Porphyromonas gingivalis B cell Antigen Epitope Vaccine, pIRES-ragB'-mGITRL, Promoted RagB-Specific Antibody Production and Tfh Cells Expansion.

The outer membrane protein RagB is one of the major virulence factors of Porphyromonas gingivalis (P. gingivalis). To prevent periodontitis and associated systemic diseases induced by P. gingivalis, we built B cell antigen epitope vaccine characterized by pIRES-ragB'-mGITRL to induce a protective immune responses. The B cell antigen epitope and scrambled peptide of ragB were predicted, cloned into pIRES and constructed pIRES-ragB', pIRES-scrambled epitopes and pIRES-ragB'-mGITRL. pIRES-ragB'-mGITRL was transfected into COS-7 cells. Subsequently, the 6-week-old female BALB/c mice were challenged by P. gingivalis following three time immunization by pIRES, pIRES-ragB', pIRES-scrambled epitopes and pIRES-ragB'-mGITRL. The levels of RagB-specific antibody in the serum and Tfh cells in the spleen were measured by ELISA and flow cytometry, respectively. And higher levels of RagB-specific IgG were produced in the immunized mice with pIRES-ragB'-mGITRL. Additionally, the number of Tfh cells was also expanded and lesions were diminished in pIRES-ragB'-mGITRL mice comparing with control groups. Our results clearly demonstrated that P. gingivalis B cell antigen epitope vaccine, pIRES-ragB'-mGITRL, could induce protective immune responses. Furthermore, our data also indicated that pIRES-ragB'-mGITRL was a potential therapeutic vaccine against P. gingivalis.

Inhibitors of histone deacetylases in class I and class II suppress human osteoclasts in vitro.

Histone deacetylase inhibitors (HDACi) suppress cancer cell growth, inflammation, and bone resorption. The aim of this study was to determine the effect of inhibitors of different HDAC classes on human osteoclast activity in vitro. Human osteoclasts generated from blood mononuclear cells stimulated with receptor activator of nuclear factor kappa B (RANK) ligand were treated with a novel compound targeting classes I and II HDACs (1179.4b), MS-275 (targets class I HDACs), 2664.12 (targets class II HDACs), or suberoylanilide hydroxamic acid (SAHA; targets classes I and II HDACs). Osteoclast differentiation was assessed by expression of tartrate resistant acid phosphatase and resorption of dentine. Expression of mRNA encoding for osteoclast genes including RANK, calcitonin receptor (CTR), c-Fos, tumur necrosis factor (TNF) receptor associated factor (TRAF)6, nuclear factor of activated T cells (NFATc1), interferon-ß, TNF-like weak inducer of apoptosis (TWEAK), and osteoclast-associated receptor (OSCAR) were assessed. Expression of HDACs 1-10 during osteoclast development was also assessed. 1179.4b significantly reduced osteoclast activity (IC(50) < 0.16 nM). MS-275 (IC(50) 54.4 nM) and 2664.12 (IC(50) > 100 nM) were markedly less effective. A combination of MS-275 and 2664.12 inhibited osteoclast activity similar to 1179.4b (IC(50) 0.35 nM). SAHA was shown to suppress osteoclast activity (IC(50) 12 nM). 1179.4b significantly (P < 0.05) reduced NFATc1, CTR, and OSCAR expression during the later stages of osteoclast development. Class I HDAC 8 and Class II HDAC5 were both elevated (P < 0.05) during osteoclast development. Results suggest that inhibition of both classes I and II HDACs may be required to suppress human osteoclastic bone resorption in vitro.

Transient TWEAK overexpression leads to a general salivary epithelial cell proliferation.

