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Combining the electrophoresis and conventional Coulter methods, we previously proposed the electrophoretic Coulter method (ECM), enabling simultaneous analysis of the size, number, and zeta potential of individual specimens. We validated the ECM experimentally using standard polystyrene particles and red blood cells (RBCs) from sheep; the latter was the first ECM application to biological particles in biotechnology research. However, specimens are prevented from passing through the ECM module aperture, which prevents accurate determination of the zeta potential of each specimen. This problem is caused by electro-osmotic flow (EOF) due to the high zeta potential at the ECM microchannel surfaces. To significantly improve ECM feasibility for biomedicine, we here propose a method to estimate the zeta potential at the ECM microchannel surfaces separate from the zeta potential of each specimen, by investigating the electric-field dependence of the specimen's experimental electrophoretic velocity. We minimize the zeta potential at the microchannel surfaces by applying an organic-molecule coating, and we suppress the surface zeta potential and its resultant EOF by optimizing the microchannel geometry. We demonstrate that the ECM can distinguish between different biological cells using the differences in zeta potential values and/or sizes. We also demonstrate that the ECM can determine the number of biomolecules attached to individual cells and identify whether the average cell state in an analyzed vial is alive or dead. The high-performance ECM can detect cellular morphology alterations, improve immunologic test sensitivity, and identify cell states (living, dying, and dead); this information is clinically useful for early diagnosis and its follow-up.

Purpose: We compared the results of prospective and retrospective cohort studies in the field of digestive surgery to clarify whether the results of prospective cohort studies were more similar to those of randomized controlled trials (RCTs)., Methods: We conducted a secondary analysis of the results to compare the results of RCTs with those of cohort studies in meta-analyses of 18 digestive surgical topics. The data from the prospective and retrospective cohort studies were combined. The summary estimates of each design were compared with those of RCTs. We used the Z score to investigate discrepancies., Results: Twenty-nine outcomes of 11 topics were investigated in 289 cohort studies (prospective, n = 69; retrospective, n = 220). These were compared with the outcomes of 123 RCTs. In comparison to retrospective studies, the summary estimates of the prospective cohort studies were more similar to those of the RCTs [19/29 (prospective) vs. 10/29 (retrospective), P = 0.035). Five of the 29 outcomes of prospective studies and 6 of 29 outcomes of retrospective studies (P = 0.99) showed significant discrepancies in comparison to RCTs., Conclusions: In the digestive surgical field, the results of prospective cohort studies tended to be more similar to those of RCTs than retrospective studies; however, there were no significant discrepancies between the two types of cohort study.

Magnetic nanoparticles are of great importance particularly in the field of biomedicine as well as nanotechnology and nano materials science and technology. Here, we synthesise magnetic alloy-filling carbon nanoparticles (MA@C NPs) via the following two-step procedure; (1) Irradiation of a laser beam of 266 nm wavelength into super-critical benzene, in which both ferrocene and cobaltocene are dissolved, at 290 °C; and (2) annealing of the particles at 600 and 800 °C. We find that the core particles are composed of cobalt (Co), iron (Fe) and oxygen (O) and covered with carbon layers. The structure of the core particles as-synthesised, and annealed at 600 and 800 °C, is, respectively, amorphous, CoFe 2 O 4 and FeCo. We also investigate the viability of L929 cells in the presence of MA@C NPs and find that there is no serious advert effect of the MA@C NPs on the cell viability thanks to the carbon layers covering the core particles. The magnetic properties are well characterised. The saturation and remnant magnetisation and coercivity increase and as a result, the hyperthermic efficiency becomes higher with an increase in the annealing temperature. The further modification of the surface of the present particles with several functional molecules becomes easier due to the carbon layers, which makes the present particles more valuable. It is therefore supposed that the presently synthesised MA@C NPs may well be utilised for nanotechnology-based biomedical engineering; e.g., nano bioimaging, nano hyperthermia and nano surgery.

Dental calculus is a mineralized deposit associated with periodontitis. The bacterial components contained in dental calculus can be recognized by host immune sensors, such as Toll-like receptors (TLRs), and induce transcription of proinflammatory cytokines, such as IL-1β. Studies have shown that cellular uptake of crystalline particles may trigger NLRP3 inflammasome activation, leading to the cleavage of the IL-1β precursor to its mature form. Phagocytosis of dental calculus in the periodontal pocket may therefore lead to the secretion of IL-1β, promoting inflammatory responses in periodontal tissues. However, the capacity of dental calculus to induce IL-1β secretion in human phagocytes has not been explored. To study this, we stimulated human polymorphonuclear leukocytes (PMNs) and peripheral blood mononuclear cells (PBMCs) with dental calculus collected from periodontitis patients, and measured IL-1β secretion by ELISA. We found that calculus induced IL-1β secretion in both human PMNs and PBMCs. Calculus also induced IL-1β in macrophages from wild-type mice, but not in macrophages from NLRP3- and ASC-deficient mice, indicating the involvement of NLRP3 and ASC. IL-1β induction was inhibited by polymyxin B, suggesting that LPS is one of the components of calculus that induces pro-IL-1β transcription. To analyze the effect of the inorganic structure, we baked calculus at 250°C for 1 h. This baked calculus failed to induce pro-IL-1β transcription. However, it did induce IL-1β secretion in lipid A-primed cells, indicating that the crystalline structure of calculus induces inflammasome activation. Furthermore, hydroxyapatite crystals, a component of dental calculus, induced IL-1β in mouse macrophages, and baked calculus induced IL-1β in lipid A-primed human PMNs and PBMCs. These results indicate that dental calculus stimulates IL-1β secretion via NLRP3 inflammasome in human and mouse phagocytes, and that the crystalline structure has a partial role in the activation of NLRP3 inflammasome., Competing Interests: The authors have declared that no competing interests exist.

Background: In surgical trials, complex variables such as equipment development and surgeons' learning curve are involved. The evidence obtained in these trials can thus fluctuate over time. We explored the stability of the evidence obtained during surgery by conducting a cumulative meta-analysis of randomized controlled trials for open and laparoscopic appendectomy., Methods: We conducted a cumulative meta-analysis of randomized controlled trials comparing laparoscopic appendectomy with open appendectomy for acute appendicitis, a topic with the greatest number of trials in the gastroenterological surgical field. We searched the MEDLINE (PubMed), EMBASE, and CINAHL databases up to September 2014 and reviewed the bibliographies. Outcomes were the incidence of intra-abdominal abscess, incidence of wound infection, operative time, and length of hospital stay. We used the 95 % confidence interval (95 % CI) of effect size for the significance test., Results: Sixty-four trials were included in this analysis. Of the 51 trials addressing intra-abdominal abscesses, our cumulative meta-analysis of trials published up to and including 2001 demonstrated statistical significance in favor of open appendectomy (cumulative odds ratio [OR] 2.35, 95 % CI 1.30-4.25). The effect size in favor of open procedures began to disappear after 2001, leading to an insignificant result with an overall cumulative OR of 1.32 (95 % CI 0.84-2.10) when laparoscopic appendectomy was compared with open appendectomy., Conclusions: The evidence regarding treatment effectiveness changed over time, after treatment effectiveness became significant in trials comparing laparoscopic and open appendectomy. Observing only the 95 % confidence interval of effect size from a meta-analysis may not provide conclusive results.

