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Thrombus migration is a well-known clinical condition that occurs before mechanical thrombectomy and after intravenous thrombolysis in patients with anterior circulation strokes. Although thrombus migration from the vertebral artery (VA) can result in life-threatening basilar artery (BA) occlusion, its occurrence in the posterior circulation has rarely been discussed. Two patients with secondary BA occlusion caused by spontaneous thrombus migration from the VA are presented. A 60-year-old man with a left cerebellar infarction secondary to ipsilateral VA occlusion was admitted to our hospital 8 hours after onset, with a National Institute of Health Stroke Scale (NIHSS) score of 4. The patient became comatose 3.5 hours after arrival owing to subsequent BA occlusion. He was successfully treated with mechanical thrombectomy. A 74-year-old man with right cerebellar infarction secondary to ipsilateral VA occlusion was admitted to our hospital 26 hours after onset, with an NIHSS score of 3. He became comatose 1 hour after arrival owing to BA occlusion and was treated with thrombectomy, followed by internal and external decompression. Despite the mild symptoms of VA occlusion and consequently delayed admission to the hospital, stroke physicians should be aware that spontaneous thrombus migration from the VA to the BA can result in a life-threatening presentation., (© 2024 The Authors. Published by Elsevier Inc. on behalf of University of Washington.)

Transparent tungsten trioxide thin films, which demonstrated visible-light (Vis-light)-induced superhydrophilicity, with thicknesses of 100-120 nm, adhesion strengths greater than 49 MPa, bandgap energies of 2.8-2.9 eV, and haze values of 0.4-0.5%, were fabricated using a solution-based process on quartz glass substrates. The precursor solution was prepared by dissolving a W 6+ complex salt isolated from a reacted solution of tungstic acid, citric acid, and dibutylamine in H 2 O, in ethanol. By heating the spin-coated films in air for 30 min at temperatures higher than 500 °C, crystallized WO 3 thin films were obtained. The O/W atomic ratio was evaluated to be 2.90, based on the peak area analysis of X-ray photoelectron spectroscopy spectra of the thin-film surfaces, indicating the co-presence of W 5+ ions. The water contact angle on film surfaces, which was approximately 25° prior to light irradiation, decreased to less than 10° upon irradiation with 0.06 mW cm -2 Vis-light for only 20 min at 20-25 °C and a relative humidity (RH) of 40-50%. By comparing the contact angle changes at RH values of 20-25%, it was revealed that the interaction between ambient water molecules and the partially O-deficient WO 3 thin films plays an important role in achieving photoinduced superhydrophilicity., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Spontaneous dissection of the brachiocephalic artery is rare, and there is insufficient evidence for optimal treatment. We herein report a case of ischemic stroke due to spontaneous dissection of the brachiocephalic to the right common carotid artery. The patient was treated medically but died suddenly 18 days after the onset because of aortic dissection. Although almost all reported cases of spontaneous dissection of the brachiocephalic artery have had good outcomes with medical management, it is important to note that sudden development of aortic dissection might occur, even without initial findings suggestive of this condition.

Objective: There are few reports on endovascular treatment of tandem lesions in the posterior circulation and no consensus on treatment strategies has been reached. We report a case of tandem lesions of basilar artery occlusion and vertebral artery stenosis treated by thrombectomy and vertebral artery stenting., Case Presentation: We present the case of a 73-year-old man who developed consciousness disorder and tetraplegia. Head and neck CTA revealed tandem left vertebral artery stenosis and basilar artery occlusion. The patient was treated using a reverse technique, which involves performing thrombectomy first and then vertebral artery stenting, along with Carotid Guardwire PS. Postoperative impairment of consciousness and improvement of tetraplegia were achieved., Conclusion: The reverse technique combined with Carotid Guardwire PS may be a useful treatment strategy for tandem lesions in the posterior circulation., Competing Interests: The authors declare no conflicts of interest., (©2022 The Japanese Society for Neuroendovascular Therapy.)

A single-walled carbon nanotube (SWCNT)-silica composite thin film on a quartz glass was formed by ultraviolet irradiation (20-40 °C) onto a spin-coated precursor film. With 7.4 mass% SWCNTs, the electrical resistivity reached 7.7 × 10 -3 Ω·cm after UV-irradiation. The transmittance was >80% at 178-2600 nm, and 79%-73% at 220-352 nm. Heat treatment increased the transparency and pencil hardness, without affecting the low electrical resistivity. Raman spectroscopy and microscopic analyses revealed the excellent film morphology with good SWCNT dispersal. The low refractive index (1.49) and haze value (<1.5%) are invaluable for transparent windows for novel optoelectronic devices.

A single-walled carbon nanotube/anatase (SWCNT/anatase) composite thin film with a transmittance of over 70% in the visible-light region was fabricated on a quartz glass substrate by heat treating a precursor film at 500 °C in air. The precursor film was formed by spin coating a mixed solution of the titania molecular precursor and well-dispersed SWCNTs (0.075 mass%) in ethanol. The anatase crystals and Ti 3+ ions in the composite thin films were determined by X-ray diffraction and X-ray photoelectron spectroscopy, respectively. The effect of the heating process on the SWCNTs was analyzed using Raman spectroscopy. The composite film showed an even surface with a scratch resistance of 4H pencil hardness, as observed using field-emission scanning electron microscopy and atomic force microscopy. The electrical resistivity and optical bandgap energy of the composite thin film with a thickness of 100 nm were 6.6 × 10 -2 Ω cm and 3.4 eV, respectively, when the SWCNT content in the composite thin film was 2.9 mass%. An anodic photocurrent density of 4.2 μA cm -2 was observed under ultraviolet light irradiation (16 mW cm -2 at 365 nm) onto the composite thin film, thus showing excellent properties as a photoelectrode without conductive substrates.

Deep-ultraviolet (DUV) light-transparent conductive composite thin films, consisting of dispersed multiwalled carbon nanotubes (MWCNTs) and SiO 2 matrix composites, were fabricated on a quartz glass substrate. Transparent and well-adhered amorphous thin films, with a thickness of 220 nm, were obtained by weak ultraviolet (UV) irradiation (4 mW cm -2 at 254 nm) for more than 6 h at 20-40 °C onto the precursor films, which were obtained by spin coating with a mixed solution of MWCNT in water and Si(IV) complex in ethanol. The electrical resistivity of MWCNT/SiO 2 composite thin film is 0.7 Ω·cm, and transmittance in the wavelength region from DUV to visible light is higher than 80%. The MWCNT/SiO 2 composite thin film showed scratch resistance at pencil hardness of 8H. Importantly, the resistivity of the MWCNT/SiO 2 composite thin film was maintained at the original level even after heat treatment at 500 °C for 1 h. It was observed that the heat treatment of the composite thin film improved durability against both aqueous solutions involving a strong acid (HCl) and a strong base (NaOH).