OBJECTIVES: Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a multifunctional cytokine that has pro-apoptotic, pro-angiogenic and pro-inflammatory effects. In liver, TWEAK leads to proliferation of progenitor oval cells, but not of mature hepatocytes. This study evaluated the hypothesis that TWEAK overexpression in salivary glands would lead to the proliferation of a salivary progenitor cell. METHODS: A recombinant, serotype 5 adenoviral vector encoding human TWEAK, AdhTWEAK, was constructed, initially tested in vitro, and then administered to male Balb/c mice via cannulation of Wharton's duct. TWEAK expression in vivo was monitored as protein secreted into saliva and serum by enzyme-linked immunosorbent assays. Salivary cell proliferation was monitored by proliferating cell nuclear antigen staining and apoptosis was monitored using TUNEL staining. RESULTS: AdhTWEAK administration led to a dose-dependent, transient TWEAK protein expression, detected primarily in saliva. Salivary epithelial cell proliferation was generalized, peaking on approximately days 2 and 3. TWEAK expression had no detectable effect on apoptosis of salivary epithelial cells. CONCLUSION: Transient overexpression of TWEAK in murine salivary glands leads to a general proliferation of epithelial cells vs a selective stimulation of a salivary progenitor cell.

Repertoire of mouse ectodysplasin-A (EDA-A) isoforms.

Mutations in ectodysplasin-A (EDA) cause loss of hair, sweat glands, and teeth in man and mouse. Isoform EDA-A1 protein shows partial rescue of the affected Tabby mouse phenotypes, suggesting that other isoforms may be required for full function. We describe genomic structure for five EDA isoforms, EDA-A1', A5, A5', A6, and A6', in addition to the previously known EDA-A1, A2, A3, and A4. The novel isoforms together account for approximately 12% of total EDA transcripts. The most different, EDA-A6 and A6', which lack the critical domain for interaction with NF-kappaB-activating receptors, were nevertheless confirmed to be present in mouse and human skin tissue. Other isoforms, EDA-A5 and A5', for example, activated NF-kappaB through receptors EDAR and XEDAR. These properties make new isoforms candidates for modulators of EDA function.

No association between selected candidate gene polymorphisms and severe chronic periodontitis.

BACKGROUND: Chronic periodontitis (CP) risk is influenced by environmental and genetic factors. Using a case-control design, we tested for associations between CP and selected DNA sequence variations (single nucleotide polymorphisms [SNPs]) in or near genes coding for proteins that play a role in the pathogenesis of this disease. METHODS: DNA was analyzed from 219 whites who were examined clinically. Cases (N=137) were >or=35 years of age with eight or more teeth having >or=5 mm of proximal clinical attachment loss. Controls (N=82) were >or=45 years of age with minimal or no proximal attachment loss or pocketing. Nine diallelic polymorphisms (gene and SNP descriptor) were studied in subjects: cytotoxic T-lymphocyte antigen-4 (CTLA-4, 49 A>G), human beta-defensin-1 (DEFB1, 692 G>A), intercellular adhesion molecule-1 (ICAM-1, 1548 A>G), Fas ligand (fasL, -844 C>T), inducible costimulator (ICOS, 3990 G>T), interleukin-6 (IL-6, -174 G>C), cysteine-cysteine chemokine receptor-5 (CCR5, 59653 C>T), osteoprotegerin (OPG, 245 T>G), and osteopontin (OPN, 707 C>T). Genotypes were determined using an automated fluorogenic 5'-nuclease, polymerase chain reaction-based assay. Gender and smoking history (pack-years) were included as covariates in logistic regression analyses. RESULTS: Heavy smoking (>10 pack-years) and male gender were significantly associated with disease (P<0.001). For all SNPs tested, the allele frequencies and distributions of genotypes did not differ between cases and controls (P>0.05). No unadjusted or adjusted odds ratios (comparing genotypes in cases versus controls) were significantly different than 1.0 (P>0.05) under any additive, dominant, or recessive inheritance model. CONCLUSIONS: None of the SNPs tested were strongly associated with generalized severe chronic periodontitis in North American whites. A potentially more fruitful approach in future studies will be to test for associations between periodontitis and haplotype blocks constructed from either multiple SNPs in candidate gene regions or from panels of markers that span the entire genome.

Prioritization of candidate genes for periodontitis using multiple computational tools.