Background: T2 mapping is an MRI method particularly reflective of the collagen arrangement in the cartilage, and diffusion tensor (DT) imaging captures the diffusion of water molecules. Laser-induced photoacoustic measurement (LIPA) makes it possible to assess not only the thickness of the cartilage layer but also its viscoelastic properties. By assessing cartilage damage assessment using LIPA and 3.0 Tesla MRI (T2 mapping and DT imaging), this study investigates the usefulness of the various methods., Methods: The International Cartilage Repair Society (ICRS) classification was used to classify 29 bone cartilage pieces excised during surgical procedures. At the same time, LIPA was performed at sites matching the area of cartilage damage. MRI was performed preoperatively to measure the T2 and the apparent diffusion coefficient. In addition, tissue sections for histological assessment using the Mankin score were prepared for each ICRS grade, and the results with the various methods were compared., Results: With DT imaging, significant differences were observed in all grades (P < 0.01). With T2 mapping, significant differences were observed in all grades except for grade 1 versus grade 2 (P < 0.01). With LIPA, significant differences were observed in ICRS grade 1 versus grade 3 (P < 0.05), grade 1 versus grade 4 (P < 0.01), grade 2 versus grade 4 (P < 0.01), and grade 3 versus grade 4 (P < 0.05). With the Mankin score, significant differences were observed in ICRS grade 1 versus grade 3 (P < 0.01), grade 1 versus grade 4 (P < 0.01), grade 2 versus grade 4 (P < 0.01), and grade 3 versus grade 4 (P < 0.01). Correlations were observed in all combinations of ICRS grade with DT imaging, T2 mapping, LIPA, and Mankin score. Correlations were observed between the degree of histological degeneration and DT imaging, T2 mapping, and ICRS grade, but LIPA had a weaker correlation than MRI., Conclusions: In the assessment of knee osteoarthritis, there are instances where it is difficult to assess the damaged cartilage site with MRI alone, and we believe that it is desirable to use a combination of LIPA and MRI.

Background: Osteoclasts differentiated from bone marrow macrophages (BMMs) induced by TNF-α alone do not have resorbing activity. When BMMs are stimulated with receptor activator of NF-κB ligand (RANKL) before TNF-α stimulation, pit-forming osteoclasts are differentiated. However, the details of the effect of RANKL pretreatment on the pit-forming osteoclast differentiation by TNF-α have not been established. The aim of this study is to examine the condition of RANKL pretreatment for differentiation of pit-forming osteoclasts induced by TNF-α. Murine BMMs were stimulated with various concentrations of RANKL for 24h in the presence of M-CSF, then the medium was changed and TNF-α was added. Osteoclasts and pits formation were examined. Osteoprotegerin (OPG), decoy receptor of RANKL, was added to the culture to examine the necessity of co-existing RANKL with TNF-α on the formation of pit-forming osteoclasts. To investigate the influence of RANKL of sufficient concentration as pretreatment for pit-forming osteoclast formation by TNF-α, dose- and time-dependent changes of osteoclast formation were checked., Results: The pit formation by osteoclasts in response to TNF-α required 10ng/mL RANKL pretreatment. Stimulation with this concentration of RANKL led to the differentiation of mature osteoclasts in the 72h culture. The pit formation was not inhibited by the OPG., Conclusion: These results suggested that the concentration of RANKL pretreatment, which also alone can differentiate BMMs into osteoclasts, may be important in the differentiation of pit-forming osteoclasts by TNF-α. In addition, the effects of TNF-α after RANKL treatment might be independent of RANKL., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: The effectiveness of prophylactic antibiotics use for acute necrotizing pancreatitis has been explored and a number of systematic reviews have been published with conflicting results. The timing of antibiotics administration can be fundamental to their effectiveness, but thus far no reviews have focused on the timing of administration., Methods: A systematic review of randomized controlled trials (RCTs) of prophylactic antibiotics for acute necrotizing pancreatitis was conducted using MEDLINE (PubMed), CINAHL and Japana Centra Revuo Medicina. Trials in which antibiotics were administered within 72 h after onset of symptoms or 48 h after admission were included. Our primary outcomes were the mortality rate and the incidence of infected pancreatic necrosis, and secondary outcomes were the incidence of non-pancreatic infection and the incidence of surgical intervention., Results: The search revealed six RCTs with a total of 397 patients. The mortality rates were significantly different for those taking antibiotics (7.4%), and controls (14.4%) (odds ratio [OR], 0.48; 95% confidence interval [CI], 0.25-0.94). Also, early prophylactic antibiotics use was associated with reduced incidence of infected pancreatic necrosis (antibiotics 16.3%, controls 25.1%; OR, 0.55; 95% CI, 0.33-0.92)., Conclusion: Early use of prophylactic antibiotics for acute necrotizing pancreatitis is associated with reduced mortality and lower incidence of infected pancreatic necrosis., (© 2015 Japanese Society of Hepato-Biliary-Pancreatic Surgery.)

Background: In the present study, we measured damaged areas of cartilage with diffusion tensor (DT) imaging and T2 mapping, and investigated the extent to which cartilage damage could be determined using these techniques., Methods: Forty-one patients underwent arthroscopic knee surgery for osteoarthritis of the knee, a meniscus injury, or an anterior cruciate ligament injury. Preoperative magnetic resonance imaging of the knee was performed, including T2 mapping and diffusion tensor imaging. The presence of cartilage injury involving the medial and lateral femoral condyles and tibia plateau was assessed during surgery using the Outerbridge scale. The ADC, T2 values and fractional anisotropy of areas of cartilage injury were then retrospectively analysed., Results: The ADC results identified significant differences between Outerbridge grades 0 and 2 (P = 0.041); 0 and 3 (P < 0.001); 1 and 2 (P = 0.045); 1 and 3 (P < 0.001); and 2 and 3 (P = 0.028). The FA results identified significant differences between grades 0 and 1 (P < 0.001); 0 and 2 (P < 0.001); and 0 and 3 (P < 0.001). T2 mapping identified significant differences between Outerbridge grades 0 and 2 (P = 0.032); 0 and 3 (P < 0.001); 1 and 3 (P < 0.001); and 2 and 3 (P < 0.001). Both the T2 mapping (R(2) = 0.7883) and the ADC (R(2) = 0.9184) correlated significantly with the Outerbridge grade. The FA (R(2) = 0.6616) correlated slightly with the Outerbridge grade., Conclusions: T2 mapping can be useful for detecting moderate or severe cartilage damage, and the ADC can be used to detect early stage cartilage damage. The FA can also distinguish normal from damaged cartilage.