The molecular precursor method is an easy and simple method for coating thin carbonate-containing apatite (CA) films onto titanium surfaces. A molecular precursor solution containing ethanol, calcium-EDTA complex, and phosphate salt was dropped onto a titanium surface and then heated at 600°C for 2 h. An adherent thin CA coating was achieved. Animal implantation experiments showed that CA-coated implants had significantly higher bone-to-implant values than non-coated implants (p<0.05). The molecular precursor method was also used to coat three-dimensional titanium webs (TWs). Thin CA films could be coated inside the center area, as well as the surface of the TW, with excellent bone formation inside the CA-coated TW. Furthermore, the molecular precursor method was used to coat partially stabilized zirconia with CA. Better bone response was observed for CA-coated zirconia. From this, it is concluded that the molecular precursor method is useful for producing thin CA coatings on implant materials.

Aim: To verify the clinical utility of instrumental activities of daily life evaluated using the Tokyo Metropolitan Institute of Gerontology index of competence (TMIG-IC) as a screening tool for patients with early-phase cognitive impairment, including mild cognitive impairment (MCI) and early Alzheimer's disease (AD)., Methods: We recruited healthy subjects from our community-based cohort and consecutive subjects with MCI and AD from our clinic. The TMIG-IC was investigated in all participants and their family members. The total and subscale scores were compared among all groups. We then statistically determined the accuracy of the differentiation of MCI and AD., Results: We registered 187 normal controls (NC), 39 participants with MCI, 50 AD patients with functional assessment staging (FAST) 4, and 19 AD patients with ≥5 FAST. The family-report score was significantly lower in MCI patients than in others, followed by AD patients. The total score was able to differentiate MCI and AD with a sensitivity of 85.7% and a specificity of 90.9% (area under the curve [AUC]=0.913). Differentiation of MCI alone had a low accuracy (AUC=0.787). However, the AUC was 0.847 when only the items with inconsistent responses between self and family reports were used as indices., Conclusions: The TMIG-IC is a useful tool for evaluating the severity of AD, including early AD. These findings suggest that family-report scores can differentiate MCI and AD from cognitive normal aging with a sufficient degree of accuracy. It was also suggested that inconsistencies between self and family reports were higher when differentiating MCI than the self- and family-reports.

Background: Recent advances in high-resolution (HR) magnetic resonance angiography (MRA) using ultrahigh-field systems enable direct visualization of the lenticulostriate arteries (LSAs), which had been hardly achieved by conventional MRA. Hence, by using HR-MRA at 7 T, we attempted to assess occlusive changes in the LSAs in patients with LSA territorial infarcts., Methods: We prospectively examined 34 consecutive patients with acute ischemic stroke in the LSA territory using a 7 T scanner. We measured the lengths of the relevant LSAs on HR-MRA and the diameters/volume of the infarcts and compared these between the patients with/without occlusive changes in the LSAs., Results: On HR-MRA, occlusion of the LSAs was observed in 19 (59%) of 32 patients who were eligible for the analyses. The curved/straight lengths of the LSAs in the patients with LSA occlusion (23.1-31.1/17.8-24.3 mm) were significantly shorter than in those without apparent LSA occlusion (25.8-39.5/24.0-30.4 mm) ( P = 0.027/0.003). The anteroposterior/superoinferior diameters of the infarcts were significantly larger in the occluded-LSA group (14.5-21.4/14.9-22.2 mm) than in the intact-LSA group (10.9-16.8/10.8-16.2 mm) ( P = 0.041/0.011). In addition, the curved lengths of the relevant LSAs showed significant correlations with the superoinferior diameters of the infarcts ( r = 0.38, P = 0.034)., Conclusion: Occlusive changes in the LSAs were frequently found in patients with acute ischemic stroke within the LSA territory when using HR-MRA at 7 T and were substantially related to superoinferior extension of the infarcts.

Photo-induced super-hydrophilic thin films were fabricated on a quartz glass substrate by ultraviolet (UV) irradiation of a molecular precursor film at room temperature. A molecular precursor film exhibiting high solubility to both ethanol and water was obtained by spin-coating a solution involving a Ti(IV) complex; this complex was prepared by the reaction of Ti(IV) alkoxide with butylammonium hydrogen oxalate and hydrogen peroxide in ethanol. Transparent and well-adhered amorphous thin films of 160⁻170 nm thickness were obtained by weak UV irradiation (4 mW·cm -2 at 254 nm) of the precursor films for over 4 h at room temperature. The resultant thin films exhibiting low refractive indices of 1.78⁻1.79 were mechanically robust and water-insoluble. The chemical components of the thin films were examined by means of Fourier transform-infrared (FT-IR) and X-ray photoelectron spectroscopy (XPS) spectra, focusing on the presence of the original ligands. The super-hydrophilic properties (evaluated based on the water contact angles on the surfaces) of the thin films after being kept in a dark condition overnight emerged when the aforementioned UV-light irradiation was performed for 10 min. It was additionally clarified that the super-hydrophilicity can be photo-induced repeatedly by UV irradiation for 10 min (indicated by a contact angle smaller than 4°) even after the hydrophilic level of the thin films had once been lowered by being in a dark condition for 4 h.

Aim: Memorization comprises three stages: encoding, storage, and retrieval. Using neuropsychological tests, we investigated the stage at which encoding and storage are retained in Alzheimer's disease (AD) patients with progressive memory disorder., Methods: The target patients were an amnestic mild cognitive impairment (MCI) group (21 cases) and FAST 4 (37 cases), 5 (10 cases), and 6 (4 cases) AD groups. The neuropsychological tests performed were the Rivermead behavioral memory test and Wechsler memory scale-revised. These were carried out in the MCI group as well as in each AD stage group. We investigated the delayed recall (free recall and cued recall) based on the disease stage and raw score of the sub-items in delayed recognition., Results: The MCI group had 48% (median 0 point) correct respondents (providing ≥1 correct answer) for free recall, whereas FAST 4 and 5 groups had ≤14% correct respondents. In the verbal paired associates II evaluated in cued recall, the MCI group had 90% correct respondents, and the FAST 4, 5, and 6 groups had rates of 51%, 60%, and 50%, respectively. For the pictures and photos in the delayed recognition tasks, there were no significant differences in the percentage of correct respondents between the MCI group (100%) and the FAST 4 and 5 groups (70%-90%)., Conclusions: Given that retrieval is impossible if encoding and storage are impaired, we inferred that the encoding and retrieval abilities were retained even in moderately advanced AD.

The cervical carotid artery has been reported to show anatomical variations. We report the case of a young stroke patient with a small right-parietal-lobe infarction whose cervical carotid artery showed anatomical variation. The right internal carotid artery (ICA) originated at the C2 level of the external carotid artery with protrusion at the right carotid bifurcation. The vessel wall of the protrusion showed a high signal intensity on T1-weighted magnetic resonance carotid plaque imaging. The protrusion, considered a remnant of the ICA, possibly caused the stroke due to the formation of thrombi as a result of changes in blood flow and viscosity.

