The Roles of FOXO1 in Periodontal Homeostasis and Disease.

Periodontitis is an oral chronic inflammatory disease that is initiated by periodontal microbial communities and requires disruption of the homeostatic responses. The prevalence of periodontal disease increases with age; more than 70% of adults 65 years and older have periodontal disease. A pathogenic microbial community is required for initiating periodontal disease. Dysbiotic immune-inflammatory response and bone remodeling are characteristics of periodontitis. The transcription factor forkhead box protein O1 (FOXO1) is a key regulator of a number of cellular processes, including cell survival and differentiation, immune status, reactive oxygen species (ROS) scavenging, and apoptosis. Although accumulating evidence indicates that FOXO1 activity can be induced by periodontal pathogens, the roles of FOXO1 in periodontal homeostasis and disease have not been well documented. The present review summarizes how the FOXO1 signaling axis can regulate periodontal bacteria-epithelial interactions, immune-inflammatory response, bone remodeling, and wound healing.

Impact of Oral Commensal Bacteria on Degradation of Periodontal Connective Tissue in Mice.

BACKGROUND: Innate and adaptive immunosurveillance mechanisms in response to the normal commensal bacteria can affect periodontal innate defense status. However, it is still unclear how commensal bacteria contribute to the inflammatory responses of junctional epithelium (JE) and periodontal connective tissue (PCT). The aim of the present study is to investigate the contribution of commensal bacteria on inflammatory responses in JE and PCT in mice. METHODS: The periodontal tissue of germ-free (GF) and specific-pathogen-free (SPF) mice were compared at age 11 to 12 weeks (n = 6 per group). In this study, the number of neutrophils and expression of intercellular adhesion molecule (ICAM)-1, fibroblast growth factor receptor (FGFR)-1, matrix metalloproteinase (MMP)-1, and MMP-8 within the JE and the PCT are evaluated. The collagen density was also determined in PCT stained with picrosirius red (PSR). PSR staining combined with or without polarized light microscopy has been used to assess the organization and maturation of collagen matrix. RESULTS: In the present findings, the area of JE in SPF mice was significantly greater than that in GF mice (P <0.05). In addition, the JE and PCT in SPF mice showed greater migration of neutrophils and higher expression of ICAM-1, FGFR-1, MMP-1, and MMP-8 than those in GF mice (P <0.05). Furthermore, the density of collagen in PCT in SPF mice was lower compared to GF mice (P <0.05). CONCLUSION: These results indicate that commensal bacteria induced a low-grade inflammatory state in JE and that such conditions may contribute to degradation of collagen in PCT in mice.

Increased expression of interleukin (IL)-35 and IL-17, but not IL-27, in gingival tissues with chronic periodontitis.

BACKGROUND: Interleukin (IL)-35 plays an important role in immune regulation through the suppression of effector T-cell populations, including T-helper 17 (Th17) cells. Although Th17 cells and IL-17 are involved in the pathogenesis of periodontitis, the level of IL-35 in inflamed periodontal tissues is unclear. Here, IL-35, IL-17, and IL-27 production/expression in gingival crevicular fluid (GCF) and human gingival tissue were investigated. METHODS: GCF samples were collected from buccal (mesial, center, and distal) sites of teeth from patients with chronic periodontitis (CP) and healthy controls and were analyzed by enzyme-linked immunosorbent assay for IL-35 (periodontitis, n = 36; healthy, n = 30) and IL-17 (periodontitis, n = 16; healthy, n = 13). Gingival tissue, including sulcus/pocket epithelium and underlying connective tissue, was collected from an additional 10 healthy participants and 10 patients with CP and were analyzed by quantitative polymerase chain reaction (qPCR) for Epstein Barr virus-induced gene 3 (EBI3), IL12A, and IL17A. IL27p28 was also tested by qPCR. RESULTS: IL-35 and IL-17 were significantly higher in GCF from patients with periodontitis than healthy participants (P <0.01, P <0.05, respectively). In both healthy participants and those with periodontitis, positive correlations were found among IL-35 and probing depth and clinical attachment level (CAL) as well as between IL-17 and CAL. EBI3, IL12A (components of IL-35), and IL17A messenger RNA expression levels were significantly higher in inflamed gingival tissue than in healthy control tissues (P <0.05). IL27p28 was not detected in any sample, suggesting that IL-27 is not produced in large quantities in periodontal tissue. CONCLUSION: IL-35 and IL-17, but not IL-27, may play important roles in the pathogenesis of periodontitis.

