Enhanced phagocytosis associated with multinucleated microglia via Pyk2 inhibition in an acute ß-amyloid infusion model.

Multinucleated microglia have been observed in contexts associated with infection, inflammation, and aging. Though commonly linked to pathological conditions, the larger cell size of multinucleated microglia might enhance their phagocytic functions, potentially aiding in the clearance of brain debris and suggesting a reassessment of their pathological significance. To assess the phagocytic capacity of multinucleated microglia and its implications for brain debris clearance, we induced their formation by inhibiting Pyk2 activity using the pharmacological inhibitor PF-431396, which triggers cytokinesis regression. Multinucleated microglia demonstrate enhanced phagocytic function, as evidenced by their increased capacity to engulf ß-amyloid (Aß) oligomers. Concurrently, the phosphorylation of Pyk2, induced by Aß peptide, was diminished upon treatment with a Pyk2 inhibitor (Pyk2-Inh, PF-431396). Furthermore, the increased expression of Lamp1, a lysosomal marker, with Pyk2-inh treatment, suggests an enhancement in proteolytic activity. In vivo, we generated an acute Alzheimer's disease (AD) model by infusing Aß into the brains of Iba-1 EGFP transgenic (Tg) mice. The administration of the Pyk2-Inh led to an increased migration of microglia toward amyloid deposits in the brains of Iba-1 EGFP Tg mice, accompanied by morphological activation, suggesting a heightened affinity for Aß. In human microglia, lipopolysaccharide (LPS)-induced inflammatory responses showed that inhibition of Pyk2 signaling significantly reduced the transcription and protein expression of pro-inflammatory markers. These results suggest that Pyk2 inhibition can modulate microglial functions, potentially reducing neuroinflammation and aiding in the clearance of neurodegenerative disease markers. This highlights Pyk2 as a promising target for therapeutic intervention in neurodegenerative diseases.

A rare case of multiple brain abscesses caused by apical periodontitis of deciduous teeth in congenital heart disease: a case report.

BACKGROUND: A brain abscess is a focal infection in which abscesses form in the brain. A brain abscess is a rare but fatal disease when rupture occurs into the ventricles. We report a case of multiple brain abscesses caused by a hematogenous infection from the apical periodontitis of deciduous teeth. CASE PRESENTATION: The patient was a 7-years and 8-months-old male with congenital heart disease. The patient sought medical attention due to fever and headache, for which he was started on three antibiotics with a diagnosis of multiple brain abscesses. Given that apical periodontitis of deciduous teeth was strongly suspected as the source of the brain abscess, the deciduous teeth were extracted. Immediately after deciduous teeth extraction, the patient's headache and neurological symptoms disappeared. CONCLUSIONS: After teeth extraction, a clear shrinkage of the brain abscess was observed, and the patient was discharged from the hospital.

Patients with SATB2-associated syndrome exhibiting multiple odontomas.

Special AT-rich sequence-binding protein 2 (SATB2)-associated syndrome (SAS) is characterized by alterations of SATB2. Its clinical features include intellectual disability and craniofacial abnormalities, such as cleft palate, dysmorphic features, and dental abnormalities. Here, we describe three previously undiagnosed, unrelated patients with SAS who exhibited dental abnormalities, including multiple odontomas. Although isolated odontomas are common, multiple odontomas are rare. Individuals in families 1 and 3 underwent whole-exome sequencing. Patient 2 and parents underwent targeted amplicon sequencing. On the basis of the hg19/GRCh37 reference and the RefSeq mRNA NM_001172517, respective heterozygous mutations were found and validated in Patients 1, 2, and 3: a splice-site mutation (chr2:g.200137396C > T, c.1741-1G > A), a nonsense mutation (chr2:g.200213750G > A, c.847C > T, p.R283*), and a frame-shift mutations (chr2:g.200188589_200188590del, c.1478_1479del, p.Q493Rfs*19). All mutations occurred de novo. The mutations in Patients 1 and 3 were novel; the mutation in Patient 2 has been described previously. Tooth mesenchymal cells derived from Patient 2 showed diminished SATB2 expression. Multiple odontomas were evident in the patients in this report; however, this has not been recognized previously as a SAS-associated phenotype. We propose that multiple odontomas be considered as an occasional manifestation of SAS.

