Long-term sorbitol consumption affects the hippocampus and alters cognitive function in aged mice.

The systemic effects of the artificial sweetener sorbitol on older adult individuals have not been elucidated. We assessed the effects of sorbitol consumption on cognitive and gingival health in a mouse model. Aged mice were fed 5% sorbitol for 3 months before their behavior was assessed, and brain and gingival tissues were collected. Long-term sorbitol consumption inhibited gingival tissue aging in aged mice. However, it caused cognitive decline and decreased brain-derived neurotrophic factor (BDNF) in the hippocampus. Sorbitol consumption did not affect homeostatic function; however, it may exert effects within the brain, particularly in the hippocampus.

Premature gray hair development in the interbrow region owing to the loss of maxillary first molars in young mice.

The exact sites of premature hair graying and whether tooth loss causes this condition remain unknown. In this study, we aimed to explore the effect of reduced mastication on premature hair graying. Maxillary first molars were extracted from young mice, and the mice were observed for 3 months, along with non-extraction control group mice. After 3 months, gray hair emerged in the interbrow region of mice in the tooth extraction group but not in the control group. The expression of tyrosinase-related protein-2 (TRP-2) mRNA was lower in the interbrow tissues of young mice without maxillary molars than in those with maxillary molars. Tooth loss leads to interbrow gray hair growth, possibly because of weakened trigeminal nerve input, suggesting that reduced mastication causes premature graying. Thus, prompt prosthetic treatment after molar loss is highly recommended.

Neutrophil extracellular traps inhibit osteoclastogenesis.

Neutrophil extracellular traps (NETs) released by neutrophils upon inflammation or infection, act as an innate immune defense against pathogens. NETs also influence inflammatory responses and cell differentiation in host cells. Osteoclasts, which are derived from myeloid stem cells, are critical for the bone remodeling by destroying bone. In the present study, we explores the impact of NETs, induced by the inflammatory agent calcium ionophore A23187, on the differentiation and activation of osteoclasts, potentially through suppressing RANK expression. Our results collectively suggested that the inhibition of RANKL-mediated osteoclastogenesis by NETs might lead to the suppression of excessive bone resorption during inflammation.

Macrophage migration inhibitory factor-mediated mast cell extracellular traps induce inflammatory responses upon Fusobacterium nucleatum infection.

Mast cell extracellular traps (MCETs) released by mast cells contribute to host defense. In this study, we investigated the effects of MCETs released from mast cells after infection with a periodontal pathogen Fusobacterium nucleatum. We found that F. nucleatum induced MCET release from mast cells, and that MCETs expressed macrophage migration inhibitory factor (MIF). Notably, MIF bound to MCETs induced proinflammatory cytokine production by monocytic cells. These findings suggest that MIF expressed on MCETs, released from mast cells upon infection with F. nucleatum, promotes inflammatory responses that may be associated with the pathogenesis of periodontal disease.

Long-term course with iris changes after trabeculectomy for uveitic glaucoma associated with iris mammillation: a case report.

BACKGROUND: Iris mammillation is a rare disease characterized by the distribution of multiple nodules on the iris surface. The course of uveitic glaucoma with iris mammillation has never been reported. CASE PRESENTATION: A 56-year-old woman, who presented with unilateral decreased vision, visited our hospital for treatment of uveitic glaucoma in the right eye. Multiple nodules were scattered over the iris surface in that eye. This case was diagnosed as iris mammillation on clinical findings. After excluding malignant tumors such as melanoma, trabeculectomy was performed. The resected iris had no pathologically malignant findings. The iris nodules evolved to a sand-like appearance, and the intraocular pressure remained stable without recurrent inflammation 7 years after trabeculectomy. CONCLUSIONS: In a case of unilateral uveitic glaucoma with iris mammillation, filtration surgery was performed after excluding the presence of a malignancy, and the long-term postoperative course has been stable.

Hericium erinaceus ethanol extract and ergosterol exert anti-inflammatory activities by neutralizing lipopolysaccharide-induced pro-inflammatory cytokine production in human monocytes.