Impact of the Oral Commensal Flora on Alveolar Bone Homeostasis.

Homeostasis of healthy periodontal tissues is affected by innate and adaptive immunosurveillance mechanisms in response to the normal oral flora. Recent comparisons of germ-free (GF) and normal specific-pathogen-free (SPF) mice have revealed the impact of host immunosurveillance mechanisms in response to the normal oral flora on alveolar bone height. Prior reports that alveolar bone height is significantly less in normal SPF mice compared with their age- and strain-matched GF counterparts suggest that naturally occurring alveolar bone loss is a normal component of healthy periodontal tissue homeostasis. In this report, histomorphometric analyses confirmed increased alveolar bone loss and revealed increased numbers of TRAP+ osteoclastic cells lining the alveolar bone surface in SPF compared with GF mice. Increased numbers of RANKL+ cells and IL17+ cells in the periodontium of SPF mice demonstrate possible molecular mechanisms mediating the up-regulated osteoclastogenesis and alveolar bone loss in SPF mice compared with GF mice. Increased numbers of T-lymphocytic cells and T-helper cells in the junctional epithelium of SPF mice compared with GF mice suggest that the adaptive immune response contributes to physiologic alveolar bone loss in the healthy periodontium. This GF animal model study notably begins to elucidate the impact of host immunosurveillance mechanisms in response to the normal oral flora, mediating catabolic alveolar bone homeostasis in the healthy periodontium.

Differential expression of Toll-like receptor 4 in healthy and diseased human gingiva.

BACKGROUND AND OBJECTIVE: Lipopolysaccharide (LPS)-mediated signaling in host cells involves Toll-like receptor 4 (TLR4) accessory molecules, including LPS-binding protein (LBP), cluster of differentiation 14 (CD14) and lymphocyte antigen 96 (MD-2). However, expression of these innate defense molecules in various compartments of the human periodontium is unclear. The aim of this study was to investigate the expression profile of TLR4 in human gingiva. MATERIAL AND METHODS: Human gingival biopsies were collected from healthy gingival or chronic periodontitis tissue. Primary gingival keratinocytes and fibroblasts were cultured. Immunohistochemical analysis for TLR4 was performed. Transcripts of TLR4, MD-2, CD14 and LBP, and their protein products, were examined using RT-PCR, immunoprecipitation and immunoblotting. The interactions between these molecules in keratinocytes and fibroblasts were investigated by co-immunoprecipitation. RESULTS: TLR4 immunoreactivity was found in healthy gingival epithelium and periodontitis tissue, and appeared to be lower in junctional epithelium ( p ≤ 0.01). Fibroblasts and inflammatory cells stained more strongly for TLR4 in diseased periodontal tissues (p < 0.001). Three TLR4 splicing variants, two MD-2 splicing variants and one CD14 mRNA were expressed by gingival keratinocytes and fibroblasts. Expression of TLR4, CD14 and MD-2 proteins was detected in keratinocytes and fibroblasts in vitro. TLR4 protein from gingival keratinocytes and fibroblasts could be co-immunoprecipitated with CD14 or MD-2, suggesting an association between the related molecules in vivo. LBP transcript was detected in gingival biopsies, but not in primary cultures of gingival keratinocytes or fibroblasts. CONCLUSION: TLR4, CD14 and MD-2, but not LBP, are expressed in human gingival keratinocytes and fibroblasts. The TLR4 expression level in the junctional epithelium appeared to be lowest within the periodontal epithelial barrier.

Expression patterns of tight junction components induced by CD24 in an oral epithelial cell-culture model correlated to affected periodontal tissues.