We performed thin carbonate-containing apatite (CA) coating on titanium (Ti) by an aqueous spray coating (ASC) method that consisted of a Ca-CO₃-PO₄ complex. Two different CA coatings were produced by two different spray amounts and were heat-treated after spraying. We evaluated three-dimensional structures, adhesiveness to Ti, and durability of the CA film. In addition, we performed immersion experiments in simulated body fluid (SBF), and bone responses were evaluated after implantation into a femoral bone defect in rats. The bonding ability of ASC-coated implant into the bone was examined by push-in tests. Unique network structures with small particles were identified on CA coatings. Although heat treatment produced no significant difference in surface morphology, scratch tests revealed that heat treatment improved the adhesion of CA coatings to Ti. Crystal formation progressed on CA-coated specimens, and the sample placement direction influenced crystal formation and growth in SBF immersion. Animal implantation experiments revealed significantly greater bone-to-implant contact ratio and bone mass in both cortical and bone marrow, respectively, four weeks after implantation. Push-in tests suggested that the bonding of the CA coating to Ti is clinically acceptable. Therefore, we conclude that CA coating to Ti by the ASC method would be possible for clinical applications, including dentistry.

Coating biomaterials with a thin hydroxyapatite (HA) was proven effective in enhancing bone compatibility. Segmental bone defects are considered as the most difficult defect to repair in bone regeneration therapy. We developed submicron-thin HA-coated titanium fiber mesh scaffolds to reconstruct immediately loaded segmental mandibular defects and evaluated their bone compatibility in vitro and in vivo. Human osteoblasts attachment, proliferation, and osteocalcin expression in non- and HA-coated scaffolds were evaluated. A 10-mm long segmental bone defect in a rabbit mandibular bone was reconstructed with non- or HA-coated scaffolds, which were removed at 9 and 21 weeks, to evaluate the mechanical strength of the bone-scaffold connection and the bone formation around the scaffold. Expression of osteocalcin was greater in HA-coated scaffolds. In vivo bone formation in HA-coated scaffolds was greater than that in non-coated scaffolds at 21 weeks. Newly formed bone in HA-coated scaffolds mostly restored bone continuity. Scanning electron microscopy identified strong integration of the bone and HA-coated scaffolds. The mechanical strength of the bone-scaffold connection was 3-fold greater in HA-coated scaffolds than that in non-coated scaffolds. These results suggest that a thin HA-coated titanium fiber mesh scaffold is a bone-compatible mandibular reconstruction device in immediately loaded segmental defects., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

In Asia-Pacific countries, as elsewhere, medical device expenditures account for only between 3% and 6% of total health care spending per capita. Yet the contribution of medical devices is essential to delivering improved clinical and economic benefits for patients and health care systems, respectively. We briefly report a selective overview of reimbursement coverage and pricing systems for single-use devices (SUDs) in Japan, Taiwan, Korea, and Australia, major Asia-Pacific markets with diverse health care systems but similar challenges. Generally speaking, all four health systems are similar in their framework for managing the coverage and reimbursement of SUDs, which are usually bundled into the overall fee designated for a surgical procedure; however, funding guidelines are unclear and decision-making processes opaque. Unfortunately, this inequitable situation encourages both the dangerous practice of reuse of devices and the imposition of additional out-of-pocket costs on patients. Reimbursement pathways in all four countries need to evolve to accommodate new methods of delivering health care, with fair decision-making processes for reimbursement coverage and pricing, which assess the overall value of medical devices, including SUDs, in terms of health outcomes and/or safety., (Copyright © 2015 International Society for Pharmacoeconomics and Outcomes Research (ISPOR). Published by Elsevier Inc. All rights reserved.)

Thin carbonate-containing hydroxyapatite (CA) films coating partially stabilized zirconia (Y-TZP) were prepared (CA-Y-TZP) to establish a metal-free implant system. CA was coated using a molecular precursor method. The CA film was deposited on the surface of Y-TZP using a precursor solution, which was a mixture of a calcium-ethylenediaminetetraacetic acid (EDTA) complex and phosphate compounds. The deposited CA film was characterized by X-ray diffraction, Fourier transform infrared spectroscopy, and energy dispersive X-ray spectroscopy measurements. A focus ion beam system technique revealed that the thickness of the CA film was less than 1.0 µm. Biological evaluations of CA-Y-TZP were performed by immersion experiments in simulated body fluid (SBF) and implantation experiments in the tibiae and femoral condyles of rabbits. In the SBF immersion experiment, apatite deposition progressed more on CA-Y-TZP at the early stage of immersion than on Y-TZP without the CA coating. Animal experiments revealed that bone formation on CA-Y-TZP was similar with than on Y-TZP. Histomorphometrical evaluations showed a significantly higher bone-to-implant contact ratio and bone mass on CA-Y-TZP after implantation into the femoral trabecular bone of rabbits. Therefore, CA-Y-TZP appears to be applicable as a metal-free implant., (© 2014 Wiley Periodicals, Inc.)

Four carbonated apatite films having average thicknesses of 1.3-0.11μm, proportions of network sizes above 10μm of 41-68%, and average border heights of the characteristic network structure of 0.98-0.29μm were fabricated on a titanium plate by aqueous spray coating. These carbonated apatite films after heat treatment showed good mineralization ability in Hanks' balanced salt solution. Assessment of initial cell attachment and calcination on these films and on the Ti plate using osteoblastic MC3T3-E1 indicated that the carbonated apatite film heat treated at 600°C, whose film thickness, proportion of network sizes above 10μm, and border height were 0.11μm, 61%, and 0.31μm, respectively, was most preferred by osteoblastic cells. Field emission scanning electron microscopic observation of the cells attached to the films showed that the wide network and low border height of the network structure on the carbonated apatite film play an important role in the development of the filopodia of the osteoblastic cells., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The influence of a thin carbonate-containing hydroxyapatite (CA) coating to tetragonal zirconia polycrystal (TZP) on osteoblastlike cell response was investigated. TZP disks were subjected to blasting and acid etching. Thin CA coatings were deposited by the molecular precursor method (TZP-CA). Initial cell adhesion of mouse osteoblast-like cells MC3T3-E1 was enhanced, and marked progress of actin filaments was observed on TZP-CA compared to on TZP. After 3, 5 or 7 days, cell proliferation on TZP-CA was significantly higher than that on TZP. Alkaline phosphatase activity was slightly lower on TZP-CA than on TZP at 7 days, and no difference was observed at 14 or 21 days. At 28 days incubation, collagenous fibers with mineral precipitants accompanied by phosphorous and amino groups were observed. These results indicate that thin CA coating with molecular precursor method offers promise as a means of enhancing cell response, particularly initial adhesion and proliferation of MC3T3-E1 cells.

