Search

Periapical bone destruction is an important pathogenic sequela of pulpal infection. Recent findings from this laboratory have demonstrated that most bone-resorbing activity in extracts of rat periapical lesions can be neutralized by an anti-interleukin (IL-1α antiserum. To further clarify pathogenic mechanisms, bone-resorptive cytokine messenger RNA (mRNA) expression was analyzed in developing rat periapical lesions. The molar teeth of 20 Sprague-Dawley rats were surgically exposed and leftopen to permit infection from the oral environment. Total cell RNA was isolated from periapical granuloma tissue obtained on days 3, 7, 15 and 30 after exposure. mRNA for IL-1α, IL-1β and tumor necrosis factor α (TNF-α) was amplified by revenue transcription polymerase chain reaction, and levels were approximated by comparison to the parallel amplification of the housekeeping gene glyceraldehyde phosphate dehydrogenase, IL-1α and TNF-α mRNA were both highly expressed beginning on day 7, increased on day 15, and declined somewhat on day 30. In contras, IL-1β mRNA was expressed at much lower levels, but with similar kinetics. The kinetics of steady state IL-1α and TNF-α mRNA levels were confirmed using the quantitative RNase protection assay, whereas IL-1β mRNA could not be detected by this technique. IL-1α mRNA-expressing cells were identified using in situ hybridization and included infiltrating macrophages, as well as resident fibroblasts, endothelial cells and osteoclasts. These results demonstrate that the IL-1α and TNF- α genes are highly expressed in developing periapical lesions in the rat and confirm previous studies at the protein level in this model. [ABSTRACT FROM AUTHOR]

Spinal cord injury causes severe bone loss. We report osteoclast resorption with severe trabecular and cortical bone loss, decreased bone mineral apposition, and growth plate abnormalities in a rodent model of contusion spinal cord injury. These findings will help elucidate the mechanisms of osteoporosis following neurological trauma. Limited understanding of the mechanism(s) that underlie spinal cord injury (SCI)-induced bone loss has led to few treatment options. As SCI-induced osteoporosis carries significant morbidity and can worsen already profound disability, there is an urgency to advance knowledge regarding this pathophysiology. A clinically relevant contusion model of experimental spinal cord injury was used to generate severe lower thoracic SCI by weight-drop (10 g × 50 mm) in adolescent male Sprague-Dawley rats. Body weight and gender-matched naïve (no surgery) rats served as controls. Bone microarchitecture was determined by micro-computed tomographic imaging. Mature osteoclasts were identified by TRAP staining and bone apposition rate was determined by dynamic histomorphometry. At 10 days post-injury we detected a marked 48% decrease in trabecular bone and a 35% decrease in cortical bone at the distal femoral metaphysis by micro-CT. A 330% increase in the number of mature osteoclasts was detected at the growth plate in the injured animals that corresponded with cellular disorganization at the chondro-osseous junction. Appositional growth studies demonstrated decreased new bone formation with a mineralization defect indicative of osteoblast dysfunction. Contusion SCI results in a rapid bone loss that is the result of increased bone resorption and decreased bone formation. [ABSTRACT FROM AUTHOR]

Abstract: Bone resorption is regulated by a complex system of hormones and cytokines that cause osteoblasts/stromal cells and lymphocytes to produce factors including RANKL, that ultimately result in the differentiation and activation of osteoclasts, the bone resorbing cells. We used a microarray approach to identify genes upregulated in RANKL-stimulated osteoclast precursor cells. Osteoclast expression was confirmed by multiple tissue Northern and in situ hybridization analysis. Gene function studies were carried out by siRNA analysis. We identified a novel gene, which we termed nha-oc/NHA2, which is strongly upregulated during RANKL-induced osteoclast differentiation in vitro and in vivo. nha-oc/NHA2 encodes a novel cation–proton antiporter (CPA) and is the mouse orthologue of a human gene identified in a database search: HsNHA2. nha-oc/NHA2 is selectively expressed in osteoclasts. NHA-oc/NHA2 protein localizes to the mitochondria, where it mediates Na+-dependent changes in mitochondrial pH and Na+ acetate induced mitochondrial passive swelling. RNA silencing of nha-oc/nha2 reduces osteoclast differentiation and resorption, suggesting a role for NHA-oc/NHA2 in these processes. nha-oc/NHA2 therefore is a novel member of the CPA family and is the first mitochondrial NHA characterized to date. nha-oc/NHA2 is also unique in that it is the first eukaryotic and tissue-specific CPA2 characterized to date. NHA-oc/NHA2 displays the expected activities of a bona fide CPA and plays a key role(s) in normal osteoclast differentiation and function. [Copyright &y& Elsevier]