BACKGROUND AND OBJECTIVE: Previously we demonstrated uniformly strong expression of CD24 in the epithelial attachment to the tooth and in the migrating epithelium of the periodontitis lesion. Titers of serum antibodies autoreactive with CD24 peptide correlated with reduced severity of periodontal disease. Ligation of CD24 expressed by oral epithelial cells induced formation of tight junctions that limited paracellular diffusion. In this study, we aimed to reveal that the lack of uniform expression of tight junction components in the pocket epithelium of periodontitis lesions is likely to contribute to increased paracellular permeability to bacterial products. This is proposed as a potential driver of the immunopathology of periodontitis. MATERIAL AND METHODS: An epithelial culture model with close correspondence for expression patterns for tight junction components in periodontal epithelia was used. Immunohistochemical staining and confocal laser scanning microscopy were used to analyse patterns of expression of gingival epithelial tight junction components. RESULTS: The minimally inflamed gingival attachment was characterized by uniformly strong staining at cell contacts for the tight junction components zona occludens-1, zona occludens-2, occludin, junction adhesion molecule-A, claudin-4 and claudin-15. In contrast, the pocket epithelium of the periodontal lesion showed scattered, uneven staining for these components. This pattern correlated closely with that of unstimulated oral epithelial cells in culture. Following ligation of CD24 expressed by these cells, the pattern of tight junction component expression of the minimally inflamed gingival attachment developed rapidly. CONCLUSION: There was evidence for non-uniform and focal expression only of tight junction components in the pocket epithelium. In the cell-culture model, ligation of CD24 induced a tight junction expression profile equivalent to that observed for the minimally inflamed gingival attachment. Ligation of CD24 expressed by gingival epithelial cells by lectin-like receptors of commensal oral streptococci could mediate the phenotype of health, whereas pathogenic organisms associated with periodontal disease might not signal effectively through CD24.

The role of Toll-like receptor 2 and 4 in gingival tissues of chronic periodontitis subjects with type 2 diabetes.

BACKGROUND AND OBJECTIVE: Diabetes is one important risk factor of chronic periodontitis. However, the roles of toll-like receptor (TLR) 2 and TLR4, which are implicated in the inflammatory process in both chronic periodontitis and diabetes, have not been studied. This study aimed to determine whether TLR2 and TLR4 might be involved in the relationship between chronic periodontitis and diabetes by examining TLR2 and TLR4 expression in gingival tissues from subjects with chronic periodontitis without diabetes (CP) and with diabetes (CP+DM) and from periodontally healthy subjects without diabetes (PH) and with diabetes (PH+DM). MATERIAL AND METHODS: Gingival tissues were collected from 23 CP subjects, 21 CP+DM subjects, 22 PH subjects and 20 PH+DM subjects. The expression of TLR2 and TLR4 in gingival tissues was determined using an immunohistochemical method. In gingival epithelium, staining patterns and intensity levels of TLR2 and TLR4 expression were studied. In connective tissues, the percentages of TLR2- and TLR4-positive cells were calculated. The intensity levels and the percentages of positive cells were statistically analyzed. RESULTS: Chronic periodontitis or diabetes showed no significant effect on TLR2 expression in the oral epithelium. However, diabetes increased the expression of TLR2 in sulcular epithelium and changed the pattern of TLR2 expression in gingival epithelium. Chronic periodontitis decreased the expression of TLR4 in gingival epithelium. In connective tissue under sulcular epithelium, CP+DM subjects showed statistically significant higher percentages of TLR2- and TLR4-positive cells compared with PH and PH+DM subjects. CONCLUSION: Our results suggest that hyperglycemia and chronic periodontitis had effects on TLR2 and TLR4 expression in gingival tissue. The differences in TLR2 and TLR4 expression could contribute to a greater inflammatory response, leading to periodontal disease initiation and progression.

Occlusal trauma accelerates attachment loss at the onset of experimental periodontitis in rats.