Background and Aim: The aim of this study was to evaluate the mid-term outcomes of a strategy for repair of coarctation of the aorta (CoA) and hypoplastic aortic arch (HAA) with a modified, extended end-to-end repair that preserves the lesser curvature of the aortic arch in neonates with intracardiac defects., Methods: We studied 21 neonates who underwent CoA repair and remote intracardiac repair (2000-2013). Fifteen patients had HAA, and six patients had no HAA. Follow-up ranged from 0.4 to 12.8 years (median, 7.5 years), and all patients underwent cardiac catheterization and blood pressure measurement in both the arms and legs., Results: The overall median age at the time of CoA repair was seven days and the median age at the time of intracardiac defect repair was 18.6 months. There were no hospital deaths and one case had recoarctation (4.8%). The overall mean pressure gradient at the latest follow-up was 3.4 ± 5.7 mmHg. Critical deformation of arch geometry was not found. No patient had hypertension or an abnormal arm-leg gradient. There was no difference in the cardiac catheterization data or blood pressure between patients with and without HAA., Conclusions: A modified, extended end-to-end repair for CoA and HAA resulted in a low rate of recoarctation, no operative mortality, maintenance of a smooth rounded arch, and normal blood pressures in the arms and legs during mid-term follow-up. These results suggest that this technique may be acceptable for repair of CoA and HAA in neonates with intracardiac defects., (© 2014 Wiley Periodicals, Inc.)

A 3-year-old boy with pulmonary atresia with ventricular septal defect, who had undergone placement of a modified Blalock-Taussig shunt, presented with a 1-week history of high fever. Computed tomography showed a pseudoaneurysm at the anastomosis between the right brachiocephalic artery and the graft. After intravenous antibiotic therapy, the pseudoaneurysm and infected graft were resected through a median sternotomy. This report describes successful management of a potentially fatal complication following placement of a modified Blalock-Taussig shunt.

We report the management of infective endocarditis after an extracardiac Fontan operation. Obstruction of the infected conduit and the associated pulmonary arterial embolism led to failure of the Fontan circulation. This was managed by completely removing both the infected conduit and emboli and replacing the conduit., (© 2013 Wiley Periodicals, Inc.)

In our previous studies, we have demonstrated effective regeneration of cartilage through the creation and application of layered cell sheets that combine both chondrocytes and synovial cells. In this study, we were able to demonstrate that cells derived from cell sheets can survive for long periods after transplantation into rat knee joints having osteochondral defects. We established a method for generating cell sheets from firefly luciferase-expressing chondrocytes obtained from transgenic Lewis rats, and carried out allogenic transplantation of these cell sheets into wild-type Lewis rats. We then administered luciferin and monitored the survival of the transplanted cells by using bioluminescence imaging (BLI). Our data showed that the transplanted cells survived and could be detected for more than 21 months, which was longer than expected. Furthermore, the BLI data showed that the transplanted cells remained in the knee joint and did not migrate to other parts of the body, thus confirming the safety of the cell sheets. In this study, we monitored the duration of survival of cell sheets composed of only chondrocytes, only synovial cells, or both chondrocytes and synovial cells, and found that all three types of cell sheets survived for an extended period of time., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Aortopulmonary window (APW) with an anomalous origin of a coronary artery is extremely rare. We report surgical management of a four-week-old infant with the association of a distal type of APW and an anomalous origin of the right coronary artery (RCA) from the pulmonary artery. Complete anatomical correction comprising division of the great arteries and transferring the RCA as an autologous flap to the aortic defect was successfully performed., (© 2013 Wiley Periodicals, Inc.)

MicroRNAs (miRNAs) are small RNAs of ∼22 base pairs that regulate gene expression. We harvested cartilage tissue from patients with polydactylism, anterior cruciate ligament injury, and osteoarthritis undergoing total knee arthroplasty and used microarrays to identify miRNAs whose expression is upregulated or downregulated with age. The results were assessed by real-time PCR and MTT assay in a mimic group, in which synthetic double-stranded RNA from the isolated miRNA was transfected to upregulate expression, and in an inhibitor group, in which the miRNA was bound specifically to downregulate expression. The expression of two miRNAs (miR-199a-3p and miR-193b) was upregulated with age and that of one miRNA (miR-320c) was downregulated with age. A real-time PCR assay showed that type 2 collagen, aggrecan, and SOX9 expression were downregulated in the miR-199a-3p mimic group but was upregulated in the inhibitor group. Similar results were observed for miR-193b. By contrast, ADAMTS5 expression was downregulated in the miR-320c mimic group and upregulated in the inhibitor group. Cell proliferative activity was upregulated significantly in the miR-193b inhibitor group compared with the control group. We believe that miR-199a-3p and miR-193b are involved in the senescence of chondrocytes, and miR-320c is involved in the juvenile properties of chondrocytes., (Copyright © 2012 Orthopaedic Research Society.)

We have previously shown that a single nucleotide polymorphism rs11536889 in the 3'-untranslated region (UTR) of TLR4 was associated with periodontitis. In this study the effects of this single nucleotide polymorphism on Toll-like receptor (TLR) 4 expression were investigated. Monocytes from subjects with the C/C genotype expressed higher levels of TLR4 on their surfaces than those from subjects with the other genotypes. Peripheral blood mononuclear cells (PBMCs) from the C/C and G/C subjects secreted higher levels of IL-8 in response to lipopolysaccharide (LPS), a TLR4 ligand, than the cells from the G/G subjects. However, there was no significant difference in TLR4 mRNA levels in PBMCs from the subjects with each genotype. After stimulation with tripalmitoylated CSK(4) (Pam(3)CSK(4)), TLR4 mRNA levels increased in PBMCs from both the C/C and G/G subjects, whereas TLR4 protein levels increased in PBMCs from the C/C but not G/G subjects. Transient transfection of a series of chimeric luciferase constructs revealed that a fragment of 3'-UTR containing rs11536889 G allele, but not C allele, suppressed luciferase activity induced by LPS or IL-6. Two microRNAs, hsa-miR-1236 and hsa-miR-642a, were predicted to bind to rs11536889 G allele. Inhibition of these microRNAs reversed the suppressed luciferase activity. These microRNA inhibitors also up-regulated endogenous TLR4 protein on THP-1 cells (the G/G genotype) after LPS stimulation. Furthermore, mutant microRNAs that bind to the C allele inhibited the luciferase activity of the construct containing the C allele. These results indicate that genetic variation of rs11536889 contributes to translational regulation of TLR4, possibly by binding to microRNAs.

