Decline in Sensory Integration in Old Age and Its Related Functional Brain Connectivity Correlates Observed during a Virtual Reality Task.

Sensory integration is an essential human function whose decline impacts quality of life, particularly in older adults. Herein, we propose an arm-reaching task based on a virtual reality head-mounted display system to assess sensory integration in daily life, and we examined whether reaching task performance was associated with resting-state functional connectivity (rsFC) between the brain regions involved in sensory integration. We hypothesized that declining sensory integration would affect performance during a reaching task with multiple cognitive loads. Using a task in which a young/middle-aged group showed only small individual differences, older adults showed large individual differences in the gap angle between the reaching hand and the target position, which was used to assess sensory integration function. Additionally, rsfMRI data were used to identify correlations between rsFC and performance in older adults, showing that performance was correlated with connectivity between the primary motor area and the left inferior temporal gyrus and temporo-occipital region. Connectivity between areas is related to visuomotor integration; thus, the results suggest the involvement of visuomotor integration in the decline of sensory integration function and the validity of the gap angle during this VR reaching task as an index of functional decline.

Surgical Treatment of Furcation Involvement Associated with Recurrence of Aggressive Periodontitis: A Case Report.

Here, we report a case of generalized chronic periodontitis with furcation involvement that was treated successfully by means of surgical intervention. The patient was a 43-year-old man requesting treatment for periodontal disease. An initial examination revealed 42% of sites with a probing depth of ≥4 mm and 42.9% of sites with bleeding on probing. The maxillary molars showed varying degrees of furcation involvement. Radiographic examination revealed bone resorption in the molar and mandibular anterior teeth regions. Microbiological examination of subgingival plaque revealed the presence of Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Tannerella forsythia. The patient's oral health-related quality of life (OHRQL) was also assessed. Based on a clinical diagnosis of severe chronic periodontitis, initial periodontal therapy was performed. Plaque control, scaling and root planing, extraction, temporary fixed restoration, occlusal adjustment, and root canal treatment were implemented. Following reevaluation, open flap debridement was performed at selected sites. Root resection was performed on the distal root of #16. Prosthetic treatment was then initiated for recovery of oral function. After confirmation of appropriate occlusion and cleanability, the patient was placed on supportive periodontal therapy. Root resection improved cleanability. This clinical improvement has been adequately maintained over a 2-year period. The patient's OHRQL score showed a slight deterioration during the supportive periodontal therapy OK period, however. This indicates the need for further careful monitoring of periodontal conditions, as well as of how they are perceived by the patient themselves.

Connective Tissue Graft for Gingival Recession in Mandibular Incisor Area: A Case Report.

We report a case of gingival recession in the mandibular incisor region requiring a connective tissue graft. The patient was a 17-year-old girl who visited the Tokyo Dental College Chiba Hospital in 2014 with the chief complaint of gingival recession in the lower incisor region. She had received orthodontic treatment for 5 years and noticed the gingival recession on completion of active orthodontic treatment in 2013. Gingival recession in tooth #31 extended 3 mm beyond the muco-gingival junction (MGJ) and was clinically diagnosed as Miller Class II recession; probing depth was 6 mm. Following initial periodontal therapy, a connective tissue graft procedure was implemented. The connective tissue was harvested from the left palate. Healing was uneventful, and the grafted site showed a favorable outcome at 6 months postoperatively. We are continuing to carefully monitor the condition of periodontal tissue.

Treponema denticola invasion into human gingival epithelial cells.

