Mineral trioxide aggregate suppresses pro-inflammatory cytokine expression via the calcineurin/nuclear factor of activated T cells/early growth response 2 pathway in lipopolysaccharide-stimulated macrophages.

AIM: To elucidate mechanisms by which mineral trioxide aggregate (MTA) suppresses pro-inflammatory cytokine mRNA expression in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. METHODOLOGY: Mineral trioxide aggregate extracts were prepared by immersing set ProRoot MTA in culture medium. RAW264.7 cells were cultured in the presence of LPS and MTA extracts. mRNA expression levels of interleukin (IL)-1α, IL-6, early growth response 2 (Egr2), suppressor of cytokine signalling 3 (Socs3) and IL-10 were quantified with reverse transcription-quantitative polymerase chain reaction. Phosphorylation of nuclear factor-kappa B (NF-κB) p65 in RAW264.7 cells was analysed by Western blotting. Intracellular calcium imaging was performed with Fluo-4 AM. The activity of nuclear factor of activated T cells (NFAT) was determined by luciferase assays. Enforced expression and silencing of Egr2 in RAW264.7 cells were carried out using an expression vector and specific RNAi, respectively. In vivo kinetics of Egr2+ cells in MTA-treated rat molar pulp tissues were examined using immunohistochemistry. Data were analysed by one-way analysis of variance, followed by the Tukey-Kramer test (P < 0.05). RESULTS: Exposure to MTA extracts resulted in reduced mRNA expression levels of IL-1α and IL-6, as well as reduced expression of phosphorylated NF-κB, in LPS-stimulated RAW264.7 cells. Exposure to MTA extracts induced Ca2+ influx, which was blocked by NPS2143, an antagonist of calcium-sensing receptor (CaSR); Ca2+ influx then triggered activation of calcineurin/NFAT signalling and enhanced mRNA expression of Egr2. Enforced expression of Egr2 in RAW264.7 cells promoted the expression of both IL-10 and Socs3. In vivo application of MTA onto rat molar pulp tissue resulted in the appearance of Egr2-expressing cells that coexpressed CD163, a typical M2 macrophage marker. CONCLUSIONS: Mineral trioxide aggregate extracts induced downregulation of IL-1α and IL-6 in LPS-stimulated RAW264.7 cells via CaSR-induced activation of calcineurin/NFAT/Egr2 signalling and subsequent upregulation of IL-10 and Socs3.

Hypoxia-inducible factor 1α promotes interleukin 1ß and tumour necrosis factor α expression in lipopolysaccharide-stimulated human dental pulp cells.

AIM: To elucidate the role of HIF1α in pro-inflammatory cytokine mRNA expression from lipopolysaccharide (LPS)-stimulated human dental pulp cells (hDPCs). METHODOLOGY: mRNA expression of interleukin (IL) 1ß and tumour necrosis factor (TNF) α in LPS-stimulated hDPCs was determined by quantitative RT-PCR. Expression of nuclear factor kappa B (NFκB) p65 and phospho-NFκB p65 was analysed by Western blotting. Activation of NFκB signalling was measured by luciferase assay using a reporter vector containing an NFκB response element. Enforced expression of HIF1α was induced by transfection of expression vectors with native or constitutively active forms of HIF1α. Expression of HIF1α protein in hDPCs was evaluated by immunocytochemistry and Western blotting. One-way analysis of variance and the Tukey-Kramer test were performed to determine a significant difference (P < 0.05). RESULTS: mRNA expression of IL1ß and TNFα, protein expression of phospho-NFκB p65 and LPS-induced NFκB signalling activity were promoted in low oxygen conditions (1% O2 ; P < 0.05). These findings were replicated following enforced expression and stabilization of HIF1α in hDPCs. Dimethyloxalylglycine, an inhibitor of prolyl hydroxylase (a HIF1α degrading enzyme), promoted IL1ß and TNFα mRNA expression and NFκB signalling in LPS-stimulated hDPCs (P < 0.05). HIF1α expression was detected in hDPCs cultured in low oxygen conditions (1% O2 ). LPS stimulation further enhanced HIF1α expression in hDPCs, especially within their nuclei. CONCLUSION: HIF1α promoted mRNA expression of IL1ß and TNFα via NFκB signalling in LPS-stimulated hDPCs, suggesting that HIF1α is involved in the progress of inflammation in dental pulp.

A series of magnon crystals appearing under ultrahigh magnetic fields in a kagomé antiferromagnet.

