Periodontitis, pathogenesis and progression: miRNA-mediated cellular responses to Porphyromonas gingivalis.

Porphyromonas gingivalis is considered a keystone pathogen in periodontitis, a disease typically driven by dysbiosis of oral inflammophilic polymicrobial pathobionts. To combat infectious agents, the natural defense response of the host is to switch on inflammatory signaling cascades, whereby microRNA (miRNA) species serve as alternative genetic inhibitory transcriptional endpoints. miRNA profiles from diseased sites differ from those detected in disease-free tissues. miRNA profiles could therefore be harnessed as potential diagnostic/prognostic tools. The regulatory role of some miRNA species (miRNA-128, miRNA-146, miRNA-203, and miRNA-584) in the innate immune system suggests these molecular signatures also have potential in therapy. P. gingivalis-associated miRNAs are likely to influence the innate immune response, whereas its lipopolysaccharide may affect the nature of host miRNAs and their mRNA targets. This mini review discusses miRNA-dependent transcriptional and regulatory phenomena ensuing immune signaling cascade switch-on with development and progression of periodontitis initiated by P. gingivalis exposure.

Anti-proliferative Properties of miR-20b and miR-363 from the miR-106a-363 Cluster on Human Carcinoma Cells.

BACKGROUND: The miR-106a-363 cluster, encoding six miRNAs (miR- 106a, miR-18b, miR-20b, miR-19b-2, miR-92-2 and miR-363), has been shown to be overexpressed in various tumours. In oral carcinoma cells, however only miR- 106a was detectable from this cluster. We have investigated how effects of transfection of oral carcinoma cells with a non-expressed member of this cluster affect mRNA transcriptomes and cellular selected functions. METHODS: Investigate effects of miR-20b and miR-363 mimics on cellular respiration, glycolysis and mobility. Effects on mRNA transcriptomes were monitored using microarrays. RESULTS: The studies show that in oral carcinoma cells transfected with miR-20b -, or miR-363-3p or miR-363-5p mimic different mRNAs were differentially expressed. Nevertheless, bioinformatics analysis suggested significant associations of differentially expressed genes to inhibition of cellular proliferation, cell cycle and cellular migration. These results were also experimentally confirmed. CONCLUSIONS: Transfection of miRNA mimics for unexpressed members of the miR-106a-363 cluster (miR20b, miR-363-3p and miR-363-5p) exhibit an anti-proliferative effect on oral carcinoma cells, although likely mediated by different regulatory mechanisms.

Mapping the global mRNA transcriptome during development of the murine first molar.

The main objective of this study was to map global gene expression in order to provide information about the populations of mRNA species participating in murine tooth development at 24 h intervals, starting at the 11th embryonic day (E11.5) up to the 7th post-natal day (P7). The levels of RNA species expressed during murine tooth development were mesured using a total of 58 deoxyoligonucleotide microarrays. Microarray data was validated using real-time RT-PCR. Differentially expressed genes (p < 0.05) were subjected to bioinformatic analysis to identify cellular activities significantly associated with these genes. Using ANOVA the microarray data yielded 4362 genes as being differentially expressed from the 11th embryonic day (E11.5) up to 7 days post-natal (P7), 1921 of these being genes without known functions. The remaining 2441 genes were subjected to further statistical analysis using a supervised procedure. Bioinformatic analysis results for each time-point studied suggests that the main molecular functions associated with genes expressed at the early pre-natal stages (E12.5-E18.5) were cell cycle progression, cell morphology, lipid metabolism, cellular growth, proliferation, senescence and apoptosis, whereas most genes expressed at post-natal and secretory stages (P0-P7) were significantly associated with regulation of cell migration, biosynthesis, differentiation, oxidative stress, polarization and cell death. Differentially expressed genes (DE) not described earlier during murine tooth development; Inositol 1, 4, 5-triphosphate receptor 3 (Itpr3), metallothionein 1(Mt1), cyclin-dependent kinase 4 (Cdk4), cathepsin D (Ctsd), keratin complex 2, basic, gene 6a (Krt2-6a), cofilin 1, non-muscle (Cfl1), cyclin 2 (Ccnd2), were verified by real-time RT-PCR.

An investigation into anti-proliferative effects of microRNAs encoded by the miR-106a-363 cluster on human carcinoma cells and keratinocytes using microarray profiling of miRNA transcriptomes.