Host cell invasion is important for periodontal pathogens in evading host defenses and spreading into deeper areas of the periodontal tissue. Treponema denticola has been implicated in a number of potentially pathogenic processes, including periodontal tissue penetration. Here we tested the ability of T. denticola strains to invade human gingival epithelial cells (HGEC). After 2 h infection, intracellular location of T. denticola cells was confirmed by confocal laser scanning microscopy (CLSM). Results from an antibiotic protection assay following [(3)H]uridine labeling indicated that invasion efficiency reached a maximum at 2 h after infection. Internalized T. denticola cells were still observed in HGEC at 24 h by CLSM. A dentilisin deficient mutant exhibited significantly decreased invasion (p < 0.05) compared with the wild-type strain. In inhibition assays, phenylmethylsulfonyl fluoride and metabolic inhibitors such as methyl-ß-cyclodextrin and staurosporine significantly reduced T. denticola invasion. Under CLSM, T. denticola colocalized with GM-1 ganglioside-containing membrane microdomains in a cholesterol-dependent manner. These results indicated that T. denticola has the ability to invade into and survive within HGECs. Dentilisin activity of T. denticola and lipid rafts on HGEC appear to play important roles in this process.

Evaluation of a rapid oral bacteria quantification system using dielectrophoresis and the impedance measurement.

To clarify the availability of the dielectrophoretic impedance measurement (DEPIM) system as the evaluator for oral care, we evaluated the usefulness of DEPIM system by comparison with the standard plate counting (SPC) method. First, the relationship between the DEPIM results and bacterial concentration measured by SPC was clarified. Next, the measurement of the microorganism number in a mixed suspension was evaluated with DEPIM and SPC. The bacterial counts with DEPIM strongly correlated with those with SPC (r(2)=0.633-0.997) and this correlation was also shown in the measurement of a mixed bacterial suspension (ranging from 10(5) to 10(8) cfu/ml) of two bacterial species. Moreover, the experiments using dissociating enzymes to eliminate the influence of the size of the bacterial aggregates demonstrated that the microbial measurement results with DEPIM are unaffected by bacterial aggregates. This study demonstrated that bacterial counts with DEPIM strongly correlated with those with SPC and were unaffected by bacterial aggregates.

Porphyromonas gingivalis entry into gingival epithelial cells modulated by Fusobacterium nucleatum is dependent on lipid rafts.

Host cell invasion by a major periodontal pathogen, Porphyromonas gingivalis, has been proposed as an important mechanism involved in host-pathogen interactions in periodontal and cardiovascular diseases. The present study sought to gain insight into the underlying mechanism(s) involved in previously demonstrated fusobacterial modulation of host cell invasion by P. gingivalis. An immortalized human gingival cell line Ca9-22 was dually infected with P. gingivalis ATCC 33277 and Fusobacterium nucleatum TDC 100, and intracellular invasion was assessed by scanning electron microscopy (SEM) and confocal scanning laser microscopy (CSLM). SEM observation showed that P. gingivalis and F. nucleatum formed consortia and were in the process of penetrating into Ca9-22 by 30-60 min after infection. In CSLM, Ca9-22 cells that contained both P. gingivalis and F. nucleatum were frequently observed after 2 h, although cells that contained exclusively P. gingivalis were also found. Infection by P. gingivalis and/or F. nucleatum revealed evident colocalization with a lipid raft marker, GM1-containing membrane microdomains. In an antibiotic protection assay, depletion of epithelial plasma membrane cholesterol resulted in a significant reduction of recovered P. gingivalis or F. nucleatum (∼33% of untreated control; p < 0.001). This inhibition was also confirmed by CSLM. Sequential infection experiments showed that timing of infection by each species could critically influence the invasion profile. Co-infection with F. nucleatum significantly enhanced host cell invasion by P. gingivalis 33277, its serine phophatase SerB mutant and complemented strains, suggesting that the SerB does not play a major role in this fusobacterial enhancement of P. gingivalis invasion. Thus, the interaction between F. nucleatum and host cells may be important in the fusobacterial enhancement of P. gingivalis invasion. Collectively, these results suggest that lipid raft-mediated process is at least one of the potential mechanisms involved in fusobacterium-modulated host cell invasion by P. gingivalis.

Exposure of P. gingivalis to noradrenaline reduces bacterial growth and elevates ArgX protease activity.

