Impact of tooth loss on oral and systemic health.

Periodontitis is a primarily bacterial infection that is common in dentate individuals, while denture stomatitis is a predominantly fungal infection that is common among denture wearers. Both infections may increase a patient's risk for chronic systemic infection dissemination, and may in turn increase the risk of chronic, inflammatory-based systemic diseases. Systemic diseases for which chronic oral infections are believed to confer attributable risk include atherosclerotic and coronary disease, stroke, chronic obstructive pulmonary disease, diabetes, and hypertension. It appears that invasive oral pathogens trigger a systemic inflammatory response via mediators released by the cardiovascular system and liver, putting the patient at increased risk for these diseases. Data comparing gene expression between denture wearers with and without denture stomatitis (and associated Candida albicans infections) has demonstrated unique up- and down-regulation patterns for a number of genes. It appears that down-regulated genes (whose functions are thereby diminished) are associated with reduced epithelial barrier integrity. By contrast, there appears to be an association between up-regulated genes (which have enhanced function) and inflammatory responses that facilitate the ability of C. albicans to bind with and penetrate the oral mucosa. Molecular biological approaches suggest that future therapeutic development could target reducing either the local inflammatory processor, the binding and attachment of C. albicans to the oral mucosa, or both. Ongoing investigations are attempting to incorporate interventions into matrices, to provide a local and sustained presence to therapeutic interventions.

Therapeutic effect of a topical CCR2 antagonist on induced alveolar bone loss in mice.

BACKGROUND AND OBJECTIVE: Chemokines are known to regulate leukocyte trafficking, recruitment and infiltration in periodontal diseases. The study objective was to determine the effect of an experimental oral/topical chemokine (C-C motif) receptor 2 (CCR2)-antagonist treatment on alveolar bone loss in a mouse model of Porphyromonas gingivalis-induced periodontitis. MATERIAL AND METHODS: Balb/C mice (n = 41) were randomly assigned to four groups. Group 1 was infected by P. gingivalis applied orally/topically for 5 wk. Group 2 was also infected and then treated with vehicle (aqueous methylcellulose) for an additional 4 wk. Group 3 was infected and orally/topically treated with CCR2 antagonist (10 mg/kg). Group 4 served as a noninfected, nontreated control group. Mice received intraperitoneal injections of Alizarin (30 mg/kg) and calcein (20 mg/kg) three times from the last day of infection to determine mineral deposition, reflecting bone dynamics. Mandibles were analysed by morphometry and confocal fluorescence microscopy. RESULTS: Alveolar bone loss was compared among groups using Tukey's test, and bone formation was qualitatively observed. Infected mice showed significantly greater alveolar bone loss than noninfected control animals (group 1 vs. 4, p < 0.01). Vehicle-treated mice (group 2) showed the largest area of alveolar bone loss (p < 0.01), while mice treated with the CCR2 antagonist showed the smallest area of alveolar bone loss and were similar to the control group (group 3 vs. 4, p = 0.14). Qualitative analysis of fluorescent dye uptake indicated increased bone formation in CCR2-antagonist-treated mice, suggesting an improved bone repairing process. CONCLUSION: The results suggested that treatment with CCR2 antagonist inhibited alveolar bone loss and improved bone formation in this model. These data support further evaluation of CCR2 antagonist as a therapeutic target for the development of new treatment modalities on bacterially induced alveolar bone resorption.

Increased TLR4 expression in murine placentas after oral infection with periodontal pathogens.