Edible mushrooms are known to exert anti-inflammatory effects. In this study, the effects of ethanol extracts from edible mushrooms, such as Hericium erinaceus, and other edible mushrooms on inflammatory responses were investigated. Experiments were conducted using the inflammatory responses of human monocytes induced by lipopolysaccharide (LPS), a bacterial component, that provokes inflammation. Notably, we demonstrated that LPS mixed with ethanol and hot water extracts derived from edible mushrooms attenuated the production of inflammatory cytokines, such as interleukin (IL)-1ß, -6, and -8, induced by LPS in human monocytic cell cultures. Moreover, we found that the ethanol extract of H. erinaceus contained ergosterol, which attenuated IL-8 production in LPS-stimulated cells. Subsequent component analysis of the ethanol extract of H. erinaceus revealed that ergosterol binds to lipid A to attenuate LPS-induced inflammation. Together, our findings suggest that ergosterol in ethanol extracts from edible mushrooms can prevent the induction of inflammation by binding to LPS.

An in vitro senescence model of gingival epithelial cell induced by hydrogen peroxide treatment.

Gingival tissue shows progressive changes with aging and an in vitro model of gingival tissue could be useful in understanding age-associated oral diseases. The present study aims to establish a hydrogen peroxide (H2O2) treatment model to induce aging in human gingival epithelial cells. In addition, fisetin, a flavonoid component studied for the anti-aging property is used to examine if it could reverse the induced senescence. Primary human gingival epithelial progenitor (HGEPp) cells were cultured and treated with different concentrations of H2O2. A cell vitality and morphology, senescence-associated beta-galactosidase (SA-ß-gal) staining, mRNA and protein expression analysis of known senescence markers p16, p21, and p53, and cell cycle assay were performed. The cells showed dose-dependent changes in vitality and morphology, SA-ß-gal staining, relative mRNA and protein expression, and cell cycle assay after H2O2 treatment. Based on these results, 400 µM H2O2 was considered as an optimal concentration to induce senescence. Treatment of senescence-induced cells with fisetin downregulated all the senescence markers used in this study. In conclusion, a senescence model of gingival epithelial cells induced by hydrogen peroxide treatment was established which could be employed to study age-related periodontal diseases.

Efficacy and safety of 5-fluorouracil in infrared monitor guided bleb revision.

PURPOSE: Infrared monitor-guided bleb revision (IRGBR), an alternative needling system, visualizes anterior-segment tissues around the bleb not visible during needle revision after trabeculectomy. This study determined the safety and efficiency of 5-fluorouracil (5-FU) as an adjunctive anti-metabolite in IRGBR. METHODS: We retrospectively analyzed 43 consecutive eyes (40 patients; 14 eyes, primary open-angle; 29 eyes, secondary glaucoma) treated with IRGBR for failing filtering blebs. The patients were divided into two groups. The first one had IRGBR without adjunctive 5-FU subconjunctival injection, and the second one had IRGBR with 5-FU. We performed Kaplan-Meier survival analysis using log-rank tests after 2 years of follow-up and Cox proportional hazards regression model to analyze the dependence of the survival time on predictor variables. Two failure criteria were defined as the need for additional surgery for intraocular pressure (IOP) reduction and the IOP at two consecutive follow-up visits based on definition 1, IOP ≧22 mmHg and definition 2, IOP ≧17 mmHg. RESULTS: Thirty eyes (29 cases) underwent IRGBR with subconjunctival 5-FU injection (group A in the second term) and 13 eyes (11 cases) without 5-FU (group B in the first term). The success rates 24 months after IRGBR were 73.3 and 23.1%, respectively, in groups A and B based on the definition 1 failure and 56.7 and 7.7% based on the definition 2 failure. Complications included transient bleb leaks (group A, 3 eyes; group B, none) and choroidal detachment (group A, 1 eye; group B, none). No use of 5-FU and IOPs ≧10 mmHg 1 week after IRGBR were significant risk factors. CONCLUSIONS: Adjunctive 5-FU in IRGBR achieved a better success rate for failing trabeculectomy blebs.

Complete Visual Recovery From Severe Outer Retinitis After Tonsillitis.