Introduction: The present study examined whether induction of an adaptive immune response to orally colonizing non-pathogenic Pasteurella pneumotropica by immunization with the phylogenetically closely related bacterium, Actinobacillus actinomycetemcomitans, can result in periodontal bone loss in mice. Methods: BALB/c mice harboring P. pneumotropica ( P. pneumotropica+ mice) in the oral cavity or control P. pneumotropica-free mice were immunized with fixed A. actinomycetemcomitans. The animals were sacrificed on day 30, and the following measurements were carried out: (i) serum immunoglobulin G and gingival T-cell responses to A. actinomycetemcomitans and P. pneumotropica; (ii) periodontal bone loss; and (iii) identification of receptor activator of nuclear factor-κB ligand (RANKL) -positive T cells in gingival tissue. Results: Immunization with A. actinomycetemcomitans induced a significantly elevated serum immunoglobulin G response to the 29-kDa A. actinomycetemcomitans outer membrane protein (Omp29), which showed strong cross-reactivity with P. pneumotropica OmpA compared to results in the control non-immunized mice. The A. actinomycetemcomitans-immunized P. pneumotropica+ mice developed remarkable periodontal bone loss in a RANKL-dependent manner, as determined by the abrogation of bone loss by treatment with osteoprotegerin-Fc. The T cells isolated from the gingival tissue of A. actinomycetemcomitans-immunized P. pneumotropica+ mice showed an in vitro proliferative response to both A. actinomycetemcomitans and P. pneumotropica antigen presentation, as well as production of soluble(s)RANKL in the culture supernatant. Double-color confocal microscopy demonstrated that the frequency of RANKL+ T cells in the gingival tissue of A. actinomycetemcomitans-immunized P. pneumotropica+ mice was remarkably elevated compared to control mice. Conclusion: The induction of an adaptive immune response to orally colonizing non-pathogenic P. pneumotropica results in RANKL-dependent periodontal bone loss in mice. [ABSTRACT FROM AUTHOR]

Foschi F, Cavrini F, Montebugnoli L, Stashenko P, Sambri V, Prati C. Detection of bacteria in endodontic samples by polymerase chain reaction assays and association with defined clinical signs in Italian patients. Oral Microbiol Immunol 2005: 20: 289–295.© Blackwell Munksgaard, 2005. The presence of selected bacteria ( Enterococcus faecalis, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythensis, Treponema denticola) in infected root canals was studied using polymerase chain reaction (PCR) assays, and the association of bacteria with clinical signs of endodontic disease was assessed. The null hypothesis, that no difference could be observed between clinical signs of apical periodontitis and a specific bacterial strain, was tested. Microbial samples were obtained from 62 teeth in 54 patients with endodontic disease. For each tooth, clinical data including patient symptoms were collected. Teeth were categorized by diagnosis as having acute apical periodontitis (AAP, teeth with clinical symptoms but no periapical radiolucency, n = 22), chronic apical periodontitis (CAP, teeth with radiolucency but no clinical symptoms, n = 15) or exacerbated apical periodontitis (EAP, teeth with symptoms and radiolucency, n = 25). Seventy-one percent of cases were primary endodontic infections, and 29% were recurrent (‘secondary’) endodontic infections (failing cases). PCR assays were used to detect the presence of the selected bacteria. T. denticola and E. faecalis were each detected in 15 of 62 samples (24%), P. gingivalis in 8 samples (13%), P. intermedia in 5 samples (8%), and T. forsythensis in 4 samples (7%). T. denticola was detected in 56% of teeth with EAP. E. faecalis was found in 60% of teeth with CAP and in 72% of teeth with secondary infection. Statistical analysis demonstrated an association of CAP and secondary endodontic infection with the presence of E. faecalis. ( P < 0.01). EAP was associated with the presence of T. denticola ( P < 0.01). T. denticola was associated with symptomatic endodontic disease in the presence of apical bone resorption. E. faecalis was associated with treatment failures. We suggest that these species may play critical roles in endodontic pathology. [ABSTRACT FROM AUTHOR]

