Consecutive intra-gingival injections of lipopolysaccharide and butyric acid to mice induce abnormal behavior and changes in cytokine concentrations.

BACKGROUND: Periodontopathic bacteria such as Porphyromonas gingivalis produce several metabolites, including lipopolysaccharide (LPS) and n-butyric acid (BA). Past work suggested that periodontal infection may cause cognitive impairment in mice. AIMS: To elucidate the mechanisms by which metabolites such as LPS and BA, resulting from Porphyromonas gingivalis activity, induce immunological and physiological abnormalities in mice. METHODS: In the present work, 28 male ICR mice were placed in an open-field arena and the total distance (cm/600 s) they covered was recorded. Based on their moving distances, mice were divided into 4 groups (n = 7) and injected the following substances into their gingival tissues for 32 consecutive days: saline (C), 5 mmol/L of BA (B), 1 µg/mouse of LPS (L), and BA-LPS (BL) solutions. Distances covered by mice were also measured on days 14 and 21, with their habituation scores considered as "(moving distance on day 14 or 21)/(moving distance on day 0)". Afterwards, mice were dissected, and hippocampal gene expression and the concentrations of short-chain fatty acids, neurotransmitters and cytokines in their blood plasma and brains were analyzed. In addition, mouse brain and liver tissues were fixed and visually assessed for histopathological abnormalities. RESULTS: Group BL had significantly higher habituation scores than C and B on day 14. LPS induced higher habituation scores on day 21. LPS induced significant decreases in the mRNA levels of interleukin (IL)-6 and brain-derived neurotrophic factors, and an increase in neurotrophic tyrosine kinase receptor type 2. In both plasma and brain, LPS induced a significant acetate increase. Moreover, LPS significantly increased acetylcholine in brain. In plasma alone, LPS and BA significantly decreased monocyte chemoattractant protein 1 (MCP-1). However, while LPS significantly decreased tyrosine, BA significantly increased it. Lastly, LPS significantly decreased IL-6 and tumor necrosis factor in plasma. No histopathological abnormalities were detected in liver or brain tissues of mice. CONCLUSION: We showed that injections of LPS and/or BA induced mice to move seemingly tireless and that both LPS and BA injections strongly induced a reduction of MCP-1 in blood plasma. We concluded that LPS and BA may have been crucial to induce and/or aggravate abnormal behavior in mice.

Varying butyric acid amounts induce different stress- and cell death-related signals in nerve growth factor-treated PC12 cells: implications in neuropathic pain absence during periodontal disease progression.

Neuropathic pain is absent from the early stages of periodontal disease possibly due to neurite retraction. Butyric acid (BA) is a periodontopathic metabolite that activates several stress-related signals and, likewise, induce neurite retraction. Neuronal cell death is associated to neurite retraction which would suggest that BA-induced neurite retraction is ascribable to neuronal cell death. However, the underlying mechanism of BA-related cell death signaling remains unknown. In this study, we exposed NGF-treated PC12 cells to varying BA concentrations [0 (control), 0.5, 1.0, 5.0 mM] and determined selected stress-related (H2O2, glutathione reductase, calcium (Ca(2+)), plasma membrane Ca(2+) ATPase (PMCA), and GADD153/CHOPS) and cell death-associated (extrinsic: FasL, TNF-α, TWEAK, and TRAIL; intrinsic: cytochrome C (CytC), NF-kB, CASP8, CASP9, CASP10, and CASP3) signals. Similarly, we confirmed cell death execution by chromatin condensation. Our results showed that low (0.5 mM) and high (1.0 and 5.0 mM) BA levels differ in stress and cell death signaling. Moreover, at periodontal disease-level BA concentration (5 mM), we observed that only FasL amounts were affected and occurred concurrently with chromatin condensation insinuating that cells have fully committed to neurodegeneration. Thus, we believe that both stress and cell death signaling in NGF-treated PC12 cells are affected differently depending on BA concentration. In a periodontal disease scenario, we hypothesize that during the early stages, low BA amounts accumulate resulting to both stress- and cell death-related signals that favor neurite non-proliferation, whereas, during the later stages, high BA amounts accumulate resulting to both stress- and cell death-related signals that favor neurodegeneration. More importantly, we propose that neuropathic pain absence at any stage of periodontal disease progression is ascribable to BA accumulation regardless of amount.