BACKGROUND: Both genetic and environmental factors contribute to the development of periodontitis. Genetic studies identified a variety of candidate genes for periodontitis. The aim of the present study is to identify the most promising candidate genes for periodontitis using an integrative gene ranking method. METHODS: Seed genes that were confirmed to be associated with periodontitis were identified using text mining. Three types of candidate genes were then extracted from different resources (expression profiles, genome-wide association studies). Combining the seed genes, four freely available bioinformatics tools (ToppGene, DIR, Endeavour, and GPEC) were integrated for prioritization of candidate genes. Candidate genes that identified with at least three programs and ranked in the top 20 by each program were considered the most promising. RESULTS: Prioritization analysis resulted in 21 promising genes involved or potentially involved in periodontitis. Among them, IL18 (interleukin 18), CD44 (CD44 molecule), CXCL1 (chemokine [CXC motif] ligand 1), IL6ST (interleukin 6 signal transducer), MMP3 (matrix metallopeptidase 3), MMP7, CCR1 (chemokine [C-C motif] receptor 1), MMP13, and TLR9 (Toll-like receptor 9) had been associated with periodontitis. However, the roles of other genes, such as CSF3 (colony stimulating factor 3 receptor), CD40, TNFSF14 (tumor necrosis factor receptor superfamily, member 14), IFNB1 (interferon-ß1), TIRAP (toll-interleukin 1 receptor domain containing adaptor protein), IL2RA (interleukin 2 receptor α), ETS1 (v-ets avian erythroblastosis virus E26 oncogene homolog 1), GADD45B (growth arrest and DNA-damage-inducible 45 ß), BIRC3 (baculoviral IAP repeat containing 3), VAV1 (vav 1 guanine nucleotide exchange factor), COL5A1 (collagen, type V, α1), and C3 (complement component 3), have not been investigated thoroughly in the process of periodontitis. These genes are mainly involved in bacterial infection, immune response, and inflammatory reaction, suggesting that further characterizing their roles in periodontitis will be important. CONCLUSIONS: A combination of computational tools will be useful in mining candidate genes for periodontitis. These theoretical results provide new clues for experimental biologists to plan targeted experiments.

Non-syndromic tooth agenesis associated with a nonsense mutation in ectodysplasin-A (EDA).

Mutations in the ectodysplasin-A (EDA) gene have been generally associated with X-linked hypohidrotic ectodermal dysplasia (XLHED). Recently, missense mutations in EDA have been reported to cause familial non-syndromic tooth agenesis. In this study, we report a novel EDA mutation in an Estonian family segregating non-syndromic tooth agenesis with variable expressivity. Affected individuals had no associated defects in other ectodermal organs. Using whole-exome sequencing, we identified a heterozygous nonsense mutation c.874G>T (p.Glu292X) in the TNF homology domain of EDA in all affected female patients. This protein-altering variant arose de novo, and the potentially causative allele was transmitted to affected offspring from the affected mother. We suggest that the dental phenotype variability described in heterozygous female carriers of EDA mutation may occur because of the differential pattern of X-chromosome inactivation, which retains reduced levels of EDA-receptor signaling in tissues involved in tooth morphogenesis. This results in selective tooth agenesis rather than XLHED phenotype. The present study broadens the mutation spectrum for this locus and demonstrates that EDA mutations may result in non-syndromic tooth agenesis in heterozygous females.

Novel EDA p.Ile260Ser mutation linked to non-syndromic hypodontia.

Hypodontia, a tooth developmental disease, can affect chewing and pronunciation. Mutations in the ectodysplasin-A (EDA) gene can lead to both X-linked hypohidrotic ectodermal dysplasia (XLHED) and non-syndromic hypodontia (NSH). However, the mechanism by which these 2 related but different disorders are caused by the distinct mutations in EDA is unknown. In this study, we identified a novel missense mutation (c.779 T>G) in a Chinese family with NSH via a direct sequencing approach. This mutation results in an Ile260Ser substitution in the tumor necrosis factor (TNF) homology domain. Homology modeling suggests that this alteration may induce a conformational change in the hydrophobic center of the TNF homology domain. Furthermore, by exploring systematic 3D conformation analysis and calculation of residue relative solvent accessibility (RSA) for all the reported mutated amino acid sites on EDA's TNF homology domain, we found that the site mutations at the interior may be linked to XLHED, while those at the surface are more likely to be associated with NSH. These findings may aid in the discovery of unidentified functionally significant mutation sites in the EDA gene and provide a new way to clarify the mechanisms by which the XLHED and NSH phenotypes arise from mutations in the same gene.

