Hypercholesterolemic Dysregulation of Calpain in Lymphatic Endothelial Cells Interferes With Regulatory T-Cell Stability and Trafficking.

BACKGROUND: Although hypercholesterolemia reportedly counteracts lymphocyte trafficking across lymphatic vessels, the roles of lymphatic endothelial cells (LECs) in the lymphocyte regulations remain unclear. Previous studies showed that calpain-an intracellular modulatory protease-interferes with leukocyte dynamics in the blood microcirculation and is associated with hypercholesterolemic dysfunction in vascular endothelial cells. METHODS: This study investigated whether the calpain systems in LECs associate with the LEC-lymphocyte interaction under hypercholesterolemia using gene-targeted mice. RESULTS: Lipidomic analysis in hypercholesterolemic mice showed that several lysophospholipids, including lysophosphatidic acid, accumulated in the lymphatic environment. Lysophosphatidic acid enables the potentiation of calpain systems in cultured LECs, which limits their ability to stabilize regulatory T cells (Treg) without altering Th1/Th2 (T helper type1/2) subsets. This occurs via the proteolytic degradation of MEKK1 (mitogen-activated protein kinase kinase kinase 1) and the subsequent inhibition of TGF (transforming growth factor)-ß1 production in LECs. Targeting calpain systems in LECs expanded Tregs in the blood circulation and reduced aortic atherosclerosis in hypercholesterolemic mice, concomitant with the reduction of proinflammatory macrophages in the lesions. Treg expansion in the blood circulation and atheroprotection in calpain-targeted mice was prevented by the administration of TGF-ß type-I receptor inhibitor. Moreover, lysophosphatidic acid-induced calpain overactivation potentiated the IL (interleukin)-18/NF-κB (nuclear factor κB)/VCAM1 (vascular cell adhesion molecule 1) axis in LECs, thereby inhibiting lymphocyte mobility on the cells. Indeed, VCAM1 in LECs was upregulated in hypercholesterolemic mice and human cases of coronary artery disease. Neutralization of VCAM1 or targeting LEC calpain systems recovered afferent Treg transportation via lymphatic vessels in mice. CONCLUSIONS: Calpain systems in LECs have a key role in controlling Treg stability and trafficking under hypercholesterolemia.

In vitro investigation of the effects of Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 exopolysaccharides on tight junction damage caused by influenza virus infection.

It is a problem that influenza virus infection increases susceptibility to secondary bacterial infection in lungs leading to lethal pneumonia. We previously reported that exopolysaccharides (EPS) derived from Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 (OLL1073R-1) could prevent against influenza virus infection followed by secondary bacterial infection in vitro. Therefore, the present study assessed whether EPS derived OLL1073R-1 protects the alveolar epithelial barrier disfunction caused by influenza virus infection. After A549 cells treated with EPS or without EPS were infected influenza virus A/Puerto Rico/8/34 (IFV) for 12 h, the levels of tight junction genes expression and inflammatory genes expression were measured by reverse transcription polymerase chain reaction. As results, EPS treatment could protect against low-titer IFV infection, but not high-titer IFV infection, followed by suppression of the increased expression of inflammatory cytokine gene levels and recovery of the decrease in the expression level of ZO-1 gene that was caused by low-titer IFV infection, leading to an improvement trend in the barrier function. Our findings showed that EPS derived from OLL1073R-1 could inhibit low-titer IFV infection leading to maintenance of the epithelial barrier function through the suppression of inflammatory cytokine genes expression.

Fast-track preparation of lung specimens for electron microscope observations of the pulmonary endothelial glycocalyx.

The glycocalyx (GCX) covers the luminal surface of blood vessels and regulates vascular permeability. As GCX degradation predicts various types of vasculopathy, confirming the presence of this structure is useful for diagnosis. Since the GCX layer is very fragile, careful fixation is necessary to preserve its structure. We explored appropriate and feasible methodologies for visualizing the GCX layer using lung tissue specimens excised from anesthetized mice. Each specimen was degassed and immersed in Alcian blue (ALB) fixative solution, and then observed using electron microscopy. Specimens from septic mice were prepared as negative GCX controls. Using these immersion-fixed specimens, the GCX layer was successfully observed using both transmission and scanning electron microscopy; these observations were similar to those obtained using the conventional method of lanthanum perfusion fixation. Spherical aggregates of GCX were observed in the septic mouse specimens, and the GCX density was lower in the septic specimens than in the non-septic specimens. Of note, the presently reported methodology reduced the specimen preparation time from 6 to 2 days. We, therefore, concluded that our novel method could be applied to human lung specimens and could potentially contribute to the further elucidation of vasculopathies.