Transfection of human oral squamous carcinoma cells (clone E10) with mimics for unexpressed miR-20b or miR-363-5p, encoded by the miR-106a-363 cluster (miR-20b, miR-106a, miR-363-3p, or miR-363-5p), caused 40-50% decrease in proliferation. Transfection with mimics for miR-18a or miR-92a, encoded by the miR-17-92 cluster (all members being expressed in E10 cells), had no effect on proliferation. In contrast, mimic for the sibling miRNA-19a yielded about 20% inhibition of proliferation. To investigate miRNA involvement profiling of miRNA transcriptomes were carried out using deoxyoligonucleotide microarrays. In transfectants for miR-19a, or miR-20b or miR-363-5p most differentially expressed miRNAs exhibited decreased expression, including some miRNAs encoded in paralogous miR-17-92-or miR-106b-25 cluster. Only in cells transfected with miR-19a mimic significantly increased expression of miR-20b observed-about 50-fold as judged by qRT-PCR. Further studies using qRT-PCR showed that transfection of E10 cells with mimic for miRNAs encoded by miR-17-92 - or miR-106a-363 - or the miR-106b-25 cluster confirmed selective effect on expression on sibling miRNAs. We conclude that high levels of miRNAs encoded by the miR-106a-363 cluster may contribute to inhibition of proliferation by decreasing expression of several sibling miRNAs encoded by miR-17-92 or by the miR-106b-25 cluster. The inhibition of proliferation observed in miR-19a-mimic transfectants is likely caused by the miR-19a-dependent increase in the levels of miR-20b and miR-106a. Bioinformatic analysis of differentially expressed miRNAs from miR-106a, miR-20b and miR-363-5p transfectants, but not miR-92a transfectants, yielded significant associations to "Cellular Growth and Proliferation" and "Cell Cycle." Western blotting results showed that levels of affected proteins to differ between transfectants, suggesting that different anti-proliferative mechanisms may operate in these transfectants.

Expression of Clu and Tgfb1 during murine tooth development: effects of in-vivo transfection with anti-miR-214.

Expression of clusterin (Clu) in the murine first molar tooth germ was markedly increased at postnatal developmental stages. The time-course of expression of this gene paralleled those of other genes encoding proteins involved during the secretory phase of odontogenesis, as described previously. Immunohistochemical studies of clusterin in murine molar tooth germs suggested this protein to be located in outer enamel epithelium, regressing enamel organ, secretory ameloblasts, and the dental epithelium connecting the tooth to the oral epithelium at an early eruptive stage. Immunolabelling of transforming growth factor beta-1 (TGF-ß1) revealed it to be located close to clusterin. The levels of expression of Clu and Tgfb1 were markedly decreased following in-vivo transfection with anti-miR-214. In contrast, the expression of several genes associated with regulation of growth and development were increased by this treatment. We suggest that clusterin has functions during secretory odontogenesis and the early eruptive phase. Bioinformatic analysis after treatment with anti-miR-214 suggested that, whilst cellular activities associated with tooth mineralization and eruption were inhibited, activities associated with an alternative developmental activity (i.e. biosynthesis of contractile proteins) appeared to be stimulated. These changes probably occur through regulation mediated by a common cluster of transcription factors and support suggestions that microRNAs (miRNAs) are highly significant as regulators of differentiation during odontogenesis.

Gene Expression Profiling during Murine Tooth Development.

The aim of this study was to describe the expression of genes, including ameloblastin (Ambn), amelogenin X chromosome (Amelx), and enamelin (Enam) during early (pre-secretory) tooth development. The expression of these genes has predominantly been studied at post-secretory stages. Deoxyoligonucleotide microarrays were used to study gene expression during development of the murine first molar tooth germ at 24 h intervals, starting at the 11th embryonic day (E11.5), and up to the 7th day after birth (P7). The profile search function of Spotfire software was used to select genes with similar expression profile as the enamel genes (Ambn, Amelx, and Enam). Microarray results where validated using real-time reverse transcription-polymerase chain reaction (real-time RT-PCR), and translated proteins identified by Western-blotting. In situ localization of the Ambn, Amelx, and Enam mRNAs were monitored from E12.5 to E17.5 using deoxyoligonucleotide probes. Bioinformatics analysis was used to associate biological functions with differentially expressed (DE; p ≤ 0.05) genes. Microarray results showed a total of 4362 genes including Ambn, Amelx, and Enam to be significant DE throughout the time-course. The expression of the three enamel genes was low at pre-natal stages (E11.5-P0) increasing after birth (P1-P7). Profile search lead to isolation of 87 genes with significantly similar expression to the three enamel proteins. These mRNAs were expressed in dental epithelium and epithelium derived cells. Although expression of Ambn, Amelx, and Enam were lower during early tooth development compared to secretory stages enamel proteins were detectable by Western-blotting. Bioinformatic analysis associated the 87 genes with multiple biological functions. Around 35 genes were associated with 15 transcription factors.

