Mevalonates restore zoledronic acid-induced osteoclastogenesis inhibition.

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is likely to be caused by continuous imperfection of bone healing after surgical treatments in patients with long-term administration of nitrogen-containing bisphosphonates (NBPs). NBPs inhibit osteoclastic bone resorption by impairing the mevalonic acid sterol pathway in osteoclasts. Thus, we hypothesized that exogenous mevalonic acid metabolites restore the inhibitory effects of NBPs on osteoclastogenesis and bone remodeling. To clarify the effects of mevalonic acid metabolites, especially geranylgeranyl pyrophosphate (GGPP) and geranylgeranyl transferase substrate geranylgeranyl acid (GGOH), we examined the effects of zoledronic acid with or without GGOH or GGPP on osteoclast differentiation, multinucleation, and bone mineral deposition in tooth-extracted sockets. Zoledronic acid decreased the number of tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells derived from mouse osteoclast precursors treated with receptor activator of nuclear factor-κB ligand and macrophage colony-stimulating factor. Zoledronic acid simultaneously suppressed not only the expressions of osteoclastic differentiation-related molecules such as TRAP, cathepsin K, calcitonin receptor, and vacuolar H-ATPase but also those of multinucleation-related molecules such as dendrocyte-expressed 7 transmembrane proteins and osteoclast stimulatory transmembrane protein. Treatment with GGOH or GGPP, but not farnesyl acid, restored the zoledronic acid-inhibited number of TRAP-positive multinuclear cells together with the expressions of these molecules. Although intraperitoneal administration of zoledronic acid and lipopolysaccharide into mice appeared to induce BRONJ-like lesions with empty bone lacunae and decreased mineral deposition in tooth-extracted socket, both GGOH and GGPP partially restored the inhibitory effects on zoledronic acid-related mineral deposition. These results suggest the potential of mevalonic acid metabolites as therapeutic agents for BRONJ.

PTHrP induces Notch signaling in periodontal ligament cells.

Periodontal ligament (PDL) cells are known to play important roles in tooth eruption and alveolar bone metabolism. We previously reported that PTHrP increases RANKL expression in human PDL cells, suggesting that it promotes odontoclastic root resorption during tooth eruption. While it is known that Notch-related genes play a key role during bone development, the role of the Notch signaling pathway in PDL cells during tooth and bone resorption is less clear. We hypothesized that PTHrP induces a Notch ligand in PDL cells and thereby regulates osteo- and odontoclastogenesis. We found that PTHrP increased Notch1 ligand Jagged1 expression in human PDL cells in a dose- and time-dependent manner. PTHrP-induced Jagged1 up-regulation was mediated by PKA activation, but not by PKC. Jagged1 also promoted RANKL-induced osteoclastogenesis. These results demonstrate that PTHrP induces Jagged1 expression in PDL cells, leading to osteo- and odontoclastogenesis, and thus likely promoting tooth and alveolar bone resorption.

The validity of the mangled extremity severity score in the assessment of upper limb injuries.

The Mangled Extremity Severity Score (MESS) may be used to decide whether to perform amputation in patients with injuries involving a limb. A score of 7 points or higher indicates the need for amputation. We have treated three patients with a MESS of 7 points or higher, in two of which the injured limb was salvaged. This scoring system was originally devised to assess injuries to the lower limb. However, a MESS of 7 points as a justification for amputation does not appear appropriate when assessing injuries to the major vessels in the upper limb.

A case of chondroma of the tongue.

Chondroma of the tongue represents a very rare benign tumor at this site, with very few reports of pure chondroma (no components of adipose, fibrous or bony tissue) existing. This report presents the case of a 17-year-old man with chondroma arising at the center of the tongue.

Evaluation of swallowing function after intraoral soft tissue reconstruction with microvascular free flaps.

This study assessed swallowing function after tumour resection and reconstruction utilizing free vascularized flap closures in patients with oral cancer. Swallowing function was evaluated postoperatively in 23 patients (21 men and 2 women) who had undergone reconstruction with either a lateral upper arm free flap (LUFF, n=16) or a radial forearm free flap (RFFF, n=7). Videofluoroscopy was used to assess tongue mobility and abnormalities of swallowing function. All patients who underwent reconstruction with LUFF or RFFF free flaps had decreased tongue mobility, except for the tip of the tongue. Patients who underwent anterior or posterior resection had greater decreases in tongue mobility than those who underwent medial resection. Swallowing impairment was similar in patients with LUFFs and those with RFFFs. Anterior resection of the oral cavity had a significant negative effect on swallowing function. Silent aspiration occurred in five patients. In conclusion the resection site affected swallowing function, but the type of flap did not, in patients with oral carcinoma, who underwent tumour resection with reconstruction

The binding of thymus leukemia (TL) antigen tetramers to normal intestinal intraepithelial lymphocytes and thymocytes.