Enhancing specific-antibody production to the ragB vaccine with GITRL that expand Tfh, IFN-γ(+) T cells and attenuates Porphyromonas gingivalis infection in mice.

The outer membrane protein RagB is one of the major virulence factors of the periodontal pathogen Porphyromonas gingivalis (P. gingivalis). In order to induce protective immune response against P. gingivalis infection, an mGITRL gene-linked ragB DNA vaccine (pIRES-ragB-mGITRL ) was constructed. Six-week-old female BALB/c mice were immunized with pIRES-ragB-mGITRL through intramuscular injection and then challenged by subcutaneous injection in the abdomen with P. gingivalis. RagB-specific antibody-forming cells were evaluated by an Enzyme-linked immunosorbent spot, and specific antibody was determined by enzyme-linked immunosorbent assay. In addition, the frequencies of Tfh and IFN-γ(+) T cells in spleen were measured using flow cytometer, and the levels of IL-21 and IFN-γ mRNA or proteins were detected by real time RT-PCR or ELISA. The data showed that the mGITRL-linked ragB DNA vaccine induced higher levels of RagB-specific IgG in serum and RagB-specific antibody-forming cells in spleen. The frequencies of Tfh and IFN-γ(+) T cells were obviously expanded in mice immunized by pIRES-ragB-mGITRL compared with other groups (pIRES or pIRES-ragB ). The levels of Tfh and IFN-γ(+) T cells associated cytokines were also significantly increased in pIRES-ragB-mGITRL group. Therefore, the mice immunized with ragB plus mGITRL showed the stronger resistant to P. gingivalis infection and a significant reduction of the lesion size caused by P. gingivalis infection comparing with other groups. Taken together, our findings demonstrated that intramuscular injection of DNA vaccine ragB together with mGITRL induced protective immune response dramatically by increasing Tfh and IFN-γ(+) T cells and antibody production to P. gingivalis.

[Construction of eukaryotic co-expression vector of Porphyromonas gingivalis outer membrane protein ragB and glucocorticoid-induced tumor necrosis factor receptor ligand (GITRL) and its immunogenic analysis].

AIM: To construct eukaryotic co-expression vector of Porphyromonas gingivalis outer membrane protein ragB and mouse glucocorticoid-induced tumor necrosis factor receptor ligand (mGITRL) and to analyze its immunogenicity in vivo. METHODS: The ragB gene was obtained from pMD18-T-ragB, and then cloned into the eukaryotic expression vector pIRES and pIRES-mGITRL, respectively. The eukaryotic expression vectors: pIRES-ragB and pIRES-ragB-mGITRL were identified by double enzyme digestion and DNA sequencing, then transfected into COS7 cells by Lipofectamine(TM);2000. The expressions of ragB or mGITRL in COS7 cells were detected by Western blotting. The mice were immunized with the recombinant pIRES-ragB-mGITRL plasmid. The serum antibody level was determined by ELISA. RESULTS: pIRES-ragB and pIRES-ragB-mGITRL plasmids were successfully constructed. Western blotting showed that the targeted gene was over-expressed in COS7 cells and skeletal muscle cells, respectively. The high titers of antibodies against RagB were detected in mouse serum. CONCLUSION: The construction of pIRES-ragB-mGITRL co-expression vector provides the experimental basis for Porphyromonas gingivalis vaccine research, prevention and treatment of periodontitis.

Revisiting the supernumerary: the epidemiological and molecular basis of extra teeth.

Supernumerary teeth are a common clinical and radiographic finding and may produce occlusal and dental problems. The aetiological basis of extra teeth is poorly understood in human populations; however, the mouse provides a useful model system to investigate the complex genetics of tooth development. This article describes recent advances in our understanding of the genetic basis of supernumerary teeth. We have reviewed biological evidence that provides insight into why supernumerary tooth formation may occur. Indeed, many of the molecular signalling pathways known to be involved in normal development of the tooth germ can also give rise to additional teeth if inappropriately regulated. These include components of the Hedgehog, FGF, Wnt, TNF and BMP families, which provide a useful resource of candidate genes that may potentially play a role in human supernumerary tooth formation.

A novel role for Glucocorticoid-Induced TNF Receptor Ligand (Gitrl) in early embryonic zebrafish development.

