Daisuke Omagari

Nerve Growth Factor Contribution via Transient Receptor Potential Vanilloid 1 to Ectopic Orofacial Pain

It is well known that oral inflammation causes tenderness in temporomandibular joints or masseter muscles. The exact mechanism of such an orofacial ectopic hyperalgesia remains unclear. Here, we investigated the functional significance of interaction of nerve growth factor (NGF) and transient receptor potential vanilloid 1 (TRPV1) in relation to heat hyperalgesia in the whisker pad skin caused by complete Freunds adjuvant (CFA) injection into the lower lip. CFA injection induced heat hyperalgesia of the ipsilateral whisker pad skin. Moreover, it leads to enhancement of spontaneous activity and heat responses in trigeminal ganglion (TG) neurons that was elicited by heat stimulation of the whisker pad skin. The heat hyperalgesia was dose-dependently reversed by intraperitoneal TRPV1 antagonist administration, also diminished by neutralizing anti-NGF antibody administration into the lower lip and intraganglionic administration of K252a, a tyrosine kinase receptor inhibitor. Nerve fibers in bundle of mandibular nerve and TG neurons that innervates the whisker pad skin and lower lip both expressed labeled NGF, which was administrated into the lower lip. Moreover, the NGF concentrations in ophthalmic-maxillary and mandibular divisions of the TG increased after CFA injection into the lower lip. The number of TRPV1-positive neurons that innervates the whisker pad skin and lower lip was increased after CFA injection into the lower lip, and this increase was annulled by anti-NGF administration. The present findings suggest that inflammation in the lower lip induces release of NGF that regulates TRPV1 expression in TG neurons. This TRPV1 overexpression may underlie ectopic heat hyperalgesia in the whisker pad skin.

Poly I:C-induced expression of intercellular adhesion molecule-1 in intestinal epithelial cells

Intercellular adhesion molecul‐1 (ICAM‐1) is a transmembrane glycoprotein belonging to the immunoglobulin superfamily of adhesion molecules and plays perdominant roles in recruitment and trafficking of leucocytes to sites of inflammation. ICAM‐1 expression in intestinal epithelial cells (IECs) is enhanced by several stimuli, such as proinflammatory cytokines, bacterial infections or pathogen‐associated molecular patterns. One of these stimuli, double‐stranded RNA (dsRNA), is a by‐product of viral replication and can be recognized by its cognate receptor Toll‐like receptor 3 (TLR‐3). In spite of expression of both TLR‐3 and ICAM‐1 in IECs, correlation between TLR‐3‐signalling and ICAM‐1 expression has never been examined in IECs. In the present study, we investigated whether poly I:C, an analogue of dsRNA, can stimulate the expression of ICAM‐1 in IEC line, HT‐29. Poly I:C‐stimulation up‐regulated the expression of ICAM‐1 mRNA by real‐time polymerase chain reaction. Enhanced expression of ICAM‐1 was confirmed in protein level by immunofluoresense cell staining and enzyme‐linked immunosorbent assay by measuring the released soluble ICAM‐1 in culture supernatant. As the stimulation effect was reduced by pre‐treatment of the cells with anti‐TLR‐3 antibody, poly I:C‐binding signal was thought to be sensed by TLR‐3 on the surface of HT‐29. The results of luciferase assay and nuclear factor kappa‐b (NF‐kB) inhibitor treatment experiments indicated that the downstream signal was mainly transduced by transcription factor, NF‐kB. All these results demonstrated the connection between TLR‐3 signalling and ICAM‐1 expression in HT‐29 cells and indicated the importance of coordinated function of both innate and adaptive immunity against viral infections.

Estimation of trace metal elements in oral mucosa specimens by using SR-XRF, PIXE, and XAFS

The effects of dissolved elements from metal dental restorations are a major concern in lesions of the oral mucosa, and the evaluation of accumulated metal elements, especially their distribution and chemical state, is essential for determining the precise effects of trace metals. In this study, X-ray fluorescence with synchrotron radiation (SR-XRF) and particle-induced X-ray emission (PIXE) were applied for distribution analysis of the trace metal elements contained in the oral mucosa, and the chemical states of the elements were estimated using X-ray absorption fine structure (XAFS) analysis. Appropriate combination of these analysis techniques, particularly SR-XRF and PIXE, to visualize the distributions of the elements in the oral mucosa allowed for the observation and evaluation of accumulated metal ions and debris. Importantly, the analyses in this study could be carried out using conventional histopathological specimens without damaging the specimens. Therefore, this method would be applicable for the detection of accumulated trace metal elements in biopsy specimens from the oral mucosa.