Geometrical frustration and a high magnetic field are two key factors for realizing unconventional quantum states in magnetic materials. Specifically, conventional magnetic order can potentially be destroyed by competing interactions and may be replaced by an exotic state that is characterized in terms of quasiparticles called magnons, the density and chemical potential of which are controlled by the magnetic field. Here we show that a synthetic copper mineral, Cd-kapellasite, which comprises a kagomé lattice consisting of corner-sharing triangles of spin-1/2 Cu2+ ions, exhibits an unprecedented series of fractional magnetization plateaus in ultrahigh magnetic fields of up to 160 T. We propose that these quantum states can be interpreted as crystallizations of emergent magnons localized on the hexagon of the kagomé lattice.

Serum Amyloid A Contributes to Chronic Apical Periodontitis via TLR2 and TLR4.

In the current concept of bacterial infections, pathogen-associated molecular patterns (PAMPs) derived from pathogens and damage-associated molecular patterns (DAMPs) released from damaged/necrotic host cells are crucial factors in induction of innate immune responses. However, the implication of DAMPs in apical and marginal periodontitis is unknown. Serum amyloid A (SAA) is a DAMP that is involved in the development of various chronic inflammatory diseases, such as rheumatoid arthritis. In the present study, we tested whether SAA is involved in the pathogenesis of periapical lesions, using human periapical surgical specimens and mice deficient in SAA and Toll-like receptors (TLR). SAA1/2 was locally expressed in human periapical lesions at the mRNA and protein levels. The level of SAA protein appeared to be positively associated with the inflammatory status of the lesions. In the development of mouse periapical inflammation, SAA1.1/2.1 was elevated locally and systemically in wild-type (WT) mice. Although SAA1.1/2.1 double-knockout and SAA3 knockout mice had redundant attenuation of the extent of periapical lesions, these animals showed strikingly improved inflammatory cell infiltration versus WT. Recombinant human SAA1 (rhSAA1) directly induced chemotaxis of WT neutrophils in a dose-dependent manner in vitro. In addition, rhSAA1 stimulation significantly prolonged the survival of WT neutrophils as compared with nonstimulated neutrophils. Furthermore, rhSAA1 activated the NF-κB pathway and subsequent IL-1α production in macrophages in a dose-dependent manner. However, TLR2/TLR4 double deficiency substantially diminished these SAA-mediated proinflammatory responses. Taken together, the SAA-TLR axis plays an important role in the chronicity of periapical inflammation via induction of inflammatory cell infiltration and prolonged cell survival. The interactions of PAMPs and DAMPs require further investigation in dental/oral inflammation.

Economic evaluation of infection control activities.

BACKGROUND: Healthcare-associated infections by drug-resistant bacteria affect a patient's prognosis. Infection control activities at medical institutions in Japan are increasingly focused on the threat from these bacteria. AIM: To undertake a full cost analysis that included the costs of consumables and labour required for infection control activities. METHODS: The cost of infection control activities undertaken by the infection control team (ICT) at Nishimino Kosei Hospital in Japan was surveyed from January 2013 to December 2015. The evaluation index of infection control activities used the meticillin-resistant Staphylococcus aureus detection rate. The cost:effectiveness ratio (CER) of each intervention was calculated. FINDINGS: Consumables and labour costs increased over time, as did the ratio of labour cost to total cost over time. However, the CER of interventions was found to have decreased, from ¥164,177 in 2014 to ¥57,989 in 2015. There were increases not only in the amount of consumables, but also in ICT time, suggesting the possibility of improvements in the economic efficiency of infection control. CONCLUSION: Increasing the amount of consumables and the time input of the ICT could help improve the economic efficiency of infection control. Our research suggests the possibility for improvements in the economic efficiency of infection control.

Increase of bone marrow macrophages and CD8+ T lymphocytes predict graft failure after allogeneic bone marrow or cord blood transplantation.

Graft failure (GF) remains an obstacle to survival after allogeneic hematopoietic stem cell transplantation. However, differentiating GF from delayed engraftment (DE) can be difficult. Host CD8+ lymphocytes have been reported to mediate graft rejection, but the impact of macrophages on DE or GF is yet to be clarified. Peri-engraftment bone marrow (BM) specimens of 32 adult patients with normal engraftment, DE or GF were retrospectively evaluated to identify the potential associations of CD163+ macrophage and CD8+ lymphocyte infiltration into BM. The macrophage or CD8+ lymphocyte number/total nucleated cell number was defined as the Mac ratio and CD8 ratio, respectively. Both DE and GF groups had significantly higher Mac ratios at day 14 than the normal group (P<0.0001), but no significant difference was observed between the DE and GF groups (P=1.000). The CD8 ratio at day 14 was significantly higher in the GF than in the normal group (P=0.005), whereas the CD8 ratios of the DE and normal groups were similar (P=0.07). A high Mac ratio at day 14 was associated with a risk of DE or subsequent GF. Patients with increased CD8 ratio at day 14 had a further risk of GF. The Mac ratio and the CD8 ratio appear to be well suited for predicting engraftment status.