Re-discovering periodontal butyric acid: New insights on an old metabolite.

The oral microbiome is composed of detrimental and beneficial microbial communities producing several microbial factors that could contribute to the development of the oral microbiome and, likewise, may lead to the development of host diseases. Metabolites, like short-chain fatty acids, are commonly produced by the oral microbiome and serve various functions. Among the periodontal short-chain fatty acids, butyric acid is mainly produced by periodontopathic bacteria and, attributable to the butyrate paradox, is postulated to exhibit a dual function depending on butyric acid concentration. A better understanding of the interconnecting networks that would influence butyric acid function in the oral cavity may shed a new light on the current existing knowledge and view regarding butyric acid.

Periodontal disease level-butyric acid amounts locally administered in the rat gingival mucosa induce ER stress in the systemic blood.

Periodontal diseases have long been postulated to contribute to systemic diseases and, likewise, it has been proposed that periodontal disease treatment may ameliorate certain systemic diseases. Short-chain fatty acids (SCFA) are major secondary metabolites produced by oral anaerobic bacteria and, among the SCFAs, butyric acid (BA) in high amounts contribute to periodontal disease development. Periodontal disease level-butyric acid (PDL-BA) is found among patients suffering from periodontal disease and has previously shown to induce oxidative stress, whereas, oxidative stress is correlated to endoplasmic reticulum (ER) stress. This would imply that PDL-BA may likewise stimulate ER stress, however, this was never elucidated. A better understanding of the correlation between PDL-BA and systemic ER stress stimulation could shed light on the possible systemic effects of PDL-BA-related periodontal diseases. Here, PDL-BA was injected into the gingival mucosa and the systemic blood obtained from the rat jugular was collected at 0, 15, 60, and 180 min post-injection. Collected blood samples were purified and only the blood cytosol was used throughout this study. Subsequently, we measured blood cytosolic GADD153, Ca(2+), representative apoptotic and inflammatory caspases, and NF-κB amounts. We found that PDL-BA presence increased blood cytosolic GADD153 and Ca(2+) amounts. Moreover, we observed that blood cytosolic caspases and NF-κB were activated only at 60 and 180 min post-injection in the rat gingival mucosa. This suggests that PDL-BA administered through the gingival mucosa may influence the systemic blood via ER stress stimulation and, moreover, prolonged PDL-BA retention in the gingival mucosa may play a significant role in ER stress-related caspase and NF-κB activation. In a periodontal disease scenario, we propose that PDL-BA-related ER stress stimulation leading to the simultaneous activation of apoptosis and inflammation may contribute to periodontal disease pathogenesis.

Varying hemin concentrations affect Porphyromonas gingivalis strains differently.

Porphyromonas gingivalis requires heme to grow, however, heme availability and concentration in the periodontal pockets vary. Fluctuations in heme concentration may affect each P. gingivalis strain differently, however, this was never fully demonstrated. Here, we elucidated the effects of varying hemin concentrations in representative P. gingivalis strains. Throughout this study, representative P. gingivalis strains [FDC381 (type I), MPWIb-01 (type Ib), TDC60 (type II), ATCC49417 (type III), W83 (type IV), and HNA99 (type V)] were used and grown for 24 h in growth media under varying hemin concentrations (5 × , 1 × , 0.5 × , 0.1 × ). Samples were lysed and protein standardized. Arg-gingipain (Rgp), H2O2, and superoxide dismutase (SOD) levels were subsequently measured. We focused our study on 24 h-grown strains which excluded MPWIb-01 and HNA99. Rgp activity among the 4 remaining strains varied with Rgp peaking at: 1 × for FDC381, 5 × for TDC60, 0.5 × for ATCC49417, 5 × and 0.5 × for W83. With regards to H2O2 and SOD amounts: FDC381 had similar H2O2 amounts in all hemin concentrations while SOD levels varied; TDC60 had the lowest H2O2 amount at 1 × while SOD levels became higher in relation to hemin concentration; ATCC49417 also had similar H2O2 amounts in all hemin concentrations while SOD levels were higher at 1 × and 0.5 × ; and W83 had statistically similar H2O2 and SOD amounts regardless of hemin concentration. Our results show that variations in hemin concentration affect each P. gingivalis strain differently.