In this study, we investigate the effects of treatment with layered chondrocyte sheets and synovial cell transplantation. An osteochondral defect was created of 48 Japanese white rabbits. In order to determine the effects of treatment, the following 6 groups were produced: (A) synovial cells (1.8 × 10(6) cells), (B)layered chondrocyte sheets (1.7 × 10(6) cells), (C) synovial cells (3.0 × 10(5) cells) + layered chondrocyte sheets, (D)synovial cells (6.0 × 10(5) cells) + layered chondrocyte sheets, (E)synovial cells (1.2 × 10(6) cells) + layered chondrocyte sheets, (F) osteochondral defect. Layered chondrocyte sheets and synovial cells were transplanted, sacrificed four and 12 weeks postoperatively. An incapacitance tester (Linton) was used to find trends in the weight distribution ratio of the damaged limbs after surgery. Sections were stained with Safranin-O. Repair sites were evaluated using ICRS grading system. In groups (A) to (E), the damaged limb weight distribution ratio had improved. The repair tissue stained positively with Safranin-O. Four and 12 weeks after surgery, groups (A) to (E) exhibited significantly higher scores than group (F), and groups (D) and (E) exhibited significantly higher scores than groups (A) and (B). This suggests the efficacy of combining layered chondrocyte sheets with synovial cells., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Lacking a blood supply and having a low cellular density, articular cartilage has a minimal ability for self-repair. Therefore, wide-ranging cartilage damage rarely resolves spontaneously. Cartilage damage is typically treated by chondrocyte transplantation, mosaicplasty or microfracture. Recent advances in tissue engineering have prompted research on techniques to repair articular cartilage damage using a variety of transplanted cells. We studied the repair and regeneration of cartilage damage using layered chondrocyte sheets prepared on a temperature-responsive culture dish. We previously reported achieving robust tissue repair when covering only the surface layer with layered chondrocyte sheets when researching partial-thickness defects in the articular cartilage of domestic rabbits. The present study was an experiment on the repair and regeneration of articular cartilage in a minipig model of full-thickness defects. Good safranin-O staining and integration with surrounding tissues was achieved in animals transplanted with layered chondrocyte sheets. However, tissue having poor safranin-O staining-not noted in the domestic rabbit experiments-was identified in some of the animals, and the subchondral bone was poorly repaired in these. Thus, although layered chondrocyte sheets facilitate articular cartilage repair, further investigations into appropriate animal models and culture and transplant conditions are required., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background and Objective: T cells infiltrate the inflammatory site of periodontitis and consequently stimulate the loss of periodontal bone. We previously reported that T cells from lipopolysaccharide (LPS)-injected mice (LPS-T cells) accelerated osteoclastogenesis in the presence of LPS. Ηowever, the detailed mechanism of this acceleration is still unclear. In this study, we analyzed the mechanism of osteoclastogenesis accelerated by LPS-T cells., Material and Methods: We examined the mechanism of osteoclastogenesis acceleration. First, to determine the effect of cell-to-cell contact, we co-cultured T cells and bone marrow macrophages, prestimulated with RANKL for 48 h (R-BMMs), in the presence of LPS for 24 h, in a Transwell. Second, to determine the effect of CD40 ligand (CD40L), we co-cultured T cells and R-BMMs in the presence of LPS and anti-CD40L immunoglobulin. Third, we examined the effect of recombinant mouse CD40L (rCD40L) in the presence of LPS in vitro and in vivo. Lastly, we examined the expression of membrane-bound CD40L (mCD40L) by fluorescence-activated cell sorting (FACS)., Results: Blocking cell-to-cell contact between LPS-T cells and R-BMMs completely inhibited the acceleration of osteoclastogenesis. Anti-CD40L immunoglobulin also completely inhibited the acceleration of osteoclastogenesis. Moreover, rCD40L accelerated osteoclastogenesis in the presence of LPS in vitro and in vivo. Finally, the expression of mCD40L on LPS-T cells was higher than that on T cells isolated from mice not injected with LPS., Conclusion: The results demonstrate that CD40L accelerates osteoclastogenesis in the presence of RANKL and LPS. The results also suggest that mCD40L on LPS-T cells accelerates osteoclastogenesis., (© 2011 John Wiley & Sons A/S.)

Background and Objective: Interferon-γ (IFN-γ) potently inhibits RANKL-induced osteoclastogenesis in vitro. In contrast, previous studies have shown that an increase in IFN-γ expression is correlated with an increase in lipopolysaccharide (LPS)-induced bone loss in vivo. However, it is not clear whether local IFN-γ accelerates osteoclastogenesis or not in vivo. Therefore, the aim of this study was to clarify the role of local IFN-γ in LPS-induced osteoclastogenesis., Materials and Methods: We induced bone loss in calvaria by injecting LPS. One group of mice received an IFN-γ injection together with LPS injection, while another group received IFN-γ 2 d after LPS injection. Bone resorption was observed histologically. Next, we stimulated murine bone marrow macrophages with macrophage-colony stimulating factor and RANKL in vitro. We added different doses of IFN-γ and/or LPS at 0 or 48 h time points. Cells were stained with tartrate-resistant acid phosphatase at 72 h., Results: Local administration of IFN-γ together with LPS injection did not affect osteoclast formation. However, IFN-γ injected after LPS injection accelerated osteoclast formation. Also, we confirmed that IFN-γ added at 0 h inhibited RANKL-induced osteoclastogenesis in vitro. However, inhibition by IFN-γ added at 48 h was reduced compared with that by IFN-γ added at 0 h. Interestingly, IFN-γ together with a low concentration of LPS accelerated osteoclast formation when both were added at 48 h compared with no addition of IFN-γ., Conclusion: The results suggest that local IFN-γ accelerates osteoclastogenesis in certain conditions of LPS-induced inflammatory bone loss., (© 2011 John Wiley & Sons A/S.)