Disease specificity of anti-tryptophan hydroxylase-1 and anti-AIE-75 autoantibodies in APECED and IPEX syndrome.

Autoantibodies to autoimmune enteropathy-related 75 kDa antigen (AIE-75) and villin are disease markers of immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome which is characterized by a peripheral tolerance defect. On the other hand, anti-tryptophan hydroxylase-1 (TPH-1) antibodies are detected in autoimmune polyendocrinopathy, candidiasis, ectodermal dystrophy (APECED), a central tolerance defect, especially when complicated with gastrointestinal dysfunction. However, to date, anti-AIE-75 and anti-villin antibodies or anti-TPH-1 antibodies have not been tested in APECED or IPEX syndrome, respectively. In the present study, we confirmed the disease specificity of both anti-AIE-75 and anti-TPH-1, although anti-villin antibodies were detected in some patients with APECED. Our observation suggests that immunotolerance to AIE-75 depends on the peripheral mechanism, whereas the tolerance to TPH-1 depends on the central mechanisms.

Photophysical properties and biocompatibility of Photoluminescent Y2O3:Eu nanoparticles in polymethylmetacrylate matrix.

In this study, we produced europium-doped yttoria (Y2O3:Eu) nanoparticles and investigated their photoluminescent properties and biocompatibility. The Y2O3:Eu nanoparticles showed excellent photoluminescent properties and cytocompatibility. We also analyzed the photophysical properties of the nanoparticles in PMMA films. When the Y2O3:Eu nanoparticles were incorporated in the polymer film, they showed a strong red emission spectrum, similar to that seen with the particles alone. Energy dispersive X-ray spectroscopy (EDS) measurements indicated that the particles were distributed homogeneously in the PMMA film. Such materials could be applied not only to optoelectronic devices but also to biomedical applications such as bioimaging tools or luminescent medical/dental adhesive materials.

Incremental lines in human cellular cementum: A histological study.

OBJECTIVES: Human cellular cementum has incremental lines that demarcate individual cementum lamellae. The structural and functional details of the lines remain poorly understood. This study was designed to examine human cellular cementum using light microscopy, scanning electron microscopy, and contact microradiography and to elucidate the ultrastructure of incremental lines and their significance in cellular cementogenesis. METHODS: Longitudinal paraffin and ground sections of human mandibular molars were prepared. Paraffin sections were stained with hematoxylin, or hematoxylin and eosin, or impregnated with silver. Hematoxylin-stained sections were observed via scanning electron microscopy using NaOH maceration. Silver-impregnated sections were further stained with hematoxylin. Hematoxylin-stained ground sections were examined using contact microradiography. RESULTS: The incremental lines were found to be collagen fibril-poor layers. The outer area of each cementum lamella consisted of highly mineralized fibrils involved in constructing an alternating lamellar structure, whereas the inner area consisted of irregularly arranged, less highly mineralized, fibrils. The incremental lines corresponded with the innermost sites of the inner area. CONCLUSIONS: Based on the obtained findings, we suggest that cellular cementogenesis progresses as follows. (1) Cementoblasts alternate between low-to high-activity states. (2) In the earliest low-activity stage, cementoblasts generate poorly mineralized, fibril-poor, incremental lines. (3) As cementoblasts recover activity, fibril-organization and mineralization advance in the cementum. (4) In the high-activity stage, cementoblasts reach full activity and construct the highly mineralized, alternating lamellar structure. (5) Cementoblasts revert back to the low-activity stage. (6) The above processes are repeated, thus, alternately generating the incremental lines and cementum lamellae.

Deep fibrous histiocytoma of the index finger: a case report.

Our patient presented with an elastic soft mass of his left index finger. Hematoxylin and eosin staining showed a high cellular density with spindle-shaped cells in a storiform pattern. Immunohistochemical staining was positive for CD68, factor XIIIa and α-smooth muscle actin, and negative for CD34, STAT6, S100 protein, and desmin.