Maternal periodontitis has emerged as a putative risk factor for preterm births in humans. The periodontitis-associated dental biofilm is thought to serve as an important source of oral bacteria and related virulence factors that hematogenously disseminate and affect the fetoplacental unit; however the underlying biological mechanisms are yet to be fully elucidated. This study hypothesized that an oral infection with the human periodontal pathogens Campylobacter rectus and Porphyromonas gingivalis is able to induce fetal growth restriction, placental inflammation and enhance Toll-like receptors type 4 (TLR4) expression in a murine pregnancy model. Female Balb/C mice (n = 40) were orally infected with C. rectus and/or P. gingivalis over a 16-week period and mated once/week. Pregnant mice were sacrificed at embryonic day (E) 16.5 and placentas were collected and analyzed for TLR4 mRNA levels and qualitative protein expression by real-time PCR and immunofluorescence. TLR4 mRNA expression was found to be increased in the C. rectus-infected group (1.98 +/- 0.886-fold difference, P < 0.01, ANOVA) compared to controls. Microscopic analysis of murine placentas showed enhanced immunofluorescence of TLR4 in trophoblasts, mainly in the placental labyrinth layer. Also, combined oral infection with C. rectus and P. gingivalis significantly reduced the overall fecundity compared to controls (16.7% vs. 75%, infected vs. non-infected mice respectively, P = 0.03, Kaplan-Meier). The results supported an enhanced placental TLR4 expression after oral infection with periodontal pathogens. The TLR4 pathway has been implicated in the pathogenesis of preterm births; therefore the abnormal regulation of placental TLR4 may give new insights into how maternal periodontitis and periodontal pathogens might be linked to placental inflammation and preterm birth pathogenesis.

Parathyroid hormone administration may modulate periodontal tissue levels of interleukin-6, matrix metalloproteinase-2 and matrix metalloproteinase-9 in experimental periodontitis.

BACKGROUND AND OBJECTIVE: Intermittent administration of the parathyroid hormone (1-34) has an anabolic effect on bone and it has been shown to reduce alveolar bone loss in experimental periodontitis models. The aim of the present study was to investigate the effect of parathyroid hormone on tissue degradation-related factors in an experimental periodontitis model in rats. MATERIAL AND METHODS: Periodontitis was induced in seventy-six male Wistar rats using ligature around the lower right first molars. The animals were then treated with parathyroid hormone (1-34) (T-group) or vehicle (C-group), three times a week for 15 d (C15, T15) or 30 d (C30, T30). At each experimental time-point, the 19 rats were killed in each group and the gingival tissue around the first lower molar was removed and prepared for the following analyses: mRNA expression of interleukin-1 beta, interleukin-6, matrix metalloproteinase (MMP)-2 and MMP-9, and gelatinolytic activity of MMP-2 and MMP-9. Hemimandibles were decalcified, and serial sections were processed and analyzed for interleukin-6 immohistochemistry. Samples were also histochemically stained by tartrate-resistant acid phosphatase (TRAP) to evaluate the number of osteoclasts present. RESULTS: Parathyroid hormone-treated samples showed decreased of levels of mRNA for interleukin-6 in the T30 group (p < 0.01) and of MMP-2 in the T15 and T30 groups (p < 0.05). Zymography assays demonstrated that treatment with parathyroid hormone led to a decrease in MMP-9 activity (p < 0.01). TRAP staining of alveolar bone revealed that osteoclasts were present in higher numbers (p < 0.05) in the groups not treated with parathyroid hormone. CONCLUSION: These data suggest that intermittent administration of parathyroid hormone can down-regulate the expression of biomarkers responsible for connective tissue breakdown and bone resorption, and potentially affect alveolar bone resorption activity.

Levels of serum interleukin (IL)-6 and gingival crevicular fluid of IL-1beta and prostaglandin E(2) among non-smoking subjects with gingivitis and type 2 diabetes.