Expression of delta-like 1 homologue and insulin-like growth factor 2 through epigenetic regulation of the genes during development of mouse molar.

Delta-like 1 homolog (Dlk1) and insulin-like growth factor 2 (Igf2) are two of six well-studied mouse imprinted gene clusters that are paternally expressed. Their expression is also linked to their maternally expressed non-coding RNAs, encoded by Gene trap locus 2 (Gtl2) and Imprinted maternally expressed transcript (H19), co-located as imprinted gene clusters. Using deoxyoligonucleotide microarrays and real-time RT-PCR analysis we showed Dlk1 and Gtl2 to exhibit a time-course of expression during tooth development that was similar to that of Igf2 and H19. Western blot analysis of proteins encoded by Dlk1 and Igf2 suggested that the levels of these proteins reflected those of the corresponding mRNAs. Immunohistochemical studies of DLK1 in murine molars detected the protein in both epithelial and mesenchymal regions, in developing cusp mesenchyme, and in newly synthesized enamel and dentin tubules. IGF2 protein was detected primarily at prenatal stages, suggesting that it may be active before birth. Analysis of methylation of cytosine-phosphate-guanine (CpG) islands in both Dlk1 and Igf2 suggested the presence of an increasing fraction of hypermethylated bases with increasing time of development. The increased levels of hypermethylation coincided both with the diminished levels of expression of Dlk1 and Igf2 and with decreased levels of DLK1 and IGF2 proteins in the tooth germ, suggesting that their expression is regulated via methylation of CpG islands present in these genes.

Deoxyoligonucleotide microarrays for gene expression profiling in murine tooth germs.

The use of deoxyoligonucleotide microarrays facilitates rapid expression profiling of gene expression using samples of about 1 µg of total RNA. Here are described practical aspects of the procedures involved, including essential reagents. Analysis of results is discussed from a practical, experimental, point of view together with software required to carry out the required statistical analysis to isolate populations of differentially expressed genes.

Expression of members of the miRNA17-92 cluster during development and in carcinogenesis.

The six microRNAs (miRNA) encoded by the miR-17-92 cluster, also named oncomir-1, have been associated with carcinogenesis and typically exhibit-increased expression in tumors. Despite the well-established role for the miR-17-92 cluster in an oncogenic network, the physiological function of these miRNAs in normal tissues remains unresolved. In order to investigate whether there are similar patterns of miR-17-92 expression during embryogenesis and carcinogenesis, we have preformed a systematic study of the expression in cultured carcinoma cells, cultured primary human keratinocytes (KC), and during development of some murine tissues. Both levels of expression of the primary transcript (pri-miRNA) and levels of expression of the individual members of the cluster were monitored. Irrespectively of tissue examined we found that the level of expression decreased markedly during development. With cultured primary human KCs their levels of expression of some of these microRNAs decreased as the number of cell passages increased. Their levels of expression in cultured carcinoma cells, in contrasts, increased, or remained unchanged, with increasing number of cell passages. The results suggest these microRNAs are involved in the regulation of foetal development and that they may promote proliferation and inhibit differentiation during embryogenesis and carcinogenesis. Additionally, the six microRNAs exhibit variable tissue expression, suggesting selective processing of these microRNAs.

Effects of in vivo transfection with anti-miR-214 on gene expression in murine molar tooth germ.