Thymus leukemia (TL) Ags belong to the family of nonclassical MHC class I Ags and can be recognized by both TCRalphabeta and TCRgammadelta CTL with TL, but not H-2 restriction. We previously reported that the CTL epitope is TAP independent, but the antigenic molecule(s) presented by TL has yet to be determined. In the present study, TL tetramers were prepared with T3(b)-TL and murine beta(2)-microglobulin, not including antigenic peptides, and binding specificity was studied. CTL clones against TL Ags were stained with the T3(b)-TL tetramer, and the binding shown to be CD3 and CD8 dependent. Normal lymphocytes from various origins were also studied. Surprisingly, most CD8(+) intraepithelial lymphocytes derived from the small intestines (iIEL), as well as CD8(+) and CD4(+)CD8(+) thymocytes, were stained, while only very minor populations of CD8(+) cells derived from other peripheral lymphoid tissues, such as spleen and lymph nodes, were positive. The binding of T3(b)-TL tetramers to CD8(+) iIEL and thymocytes was CD8 dependent, but CD3 independent, in contrast to that to TL-restricted CTL. These results altogether showed that TL-restricted CTL can be monitored by CD3-dependent binding of T3(b)-TL tetramers. In addition, CD3-independent T3(b)-TL tetramer binding to iIEL and thymocytes may imply that TL expressed on intestinal epithelium and cortical thymocytes has a physiological function interacting with these tetramer(+)CD8(+) T lymphocytes.

A novel C1-using denitrifier alcaligenes sp. STC1 and its genes for copper-containing nitrite reductase and azurin.

A novel denitrifier Alcaligenes sp. STC1 was identified. The strain efficiently denitrifies under an atmosphere of 10% oxygen (O2) where Paracoccus denitrificans, one of the most studied aerobic denitrifiers, had less denitrifying activity, indicating that the strain has an O2-torelant denitrifying system. It denitrified by using C1-carbon sources such as formate and methanol as well as glucose, glycerol, and succinate. The genes for the copper-containing nitrite reductase and azurin of this C1-using denitrifier were cloned. Their predicted products of them were similar to those of their counterparts and the maximum similarities were 90% and 92%, respectively.

Influence of thoracic radiotherapy on exhaled nitric oxide levels in patients with lung cancer.

BACKGROUND: To determine the physiological role of exhaled nitric oxide (NO) in patients with lung cancer. METHODS: We investigated changes in exhaled NO levels in 29 patients undergoing thoracic radiation therapy with or without chemotherapy. The exhaled NO level was assessed using a chemiluminescence analyzer. RESULTS: The level of exhaled NO was higher in patients with lung cancer before treatment than in controls. With radiotherapy, the exhaled NO level decreased for patients undergoing 40 Gy irradiation and post-radiotherapy. However, five patients showed elevated levels of exhaled NO three times or more than that before radiotherapy. Three of these patients showed signs of radiation pneumonitis. However, none of the other patients showed signs of radiation pneumonitis (p = 0.002). CONCLUSION: Radiation therapy can lower exhaled levels of NO and the levels of exhaled NO may be a useful index for the early prediction of radiation pneumonitis.

State-dependent inhibition of L-type Ca2+ channels in A7r5 cells by cilnidipine and its derivatives.

Using a whole-cell patch-clamp technique, state-dependent inhibition of dihydropyridines (DHP)s was investigated on L-type channels in A7r5 cells. Cilnidipine, its derivatives (D-342 and D-69) and nimodipine inhibited the Ba2+ current. However, cilnidipine and D-342, but not D-69 or nimodipine, accelerated current decay. The apparent rank order for the effects on the DHP-sensitive decaying component was different from that obtained for inhibition of the peak current. The dissociation constants for cilnidipine in the resting and inactivated states were estimated to be 190 and 12 nM, respectively. Cilnidipine, but not other DHP derivatives, created a faster and voltage-independent component (r= 37 ms). The linear relationship between the tau(-1) of the current decay and the cilnidipine concentration provided a value of 471 nM for the dissociation constant in the open state, suggesting that the current decay is mediated by one-to-one lower affinity binding of cilnidipine molecules to their binding site. The present study demonstrates that structurally related DHPs act in distinct ways to inhibit the L-type channel in the resting, open and inactivated states. Cilnidipine and some related DHPs probably exert their blocking action on the open channel by binding to a receptor distinct from the known DHP-binding site.