Castro GF, Souza IPR, Lopes S, Stashenko P, Teles RP. Salivary IgA to cariogenic bacteria in HIV-positive children and its correlation with caries prevalence and levels of cariogenic microorganisms. Oral Microbiol Immunol 2004: 19: 281–288© Blackwell Munksgaard, 2004. The interrelationship of HIV infection, dental caries and mucosal immune responses remains controversial. In our study population of 40 HIV-infected and 40 healthy control children (ages 2–5 years) there was a significantly higher prevalence of dental caries in HIV-infected children ( P<0.05). The extent of caries correlated with the severity of HIV disease. To determine whether the immunosuppression that ensues after HIV infection could contribute to the increased caries prevalence, the concentrations of total IgA and IgA specific to cariogenic bacteria ( Streptococcus mutans, Streptococcus sobrinus and Lactobacillus acidophilus) were determined in whole saliva by enzyme-linked immunosorbent assay . Levels of the same bacteria were also quantified in saliva using checkerboard DNA–DNA hybridization. A significantly increased level of total salivary IgA was found in the HIV-positive population ( P < 0.05), but there were comparable titers of specific IgA to cariogenic bacteria in HIV-positive and healthy controls. The microbiological assessment also demonstrated similar levels of cariogenic microorganisms in both groups. We conclude that HIV-positive children appear to maintain the capacity to mount a mucosal immune response to cariogenic microorganisms, at least until late stages of disease. [ABSTRACT FROM AUTHOR]

Abstract Background: Retrospective and correlation studies suggest that early-onset periodontal disease may be due to a deficiency in phagocyte function, a pathogenic oral biofilm, and/or dysregulated gingival cytokine expression. Increased susceptibility to periodontal disease is therefore thought to result from multiple risk factors. Methods: We tested this hypothesis prospectively using P/E-selectin adhesion molecule deficient mice that mimic the human syndrome leukocyte adhesion deficiency II. Results: Our studies demonstrate that, in comparison to wild type animals, P/E-/- mice exhibit: spontaneous, early onset alveolar bone loss which is significant by 6 weeks of age; a 10-fold elevation in bacterial colonization of their oral cavities; and elevated gingival tissue levels of the bone resorptive cytokine IL-1α. Alveolar bone loss is completely prevented by prophylactic antibiotic therapy. Conclusions: These experiments provide the first prospective evidence for the multiple risk factor hypothesis of periodontal disease, and validate the first animal model for early onset periodontitis in which both the microbiota and host response can be systematically manipulated. P/E-/- animals should be useful in testing the virulence of putative periodontal pathogens, in determining the role of host resistance factors in periodontitis, in exploring the proposed relationship(s) between infection mediated alveolar bone loss and systemic health disorders, and exploring their genetic relationships. [ABSTRACT FROM AUTHOR]

Introduction: This study aimed to assess growth-related dental and symmetry changes in the dental arch within and between identical and fraternal twins in mixed and permanent dentition., Methods: Three-dimensional scanned dental models of eligible subjects were selected from the Forsyth-Moorrees Twin Study sample. This retrospective cohort study was carried out on 36 identical (18 pairs) and 28 fraternal (14 pairs) twins in mixed dentition and 36 identical (18 pairs) and 38 fraternal (19 pairs) twins in permanent dentition stages on the basis of the availability of the dental casts scanned each year from each group (Table I). Linear measurements from dental casts were performed in patients aged 8-16 years. Student t test and Pearson's correlation were used to compare the symmetry between and within the identical and fraternal twins. The resemblance and heritability patterns were retrospectively obtained from the Pearson correlation coefficient and Falconer's heritability test (H 2  = 2 × b). Adjusted mixed-effects estimates and 95% confidence intervals were calculated to test the association between age and dental parameters for both mixed and permanent dentition groups., Results: Intercanine and intermolar widths significantly increased (P <0.05) during the mixed dentition but became stable after 13 years old. No statistically significant differences were found in arch symmetry between the 2 groups (ie, identical and fraternal) in any of the included measurements. Evaluation of the resemblance and heritability pattern showed nonsignificant results for all variables measured (H 2 range, -0.67 to 0.56)., Conclusions: The dental arch becomes wider at a higher rate in the canine region than the molar region in both the mixed and early permanent dentition. The dental arches of twins develop symmetrically, and their growth is not mainly affected by genetics. Asymmetrical teeth will maintain their relative position to reference planes throughout growth., (Copyright © 2022. Published by Elsevier Inc.)