Virological analysis of inherited chromosomally integrated human herpesvirus-6 in three hematopoietic stem cell transplant patients.

We analyzed 3 hematopoietic stem cell transplant (HSCT) recipients with inherited chromosomally integrated human herpesvirus-6 (inherited CIHHV-6). Cases 1 (inherited CIHHV-6A) and 2 (inherited CIHHV-6B) were inherited CIHHV-6 recipients. Case 3 received bone marrow from a donor with inherited CIHHV-6B. Following HSCT, HHV-6B was isolated from Case 1. HHV-6A and -6B messenger RNAs were detected in Cases 1 and 3.

Mechanical and neural changes in plantar-flexor muscles after spinal cord injury in humans.

STUDY DESIGN: Cross-sectional study. OBJECTIVES: To determine the effect of injury duration on plantar-flexor elastic properties in individuals with chronic spinal cord injury (SCI) and spasticity. SETTING: National Rehabilitation Center for Persons with Disabilities, Japan. METHODS: A total of 16 chronic SCI patients (age, 33±9.3 years; injury localization, C6-T12; injury duration, 11-371 months) participated. Spasticity of the ankle plantar-flexors was assessed using the Modified Ashworth Scale (MAS). The calf circumference and muscle thickness of the medial gastrocnemius (MG), lateral gastrocnemius and soleus were assessed using tape measure and ultrasonography. In addition, the ankle was rotated from 10° plantar-flexion to 20° dorsiflexion at 5 deg s(-1) with a dynamometer, and the ankle angle and torque were recorded. After normalizing the data (the initial points of angle and torque were set to zero), we calculated the peak torque and energy. Furthermore, angle-torque data (before and after normalization) were fitted with a second- and fourth-order polynomial, and exponential (Sten-Knudsen) models, and stiffness indices (SISOP, SIFOP, SISK) and AngleSLACK (the angle at which plantar-flexor passive torque equals zero) were calculated. The stretch reflex gain and offset were determined from 0-10° dorsiflexion at 50, 90, 120 and 150 deg s(-1). After logarithmic transformation, Pearson's correlation coefficients were calculated. RESULTS: MAS, calf circumference, MG thickness, peak torque and SIFOP significantly decreased with injury duration (r log-log=-0.63, -0.69, -0.63, -0.53 and -0.55, respectively, P<0.05). The peak torque and SIFOP maintained significant relationships even after excluding impacts from muscle morphology. CONCLUSION: Plantar-flexor elasticity in chronic SCI patients decreased with increased injury duration.

A review of the structure, and fundamental mechanisms and kinetics of the leaching of chalcopyrite.