Mitochondrial genotypes and radiation-induced micronucleus formation in human osteosarcoma cells in vitro.

We investigated whether or not the mitochondrial genotypes affect radiation-induced micronucleus (MN) formation. For that purpose, the rho+, KT1 and rho0 human osteosarcoma cell lines were used, which carry the wild-type mitochondrial DNA (mtDNA), the tRNALys mutant mtDNA and no mtDNA, respectively. Despite no significant difference in the clonogenic radiosensitivity, the rho+, KT1 and rho0 cells exhibited high, intermediate and low radiosensitivities, respectively, to the MN induction in cytokinesis-blocked binucleated cells. Such differential MN inductions were correlated with high, intermediate and low levels of cellular ATP in the rho+, KT1 and rho0 cells, respectively, but not exactly with ROS production. Antimycin A that inhibits the respiratory complex III reduced the rate of radiation-induced MN induction in the rho+ and KT1, but not rho0 cells. Thus, the functional status of the mtDNA to produce ATP appears to play a significant role for radiation-induced MN.

Comparison of anti-tumor responses against TL positive lymphoma induced by skin grafting and dendritic cell immunization.

When the skin of Tg.Con.3-1 transgenic mice expressing the TL (thymus leukemia) antigen in most tissues is grafted on syngeneic C3H mice, it is rejected, and a cytotoxic T cell (CTL) response against the TL antigen is induced. In this study, we first demonstrated that growth of TL positive lymphoma is suppressed in mice immunized by skin grafting. Immunization with bone marrow derived dendritic cells (DCs) from Tg.Con.3-1, was also found to be associated with an anti-tumor response, but less potent than skin grafting. Relative CTL precursor frequency with DC immunization was also approximately only one third that of skin grafting. The numbers of IFN-gamma producing cells in responder CD8 and CD4 T cell populations were higher with DC immunization than with skin grafting. However, DC immunization seems to induce non-specific immune responses, as re-stimulation with TL negative C3H spleen cells resulted in induction of almost half the number observed with TL positive cells. Thus, the actual number of IFN-gamma producing cells in specific responses to TL is not necessarily larger than with skin grafting immunization. The present results altogether suggest that DC immunization is capable of inducing an anti-tumor reaction, but also possibly unwanted immune responses. In vitro monitoring of specific and non-specific responses in the immune system, thus, is of particular importance for future development of cancer immunotherapy.

The epitope detected by cytotoxic T lymphocytes against thymus leukemia (TL) antigen is TAP independent.

Thymus leukemia (TL) antigens belong to the family of MHC class Ib antigens. We have shown in our previous studies that they serve as transplantation antigens, and can be recognized by both TCR alpha beta and TCR gamma delta cytotoxic T lymphocytes (CTL) with TL but not H-2 restriction. Although TL are known to be expressed TAP independently, it is unclear whether peptide loading on TL molecules is necessary for the formation of CTL epitopes. In the present study, we first showed that TL expression is beta(2)-microglobulin (beta(2)m)-dependent but TAP1 independent by flow cytometric analysis of thymocytes from beta(2)m- or TAP1-deficient mice crossed with TL transgenic mice expressing Tla(a)-3-TL on their thymocytes. Subsequently, we investigated the epitope recognized by CTL derived from C3H mice immunized with skin from a transgenic mouse expressing T3(b)-TL ubiquitously. Bulk CTL lines against TL from primary mixed lymphocyte cultures showed comparable cytotoxicity against T3(b)-TL transfectants of TAP2-deficient murine RMA-S grown at 37 degrees C to that against those grown at 25 degrees C. Furthermore, TCR alpha beta and TCR gamma delta CTL clones against TL recognized TL expressed on T3(b)-TL transfectants of RMA-S and Drosophila melanogaster cells having broad defects in peptide loading of MHC, and lysed these target cells. These results together indicate that TL-specific CTL populations primarily recognize epitopes expressed TAP independently.

Incidence of human papillomavirus 16 and 18 infection and p53 mutation in patients with oral squamous cell carcinoma in Japan.