BACKGROUND: The goal of this study was to assess whether non-smoking patients with type 2 diabetes present with increased levels of local and systemic proinflammatory mediators and, if so, whether such an increase is associated with enhanced clinical gingival inflammation compared to non-smoking patients without diabetes. METHODS: We used a cross-sectional database consisting of 725 self-reported lifelong non-smokers aged 53 to 74 years. Gingival crevicular fluid (GCF) levels of interleukin (IL)-1beta and prostaglandin E(2) (PGE(2)) and serum levels of IL-6 were measured using enzyme-linked immunosorbent assay. No participant had probing depth >3 mm. Participants with bleeding on probing (BOP) in <10% of sites were classified as healthy, whereas those with BOP in >or=10% of sites were defined as having biofilm-gingival interface (BGI) gingivitis. RESULTS: Approximately 53% (n = 385) and 11% (n = 80) of the sample had BGI gingivitis and type 2 diabetes, respectively. The mean age-adjusted level of GCF IL-1beta was significantly elevated in the diabetic group compared to the non-diabetic group (P = 0.048), but serum IL-6 (P = 0.14) and GCF PGE(2) were not (P = 0.98). The mean GCF IL-1beta and PGE(2) levels were significantly elevated in subjects with BGI gingivitis (136.2 +/- 112.9 ng/ml and 277.2 +/- 187.2 ng/ml, respectively) compared to subjects with gingival health (95.9 +/- 82.9 ng/ml and 205.7 +/- 149.6 ng/ml, respectively), regardless of diabetic status (P <0.001 for both). However, serum IL-6 was elevated in subjects with BGI gingivitis compared to subjects with gingival health only among subjects with diabetes (2.9 +/- 3.2 pg/ml versus 1.5 +/- 1.4 pg/ml; P = 0.008). With the exception of serum IL-6 in subjects without diabetes, an increase in the levels of proinflammatory mediators was associated with increased odds of having BGI gingivitis. The associations were stronger in the diabetic group. CONCLUSIONS: Type 2 diabetes may increase the host inflammatory response to oral biofilm, which, in turn, may exacerbate preconditions associated with gingivitis in susceptible individuals. Furthermore, systemic inflammation, as demonstrated by the increased level of serum IL-6, is associated with BGI gingivitis among non-smoking patients with diabetes.

Transcriptomic profiling of tantalum metal implant osseointegration in osteopenic patients.

OBJECTIVES: The long-term success of dental implants is established by literature. Although clinically well defined, the complex genetic pathways underlying osseointegration have not yet been fully elucidated. Furthermore, patients with osteopenia/osteoporosis are considered to present as higher risk for implant failure. Porous tantalum trabecular metal (PTTM), an open-cell porous biomaterial, is suggested to present enhanced biocompatibility and osteoconductivity. The goal of this study was to evaluate the expression patterns of a panel of genes closely associated with osteogenesis and wound healing in osteopenic patients receiving either traditional titanium (Ti) or PTTM cylinders to assess the pathway of genes activation in the early phases of osseointegration. MATERIAL AND METHODS: Implant cylinders made of Ti and PTTM were placed in osteopenic volunteers. At 2- and 4 weeks of healing, one Ti and one PTTM cylinder were removed from each subject for RT-PCR analysis using osteogenesis PCR array. RESULTS: Compared to Ti, PTTM-associated bone displayed upregulation of bone matrix proteins, BMP/TGF tisuperfamily, soluble ligand and integrin receptors, growth factors, and collagen genes at one or both time points. Histologically, PTTM implants displayed more robust osteogenesis deposition and maturity when compared to Ti implants from the same patient. CONCLUSIONS: Our results indicate that PTTM properties could induce an earlier activation of genes associated with osteogenesis in osteopenic patients suggesting that PTTM implants may attenuate the relative risk of placing dental implants in this population.

Bacterial infection promotes DNA hypermethylation.

Maternal oral infection, caused by bacteria such as C. rectus or P. gingivalis, has been implicated as a potential source of placental and fetal infection and inflammatory challenge, which increases the relative risk for pre-term delivery and growth restriction. Intra-uterine growth restriction has also been reported in various animal models infected with oral organisms. Analyzing placental tissues of infected growth-restricted mice, we found down-regulation of the imprinted Igf2 gene. Epigenetic modification of imprinted genes via changes in DNA methylation plays a critical role in fetal growth and development programming. Here, we assessed whether C. rectus infection mediates changes in the murine placenta Igf2 methylation patterns. We found that infection induced hypermethylation in the promoter region-P0 of the Igf2 gene. This novel finding, correlating infection with epigenetic alterations, provides a mechanism linking environmental signals to placental phenotype, with consequences for development.

Periodontal disease at the biofilm-gingival interface.