BACKGROUND AND OBJECTIVE: Occlusal trauma is an important factor that influences the progression of periodontitis, but it is unclear whether occlusal trauma influences periodontal destruction at the onset of periodontitis. We established an experimental periodontitis model with both site-specific loss of attachment and alveolar bone resorption. The purpose of the present study was to investigate the effects of occlusal trauma on periodontal destruction, particularly loss of attachment, at the onset of experimental periodontitis. MATERIAL AND METHODS: Sixty rats were used in the present study. Forty-eight rats immunized with lipopolysaccharide (LPS) intraperitoneally were divided into four groups. In the trauma (T) group, occlusal trauma was induced by placing an excessively high metal wire in the occlusal surface of the mandibular right first molar. In the inflammation (I) group, periodontal inflammation was induced by topical application of LPS into the palatal gingival sulcus of maxillary right first molars. In the trauma + inflammation (T+I) group, both trauma and periodontal inflammation were simultaneously induced. The PBS group was administered phosphate-buffered saline only. Another 12 nonimmunized rats (the n-(T+I) group) were treated as described for the T+I group. All rats were killed after 5 or 10 d, and their maxillary first molars with surrounding tissues were observed histopathologically. Loss of attachment and osteoclasts on the alveolar bone crest were investigated histopathologically. To detect immune complexes, immunohistological staining for C1qB was performed. Collagen fibers were also observed using the picrosirius red-polarization method. RESULTS: There were significant increases in loss of attachment and in the number of osteoclasts in the T+I group compared with the other groups. Moreover, widespread distribution of immune complexes was observed in the T + I group, and collagen fibers oriented from the root surface to the alveolar bone crest had partially disappeared in the T, T+I and n-(T+I) groups. CONCLUSION: When inflammation was combined with occlusal trauma, immune complexes were confirmed in more expanding areas than in the area of the I group without occlusal trauma, and loss of attachment at the onset of experimental periodontitis was increased. Damage of collagen fibers by occlusal trauma may elevate the permeability of the antigen through the tissue and result in expansion of the area of immune-complex formation and accelerating inflammatory reaction. The periodontal tissue destruction was thus greater in the T+I group than in the I group.

Defensins and LL-37: a review of function in the gingival epithelium.

Antimicrobial peptides represent an important aspect of the innate defense system that contributes to the control of bacterial colonization and infection. As studies have progressed it has become clear that antimicrobial peptides manifest other functions in addition to their antimicrobial effects. These functions include chemotaxis of numerous types of host cells involved in both the innate and adaptive immune responses. In this review, the antimicrobial activity, the regulation and the contribution to host homeostasis of alpha-defensins and LL-37, as well as of beta-defensins, are discussed in the context of their specific tissue locations in the junctional epithelium and oral epithelium, respectively.

Comparison of the distribution of dendritic cells in peri-implant mucosa and healthy gingiva.

PURPOSE: The host response to infection differs between peri-implantitis and periodontitis, but the mechanisms underlying these differences are not understood. In this study, the distribution of dendritic cell subpopulations in healthy peri-implant mucosa (HPIM) was compared to that of healthy gingiva (HG). MATERIALS AND METHODS: HPIM and HG specimens were obtained from nonsmoking, systemically healthy subjects. Immunohistochemistry was used to quantify the number of Langerhans cells (LCs) (CD1a+) and interstitial dendritic cells (IDCs) (factor XIIIa+) in the oral epithelium, sulcular/junctional epithelia, and lamina propria without inflammatory infiltration and with inflammatory infiltration. RESULTS: Fourteen HPIM and 13 HG specimens were obtained from subjects aged 29 to 55 years. The lamina propria of the HPIM had fewer LCs than that of the HG (HPIM: 7.99 ± 10.76, HG: 25.68 ± 16.98; P = .003). There was no significant difference in the number of CD1a+ cells in the oral epithelium or the sulcular/junctional epithelia between the HPIM and the HG (P ≥ .23). A greater number of IDCs was observed in the lamina propria with inflammatory infiltration of the HPIM compared to the HG (HPIM: 57.02 ± 35.70, HG: 33.89 ± 26.98; P = .06). CONCLUSIONS: In the lamina propria of HPIM, fewer LCs and more IDCs were observed. These differences may be associated with reduced stimulation of the innate and acquired immune responses initiated by LCs and the greater matrix remodeling of peri-implant tissue associated with IDCs.

Gram-positive bacteria as an antigen topically applied into gingival sulcus of immunized rat accelerates periodontal destruction.

