor response to neoadjuvant therapy for head and neck squamous cell carcinomas.

The effect of neoadjuvant therapy on the 5-fluorouracil metabolic and relative enzymes of oral squamous cell carcinoma.

To assess the effect of neoadjuvant therapy using tegafur/uracil (UFT) and radiation therapy on the 5-fluorouracil (5-FU) metabolic and relative enzymes, thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), orotate phosphoribosyl transferase (OPRT) and thymidine phosphorylase (TP) in oral squamous cell carcinoma (OSCC), we examined the mRNA expression and immunohistochemical staining status of these enzymes using 17 surgical specimens. Seven patients did not receive any neoadjuvant therapy and 10 were treated with UFT and local irradiation therapy. Our result showed that the mRNA expression of these enzymes in neoadjuvant group was not significantly different from that of non-treated group using real-time quantitative PCR. To confirm the protein expression, we also carried out immunohistological staining of TS and DPD two key enzymes in the 5-FU metabolism, using the same specimens. Immunohistological staining status did not correspond to the results of mRNA analysis completely, though no significant difference between the groups was observed. Furthermore, no significant relationship between the UFT administration period and mRNA expression of the 5-FU metabolic enzymes was observed in neoadjuvant therapy group and also the distribution of the enzyme mRNA expression levels was similar to that of non-treated group. The results suggested that the neoadjuvant therapy of OSCC might not affect the expression status of 5-FU metabolic and relative enzymes in surgical tumor samples and the tumor tissues might serve as a useful specimen source to analyze the expression status of the 5-FU metabolic and relative enzymes and to determine the prospective efficiency of 5-FU-based adjuvant chemotherapy.

Chemopreventive effect of fermented brown rice and rice bran on 4-nitroquinoline 1-oxide-induced oral carcinogenesis in rats.

The preventive effects of the dietary administration of brown rice and rice bran fermented with Aspergillus oryzae (FBRA) on oral carcinogenesis induced by 4-nitroquinoline 1-oxide (4-NQO) were investigated in male F344 rats. At 7 weeks of age, the animals were given 20 ppm 4-NQO in their drinking water for 8 weeks to induce tongue neoplasms. Groups of rats were fed diets containing 5 or 10% FBRA during the initiation or postinitiation phases of the 4-NQO-induced oral carcinogenesis. The other groups consisted of rats fed 10% FBRA or untreated rats. At the termination of the study (week 32), the incidences, multiplicities of tongue lesions (pre-neoplasms and neoplasms) and the cell proliferation activity estimated by the 5-bromodeoxyuridine (BrdU)-labeling index were compared among the groups. Feeding of 5% FBRA during the initiation phase significantly decreased the incidence (68.2 vs 36.8%; p<0.05) and multiplicity (1.05+/-0.84 vs 0.37+/-0.50; p<0.005) of the tongue carcinoma. When feeding of 10% FBRA occurred after the 4-NQO exposure, the multiplicity of tongue carcinoma was also reduced (1.05+/-0.84 vs 0.52+/-0.60; p<0.05). In addition, the dietary administration of FBRA at both doses significantly decreased the BrdU-labeling index in the oral squamous epithelium (p<0.05). Although a dose-dependent response was not observed, FBRA is effective in suppressing the development of 4-NQO-induced oral carcinogenesis by its concurrent exposure to the carcinogen. The inhibitory effect could be related to the suppression of the hyperproliferation of cells in the tongue epithelium and the radical scavenging activity of FBRA.

RORγt-expressing cells attenuate cardiac remodeling after myocardial infarction.