Gemcitabine alters sialic acid binding of the glycocalyx and induces inflammatory cytokine production in cultured endothelial cells.

Gemcitabine (GEM) is an anticancer drug inhibiting DNA synthesis. Glomerular thrombotic microangiopathy (TMA) has been reported as an adverse effect. However, the precise mechanism of GEM-induced endothelial injury remains unknown. Cultured human umbilical vein endothelial cells (HUVECs) in the confluent phase were exposed to GEM (5-100 µM) for 48 h and evaluated cell viability and morphology, lectin binding concerning sialic acid of endothelial glycocalyx (GCX), and immunofluorescent staining of platelet-endothelial cell adhesion molecule (PECAM) and vascular endothelial growth factor receptor 2 (VEGFR2). The mRNA expression of α2,6-sialyltransferase (ST6Gal1), sialidase (neuraminidase-1: NEU-1), and interleukin (IL)-1ß and IL-6 was also evaluated. GEM exposure at 5 µM induced cellular shrinkage and intercellular dissociation, accompanied by slight attenuation of PECAM and VEGFR2 immunostaining, although cell viability was still preserved. At this concentration, lectin binding showed a reduction of terminal sialic acids in endothelial GCX, probably associated with reduced ST6Gal1 mRNA expression. IL-1ß and IL-6 mRNA expression was significantly increased after GEM exposure. GEM reduced terminal sialic acids in endothelial GCX through mRNA suppression of ST6Gal1 and induced inflammatory cytokine production in HUVECs. This phenomenon could be associated with the mechanism of GEM-induced TMA.

Three-dimensional Maxillofacial Morphology Measurements in Japanese Adults with Normal Occlusion.

Accurate orthodontic analysis and diagnosis based on anatomical landmarks is essential to the success of orthodontic treatment. Helical computed tomography (CT) has evolved markedly, and dentists can now quickly obtain 3-dimensional (3D) reconstruction data using this imaging modality. The planning of orthodontic treatment had traditionally been based on cephalometric analysis using 2D landmarks. This study aimed to collect 3D morphological data using CT images to establish new landmarks for analysis and diagnosis in orthodontic treatment. Twenty male and 20 female adult Japanese dry skulls with of normal occlusion were selected. The skulls were scanned using a multidetector helical CT system (SIEMENS, Volume Zoom Plus 4, Germany). Models were reconstructed using 3D measurement software (Simplant, Dentsply Sirona, Tokyo, Japan) and 45 landmarks determined. Three-dimensional measurement for a total of 30 items representing these landmarks was then performed. The results provided 3D standard values for maxillofacial morphology in adult Japanese individuals with normal occlusion. These measurement items should allow the disadvantages of 2D cephalometric analysis to be overcome.

A morphological study of adipose-derived stem cell sheets created with temperature-responsive culture dishes using scanning electron microscopy.

Adipose-derived stem cell (ADSC) sheets have potential to be effective in various therapies. In this study, we first demonstrated that a cell sheet composed of human ADSCs could be created using a new temperature-responsive culture dish from the DIC Corporation. The dish can cause detachment of adherent cells due to temperature changes, but a few morphological analyses have evaluated the presence or absence of damage on the detached surface of cell sheet. To characterize our ADSC sheet, we tried to observe the surface of ADSC sheets with scanning electron microscope (SEM) using the ionic liquid, which enables the rapid preparation of samples. No damage was found on the surface of the ADSC sheets on the side that had been in contact with the surface of the culture dishes. In addition, when the transcriptomes of the harvested cell sheets were compared with those of monolayer cultures, no up-regulation of cell death related genes were detected. These results propose that the detachment from temperature-responsive culture dish causes no serious damage on the prepared ADSC sheet. It is also suggested that the SEM with ionic liquids is a useful and rapid method for the analysis of ADSC sheets for therapy.