BACKGROUND AND OBJECTIVE: Periodontitis is generally accepted to relate to gram-negative bacteria, and the host defense system influences its onset and progression. However, little is known about the relation between gram-positive bacteria and periodontitis. In this study, we topically applied gram-positive and gram-negative bacterial suspensions to the gingival sulcus in rats after immunization, and then histopathologically examined their influence on periodontal destruction. MATERIALS AND METHODS: Rats previously immunized with heat-treated and sonicated Staphylococcus aureus or Aggregatibacter actinomycetemcomitans were used as immunized groups. The non-immunized group received only sterile phosphate-buffered saline. In each animal, S. aureus or A. actinomycetemcomitans suspension was applied topically to the palatal gingival sulcus of first molars every 24 h for 10 d. Blood samples were collected and the serum level of anti-S. aureus or anti-A. actinomycetemcomitans immunoglobulin G (IgG) antibodies was determined by enzyme-linked immunosorbent assay. The first molar regions were resected and observed histopathologically. Osteoclasts were stained with tartrate-resistant acid phosphatase (TRAP). The formation of immune complexes was confirmed by immunohistological staining of C1qB. RESULTS: Serum levels of anti-S. aureus and anti-A. actinomycetemcomitans IgG antibodies in the immunized groups were significantly higher than those in the non-immunized groups were. The loss of attachment, increase in apical migration of the junctional epithelium, and decreases in alveolar bone level and number of TRAP-positive multinuclear cells in each immunized group were significantly greater than in each non-immunized group. The presence of C1qB was observed in the junctional epithelium and adjacent connective tissue in the immunized groups. CONCLUSIONS: Heat-treated and sonicated S. aureus and A. actinomycetemcomitans induced attachment loss in rats immunized with their suspensions. Our results suggest that not only gram-negative but also gram-positive bacteria are able to induce periodontal destruction.

Distribution of carcinoembryonic antigen-related cellular adhesion molecules in human gingiva.

Carcinoembryonic antigen-related cellular adhesion molecules (CEACAMs) are glycoproteins produced in epithelial, endothelial, lymphoid, and myeloid cells. Carcinoembryonic antigen-related cellular adhesion molecules mediate cell-cell contact and host-pathogen interactions. The aims of this study were to map the distribution and examine the regulation of CEACAMs in human gingival sites. Quantitative real-time PCR performed on human gingival biopsies from periodontitis sites revealed mRNA coding for CEACAM1, -5, -6, and -7. Immunohistochemistry showed that CEACAMs were not found in oral gingival epithelium, except for CEACAM5 in periodontitis. Carcinoembryonic antigen-related cellular adhesion molecules 1, 5, and 6 were present in the oral sulcular epithelium of periodontitis but not in that of healthy gingiva. In junctional epithelium, all three molecules were present in healthy gingiva, but in periodontitis only CEACAM1 and -6 were detected. Staining for CEACAM1 and -6 was also seen in the inflammatory cell infiltrate in periodontitis. No staining for CEACAM7 was found. Proinflammatory mediators, including lipopolysaccharide (LPS), tumour necrosis factor-α (TNF-α)/interleukin-1ß (IL-1ß), and interferon-γ (IFN-γ), increased the expression of CEACAM1 and CEACAM6 mRNAs in cultured human oral keratinocytes. CEACAM1 and CEACAM6 mRNAs were also strongly up-regulated upon stimulation with lysophosphatidic acid. In conclusion, the distribution of different CEACAMs was related to specific sites in the gingiva. This might reflect different functional roles in this tissue.

Role of the junctional epithelium in periodontal innate defense and homeostasis.

BACKGROUND AND OBJECTIVE: The junctional epithelium provides the front-line defense against periodontal bacterial infection. The migration of neutrophils into the junctional epithelium might represent a protective reaction against bacterial infections. However, neutrophils penetrate into the junctional epithelium even under sterile conditions. In this study, we analyzed and compared the number of neutrophils and the cytokine expression related to neutrophil migration in the junctional epithelium in conventional and germ-free mice. MATERIAL AND METHODS: Germ-free and conventional ICR mice were used at 12 wk of age. Frozen sections were used for the detection of Gr-1, macrophage inflammatory protein-2 (MIP-2/CXCL2) and proliferating cell nuclear antigen-positive cells in the two groups of mice. Laser capture microdissection and RT-PCR analysis were used to evaluate the expression of keratinocyte-derived chemokine (KC/CXCL1), MIP-2, interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) mRNAs in the two groups of mice. RESULTS: Morphometric examination indicated an increase in the area of the junctional epithelium upon bacterial infection. Immunohistochemical studies also detected an increased number of neutrophils in the junctional epithelium upon bacterial infection. Higher up-regulation of KC and MIP-2 were detected in the junctional epithelium of conventional mice than in germ-free mice, whereas the expression of Il-1ß and Tnfα mRNAs was not affected. CONCLUSION: Junctional epithelium cells constitutively expressed several types of chemokines and cytokines and the expression of chemokines was augmented by bacterial infection. Therefore, the constitutive expression of cytokines in junctional epithelium might be related to the morphological and functional homeostasis of the junctional epithelium in addition to the defense against the bacterial infection.