MicroRNAs (miRNAs) are an abundant class of noncoding RNAs that are believed to be important in many biological processes through regulation of gene expression. Little is known of their function in tooth morphogenesis and differentiation. MicroRNA-214 (miR-214), encoded by the polycistronic Dnm30os gene, is highly expressed during development of molar tooth germ and was selected as a target for silencing with anti-miR-214. Mandibular injection of 1-100 pmol of anti-miR-214 close to the developing first molar in newborn mice resulted in significant decrease in expression of miR-214, miR-466h, and miR-574-5p in the tooth germ. Furthermore, levels of miR-199a-3p, miR-199a-5p, miR-690, miR-720, and miR-1224 were significantly increased. Additionally, the expression of 863 genes was significantly increased and the expression of 305 genes was significantly decreased. Among the genes with increased expression was Twist-1 and Ezh2, suggested to regulate expression of miR-214. Microarray results were validated using real-time RT-PCR and Western blotting. Among genes with decreased expression were Amelx, Calb1, Enam, and Prnp; these changes also being reflected in levels of corresponding encoded proteins in the tooth germ. In the anti-miR-214-treated molars the enamel exhibited evidence of hypomineralization with remnants of organic material and reduced surface roughness after acid etching, possibly due to the transiently decreased expression of Amelx and Enam. In contrast, several genes encoding contractile proteins exhibited significantly increased expression. mRNAs involved in amelogenesis (Ambn, Amelx, Enam) were not found among targets of miRNAs that were differentially expressed following treatment with anti-miR-214. It is therefore suggested that effects of miR-214 on amelogenesis are indirect, perhaps mediated by the observed miR-214-dependent changes in levels of expression of numerous transcription factors.

Expression of prion gene and presence of prion protein during development of mouse molar tooth germ.

In order to gain insight into possible cellular functions of the prion protein (PrP) during normal development, the expression of Prnp (encoding the PrP) and the distribution of the PrP were studied in murine tooth germs. Expression of Prnp in the mouse first molar tooth germ was highly dynamic, increasing several-fold during the secretory phase of odontogenesis, exhibiting a time-course of expression similar to that of genes coding for other extracellular proteins [e.g. enamel matrix proteins (Amelx, Ambn, Enam), Aplp1, Clstn1, and Clu]. Western blot analysis suggested that the amounts of PrP and amyloid beta (A4) precursor-like protein 1 (APLP1) in the tooth germ followed time-courses similar to those of the corresponding mRNAs. Immunohistochemical studies of the distribution of PrP in murine molar and incisor tooth germs at embryonic day (E)18.5 suggested that this protein was located in the cervical loop, outer enamel epithelium, pre-ameloblasts, and dental papilla. Different degrees of immunolabelling of pre-ameloblasts on the mesial and distal aspects of a lower molar cusp may be related to different enamel configurations on the two aspects. It is concluded that the dynamic patterns of expression of Prnp, and of distribution of PrP, suggest that PrP may have functions during secretory odontogenesis, perhaps in relation to amelogenesis.

Gene expression and dental enamel structure in developing mouse incisor.

At the mouse incisor tip the initially differentiated ameloblasts produce a thin, prism-free enamel, while further apically, in the immediate adjacent segment, the enamel thickness increases and the four-layered enamel of mouse incisor is formed. Comparative gene-expression profiling was carried out on RNA isolated from these two segments of incisor tooth germs at embryonic day (E)17.5 and at postnatal days (P)0, 1, 2, and 10 using microarrays to measure messenger RNA (mRNA) and microRNA (miRNA) species present in the segments. Validation of expression data was achieved using real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blotting. Bioinformatic data suggested enhanced cellular apoptosis in the incisal tip segment, which, together with diminished expression of the Amelx and Enam genes, may contribute to the production of the thin enamel seen in this tooth segment. For genes exhibiting higher levels of expression in the adjacent segment where complex enamel is being formed, bioinformatic analysis suggested significant associations with cellular functions involving the actin cytoskeleton, cellular development, morphology, and movement. This is suggested to reflect that ameloblasts with Tomes' process are being organized in transverse rows, facilitating the transverse movement that results in prism decussation in the inner enamel of the adjacent segment. Bioinformatic analysis of miRNA expression data lends support to these suggestions.

Signal transduction and gene transcription induced by TFF3 in oral keratinocytes.

Trefoil factor family 3 (TFF3) is secreted in saliva. The peptide improves the mechanical and chemical resistance of mucins, and it may act as a motility signal for oral keratinocytes during wound healing. This study aimed to identify novel functions of TFF3 in oral keratinocytes. To achieve this, we used phosphoprotein and messenger RNA (mRNA) arrays to compare TFF3-treated and untreated oral keratinocytes. Analysis of the phosphoprotein array indicated that TFF3 signals through the mitogen-activated protein kinases (MAPKs) c-Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (ERK1/2), and through the phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB) pathway. Microarray analysis of mRNA showed that TFF3 stimulation induced changes in the expression of genes functionally related to cell death/survival, cell growth and proliferation, and cell movement. The reverse transcription-polymerase chain reaction (RT-PCR) results indicated that the transcription of some immediate-early genes (IEGs) was downregulated, whereas the IEGs FBJ osteosarkoma oncogene (FOS) and C-MYC binding protein (MYCBP2) were transiently upregulated by TFF3 stimulation. Together, the results of the arrays indicate that TFF3 is a modifying factor in pathways regulating cell survival, cell growth and proliferation, and cell migration of oral keratinocytes. Trefoil factor family 3 may therefore promote oral wound healing and it should be considered for the treatment of oral ulcerating diseases, or of other diseases.