Three-dimensional electron microscopy for endothelial glycocalyx observation using Alcian blue with silver enhancement.

Glycocalyx (GCX) is a thin layer of negatively charged glycoproteins that covers the vascular endothelial surface and regulates various biological processes. Because of the delicate and fragile properties of this structure, it is difficult to detect GCX morphologically. We established a simple method for a three-dimensional visualization of endothelial GCX using low-vacuum scanning electron microscopy (LVSEM) on formalin-fixed paraffin-embedded (FFPE) sections. Mouse kidney tissue was fixed with 10% buffered formalin containing 1% Alcian blue (ALB) via perfusion and immersion. FFPE sections were observed by light microscopy (LM) and LVSEM, and formalin-fixed epoxy resin-embedded ultrathin sections were observed by transmission electron microscopy (TEM). The endothelial GCX from various levels of kidney blood vessels was stained blue in LM and confirmed as a thin osmiophilic layer in TEM. In LVSEM, the sections stained by periodic acid methenamine silver (PAM) revealed the endothelial GCX as a layer of dense silver-enhanced particles, in both the samples fixed via perfusion and immersion. Correlative light and electron microscopy (CLEM) revealed the fine visible structure of endothelial GCX. This simple method using FFPE samples with ALB will enable the three-dimensional evaluation of endothelial GCX alterations in various human diseases associated with endothelial injury in future studies.

Three-Dimensional Analysis of Soft and Hard Tissue Changes following Orthognathic Surgery.

Change in soft tissue in relation to that in hard tissue following orthognathic surgery was evaluated. Twenty-five patients were enrolled in the study. The diagnosis in all was jaw deformity (maxillary retrusion and mandibular protrusion) and all underwent a Le Fort I osteotomy and bilateral sagittal splitting ramus osteotomy. Three-dimensional (3D) computer-aided design (CAD) models (polygon models) of the hard and soft tissue of the maxilla and mandible were constructed and superimposed. Reference points were established on the pre- and postoperative hard and soft tissues. Specific elements of each reference point were divided into X, Y, and Z components, respectively, and the distances in each direction and 3D distance (normal distance) measured. The Wilcoxon signed-rank test was used to determine differences in the mean values for the distance moved of each element as the error between pre- and postoperatively. The results revealed statistically significant differences in the Y-direction in the maxilla and the X- and Z-directions in the mandible. A significant difference was also observed in the 3D distances of the maxilla and mandible. Little evidence was found of linearity between the amount of hard and soft tissue movement in the X- and Z-directions in the maxilla. This means that 3D movement in the maxilla was masked more by changes in the morphology of the soft tissue than in the mandible, making it less evident. The results of this study suggest that the 3D analysis method used enables changes in hard and soft tissues to be understood qualitatively, and that it can be used in diagnosis and treatment in orthognathic surgery. It may also be useful in simulation of morphological change in soft tissue.

Three-Dimensional Evaluation of Pharyngeal Morphology in Bimaxillary Surgery with and without Horseshoe Osteotomy in Skeletal Class III Cases.

Repositioning of the jaw in orthognathic treatment generates changes in the soft tissues of the maxillofacial region, with consequent changes in the airway. The purpose of this study was to determine how type of orthognathic surgical procedure affected the 3-dimensional morphology of the upper airway. Forty patients were divided into the following 2 groups according to the type of surgical procedure used: a horseshoe osteotomy (HS) group (20 patients, comprising 11 men and 9 women; average age 24.3±4.5 years) who underwent bimaxillary surgery; and a LeFort I osteotomy (LF) group (20 patients, comprising 8 men and 12 women; average age 22.5±4.6 years) who also underwent bimaxillary surgery. Cephalometric measurements were taken and 3-dimensional pharyngeal morphology evaluated in each group. The amounts of maxilla rotation, posterior maxilla impaction, and mandibular setback all revealed a significantly larger value in the HS group. Evaluation of pharyngeal volume revealed a significant decrease in the upper pharyngeal segment in the LF group. A significant decrease in the lower pharyngeal segment was observed in both groups. Differences were noted in postoperative pharyngeal morphology between the two groups. The results of this study suggest that HS has less effect on the upper pharyngeal segment, regardless of the amount of posterior maxilla impaction.