Eighty-one patients with oral squamous cell carcinoma (OSCC) received oral fluoropyrimidine UFT and radiotherapy (RT) with or without an immunotherapeutic agent OK-432. Both overall survival and progression-free survival of patients who received RT + UFT + OK-432 were significantly longer than those of patients who received RT + UFT (P = .0075 and P = .0175, respectively). Clinical response was also more favorable in RT + UFT + OK-432 group than in RT + UFT group (P = .0066). Next, in vitro experiments were conducted to examine the effect of 5-fluorouracil (5-FU) and X-ray irradiation in OK-432-induced immunity. Human peripheral blood mononuclear cells stimulated with OK-432 produced helper T cell 1 (Th1)-type cytokines as well as interleukin-10 (IL-10) and transforming growth factor-β (TGF-β), which are produced by Th2 and regulatory T cells (Tregs), respectively, and are inhibitory in antitumor immunity. OK-432-induced IL-10 and TGF-β but not Th1 cytokines were significantly inhibited by 5-FU and/or X-ray. 5-FU and X-ray also inhibited the expression of mRNAs for GATA-3 and Foxp3, which are transcription factors for Th2 and Tregs, respectively, but not for T-bet, a transcription factor for Th1. In addition, 5-FU and X-ray decreased the expression of mRNAs for suppressor of cytokine signaling 1 (SOCS1) and SOCS3. Antisense oligonucleotides for SOCS1 and SOCS3 markedly reduced OK-432-induced IL-10 and TGF-β. This is the first report clearly demonstrating that OK-432-based immunotherapy significantly enhanced the therapeutic effects of chemoradiotherapy in patients with OSCC as well as elucidating the mechanism of the synergistic effect of immunochemoradiotherapy in which 5-FU and radiation enhanced OK-432-induced Th1 response mediated by the inhibition of SOCS1 and SOCS3 gene expression.

The fabrication and characterization of a carbonate-containing apatite film deposited on a Ti plate via an aqueous spray method is described. The mist of the spray solution emitted from a perpendicularly oriented airbrush was made to strike a warmed Ti substrate. The thicknesses of the sprayed film and those heat-treated at 400 °C-700 °C under Ar gas flow were in the range 1.21-1.40 μm. The results of elemental analyses and Fourier transform infrared spectroscopy of the powders that were mechanically collected from the surface of the sprayed film suggest that the film was Ca(10)(PO4)6(CO3) · 2CO2 · 3H2O. The presence of the carbonate ion and the lattice CO2 molecule was confirmed via the aforementioned analyses; the finding was also consistent with the X-ray diffraction patterns of the films and the chemical identity of the sprayed and heat-treated films that were measured using X-ray photoelectron spectroscopy. The sprayed film comprises a characteristic network structure, which contains round particles within the networks, as was observed by field-emission scanning electron microscopy. A scratch test indicated that the shear stress of the sprayed film (21 MPa) significantly improved to 40 and >133 MPa after heat-treatment at 600 °C and 700 °C, respectively, under Ar gas flow for 10 min., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Antitumor functions of the host immune system are frequently compromised in patients with malignancies. In the current study, we evaluated the relationship between expression ratio of mRNAs for the antiapoptotic protein Bcl-2 and the proapoptotic protein Bax (the Bcl-2/Bax ratio) in peripheral blood mononuclear cells and clinical outcomes in patients with head and neck carcinomas. The overall survival (OS) time of patients with Bcl-2/Bax ratios ≥ 1.2 tended to be longer than that of patients with Bcl-2/Bax ratios < 1.2 but not significantly so (P = .084, n = 61). Disease-free survival (DFS) of patients with Bcl-2/Bax ratios ≥ 1.2 was statistically significantly longer than that of patients with Bcl-2/Bax ratios < 1.2 (P = .001, n = 76). All of the patients whose Bcl-2/Bax ratio is ≥ 2.0 were alive after 36 months and survived without any evidence of disease for 24 months (Bcl-2/Bax ≥ 2.0 versus Bcl-2/Bax < 2.0; P = .035, n = 61 in OS, P < .001, n = 76 in DFS, respectively). In 56 patients who received immunochemoradiotherapy using UFT and OK-432 in combination with radiotherapy, a statistically significant relationship between the Bcl-2/Bax ratio and the therapeutic effect estimated using Response Evaluation Criteria in Solid Tumors was observed, as well as a relation with interferon-γ (IFN-γ) induction in response to the therapy [P = .002 in complete response versus partial response + stable disease; P = .046 in IFN-γ(+) versus IFN-γ(-)]. In addition, there were significant correlations of the Bcl-2/Bax ratio with both the absolute number of CD4(+) T cells and the rate of CD4(+) T cell and natural killer cell activity. These findings strongly suggest that the balance of expression of Bcl-2 and Bax genes in circulating immune cells has a high prognostic value in head and neck cancer patients.

This study investigated the bone regeneration properties of titanium fibre mesh as a tissue engineering material. A thin hydroxyapatite (HA) coating on the titanium fibre web was created using the developed molecular precursor method without losing the complex interior structure. HA-coated titanium fibre mesh showed apatite crystal formation in vitro in a human osteoblast culture. Titanium fibre mesh discs with or without a thin HA coating were implanted into rat cranial bone defects, and the animals were killed at 2 and 4 weeks. The in vivo experience revealed that the amount of newly formed bone was significantly higher in the HA-coated titanium fibre mesh than in the non-coated titanium fibre mesh 2 weeks after implantation. These results suggest that thin HA coating enhances osteoblast activity and bone regeneration in the titanium fibre mesh scaffold. Thin HA-coating improved the ability of titanium fibre mesh to act as a bone regeneration scaffold., (Copyright © 2012 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.)

Different approaches towards making 3-dimensional (3-D) bioengineered tooth for future replacement therapy have been developed including scaffold-based tooth regeneration. However, selection of optimal scaffold for future clinical application remains a challenge. In the present study, we tested biocompatibility of four different types of 3-D scaffolds for tooth-tissue regeneration, including a pure poly(lactide-co-glycolide) (PLGA) (70/30, mol/mol) scaffold and three types of calcium phosphate contained composites scaffolds that were 50 wt% of PLGA combined with 50 wt% of hydroxyapatite (HA), tricalcium phosphate (TCP) or calcium carbonate hydroxyapatite (CDHA) respectively. These scaffolds were fabricated by the particle leaching in combination with phase separation technology. Surface modification of these scaffolds was further performed by an ammonia plasma treatment and anchorage of collagen technology. Effect of composition of the composite scaffolds on proliferation of human dental pulp stem cells (DPSCs) was accessed using in vitro MTT assay and in vivo BrdU labeling. Differentiation capability of the DPSCs in the scaffolds was analyzed by measurement of the levels of calcified tissue formation and ALP activity. Our results showed that while the calcium phosphate contained compound is able to support regeneration of tooth tissue effectively, the PLGA/TCP scaffold is more appropriate for the proliferation and differentiation of DPSCs. Furthermore, seeding of dissociated 4-dpn rat tooth bud cells on the PLGA/TCP scaffold generated dentin- and pulp-like tissues. Our results demonstrate that the PLGA/TCP scaffold is superior to the other three scaffolds for tooth-tissue regeneration, especially for dentin formation., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Purpose: The aim of this study was to evaluate human osteoblast activity on thin hydroxyapatite (HA)-coated three-dimensional scaffolds made of titanium fiber web., Materials and Methods: A thin HA film was coated on a titanium fiber web by the molecular precursor method. Human osteoblasts were disseminated onto the uncoated and HA-coated titanium fiber web, and osteoblast activity was observed at days 3, 7, 14, and 21 of culture., Results: Proliferation activities of osteoblasts were significantly higher in the uncoated titanium fiber web. Osteoblasts in the uncoated titanium fiber web showed a typical expression pattern, but those in the HA-coated titanium fiber web showed rapid osteocalcin expression and calcification at an early stage of culture. Moreover, osteocalcin expression per osteoblast was significantly higher in the HA-coated group., Conclusion: These results suggest that HA coating with the molecular precursor method accelerated osteoblast functional activity.