AIMS: Retinoic acid receptor-related orphan nuclear receptor γt (RORγt) is a transcriptional factor responsible for IL-17-producing T-cell differentiation. Although it was demonstrated that RORγt plays essential roles in the onset of autoimmune myocarditis, pathophysiological significance of RORγt in cardiac remodeling after myocardial infarction (MI) remains to be fully elucidated. METHODS AND RESULTS: MI was generated by ligating coronary artery. The expression of RORγt and IL-17A transcripts increased in murine hearts after MI. Additionally, immunohistochemical staining revealed that RORγt-expressing cells infiltrated in the border zone after MI. Flow cytometric analysis showed that RORγt-expressing cells were released from the spleen at day 1 after MI. Though RORγt-expressing cells in spleen expressed γδTCR or CD4, γδTCR+ cells were major population of RORγt-expressing cells that infiltrated into post-infarct myocardium. To address the biological functions of RORγt-expressing cells in infarcted hearts, we used mice with enhanced GFP gene heterozygously knocked-in at RORγt locus (RORγt+/- mice), which physiologically showed reduced expression of RORγt mRNA in thymus. Kaplan-Meier analysis showed that MI-induced mortality was higher in RORγt+/- mice than wild-type (WT) mice. Masson's trichrome staining demonstrated that cardiac injury was exacerbated in RORγt+/- mice 7 days after MI (Injured area: RORγt+/-; 42.1±6.5%, WT; 34.0±3.7%, circumference of injured myocardium: RORγt+/-; 61.8±4.8%, WT; 49.6±5.1%), accompanied by exacerbation of cardiac function (fractional shortening: RORγt+/-; 32.9±2.9%, WT; 38.3±3.6%). Moreover, immunohistochemical analyses revealed that capillary density in border zone was significantly reduced in RORγt+/- mice after MI, compared with WT mice, associated with the reduced expression of angiopoietin 2. Finally, the mRNA expression of RORγt, IL-17A, IL-17F and IL-23 receptor (IL-23R) mRNA and protein expression of IL-10 were decreased in RORγt+/- hearts. CONCLUSIONS: Heterozygous deletion of RORγt gene resulted in aggravated cardiac remodeling, accompanied by reduced capillary density, after MI, suggesting that RORγt-expressing cells contribute to tissue repair in infarcted myocardium.

Adult murine cardiomyocytes exhibit regenerative activity with cell cycle reentry through STAT3 in the healing process of myocarditis.

Mammalian cardiomyocytes substantially lose proliferative capacity immediately after birth, limiting adult heart regeneration after injury. However, clinical myocarditis appears to be self-limiting with tissue-reparative properties. Here, we investigated the molecular mechanisms underlying the recovery from myocarditis with regard to cardiomyocyte proliferation using an experimental autoimmune myocarditis (EAM) model. Three weeks after EAM induction (EAM3w), cardiac tissue displayed infiltration of inflammatory cells with cardiomyocyte apoptosis. However, by EAM5w, the myocardial damage was remarkably attenuated, associated with an increase in cardiomyocytes that were positively stained with cell cycle markers at EAM3w. Cardiomyocyte fate mapping study revealed that the proliferating cardiomyocytes primarily derived from pre-existing cardiomyocytes. Signal transducer and activator of transcription 3 (STAT3) was robustly activated in cardiomyocytes during inflammation, accompanied by induction of interleukin-6 family cytokines. Cardiomyocyte-specific ablation of STAT3 gene suppressed the frequency of cycling cardiomyocytes in the recovery period without influencing inflammatory status, resulting in impaired tissue repair and cardiac dysfunction. Finally, microarray analysis revealed that the expression of regeneration-related genes, metallothioneins and clusterin, in cardiomyocytes was decreased by STAT3 gene deletion. These data show that adult mammalian cardiomyocytes restore regenerative capacity with cell cycle reentry through STAT3 as the heart recovers from myocarditis-induced cardiac damage.

Aberrant promoter hypermethylation of p16 and MGMT genes in oral squamous cell carcinomas and the surrounding normal mucosa.