BACKGROUND: A molecular epidemiologic study provided the opportunity to characterize the biology of the biofilm-gingival interface (BGI) in 6,768 community-dwelling subjects. METHODS: Disease classifications and multivariable models were developed using clinical, microbial, inflammatory, and host-response data. The purpose was to identify new clinical categories that represented distinct biologic phenotypes based upon DNA checkerboard analyses of eight plaque bacteria, serum immunoglobulin G (IgG) titers to 17 bacteria, and the gingival crevicular fluid (GCF) levels of 16 inflammatory mediators. Five BGI clinical conditions were defined using probing depths (PDs) and bleeding on probing (BOP) scores. Subjects with all PDs < or = 3 mm were grouped as BGI-healthy (14.3% of sample) or BGI-gingivitis (BGI-G, 15.1%). Subjects with one or more PDs > or = 4 mm [deep lesion (DL)] were divided into low BOP (18.0%), moderate BOP (BGI-DL/MB, 39.7%), and severe BOP (BGI-DL/SB, 12.9%). RESULTS: Subjects with BGI-G had increased levels of Campylobacter rectus-specific serum IgG levels (P = 0.01), and those with BGI-DL/SB had increased IgG levels to Porphyromonas gingivalis (P < 0.0003) and C. rectus (P < 0.01). BGI-DL/SB subjects had an excessive GCF interleukin (IL)-1beta and prostaglandin E2 response and an enhanced chronic inflammatory response with significant increases in GCF IL-6 and monocyte chemotactic peptide-1. Within BGI-DL/SB subjects, more severe pocketing and BOP were associated with higher levels of GCF IL-1beta, not higher microbial counts or plaque scores. CONCLUSIONS: New BGI classifications create categories with distinct biologic phenotypes. The increased titers of C. rectus IgG among 68.5% of the BGI-G subjects and elevated P. gingivalis titers among BGI-DL/MB and BGI-DL/SB subjects (63.8% and 75.7%, respectively) are strongly supportive of the microbial specificity of pathogenesis for BGI categories.

Parathyroid hormone intermittent administration promotes delay on rat incisor eruption.

OBJECTIVE: This study evaluated the influence of parathyroid hormone (PTH) (1-34) intermittent administration on rat eruption rates of lower incisors under normo, hyper and hypofunctional conditions, Sharpey fibers insertion, and alveolar bone formation. MATERIALS AND METHODS: Wistar male rats received PTH (1-34) three times a week during the entire experimental period, 31days. Control animals received the same concentration of the vehicle solution during the same period. Three injections of alizarin were also performed. The experiment evaluated the eruptive rate, the alveolar bone formation and also the morphology, and the area density of Sharpey fibers. After the sacrifice, the mandibles were dissected and samples were prepared for fluorescence and scanning electron microscopy observations. RESULTS: PTH-treated animals showed significantly reduced eruption rates in all different functional conditions. Analysis evidenced that PTH-treated rats present an increase in bone formation and area density of the Sharpey fibers. CONCLUSION: We concluded that the PTH (1-34) intermittent administration reduced the eruptive process rates, through bone formation enhancement and increase in the area density of Sharpey fibers.

Effects of maternal Campylobacter rectus infection on murine placenta, fetal and neonatal survival, and brain development.

BACKGROUND: Maternal periodontal infection has been associated with increased risk of prematurity and low birthweight. Infection and inflammatory pathways that mediate prematurity have also been implicated in neonatal developmental impairments. The objective of this study was to determine whether maternal Campylobacter rectus infection that induces fetal growth restriction in a mouse model also compromises neonatal pup survival, growth, and neurodevelopment. METHODS: Timed pregnant mice were challenged with C. rectus on gestation day 7.5. One group of animals was sacrificed on embryonic day 16.5 for placental histology and measurement of fetal brain mRNA expression of tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma. Another group of animals was allowed to deliver to follow pup survival, growth, and brain structure at day 9. RESULTS: C. rectus challenge resulted in abnormal placental architecture with inflammation and a 2.8-fold increase in fetal brain expression of IFN-gamma (P = 0.04). Pup birthweight was unaffected by C. rectus exposure, but lethality was 3.9-fold higher after 1 week. Ultrastructurally, the 9-day neonatal brain tissue displayed cellular and myelin alterations consistent with white matter damage. CONCLUSIONS: Maternal C. rectus infection induces placental inflammation and decidual hyperplasia as well as concomitant increase in fetal brain IFN-gamma. Maternal infection increased pup mortality, and preliminary findings demonstrate ultrastructural changes in the hippocampal region of the neonatal brain, in a manner analogous to the effects of maternal infection on white matter damage seen in humans. Thus, the threat of maternal oral infectious exposure during pregnancy may not be limited to the duration of gestation, but may also potentially affect perinatal neurological growth and development.