Most investigators regard CuFeS2 as having the formal oxidation states of Cu(+)Fe(3+)(S(2-))2. However, the spectroscopic characterisation of chalcopyrite is clearly influenced by the considerable degree of covalency between S and both Fe and Cu. The poor cleavage of CuFeS2 results in conchoidal surfaces. Reconstruction of the fractured surfaces to form, from what was previously bulk S(2-), a mixture of surface S(2-), S2(2) and S(n)(2-) (or metal deficient sulfide) takes place. Oxidation of chalcopyrite in air (i.e. 0.2 atm of O2 equilibrated with atmospheric water vapour) results in a Fe(III)-O-OH surface layer on top of a Cu rich sulfide layer overlying the bulk chalcopyrite with the formation of Cu(II) and Fe(III) sulfate, and Cu(I)-O on prolonged oxidation. Cu2O and Cu2S-like species have also been proposed to form on exposure of chalcopyrite to air. S2(2-), S(n)(2-) and S(0) form on the chalcopyrite surface upon aqueous leaching. The latter two of these species along with a jarosite-like species are frequently proposed to result in surface leaching passivation. However, some investigators have reported the formation of S(0) sufficiently porous to allow ion transportation to and from the chalcopyrite surface. Moreover, under some conditions both S(n)(2-) and S(0) were observed to increase in surface concentration for the duration of the leach with no resulting passivation. The effect of a number of oxidants, e.g. O2, H2O2, Cu(2+), Cr(6+) and Fe(3+), has been examined. However, this is often accompanied by poor control of leach parameters, principally pH and E(h). Nevertheless, there is general agreement in the literature that chalcopyrite leaching is significantly affected by solution redox potential with an optimum E(h) range suggesting the participation of leach steps that involve both oxidation and reduction. Three kinetic models have generally been suggested by researchers to be applicable: diffusion, chemical reaction and a mixed model containing diffusion and chemical components which occur at different stages of leaching. Passivation effects, due to surface diffusion rate control, may be affected by leach conditions such as pH or E(h). However, only initial conditions are generally described and these parameters are not controlled in most studies. However, at fixed pH, E(h) and temperature, it appears most likely that leaching in sulfuric acid media in the presence of added Fe(3+) is surface reaction rate controlled with some initial period, depending on leach conditions, where the leach rate is surface layer diffusion controlled. Although bioleaching of some copper ores has been adopted by industry, bioleaching has yet to be applied to predominantly chalcopyrite ores due to the slow resulting leach rates. Mixed microbial strains usually yield higher leach rates, as compared to single strains, as different bacterial strains are able to adapt to the changing leach conditions throughout the leach process. As for chemical leaching, passivation is also observed on bioleaching with jarosite being likely to be the main contributor. In summary, whilst much has been observed at the macro-scale regarding the chalcopyrite leach process it is clear that interpretation of these phenomena is hampered by lack of understanding at the molecular or atomic scale. Three primary questions that require elucidation, before the overall mechanism can be understood are: 1. How does the surface of chalcopyrite interact with solution or air borne oxidants? 2. How does the nature of these oxidants affect the surface products formed? 3. What determines whether the surface formed will be passivating or not? These can only realistically be tackled by the application of near atomic-scale analytical approaches, which may include quantum chemical modelling, PEEM/SPEM, TEM, AFM etc.

Requirement of integrin ß3 for iron transportation during enamel formation.

Rodent incisors exhibit pigmentation on their labial surfaces. Although previous studies have shown that this pigment is composed of iron, the existence of other elements has not been investigated. This study found that the lower incisors of CD61, also known as integrin ß3, null mice (CD61(-/-)) lacked pigmentation. Although ameloblasts differentiated and formed enamel normally, no ferric ion accumulation was observed in maturation-stage ameloblasts in CD61(-/-) mice. Surface elements of control and CD61-/- lower incisors were compared by x-ray photoelectron spectroscopy (XPS). XPS analysis detected C, Ca, N, O, and P on the labial surfaces of lower incisors of both mice, whereas Fe was detected only in control samples. No peak of non-ferrous metal or other element was detected in either group. Quantitative RT-PCR analysis of 18 iron-transportation-related genes with mRNA from maturation-stage ameloblasts and ALC, a pre-ameloblastic cell line, was performed. The results suggested that CD61 regulates the expressions of Slc11a2 and Slc40a1, both of which are involved in iron transportation in epithelial tissues. These results suggested that the pigment on the labial surface of mouse incisors is composed of Fe and that both anemia and reduction of iron-transporting proteins may cause the loss of pigmentation in CD61(-/-) mice.

Initial fibroblast attachment to Erbium:YAG laser-irradiated dentine.

AIMS: To evaluate the effects of Erbium (Er):YAG laser irradiation on the morphology of resected dentine surfaces, and to investigate fibroblast attachment to laser-irradiated dentine surfaces. METHODOLOGY: Dentine blocks obtained from single-rooted human teeth were divided into the following groups after sterilization in an autoclave: (i) Laser group treated with Er:YAG laser irradiation (30 mJ per pulse, 10 pps, 60 s); (ii) L-MTAD group treated with laser irradiation as in (i) plus a mixture of doxycycline, tetracycline isomer and citric acid; (iii) RC-Prep group treated with EDTA gel or cream (RC-Prep) and (iv) Control group left untreated. After each treatment, the dentine blocks were incubated with NIH/3T3 fibroblasts cultured to subconfluency in Dulbecco's modified Eagle's medium supplemented with 10% foetal bovine serum and antibiotics. The number of attached cells amongst the groups was analysed statistically at the 5% significance level. The dentine surface morphologies and cell attachments were evaluated by counting assays, histological observations and scanning electron microscopy (SEM). RESULTS: The number of attached cells was significantly higher (P < 0.05) in the Laser group than in the RC-Prep and Control groups at 16 h. Dendritic cell extension of the fibroblasts was only observed in the Laser group at 8 h by SEM. In the histological analyses, significantly more attached cells were found on the dentine surfaces treated with laser irradiation. CONCLUSIONS: Er:YAG laser irradiation induced morphological alterations in dentine surfaces, which may improve the attachment of fibroblasts to dentine.