Prevalence and quantitative analysis of Epstein-Barr virus DNA and Porphyromonas gingivalis associated with Japanese chronic periodontitis patients.

OBJECTIVE: A number of studies have recently suggested Epstein-Barr virus (EBV) involvement in the pathogenesis of periodontitis. In this study, we investigated the association between major periodontopathic bacteria Porphyromonas gingivalis (P. gingivalis) and EBV in Japanese chronic periodontitis (CP) patients. MATERIALS AND METHODS: A group of 25 patients with CP participated in the study along with 13 individuals without periodontitis. Subgingival samples were obtained with paper points. Quantitative real-time polymerase chain reaction (PCR) was used to detect EBV DNA and P. gingivalis. RESULTS: In the CP patients, EBV DNA and P. gingivalis were detected in both 80 % of sites with probing pocket depths (PPD) of ≥5 mm and in 40 and 36 % of sites with PPD ≤3 mm, respectively. EBV DNA and P. gingivalis were detected in 50 and 27 % of the sites in periodontally healthy individuals. Coexistence of EBV DNA and P. gingivalis was significantly higher in the deeper PPD sites of CP patients (68 %) than in the PPD sites of the healthy controls (15 %) and shallow PPD sites of CP patients (12 %). PCR-positive deeper PPD sites of CP patients for EBV DNA and P. gingivalis range between 3.74 × 10(3)∼2.83 × 10(9) and 2.73 × 10(5)∼6.65 × 10(9) (copies/ml), respectively. CONCLUSION: These results suggest an association between EBV DNA, P. gingivalis, and CP in Japanese individuals. Further studies are required to clarify this association; however, we believe that our enhanced understanding of the pathogenesis of periodontal diseases involving viral infections will lead to new treatments.

Similar physiological effects in Porphyromonas gingivalis ATCC 33277 under hemin-excess and hemin-limited concentrations are putatively associated to different hydrogen peroxide function.

Porphyromonas gingivalis requires optimal hemin to grow while non-optimal hemin hampers growth. Hemin induces H2O2 production while H2O2 has a dual function. In P. gingivalis ATCC 33277, we found similar physiological effects under hemin-excess and hemin-limited concentrations which we propose is related to two different functions of the H2O2 molecule.

Bactericidal effect of hydroxyl radicals generated from a low concentration hydrogen peroxide with ultrasound in endodontic treatment.

One approach to enhance the disinfection of root canals in endodontic treatment is ultrasonic irrigation with sodium hypochlorite. Reactive oxygen species, such as hydroxyl radical, are generated by biological defense systems to kill invading bacteria. Ultrasonic irrigation with hydrogen peroxide may be a promising option to increase hydroxyl radical generation. We examined the bactericidal effects of hydroxyl radical generated from low concentration hydrogen peroxide with ultrasound in vitro. An ultrasonic tip was submerged in 0.5 or 1.0 M hydrogen peroxide in a microfuge tube. hydrogen peroxide was irradiated with the ultrasound, the tip of which was maintained centered in the tube to mimic ultrasonic irrigation. Hydroxyl radical generation was assessed by electron spin resonance spectroscopy. Subsequently, Enterococcus faecalis suspension in hydrogen peroxide was prepared and irradiated as described above. Bactericidal effects were assessed by viable counting. Electron spin resonance measurements showed that hydroxyl radical generation increased significantly in a time- and dose-dependent manner (two-way analysis of variance and Tukey's test, p<0.05). Moreover, the bactericidal effects of hydrogen peroxide against Enterococcus faecalis were enhanced by ultrasonic irradiation in a time- and dose-dependent manner. These results suggest that ultrasonic irrigation in the presence of low concentration hydrogen peroxide can serve as a disinfection strategy in endodontic treatment.

Enzymatic measurement of short-chain fatty acids and application in periodontal disease diagnosis.