Insulin Response Genes in Different Stages of Periodontal Disease.

Bacterial infections are known to alter glucose metabolism within tissues via mechanisms of inflammation. We conducted this study to examine whether insulin response genes are differentially expressed in gingival tissues, comparing samples from experimental gingivitis and periodontitis subjects to those from healthy individuals. Total RNA was extracted from gingival biopsies from 26 participants: 8 periodontally healthy, 9 experimental gingivitis, and 9 periodontitis subjects. Gene expression patterns were evaluated with a polymerase chain reaction array panel to examine 84 candidate genes involved with glucose metabolism, insulin resistance, and obesity. Array data were evaluated with a t test adjusted by the false discover rate (P < 0.05), and ingenuity pathway analysis was performed for statistical testing of pathways. Although tissue samples were not sufficient to enable protein quantification, we confirmed the upregulation of the key gene using lipopolysaccharide-stimulated primary gingival epithelial cells by Western blot. The mRNA expression patterns of genes that are associated with insulin response and glucose metabolism are markedly different in experimental gingivitis subjects compared with healthy controls. Thirty-two genes are upregulated significantly by at least 2-fold, adjusted for false discover rate (P < 0.05). Periodontitis subjects show similar but attenuated changes in gene expression patterns, and no genes meet the significance criteria. Ingenuity pathway analysis demonstrates significant activation of the carbohydrate metabolism network in experimental gingivitis but not in periodontitis. G6PD protein increases in response to lipopolysaccharide stimulation in primary gingival epithelial cells, which is in the same direction as upregulated mRNA in tissues. Acute gingival inflammation may be associated with tissue metabolism changes, but these changes are not evident in chronic periodontitis. This study suggests that acute gingival inflammation may induce localized changes that modify tissue insulin/glucose metabolism.

Association of Synergistetes and Cyclodipeptides with Periodontitis.

The purpose of this study was to evaluate the microbial community (MC) composition as it relates to salivary metabolites and periodontal clinical parameters in a 21-d biofilm-overgrowth model. Subjects (N = 168) were enrolled equally into 5 categories of periodontal status per the biofilm-gingival interface classification. Microbial species within subgingival plaque samples were identified by human microbiome identification microarray. Whole saliva was analyzed by liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry for metabolite identification. Phylum was grouped into MCs according to principal component analysis. Generalized linear and regression models were used to examine the association among MC, species, periodontal clinical parameters, and salivary metabolome. Multiple comparisons were adjusted with the false discovery rate. The study population was distributed into 8 distinct MC profiles, designated MC-1 to MC-8. MC-2 explained 14% of the variance and was dominated by Synergistetes and Spirochaetes. It was the only community structure significantly associated with high probing depth (P = 0.02) and high bleeding on probing (P = 0.008). MC-2 was correlated with traditional periodontal pathogens and several newly identified putative periodontal pathogens: Fretibacterium fastidiosum, Fretibacterium sp. OT360/OT362, Filifactor alocis, Treponema lecithinolyticum, Eubacterium saphenum, Desulfobulbus sp./OT041, and Mogibacterium timidum. Synergistetes phylum was strongly associated with 2 novel metabolites-cyclo (-leu-pro) and cyclo (-phe-pro)-at 21 d of biofilm overgrowth (P = 0.02). In subjects with severe periodontitis (P2 and P3), cyclo (-leu-pro) and cyclo (-phe-pro) were significantly associated with increased changes in probing depth at 21 d of biofilm overgrowth (P ≤ 0.05). The analysis identified a MC dominated by Synergistetes, with classic and putative newly identified pathogens/pathobionts associated with clinical disease. The metabolomic discovery of 2 novel cyclodipeptides that have been reported to serve as quorum-sensing and/or bacteriocidal/bacteriostatic molecules, in association with Synergistetes, suggests a potential role in periodontal biofilm dysbiosis and periodontal disease that warrants further investigation.