Biodistribution of aqueous suspensions of carbon nanotubes in mice and their biocompatibility.

In this study, we prepared two-types of water-dispersible carbon nanotubes (CNTs) and investigated their biodistribution in mice as well as bio-/cyto-compatibility. After administration, their organs were excised at various post-injection times, then observed using both optical and transmission electron microscopy (TEM). The color of the liver and lung markedly darkened, suggesting that administered CNTs reached these organs. By TEM observation, the CNTs were found in the liver and lung. They were observed even in the kidney and spleen, though their distributions in those organs were very low compared with that in liver and lung. Therefore, most of the administered CNTs would be accumulated in the liver or lung. However, the time profile of the body weight of CNT-administered mice was close to that of control mice. In addition, we estimated the cytocompatibility of the water-dispersible CNTs for hepatocytes. According to a TNF-alpha assay of the cells cultured with CNTs, the expression level was almost the same as that of the control. These results suggested that the water-dispersible CNTs have good bio-/cyto-compatibility under this condition.

Bone resorption improvement by conditioned medium of stem cells from human exfoliated deciduous teeth in ovariectomized mice.

Stem cells from human exfoliated deciduous teeth (SHED) are mesenchymal stem cells with multipotent differentiation potential present in the dental pulp tissue of the deciduous teeth. SHED produce secretions that have immunomodulatory and regenerative functions. In the present study, we investigated the effects of SHED-conditioned medium (SHED-CM) on osteopenia induced by the ovariectomy (OVX) phenotype and its corresponding immunological changes. Eleven-week-old female C3H/HeJ mice were subjected to OVX. SHED-CM was administered intraperitoneally in these mice for 4 weeks starting immediately after OVX. SHED-CM improved bone mass after OVX and elevated the polarization of M2 macrophages in the peritoneal cavity. SHED-CM also suppressed an OVX-induced increase in interferon-γ (INF-γ) and interleukin-17 (IL-17) concentrations in the peripheral blood. Inhibition of M2 macrophage polarization with neutralizing antibodies did not reduce the concentration of IFN-γ and IL-17 in peripheral blood, which were increased by OVX, and did not alleviate osteopenia induced by the OVX phenotype. Mechanistically, these findings suggest that SHED-CM alleviates bone resorption by suppressing the activation of IFN-γ and IL-17 cells by polarizing M2 macrophages. In conclusion, our data indicate that SHED-CM contains active secretions that may have promising efficacy to ameliorate OVX-induced osteopenia. We suggest that SHED-CM has the potential to be used as a novel therapeutic agent to inhibit osteoporosis.

N-acetylcysteine attenuates PGE2 and ROS production stimulated by 4-META/MMA-based resin in murine osteoblastic cells.

This study examined the effects of N-acetylcysteine (NAC) on the inflammatory reactions of murine osteoblastic cells cultured on the 4-methacryloxyethyl trimellitate anhydride/methyl methacrylate (4-META/MMA)-based resin. Superbond C&B (SB) was used as the 4-META/MMA-based resin and placed in a 48-well cell culture plate. The cells were cultured in αMEM (control) as well as on SB and SB in αMEM with NAC (SB+NAC). They were examined using the WST-1 proliferation assay, real-time PCR, enzyme-linked immunosorbent assay (ELISA), intracellular reactive oxygen species (ROS) measurements, and cellular glutathione (GSH) detection. COX-2 and IL-6 gene expressions were upregulated in SB; however, they were suppressed by NAC. Furthermore, PGE2 production in the culture medium was increased in SB, whereas NAC decreased the PGE2 production. NAC lowered the ROS level in the culture medium and significantly increased the intracellular GSH level. The present in vitro study demonstrated that NAC might be effective for dental material detoxification.

Bone morphogenetic protein-2 enhances Wnt/beta-catenin signaling-induced osteoprotegerin expression.