OBJECTIVE: Periodontitis, an infectious disease caused by periodontopathic bacteria, including Porphyromonas gingivalis, is reported to be accelerated by stress, under which noradrenaline levels are increased in the bloodstream. The purpose of this study was to evaluate the effects of noradrenaline on P. gingivalis. DESIGN: P. gingivalis was incubated in the presence of 25µM, 50µM, or 100µM adrenaline or noradrenaline at 37°C for 12, 24 or 36h and growth was evaluated by OD(660). Auto-inducer-2 (AI-2) was measured by luminescence of Vibrio harveyi BB 170. Expression of P. gingivalis genes was evaluated using a microarray and RT-PCR. Rgp activity of arg-gingipainA and B (Rgp) was measured with a synthetic substrate. RESULTS: Growth of P. gingivalis FDC381 was inhibited by noradrenaline at 24 and 36h. Growth inhibition by noradrenaline increased dose-dependently. Inhibition of growth partially recovered with addition of propranolol. AI-2 production from P. gingivalis showed a marked decrease with addition of noradrenaline compared with peak production levels in the control group. Microarray analysis revealed an increase in expression in 18 genes and a decrease in expression in 2 genes. Amongst these genes, expression of the protease arg-gingipainB (RgpB) gene, a major virulence factor of P. gingivalis, was further analysed. Expression of rgpB showed a significant increase with addition of noradrenaline, which was partially reduced by addition of propranolol. Cell-associated Rgp activity also increased with addition of noradrenaline. CONCLUSIONS: These results suggest that stressors influence the expression of the virulence factors of P. gingivalis via noradrenaline.

Dentipain, a Streptococcus pyogenes IdeS protease homolog, is a novel virulence factor of Treponema denticola.

Treponema denticola is a major pathogen of chronic periodontitis. Analysis of the T. denticola genome revealed a gene orthologous with a cysteine protease-encoding gene from Streptococcus pyogenes (IdeS). IdeS interferes with IgG-dependent opsonophagocytosis by specific cleavage of IgG molecules. Analysis of this gene (termed ideT) revealed it to encode a two-domain protein whose N-terminus is composed of tandem immunoglobulin-like domains followed by a C-terminal IdeS-like protease domain. In this study we show that during secretion the IdeT protein is processed into an N-terminal fragment which remains associated with the cell, and a C-terminal part released into the medium. Although the secreted domain of IdeT, termed dentipain, shows only 25% identity to the IdeS protease, the putative catalytic cysteine and histidine residues are strongly conserved. Recombinant dentipain cleaves the insulin ß-chain, an activity which is inhibited by E-64, a diagnostic inhibitor of cysteine proteases. Apart from insulin no cleavage of other protein substrates was detected, suggesting that dentipain has oligopeptidase activity. A mutant strain was constructed expressing a modified IdeT variant, the dentipain domain of which was deleted. This strain was found to be significantly reduced in its abscess-forming activity compared with the parental strain in a murine abscess model, suggesting that dentipain contributes to the virulence of T. denticola.

Differential ability of periodontopathic bacteria to modulate invasion of human gingival epithelial cells by Porphyromonas gingivalis.

Periodontitis is a polymicrobial infection caused by selected gram-negative bacteria including Porphyromonas gingivalis. Host cell invasion by P. gingivalis has been proposed as a possible mechanism of pathogenesis in periodontitis. The aim of the present study was to assess the influence of periodontopathogens on P. gingivalis invasion of gingival epithelial cells in polymicrobial infection. P. gingivalis was tested for its ability to invade a human gingival epithelial cell line Ca9-22 in co-infection with periodontopathogens, using an antibiotic protection assay. Among the pathogens tested, only Fusobacterium nucleatum demonstrated the ability to significantly promote P. gingivalis invasion (P<0.01). This increased invasion was confirmed by confocal scanning laser microscopy utilizing a dual labeling technique. In contrast, co-infection with Aggregatibacter actinomycetemcomitans or Tannerella forsythia attenuated P. gingivalis invasion. The fusobacterial enhancement of host cell invasion was not observed in co-incubation with other periodontopathogens tested. These results suggested that complex synergistic or antagonistic physiologic mechanisms are intimately involved in host cell invasion by P. gingivalis in polymicrobial infection.

The OxyR homologue in Tannerella forsythia regulates expression of oxidative stress responses and biofilm formation.