Periodontal disease is a chronic inflammatory condition caused by periodontal pathogens in the gingival sulcus. Short-chain fatty acids (SCFAs) produced by causal bacteria are closely related to the onset and progression of periodontal disease and have been reported to proliferate in the periodontal sulcus of patients experiencing this pathology. In such patients, propionic acid (C3), butyric acid (C4), isobutyric acid (IC4), valeric acid (C5), isovaleric acid (IC5), and caproic acid (C6), henceforth referred to as [C3-C6], has been reported to have a detrimental effect, while acetic acid (C2) exhibits no detrimental effect. In this study, we established an inexpensive and simple enzymatic assay that can fractionate and measure these acids. The possibility of applying this technique to determine the severity of periodontal disease by adapting it to specimens collected from humans has been explored. We established an enzyme system using acetate kinase and butyrate kinase capable of measuring SCFAs in two fractions, C2 and [C3-C6]. The gingival crevicular fluid (GCF) and saliva of 10 healthy participants and 10 participants with mild and severe periodontal disease were measured using the established enzymatic method and conventional gas chromatography-mass spectrometry (GC-MS). The quantification of C2 and [C3-C6] in human GCF and saliva was well correlated when using the GC-MS method. Furthermore, both C2 and [C3-C6] in the GCF increased with disease severity. However, while no significant difference was observed between healthy participants and periodontal patients when using saliva, [C3-C6] significantly differed between mild and severe periodontal disease. The enzymatic method was able to measure C2 and [C3-C6] separately as well as using the GC-MS method. Furthermore, the C2 and [C3-C6] fractions of GCF correlated with disease severity, suggesting that this method can be applied clinically. In contrast, the quantification of C2 and [C3-C6] in saliva did not differ significantly between healthy participants and patients with periodontal disease. Future studies should focus on inflammation rather than on tissue destruction.

Inhibition of Streptococcus mutans biofilm formation by Streptococcus salivarius FruA.

The oral microbial flora consists of many beneficial species of bacteria that are associated with a healthy condition and control the progression of oral disease. Cooperative interactions between oral streptococci and the pathogens play important roles in the development of dental biofilms in the oral cavity. To determine the roles of oral streptococci in multispecies biofilm development and the effects of the streptococci in biofilm formation, the active substances inhibiting Streptococcus mutans biofilm formation were purified from Streptococcus salivarius ATCC 9759 and HT9R culture supernatants using ion exchange and gel filtration chromatography. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis was performed, and the results were compared to databases. The S. salivarius HT9R genome sequence was determined and used to indentify candidate proteins for inhibition. The candidates inhibiting biofilms were identified as S. salivarius fructosyltransferase (FTF) and exo-beta-d-fructosidase (FruA). The activity of the inhibitors was elevated in the presence of sucrose, and the inhibitory effects were dependent on the sucrose concentration in the biofilm formation assay medium. Purified and commercial FruA from Aspergillus niger (31.6% identity and 59.6% similarity to the amino acid sequence of FruA from S. salivarius HT9R) completely inhibited S. mutans GS-5 biofilm formation on saliva-coated polystyrene and hydroxyapatite surfaces. Inhibition was induced by decreasing polysaccharide production, which is dependent on sucrose digestion rather than fructan digestion. The data indicate that S. salivarius produces large quantities of FruA and that FruA alone may play an important role in multispecies microbial interactions for sucrose-dependent biofilm formation in the oral cavity.

Reactivation of latent HIV-1 infection by the periodontopathic bacterium Porphyromonas gingivalis involves histone modification.

Latently infected cells harbor the HIV-1 proviral DNA genome primarily integrated into heterochromatin, allowing the persistence of transcriptionally silent proviruses. Hypoacetylation of histone proteins by histone deacetylases (HDAC) is involved in the maintenance of HIV-1 latency by repressing viral transcription. In addition, periodontal diseases, caused by polymicrobial subgingival bacteria including Porphyromonas gingivalis, are among the most prevalent infections of mankind. Here we demonstrate the effects of P. gingivalis on HIV-1 replication. This activity could be ascribable to the bacterial culture supernatant but not to other bacterial components such as fimbriae or LPS. We found that this HIV-1-inducing activity was recovered in the lower molecular mass (<3 kDa) fraction of the culture supernatant. We also demonstrated that P. gingivalis produces high concentrations of butyric acid, acting as a potent inhibitor of HDACs and causing histone acetylation. Chromatin immunoprecipitation assays revealed that the corepressor complex containing HDAC1 and AP-4 was dissociated from the HIV-1 long terminal repeat promoter upon stimulation with bacterial culture supernatant concomitantly with the association of acetylated histone and RNA polymerase II. We thus found that P. gingivalis could induce HIV-1 reactivation via chromatin modification and that butyric acid, one of the bacterial metabolites, is responsible for this effect. These results suggest that periodontal diseases could act as a risk factor for HIV-1 reactivation in infected individuals and might contribute to the systemic dissemination of the virus.