PURPOSE: Several genetic alterations have been reported to contribute to the development of oral squamous cell carcinoma (OSCC). Recent studies have shown roles of promoter hypermethylation of tumor suppressor genes, including p16 and MGMT, in several types of cancers. The purpose of this study is to examine the hypermethylation status of p16 and MGMT genes in both oral cancers and normal mucosa, surrounding the cancers. METHODS: Promoter hypermethylation status of p16 and MGMT genes were examined by the methylation-specific PCR (MSP) in OSCC (n = 51), verrucous carcinoma (n = 2), and carcinoma in situ (n = 2) tissues. Moreover, normal mucosa surrounding the cancers were also examined in 22 cases out of the 51 OSCCs. As a normal control, oral mucosa from healthy volunteers (n = 18) was used. RESULTS: Aberrant promoter hypermethylation of p16 and MGMT genes was detected in 50.9% (28 of 55) and 56.4% (31 of 55) of the total malignant cases, respectively. As for the 22 OSCC cases, in which paired cancerous tissues and the surrounding normal mucosa were examined simultaneously, promoter hypermethylation of p16 and MGMT genes was confirmed in 72.73% (16 of 22) and 68.18% (15 of 22), respectively. In contrast, as for the surrounding normal mucosa, promoter hypermethylation of p16 and MGMT genes was recognized in 27.27% (6 of 22) cases and 40.91% (9 of 22), respectively. CONCLUSIONS: Hypermethylation of both p16 and MGMT genes was frequently detected in not only OSCC tissues, but also the surrounding normal mucosa around the cancerous tissues. Thus promoter hypermethylation of p16 and MGMT genes are an important, probably early event in oral carcinogenesis.

Preoperative radiotherapy contributes to induction of proliferative activity of CD8+ tumor-infiltrating T-cells in oral squamous cell carcinoma.

In order to evaluate host immune response to cancer, many methods have been applied. However, in the field of oral squamous cell carcinoma, evaluation of host immune response on the basis of proliferative activity of tumor-infiltrating T-cells (TIL) has not been reported. Therefore, we applied double immunohistochemical staining of proliferation markers Ki-67 and CD8 to surgically resected and paraffin-embedded tissue sections for 35 cases of oral squamous cell carcinoma. With this method, there was a significant correlation between the percentage of Ki-67+CD8+TIL and the intra-tumor epithelium infiltration rate of CD8+TIL (P=0.0237). In the process of analysis, we found that the proliferative activity of CD8+TIL tended to correlate (P=0.0859) with clinical N factor (lymph node metastasis), which was previously reported to suppress host immune response. We therefore assumed there was another factor inducing host immune response. The proliferative activity of CD8+TIL was well correlated with preoperative radiotherapy (P=0.0200) while there was no significant correlation between the proliferative activity of CD8+TIL and other clinical factors; age, tumor size, clinical stage, pathological N factor (P=0.5410, 0.7769, 0.1041, and 0.1072, respectively). Our present results strongly imply that preoperative radiotherapy is a very important factor in oral squamous cell carcinoma inducing host immune response regardless of the clinical factors present.

Inhibitory effects of fermented brown rice and rice bran on the development of acute hepatitis in Long-Evans Cinnamon rats.

To investigate the effects of fermented brown rice (FBRA) on the development of hereditary hepatitis in Long-Evans Cinnamon (LEC) rats, we compared the incidence and grades of acute hepatitis among rats fed 5% and 10% FBRA in the conventional diet and the conventional diet alone. Both the 5% and 10% FBRA-supplemented diets indicated a tendency to prevent the development of hepatitis, and the significant effect of 10% FBRA was observed until 16-17 weeks of age in the accumulated incidence and survival ratio compared with the unsupplemented conventional diet, although no significant difference was observed between 5% and 10% FBRA-supplemented diets. At the age of 12 weeks, which is just before the rats develop hepatitis, serum copper levels in rats fed either of the test diets were similar to those in rats fed the conventional diet. Furthermore, the copper concentration in liver tissue at 12 weeks of age was not changed by the test diet. These results suggest that FBRA has preventive effects on the development of hepatitis in LEC rats and may play an important role in protecting the liver against the free radicals induced by copper accumulation in the liver.

Comparison of the prevalence and characteristics of food hypersensitivity among adolescent and older women.