Family-with-sequence-similarity-46, member A (Fam46a) gene is expressed in developing tooth buds.

OBJECTIVE: In search for possible novel genes that may be involved in tooth development, we analysed the genome-wide transcriptome of developing mandibular tooth germs of mouse during embryonic and early life and selected family-with-sequence-similarity-46, member A (Fam46a) gene for further expression analysis. METHODS: We applied microarray, quantitative real time polymerase chain reaction and in situ hybridisation methods for the expression study of the mouse Fam46a gene. RESULTS: We found the family-with-sequence-similarity-46, member A (Fam46a) gene to be highly expressed and further verify its temporo-spatial expression in the mouse tooth. CONCLUSION: We have shown that Fam46a is expressed in ameloblasts' nuclei of tooth germs and hypothesise that it might act together with morphogenetic factors important for the formation of enamel in mouse tooth.

Differential gene expression profiling of the molar tooth germ in peroxisome proliferator-activated receptor-alpha (PPAR-alpha) knockout mouse and in wild-type mouse: molar tooth phenotype of PPAR-alpha knockout mouse.

Gene expression profiling of the first molar tooth germ at embryonic days (E)17.5 and 18.5, and at postnatal days (P)0, 2, and 6 from peroxisome proliferator-activated receptor-alpha (PPAR-alpha) knockout mouse and from wild-type mouse was carried out using microarrays and validated using real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blotting. When comparing expression profiles at each time-point, a total of 1,235 genes showed significantly different expression, 772 of which exhibited significantly decreased expression in tooth germ from knockout mouse. With genes exhibiting significantly decreased levels of expression in tooth germ from PPAR-alpha knockout mouse, bioinformatic analysis using ingenuity pathway analysis yielded significant associations to cellular functions related to cellular growth/proliferation and to networks related to regulation of calcium homeostasis. Using scanning electron microscopy to investigate molars from adult PPAR-alpha knockout mouse, the molar size was found to be slightly reduced, the enamel structure was found to be normal, but cervical molar enamel exhibited evidence suggesting hypomineralization. Although the PPAR-alpha knockout had no significant effect on molar morphology, the results suggest that active PPAR-alpha signaling is required to achieve normal mineralization of molar enamel, most probably through regulation of calcium homeostasis and metabolism of vitamin D. Cyp27b1 was expressed in tooth germ, suggesting that tooth germ can synthesize active vitamin D. Expression of Cyp27b1 was significantly enhanced in postnatal PPAR-alpha knockout tooth germ.

Comparative hepatic gene expression profiling of rats treated with 1H,1H,2H,2H-heptadecafluorodecan-1-ol or with pentadecafluorooctanoic acid.

Pentadecafluorooctanoic acid is an established peroxisome proliferator. Little is known about effects of treatment with 1H,1H,2H,2H-heptadecafluorodecan-1-ol, which is metabolized to pentadecafluorooctanoic acid. We compared effects of various dosages (3, 10, or 25 mg/kg body wt) of each of these compounds on hepatic gene expression in rats with microarrays. Microarray data were validated by real-time RT-PCR. Expression data were also correlated with hepatic activities of selected enzymes and with hepatic levels of pentadecafluorooctanoic acid and 1H,1H,2H,2H-heptadecafluorodecan-1-ol. Pentadecafluorooctanoic acid caused the more powerful change in gene expression, in terms of both number of genes affected and extent of change in expression. Across the dosages used pentadecafluorooctanoic acid and 1H,1H,2H,2H-heptadecafluorodecan-1-ol caused significant (P < or = 0.05) changes in expression for 441 and 105 genes, respectively. With 1H,1H,2H,2H-heptadecafluorodecan-1-ol approximately 38% of the 105 genes exhibited decreased expression with a dose of 25 mg/kg body wt, these genes also appearing less responsive to treatment at the lower dosages. Bioinformatic analysis suggested that these genes are associated with regulatory functions. With pentadecafluorooctanoic acid, increasing dosage up to 10 mg/kg body wt brought about progressive increase in expression of affected genes. Pathways analysis suggested similar effects of the two compounds on lipid and amino acid metabolism. Marked differences were also found, particularly with respect to effects on genes related to oxidative phosphorylation, oxidative metabolism, free radical scavenging, xenobiotic metabolism, and complement and coagulation cascades.