Tannerella forsythia is an anaerobic periodontal pathogen that encounters constant oxidative stress in the human oral cavity due to exposure to air and reactive oxidative species from coexisting dental plaque bacteria as well as leukocytes. In this study, we sought to characterize a T. forsythia ORF with close similarity to bacterial oxidative stress response sensor protein OxyR. To analyse the role of this OxyR homologue, a gene deletion mutant was constructed and characterized. Aerotolerance, survival after hydrogen peroxide challenge and transcription levels of known bacterial antioxidant genes were then determined. Since an association between oxidative stress and biofilm formation has been observed in bacterial systems, we also investigated the role of the OxyR protein in biofilm development by T. forsythia. Our results showed that aerotolerance, sensitivity to peroxide challenge and the expression of oxidative stress response genes were significantly reduced in the mutant as compared with the wild-type strain. Moreover, the results of biofilm analyses showed that, as compared with the wild-type strain, the oxyR mutant showed significantly less autoaggregation and a reduced ability to form mixed biofilms with Fusobacterium nucleatum. In conclusion, a gene annotated in the T. forsythia genome as an oxyR homologue was characterized. Our studies showed that the oxyR homologue in T. forsythia constitutively activates antioxidant genes involved in resistance to peroxides as well as oxygen stress (aerotolerance). In addition, the oxyR deletion attenuates biofilm formation in T. forsythia.

Fusobacterium nucleatum enhances invasion of human gingival epithelial and aortic endothelial cells by Porphyromonas gingivalis.

Invasion by Porphyromonas gingivalis has been proposed as a possible mechanism of pathogenesis in periodontal and cardiovascular diseases. Porphyromonas gingivalis have direct access to the systemic circulation and endothelium in periodontitis patients by transient bacteremia. Periodontitis can be described as one of the predominant polymicrobial infections of humans. In the present study, P. gingivalis strains were tested for their ability to invade a human gingival epithelial cell line (Ca9-22) and human aortic endothelial cells in coinfection with Fusobacterium nucleatum using antibiotic protection assays. Coinfection with F. nucleatum resulted in 2-20-fold increase in the invasion of host cells by P. gingivalis strains. The invasive abilities of P. gingivalis strains were significantly greater when incubated with a F. nucleatum clinical isolate (which possesses strong biofilm-forming ability), than when incubated with a F. nucleatum-type strain. In inhibition assays with metabolic inhibitors, a difference in inhibition profiles was observed between mono- and polymicrobial infections. Collectively, our results suggest that F. nucleatum facilitates invasion of host cells by P. gingivalis. Investigations of polymicrobial infection of host cells should improve our understanding of the role of P. gingivalis in periodontal infection and proatherogenic mechanisms.

Role of a Tannerella forsythia exopolysaccharide synthesis operon in biofilm development.

Tannerella forsythia is a Gram-negative oral anaerobe implicated in the development of periodontitis, a chronic inflammatory disease induced by bacterial infections which leads to tooth loss if untreated. Since biofilms formed by periodontal bacteria are considered important in disease progression and pose difficulties in treatment, we sought to investigate the underlying mechanisms of T. forsythia biofilm formation. This was carried out by screening random insertion mutants of T. forsythia for alterations in biofilm development. This approach lead to the identification of an operon involved in exopolysaccharide (EPS) synthesis. An isogenic mutant of one of the genes, wecC, contained within the operon was constructed. The isogenic wecC mutant showed increased ability to form biofilms as compared to the parent strain. The wecC mutant also formed aggregated microcolonies and showed increased cell-surface associated hydrophobicity as compared to the parent strain. Moreover, biochemical characterization of the wecC mutant indicated that glycosylation of surface glycoproteins was reduced. Therefore, our results suggest that the wecC operon is associated with glycosylation of surface-glycoprotein expression and likely plays an inhibitory role in T. forsythia biofilm formation.

Porphyromonas gingivalis vesicles enhance attachment, and the leucine-rich repeat BspA protein is required for invasion of epithelial cells by "Tannerella forsythia".