MEPE activated by furin promotes pulpal cell adhesion.

UNLABELLED: Matrix extracellular phosphoglycoprotein (MEPE) is predominantly expressed in osteoblasts, osteocytes, and odontoblasts and plays key biological roles in bone and dentin metabolism. Post-translational modifications are essential for its activation. This study tested the hypothesis that MEPE is activated through proteolytic processing by furin in dental pulp. MEPE was present in three sizes, 1 full-length and 2 cleaved fragments; the cleavage site was 146R↓147. The proprotein convertase family, particularly furin, was a candidate enzyme. Introducing a substitution at the cleavage site inhibited hydrolysis, but there was no cleavage of MEPE expressed in furin-deficient LoVo cells. Therefore, furin is a strong candidate for the proteolytic cleavage of MEPE. The C-terminal cleavage product promoted cell adhesion via its RGD motif. These results indicate that proteolytic processing by furin may activate MEPE during its secretion from odontoblasts and may play important roles in dentinogenesis and pulpal homeostasis. ABBREVIATIONS: MEPE, matrix extracellular phosphoglycoprotein; PTM, post-translational modifications; OLC, odontoblast-lineage cells.

Amelogenin positive cells scattered in the interstitial component of odontogenic fibromas.

BACKGROUND: Odontogenic tumours are often biphasic, consisting of epithelial and interstitial components, with an origin that is not well understood. Odontogenic fibromas are rich in mesenchymal component, but also have many epithelial nests. AIMS: To investigate the origin of this tumour by immunohistochemistry. METHODS: The expression of several odontogenic and epithelial markers, including amelogenin, was investigated by immunofluorescent studies. RESULTS: Immunohistochemical analysis showed that epithelial nests exhibited E-cadherin expression, but not amelogenin. Amelogenin positive cells were scattered in the fibrous tissue, which did not exhibit epithelial marker expression except for epithelial membrane antigen. In one case that had received a test biopsy before whole resection of tumour, amelogenin positive cells were distributed in the regenerating mucosal epithelium or subepithelial tissue. CONCLUSIONS: Results indicate that amelogenin positive cells of odontogenic fibromas have an epithelial origin and may have the potential for epithelial mesenchymal transition, which has not to date been investigated in benign tumours.

A two-degree-of-freedom motor-powered gait orthosis for spinal cord injury patients.

A number of orthoses have been developed to restore stance and walking in paraplegic subjects. Compliance, however, has been limited, mainly owing to walking effort. Use of the forces produced by actuators is an effective way to solve the problem of the considerable effort required for orthotic gait, namely high muscular effort and high energy expenditure. The purpose of the present study was to investigate the effects of assistance by external actuators on the orthotic gait of spinal cord injury (SCI) patients. Two kinds of linear actuator were developed by using direct current (d.c.) motors for assisting the knee and hip joint of a gait orthosis. They were mounted on the knee and hip joint of a commercial advanced reciprocating gait orthosis (ARGO), and a new two-degree-of-freedom externally powered gait orthosis was thus developed. The orthosis was assessed through inter-subject experiments on five male adult complete SCI patients. Owing to the short training period available for the assisted gait, simultaneous operation of both joint actuators was not conducted: either the knee actuation or the hip actuation was executed only. Thus, the knee actuator and the hip actuator were assessed with a T12 subject and with subjects for T5, T8, T11, and T12 respectively. The motions of the gaits, assisted by the linear actuators, were measured by a Vicon 370 system, and the general gait parameters and compensatory motions were evaluated. Results demonstrated that (a) all subjects could walk without falling, assisted either by the knee or the hip actuator; (b) both the knee and hip joint actuator increased the gait speed and the step length; (c) the knee flexion produced by the orthosis improved the dynamic cosmesis of walking; and (d) lateral compensatory motions as well as vertical ones tended to decrease when the hip joint was assisted, which could contribute to a reduction in walking effort.

Geometric frustration and dimensional reduction at a quantum critical point.

We show that the spatial dimensionality of the quantum critical point associated with Bose-Einstein condensation at T=0 is reduced when the underlying lattice comprises layers coupled by a frustrating interaction. Our theoretical predictions for the critical behavior correspond very well with recent measurements in BaCuSi(2)O(6) [ S. E. Sebastian et al., Nature (London) 441, 617 (2006)].