BACKGROUND: To report a case of severe acute bilateral outer retinitis after tonsillitis and rapid morphologic and functional recovery after steroid treatment. METHODS: Observational case report. RESULTS: A 26-year-old woman with acute bilateral blurred vision that developed after tonsillitis underwent spectral-domain optical coherence tomography (SD-OCT) that showed photoreceptor outer segment damage. Full-field electroretinography (ERG) and multifocal ERG were nonrecordable. The patient had a remarkable anatomic and functional recovery in response to steroid treatment; however, partial damage remained around the macula on SD-OCT, and an adaptive optics imaging system showed damaged cone photoreceptors. CONCLUSIONS: Prednisolone is an effective treatment for a disease that is believed to be due to suspicious involvement of the autoimmune system. Even severe outer retinitis can recover completely with rapid diagnosis and treatment.

Chemokines Up-Regulated in Epithelial Cells Control Senescence-Associated T Cell Accumulation in Salivary Glands of Aged and Sjögren's Syndrome Model Mice.

Immunosenescence is characterized by age-associated changes in immunological functions. Although age- and autoimmune-related sialadenitis cause dry mouth (xerostomia), the roles of immunosenescence and cellular senescence in the pathogenesis of sialadenitis remain unknown. We demonstrated that acquired immune cells rather than innate immune cells infiltrated the salivary glands (SG) of aged mice. An analysis of isolated epithelial cells from SG revealed that the expression levels of the chemokine CXCL13 were elevated in aged mice. Senescence-associated T cells (SA-Ts), which secrete large amounts of atypical pro-inflammatory cytokines, are involved in the pathogenesis of metabolic disorders and autoimmune diseases. The present results showed that SA-Ts and B cells, which express the CXCL13 receptor CXCR5, accumulated in the SG of aged mice, particularly females. CD4+ T cells derived from aged mice exhibited stronger in vitro migratory activity toward CXCL13 than those from young mice. In a mouse model of Sjögren's syndrome (SS), SA-Ts also accumulated in SG, presumably via CXCL12-CXCR4 signaling. Collectively, the present results indicate that SA-Ts accumulate in SG, contribute to the pathogenesis of age- and SS-related sialadenitis by up-regulating chemokines in epithelial cells, and have potential as therapeutic targets for the treatment of xerostomia caused by these types of sialadenitis.

Porphyromonas gingivalis induces the production of interleukin-31 by human mast cells, resulting in dysfunction of the gingival epithelial barrier.

Interleukin (IL)-31 is important for innate immunity in mucosal tissues and skin, and increased IL-31 expression participates in the pathogenesis of chronic inflammatory diseases affecting the skin, airways, lungs, and intestines. We investigated the contribution of mast cells to the induction of IL-31 production following infection with the periodontal pathogen, Porphyromonas gingivalis. We found that oral infection with P. gingivalis increased IL-31 expression in the gingival tissues of wild-type mice but not in those of mast cell-deficient mice. The P. gingivalis-induced IL-31 production by human mast cells occurred through the activation of the JNK and NF-κB signalling pathways and was dependent on the P. gingivalis lysine-specific protease gingipain-K. P. gingivalis infection induced IL-31 receptor α and oncostatin M receptor ß expression in human gingival epithelial cells. Notably, the P. gingivalis-induced IL-31 production by mast cells led to the downregulation of claudin-1, a tight junction molecule, in gingival epithelial cells, resulting in an IL-31-dependent increase in the paracellular permeability of the gingival epithelial barrier. These findings suggest that IL-31 produced by mast cells in response to P. gingivalis infection causes gingival epithelial barrier dysfunction, which may contribute to the chronic inflammation observed in periodontitis.

Association of the CAV1-CAV2 locus with normal-tension glaucoma in Chinese and Japanese.

BACKGROUND: The CAV1-CAV2 locus has been associated with primary open-angle glaucoma (POAG) and intraocular pressure. However, its association with normal-tension glaucoma (NTG) was inconclusive. Therefore, we evaluated this association in Chinese and Japanese. METHODS: Two single-nucleotide polymorphisms (SNPs, rs4236601 and rs1052990) from previous genome-wide association studies of POAG were genotyped in a total of 2220 study subjects: a Hong Kong Chinese cohort of 537 NTG patients and 490 controls, a Shantou Chinese cohort of 102 NTG and 731 controls and an Osaka Japanese cohort of 153 NTG and 207 controls. Subgroup analysis by gender was conducted. Outcomes from different cohorts were combined using meta-analysis. RESULTS: SNP rs4236601 was significantly associated with NTG in the two Chinese cohorts (Pmeta = .0019, OR = 4.55, I2 = 0). In contrast, rs4236601 was monomorphic in the Osaka cohort. The association of rs1052990 was insignificant in a meta-analysis combining Chinese and Japanese cohorts (Pmeta = .81, OR = 1.05; I2 = 64%), and the OR tended towards opposite directions between Chinese (OR = 1.26) and Japanese (OR = 0.69). Gender-specific effects of the SNPs were not statistically significant in the logistic regression or Breslow-day tests of ORs (P > .05), although rs4236601 was significant in males (P = .0068; OR = 10.30) but not in females (P = .14; OR = 2.65) in the meta-analysis of Chinese subjects. CONCLUSIONS: In this study, we confirmed the association of rs4236601 at the CAV1-CAV2 locus with NTG in Chinese. SNP rs4236601 is monomorphic, and rs1052990 tends towards a different direction in the Japanese cohort. Further studies are warranted to verify the ethnic difference and gender-specific effects of this locus.