The human oral cavity harbors more than 500 species of bacteria. Periodontitis, a bacterially induced inflammatory disease that leads to tooth loss, is believed to result from infection by a select group of gram-negative periodontopathogens that includes Porphyromonas gingivalis, Treponema denticola, and "Tannerella forsythia" (opinion on name change from Tannerella forsythensis pending; formerly Bacteroides forsythus). Epithelial cell invasion by periodontopathogens is considered to be an important virulence mechanism for evasion of the host defense responses. Further, the epithelial cells with invading bacteria also serve as reservoirs important in recurrent infections. The present study was therefore undertaken to address the epithelial cell adherence and invasion properties of T. forsythia and the role of the cell surface-associated protein BspA in these processes. Further, we were interested in determining if P. gingivalis, one of the pathogens frequently found associated in disease, or its outer membrane vesicles (OMVs) could modulate the epithelial cell adherence and invasion abilities of T. forsythia. Here we show that epithelial cell attachment and invasion by T. forsythia are dependent on the BspA protein. In addition, P. gingivalis or its OMVs enhance the attachment and invasion of T. forsythia to epithelial cells. Thus, interactions between these two bacteria may play important roles in virulence by promoting host cell attachment and invasion.

Production of protective antibodies against Porphyromonas gingivalis strains by immunization with recombinant gingipain domains.

We studied the effect of antibodies against Porphyromonas gingivalis gingipain domains, preparing them against three recombinant fragments of RgpA (catalytic domain, r-Rgp CAT; hemagglutinin domains, r-Rgp 44 and r-Rgps 15-27) and one fragment of Kgp (catalytic domain, r-Kgp CAT). Enhancement of opsonization and killing by human polymorphonuclear leukocytes were measured in the noninvasive FDC 381 and invasive W50 strains of P. gingivalis. Anti-r-Rgp 44 was the most effective in both strains of P. gingivalis. The present findings lead us to recommend RgpA 44 as a candidate immunogen for vaccines against P. gingivalis.

Vascular changes associated with chorioretinal and optic nerve colobomas in rats (Crj: CD(SD), IGS).

OBJECTIVE: Three female adult rats (Crj: CD(SD) IGS) with colobomatous anomalies were investigated. MATERIALS AND METHODS: The microvascular changes of the coloboma were studied using the techniques of fluorescein angiography, histology and scanning electron microscopy (SEM) of vascular corrosion casts. RESULTS: Fluorescein angiography revealed the pits of the optic disk as a dark hole with some abnormalities in vessel arrangement. Light microscopy confirmed the presence of attenuated lamina cribrosa, retinal dysplasia and marked dilation of the retinal veins. SEM revealed that the optic disk coloboma formed a crater-like pit and that central retinal vessels ran a tortuous course along the bottom and side of the crater. Capillaries in the optic nerve head were missing in the affected area. The central retinal veins were thick and had various changes such as strangulation, rough surface structures, mural voids and evaginations, which represent loss of integrity of the vascular wall. CONCLUSIONS: These vascular changes that are associated with colobomatous anomalies may impede the retinal circulation and be responsible for the fluctuating fluorescein pattern during fluorangiogram of affected animals. The lesions of the vascular wall may increase the subretinal fluid due to the leakage of fluid, thus causing the maculopathy or serous retinopathy, which is frequently associated with posterior pole coloboma.

Contact-dependent regulation of a Tannerella forsythia virulence factor, BspA, in biofilms.

Tannerella forsythia is one of the periodontal organisms implicated in the development of periodontal diseases. The surface associated and secreted protein, BspA (encoded by the bspA gene), of this bacterium is an important virulence factor. The present study was carried out to examine the regulation of the bspA gene during biofilm growth and contact stimuli encountered in interbacterial interactions. The expression levels of the bspA transcript were determined by real-time RT-PCR approach. The levels of bspA transcript were found to be significantly reduced as a result of contact stimulus and in biofilm cells relative to planktonic cells. The results of our study suggest that the likely downregulation of the BspA protein in biofilms and following contact may have implications in pathogenesis as a plausible mechanism of evasion of host immune responses.