Lipopolysaccharide induces rapid loss of follicular dendritic cell-secreted protein in the junctional epithelium.

UNLABELLED: Oshiro A, Iseki S, Miyauchi M, Terashima T, Kawaguchi Y, Ikeda Y, Shinomura T. Lipopolysaccharide induces rapid loss of follicular dendritic cell-secreted protein in the junctional epithelium. J Periodont Res 2012; 47: 689-694. © 2012 John Wiley & Sons A/S Background and Objective: We have previously reported that mRNA encoding follicular dendritic cell-secreted protein (FDC-SP) is expressed specifically in the junctional epithelium at the gingival crevice. Other tissues, such as tonsil, prostate gland and trachea, also express high levels of FDC-SP. These tissues participate in a range of functions closely related to innate immunity. Therefore, it is hypothesized that FDC-SP plays a crucial role in close association with the host defense system within the gingival crevice. Accordingly, the main aim of this study was to investigate the expression and localization of FDC-SP in and around the junctional epithelium and to observe the dynamic changes of FDC-SP in experimental inflammation. MATERIAL AND METHODS: We examined, immunohistochemically, the expression of FDC-SP in the junctional epithelium using a specific antibody raised in rabbit after immunization with a synthetic peptide derived from the hydrophilic region of FDC-SP. Experimental inflammation was induced in the upper molars of Wistar rats by applying bacterial lipopolysaccharide (LPS; 5 mg/mL in sterile saline) for 1 h. RESULTS: We confirmed that FDC-SP is present in the junctional epithelium in a pattern that is consistent with the expression of FDC-SP mRNA. Of special interest is that no FDC-SP was detectable in the junctional epithelium 3 h after transient topical treatment with LPS. CONCLUSION: The presence of FDC-SP in the junctional epithelium and its loss after LPS treatment strongly support our hypothesis of FDC-SP playing a crucial role in close association with the host defense system within the gingival crevice.

A.actinomycetemcomitans-induced periodontal disease promotes systemic and local responses in rat periodontium.

AIM: To characterize the histologic and cellular response to A. actinomycetemcomitans (Aa) infection. MATERIAL & METHODS: Wistar rats infected with Aa were evaluated for antibody response, oral Aa colonization, loss of attachment, PMN recruitment, TNF-α in the junctional epithelium and connective tissue, osteoclasts and adaptive immune response in local lymph nodes at baseline and 4, 5 or 6 weeks after infection. Some groups were given antibacterial treatment at 4 weeks. RESULTS: An antibody response against Aa occurred within 4 weeks of infection, and 78% of inoculated rats had detectable Aa in the oral cavity (p < 0.05). Aa infection significantly increased loss of attachment that was reversed by antibacterial treatment (p < 0.05). TNF-α expression in the junctional epithelium followed the same pattern. Aa stimulated high osteoclast formation and TNF-α expression in the connective tissue (p < 0.05). PMN recruitment significantly increased after Aa infection (p < 0.05). Aa also increased the number of CD8(+) T cells (p < 0.05), but not CD4(+) T cells or regulatory T cells (Tregs) (p > 0.05). CONCLUSION: Aa infection stimulated a local response that increased numbers of PMNs and TNF-α expression in the junctional epithelium and loss of attachment. Both TNF-α expression in JE and loss of attachment was reversed by antibiotic treatment. Aa infection also increased TNF-α in the connective tissue, osteoclast numbers and CD8(+) T cells in lymph nodes. The results link Aa infection with important characteristics of periodontal destruction.