Circulating oxidized LDL, increased in patients with acute myocardial infarction, is accompanied by heavily modified HDL.

Oxidized LDL (oxLDL) is a known risk factor for atherogenesis. This study aimed to reveal structural features of oxLDL present in human circulation related to atherosclerosis. When LDL was fractionated on an anion-exchange column, in vivo-oxLDL, detected by the anti-oxidized PC (oxPC) mAb, was recovered in flow-through and electronegative LDL [LDL(-)] fractions. The amount of the electronegative in vivo-oxLDL, namely oxLDL in the LDL(-) fraction, present in patients with acute MI was 3-fold higher than that observed in healthy subjects. Surprisingly, the LDL(-) fraction contained apoA1 in addition to apoB, and HDL-sized particles were observed with transmission electron microscopy. In LDL(-) fractions, acrolein adducts were identified at all lysine residues in apoA1, with only a small number of acrolein-modified residues identified in apoB. The amount of oxPC adducts of apoB was higher in the LDL(-) than in the L1 fraction, as determined using Western blotting. The electronegative in vivo-oxLDL was immunologically purified from the LDL(-) fraction with an anti-oxPC mAb. The majority of PC species were not oxidized, whereas oxPC and lysoPC did not accumulate. Here, we propose that there are two types of in vivo-oxLDL in human circulating plasma and the electronegative in vivo-oxLDL accompanies oxidized HDL.

Extracellular Vesicles Produced by Bifidobacterium longum Export Mucin-Binding Proteins.

Extracellular proteins are important factors in host-microbe interactions; however, the specific factors that enable bifidobacterial adhesion and survival in the gastrointestinal (GI) tract are not fully characterized. Here, we discovered that Bifidobacterium longum NCC2705 cultured in bacterium-free supernatants of human fecal fermentation broth released a myriad of particles into the extracellular environment. The aim of this study was to characterize the physiological properties of these extracellular particles. The particles, approximately 50 to 80 nm in diameter, had high protein and double-stranded DNA contents, suggesting that they were extracellular vesicles (EVs). A proteomic analysis showed that the EVs primarily consisted of cytoplasmic proteins with crucial functions in essential cellular processes. We identified several mucin-binding proteins by performing a biomolecular interaction analysis of phosphoketolase, GroEL, elongation factor Tu (EF-Tu), phosphoglycerate kinase, transaldolase (Tal), and heat shock protein 20 (Hsp20). The recombinant GroEL and Tal proteins showed high binding affinities to mucin. Furthermore, the immobilization of these proteins on microbeads affected the permanence of the microbeads in the murine GI tract. These results suggest that bifidobacterial exposure conditions that mimic the intestine stimulate B. longum EV production. The resulting EVs exported several cytoplasmic proteins that may have promoted B. longum adhesion. This study improved our understanding of the Bifidobacterium colonization strategy in the intestinal microbiome.IMPORTANCEBifidobacterium is a natural inhabitant of the human gastrointestinal (GI) tract. Morphological observations revealed that extracellular appendages of bifidobacteria in complex microbial communities are important for understanding its adaptations to the GI tract environment. We identified dynamic extracellular vesicle (EV) production by Bifidobacterium longum in bacterium-free fecal fermentation broth that was strongly suggestive of differing bifidobacterial extracellular appendages in the GI tract. In addition, export of the adhesive moonlighting proteins mediated by EVs may promote bifidobacterial colonization. This study provides new insight into the roles of EVs in bifidobacterial colonization processes as these bacteria adapt to the GI environment.

Surgical Orthodontic Treatment in Case of Severe High Angle Skeletal Class II Malocclusion and Mandibular Retrusion.