Apical periodontitis (AP) develops as a result of an immune response to pulpal bacterial infection, and various cytokines are involved in the pathogenesis of AP, with Interleukin (IL)-1 being considered a key cytokine. The role of IL-1 in the pathogenesis of AP has been well studied. It is known that IL-1 expression in periapical lesions correlates closely with the development of AP. IL-1 is a potent bone-resorptive cytokine that induces osteoclast formation and activation. Hence, inhibiting its signaling with IL-1 receptor antagonist (IL-1RA) results in a reduction in periapical lesion size. On the other hand, IL-1 is also a central cytokine that combats bacterial infection by activating innate immune responses. Therefore, a complete loss of IL-1 signaling leads to a failure to limit bacterial dissemination and consequently exacerbates AP. In vivo , IL-1 expression is tightly regulated and its signaling is modulated to optimize the immune response. Obesity causes systemic low-grade chronic inflammation and increases the risk of cardiovascular, renal, and other disorders. In experimentally induced AP, obesity significantly increases periapical bone loss, albeit the underlying mechanism remains unclear. Recent technological innovations have enabled more comprehensive and detailed analyses than previously, leading to new insights into the role of IL-1RA in regulating IL-1 signaling, and modulating apical lesion progression in obesity. In this review, we provide a brief overview of the function of IL-1 in AP development, with special emphasis on the latest findings in normal weight and obese states., Competing Interests: Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Oral squamous cell carcinoma (OSCC) is the most common malignancy of the head and neck worldwide. Dysbiosis of the microbiome has increasingly been linked to the development of different kinds of cancer. Applying 16S rRNA gene sequence analysis and metatranscriptomic analyses, we characterized the longitudinal changes in the profiles and the function of the oral microbiome in a 4-nitroquinoline-1-oxide (4-NQO)-induced model of OSCC in gnotobiotic mice. We characterized the dynamics of the oral microbiome in this model using two different microbiome inocula: one from healthy mice and the other from mice bearing a 4-NQO-induced tumor. Mice colonized with different oral microbiomes and exposed to 4-NQO had increased tumor numbers and sizes compared to controls exposed to 4-NQO but lacking a microbiome. We observed an overall increase in diversity in the tumorigenic samples compared to that in the nontumor group not exposed to 4-NQO. Despite the variability in community dynamics, specific patterns emerged during the progression of the disease. In the two groups that were inoculated with the OSCC-associated microbiome, we observed opposite profiles of abundance in Parabacteroides and Corynebacterium While the percentage of Parabacteroides bacteria decreased in the control group, it increased in the OSCC group, and the opposite was observed for Corynebacterium The metatranscriptomic analysis revealed overexpression of the same metabolic signatures associated with OSCC regardless of the community profile. These included nitrogen transport, response to stress, interspecies interactions, Wnt pathway modulation, and amino acid and lipid biosynthesis. Thus, these results seem to suggest that certain collective physiological activities are critical for microbiome-mediated OSCC progression. IMPORTANCE There is growing evidence that changes in the microbiome are associated with carcinogenesis. To date, no consistent oral microbiome composition associated with OSCC has been identified. Longitudinal and functional studies like the study presented here should yield a better understanding of the role that the oral microbiome plays in OSCC. Our findings, obtained using a germ-free mouse model, indicate that the presence of different oral microbiomes enhances tumorigenesis and increases the final number of tumors in mice. By studying community-wide expression profiles, we found that regardless of the phylogenetic composition of the microbiome, the same metabolic activities were consistently associated with OSCC. Therefore, due to the functional redundancy of the microbiome, the critical element in explaining the contribution of the microbiota in OSCC is the collective physiological activity of the community, thus accounting for the previous inability to identify a consensus community profile or etiologic agents for OSCC., (Copyright © 2019 Stashenko et al.)