Excessive fluoride intake alters the MMP-2, TIMP-1 and TGF-ß levels of periodontal soft tissues: an experimental study in rabbits.

OBJECTIVES: This study evaluated the influence of fluoride on periodontal soft tissues by investigating any alterations in their MMP-2, TIMP-1 and TGF-ß profiles secondary to excessive fluoride intake. MATERIAL AND METHODS: Fluorosis was induced in 18 rabbits (test group) through consumption of fluoride added to drinking water, whereas 10 rabbits consumed regular tap water as daily supply (control group). Following fluorosis verification, animals were sacrificed and their 1st mandibular molar teeth were utilized in the assessments. MMP-2, TIMP-1 and TGF-ß were separately investigated for gingival epithelium (GE), gingival connective tissue (GC) and periodontal ligament (PL) to evaluate periodontal soft tissues. Histological sections were prepared from the groups, the parameters were determined by immunohistochemistry, and their levels were calculated by quantification of the immunostainings. RESULTS: Staining intensity of MMP-2 in GC and PL (p < 0.01); TIMP-1 and TGF-ß of GE, GC and PL (p < 0.01) were higher in the test group compared to those of the control group. Intra-group staining of TIMP-1 was higher than MMP-2 in all test group compartments (p < 0.01) and in the control group GE (p < 0.01). TIMP-1 was also higher than TGF-ß in the GE and PL of the test group (p < 0.05) and in the GE of the control group (p < 0.01). CONCLUSION: These results suggest that excessive fluoride intake may affect periodontal soft tissues by increasing MMP-2, TIMP-1 and TGF-ß, and thereby altering the MMP-2/TIMP-1 and TIMP-1/TGF-ß ratios. CLINICAL RELEVANCE: Excessive fluoride consumption may alter the periodontal tissue homeostasis which may be detrimental in the maintenance of periodontal health.

Gene expression profiling of early-phase Sjögren's syndrome in C57BL/6.NOD-Aec1Aec2 mice identifies focal adhesion maturation associated with infiltrating leukocytes.

PURPOSE: Despite considerable efforts, the molecular and cellular events in lacrimal gland tissues initiating inflammatory responses leading to keratoconjunctivitis sicca (KCS), autoimmunity, and Sjögren's syndrome (SjS) have yet to be defined. To determine whether altered glandular homeostasis occurs before the onset of autoimmunity, a temporal transcriptome study was carried out in an animal model of primary SjS. METHODS: Using oligonucleotide microarrays, gene expression profiles were generated for lacrimal glands of C57BL/6.NOD-Aec1Aec2 mice 4 to 20 weeks of age. Pairwise analyses identified genes differentially expressed, relative to their 4-week expression, during the development of SjS-like disease. Statistical analyses defined differentially and coordinately expressed gene sets. The PANTHER (Protein ANalysis THrough Evolutionary Relationships) classification system was used to define annotated biological processes or functions. RESULTS: Temporal transcript expression profiles of C57BL/6.NOD-Aec1Aec2 lacrimal glands before, or concomitant with, the first appearance of inflammatory cells revealed a highly restricted subset of differentially expressed genes encoding interactive extracellular matrix proteins, fibronectin, integrins, and syndecans. In contrast, genes encoding interepithelial junctional complex proteins defined alterations in tight junctions (TJ), adherens, desmosomes, and gap junctions, suggesting perturbations in the permeability of the paracellular spaces between epithelial barriers. Correlating with this were gene sets defining focal adhesion (FA) maturation and Ras/Raf-Mek/Erk signal transduction. Immunohistochemically, FAs were associated with infiltrating leukocytes and not with lacrimal epithelial cells. CONCLUSIONS: For the first time, FA maturations are implicated as initial biomarkers of impending autoimmunity in lacrimal glands of SjS-prone mice. Changes in TJ complex genes support an increased movement of cells through paracellular spaces.

Interaction of oral bacteria with gingival epithelial cell multilayers.