BACKGROUND AND OBJECTIVES: Although food hypersensitivity is a public health concern, its documentation among the elderly is limited. The current study aims to compare the prevalence and characteristics of food hypersensitivity among adolescent women between aged 18-24 with among older women >50 years of age. METHODS AND STUDY DESIGN: 660 female university students between the ages of 18 and 24 who volunteered were enrolled as adolescent subjects. 470 women >50 years old who visited the Health Care Centre of Kyoto Katsura Hospital for health check-ups were enrolled as the older subjects. A questionnaire created by ourselves asking the presence of food hypersensitivity, symptoms, causative food, personal or family history of other allergic disorders was distributed. RESULTS: The prevalence of food hypersensitivity was statistically similar between adolescent (8.2%) and older women (8.9%). Among them, only 24.1% of the adolescent women and 26.2% of the older women had been diagnosed by physicians as having food allergy. The main causative foods (fruits, shellfish and fish) and the manifestations relating to food hypersensitivity were almost identical between adolescent and older women. In both adolescent and older women, food hypersensitivity positive group showed significantly higher prevalence of personal or family history of allergic disorders than that in food hypersensitivity negative group. CONCLUSIONS: These data indicate that food hypersensitivity in older women should be given more attention because the prevalence of this condition was as common as that in adolescent women.

Impacts of Dams and Global Warming on Fish Biodiversity in the Indo-Burma Hotspot.

Both hydropower dams and global warming pose threats to freshwater fish diversity. While the extent of global warming may be reduced by a shift towards energy generation by large dams in order to reduce fossil-fuel use, such dams profoundly modify riverine habitats. Furthermore, the threats posed by dams and global warming will interact: for example, dams constrain range adjustments by fishes that might compensate for warming temperatures. Evaluation of their combined or synergistic effects is thus essential for adequate assessment of the consequences of planned water-resource developments. We made projections of the responses of 363 fish species within the Indo-Burma global biodiversity hotspot to the separate and joint impacts of dams and global warming. The hotspot encompasses the Lower Mekong Basin, which is the world's largest freshwater capture fishery. Projections for 81 dam-building scenarios revealed progressive impacts upon projected species richness, habitable area, and the proportion of threatened species as generating capacity increased. Projections from 126 global-warming scenarios included a rise in species richness, a reduction in habitable area, and an increase in the proportion of threatened species; however, there was substantial variation in the extent of these changes among warming projections. Projections from scenarios that combined the effects of dams and global warming were derived either by simply adding the two threats, or by combining them in a synergistic manner that took account of the likelihood that habitat shifts under global warming would be constrained by river fragmentation. Impacts on fish diversity under the synergistic projections were 10-20% higher than those attributable to additive scenarios, and were exacerbated as generating capacity increased-particularly if CO2 emissions remained high. The impacts of dams, especially those on river mainstreams, are likely to be greater, more predictable and more immediately pressing for fishes than the consequences of global warming. Limits upon dam construction should therefore be a priority action for conserving fish biodiversity in the Indo-Burma hotspot. This would minimize synergistic impacts attributable to dams plus global warming, and help ensure the continued provision of ecosystem services represented by the Lower Mekong fishery.

Analysis of ameloblastomas by comparative genomic hybridization and fluorescence in situ hybridization.

In order to characterize the chromosomal alterations in ameloblastomas, a combination of comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) techniques was performed on 9 tumors. Chromosomal alterations including a gain at 1q and losses at 1pter, 10q, and 22q could be detected by CGH only in 1 tumor. Interphase FISH analysis, using centromeric probes for chromosomes 1, 10, and 22 as well as region-specific probes for 1p36 and 10q26, revealed the most frequent alterations to exist in the tumor with the abnormal CGH profile. These alterations included marked to slight increases of monosomic cells for chromosome 10 (91.5%), 10q26 (35.8%), 1p36 (24.4%), and chromosome 22 (18.8%), as well as significant elevations of trisomic cells for chromosome 1 (41.2%). Moreover, FISH analysis revealed a frequent loss of chromosome 22 in all tumors examined, except for one lesion, indicating that loss of the entire or a part of this chromosome is a common event in ameloblastomas, possibly being a predisposing factor to ameloblastoma tumorigenesis.

Rho regulates the hepatocyte growth factor/scatter factor-stimulated cell motility of human oral squamous cell carcinoma cells.