Antimicrobial activity of Gel-entrapped catechins toward oral microorganisms.

The oral cavity contains almost half of the commensal bacterial population present in the human body. An increase in the number of these microorganisms may result in systemic diseases such as infective endocarditis and aspiration pneumonia as well as oral infections. It is essential to control the total numbers of these microorganisms in order to suppress disease onset. Thus, we examined the antimicrobial activity of a newly developed gel-entrapped catechin (GEC) preparation against oral microorganisms. The minimum inhibitory concentration (MIC) of GEC was determined based on the relationship between a modified agar diffusion method and a broth microdilution method. GEC inhibited the growth of the Actinomyces, periodontopathic bacteria and Candida strains tested, but did not inhibit the growth of the oral streptococci that are important in the normal oral flora. Commercially available moisture gels containing antimicrobial components showed antimicrobial activity against all of the tested strains. After a series of washes and after a 24-h incubation, GEC retained the antimicrobial activity of the catechins. Catalase prevented GEC-induced growth inhibition of Actinomyces naeslundii and Streptococcus mutans suggesting that hydrogen peroxide may be involved in the antimicrobial activity of catechins. These results suggest that GEC may be useful for controlling oral microorganism populations and reducing the accumulation of dental plaque, thereby helping to prevent periodontal disease and oral candidiasis.

Glycosylation of the OMP85 homolog of Porphyromonas gingivalis and its involvement in biofilm formation.

OMP85 is a highly conserved outer membrane protein in all Gram-negative bacteria. We studied an uncharacterized OMP85 homolog of Porphyromonas gingivalis, a primary periodontal pathogen forming subgingival plaque biofilms. Using an outer-loop peptide antibody specific for the OMP85 of P. gingivalis, loop-3 Ab, we found a difference in the mobility of OMP85 on SDS-PAGE gel between the P. gingivalis wild-type and the isogenic galE mutant, a deglycosylated strain, suggesting that OMP85 naturally exists in a glycosylated form. This was also supported by a shift in OMP85 PAGE mobility after chemical deglycosylation treatment. Further, loop-3 Ab cross-reacted with the galE mutant stronger than the wild-type strain; and could inhibit biofilm formation in the galE mutant more than in the wild-type strain. In conclusion, this is the first report providing the evidence of OMP85 glycosylation and the involvement of OMP85 in biofilm formation.

Effects of sodium bicarbonate on butyric acid-induced epithelial cell damage in vitro.

Butyric acid is detected in periodontal pockets and is thought to be involved in the initiation and progression of periodontal disease. We examined the effects of sodium bicarbonate on the butyric acid-induced epithelial cell damage. The human gingival carcinoma cell line Ca9-22 was cultured in medium that contained butyric acid with or without sodium bicarbonate. The viability of cells treated with sodium bicarbonate was significantly higher than that of cells treated with butyric acid alone. The effects of butyric acid on ICAM-1 expression were significantly improved by sodium bicarbonate. Within the limitations of this in vitro study, sodium bicarbonate was indicated to be a useful therapeutic agent to reduce the butyric acid-induced periodontal tissue damage.

Gingival Periodontal Disease (PD) Level-Butyric Acid Affects the Systemic Blood and Brain Organ: Insights Into the Systemic Inflammation of Periodontal Disease.