Association of the SIX6 locus with primary open angle glaucoma in southern Chinese and Japanese.

The purpose of the study was to evaluate the association profiles of the SIX6 locus with primary open-angle glaucoma (POAG) in southern Chinese and Japanese. In this study, we tested single marker and haplotype-based associations of 11 tagging single nucleotide polymorphisms (SNPs) covering the SIX6 locus with POAG in a Hong Kong Chinese cohort (N = 1402). A novel SNP (i.e., rs12436579) and two SNPs (i.e., rs33912345 and rs10483727) from previous genome-wide association studies were further tested in a Chinese cohort from Shantou (N = 888) and a Japanese cohort from Osaka (N = 463). Results from the three cohorts were meta-analysed using a random-effect model. We found rs12436579, which has not been previously reported, was associated with POAG in Hong Kong and Shantou Chinese (Pcombined = 4.3 × 10-5, OR = 0.72, I2 = 0). Additionally, we replicated the association of one known SNP, rs33912345 (Pcombined = 0.0061, OR = 0.69, I2 = 45%), with POAG in the Chinese cohorts but not in the Japanese cohort (P > 0.6). Another known SNP, rs10483727, was nominally associated with POAG in the two Chinese cohorts (Pcombined = 0.017, OR = 0.70, I2 = 53%). All these three SNPs were significantly associated with POAG when the three cohorts were combined in meta-analysis (Pcombined<0.005). Furthermore, two haplotypes, C-C (Pcombined = 1.13 × 10-5, OR = 1.41, I2 = 0) and A-A (Pcombined = 0.045, OR = 0.68, I2 = 70%), defined by rs33912345-rs12436579 were associated with POAG in Chinese but not in Japanese. In conclusion, this study confirmed the association between two GWAS SNPs in SIX6 (rs33912345 and rs10483727) and POAG. Also, a SNP, rs12436579, not associated with POAG before, was found to be associated with POAG in Chinese. Further studies are warranted to elucidate the role of this novel SNP in POAG.

Anterior segment Scheimpflug imaging for detecting primary angle closure disease.

PURPOSE: To evaluate the capability of anterior segment Scheimpflug imaging for detecting primary angle closure disease (PACD): primary angle closure suspect, primary angle closure, and primary angle closure glaucoma, using cutoff points derived from reference databases of healthy subjects. METHODS: Eighty-seven patients with PACD and 49 age-matched control subjects were included. We evaluated the sensitivity and specificity of anterior chamber depth (ACD), anterior chamber volume (ACV), and anterior chamber angle (ACA) to differentiate patients with PACD from controls. Additionally, the study's raw data was analyzed via receiver operating characteristic curves for comparison. RESULTS: One standard deviation from the normative data's mean values was used as the cutoff point and yielded a sensitivity and specificity of 96.2% and 92.6% for ACD, 97.1% and 75.9% for ACV, and 93.3% and 72.2% for ACA, respectively. Receiver operating characteristic analysis of the raw data showed the area under the curve to be 0.984, 0.975, and 0.931 for ACD, ACV, and ACA, respectively. CONCLUSIONS: Our study demonstrated that the parameters of anterior segment Scheimpflug imaging, particularly ACD, accurately discriminate PACD. This was the first study to validate the device's normative data in a separate population. With its high reproducibility, ease of use, non-invasiveness, and speed, anterior segment Scheimpflug imaging is a potentially powerful screening tool for PACD.