The aim of this study was to develop in situ gellable hydrogels composed of periodate oxidized citrus pectin (OP) for localized anticancer drug delivery and evaluate the potential of OP to inhibit cancer metastasis. Doxorubicin (Dox) was coupled to OP by imine bonds. Adipic dihydrazide (ADH) was used for cross-linking of the Dox-OP conjugates. The Dox-OP conjugate solution gelled within 2 min after addition of ADH. The release rate of Dox from the hydrogels was controllable by an additive amount of ADH. The released Dox retained anticancer activity. OP was shown to have a potency to prevent homotypic cancer cell aggregation compared to unmodified citrus pectin, strongly suggesting that OP released from hydrogels in vivo will inhibit cancer metastasis. These results indicate that OP hydrogels have the potential to prevent progression of primary cancer by the released Dox and generation of metastatic cancer by the released OP.

The cortical bone response towards poly(lactide-co-glycolide) (70/30) (PLGA) (70/30)/apatite complex scaffolds with different levels of crystallinity was investigated. Apatite with different levels of crystallinity, Ca-deficient hydroxyapatite (CDHA), which has a low crystallinity, and a mixture of carbonated hydroxyapatite (CHA) and CDHA, which has a higher crystallinity, were prepared from an aqueous mixture of Ca-EDTA complex, H(2)O(2), H(3)PO(4), and NH(4)OH. Two porous PLGA(70/30)/apatite composite scaffolds, composite scaffold A (containing low crystallinity CDHA) and composite scaffold B (containing the higher crystallinity CHA/CDHA mixture), were prepared. Afterwards, pure porous PLGA and the two composite scaffolds were implanted into the cortical bone of rabbit tibiae for 12 weeks. High-resolution microfocus X-ray computed tomography and histological examinations revealed a better bone response for composite scaffold A compared with PLGA and composite scaffold B. For composite scaffold A, the original bone defect was almost filled with new bone. Quantitative analysis revealed that composite scaffold A produced a significantly greater amount of new bone. The present study demonstrated that the level of apatite crystallinity influences bone response. A PLGA/apatite porous composite with a low level of apatite crystallinity shows promise as a bone substitute or scaffold material for bone tissue engineering.

Apatite compounds with different levels of crystallinity were prepared using Ca-EDTA complexes. Ca-deficient hydroxyapatite (CDHA) with low crystallinity was synthesized by ultrasonic stirring of a mixture of Ca-EDTA complex, phosphoric acid, and ammonium hydroxide in hydrogen peroxide aqueous solution. Mixtures of carbonate hydroxyapatite (HA) and CDHA with higher crystallinity were also prepared from a solution involving the same complex. The porous composites with lower or higher crystallinity apatite with a copolymer of poly(L-lactide-co-glycilide)(70/30) (PLGA(70/30)) were fabricated by a solution-casting/particles leaching method. The apatites and porous composites were characterized, and it was found that the degradation of composites of apatite with a low level of crystallinity was fastest in phosphate-bufferd saline (PBS) solution compared with other apatite composites with higher levels of crystallinity; however, the rate was smaller than that of PLGA alone. Plasma treatment influenced the degradation of composites in PBS and apatite precipitation in simulated body fluid (SBF). Hydroxyapatite deposition on the PLGA composite with the low crystallinity occurred six times faster than that on PLGA alone after immersion in SBF. The incorporation of apatite into the PLGA matrix did not cause any adverse effects on cell attachment in an assay employing human gingival fibroblasts. This study suggested that the current apatite and PLGA porous composite will be a promising scaffold material for tissue engineering., ((c) 2008 Wiley Periodicals, Inc.)

The influence of thin carbonate-containing apatite (CA) coating on trabecular bone response to cylindrical titanium fiber mesh (porosity of 85%, pore size of 200-300 microm, 2.8 mm diameter x 6 mm length) implants was investigated. Thin CA coatings were deposited by the so-called molecular precursor method. Molecular precursor solution was obtained by adding dibutylammonium diphosphate salt to Ca-EDTA/amine ethanol solution by adjusting Ca/P = 1.67. Sintered cylindrical titanium fiber mesh was immersed into molecular precursor solution and then tempered at 600 degrees C for 2 h. The immersion and tempering process was repeated three times. An adherent thin CA film could be deposited on the inside of titanium fiber mesh. After the immersion of a CA-coated titanium fiber mesh in simulated body fluid, apatite crystals precipitated on the titanium fiber mesh. Uncoated and CA-coated titanium fiber mesh was inserted into the trabecular bone of the left and right femoral condyles of rabbits. Histological and histomorphometrical evaluation revealed a significantly greater amount of bone formation inside the porous area of the CA-coated titanium fiber mesh after 12 weeks of implantation. The present results suggested that a thin CA-coated titanium mesh has better osteoconductivity and will be useful for a three-dimensional scaffold.

The aim of this study was to investigate the efficacy and safety of an oral fluoropyrimidine anticancer agent, S-1, in patients with oral squamous cell carcinoma. Patients with pathologically confirmed squamous cell carcinoma and at least one measurable lesion were enrolled. Oral administration of S-1 (40 mg/m2 twice daily) for 28 days was followed by a 14-day rest period. A total of 41 consecutive eligible patients were enrolled in the study between October 2002 and August 2004. The sites of the primary tumor were the gingiva (n=18), the tongue (n=12), the palate (n=5), the oral floor (n=4), the buccal mucosa (n=1), and the labial mucosa (n=1). A median of two cycles of treatment (range, 1-5) was administered. A complete response was achieved in nine patients and a partial response in eight patients, for an overall response rate of 41.5% (95% confidence interval, 26.4-56.5%). The 3-year survival rate was 76.4% (95% confidence interval, 62.8-90.0%). Although grade 3 anemia and anorexia occurred in two patients each (4.9%), and grade 3 neutropenia, thrombocytopenia, nausea, vomiting, stomatitis, and diarrhea in one patient each (2.4%), no grade 4 toxicities were observed. S-1 exhibits definite antitumor activity in patients with oral squamous cell carcinoma and is well tolerated.