A Novel Loop-Mediated Isothermal Amplification Assay for Serogroup Identification of Neisseria meningitidis in Cerebrospinal Fluid

We have developed a novel Neisseria meningitidis serogroup-specific loop-mediated isothermal amplification (LAMP) assay for six of the most common meningococcal serogroups (A, B, C, W, X, and Y). The assay was evaluated using a set of 31 meningococcal LAMP assay positive cerebrospinal fluid (CSF) specimens from 1574 children with suspected meningitis identified in prospective surveillance between 1998 and 2002 in Vietnam, China, and Korea. Primer specificity was validated using 15 N. meningitidis strains (including serogroups A, B, C, E, W, X, Y, and Z) and 19 non-N. meningitidis species. The N. meningitidis serogroup LAMP detected down to ten copies and 100 colony-forming units per reaction. Twenty-nine CSF had N. meningitidis serogroup identified by LAMP compared with two CSF in which N. meningitidis serogroup was identified by culture and multi-locus sequence typing. This is the first report of a serogroup-specific identification assay for N. meningitidis using the LAMP method. Our results suggest that this assay will be a rapid, sensitive, and uniquely serogroup-specific assay with potential for application in clinical laboratories and public health surveillance systems.

Nuclear factor kappa B plays a pivotal role in polyinosinic‐polycytidylic acid‐induced expression of human β‐defensin 2 in intestinal epithelial cells

Intestinal epithelial cells (IECs) play an important role in protecting the intestinal surface from invading pathogens by producing effector molecules. IECs are one of the major sources of human beta‐defensin 2 (hBD‐2), and can produce it in response to a variety of stimuli. Although IECs express Toll‐like receptor 3 (TLR‐3) and can respond to its ligand, double‐stranded RNA (dsRNA), hBD‐2 expression in response to dsRNA has not been elucidated. In the present study, using an artificial analogue of dsRNA, polyinosinic‐polycytidylic acid (poly I:C), we investigated whether the human IEC line, HT‐29, can produce hBD‐2 in response to poly I:C. HT‐29 cells can express hBD‐2 mRNA only when stimulated with poly I:C. The induction of hBD‐2 mRNA expression was observed at 3 h after stimulation and peaked at 12 h of post‐stimulation. Pre‐incubation of the cells with nuclear factor kappa B (NF‐κB)‐specific inhibitor, l‐1–4′‐tosylamino‐phenylethyl‐chloromethyl ketone (TPCK) and isohelenine abolished the expression of hBD‐2. Detection of the poly I:C signal by TLR‐3 on the surface of HT‐29 cells was revealed by pre‐incubating the cells with anti‐TLR‐3 antibody. The 5′‐regulatory region of the hBD‐2 gene contains two NF‐κB binding sites. A luciferase assay revealed the importance of the proximal NF‐κB binding site for poly I:C‐induced expression of hBD‐2. Among NF‐κB subunits, p65 and p50 were activated by poly I:C stimulation and accumulated in the nucleus. Activation of the p65 subunit was investigated further by determining its phosphorylation status, which revealed that poly I:C stimulation resulted in prolonged phosphorylation of p65. These results indicate clearly that NF‐κB plays an indispensable role in poly I:C induced hBD‐2 expression in HT‐29 cells.

Novel metal allergy patch test using metal nanoballs

BackgroundPatch tests are often used in the clinical diagnosis of metal allergies. In currently available patch tests, high concentrations of metal salt solutions are used. However, diagnosis accuracy can be influenced not only by acute skin reactions to high concentrations of metal salt, but also by skin reactions to other components present in the patch or to pH changes. In this study, we developed Ni nanoparticles (termed “nanoballs”) for use in patch-test solutions.FindingsHighly soluble, spherical Ni nanoballs were prepared using plasma electrolysis. The Ni released from the nanoballs permeated through a dialysis membrane, and the nanoball-containing solution’s pH was maintained constant. Ni ions were released slowly at low concentrations in a time-dependent manner, which contrasted the rapid release observed in the case of a commercial patch test. Consequently, in the new test system, reactions caused by high concentrations of metal salts were avoided.ConclusionsBy exploiting the high specific surface area of Ni nanoballs, we obtained an effective dissolution of Ni ions that triggered Ni allergy in the absence of direct contact between the nanoballs and mouse skin. This novel patch system can be applied to other metals and alloys for diagnosing various types of metal-induced contact dermatitis.

Characterization of human dental pulp-derived cell lines.