Topical Glucose Induces Claudin-1 and Filaggrin Expression in a Mouse Model of Atopic Dermatitis and in Keratinocyte Culture, Exerting Anti-inflammatory Effects by Repairing Skin Barrier Function.

Atopic dermatitis (AD) is a chronic inflammatory skin disease. Corticosteroids, which are widely used for AD treatment, have adverse effects, and alternative treatments are urgently needed. This study examined the effect of topical application of high-dose glucose on inflamed skin in a murine model of AD. High-dose glucose treatment on the ear reduced dermatitis scores and ear thicknesses in mite antigen-treated NC/Nga mice. The levels of thymus and activation-regulated chemokine (TARC), Th cytokines (interleukin (IL)-4, IL-5, IL-12, IL-13, and (interferon) IFN-γ), and IgE were decreased in the serum of high-dose glucose-treated mice. Expression of claudin-1 and filaggrin was reduced in the ear epithelium in the NC/Nga mice. However, the reduced expression was restored by topical treatment with high-dose glucose. High-dose glucose also induced the expression of claudin-1 and filaggrin in cultured human skin keratinocytes. Co-stimulation with IL-4, IL-13, and thymic stromal lymphoprotein downregulated the expression of filaggrin in culture. However, high-dose glucose treatment restored the reduced expression of filaggrin. These results suggest that high-dose glucose treatment suppresses inflammation in the skin lesions by improving the skin barrier function.

Modification of Tau by 8-Nitroguanosine 3',5'-Cyclic Monophosphate (8-Nitro-cGMP): EFFECTS OF NITRIC OXIDE-LINKED CHEMICAL MODIFICATION ON TAU AGGREGATION.

Neurofibrillar tangles caused by intracellular hyperphosphorylated tau inclusion and extracellular amyloid ß peptide deposition are hallmarks of Alzheimer's disease. Tau contains one or two cysteine residues in three or four repeats of the microtubule binding region following alternative splicing of exon 10, and formation of intermolecular cysteine disulfide bonds accelerates tau aggregation. 8-Nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) acts as a novel second messenger of nitric oxide (NO) by covalently binding cGMP to cysteine residues by electrophilic properties, a process termed protein S-guanylation. Here we studied S-guanylation of tau and its effects on tau aggregation. 8-Nitro-cGMP exposure induced S-guanylation of tau both in vitro and in tau-overexpressed HEK293T cells. S-guanylated tau inhibited heparin-induced tau aggregation in a thioflavin T assay. Atomic force microscopy observations indicated that S-guanylated tau could not form tau granules and fibrils. Further biochemical analyses showed that S-guanylated tau was inhibited at the step of tau oligomer formation. In P301L tau-expressing Neuro2A cells, 8-nitro-cGMP treatment significantly reduced the amount of sarcosyl-insoluble tau. NO-linked chemical modification on cysteine residues of tau could block tau aggregation, and therefore, increasing 8-nitro-cGMP levels in the brain could become a potential therapeutic strategy for Alzheimer's disease.

Bactericidal effects of 310 nm ultraviolet light-emitting diode irradiation on oral bacteria.

BACKGROUND: Ultraviolet (UV) light is used for phototherapy in dermatology, and UVB light (around 310 nm) is effective for treatment of psoriasis and atopic dermatitis. In addition, it is known that UVC light (around 265 nm) has a bactericidal effect, but little is known about the bactericidal effect of UVB light. In this study, we examined the bactericidal effects of UVB-light emitting diode (LED) irradiation on oral bacteria to explore the possibility of using a 310 nm UVB-LED irradiation device for treatment of oral infectious diseases. METHODS: We prepared a UVB (310 nm) LED device for intraoral use to examine bactericidal effects on Streptococcus mutans, Streptococcus sauguinis, Porphyromonas gingivalis, and Fusobacterium nucleatum and also to examine the cytotoxicity to a human oral epithelial cell line (Ca9-22). We also examined the production of nitric oxide and hydrogen peroxide from Ca9-22 cells after irradiation with UVB-LED light. RESULTS: Irradiation with the 310 nm UVB-LED at 105 mJ/cm2 showed 30-50% bactericidal activity to oral bacteria, though 17.1 mJ/cm2 irradiation with the 265 nm UVC-LED completely killed the bacteria. Ca9-22 cells were strongly injured by irradiation with the 265 nm UVC-LED but were not harmed by irradiation with the 310 nm UVB-LED. Nitric oxide and hydrogen peroxide were produced by Ca9-22 cells with irradiation using the 310 nm UVB-LED. P. gingivalis was killed by applying small amounts of those reactive oxygen species (ROS) in culture, but other bacteria showed low sensitivity to the ROS. CONCLUSIONS: Narrowband UVB-LED irradiation exhibited a weak bactericidal effect on oral bacteria but showed low toxicity to gingival epithelial cells. Its irradiation also induces the production of ROS from oral epithelial cells and may enhance bactericidal activity to specific periodontopathic bacteria. It may be useful as a new adjunctive therapy for periodontitis.