We have previously shown that a stromal cell-derived factor-1 (SDF-1; CXCL12)/CXCR4 system is involved in the establishment of lymph node metastasis, but not in that of distant metastasis, in oral squamous cell carcinoma (SCC). In this study, we investigated the role of the autocrine SDF-1/CXCR4 system, with a focus on distant metastasis in oral SCC cells. The immunohistochemical staining of SDF-1 and CXCR4 using primary oral SCCs and metastatic lymph nodes showed a significantly higher number of SDF-1-positive cases among the metastatic lymph nodes than among the primary oral SCCs, which was associated with a poor survival rate among those of the former group. The forced expression of SDF-1 in B88 cells, which exhibit functional CXCR4 and lymph node metastatic potential (i.e., the autocrine SDF-1/CXCR4 system), conferred enhanced cell motility and anchorage-independent growth potential onto the cells. Orthotopic inoculation of the transfectant into nude mice was associated with an increase in the number of metastatic lymph nodes and more aggressive metastatic foci in the lymph nodes. Furthermore, the SDF-1 transfectant (i.e., the autocrine SDF-1/CXCR4 system) exhibited dramatic metastasis to the lung after i.v. inoculation, whereas the mock transfectant (i.e., the paracrine SDF-1/CXCR4 system) did not. Under the present conditions, AMD3100, a CXCR4 antagonist, significantly inhibited the lung metastasis of the SDF-1 transfectant, ameliorated body weight loss, and improved the survival rate of tumor-bearing nude mice. These results suggested that, in cases of oral SCC, the paracrine SDF-1/CXCR4 system potentiates lymph node metastasis, but distant metastasis might require the autocrine SDF-1/CXCR4 system.

It has been reported that S-1 can exert antitumor effects on various human cancers including oral squamous cell carcinoma (OSCC). However, little is known about the detailed mechanisms of the antitumor activity of S-1. In the present study, we determined whether S-1 could suppress the angiogenesis and growth of human OSCC cells in vitro and in vivo. The S-1 component (5-FU plus CDHP) significantly suppressed the growth and migration of OSCC cells and BAEC, which inhibited tubule formation in HUVECs in vitro. Also, S-1 inhibited the nuclear factor-kappaB (NF-kappaB) activity in human OSCC cells in vitro. Moreover, S-1 inhibited the expression of survival signal, phosphorylated Akt (p-Akt), and of two major proangiogenic molecules, vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2), in cells implanted into the subcutaneous tissue of nude mice. The decreased expression of p-Akt, VEGF and FGF-2 correlated with decreased tumorigenicity and decreased vascularization of lesions in vivo. These findings suggest that S-1 can suppress the angiogenesis and growth of OSCC cells by inhibiting the expression of p-Akt, VEGF and FGF-2 involved in the blockade of Akt/NF-kappaB pathway.

Purpose: The influence of thin carbonate-containing apatite (CA) coating on the trabecular bone response to titanium implants was investigated., Materials and Methods: Thin CA coatings were deposited by a new method known as the molecular precursor method. Using a precursor solution composed of an EDTA-calcium complex, a CA film was deposited on the titanium surfaces. Uncoated and CA-coated titanium were placed in the trabecular bone of the left and right femoral condyles of 16 rabbits. After implantation periods of 2, 4, 8, and 12 weeks, the bone-implant interface was evaluated histologically and histomorphometrically., Results: Histologic evaluation revealed new bone formation around the uncoated and CA-coated implant surfaces after only 4 weeks of implantation. After 12 weeks, mature trabecular bone surrounded all implants. At 4 and 8 weeks of implantation, no difference existed in bone contact between uncoated and CA-coated implants. After 12 weeks of implantation, the CA-coated implant group showed a significantly higher percentage of bone contact than the uncoated implant group., Discussion and Conclusion: The present study demonstrated that thin CA coatings applied using the molecular precursor method showed greater bone-to-implant contact during the healing phase than uncoated controls. The results were similar to those observed with implants with calcium phosphate coatings deposited with a physical vapor deposition technique.

We have investigated the relationship between gene expression of Bcl 2 and Bax and the therapeutic effect in oral cancer patients. A significant correlation between Bcl-2/Bax gene expression ratio in the peripheral blood mononuclear cells (PBMCs) from the patients, and the therapeutic effect of radiation therapy in combination with UFT and OK-432, as well as survival rate, was observed. In addition, the statistically significant correlation was also observed between Bcl-2/Bax ratio and IFN-gamma and NK activity induced with OK-432. These findings suggest that Bcl-2 and Bax gene expression in PBMCs may be useful as a prognostic factor in oral cancer patients.

Epithelial-mesenchymal transition (EMT) refers to critical events occasionally observed during tumor progression, including invasion and metastasis, by which cancer cells acquire a fibroblast-like phenotype. Since the stromal cell-derived factor-1 (SDF-1)/CXCR4 system can facilitate lymph node metastasis in oral squamous cell carcinoma (SCC), we have explored the possibility that this system might be involved in EMT. Oral SCC cells, B88 and HNt, which have functional CXCR4 and lymph node metastatic potential, were found to lose their epithelial cell morphology due to SDF-1. In this context, the downregulation of epithelial markers, cytokeratin, E-cadherin and beta-catenin, and the upregulation of mesenchymal marker, vimentin and snail were detected. Furthermore, upregulation of vimentin by treatment with SDF-1 was impaired by phosphatidylinositol 3 kinase (PI3K) inhibitor Wortmannin, but not by mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor U0126. In the type I collagen embedding culture, SDF-1-treated B88 cells formed protruding extensions, but the effect was impaired by treatment with Wortmannin. These results suggested that EMT induced by the SDF-1/CXCR4 system might be involved in the lymph node metastasis of oral SCCs via activation of PI3K-Akt/PKB pathway.

Bacterial stimulation may serve to control atopic disorders such as atopic dermatitis (AD) through inducement of Th1 cell-mediated immune response. The lipoteichoic acid (LTA)-related molecule (okLTA) from streptococcal preparation, OK-432, has been shown to be a potent Th1 inducer through the action of IL-12. Examination was made of the therapeutic effects of this okLTA injected intra- and/or subcutaneously into AD-like lesions in NC/Nga mice, particularly in the vicinity of the suppressor of cytokine signaling (SOCS) regulatory pathways. Using immunohistochemical staining with IL-4/IL-12p40 and phosphorylated STAT6/p-STAT4 and RT-PCR for IL-4/IL-12p40, STAT6/STAT4 and mRNA expression and in situ hybridization of SOCS3 and 5, evaluation was made of the immunoregulatory effects of this okLTA in the treatment of spontaneous AD-like lesions in NC/Nga mice. Following the injection of okLTA, remarkable improvement in the lesions of NC/Nga mice was noted. In okLTA-treated skin, IL-12p40/p-STAT4 positive cellular infiltration was extensive while IL-4/p-STAT6 positive cell infiltration was seen to diminish considerably, compared to untreated NC mice. SOCS3 in situ expression in okLTA-treated mice was noted to be significantly less compared to untreated NC mice, in which the expression was prominent. SOCS5 in situ expression was rather, though not significantly, strong in okLTA-treated mice. okLTA treatment is clearly shown to induce Th1 cellular response and down-regulate immune response in the Th2 pathway through SOCS3 reduction in AD-like lesions of NC/Nga mice. The present results demonstrate that bacterial wall components such as okLTA should serve as an effective new therapeutic approach for treating AD.