MicroRNA expression profiling of the developing murine molar tooth germ and the developing murine submandibular salivary gland.

Using microarrays, miRNA expression profiles have been established at selected times during development (E15.5, P0 and P5) of the murine first molar mandibular tooth germ and the right submandibular salivary gland (E15.5, P0, P5 and P25). Microarray data was validated using real-time PCR, also facilitating RT-PCR profiling of nine selected miRNAs. In general, good agreement between microarray data and real-time PCR data was found. Further, miRNA expression profiles of foetal and adult liver were also investigated, and found to agree with published data. In tooth germ and salivary gland up to 88 different miRNAs were detected. In all tissues examined miRNA expression was highly dynamic; miRNA profiles changing extensively with time of development. Additionally, the expression of some miRNAs was tissue-specific. Bioinformatic analysis of clusters of miRNAs was attempted using the miRGate software, the results suggesting miRNAs to be involved in the regulation of essential developmental processes, e.g., epithelical cell proliferation, mesodermal cell fate determination and salivary gland morphogenesis.

Changes in gene-expression during development of the murine molar tooth germ.

In a matter of a few days the murine tooth germ develops into a complex, mineralized, structure. Murine 30K microarrays were used to examine gene expression in the mandibular first molar tooth germs isolated at 15.5dpc and at 2DPN. Microarray results were validated using real-time RT-PCR. The results suggested that only 25 genes (3 without known functions) exhibited significantly higher expression at 15.5dpc compared to 2DPN. In contrast, almost 1400 genes exhibited significantly (P<0.015) higher expression at 2DPN compared to 15.5dpc, about half of which were genes with unknown functions. More than 50 of the 783 known genes exhibited higher than 10-fold increase in expression at 2DPN, amongst these were genes coding for enamel matrix proteins which were expressed several 100-fold higher at 2DPN. GO and KEGG analysis showed highly significant associations between families of the 783 known genes and cellular functions relating to energy metabolism, protein metabolism, regulation of cell division, cell growth and apoptosis. The use of bioinformatics analysis therefore yielded a functional profile in agreement with known differences in tissue morphology and cellular composition between these two stages. Such data is therefore useful in directing attention towards genes, or cellular activities, which likely are worthy of further studies as regards their involvement in odontogenesis.

Enhanced susceptibility to periodontitis in an animal model of depression: reversed by chronic treatment with the anti-depressant tianeptine.

OBJECTIVE: To test the hypothesis that the olfactory bulbectomy model of depression in rats could influence susceptibility to ligature-induced periodontitis, and that chronic treatment with the anti-depressant drug tianeptine could attenuate this effect. MATERIAL AND METHODS: Tianeptine was given twice daily (10 mg/kg, i.p.) during the entire experiment, starting 29 days before induction of olfactory bulbectomy and periodontitis. Olfactory bulbectomized (OB) rats and sham-operated rats were given saline in a similar manner. Periodontal disease was assessed when the ligatures had been in place for 21 days. Two hours before decapitation, rats were injected with lipopolysaccharide (LPS;100 microg/kg, i.p.) to induce a robust immune and stress response. RESULTS: Compared with sham-operated controls, OB rats developed significantly more periodontal bone loss, exhibited characteristic behavioural responses in a novel open field test, and showed a decreased expression of glucocorticoid receptors (GRs) in the hippocampus. LPS provoked a significantly larger increase in circulating levels of the stress hormone corticosterone and the cytokine transformation growth factor (TGF)-1beta but smaller tumour necrosis factor (TNF)-alpha levels. Tianeptine treatment of OB rats significantly inhibited peridodontal bone loss, normalized behavioural responses, enhanced TGF-1beta levels, and abolished TNF-alpha decrease, but did not attenuate the increased corticosterone response and the decreased hippocampal GR expression. CONCLUSIONS: These experimental results are consistent with an emerging literature showing that life stress, anxiety, depression, pathological grief, and poor coping behaviour may dysregulate regulatory mechanisms within the brain involved in immune regulation, and thereby alter immune responses and influence the susceptibility/resistance to inflammatory disorders.