Butyric acid (BA) is produced by periodontopathic bacterial pathogens and contributes to periodontal disease (PD) induction. Moreover, PD has been associated with detrimental effects which subsequently may lead to systemic disease (SD) development affecting certain organs. Surprisingly, the potential systemic manifestations and organ-localized effects of BA have never been elucidated. Here, we simulated BA-based oral infection among young (20-week-old) rats and isolated blood cytosol to determine BA effects on stress network-related signals [total heme, hydrogen peroxide (H2O2), catalase (CAT), glutathione reductase (GR), free fatty acid (FFA), NADP/NADPH], inflammation-associated signals [caspases (CASP12 and CASP1), IL-1ß, TNF-α, metallomatrix proteinase-9 (MMP-9), and toll-like receptor-2 (TLR2)], and neurological blood biomarkers [presenilin (PS1 and PS2) and amyloid precursor protein (APP)]. Similarly, we extracted the brain from both control and BA-treated rats, isolated the major regions (hippocampus, pineal gland, hypothalamus, cerebrum, and cerebellum), and, subsequently, measured stress network-related signals [oxidative stress: total heme, NADPH, H2O2, GR, and FFA; ER stress: GADD153, calcium, CASP1, and CASP3] and a brain neurodegenerative biomarker (Tau). In the blood, we found that BA was no longer detectable. Nevertheless, oxidative stress and inflammation were induced. Interestingly, amounts of representative inflammatory signals (CASP12, CASP1, IL-1ß, and TNF-α) decreased while MMP-9 levels increased which we believe would suggest that inflammation was MMP-9-modulated and would serve as an alternative inflammatory mechanism. Similarly, TLR2 activity was increased which would insinuate that neurological blood biomarkers (APP, PS1, and PS2) were likewise affected. In the brain, BA was not detected, however, we found that both oxidative and ER stresses were likewise altered in all brain regions. Interestingly, tau protein amounts were significantly affected in the cerebellar and hippocampal regions which coincidentally are the major brain regions affected in several neurological disorders. Taken together, we propose that gingival BA can potentially cause systemic inflammation ascribable to prolonged systemic manifestations in the blood and localized detrimental effects within the brain organ.

Ions released from a S-PRG filler induces oxidative stress in Candida albicans inhibiting its growth and pathogenicity.

Candida albicans causes opportunistic fungal infections usually hidden among more dominant bacteria and does not exhibit high pathogenicity in vivo. Among the elderly, due to reduced host resistance to pathogens attributable to immunoscenesence, oral candidiasis is more likely to develop often leading to systemic candidiasis. Surface pre-reacted glass ionomer filler (S-PRG filler) is an ion-releasing functional bioactive glass that can release and recharge six ions which in turn strengthens tooth structure, inhibits demineralization arising from dental caries, and suppresses dental plaque accumulation. However, its effects on C. albicans have never been elucidated. Here, we evaluated the effects of ion released from S-PRG filler on C. albicans. Results show that extraction liquids containing released ions (ELIS) decreased the amount of hydrogen peroxide and catalase activity in C. albicans. Moreover, ELIS presence was found to affect C. albicans: (1) suppression of fungal growth and biofilm formation, (2) prevent adherence to denture base resin, (3) inhibit dimorphism conversion, and (4) hinder the capability to produce secreted aspartyl proteinase. Taken together, our findings suggest that ELIS induces oxidative stress in C. albicans and suppresses its growth and pathogenicity. In this regard, we propose that ELIS has the potential to be clinically used to help prevent the onset and inhibition of oral candidiasis among the elderly population.

Periodontal disease level-butyric acid putatively contributes to the ageing blood: A proposed link between periodontal diseases and the ageing process.

Periodontal diseases are partly attributable to periodontopathic bacteria found in the host, whereas, butyric acid (BA) is a common secondary metabolite produced by periodontopathic bacterial pathogens. BA has been linked to oxidative stress induction while oxidative stress has long been associated with the ageing process. However, the possible link between BA-induced oxidative stress and the ageing process has never been elucidated. Here, we attempted to show the possible role of periodontal diseaselevel-BA (PDL-BA) in influencing the rat blood ageing process. We injected PDL-BA into the young rat gingiva and, after 24h, heart blood extraction was performed. Blood obtained from PDL-BA-treated young rats was compared to untreated young and middle-aged rats. We found that cytosolic, but not mitochondrial, heme was affected 24h post-injection. In addition, we observed that PDL-BA treatment altered blood NOX activation, NADPH-related oxidative stress components (H2O2 and GR), calcium homeostasis, cell death signals (CASP3 and CASP1), and age-related markers (SIRT1 and mTOR) in young rats, with some components more closely mimicking levels found in middle-aged rats. In this regard, we propose that PDL-BA may play a role in contributing to the rat blood ageing process.

Porphyromonas gingivalis Suppresses Trophoblast Invasion by Soluble Factors.