Three C-17 diacetylenic compounds (1-3), one monoterpenoid (4), seven ceramides (leucoceramides A-G, 5a-g), six cerebrosides (leucocerebrosides A-F, 6a-f) and nine known compounds were isolated from the methanolic extract of Hydrocotyle leucocephala. Their structures were established by spectroscopic methods. The isolated compounds 1-3, 5a-g, 6a-f and 7 were shown to be active in the lipopolysaccharide (LPS) induced cytokine production assay for IL-10, IL-12, and TNF-alpha.

The purpose of this research was to evaluate the predictive value of expression of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), thymidine phosphorylase (TP), or orotate phosphoribosyltransferase (OPRT) genes for response to S-1. Twenty-five patients with oral squamous cell carcinoma (OSCC) received S-1 80 mg/m2/day. Pretreatment tumor biopsies were analyzed for TS, DPD, TP or OPRT mRNA expression by real-time reverse transcription-PCR. TS protein expression was evaluated by immunohistochemistry using a polyclonal TS antibody. Twenty-five patients were evaluable for response and gene expression. Six of the 25 (24%) achieved complete remission and 4 of the 25 (16%) had a partial response. Median TS/beta-actin was 2.51 (range 0.98-7.07). Median TS/beta-actin was 1.26 in responding patients and 3.43 in non-responders (P=0.0001). Ten of 11 patients with TS/beta-actin <1.80 and 0 of 15 with higher values responded (P<0.0001). Overall survival was 29.7 months in patients with TS/beta-actin <1.80 and 41.7 months in patients with higher values (P=0.0013). No correlations were seen between expression of DPD, TP or OPRT mRNA and response or survival. Weak TS staining was seen in 6 of 25 tumors evaluable for immunohistochemistry, including 5 responders. All 4 of the patients with both weak staining and TS/beta-actin <1.80 responded. High TS mRNA expression predicts non-response to S-1. On the other hand, high levels of DPD or TP mRNA and low levels of OPRT mRNA are not associated with S-1 resistance. TS mRNA expression is considered to be a useful prognostic factor in OSCC patients with S-1 single-agent therapy.

S-1 is a novel oral fluoropyrimidine inhibitory for dihydropyrimidine dehydrogenase (DPD). In the present study, we have examined the appropriate dose of S-1 in the combination with radiation and the safety and clinical efficacy. Radiation was given (2 Gy/day; 5 days/week) for a total of 60 Gy. S-1 was given orally every day for 2 weeks and then S-1 was stopped for 1 week. The levels were divided accordingly to the S-1 application as follows; level 0, 50 mg/m2/day; level 1, 65 mg/m2/day; level 2, 80 mg/m2/day. grade 3 toxicity of anorexia and the grade 3 toxicity increase in bilirubin level were observed in 2 cases of level 2. We decided that level 1 (65 mg/m2/day) was the recommended dose of the S-1 application as observed in compliance and efficacy. This therapy is a useful concurrent chemo-radiotherapy which may improve the response rate and quality of life (QOL) of patients with oral squamous cell carcinoma.

We examined the role of nitric oxide (NO) induced by OK-432, a streptococcal immunotherapeutic agent, in anti-tumor effects of the OK-432 by in vitro and in vivo experiments using an NO synthase inhibitor, N-monomethyl-l-arginine acetate (NMA). The in vitro treatment of mouse splenocytes with OK-432 increased the expression of inducible NO synthase (iNOS) gene and NO production in a dose-dependent manner. Although it is well known that OK-432 induces cytokines such as interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha, both of which are known to be potent NO inducers, we observed only a partial reduction of OK-432-induced NO production with the addition of anti-IFN-gamma and/or anti-TNF-alpha neutralizing antibodies. The cytotoxicity of the splenocytes increased by in vitro OK-432 stimulation was almost completely inhibited by the treatment with NMA. OK-432 administration resulted in a marked prolongation of survival and a significant inhibition of tumor growth in syngeneic tumor-bearing mice, whereas NMA significantly inhibited the anti-tumor effects of OK-432. Although the increased cytotoxicity of adherent splenocytes derived from OK-432-treated tumor-bearing mice was almost completely inhibited by NMA, only partial inhibition by NMA was observed in the cytotoxicity of the nonadherent splenocytes. These findings strongly suggest that the iNOS/NO induced by OK-432 is intimately involved in the anti-tumor effects of OK-432.

Objective: Our previous study suggested that suppression by cepharanthin of tumor necrosis factor-a (TNF-a)-induced matrix metalloproteinase-9 (MMP-9) could prevent destruction of the acinar structure in the salivary glands of patients with Sjögren's syndrome (SS). In this study, we observed that in vivo administration of cepharanthin prevented severe damage to acinar tissues in the murine model of human SS., Methods: Cepharanthin was intraperitoneally administered to thymectomized female NFS/sld mice. Inflammatory lesions in the salivary and lacrimal glands were then examined histologically. Expression of phosphorylated IkB-a, MMP-9, and type IV collagen was analyzed immunohistochemically. The apoptotic cell death of acinar cells was determined., Results: Although extensive mononuclear cell infiltration and destruction of acinar tissue in salivary and lacrimal glands were observed in control mice, significant improvement of these lesions was evident in mice treated with cepharanthin. Immunohistochemical analysis revealed that p65, phosphorylated IkB-a, and MMP-9 were more strongly stained in the acinar cells of control mice than in cepharanthin-treated mice. Although no staining for type IV collagen was observed in the acinar tissues of control mice, continuity of staining for type IV collagen was observed in acinar tissues of cepharanthin-treated mice. Destruction of acinar tissues was attributed to the induction of apoptosis, suggesting that cepharanthin inhibits apoptosis by suppressing phosphorylation of IkB-a, followed by prevention of MMP-9 activation., Conclusion: Our findings suggest that cepharanthin may be a promising agent for use in preventing destruction of acinar tissues in murine SS.

This study was designed to disclose detailed genetic mechanisms in salivary gland tumors (SGTs) for development of novel independent marker. We constructed an in-house cDNA microarray carrying 2,201 cDNA clones derived from SGT and oral squamous cell carcinoma cDNA libraries. Four cell lines that originated from the SGT-derived cell lines were analyzed using this microarray system. The genes identified by our microarray system were further analyzed at the mRNA or protein expression level in other types of human cancer cell lines and clinical samples (ten normal salivary glands [NSGs], eleven pleomorphic adenomas, ten adenoid cystic carcinomas and three adenocarcinomas). Two up-regulated genes and six down-regulated genes were identified in common when compared with the control RNA. Of the up-regulated genes, WISP-2, which plays an important role in breast carcinogenesis, was selected for further analyses. We found a higher expression of the WISP-2 gene in the SGT-derived cell lines compared with other types of human cancer cell lines. Furthermore, WISP-2 mRNA and protein expression levels in NSGs were significantly higher than those in SGTs. These results suggest that WISP-2 could be a reliable independent marker and that down-regulation or loss of the WISP-2 gene may be associated with the development of SGTs.