Cementoblast gene expression is regulated by Porphyromonas gingivalis lipopolysaccharide partially via toll-like receptor-4/MD-2.

Lipopolysaccharides are potent inflammatory mediators considered to contribute to destruction of periodontal tissues. Here, we hypothesized that Porphyromonas gingivalis lipopolysaccharide (P-LPS) treatment would regulate gene expression in murine cementoblasts through Toll-like receptor 4. Real-time (RT)-PCR and Northern blot analysis indicated that P-LPS decreased expression of transcripts for osteocalcin (OCN) and receptor activator of nuclear factor kappaB ligand (RANKL). In contrast, a dose-dependent up-regulation in mRNA levels for osteopontin (OPN) and osteoprotegerin (OPG) was observed. Similarly, ELISA demonstrated decreased RANKL and increased OPG levels. A monoclonal antibody specific for mouse TLR-4/MD-2 partially neutralized the P-LPS effect on cementoblasts. These results indicate that exposure of cementoblasts to P-LPS can alter cell function by regulating markers of osteoclastic activity (e.g., RANKL/OPG), thereby potentially affecting the inflammation-associated resorption of mineralized tissues.

Parathyroid hormone protects against periodontitis-associated bone loss.

Parathyroid hormone (PTH) functions as a major mediator of bone remodeling and as an essential regulator of calcium homeostasis. In addition to the well-established catabolic effects (activation of bone resorption) of PTH, it is now recognized that intermittent PTH administration has anabolic effects (promotion of bone formation). The aim of this study was to investigate whether intermittent administration of PTH in rodents would block the alveolar bone loss observed in rats when a ligature model of periodontitis is used. Morphometric analysis showed that intermittent PTH administration (40 microg/kg) was able to protect the tooth site from periodontitis-induced bone resorption. In addition, there was a significant reduction in the number of inflammatory cells at the marginal gingival area in sections obtained from animals receiving PTH compared with control animals. These findings demonstrated that intermittent PTH administration was able to protect against periodontitis-associated bone loss in a rodent model.

The influence of nicotine on the bone loss rate in ligature-induced periodontitis. A histometric study in rats.

BACKGROUND: The present study investigated the possible influence of nicotine on the bone loss rate in the furcation region due to ligature-induced periodontitis in rats. METHODS: Twenty adult male Wistar rats were included. After anesthesia, the tooth was randomly assigned to receive the cotton ligature in the sulcular area, while the contralateral tooth was left unligated. The animals were randomly assigned to one of the following treatments, including daily intraperitoneal injections: group A, 2 microl/g body weight of saline solution; group B, 2 microl/g body weight of a nicotine solution with 0.13 microl of nicotine/ml of saline solution; group C, 2 microl/g body weight of a nicotine solution with 0.19 microl of nicotine/ml of saline solution; and group D, 2 microl/g body weight of a nicotine solution with 0.26 microl of nicotine/ml of saline solution. Thirty days later, the animals were sacrificed and the specimens routinely processed for serial decalcified sections. RESULTS: Intergroup analysis revealed greater bone loss in the ligated teeth of group B (1.01 +/- 0.61 mm2), group C (1.14 +/- 0.72 mm2), and group D (1.36 +/- 0.60 mm2) when compared with group A (0.64 +/- 0.62 mm2) (P <0.01). However, no statistically significant differences in bone loss were found among groups B, C, and D. In addition, no bone loss was observed for unligated teeth (P>0.01). CONCLUSIONS: Within the limits of the present study, nicotine enhanced the effects of the local components of periodontal disease in a non-dose-dependent way; nevertheless, the administration of nicotine did not produce periodontal bone loss by itself.

Ultrastructural aspects of connective tissue in hereditary gingival fibromatosis.