AIM To establish and characterize different types of fibroblastic cell lines derived from dental pulp tissue. METHODOLOGY Human dental pulp tissue-derived cells were transfected with SV40 large T antigen by Lipofectamine transfection method. Geneticin (G418)-resistant cells were selected and different cell lines were established by a limiting dilution method. To characterize the lineages of cells, each clone was immunofluorescently stained by anti-fibroblast, anti-vimentin, anti-collagen type I and type III antibodies. Total RNA was extracted from each clone and subjected to a differential display experiment. RESULTS By transfecting SV40 large T antigen, nine different cell clones were obtained. All these cell clones were positively stained by anti-fibroblast, anti-vimentin, anti-collagen type I and type III antibodies. With differential display experiment, eight different genes, the expression levels of these genes were varied amongst each cell clone, were detected. After sequencing and database search, one gene was revealed to be identical to T-cell marker, Thy-1. Thy-1 expression in dental pulp tissue was confirmed by immunohistochemical staining. CONCLUSION Fibroblastic cell lines derived from human dental pulp tissue possessed different gene expression profiles suggesting the existence of subpopulations.

Detection of trace metallic elements in oral lichenoid contact lesions using SR-XRF, PIXE, and XAFS

Oral lichen planus (OLP) and oral lichenoid contact lesions (OLCL) are chronic inflammatory mucocutaneous reactions with a risk of malignant transformation that alter the epithelium. OLP and OLCL have similar clinical and histopathological features and it is difficult to distinguish one from the other. Metallic restorations are suspected to generate OLCLs. Trace metal analysis of OLCL specimens may facilitate the discrimination of symptoms and identification of causative metallic restorations. The purpose of this study was to assess OLCL tissue samples for the prevalence of metallic elements derived from dental restorations, and to discriminate OLCL from OLP by using synchrotron radiation-excited X-ray fluorescence analysis (SR-XRF), particle-induced X-ray emission (PIXE), and X-ray absorption fine structure (XAFS). Typical elements of dental materials were detected in the OLCL, whereas no obvious element accumulation was detected in OLP and negative control specimens. The origin of the detected metallic elements was presumed to be dental alloys through erosion. Therefore, our findings support the feasibility of providing supporting information to distinguish OLCL from OLP by using elemental analysis.

Nickel Ion Inhibits Nuclear Factor-Kappa B Activity in Human Oral Squamous Cell Carcinoma

Background The spontaneous IL-8 secretion observed in OSCC is partially dependent on the disregulated activity of transcription factor NF-κB. Nickel compounds are well established human carcinogens, however, little is known about the influence of nickel on the spontaneous secretion of IL-8 in oral squamous cell carcinoma (OSCC) cells. The aim of the present study was to investigate whether Ni2+ ions can influence on IL-8 secretion by OSCC. Methods and Results The IL-8 secretion was measured by ELISA. The expression of IL-8 mRNA was examined by real-time PCR. The NF-κB activity was measured by luciferase assay. The phosphorylation status and nuclear localization of NF-κB subunits were examined by Western blotting or Transfactor kit and immunofluorescence staining, respectively. The interaction of NF-κB p50 subunit and Ni2+ ions was examined by Ni2+-column pull down assay. The site-directed mutagenesis was used to generate a series of p50 mutants. Scratch motility assay was used to monitor the cell mobility. Our results demonstrated that, on the contrary to our expectations, Ni2+ ions inhibited the spontaneous secretion of IL-8. As IL-8 reduction was observed in a transcriptional level, we performed the luciferase assay and the data indicated that Ni2+ ions reduced the NF-κB activity. Measurement of p50 subunit in the nucleus and the immunofluorescence staining revealed that the inhibitory effect of Ni2+ ions was attributed to the prevention of p50 subunit accumulation to the nucleus. By Ni2+-column pull down assay, Ni2+ ions were shown to interact directly with His cluster in the N-terminus of p50 subunit. The inhibitory effect of Ni2+ ions was reverted in the transfectant expressing the His cluster-deleted p50 mutant. Moreover, Ni2+ ions inhibited the OSCC mobility in a dose dependent fashion. Conclusions Taken together, inhibition of NF-κB activity by Ni2+ ion might be a novel therapeutic strategy for the treatment of oral cancer.

Anaplastic sarcoma of the kidney: Case report and literature review

Anaplastic sarcoma of the kidney (ASK) is a relatively newly recognized pediatric renal tumor. The present patient, a 13‐year‐old boy with a large renal mass, underwent surgery. Pathological findings showed proliferation of short spindle‐shaped cells with anaplastic features including multiple foci in hyaline cartilage. Complex chromosomal abnormalities were detected in the tumor cells. Postoperative chemotherapy with the regimen for Ewings sarcoma achieved complete remission but the tumor recurred and the patient died during re‐induction chemotherapy. Autopsy indicated the cause of death as duodenal hemorrhage. Because there were no viable tumor cells, the recurrent tumor was considered to have been completely cured by chemotherapy. ASK is a very rare tumor, of unknown pathogenesis, and no standard treatment has yet been established, but the tumor cells may be responsive to chemotherapy. Further study is needed to establish the optimal treatment strategy.