Wnt/beta-catenin signaling plays an important role in the developing skeletal system. Our previous studies demonstrated that Wnt/beta-catenin signaling inhibits the ability of bone morphogenetic protein (BMP)-2 to suppress myotube formation in the multipotent mesenchymal cell line C2C12 and that this inhibition is mediated by Id1. In this study, we examined the role of intracellular signaling by Wnt/beta-catenin and BMP-2 in regulating the expression of osteoprotegerin (OPG) and of the receptor activator of NFkappaB ligand (RANKL). OPG expression was induced by Wnt/beta-catenin signaling in C2C12 cells and osteoblastic MC3T3-E1 cells. Silencing of glycogen synthase kinase-3beta also increased OPG expression. In contrast, R expression was suppressed by Wnt/beta-catenin signaling. In a transfection assay, beta-catenin induced the activity of a reporter gene, a 1.5 kb fragment of the 5'-flanking region of the OPG gene. Deletion and mutation analysis revealed that Wnt/beta-catenin signaling regulates transcription of OPG via a promoter region containing two Wnt/beta-catenin responsive sites. BMP-2 enhanced Wnt/beta-catenin-dependent transcriptional activation of the OPG promoter. In response to BMP-2 stimulation, Smad 1 and 4 interacted with Wnt/beta-catenin responsive sites. These results show that the regulation of OPG expression is mediated through two transcription pathways that involve the OPG promoter.

Bone morphogenetic protein-2 down-regulates miR-206 expression by blocking its maturation process.

MicroRNAs (miRNAs) are small non-coding RNAs that are emerging as important post-transcriptional gene regulators. miR-206 is unique in that it is expressed only in skeletal muscle, including the myoblastic C2C12 cell line. In C2C12 cells, miR-206 expression was reduced dramatically after bone morphogenetic protein (BMP)-2 treatment. The down-regulation of miR-206 expression was also observed after co-transfection with constitutively-active Smad1 and Smad4, which are the intracellular signaling molecules of the BMP pathway. BMP-2 also reduced miR-206 expression in the presence of alpha-amanitin in a similar manner to that in the absence of alpha-amanitin. Moreover, the expression of pri-miR-206 was increased upon BMP-2 treatment for 6h compared to that in the absence of BMP-2. These results suggested that BMP-2 down-regulates miR-206 expression at the post-transcriptional level, by inhibiting the processing of pri-miR-206 into mature miR-206, and that BMP-2 could regulate miRNA biogenesis by a novel mechanism.

Occlusal disharmony increases stress response in rats.

Repeated or chronic stress is known to produce structural and functional changes in the rat brain, and in particular, alter the response of the hypothalamic -- pituitary -- adrenal (HPA) axis to subsequent new stress. Occlusal disharmony via placement of acryl cap on the lower incisors of rats is perceived as chronic stress. To determine the response of the HPA axis to subsequent new stress in rats with occlusal disharmony, we measured plasma corticosterone levels in these rats after subjecting them to new stress. Plasma corticosterone levels in rats with and without incisal cap increased and reached a peak 30 min after exposure to the new stress. However, a later decrease in plasma corticosterone levels from peak levels was found in rats with incisal cap compared with rats without incisal cap. This finding suggests that occlusal disharmony alters the response of the HPA axis to subsequent new stress.

Possible application of flow cytometry for evaluation of the structure and functional status of WASP in peripheral blood mononuclear cells.

The Wiskott-Aldrich syndrome protein (WASP), which is defective in Wiskott-Aldrich syndrome (WAS) patients, is an intracellular protein expressed in non-erythroid hematopoietic cells. Previously, we have established methods to detect intracellular WASP expression in peripheral blood mononuclear cells (PBMNCs) using flow cytometric analysis (FCM-WASP) and have revealed that WAS patients showed absent or very low level intracellular WASP expression in lymphocytes and monocytes, while a significant amount of WASP was detected in those of normal individuals. We applied these methods for diagnostic screening of WAS patients and WAS carriers, as well as to the evaluation of mixed chimera in WAS patients who had previously undergone hematopoietic stem cell transplantation. During these procedures, we have noticed that lymphocytes from normal control individuals showed dual positive peaks, while their monocytes invariably showed a single sharp WASP-positive peak. To investigate the basis of the dual positive peaks (WASP(low-bright) and WASP(high-bright)), we characterized the constituent linage lymphocytes of these two WASP-positive populations. As a result, we found each WASP(low/high) population comprised different linage PBMNCs. Furthermore, we propose that the difference between the two WASP-positive peaks did not result from any difference in WASP expression in the cells, but rather from a difference in the structural and functional status of the WASP protein in the cells. It has been shown that WASP may exist in two forms; an activated or inactivated form. Thus, the structural and functional WASP status or configuration could be evaluated by flow cytometric analysis.