OK-432 is a Streptococcus-derived immunotherapeutic agent for malignancies. Our group has tried to identify the effective components of OK-432 and has succeeded in isolating a lipoteichoic acid-related preparation designated as OK-PSA, which is a strong inducer of T helper 1 (T(H)1) cells, and elicits an anticancer effect via Toll-like receptor (TLR) 4. Conversely, bacterial DNA with unmethylated CpG motifs can stimulate a T(H)1-type host response via TLR9. The unmethylated CpG DNA contained in OK-432 may play a role in its anticancer effect. In the current study, we investigated the effect of OK-432-derived DNA (OK-DNA) in augmenting the anticancer immune response. Analysis of OK-DNA with the restriction enzymes Hpa II and MspI revealed that OK-DNA contained unmethylated CpG motifs. OK-DNA induced TH1-type cytokines such as interferon-gamma, tumor necrosis factor-alpha, interleukin (IL)-12, and IL-18 and augmented killer cell activities in vitro on human peripheral blood mononuclear cells, whereas the methylated OK-DNA did not. Cytokines were also produced by OK-DNA-stimulated splenocytes derived from wild-type mice but not from TLR9-deficient mice. In the in vivo study, peritumoral administration of OK-DNA resulted in a significant inhibition of tumor growth in syngeneic tumor-bearing wild-type and TLR4-deficient mice but not in TLR9-deficient mice. The antitumor effect of OK-432 in TLR9-deficient mice was significantly but partially reduced compared with that in wild-type mice, whereas the effect of OK-432 was almost completely eliminated in TLR4-deficient mice. These findings suggest that unmethylated CpG DNA in OK-432 functions as an active component in OK-432-induced anticancer immunity via TLR9.

Low expression of p27Kip1 is associated with disease progression and an unfavorable outcome in several malignancies, including oral squamous cell carcinoma (OSCC). In addition, the p27Kip1 protein is thought to be degraded by the Jun activation domain-binding protein 1 (Jab1). The purpose of this study was to examine whether Jab1 expression can be a useful prognostic factor in OSCC patients treated by 1 M Tegafur and 4 M uracil (UFT) in combination with radiation. Jab1 expression was investigated by immunohistochemistry in biopsy samples from 102 OSCC patients who were treated by UFT in combination with radiation. The associations of each expression with the clinicopathological characteristics and patient survival were also analyzed. A significant association was found between Jab1 expression and cervical lymph node metastasis (p=0.0004), stage of disease (p=0.0011), therapeutic effect (p=0.0133) and patient outcome (p=0.0095). The 5-year survival rates of Jab1 high- and low-expression tumors were 53.0% and 80.6%, respectively and this difference was significant (p=0.0053) by the log-rank test. Multivariate analysis revealed that reduced term survival was related to high levels of Jab1 expression (p=0.0082). These results suggest that Jab1 may be a useful prognostic factor in OSCC patients treated by UFT in combination with radiation.

The human salivary glands have a variety of histologic features such as intercalated duct cells, myoepithelial cells and acinar cells. A neoplastic human salivary intercalated duct cell line (HSG) and its derivatives, HSG with a myoepithelial cell phenotype (HSG-AZA1) and HSG with an acinar cell phenotype (HSG-AZA3) induced by 5-aza-2'-dC treatment of HSG cells, have been reported. To identify characterization of intercalated duct cells, myoepithelial cells and acinar cells in the salivary gland, we selected HSG, HSG-AZA1 and HSG-AZA3 cell lines to perform two-dimensional electrophoresis analysis. We used a fluorescent two-dimensional differential in-gel electrophoresis (2-D-DIGE) for comparative proteomics, which improved the reproducibility and reliability of differential protein expression analysis between the samples. Furthermore, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) peptide mass fingerprinting (PMF) was used to identify the proteins. These methods were combined to approach the protein profiles associated with characterization between HSG, HSG-AZA1 and HSG-AZA3 cells. Using these strategies, we identified seven HSG associated proteins, such as actin-beta, hydrocephalus inducing protein, L-plastin, KIAA0657 protein, septin 6 isoform A, lamin A/C isoform 2 and superoxide dismutase 2, three HSG-AZA1 associated proteins such as ubiquitin carboxyl-terminal esterase L1, myosin light chain 2 and muscle creatine kinase, and two HSG-AZA3 associated proteins, microtubule-associated protein 6 and Annexin A3. These results suggest that the proteins are associated with characterization of the salivary gland.

It has previously been reported by our group that Toll-like receptor (TLR) 4 is involved in anticancer immunity induced by OK-432, a Streptococcus-derived immunotherapeutic agent. However the detailed mechanism of the OK-432-induced immune response via TLR4 remained uncertain, because it may not be possible for OK-432, which consists of whole bacterial bodies, to bind directly to TLR4. In the current study, we conducted in vitro and in vivo experiments to investigate the hypothesis that OK-432 may first be captured and dissolved by phagocytes and that the active components released by the cells may then induce host responses via TLR4. TS-2 monoclonal antibody, which recognizes an active component of OK-432 designated OK-PSA was used in the current study. First, it was observed that OK-432-induced cytokine production by dendritic cells (DCs) and macrophages was significantly inhibited in vitro by cytochalasin B, a phagocytosis inhibitor. Immunofluorescence staining using TS-2 demonstrated that OK-432 was captured and dissolved by phagocytes. OK-PSA was detected in the supernatants derived from OK-432-treated DC culture by enzyme-linked immunosorbent assay using TS-2. Supernatants from OK-432-treated DC culture increased nuclear factor (NF)-kappaB activity in TLR4-expressing cells, and the increased activity was inhibited by TS-2 antibody. OK-432 itself did not activate NF-kappaB in these cells. In in vivo experiments, the anticancer effect of OK-432 was significantly inhibited by suppression of phagocytosis activity by cytochalasin B. In this case, the amount of OK-PSA, an active component of OK-432, in the sera was also reduced by cytochalasin B. These findings elucidated the mechanism mediated by phagocytosis and TLR4 signaling in the immune effect of OK-432.

Vesnarinone is a synthesized positive oral inotropic agent that has multiple biological activities on mammalian cells both in vitro and in vivo. This agent has been reported in relation to its antitumor effect with apoptosis-inducing activity. In the present study, we determined whether vesnarinone could suppress angiogenesis and growth of human oral squamous cell carcinoma cells in vitro and in vivo. Vesnarinone significantly inhibited the in vitro and in vivo expression of two major proangiogenic molecules, vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8), in cultured cells and in cells implanted into the subcutaneous tissue of nude mice. Also, vesnarinone inhibited the nuclear factor-kappa B (NF-kappaB) activity in human oral squamous cell carcinoma cells in vitro. The decreased expression of VEGF and IL-8 correlated with decreased tumorigenicity and decreased vascularization of lesions in vivo. These findings suggest that vesnarinone can suppress the angiogenesis and growth of oral squamous cell carcinoma cells by inhibiting the expression of VEGF and IL-8 involved in blockade of NF-kappaB activity.