Tumour necrosis factor ligand and receptor superfamily (TNFSF and TNFRSF) members have diverse and well-studied functions in the immune system. Additional, non-immunological roles, such as in the morphogenesis of bone, tooth, hair and skin have also been described for some members. GITRL and its receptor GITR are well-described as co-regulators of the mammalian immune response. Here, we describe the identification and cloning of their zebrafish homologues and demonstrate a novel role for the ligand, but not the receptor, in early vertebrate development. The assignment of zebrafish Gitrl and Gitr was supported by homology and phylogenetic analysis. The ligand exhibited an oscillating pattern of mRNA expression during the first 36 hours post fertilization, during which time gitr mRNA was not detected, and morpholino oligonucleotide-mediated knock-down of gitrl, but not of gitr, resulted in disruption of early embryogenesis, most clearly revealed during gastrulation, which corresponded to the earliest peak in gitrl mRNA expression (5.25-10 hpf). We found Stat3 signalling to be altered in the gitrl-morphants, suggesting that one possible role for Gitrl during embryogenesis may be modulation of Jak/Stat signalling.

A novel missense mutation of the EDA gene in a Mongolian family with congenital hypodontia.

X-linked hypohidrotic ectodermal dysplasia (HED) is a rare disease characterized by the hypoplasia or absence of eccrine glands, dry skin, scant hair, and dental abnormalities. Here, we report a Mongolian family with congenital absence of teeth inherited in an X-linked fashion. The affected members of the family did not show other HED characteristics, except hypodontia. We successfully mapped the affected locus to chromosome Xq12-q13.1, and then found a novel missense mutation, c.193C>G, in the ectodysplasin A (EDA) gene in all affected males and carrier females. The mutation causes arginine to be replaced by glycine in codon 65 (R65G) in the juxtamembrane region of EDA. In addition, 33% (3/9) of female carriers have a skewed X-chromosome inactivation pattern. Our result strongly suggests that the c.193C>G mutation is the disease-causing mutation in this family.

Proinflammatory effects of tumour necrosis factor-like weak inducer of apoptosis (TWEAK) on human gingival fibroblasts.

Tumour necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK), a member of the TNF family, is a multi-functional cytokine that regulates cellular proliferation, angiogenesis, inflammation and apoptosis. In this study, we investigated TWEAK expression in periodontally diseased tissues and the effect of TWEAK on human gingival fibroblasts (HGF). Reverse transcription-polymerase chain reaction (RT-PCR) analysis and immunohistochemistry revealed that TWEAK and the TWEAK receptor, fibroblast growth factor-inducible 14 (Fn14), mRNA and protein were expressed in periodontally diseased tissues. HGF expressed Fn14 and produced interleukin (IL)-8 and vascular endothelial growth factor (VEGF) production upon TWEAK stimulation in a dose-dependent manner. The IL-8 and VEGF production induced by TWEAK was augmented synergistically by simultaneous stimulation with transforming growth factor (TGF)-beta1 or IL-1beta. IL-1beta and TGF-beta1 enhanced Fn14 expression in a dose-dependent manner. Moreover, TWEAK induced intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression on HGF in a dose-dependent manner. The ICAM-1 expression induced by TWEAK was augmented by TGF-beta1. On the other hand, the TWEAK-induced VCAM-1 expression was inhibited by TGF-beta1. Phosphatidylinositol 3-kinase (PI3K) and nuclear factor-kappaB (NF-kappaB) inhibitor inhibit both ICAM-1 and VCAM-1 expression induced by TWEAK. However, mitogen-activated protein kinase (MEK) and c-Jun NH2-terminal kinase (JNK) inhibitor enhanced only VCAM-1 expression on HGF. These results suggest that TWEAK may be involved in the pathophysiology of periodontal disease. Moreover, in combination with IL-1beta or TGF-beta1, TWEAK may be related to the exacerbation of periodontal disease to induce proinflammatory cytokines and adherent molecules by HGF.

Ectodysplasin regulates the lymphotoxin-beta pathway for hair differentiation.