OBJECTIVE: The purpose of this study was to analyze the ultrastructure of gingival connective tissue from patients in one family affected by hereditary gingival fibromatosis (HGF). STUDY DESIGN: Electron microscopic examination was performed with gingival tissue from 10 patients from a Brazilian family with 132 members. Fifty of 96 persons at risk for this disorder were affected, which is consistent with an autosomal dominant pattern of inheritance. RESULTS: The extracellular matrix showed flocculent material and collagen fibrils with structural abnormalities and variation in diameter. Increased numbers of oxytalan fibers were identified; however, elastic fibers were rare in the analyzed areas. CONCLUSIONS: The structural alterations found in HGF appear similar to those described in certain other heritable collagen disorders, suggesting that HGF should be included in the group of hereditary diseases in which connective tissue alterations have a distinct pattern, in contrast to reactive fibrotic gingival enlargements with no genetic component.

Lactobacillus acidophilus NCFM affects colonic mucosal opioid receptor expression in patients with functional abdominal pain - a randomised clinical study.

BACKGROUND: In a recent double-blinded clinical trial, the probiotic combination of Lactobacillus acidophilus NCFM (L-NCFM) and B-LBi07 reduced bloating symptoms in patients with functional bowel disorders; an effect more evident in those who reported abdominal pain. In mice, L-NCFM but not B-LBi07 induced colonic mu-opioid receptor (MOR) and cannabinoid receptor 2 (CB2) expression, and reduced visceral sensitivity. AIMS: To determine if L-NCFM was the active component in the clinical trial and to investigate the mechanism of action in humans with mild to moderate abdominal pain. METHODS: Caucasian women (n = 20) 18-70 years with mild to moderate abdominal pain were enrolled in a double-blind, two-armed, single-centre study. Patients were given either L-NCFM alone or in combination with B-LBi07 for 21 days at a total dose of 2 × 10(10) CFU b.d. Colonic biopsies were collected during unsedated, unprepped flexible sigmoidoscopy before and at the end of probiotic consumption. mRNA and immunostaining were then performed on these biopsies. Patients kept symptom diaries for the 7 days prior to starting probiotic therapy and for the last 7 days of therapy. RESULTS: L-NCFM alone, but not with B-LBi07, induced colonic MOR mRNA and protein expression, as well as downstream signalling, as measured by enterocyte STAT3-phosphorylation. In contrast, CB2 expression was decreased. Both treatment groups trended towards improvement in symptoms, but the study was insufficiently powered to draw meaningful conclusions. CONCLUSIONS: Lactobacillus acidophilus NCFM modulates mu-opioid receptor expression and activity, while the combination of L-NCFM and B-LBi07 does not. This study provides a possible mechanism for action by which probiotics modulates pain sensation in humans (Clinical Trial Number: NCT01064661).

Chronic periodontitis genome-wide association studies: gene-centric and gene set enrichment analyses.

Recent genome-wide association studies (GWAS) of chronic periodontitis (CP) offer rich data sources for the investigation of candidate genes, functional elements, and pathways. We used GWAS data of CP (n = 4,504) and periodontal pathogen colonization (n = 1,020) from a cohort of adult Americans of European descent participating in the Atherosclerosis Risk in Communities study and employed a MAGENTA approach (i.e., meta-analysis gene set enrichment of variant associations) to obtain gene-centric and gene set association results corrected for gene size, number of single-nucleotide polymorphisms, and local linkage disequilibrium characteristics based on the human genome build 18 (National Center for Biotechnology Information build 36). We used the Gene Ontology, Ingenuity, KEGG, Panther, Reactome, and Biocarta databases for gene set enrichment analyses. Six genes showed evidence of statistically significant association: 4 with severe CP (NIN, p = 1.6 × 10(-7); ABHD12B, p = 3.6 × 10(-7); WHAMM, p = 1.7 × 10(-6); AP3B2, p = 2.2 × 10(-6)) and 2 with high periodontal pathogen colonization (red complex-KCNK1, p = 3.4 × 10(-7); Porphyromonas gingivalis-DAB2IP, p = 1.0 × 10(-6)). Top-ranked genes for moderate CP were HGD (p = 1.4 × 10(-5)), ZNF675 (p = 1.5 × 10(-5)), TNFRSF10C (p = 2.0 × 10(-5)), and EMR1 (p = 2.0 × 10(-5)). Loci containing NIN, EMR1, KCNK1, and DAB2IP had showed suggestive evidence of association in the earlier single-nucleotide polymorphism-based analyses, whereas WHAMM and AP2B2 emerged as novel candidates. The top gene sets included severe CP ("endoplasmic reticulum membrane," "cytochrome P450," "microsome," and "oxidation reduction") and moderate CP ("regulation of gene expression," "zinc ion binding," "BMP signaling pathway," and "ruffle"). Gene-centric analyses offer a promising avenue for efficient interrogation of large-scale GWAS data. These results highlight genes in previously identified loci and new candidate genes and pathways possibly associated with CP, which will need to be validated via replication and mechanistic studies.