Antibody responses of periodontitis patients to gingipains of Porphyromonas gingivalis.

BACKGROUND: Arginine- and lysine-specific cysteine proteinases (arg-gingipain: Rgp, lys-gingipain: Kgp) are major virulence factors of Porphyromonas gingivalis. Recent reports have suggested that antibodies against gingipains can play a protective role against infection by P. gingivalis. The purpose of this study was to evaluate the IgG responses of patients with periodontitis to functional domains of gingipains. METHODS: A group of 29 periodontitis patients and 10 periodontally healthy subjects (control group) were recruited into this study. We prepared three recombinant fragments of rgp A (catalytic domain; r-Rgp CAT) and two hemagglutinin domains (r-Rgp 44, and r-Rgps 15-27) corresponding to amino acid residues 228 to 719, 720 to 1136, and 1137 to 1704, respectively. One fragment of the Kgp catalytic domain (r-Kgp CAT) corresponding to amino acid residues 229 to 737 and expressed in Escherichia coli was also used. IgG antibody levels to these recombinant proteins in sera from the subjects were determined by an enzyme-linked immunosorbent assay (ELISA). RESULTS: We found that IgG levels against r-Rgp 44 and r-Rgps 15-27 in sera obtained from the patients were significantly higher than those in the healthy group (P<0.01). In contrast, no significant differences in IgG levels against r-Rgp CAT and r-Kgp CAT were found between the control and patient groups. The IgG responses to P. gingivalis sonic extracts, r-Rgp 44 and r-Rgps 15-27, were related to probing depth in sera from patients, but those to r-Rgp CAT and r-Kgp CAT were not. CONCLUSION: The present findings suggest that the low responsiveness of IgG antibody against the catalytic domains of gingipain, r-Rgp CAT, and r-Kgp CAT is a key factor in infection by P. gingivalis.

Lens formation in the absence of optic cup in rat embryos irradiated with soft X-ray.

In order to investigate the effect of soft X-ray irradiation on ocular development, pregnant rats were exposed to a single 12.5 Gy irradiation on embryonic day 9 (ED 9). The embryos obtained by laparotomy on ED 12 and 21 were examined for ocular abnormalities under a binocular stereo-microscope and a light microscope. The ED 12 embryos were stained with osmium tetroxide to facilitate the observation. The stereo-microscopic examination on ED 12 and 21 revealed various types of ocular abnormalities characterized primarily by aplasia or hypoplasia of the optic cup and invaginated lens placode. The light microscopic examination further confirmed these findings histomorphologically, and the hypoplastic abnormalities were classified into three types: (1) hypoplasia of the optic cup and invaginated lens placode, (2) complete malformation of the optic cup and hypoplasia of the invaginated lens placode, and (3) complete malformation of the optic cup and invaginated lens placode. Because the lens was formed in the complete absence of the retina, the development and differentiation of the retina and lens do not seem to be tightly synchronized. Thus, this sequential analysis on ocular abnormalities during the early stage of development supports the notion that the presence of the retina is not always necessary for the development of the lens.

Examination of the rat eye at the early stage of development with osmium tetroxide staining.

This communication describes the benefit of osmium tetroxide (OsO4) staining on the examination of the eye during the early stage of organogenesis of rat embryos. The embryos were obtained by laparotomy on embryonic day 12 (ED 12) and were stained with OsO4 for examination of the ocular tissues with a binocular stereo-microscope, light microscope and scanning electron microscope. At the binocular stereo-microscopic level, the invaginated lens placode, lens pit and optic cup were clearly distinguished. The osmium-stained lens placode and the optic cup were light brown and dark brown in color, respectively. Light microscopic examination revealed that OsO4 postfixation could provide superior paraffin-embedded embryonic sections. Scanning electron microscopic examination revealed the lens pit as a round opening between the lateral nasal prominence and maxillary prominence. Thus, a rapid technique by which the ocular tissues of rat embryos can be examined under a binocular stereo-microscope was developed. This OsO4 staining method will provide a useful tool for research on organogenesis and ocular development.