Mutations in the EDA gene cause anhidrotic/hypohidrotic ectodermal dysplasia, a disorder characterized by defective formation of hair, sweat glands, and teeth in humans and in a mouse model, "Tabby" (Ta). The gene encodes ectodysplasin, a TNF ligand family member that activates the NF-kappaB-signaling pathway, but downstream targets and the mechanism of skin appendage formation have been only partially analyzed. Comparative transcription profiling of embryonic skin during hair follicle development in WT and Ta mice identified critical anhidrotic/hypohidrotic ectodermal dysplasia (EDA) effectors in four pathways, three already implicated in follicle formation. They included Shh and its effectors, as well as antagonists for the Wnt (Dkk4) and BMP (Sostdc1) pathways. The fourth pathway was unexpected, a variant NF-kappaB-signaling cascade based on lymphotoxin-beta (LTbeta)/RelB. Previously known to participate only in lymphoid organogenesis, LTbeta was enriched in developing hair follicles of WT but not in Ta mice. Furthermore, in mice lacking LTbeta, all three types of mouse hair were still formed, but all were structurally abnormal. Guard hairs became wavy and irregular, zigzag/auchen hairs lost their kinks, and in a phenocopy of features of Ta animals, the awl hairs doubled in number and were characteristically distorted and pinched. LTbeta-null mice that received WT bone marrow transplants maintained mutant hair phenotypes, consistent with autonomous LTbeta action in skin independent of its expression in lymphoid cells. Thus, as an EDA target, LTbeta regulates the form of hair in developing hair follicles; and when EDA is defective, failure of LTbeta activation can account for part of the Ta phenotype.

Mutation identification in a canine model of X-linked ectodermal dysplasia.

X-linked hypohidrotic ectodermal dysplasia (XHED), an inherited disease recognized in humans, mice, and cattle, is characterized by hypotrichosis, a reduced number or absence of sweat glands, and missing or malformed teeth. In a subset of affected individuals and animals, mutations in the EDA gene (formerly EDI), coding for ectodysplasin, have been found to cause this phenotype. Ectodysplasin is a homotrimeric transmembrane protein with an extracellular TNF-like domain, which has been shown to be involved in the morphogenesis of hair follicles and tooth buds during fetal development. Some human XHED patients also have concurrent immunodeficiency, due to mutations in the NF-kappaB essential modulator protein (IKBKG; formerly NEMO), which is also encoded on the X chromosome. In a breeding colony of dogs with XHED, immune system defects had been suspected because of frequent pulmonary infections and unexpected deaths resulting from pneumonia. To determine if defects in EDA or IKBKG cause XHED in the dogs, linkage analysis and sequencing experiments were performed. A polymorphic marker near the canine EDA gene showed significant linkage to XHED. The canine EDA gene was sequenced and a nucleotide substitution (G to A) in the splice acceptor site of intron 8 was detected in affected dogs. In the presence of the A residue, a cryptic acceptor site within exon 9 is used, leading to a frame shift and use of a premature stop codon that truncates the translation of both isoforms, EDA-A1 and EDA-A2, resulting in the absence of the TNF-like homology domain, the receptor-binding site of ectodysplasin.

Edar signaling in the control of hair follicle development.

Ectodysplasin receptor Edar and its ligand Eda A1, as well as their related receptor Xedar and ligand Eda A2, are recently discovered members of the tumor necrosis factor superfamily that signal predominantly through the nuclear factor-kappaB and c-jun N-terminal kinases pathways. Mutations in genes that encode proteins involved in Edar signaling pathway cause hypohidrotic ectodermal displasias in humans and mice and characterized by severe defects in development of ectodermal appendages including hairs, teeth, and exocrine glands. Here, we summarize the current knowledge of molecular mechanisms underlying the involvement of Edar signaling pathway in controlling hair follicle (HF) development and cycling. Genetic and experimental studies suggest that Edar signaling is involved in the control of cell fate decision in embryonic epidermis, as well as in the regulation of cell differentiation programs in the HF. Loss or gain of Edar signaling affects the initiation of several HF types (guard and zig-zag HF), hair shaft formation, as well as sebaceous gland morphology. We also review data on the cross-talk between Edar and Wnt, transforming growth factor-beta/bone morphogenic protein/activin, and Shh signaling pathways in the control of HF development and cycling.