Introduction: In general, mice develop chronic and nonhealing periapical lesions after endodontic infection. Surprisingly, we recently found that toll-like receptor 2 (TLR2)/interleukin 10 (IL-10) double-knockout (dKO) mice exhibited acute but resolving osteomyelitislike inflammation. In this study, we examined the kinetics of endodontic infection-induced inflammation in TLR2/IL-10 dKO mice and explored a potential mechanism of periapical wound healing mediated by the hypoxia-inducible factor 1 alpha (HIF-1α) subunit and arginase 1., Methods: TLR2/IL-10 dKO and wild-type C57BL/6J mice were subjected to endodontic infection in the mandibular first molars. Mice were sacrificed on days 0 (noninfected), 10, and 21 postinfection. The extent of bone destruction, inflammation, bone deposition, and gene expression were determined by micro-computed tomographic imaging, histology, bone polychrome labeling, and microarray analysis. In addition, the effect of blocking endogenous HIF-1α was tested in infected TLR2/IL-10 dKO mice using the specific inhibitor YC-1., Results: Infected TLR2/IL-10 dKO mice exhibited extensive bone destruction and inflammation on day 10 followed by spontaneous periapical wound healing including bone formation and resolution of inflammation by day 21 postinfection. In contrast, WT mice developed increasing chronic periapical inflammation over the 21-day observation period. Gene expression analyses and immunohistochemistry revealed that HIF-1α and arginase 1 were up-regulated in spontaneous wound healing in TLR2/IL-10 dKO mice. Blocking of HIF-1α in TLR2/IL-10 dKO mice using YC-1 resulted in significant inhibition of regenerative bone formation., Conclusions: The TLR2/IL-10 dKO mouse is a novel model resembling osteomyelitis of the jaws in which HIF-1α and arginase 1 appear to be crucial factors in spontaneous wound healing and bone repair., (Copyright © 2018 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

In the current concept of bacterial infections, pathogen-associated molecular patterns (PAMPs) derived from pathogens and damage-associated molecular patterns (DAMPs) released from damaged/necrotic host cells are crucial factors in induction of innate immune responses. However, the implication of DAMPs in apical and marginal periodontitis is unknown. Serum amyloid A (SAA) is a DAMP that is involved in the development of various chronic inflammatory diseases, such as rheumatoid arthritis. In the present study, we tested whether SAA is involved in the pathogenesis of periapical lesions, using human periapical surgical specimens and mice deficient in SAA and Toll-like receptors (TLR). SAA1/2 was locally expressed in human periapical lesions at the mRNA and protein levels. The level of SAA protein appeared to be positively associated with the inflammatory status of the lesions. In the development of mouse periapical inflammation, SAA1.1/2.1 was elevated locally and systemically in wild-type (WT) mice. Although SAA1.1/2.1 double-knockout and SAA3 knockout mice had redundant attenuation of the extent of periapical lesions, these animals showed strikingly improved inflammatory cell infiltration versus WT. Recombinant human SAA1 (rhSAA1) directly induced chemotaxis of WT neutrophils in a dose-dependent manner in vitro. In addition, rhSAA1 stimulation significantly prolonged the survival of WT neutrophils as compared with nonstimulated neutrophils. Furthermore, rhSAA1 activated the NF-κB pathway and subsequent IL-1α production in macrophages in a dose-dependent manner. However, TLR2/TLR4 double deficiency substantially diminished these SAA-mediated proinflammatory responses. Taken together, the SAA-TLR axis plays an important role in the chronicity of periapical inflammation via induction of inflammatory cell infiltration and prolonged cell survival. The interactions of PAMPs and DAMPs require further investigation in dental/oral inflammation.