BACKGROUND: Periodontitis is a common chronic inflammatory disorder that affects supporting tissues of the teeth. An increased risk of pregnancy complications has been reported in patients with periodontitis; however, its pathophysiology remains uncharacterized to date. In addition, Porphyromonas gingivalis (Pg) is detectable in a few placentae derived from diseased pregnancies. Thus, the aim of this study is to examine the roles of soluble factors produced by Pg on trophoblast invasion in vitro to clarify the remote effects of periodontitis on pregnancy outcomes. METHODS: The immortalized trophoblast cell line HTR-8/SVneo was cultured on plates or via hanging drop to evaluate viability, apoptosis, and morphology in the presence of the culture supernatant of Pg (PG-sup). Cells were plated on solubilized extracellular matrix rich membrane preparation plates to evaluate cell invasion. Morphologic changes were evaluated via stereomicroscopy, optical microscopy, and transmission electron microscopy. RESULTS: After 24 hours of culture, cell invasion was inhibited by PG-sup in a dose-dependent manner. Although cell viability and apoptotic counts were not affected by PG-sup, spheroid formation in hanging drop culture was inhibited. Spheroids became fragile and irregular in the presence of PG-sup. Transmission electron microscopy revealed shortened microvilli and increased intracellular spaces. CONCLUSIONS: PG-sup inhibits trophoblast invasion and affects trophoblast morphology without direct cytotoxicity. These results indicate that Pg produces soluble factor(s) that suppress trophoblast invasion and subsequent vascular remodeling, which affect placental growth and fetal well-being. It is expected that the current findings will explain the increased prevalence of pregnancy complications in patients with periodontitis.

Utilizing the age-related widening of the gingival crevice as a potential non-invasive vaccination route: Prospects for elderly vaccination.

Gingival crevice (GC) increases with age allowing periodontopathic bacteria and its products to enter. We hypothesize that by mimicking this event we can utilize the GC as a potential vaccination route. Here, we used 20 wk-old (young) and 77 wk-old (old) Sprague-Dawley rats. Initially, we elucidated the difference between oral-administration and oral-supplementation in both young and old rats and, subsequently, we determined the optimal component concentration for xanthan gel-encapsulation. Next, through molecular docking, we simulated xanthan gel-encapsulation of a representative antigen (for this study we used influenza H5N1 hemagglutinin) in order to verify that target epitopes were not blocked. Lastly, we compared the antibody titer among gingival-vaccinated rats (old and young) and, likewise, we evaluated the antibody titer produced via the gingival route as compared to other vaccination routes (intradermal, oral, sublingual). Rat blood serum was collected for further downstream analyses. Throughout the study, we were able to establish the following conditions: higher target components enter old rats via oral-supplementation; 100 µg mL(-1) is the optimal component concentration for xanthan gel-encapsulation; and xanthan gel-encapsulation leaves antibody epitopes exposed. More importantly, we observed that gingival-vaccinated old rats have higher antibody titer as compared to young rats and, likewise, we found that antibody titer elicited via gingival vaccination is comparable to other mucosal vaccination routes. Thus, we propose that the GC has the potential to serve as a non-invasive vaccination route.

High butyric acid amounts induce oxidative stress, alter calcium homeostasis, and cause neurite retraction in nerve growth factor-treated PC12 cells.

Butyric acid (BA) is a common secondary metabolite by-product produced by oral pathogenic bacteria and is detected in high amounts in the gingival tissue of patients with periodontal disease. Previous works have demonstrated that BA can cause oxidative stress in various cell types; however, this was never explored using neuronal cells. Here, we exposed nerve growth factor (NGF)-treated PC1(2) cells to varying BA concentrations (0.5, 1.0, 5.0 mM). We measured total heme, H(2)O(2), catalase, and calcium levels through biochemical assays and visualized the neurite outgrowth after BA treatment. Similarly, we determined the effects of other common periodontal short-chain fatty acids (SCFAs) on neurite outgrowth for comparison. We found that high (1.0 and 5.0 mM) BA concentrations induced oxidative stress and altered calcium homeostasis, whereas low (0.5 mM) BA concentration had no significant effect. Moreover, compared to other SCFAs, we established that only BA was able to induce neurite retraction.