We investigate the cluster-cluster aggregations of superparamagnetic particles in a rotational magnetic field numerically by the Brownian dynamics method, focusing on the cases of ϕ = 0.01 and 0.03 and Ma = 0, 0.001, 0.01, and 0.1, where ϕ is the area fraction of superparamagnetic particles and Ma is the Mason number, i.e., the ratio of viscous drag to magnetic force acting on a magnetic particle. We clarify the effect of ϕ and Ma on the cluster-cluster aggregation process from the point of view of dynamic scaling law.

Indenes are important core structures in organic chemistry. Few simple arylallenes have been used to construct indene skeletons by Friedel-Crafts reaction. Lewis acid catalyzed reaction of ethenetricarboxylates 1 and arylallenes has been examined in this study. The reaction of arylallenes and ethenetricarboxylate triesters with SnCl(4) gave indene derivatives efficiently, via a conjugate addition/Friedel-Crafts cyclization reaction. On the other hand, the reactions of 1,1-diethyl 2-hydrogen ethenetricarboxylate and arylallenes or alkylallenes with SnCl(4) at -78 degrees C or room temperature and subsequent treatment with Et(3)N gave gamma-lactones. The reactions of triethyl ethenetricarboxylate and 1,1-dialkylallenes with SnCl(4) at room temperature also gave gamma-lactones.

Background and Objective: Bone resorption is positively regulated by receptor activator of nuclear factor-kappaB ligand (RANKL). Pro-inflammatory cytokines, such as interleukin (IL)-1beta, promote RANKL expression by stromal cells and osteoblasts. Green tea catechin (GTC) has beneficial effects on human health and has been reported to inhibit osteoclast formation in an in vitro co-culture system. However, there has been no investigation of the effect of GTC on periodontal bone resorption in vivo. We therefore investigated whether GTC has an inhibitory effect on lipopolysaccharide (LPS)-induced bone resorption., Material and Methods: Escherichia coli (E. coli) LPS or LPS with GTC was injected a total of 10 times, once every 48 h, into the gingivae of BALB/c mice. Another group of mice, housed with free access to water containing GTC throughout the experimental period, were also injected with LPS in a similar manner., Results: The alveolar bone resorption and IL-1beta expression induced by LPS in gingival tissue were significantly decreased by injection or oral administration of GTC. Furthermore, when GTC was added to the medium, decreased responses to LPS were observed in CD14-expressing Chinese hamster ovary (CHO) reporter cells, which express CD25 through LPS-induced nuclear factor-kappaB (NF-kappaB) activation. These findings demonstrated that GTC inhibits nuclear translocation of NF-kappaB activated by LPS. In addition, osteoclasts were generated from mouse bone marrow macrophages cultured in a medium containing RANKL and macrophage colony-stimulating factor with or without GTC. The number of osteoclasts was decreased in dose-dependent manner when GTC was added to the culture medium., Conclusion: These results suggest that GTC suppresses LPS-induced bone resorption by inhibiting IL-1beta production or by directly inhibiting osteoclastogenesis.

We have developed a simple fabrication method of a highly sensitive direct electron transfer-type electrochemical biosensor for hydrogen peroxide by use of cup-stacked carbon nanotubes (CSCNTs). The CSCNTs, formed by stacking of cup-shaped carbon units, has larger internal space and more hydrophilic edges, thanks to the presence of functional groups containing oxygen (e.g., -COOH, -OH), than multi-walled carbon nanotubes (MWCNTs). When the CSCNTs suspension was cast, the CSCNTs were dispersed homogeneously onto a glassy carbon (GC) electrode, and horseradish peroxidase (HRP) was immobilized firmly by physical adsorption without any chemical reactions. The flow injection analysis (FIA) system with the HRP/CSCNTs/GC electrode has superior sensitivity and stability to the HRP/MWCNTs/GC electrode. The detection limit was 0.75 nM (S/N = 3) and the activity was maintained over 85% for 21 days. Further, when the glutamate oxidase (GlOD)-immobilized reactor was set into the proposed FIA system, L-glutamate could be detected repeatedly with a detection limit of 1.2 nM (S/N = 3).

Studies in humans have established that polymorphisms in genes encoding the innate immune Toll-like receptors (TLRs) are associated with inflammatory atherosclerosis. In hyperlipidemic mice, TLR2 and TLR4 have been reported to contribute to atherosclerosis progression. Human and mouse studies support a role for the oral pathogen Porphyromonas gingivalis in atherosclerosis, although the mechanisms by which this pathogen stimulates inflammatory atherosclerosis via innate immune system activation is not known. Using a genetically defined apolipoprotein E-deficient (ApoE(-/-)) mouse model we demonstrate that pathogen-mediated inflammatory atherosclerosis occurs via both TLR2-dependent and TLR2-independent mechanisms. P. gingivalis infection in mice possessing functional TLR2 induced the accumulation of macrophages as well as inflammatory mediators including CD40, IFN-gamma and the pro-inflammatory cytokines IL-1 beta, IL-6 and tumor necrosis factor-alpha in atherosclerotic lesions. The expression of these inflammatory mediators was reduced in atherosclerotic lesions from P. gingivalis-infected TLR2-deficient (TLR2(-/-)) mice. These studies provide a mechanistic link between an innate immune receptor and pathogen-accelerated atherosclerosis by a clinically and biologically relevant bacterial pathogen., ((c) 2010 S. Karger AG, Basel.)

T cells play important roles in bone destruction and osteoclastogenesis and are found in chronic destructive bone lesions. Lipopolysaccharide (LPS) is one of several pathological factors involved in inflammatory bone destruction. We previously described the importance of T cells in the inflammatory bone resorption that occurs after repeated LPS administration. However, whether local or systemic T cells are important for inflammatory bone resorption and whether immunization of host animals influences bone resorption remain unclear. The present study examines the effects of local extant T cells from LPS-immunized mice on LPS-induced bone resorption. T cells from LPS-immunized or non-immunized mice were injected together with LPS into the gingival tissues of mice with severe combined immunodeficiency disease that lack both T and B cells. We histomorphometrically evaluated bone resorption at sites of T cell injections and examined the influence of T cells from LPS-immunized mice on osteoclastogenesis in vitro. We found that locally administered T cells from LPS-immunized but not non-immunized mice accelerated LPS-induced bone resorption in vivo. Moreover, T cells from LPS-immunized mice increased osteoclastogenesis in vitro induced by receptor activator of NF-kappa B ligand and LPS and anti-tumor necrosis factor (TNF)-alpha antibody inhibited this increase. These results demonstrated that local extant T cells accelerate inflammatory bone resorption. Furthermore, T cells from LPS-immunized mice appear to elevate LPS-induced bone resorption using TNF-alpha.