Unipotent Megakaryopoietic Pathway Bridging Hematopoietic Stem Cells and Mature Megakaryocytes.

Recent identification of platelet/megakaryocyte-biased hematopoietic stem/repopulating cells requires revision of the intermediate pathway for megakaryopoiesis. Here, we show a unipotent megakaryopoietic pathway bypassing the bipotent megakaryocyte/erythroid progenitors (biEMPs). Cells purified from mouse bone marrow by CD42b (GPIbα) marking were demonstrated to be unipotent megakaryocytic progenitors (MKPs) by culture and transplantation. A subpopulation of freshly isolated CD41(+) cells in the lineage Sca1(+) cKit(+) (LSK) fraction (subCD41(+) LSK) differentiated only into MKP and mature megakaryocytes in culture. Although CD41(+) LSK cells as a whole were capable of differentiating into all myeloid and lymphoid cells in vivo, they produced unipotent MKP, mature megakaryocytes, and platelets in vitro and in vivo much more efficiently than Flt3(+) CD41(-) LSK cells, especially at the early phase after transplantation. In single cell polymerase chain reaction and thrombopoietin (TPO) signaling analyses, the MKP and a fraction of CD41(+) LSK, but not the biEMP, showed the similarities in mRNA expression profile and visible TPO-mediated phosphorylation. On increased demand of platelet production after 5-FU treatment, a part of CD41(+) LSK population expressed CD42b on the surface, and 90% of them showed unipotent megakaryopoietic capacity in single cell culture and predominantly produced platelets in vivo at the early phase after transplantation. These results suggest that the CD41(+) CD42b(+) LSK are straightforward progenies of megakaryocytes/platelet-biased stem/repopulating cells, but not progenies of biEMP. Consequently, we show a unipotent/highly biased megakaryopoietic pathway interconnecting stem/repopulating cells and mature megakaryocytes, the one that may play physiologic roles especially in emergency megakaryopoiesis.

TNF-α augmented Porphyromonas gingivalis invasion in human gingival epithelial cells through Rab5 and ICAM-1.

BACKGROUND: Tumor necrosis factor alpha (TNF-α) plays a central role in the initiation and maintenance of immune responses to periodontopathic bacteria. However, excess TNF-α leads to dysregulated immune responses and progression of periodontitis. Porphyromonas gingivalis (P. gingivalis) invades gingival epithelial cells and then multiplies and survives for a long period. Additionally, increment of TNF-α in periodontal sites is associated with a high prevalence of gram-negative anaerobes such as P. gingivalis. However, it has not been determined whether TNF-α affects invasion of P. gingivalis in periodontal tissues. RESULTS: We examined the effect of TNF-α on invasion of P. gingivalis in gingival epithelial cells and clarified the mechanism by which TNF-α augments invasion of P. gingivalis. Invasion of P. gingivalis into Ca9-22 cells was augmented by stimulation with TNF-α and it was inhibited by treatment with an antibody to TNF receptor-1. TNF-α increased production of ICAM-1, and P. gingivalis invasion was inhibited by an antibody to ICAM-1 in Ca9-22 cells. Silencing of Rab5 mRNA inhibited P. gingivalis invasion. Furthermore, the JNK inhibitor SP600125 inhibited invasion of P. gingivalis and also decreased the active form of Rab5 in Ca9-22 cells. CONCLUSION: TNF-α augments invasion of P. gingivalis in human gingival epithelial cells through increment of ICAM-1 and activation of Rab5. These phenomena may contribute to persistent infection of P. ginigvalis and prolongation of immune responses in periodontal tissues.