Toll-like receptor 4 mediates intrauterine growth restriction after systemic Campylobacter rectus infection in mice.

Campylobacter rectus is associated with fetal exposure and low birthweight in humans. C. rectus also invades placental tissues and induces fetal intrauterine growth restriction (IUGR) in mice, along with overexpression of Toll-like receptors (TLR4), suggesting that TLR4 may mediate placental immunity and IUGR in mice. To test this hypothesis we examined the effect of in vitro TLR4 neutralization on trophoblastic proinflammatory activity and studied the IUGR phenotype in a congenic TLR4-mutant mouse strain after in vivo C. rectus infection. Human trophoblasts were pretreated with TLR4 neutralizing antibodies and infected with C. rectus; proinflammatory cytokine production was assessed by cytokine multiplex assays. Neutralizing TLR4 antibodies significantly impaired the production of proinflammatory cytokines in trophoblastic cells after infection in a dose-dependent manner. We used a subcutaneous chamber model to provide a C. rectus challenge in BALB/cAnPt (TLR4(Lps-d) ) and wild-type (WT) females. Females were mated with WT or TLR4(Lps-d) males once/week; pregnant mice were infected at (E)7.5 and sacrificed at (E)16.5 to establish IUGR phenotypes. Maternal C. rectus infection significantly decreased fetal weight/length in infected WT when compared with sham WT controls (P < 0.05, analysis of variance). However, infected TLR4(Lps-d -/-) mice did not show statistically significant differences in fetal weight and length when compared with WT controls (P > 0.05). Furthermore, heterozygous TLR4(Lps-d +/-) fetuses showed IUGR phenotype rescue. We conclude that TLR4 is an important mediator of trophoblastic proinflammatory responses and TLR4-deficient fetuses do not develop IUGR phenotypes after C. rectus infection, suggesting that placental cytokine activation is likely to be mediated by TLR4 during low birthweight/preterm birth pathogenesis.

MicroRNA modulation in obesity and periodontitis.

The aim of this pilot investigation was to determine if microRNA expression differed in the presence or absence of obesity, comparing gingival biopsies obtained from patients with or without periodontal disease. Total RNA was extracted from gingival biopsy samples collected from 20 patients: 10 non-obese patients (BMI < 30 kg/m(2)) and 10 obese patients (BMI > 30 kg/m(2)), each group with 5 periodontally healthy sites and 5 chronic periodontitis sites. MicroRNA expression patterns were assessed with a quantitative microRNA PCR array to survey 88 candidate microRNA species. Four microRNA databases were used to identify potential relevant mRNA target genes of differentially expressed microRNAs. Two microRNA species (miR-18a, miR-30e) were up-regulated among obese individuals with a healthy periodontium. Two microRNA species (miR-30e, miR-106b) were up-regulated in non-obese individuals with periodontal disease. In the presence of periodontal disease and obesity, 9 of 11 listed microRNAs were significantly up-regulated (miR-15a, miR-18a, miR-22, miR-30d, miR-30e, miR-103, miR-106b, miR-130a, miR-142-3p, miR-185, and miR-210). Predicted targets include 69 different mRNAs from genes that comprise cytokines, chemokines, specific collagens, and regulators of glucose and lipid metabolism. The expression of specific microRNA species in obesity, which could also target and post-transcriptionally modulate cytokine mRNA, provides new insight into possible mechanisms of how risk factors might modify periodontal inflammation and may represent novel therapeutic targets.