Background and Objective: T cells and their cytokines are believed to be key factors in periodontal disease and bone resorption. We previously showed that T cells transferred to nude mice were related to inflammatory bone resorption in vivo. However, it has not been clarified whether T cells can induce bone resorption in the absence of B cells. In this study, we therefore investigated the ability of T cells to induce bone resorption without B cells, using both T cell- and B cell-deficient mice with severe combined immune deficiency (SCID)., Material and Methods: Escherichia coli lipopolysaccharide (LPS) was injected into the gingivae of SCID mice reconstituted by T cells (SCID + T mice). Wild-type C.B-17 mice and SCID mice were used as control animals. Alveolar bone resorption and production of cytokines in the gingivae were then compared histopathologically and immunohistologically., Results: The degree of bone resorption in SCID + T mice was significantly greater than that in SCID mice but less than that in wild-type mice. The same tendency was found for expression of receptor activator of nuclear factor kappaB ligand. The number of interferon-gamma-positive cells in SCID + T mice was the highest of the three groups. In contrast, interleukin-4-positive cells were detected in wild-type mice but not in SCID + T and SCID mice., Conclusion: The results suggest that T cells are able to promote LPS-induced bone resorption in the absence of B cells. The expressions of cytokines in the presence of B cells are quite different.

We analyze the dynamics of paramagnetic particles on a paramagnetic substrate under a rotational magnetic field. When the paramagnetic particles are subjected to a rotational magnetic field, the rotational plane of which is perpendicular to the substrate surface, the particles form chain clusters caused by the dipole-dipole interaction between the particles and these clusters display a tumbling motion under certain conditions. In this case, the angular momentum of the clusters is converted to a translational one through the force of friction acting between the particles and substrate and, as a result, the clusters move along the surface of the substrate. We analyze the conditions under which the tumbling motion occurs and the dependence of the translational velocity of a cluster on the control parameters by the Stokesian dynamics method. Based on the dynamics of magnetic particles, we propose a method of manipulating nano- and microparticles using a rotational magnetic field. We demonstrate the manipulation of magnetic and nonmagnetic particles, a carbon nanotube, and a biological cell.

The major and minor fimbriae proteins produced by the human pathogen Porphyromonas gingivalis are required for invasion of human aortic endothelial cells and for the stimulation of potent inflammatory responses. In this study, we report that native forms of both the major and minor fimbriae proteins bind to and signal through TLR2 for this response. Major and minor fimbriae bound to a human TLR2:Fc chimeric protein with an observed K(d) of 28.9 nM and 61.7 nM, respectively. Direct binding of the major and minor fimbriae to a human chimeric CD14-Fc protein also established specific binding of the major and minor fimbriae to CD14 with classic saturation kinetics. Using a P. gingivalis major and minor fimbriae mutant, we confirmed that TLR2 binding in whole cells is dependent on the expression of the major and minor fimbriae. Although we did not observe binding with the major or minor fimbriae to the TLR4-Fc chimeric protein, signaling through TLR4 for both proteins was demonstrated in human embryonic kidney 293 cells transfected with TLR4 and only in the presence MD-2. Transient transfection of dominant-negative forms of TLR2 or TLR4 reduced IL-8 production by human aortic endothelial cells following stimulation with major or minor fimbriae. The ability of two well-defined microbe-associated molecular patterns to select for innate immune recognition receptors based on accessory proteins may provide a novel way for a pathogen to sense and signal in appropriate host environments.

This study showed that gluten proteins were extracted with distilled water from dough prepared in the presence of NaCl. To elucidate the interrelationship of NaCl and gluten proteins in dough, the extracted proteins were characterized. These proteins were primarily found to be soluble gliadin monomers by N-terminal amino acid sequencing and analytical ultracentrifugation. Extracted proteins were aggregated by the addition of NaCl at concentrations of >10 mM. A decrease in beta-turn structures, which expose tryptophan residues to an aqueous environment in the presence of NaCl, was revealed by Fourier transform infrared analysis and scanning of fluorescence spectra. In addition, cross-linking experiments with disuccinimidyl tartrate showed that a large amount of protein was cross-linked in the dough only in the presence of NaCl. These results suggest that both interactions and distances between proteins were altered by the addition of NaCl.

The receptor activator of NF-kappaB ligand (RANKL) and the proinflammatory cytokines are believed to play important roles in osteoclastogenesis. We recently reported that the innate immune recognition receptor, Toll-like receptor 2 (TLR2), is crucial for inflammatory bone loss in response to infection by Porphyromonas gingivalis, the primary organism associated with chronic inflammatory periodontal disease. However, the contribution of macrophage-expressed TLRs to osteoclastogenesis has not been defined. In this study, we defined a requirement for TLR2 in tumor necrosis factor-alpha (TNF-alpha)-elicited osteoclastogenesis in response to exposure to P. gingivalis. Culture supernatant (CS) fluids from P. gingivalis-stimulated macrophages induced bone marrow macrophage-derived osteoclastogenesis. This activity was dependent on TNF-alpha and occurred independently of RANKL, interleukin-1beta (IL-1beta), and IL-6. CS fluids from P. gingivalis-stimulated TLR2(-/-) macrophages failed to express TNF-alpha, and these fluids induced significantly less osteoclast formation compared with that of the wild-type or the TLR4(-/-) macrophages. In addition, P. gingivalis exposure induced up-regulation of TLR2 expression on the cell surface of macrophages, which was demonstrated to functionally react to reexposure to P. gingivalis, as measured by a further increase in TNF-alpha production. These results demonstrate that macrophage-dependent TLR2 signaling is crucial for TNF-alpha-dependent/RANKL-independent osteoclastogenesis in response to P. gingivalis infection. Furthermore, the ability of P. gingivalis to induce the cell surface expression of TLR2 may contribute to the chronic inflammatory state induced by this pathogen.