This report describes a patient with severe high angle class II malocclusion and mandibular retrusion in whom surgical orthodontic treatment to prevent an increase in ramus height resulted in a significant improvement in esthetics and long-term stability. The patient was a woman aged 30 years 5 months who presented with the chief complaint of maxillary protrusion. She had a convex facial type, a chin button on lip sealing, and a gummy smile. Cephalometric analysis revealed a normal maxilla anterior-posterior position, but significant mandibular retrusion with pronounced clockwise rotation. The anterior maxillary tooth axis was standard, but labially inclined in the mandible. Based on these findings, the diagnosis was skeletal class II high angle malocclusion and mandibular retrusion. The proposed treatment plan comprised 2-jaw surgery with premolar extraction. Le Fort I osteotomy, in particular, was planned in the maxilla to move the ANS upward by 3.0 mm and the PNS downward by 3.0 mm. Sagittal split ramus osteotomy (SSRO) was planned to adjust the mandible and move the mandible forward by 10.0 mm. To prevent postoperative relapse, the short lingual split method was used in performing the SSRO. The mandible was split to minimize stretching of the median pterygoid muscle. Postoperatively, the ANS, PNS, and pogonion showed movement of 2.0 mm upward, 3.0 mm downward, and 8.0 mm forward, respectively. Additionally, lip closure was now natural, and the gummy smile had markedly improved. At 6 years postoperatively, there has been no change skeletally or dentally. Follow-up is being continued to monitor further progress.

Dysregulation of Intestinal Microbiota Elicited by Food Allergy Induces IgA-Mediated Oral Dysbiosis.

Food allergy is a life-threatening response to specific foods, and microbiota imbalance (dysbiosis) in gut is considered a cause of this disease. Meanwhile, the host immune response also plays an important role in the disease. Notably, interleukin 33 (IL-33) released from damaged or necrotic intestinal epithelial cells facilitates IL-2-producing CD4 helper T (Th2) responses. However, causal relationships between the gut and oral dysbiosis and food allergy remain unknown. In this study, we analyzed effects of gut and oral dysbiosis on development of food allergy. A murine model of food allergy was established via ovalbumin (OVA) injection in BALB/c mice. Viable fecal bacteria were identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). il33 expression in colon-26 mouse colon cells stimulated by isolated fecal bacteria was quantified by real-time PCR. Intestinal T cells from the mice were analyzed by flow cytometry. Salivary IgA levels were quantified by enzyme-linked immunosorbent assay (ELISA), and IgA-bound oral bacteria were detected by flow cytometry. Among fecal bacteria, the abundance of Citrobacter sp. increased in the feces of allergic mice and induced il33 expression in colon-26 cells. Orally administered Citrobacter koseri JCM1658 exacerbated systemic allergic symptoms and reduced intestinal Th17 cells. Salivary IgA and IgA-bound oral bacteria increased in the allergic mice. Based on the results described above, food allergy induced both gut and oral dysbiosis. Citrobacter sp. aggravated allergy symptoms by inducing IL-33 release from intestinal epithelial cells.

Inhibition of ß-amyloid-induced neurotoxicity by planar analogues of procyanidin B3.

Reactive oxygen species (ROS) are known to be produced during the amyloid beta (Aß) aggregation process. Both ROS production and Aß fibril formation can result in nerve cell injury. Proanthocyanidins are oligomers of catechin that can act as inhibitors of Aß aggregation. Procyanidin B3 (Cat-Cat), the dimer of (+)-catechin, can easily cross the blood-brain barrier. Previously, we synthesized two derivatives of Cat-Cat, namely Cat-PCat and PCat-PCat, in which the geometry of one or both catechin molecules in Cat-Cat was constrained to be planar. The antioxidative activities of Cat-PCat and PCat-PCat were found to be stronger than that of Cat-Cat, with PCat-PC at exhibiting the most potent activity. These compounds are predicted to protect against Aß-induced neurotoxicity via inhibition of Aß aggregation as well as by antioxidative effects toward Aß-induced intracellular ROS generation. PCat-PCat exhibited the most potent neuroprotective effects against Aß-induced cytotoxicity, which resulted from inhibition of ß-sheet structure formation during the Aß aggregation process. PCat-PCat may be a promising lead compound for the treatment of Alzheimer's disease.

Podocyte penetration of the glomerular basement membrane to contact on the mesangial cell at the lesion of mesangial interposition in lupus nephritis: a three-dimensional analysis by serial block-face scanning electron microscopy.