The purpose of this study was to investigate the prevalence of human papillomaviruses (HPVs) 16 and 18 infection, and p53 mutation in oral squamous cell carcinomas (SCCs) in Japanese patients. Our results showed a higher incidence of HPV16 and 18 infections than previous studies because we combined the findings of a consensus polymerase chain reaction (PCR), restriction fragment length polymorphism by using the restriction enzyme digestion of the PCR products and Southern blot hybridization. Each HPV16 and 18 E6/E7 DNA was detected in 9 (20%) and 25 (54%) of 46 samples. The p53 mutation in the exons from 5 to 8 were detected in 20 out of 46 samples (43%) by a PCR-single strand conformation polymorphism analysis. There was a significant relationship between HPV16 and the p53 mutation (P =0.02) suggesting that HPV16 infection has a mutagenic effect in oral SCC. However, neither HPV infection nor p53 mutation influenced survival.

Role of mitochondrial DNA in radiation exposure.

PURPOSE: To evaluate the role of mitochondrial DNA (mtDNA) following exposure to ionizing irradiation. MATERIALS AND METHODS: We examined two human osteosarcoma cell lines either lacking mtDNA (143B.rho(0)206; rho0 cells) or having normal mtDNA (143B.TK-; rho+ cells). Cell survival curves were generated by using colony formation and micronucleus assay. The delay in population doubling time after irradiation was evaluated with dye exclusion tests. RESULTS: No significant difference was seen between rho+ and rho0 cell lines in colony formation assay. In micronucleus assay, rho0 cells showed a significantly lower rate of micronucleus formation. The ratios of binucleated cells with micronuclei were 0.49 for rho+ cells and 0.25 for rho0 cells (p=0.005). In the dye exclusion test, rho0 cells revealed a delay of about 1.6 times in population doubling time compared with the control after 5 Gy of irradiation, similar to the 1.7 times of rho+ cells. CONCLUSION: In the human osteosarcoma cell line 143B.TK-, mtDNA does not influence clonogenic survival and delay of population doubling time after irradiation. However, the difference in micronucleus formation shows that mtDNA influences DNA damage after radiation exposure.

A study of the association between the prognosis of carcinoma of the mandibular gingiva and the pattern of bone destruction on computed tomography.

OBJECTIVE: To clarify whether the pattern of bone destruction seen on CT is more closely associated with the outcome of carcinoma of the mandibular gingiva than that derived from panoramic radiographs (PR). METHOD: Axial bone window CT scans and PR of 62 patients with carcinoma of the mandibular gingiva were evaluated retrospectively by two oral radiologists for the pattern of bone destruction. Patterns were classified into the three types: erosive, invasive and mixed. The relationship between these patterns with each imaging modality and cumulative recurrence rate, cumulative metastasis rate and cumulative survival rate, calculated by the Kaplan-Meier method, were statistically analysed by the log rank test. RESULTS: The pattern of bone destruction derived from CT was closely associated with the cumulative metastasis rate (P < 0.05), the cumulative recurrence rate and the cumulative survival rate. In contrast, the pattern of bone destruction based on the PR was not associated with the cumulative metastasis rate (P = 0.43), the cumulative recurrence rate (P = 0.44), or the cumulative survival rate (P = 0.5). CONCLUSION: The prognosis of patients with carcinoma of the mandibular gingiva is more closely related to a classification derived from the pattern of bone destruction on CT rather than PR. However, the number of subjects investigated in this study was not large enough to confirm our conclusions statistically. Further studies by other investigators are therefore needed.

Detection of bone invasion by gingival carcinoma of the mandible: a comparison of intraoral and panoramic radiography and computed tomography.

OBJECTIVE: To assess the diagnostic accuracy of panoramic radiography (PR), panoramic radiography combined with intraoral radiography (PR+IR), and CT in detecting the supero-inferior extent of tumor invasion of the mandible by gingival carcinoma. METHOD: PR, PR+IR, and CT images of the mandible in 37 patients with gingival carcinoma were evaluated by five oral radiologists for the supero-inferior extent of bone invasion using ROC analysis. The mean ROC curve area (Az) of each observer for the different imaging modalities was analysed by nonparametric two-way ANOVA. P<0.05 was considered statistically significant. RESULTS: The mean Az for the detection of bone invasion were 0.88+/-0.03 for PR, 0.77+/-0.12 for PR+IR, and 0.87+/-0.03 for CT (P=0.0907). The mean Az for the detection of bone invasion beyond the alveolus was 0.89+/-0.07 for PR, 0.85+/-0.08 for PR+IR, and 0.83+/-0.06 for CT (P=0.5438). The mean Az for the detection of bone invasion beneath the mandibular canal were 0.94+/-0.04 for PR, 0.94+/-0.02 for PR+IR, and 0.91+/-0. 04 for CT (P=0.2466). No statistically significant differences were observed in Az between PR, PR+IR, and CT. CONCLUSION: We consider that PR+IR should be adopted as the initial imaging modality to determine the extent of supero-inferior invasion of the mandible in gingival carcinoma.