To investigate the effects of hepatocyte growth factor/scatter factor (HGF/SF) on the invasion and metastasis of human oral squamous cell carcinoma (SCC) cells, we examined cell motility and intercellular signal transduction of a human oral SCC cell line (SAS) obtained from the primary lesion of a tongue carcinoma. HGF/SF stimulation significantly enhanced the motility of SAS cells in a dose-dependent manner. Clostridium botulinum C3 exoenzyme (C3), which is known to selectively impair the function of Ras-related small G-protein p21rho (Rho), significantly reduced the motility of SAS cells. HGF/SF stimulation also enhanced the tyrosine phosphorylation of HGF receptors (c-Met) and focal adhesion kinase (FAK) on SAS cells, but C3 completely inhibited the phosphorylation of FAK. Furthermore, it was observed that Rho A protein, normally located around the nuclear area, was translocated to the membrane and levels in the cytolysate increased following HGF/SF stimulation with no change in Rho A mRNA. These results suggest that the activation of FAK caused by phosphorylation of c-Met may mediate the HGF/SF-induced motility of human oral SCC cells, and that Rho protein regulates the tyrosine phosphorylation of FAK through translocation from the nucleus to the membrane.

Effects of smoking and alcohol consumption on 5-fluorouracil-related metabolic enzymes in oral squamous cell carcinoma.

Lifestyle, particularly smoking and alcohol consumption, may induce and/or inhibit drug metabolism. In order to reveal the effects of smoking and alcohol consumption on the 5-fluorouracil (5-FU)-related metabolic enzymes, namely thymidylate synthase, dihydropyrimidine dehydrogenase (DPD; a sole catabolic enzyme of 5-FU), orotate phosphoribosyl transferase (OPRT) and thymidine phosphorylase, in oral squamous cell carcinomas, the mRNA expression of these enzymes was investigated in 29 surgical specimens and compared by the Brinkman index and drinking years. The surgical specimens were divided into normal and tumor regions and were independently analyzed using quantitative reverse transcription-polymerase chain reaction. There was a significantly positive correlation between DPD mRNA expression in these tissues and Brinkman index/drinking years, with OPRT mRNA expression being significantly correlated to the Brinkman index in tumor tissues. These results revealed that lifestyle habits, including smoking and alcohol consumption, may vary the activity of the 5-FU-related metabolic enzymes. DPD is the initial and rate-limiting enzyme in the catabolic pathway of 5-FU. Therefore, smoking and alcohol consumption may reduce the anticancer activity of 5-FU, possibly through the induction of DPD activity.

IL-6-mediated Th17 differentiation through RORγt is essential for the initiation of experimental autoimmune myocarditis.

AIMS: Interleukin (IL)-17-producing helper T (Th17) cells have been proposed to participate in the pathogenesis of chronic inflammation, such as autoimmune myocarditis. IL-6 gene ablation confers the resistance to experimental autoimmune myocarditis (EAM). In this study, we have addressed the pathological roles of IL-6 in the regulation of Th17 cells in EAM. METHODS AND RESULTS: To induce EAM, mice were immunized twice with α-myosin heavy chain peptide. Three weeks after the first injection, the cardiac expression of the Th17-specific transcription factor, retinoic acid receptor-related orphan nuclear receptor (ROR γt), was up-regulated. Consistently, Th17 cells were recruited into EAM hearts, as analysed by flow cytometry. Using the mice with enhanced green fluorescence protein (eGFP) gene knocked-in at RORγt locus (RORγt-eGFP mice), we observed Th17 cell infiltration into inflamed lesions. Pre-treatment with IL-6 receptor (IL-6R)-blocking antibody (anti-IL-6R Ab) inhibited EAM induction in terms of disease severity score (3.5 ± 0.8; IgG vs. 0.5 ± 0.8; anti-IL-6R Ab, n = 6, P< 0.01) and suppressed the myocardial expression of IL-17 and RORγt. In contrast, the administration of anti-IL-6R Ab 7 days after the first immunization failed to show the inhibitory effects, suggesting that IL-6 plays important roles in EAM initiation. Finally, by generating RORγt-eGFP homozygous mice, we revealed that RORγt gene ablation conferred the resistance to EAM induction. CONCLUSION: IL-6-mediated induction of Th17 cells is critical for the onset of EAM, but not for its progression. IL-6/Th17 signalling could be a promising therapeutic target for the prevention of myocardial inflammation.