We report a novel method for in situ imaging of microvascular permeability in inflamed gingival tissue, using state-of-the-art Cellvizio™ intravital endoscopic technology and a mouse model of ligature-induced periodontitis. The silk ligature was first placed at the upper left second molar. Seven days later, the ligature was removed, and the animals were intravenously injected with Evans blue. Evans blue dye, which selectively binds to blood albumin, was used to monitor the level of inflammation by monitoring vascular permeability in control non-diseased and ligature-induced experimental periodontitis tissue. More specifically, leakage of Evans blue-bound albumin from the micro-capillary to connective tissue indicates the state of inflammation occurring in the specific site. Evans blue leakage from blood vessels was imaged in situ by directly attaching the endoscope (mini Z tip) of the Cellvizio™ system to the gingival tissue without any surgical incision. Evans blue emission intensity was significantly elevated in gingiva of periodontitis lesions, but not control non-ligature placed gingiva, indicating that this technology can be used as a potential minimally invasive diagnostic tool to monitor the level of inflammation at the periodontal disease site., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Apical periodontitis (periapical lesions) is an infection-induced chronic inflammation in the jaw, ultimately resulting in the destruction of apical periodontal tissue. Toll-like receptors (TLRs) are prominent in the initial recognition of pathogens. Our previous study showed that TLR4 signaling is proinflammatory in periapical lesions induced by a polymicrobial endodontic infection. In contrast, the functional role of TLR2 in regulation of periapical tissue destruction is still not fully understood. Using TLR2 deficient (KO), TLR2/TLR4 double deficient (dKO), and wild-type (WT) mice, we demonstrate that TLR2 KO mice are highly responsive to polymicrobial infection-induced periapical lesion caused by over activation of TLR4 signal transduction pathway that resulted in elevation of NF-kB (nuclear factor kappa B) and proinflammatory cytokine production. The altered TLR4 signaling is caused by TLR2 deficiency-dependent elevation of CD14 (cluster of differentiation 14), which is a co-receptor of TLR4. Indeed, neutralization of CD14 strikingly suppresses TLR2 deficiency-dependent inflammation and tissue destruction in vitro and in vivo. Our findings suggest that a network of TLR2, TLR4, and CD14 is a key factor in regulation of polymicrobial dentoalveolar infection and subsequent tissue destruction. Anat Rec, 299:1281-1292, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

The pioneer human oral commensal bacterium Streptococcus mitis has unique biologic features that make it an attractive mucosal vaccine or therapeutic delivery vector. S. mitis is safe as a natural persistent colonizer of the mouth, throat and nasopharynx and the oral commensal bacterium is capable of inducing mucosal antibody responses. A recombinant S. mitis (rS. mitis) that stably expresses HIV envelope protein was generated and tested in the germ-free mouse model to evaluate the potential usefulness of this vector as a mucosal vaccine against HIV. Oral vaccination led to the efficient and persistent bacterial colonization of the mouth and the induction of both salivary and systemic antibody responses. Interestingly, persistently colonized animals developed antigen-specific systemic T cell tolerance. Based on these findings we propose the use of rS. mitis vaccine vector for the induction of mucosal antibodies that will prevent the penetration of the mucosa by pathogens such as HIV. Moreover, the first demonstration of rS. mitis having the ability to elicit T cell tolerance suggest the potential use of rS. mitis as an immunotherapeutic vector to treat inflammatory, allergic and autoimmune diseases.

The development of vaccine approaches that induce mucosal and systemic immune responses is critical for the effective prevention of several infections. Here, we report on the use of the abundant human oral commensal bacterium Streptococcus mitis as a delivery vehicle for mucosal immunization. Using homologous recombination we generated a stable rS. mitis expressing a Mycobacterium tuberculosis protein (Ag85b). Oral administration of rS. mitis in gnotobiotic piglets resulted in efficient oral colonization and production of oral and systemic anti-Ag85b specific IgA and IgG antibodies. These results support that the commensal S. mitis is potentially a useful vector for mucosal vaccination., (Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