Daily life habits associated with eveningness lead to a higher prevalence of dental caries in children.

BACKGROUND/PURPOSE: Circadian rhythm is an endogenous daily variation observed in most physiological functions including salivary secretion. Irregular lifestyle causes many diseases such as obesity and sleep disorders. The aim of this study is to examine the effects of the timings of sleep and meal on the prevalence of dental caries. MATERIALS AND METHODS: Study was conducted at university hospital in Japan. We asked 230 children (1-16 years old) to record the following life habits for 8 days: waking time, bedtime, mealtimes, snacking frequency, and tooth brushing frequency. We analyzed sleep habits from all data and compared dental caries and life habits using data from subjects with primary (2-7 years old) or permanent (11-16 years old) dentition period. RESULTS: The number of dental caries assessed using the decay or filled teeth (dft) index correlated with bedtime, supper time, regularity of supper time, and snacking frequency in subjects with primary dentition. Multiple regression analysis revealed that bedtime and snacking frequency were mutually independent risk factors for dental caries. No correlations were found between the prevalence of dental caries and other measurement items. The number of caries correlated with the regularity of supper time and age in subjects with permanent dentition. CONCLUSION: Children with daily life habits associated with eveningness have a higher prevalence of dental caries.

Immunolocalization of podoplanin/E11/gp38, CD44, and endomucin in the odontoblastic cell layer of murine tooth germs.

In this study, we attempted to localize the immunoreactivities of podoplanin/E11/gp38 and CD44, a counterpart possessing a high affinity to podoplanin/E11/gp38, as well as endomucin-immunoreactive blood vessels in the regions of odontoblast layers and the underlying sub-odontoblastic layers in murine tooth germs. Endomucin-reactive small blood vessels were scattered throughout the dental papillae of the tooth germs at postnatal day 1 but came to be localized close to the odontoblast/sub-odontoblastic layers until day 3. After postnatal day 5, small blood vessels were seen in odontoblast cell layers, while blood vessels with relatively larger diameters were seen forming in sub-odontoblastic layers. Immunoreactivities of podoplanin/E11/gp38 and CD44 were not detectable in the cells of dental papillae facing the inner enamel epithelium at postnatal day 1. However, at around postnatal days 3-5, podoplanin/E11/gp38 was localized in the odontoblast layer but not in the sub-odontoblastic layer, whereas CD44 was observed in the sub-odontoblastic layer but not in the odontoblast layer. The exclusive immunolocalization of podoplanin/E11/gp38 and CD44 in the odontoblast layers and sub-odontoblastic layers was seen after postnatal day 3 of the tooth germs, when the mesenchymal cells of dental papillae have already differentiated into mature odontoblasts at the cusp tip. Taken together, it seems likely that endomucin-reactive small blood vessels extended to the podoplanin/E11/gp38-positive odontoblast layers, whereas endomucin-reactive large blood vessels were already present in CD44-immmunopositive sub-odontoblastic layer, indicating the cellular regulation on the vascularization of endomucin-reactive endothelial cells during odontogenesis of the tooth germs.

Repeated immobilization stress in the early postnatal period increases stress response in adult rats.