BACKGROUND: The interaction among the glomerular components plays an important role in the development of glomerular lesions; thus, investigation of the ultrastructural three-dimensional (3D) configuration of the human glomerular cells and extracellular matrix (ECM) is important for understanding the pathogenesis of glomerulosclerosis, especially glomerulonephritis. METHODS: We applied a new technique of serial block-face scanning electron microscopy (SBF-SEM), which helps to acquire serial electron microscopic images to reconstruct a 3D ultrastructure, to a human kidney biopsy specimen obtained from a 25-year-old woman with lupus nephritis. RESULTS: SBF-SEM demonstrated that the cytoplasmic processes of the podocyte penetrated into the lamina densa of the glomerular basement membrane, and was in direct contact with the cytoplasm of mesangial cells at the site of mesangial interposition. CONCLUSION: Although this is a single-case observational study, SBF-SEM revealed a unique 3D configuration, suggesting a novel mechanism of direct intercellular cross-communication between podocytes and mesangial cells, aside from the presumed paracrine communication.

Three-dimensional Analysis and Evaluation in Orthognathic Surgical Cases with Facial Asymmetry.

Two-dimensional cephalometric analysis is commonly used in planning and evaluating the outcome of orthognathic surgery. It is difficult to arrive at an accurate evaluation with this method, however, as the jaw bones overlap in profile. Therefore, the purpose of this study was to use 3-dimensional (3-D) orthognathic treatment planning software for measurement of distances and angles to evaluate change in dentofacial morphology and stability after orthognathic surgery in patients with jaw deformity and facial asymmetry. Computed tomography with SimPlant O&O® (Materialize Dental, Belgium) was used to obtain data at before surgery (T1) and at 1 month (T2) and 1-2 years postoperatively (T3). Reference points, reference planes, and evaluation items were set to measure angles and distances. The average values at T1, T2, and T3 and the standard deviations were obtained. The analyses of change in distance and angle between T1 and T2 in both the maxilla and mandible revealed that the amount of postoperative deviation and change was greatest in the mandible (p<0.05). These findings showed a correlation between postoperative change in position of the chin and symmetry. Only a minimal amount of change was observed between T2 and T3 in both the maxilla and mandible. The results also revealed a correlation between positional change in both the proximal and distal bone segments and stability. It was found to be possible to determine change in horizontal, vertical, and anterior/posterior angle in both the maxilla and mandible by such 3-D analysis. The results suggest that the chin is the most appropriate facial element for evaluation of symmetry after orthognathic surgery.

Three-dimensional Finite Element Analysis of Bone Fixation in Bilateral Sagittal Split Ramus Osteotomy Using Individual Models.

The purpose of this study was to investigate factors involved in stress on locking mini-plate/screws used in orthognathic surgery based on patient-specific 3-dimensional finite element analysis. Data were obtained from 10 patients undergoing mandibular advancement by bilateral sagittal split ramus osteotomy. All underwent osteosynthesis with 2.0-mm titanium locking mini-plate/screws. A 3-dimensional finite element model of the mandible was created for each patient and each model subjected to the same loading conditions, which produced different stress values on locking mini-plate/screws. When the contact area of the proximal and distal bone segments was narrower and bone mineral density (BMD) lower, the von Mises stress values on the plate/screws were higher (contact area, p<0.01; BMD, p<0.05). The present results suggest that bone contact area and BMD should be considered as plate stress factors.

Condition-specific Quality of Life Assessment at Each Stage of Class III Surgical Orthodontic Treatment -A Prospective Study.