Primary gingival epithelial cells were cultured in multilayers as a model for the study of interactions with oral bacteria associated with health and periodontal disease. Multilayers maintained at an air-liquid interface in low-calcium medium displayed differentiation and cytokeratin properties characteristic of junctional epithelium. Multilayers were infected with fluorescently labeled Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum or Streptococcus gordonii, and bacterial association was determined by confocal microscopy and quantitative image analysis. Porphyromonas gingivalis invaded intracellularly and spread from cell to cell; A. actinomycetemcomitans and F. nucleatum remained extracellular and showed intercellular movement through the multilayer; whereas S. gordonii remained extracellular and predominantly associated with the superficial cell layer. None of the bacterial species disrupted barrier function as measured by transepithelial electrical resistance. P. gingivalis did not elicit secretion of proinflammatory cytokines. However, A. actinomycetemcomitans and S. gordonii induced interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), IL-6 and IL-8 secretion; and F. nucleatum stimulated production of IL-1ß and TNF-α. Aggregatibacter actinomycetemcomitans, F. nucleatum and S. gordonii, but not P. gingivalis, increased levels of apoptosis after 24 h infection. The results indicate that the organisms with pathogenic potential were able to traverse the epithelium, whereas the commensal bacteria did not. In addition, distinct host responses characterized the interaction between the junctional epithelium and oral bacteria.

How has neutrophil research improved our understanding of periodontal pathogenesis?

BACKGROUND: Neutrophils are the predominant cells responsible for host defence against bacterial infection. Loss of neutrophil defence, due either to deficient number or function, strongly predisposes to bacterial infections such as periodontitis. Yet, the neutrophil oxidative and proteolytic arsenal has also been implicated in perpetrating periodontal tissue damage in periodontitis. AIM: In this review, we focus on recent developments that shed light on these two aspects of neutrophil function in periodontitis. METHODS: Primary search: using PubMed search for "neutophil", "periodontal", and "periodontitis". Secondary search: using references from the articles found in the first stage. RESULTS: Early histological studies showed that infiltrating neutrophils form a wall of cells abutting the junctional epithelium in periodontal inflammatory lesions. The chronic standoff between these neutrophils and the bacterial community suggests that bacterial evasion of neutrophil clearance is a major characteristic of periodontitis. Indeed, not all functional neutrophil deficiencies increase the risk of periodontitis, an observation that points the way towards identification of particular anti-bacterial pathways essential for protection against periodontal pathogens. The net result in the majority of periodontitis patients who exhibit normal neutrophil number and function, is that neutrophils accumulate in the periodontal tissue where they are available to participate in tissue destruction. Diminished neutrophil clearance further contributes to the persistence of activated neutrophils in the periodontal tissue. CONCLUSIONS: Data on the role of neutrophils in the pathogenesis of periodontitis are mixed. Neutrophils are a critical arm of the defence against periodontitis, but bacterial evasion of the neutrophil microbicidal machinery coupled with delayed neutrophil apoptosis may transform the neutrophil from defender to perpetrator. At this stage of knowledge, attempts to induce host modulation through neutrophil suppression or activation are premature.

Virulence of major periodontal pathogens and lack of humoral immune protection in a rat model of periodontal disease.

OBJECTIVE: This study was designed to test the hypothesis that periodontal pathogens Tannerella forsythia and Porphyromonas gingivalis are synergistic in terms of virulence potential using a model of mixed-microbial infection in rats. MATERIALS AND METHODS: Three groups of rats were infected orally with either T. forsythia or P. gingivalis in mono-bacterial infections or as mixed-microbial infections for 12 weeks and a sham-infected group were used as a control. This study examined bacterial infection, inflammation, immunity, and alveolar bone loss changes with disease progression. RESULTS: Tannerella forsythia and P. gingivalis genomic DNA was detected in microbial samples from infected rats by PCR indicating their colonization in the rat oral cavity. Primary infection induced significantly high IgG, IgG2b, IgG1, and IgG2a antibody levels indicating activation of mixed Th1 and Th2 immune responses. Rats infected with the mixed-microbial consortium exhibited significantly increased palatal horizontal and interproximal alveolar bone loss. Histological examinations indicated significant hyperplasia of the gingival epithelium with moderate inflammatory infiltration and apical migration of junctional epithelium. The results observed differ compared to uninfected controls. CONCLUSION: Our results indicated that T. forsythia and P. gingivalis exhibit virulence, but not virulence synergy, resulting in the immuno-inflammatory responses and lack of humoral immune protection during periodontitis in rats.