Value of contrast-enhanced magnetic resonance imaging in differentiation of hemangiomas from lymphangiomas in the oral and maxillofacial region.

OBJECTIVE: The purpose of this study was to investigate the ability of contrast-enhanced magnetic resonance imaging to differentiate hemangioma from lymphangioma in the oral and maxillofacial region. STUDY DESIGN: Contrast-enhanced magnetic resonance imaging was performed in 20 patients (21 masses: 17 hemangiomas and 4 lymphangiomas) through use of either a 0.2-T permanent system or a 0. 5-T superconductive system and spin-echo pulse sequences. RESULTS: After intravenous administration of contrast medium, enhancement was observed in all hemangiomas in areas corresponding to those with high signal on T(2)-weighted images. Unequivocally increased signal was observed in 10 masses, and slightly increased signal was observed in 7 masses. On the other hand, none of the lymphangiomas showed an enhancing mass on contrast-enhanced T(1)-weighted images. CONCLUSIONS: Although contrast-enhanced T(1)-weighted imaging may not improve delineation of masses in all cases, it can be used to differentiate between deep hemangiomas and lymphangiomas.

Prevalence of Epstein-Barr virus in oral squamous cell carcinoma.

Forty-six samples of oral squamous cell carcinoma (OSCC) were evaluated for the prevalence of Epstein-Barr virus (EBV) infection by the polymerase chain reaction (PCR), Southern blot hybridization, and in situ hybridization (ISH). EBV DNA was detected in 7 (15.2 per cent) out of 46 samples by a combination of PCR and Southern blot hybridization methods. All seven positive samples showed well-differentiated carcinoma, thus suggesting a possible relationship between EBV infection and the degree of differentiation of carcinoma tissue. Latent infection membrane protein 1 (LMP1) was detected immunohistochemically in six of the EBV-positive OSCCs. However, no signal of the EBV-encoded small RNA (EBER)-1 was demonstrated by the ISH method. No significant relationship was observed between EBV infection and lymph node metastasis. A follow-up study (range from 4.4 to 79 months; mean 34.9 months) showed no recurrence or death to occur in the EBV-positive patients, which thus suggested a good prognosis for EBV-positive OSCC patients.

Immunohistochemical and ultrastructural study of a papillary cystadenocarcinoma arising from the sublingual gland.

Immunohistochemical and ultrastructural findings in a rare case of papillary cystadenocarcinoma arising from the left sublingual gland of a 55-year-old Japanese man are reported. Histologically, the tumor tissue was found to be composed of various-sized cystic cavities in which papillary epithelial projections with thin fibrovascular cores were observed. The papillary projections consisted of a single layer to several layers of high columnar epithelial cells. Invasion to the surrounding fibrous tissue and into the lymphatics was observed, thus suggesting an aggressive potential in the present case. The possibility of the involvement of myoepithelial cells could be excluded based on the immunohistochemical and ultrastructural findings. The immunohistochemical and ultrastructural findings also suggested that this type of salivary gland tumor, at least the present case, may arise from striated or excretory ducts. There was positive immunostaining for tumor markers CA19-9 and CA125. However, the biological role of these carbohydrate antigens in salivary gland tumors is unclear at present. Further investigations are, therefore, called for to solve this issue.

Differences in the biological effects of in vitro irradiation by 65 MeV protons and 137cesium gamma-rays.

Using the irradiation system constructed for research in radiation biology, we have investigated the differences in the biological effects of in vitro irradiation by 65 MeV protons and by 137Cs gamma-rays. Survival curves were generated using V79 cells. The effects on biological parameters (SF2, RBE) of protons were greater than gamma-rays. Furthermore, 3H-thymidine incorporation in KOSC-3 cells, which display the p53 gene mutation, was inhibited by protons much more than by gamma-rays. On the other hand, in bleomycin-sensitive SCCKN cells, 3H-thymidine incorporation decreased more than in bleomycin-resistant SCCTF cells, however, both were inhibited by protons much more than by gamma-rays. In this study, the biological parameters and 3H-thymidine incorporation caused by 65 MeV protons were more severe than those caused by 137Cs gamma-rays.