Repeated immobilization stress tests in the early postnatal period were performed to determine the effects on the growth of developing rats as well as the response of the HPA axis to subsequent novel stress in adulthood. In addition, effects of maternal deprivation (MD) with the same period of the exposure to immobilization stress were also examined. We used 2 different types of immobilization stress and 2 different types of MD: immobilization stress for 30 min/day from postnatal day 7 (P7) to P13 (IS7-13 group); immobilization stress for 30 min on P7 (IS7 group); MD for 30 min/day from P7 to P13 (MD7-13 group); and MD for 30 min on P7 (MD7 group). Body weights were lower in the IS7-13 group than in the control group from P10 to P50, although body weight gain in the MD7-13 group was only transiently affected. Stress-induced corticosterone levels in the IS7-13 group were higher than in the control group and did not return to baseline levels until at least 120 min after the termination of stress, whereas temporal variations of stress-induced corticosterone levels did not differ between the IS, MD7-13, MD7, and control groups. Repeated immobilization stress in the early postnatal period induced long-term effects on the growth of developing rats and stress response of the HPA axis to the novel stress in adulthood, although a single immobilization stress, periodic MD, or a single MD had little effect.

Crystal growth by fluoridated adhesive resins.

OBJECTIVE: This investigation was carried out to evaluate the long-term effects of fluoride-releasing adhesive resins on structural changes in standardized fluid-filled gaps simulating microleakage between the materials and the tooth surface in vitro. METHODS: Three commercially available fluoride-releasing resin adhesives (One-Up Bond F, OptiBond Solo, and Reactmer Bond) were used in this study. Cured disks of resin adhesive were placed over flat human tooth surfaces (enamel and dentin), separated by a standardized 40microm interfacial gap and stored in distilled water for 24h (control group) or 1000 days (experimental group). After 1000 days of water storage, the resins were detached from the teeth and the opposing surfaces were examined by scanning electron microscopy (SEM). In addition, chemical structural analysis was performed by laser-Raman spectroscopy. RESULTS: The SEM microphotographs showed numerous crystal types on the enamel, dentin, and resin surfaces after 1000 days of water storage for OptiBond Solo and Reactmer Bond. However, there was no crystal formation in the control specimens and the aged specimens of One-Up Bond F. Raman analysis showed several peaks (463, 618, and 990cm(-1)) from the crystals of OptiBond Solo that were not identified in the enamel, dentin, or cured resin. SIGNIFICANCE: In conclusion, two of the three tested fluoride-release resin adhesives (OptiBond Solo and Reactmer Bond) have the ability to induce crystal growth within gaps between the adhesive and teeth in long-term water storage. These results suggest that the two adhesive resins have self-reparative ability with regard to bond leakage.

Effect of water on bonding of one-bottle self-etching adhesives.

This study evaluated the effect of water on dentin substrate bonding of one-bottle self-etching adhesives. Dentin substrates were divided into two groups: wet and dry dentin. Wet dentin is the normal substrate for bond testing whereas dry dentin was dehydrated in a desiccator for 24 hours. Bonded dehydrated teeth were then divided into two subgroups: stored in water or in desiccator for 24 hours. Microtensile bond strength of resin to dentin was measured using three one-bottle self-etching adhesives. In addition, nanoleakage evaluation was performed through the analyses of SEM and TEM micrographs. The bond strength of dry-dentin group was significantly greater than that of wet-dentin. Further, the amount of nanoleakage within the adhesive interface of dry-dentin group was less than that of wet-dentin. Results showed that bond strength and nanoleakage formation depended on the bonding substrate (wet versus dry dentin) before bonding. One-bottle self-etching adhesives might suck the water from dentinal tubules during bonding by osmosis, leading to nanoleakage formation and thus a decline in bond strength.

Calcium Charge and Release of Conventional Glass-Ionomer Cement Containing Nanoporous Silica.

The aim of this study was to evaluate calcium charge and release of conventional glass-ionomer cement (GIC) containing nanoporous silica (NPS). Experimental specimens were divided into two groups: the control (GIC containing no NPS) and GIC-NPS (GIC containing 10 wt % NPS). The specimens were immersed in calcium chloride solutions of 5 wt % calcium concentration for 24 h at 37 °C, whereupon the calcium ion release of the specimens was measured. The calcium ion release behavior of GIC-NPS after immersion in the calcium solution was significantly greater than that of the control. Scanning electron microscopy and electron-dispersive X-ray spectroscopy results indicated that calcium penetrated inside the GIC-NPS specimen, while the calcium was primarily localized on the surface of the control specimen. It was demonstrated that NPS markedly improved the calcium charge and release property of GIC.