Toll-like receptors (TLRs) are a group of pathogen-associated molecular pattern receptors, which play an important role in innate immune signaling in response to microbial infection. It has been demonstrated that TLRs are differentially up regulated in response to microbial infection and chronic inflammatory diseases such as atherosclerosis. The expression of TLRs are markedly augmented in human atherosclerotic lesions and this occurs preferentially by endothelial cells and macrophages in areas infiltrated with inflammatory cells. Furthermore polymorphisms in the human gene encoding one TLR receptor (TLR4) which attenuates receptor signaling and diminishes the inflammatory response to gram-negative pathogens, is associated with low levels of certain circulating mediators of inflammation and a decreased risk for atherosclerosis in humans. Recent advances have established a fundamental role for inflammation in mediating all stages of atherosclerosis. However, the triggers that initiate and sustain the inflammatory process have not been definitively identified. Although definitive proof of a role of infection contributing to atherogenesis is lacking, multiple investigations have demonstrated that infectious agents evoke cellular and molecular changes supportive of such a role. Evidence in humans suggesting that periodontal infection predisposes to atherosclerosis is derived from studies demonstrating that the periodontal pathogen Porphyromonas gingivalis resides in the wall of atherosclerotic vessels and seroepidemiological studies demonstrating an association between pathogen-specific IgG antibodies and atherosclerosis. Our recent work with P. gingivalis has demonstrated the effectiveness of specific intervention strategies (immunization) in the prevention of pathogen-accelerated atherosclerosis. We have also established that the inflammatory signaling pathways that P. gingivalis utilizes is dependent on the cell type and this specificity clearly influences innate immune signaling in the context of local chronic inflammation versus distant chronic inflammation. We postulate that bacterial infection mediates inflammatory responses that involve specific innate immune pathways in defined host cells. Furthermore, these inflammatory responses can be correlated with atherosclerosis and ultimately thrombotic complications.

Objective: Toll-like receptors (TLRs), a group of pathogen-associated microbial pattern recognition receptors, play an important role in innate immune signaling and are differentially regulated in chronic inflammatory diseases such as atherosclerosis. However, the involvement of TLRs in the progression of atherosclerosis is still unclear., Methods and Results: TLR2 and apolipoprotein E double knockout (Tlr2(-/-)Apoe(-/-)) mice were generated and the progressive formation of atherosclerotic plaque in the aortas was examined in mice fed a normal chow diet. We demonstrate that inactivation of TLR2 resulted in reduced progression of atherosclerosis in both male and female Apoe(-/-) mice. Likewise, TLR2 deficiency resulted in a reduction in lipid accumulation and decreased macrophage recruitment to the aortic sinus, as well as reduced monocyte chemoattractant protein-1 (MCP-1) levels. Furthermore, macrophages isolated from Tlr2(-/-)Apoe(-/-) mice demonstrated significantly reduced MCP-1 production upon stimulation with a TLR2 ligand. However, no differences in acetylated low-density lipoprotein uptake and foam cell formation were observed in macrophages isolated from Tlr2(-/-)Apoe(-/-) mice as compared to Apoe(-/-) mice., Conclusions: TLR2 plays a critical role in the progression of atherosclerosis in Apoe(-/-) mice, which is independent of dietary lipids and macrophage lipid uptake.

Background and Objective: Receptor activator of nuclear factor kappa B ligand (RANKL) is an important factor in osteoclast differentiation, activation and survival; however, its involvement in inflammatory bone resorption, with or without occlusal trauma, is unclear. The purpose of the present study was to investigate the distribution of RANKL-expressing cells in rat periodontium during lipopolysaccharide-induced inflammation with or without occlusal trauma., Material and Methods: Lipopolysaccharide was injected into rat gingiva of the lower left first molar to induce inflammation. In addition, the occlusal surface of the upper left first molar of rat was raised by placing a gold inlay to induce occlusal trauma in the lower left first molars. The distribution of RANKL-expressing cells was immunohistochemically observed., Results: In the inflammatory model, many osteoclasts were observed at the apical inter-radicular septum on day 5 and they were reduced by day 10. On the other hand, in the inflammatory model with occlusal trauma, many osteoclasts were still observed on day 10. RANKL expression was similar to the changes in osteoclast number. The expression of RANKL increased in endothelial cells, inflammatory cells and periodontal ligament cells., Conclusion: These findings clearly demonstrated that RANKL expression on endothelial cells, inflammatory cells and periodontal ligament cells is involved in inflammatory bone resorption and the expression is enhanced by traumatic occlusion. These results suggest that RANKL expression on these cells is closely involved in the increase of osteoclasts induced by occlusal trauma.

Introduction: On the morning of 25 April 2005, a Japan Railway express train derailed in an urban area of Amagasaki, Japan. The crash was Japan's worst rail disaster in 40 years. This study chronicles the rescue efforts and highlights the capacity of Japan's urban disaster response., Methods: Public reports were gathered from the media, Internet, government, fire department, and railway company. Four key informants, who were close to the disaster response, were interviewed to corroborate public data and highlight challenges facing the response., Results: The crash left 107 passengers dead and 549 injured. First responders, most of whom were volunteers, were helpful in the rescue effort, and no lives were lost due to transport delays or faulty triage. Responders criticized an early decision to withdraw rescue efforts, a delay in heliport set-up, the inefficiency of the information and instruction center, and emphasized the need for training in confined space medicine. Communication and chain-of-command problems created confusion at the scene., Conclusions: The urban disaster response to the train crash in Amagasaki was rapid and effective. The Kobe Earthquake and other incidents sparked changes that improved disaster preparedness in Amagasaki. However, communication and cooperation among responders were hampered, as in previous disasters, by the lack of a structured command system. Application of an incident command system may improve disaster coordination in Japan.

The host immune system, especially activated T cells, plays a crucial role in inflammatory bone resorption and osteoclastogenesis. Previously, we showed that T cells are involved in inflammatory bone resorption in vivo. However, little is known about whether B cells are involved in inflammatory bone resorption and how B cells take part in osteoclastogenesis. Therefore, the aim of this study was to examine whether B c ells truly influence inflammatory bone resorption in vivo. Alveolar bone resorption in normal mice, in SCID mice that lack both B and T cells, and in B cell-reconstituted SCID mice was compared histopathologically after repeated injections of lipopolysaccharide (LPS) into the gingiva. Furthermore, we examined whether the B cells that are stimulated by LPS are involved in osteoclastogenesis in vitro. As a result, the B cell-reconstituted SCID mice showed stronger inflammatory bone resorption than the SCID mice. Also, in vitro, LPS-stimulated B cells enhanced osteoclastogenesis and anti-tumor necrosis factor (TNF)-alpha antibody completely blocked osteoclastogenesis induced by LPS-stimulated B cells. These results suggest that B cells promote inflammatory bone resorption through TNF-alpha.