Surgical orthodontic treatment has been reported to improve oral health-related quality of life (OHRQL). Such treatment comprises three stages: pre-surgical orthodontic treatment; orthognathic surgery; and post-surgical orthodontic treatment. Most studies have focused on change in OHRQL between before and after surgery. However, it is also necessary to evaluate OHRQL at the pre-surgical orthodontic treatment stage, as it may be negatively affected by dental decompensation compared with at pre-treatment. The purpose of this prospective study was to investigate the influence of surgical orthodontic treatment on QOL by assessing change in condition-specific QOL at each stage of treatment in skeletal class III cases. Twenty skeletal class III patients requiring surgical orthodontic treatment were enrolled in the study. Each patient completed the Orthognathic Quality of Life Questionnaire (OQLQ), which was developed for patients with dentofacial deformity. Its items are grouped into 4 domains: "social aspects of dentofacial deformity"; "facial esthetics"; "oral function"; and "awareness of dentofacial esthetics". The questionnaire was completed at the pre-treatment, pre-surgical orthodontic treatment, and post-surgical orthodontic treatment stages. The results revealed a significant worsening in scores between at pre-treatment and pre-surgical orthodontic treatment in the domains of facial esthetics and oral function (p<0.01), and between at pre-surgical orthodontic and post-surgical orthodontic treatment in all domains except awareness of dentofacial esthetics (p<0.05, p<0.01). A significant correlation was observed between a negative change in overjet and worsening OQLQ scores at the pre-surgical orthodontic treatment stage. Significant correlations were also observed between improvement in upper and lower lip difference, soft tissue pogonion protrusion, and ANB angle and improvement in OQLQ scores at the post-surgical orthodontic treatment stage. These results indicate that morphologic change influences OHRQL in patients undergoing surgical orthodontic treatment not only after surgery, but also during pre-surgical orthodontic treatment.

Implant Treatment after Sagittal Splitting Ramus Osteotomy and Alveolar Ridge Augmentation in Patient with Mandibular Prognathism and Multiple Missing Maxillary Teeth.

An abnormal maxillomandibular ridge relationship frequently hinders oral implant treatment in patients with jaw deformities. Here, we describe a patient who was experiencing difficulty using dentures due to multiple maxillary tooth loss and mandibular prognathism. Treatment comprising sagittal splitting ramus osteotomy and alveolar ridge augmentation using bone grafts harvested from the mandibular ramus followed by implant treatment yielded good outcomes. The patient was a 47-year-old woman presenting with an unstable upper partial denture. Although prior prosthetic treatment for mandibular prognathism had resulted in normal overbite, she had since lost an increasing number of teeth due to advanced periodontal disease, impairing support for the denture. She was referred to the Department of Oral Implantology at the Tokyo Dental College Chiba Hospital in October 2008. Subsequent treatment comprised implant treatment following maxillary alveolar ridge augmentation and sagittal splitting ramus osteotomy to correct the maxillary-mandibular relationship. In January 2010, sagittal splitting ramus osteotomy and alveolar bone augmentation using a bone graft from the mandibular ramus were performed under general anesthesia. In July and August 2010, a total of 7 implants were placed in the maxilla and implant superstructure preparation started after 3 months. Taking both the patient's wishes and ease of maintenance into account, retrievable superstructures made of Auro Galvano Crown were fitted in April 2011. The jaw-to-jaw alveolar ridge relationship was improved by sagittal splitting ramus osteotomy, rendering subsequent treatment, from implant placement to superstructure preparation, feasible by conventional methods. The use of surplus bone generated during sagittal splitting ramus osteotomy for bone augmentation avoided the need to harvest bone from another area.

Rapid specimen preparation to improve the throughput of electron microscopic volume imaging for three-dimensional analyses of subcellular ultrastructures with serial block-face scanning electron microscopy.

Serial block-face imaging using scanning electron microscopy enables rapid observations of three-dimensional ultrastructures in a large volume of biological specimens. However, such imaging usually requires days for sample preparation to reduce charging and increase image contrast. In this study, we report a rapid procedure to acquire serial electron microscopic images within 1 day for three-dimensional analyses of subcellular ultrastructures. This procedure is based on serial block-face with two major modifications, including a new sample treatment device and direct polymerization on the rivets, to reduce the time and workload needed. The modified procedure without uranyl acetate can produce tens of embedded samples observable under serial block-face scanning electron microscopy within 1 day. The serial images obtained are similar to the block-face images acquired by common procedures, and are applicable to three-dimensional reconstructions at a subcellular resolution. Using this approach, regional immune deposits and the double contour or heterogeneous thinning of basement membranes were observed in the glomerular capillary loops of an autoimmune nephropathy model. These modifications provide options to improve the throughput of three-dimensional electron microscopic examinations, and will ultimately be beneficial for the wider application of volume imaging in life science and clinical medicine.