The new myofilament Ca2+ sensitizer levosimendan (LSM) is a positive inotropic and vasodilatory agent. Its beneficial effects have been demonstrated at rest in congestive heart failure (CHF). However, its effect during exercise (Ex) in CHF is unknown. We assessed the effects of LSM on left ventricular (LV) dynamics at rest and during Ex in eight conscious, instrumented dogs with pacing-induced CHF. After CHF, with dogs at rest, LSM decreased arterial elastance (Ea) and increased LV contractile performance as assessed by the slope of LV pressure-volume (P-V) relation. LSM caused a >60% increase in the peak rate of mitral flow (dV/dtmax) due to decreases in minimal LV pressure and the time constant of LV relaxation (tau). LV arterial coupling, quantified as the ratio of end-systolic elastance (Ees) to Ea, was increased from 0.47 to 0.85%. LV mechanical efficiency, determined as the ratio of stroke work to total P-V area, was improved from 0.54 +/- 0.09 to 0.61 +/- 0.07. These beneficial effects persisted during Ex after CHF. Compared with CHF Ex dogs, treatment with LSM prevented Ex-induced abnormal increases in mean left atrial pressure and end-diastolic pressure and decreased Ees/Ea. With LSM treatment during CHF Ex, the early diastolic portion of the LV P-V loop was shifted downward with decreased minimal LV pressure and tau values and a further augmented dV/dtmax. Ees/Ea improved, and mechanical efficiency further increased from 0.61 +/- 0.07 to 0.67 +/- 0.07, which was close to the value reached during normal Ex. After CHF, LSM produced arterial vasodilatation; improved LV relaxation and diastolic filling; increased contractility, LV arterial coupling, and mechanical efficiency; and normalized the response to Ex.

The elevation of plasma L-arginine levels stimulates nitric oxide (NO) synthesis, but the underlying mechanisms are not yet understood. We examined the role of physiological changes in pulmonary arteries on endogenous NO production. Male Wistar rats were divided into following groups: (1) control rats receiving normal water orally, (2) ARG rats receiving L-arginine water orally, (3) MCT rats injected with monocrotaline (MCT) on day 0 and receiving normal water orally, and (4) MCT+ARG rats injected with MCT on day 0 and receiving L-arginine water orally. The rats were studied after 23 days of dietary intervention. In MCT+ARG rats, supplemental L-arginine exhibited a significant pulmonary vasodilatory effect, as shown by a decreased pulmonary arterial pressure (PAP) (P<0.001), decreased right ventricular hypertrophy (P<0.01), and improved endothelium-dependent relaxation (P<0.01). Also L-arginine inhibited the elevation of plasma endothelin-1 (P<0.01). Oral L-arginine administration increased plasma L-arginine levels about twofold, but in only MCT+ARG rats (i.e., not in ARG rats) did the urinary nitrate excretion significantly increase (P<0.05), which is an indicator of endogenous NO formation. Oral administration of L-arginine is effective against pulmonary vascular remodeling. The data also suggest that the initial elevation of PAP is important for the induction of endogenous NO synthesis.

We investigate the patterns formed by paramagnetic particles, which are dispersed in a liquid solvent subjected to a dc magnetic field. We calculate the dynamics of paramagnetic particles by the Brownian dynamics method based on the Langevin equation. We, in particular, focus on the effect of the system height on the pattern formations. We also discuss the mechanism of the pattern formations and the dynamics of the structure creation processes.

The present study investigated the expression of Toll-like receptor (TLR) 2, TLR4, cluster of differentiation (CD) 14 and CD1a in human periodontitis gingiva using immunohistochemical methods. The specimens were classified according to the degree of inflammation into three groups (mild, moderate and severe). We established three zones in which to evaluate the ratios of TLR2-, TLR4-, CD14- and CD1a-positive cells to total cells in the connective tissues of each section. TLR2 and TLR4 were expressed in human periodontal tissues, and the ratio of TLR2-positive cells was highest overall in zone 1 (connective tissue subjacent to pocket epithelium) of the severe group and that of TLR4-positive cells was higher in the severe group than in the other groups. These results suggest that TLR2 and TLR4 participate in the innate immune response to stimulation by bacterial products in periodontal tissues. The ratio of CD14-positive cells was lowest overall in zone 1 of the severe group and that of CD1a was higher in the severe group than in the other groups. These results suggest that CD14 may be down-regulated during the development of inflammation and/or dendritic cells might infiltrate chronically inflamed gingival tissue.

A case of pancreatic pseudocyst extending into the liver via the hepatoduodenal ligament is reported. This is the first report clearly demonstrating that the hepatoduodenal ligament is another route of hepatic extension of pancreatic pseudocysts as well as the hepatogastric ligament, which is the most common. The importance of the topological anatomy of the peritoneum is emphasized in understanding the extrapancreatic extension of pancreatic pseudocysts.

It has been reported that bacterial lipopolysaccharide (LPS) induces alveolar bone resorption and that the host immune system, especially activated T cells, plays a crucial role in osteoclastogenesis. On the other hand, interferon-gamma (IFN-g), which is produced by activated T cells, suppresses bone resorption both in vitro and in vivo. Thus, the question arises as to whether or not IFN-g production increases with increasing bone resorption. We previously demonstrated that repeated injections of Escherichia coli LPS into mouse gingiva causes osteoclast formation in alveolar bone. In the present study we observed changes in the IFN-g production of infiltrating cells in concurrence with bone resorption. Mice were repeatedly injected with 5 mg LPS 26 times every 48 hours. After the 16th injection, when the alveolar bone resorption reached a plateau, the concentration of LPS was altered (25 mg LPS or PBS alone). The level of bone resorption became significantly elevated, and the number of IFN-g- and interleukin-1 beta (IL-1b)-bearing cells also increased significantly in relation to bone resorption within the 25 mg LPS-injected group. On the other hand, few tartrate-resistant acid phosphatase positive cells, or IFN-g- and IL-1b-bearing cells, were seen in the PBS-injected group. These results suggest that alteration in IFN-g-bearing cells might play a role in counterbalancing LPS-induced bone resorption resulting from osteoclast activating cytokines such as IL-1b.

The purpose was to investigate the balance between interferon (IFN)-gamma- and interleukin (IL)-4-bearing cells in various human inflamed gingiva by immunohistochemistry. Gingival tissues obtained from patients with gingivitis or periodontitis were divided into three groups based on the degree of histopathological inflammation, mild, moderate and severe. The tissues were also divided into four groups according to the clinical probing depth (PD). IFN-gamma- and IL-4-bearing cells in gingival tissues were stained immunohistologically and counted. The ratio of IL-4-bearing cells to IFN-gamma-bearing cells was calculated for each section. IFN-gamma-bearing cells were widespread in the connective tissue and their number increased significantly with the severity of inflammation and an increase of PD. IL-4-bearing cells were located beneath the pocket epithelium and their number showed no significant differences among the inflammation or PD groups. The ratios of IL-4-bearing cells to IFN-gamma-bearing cells in the severe inflammation or deep PD groups were significantly lower than those in the moderate inflammation or shallow PD groups. These results suggest that a low ratio of IL-4-bearing cells to IFN-gamma-bearing cells might be involved in the destruction of periodontal tissue.