IL-17 is a pleiotropic cytokine produced by Th17 T cells that induces a myriad of proinflammatory mediators. However, different models of inflammation report opposite functional roles of IL-17 signal in terms of its effects on bone destruction. In this study we determined the role of IL-17RA signal in bone resorption stimulated by dentoalveolar infections. Infrabony resorptive lesions were induced by surgical pulp exposure and microbial infection of mouse molar teeth. IL-17 was strongly induced in periapical tissues in wild-type (WT) mice by 7 d after the infection but was not expressed in uninfected mice. Dentoalveolar infections of IL-17RA knockout (KO) mice demonstrated significantly increased bone destruction and more abscess formation in the apical area compared with WT mice. Infected IL-17RA KO mice exhibited significantly increased neutrophils and macrophages compared with the WT littermates at day 21, suggesting a failure of transition from acute to chronic inflammation in the IL-17RA KO mice. The expression of IL-1 (both α and β isoforms) and MIP2 were significantly upregulated in the IL-17RA KO compared with WT mice at day 21 postinfection. The development of periapical lesions in IL-17RA KO mice was significantly attenuated by neutralization of IL-1β and MIP2. Taken together, these results demonstrate that IL-17RA signal seems to be protective against infection-induced periapical inflammation and bone destruction via suppression of neutrophil and mononuclear inflammation.

Dental caries is one of the most prevalent infectious diseases in the United States, affecting approximately 80% of children and the majority of adults. Dental caries may lead to endodontic disease, where the bacterial infection progresses to the root canal system of the tooth, leading to periapical inflammation, bone erosion, severe pain, and tooth loss. Periapical inflammation may also exacerbate inflammation in other parts of the body. Although conventional clinical therapies for this disease are successful in approximately 80% of cases, there is still an urgent need for increased efficacy of treatment. In this study, we applied a novel gene-therapeutic approach using recombinant adeno-associated virus (AAV)-mediated Atp6i RNA interference (RNAi) knockdown of Atp6i/TIRC7 gene expression to simultaneously target periapical bone resorption and periapical inflammation. We found that Atp6i inhibition impaired osteoclast function in vitro and in vivo and decreased the number of T cells in the periapical lesion. Notably, AAV-mediated Atp6i/TIRC7 knockdown gene therapy reduced bacterial infection-stimulated bone resorption by 80% in the mouse model of endodontic disease. Importantly, Atp6i(+/-) mice with haploinsufficiency of Atp6i exhibited protection similar to that in mice with bacterial infection-stimulated bone erosion and periapical inflammation, which confirms the potential therapeutic effect of AAV-small hairpin RNA (shRNA)-Atp6i/TIRC7. Our results demonstrate that AAV-mediated Atp6i/TIRC7 knockdown in periapical tissues can inhibit endodontic disease development, bone resorption, and inflammation, indicating for the first time that this potential gene therapy may significantly improve the health of those who suffer from endodontic disease.

Periodontal disease affects about 80% of adults in America, and is characterized by oral bacterial infection-induced gingival inflammation, oral bone resorption, and tooth loss. Periodontitis is also associated with other diseases such as rheumatoid arthritis, diabetes, and heart disease. Although many efforts have been made to develop effective therapies for this disease, none have been very effective and there is still an urgent need for better treatments and preventative strategies. Herein we explored for the first time the possibility that adeno-associated virus (AAV)-mediated RNAi knockdown could be used to treat periodontal disease with improved efficacy. For this purpose, we used AAV-mediated RNAi knockdown of Atp6i/TIRC7 gene expression to target bone resorption and gingival inflammation simultaneously. Mice were infected with the oral pathogen Porphyromonas gingivalis W50 (P. gingivalis) in the maxillary periodontium to induce periodontitis. We found that Atp6i depletion impaired extracellular acidification and osteoclast-mediated bone resorption. Furthermore, local injection of AAV-shRNA-Atp6i/TIRC7 into the periodontal tissues in vivo protected mice from P. gingivalis infection-stimulated bone resorption by >85% and decreased the T-cell number in periodontal tissues. Notably, AAV-mediated Atp6i/TIRC7 knockdown also reduced the expression of osteoclast marker genes and inflammation-induced cytokine genes. Atp6i(+/-) mice with haploinsufficiency were similarly protected from P. gingivalis infection-stimulated bone loss and gingival inflammation. This suggests that AAV-shRNA-Atp6i/TIRC7 therapeutic treatment may significantly improve the health of millions who suffer from P. gingivalis-mediated periodontal disease.

Spinal cord injury is associated with rapid bone loss and arrested long bone growth due to mechanisms that are poorly understood. In this study, we sought to determine the effects of severe T10 contusion spinal cord injury on the sublesional bone microenvironment in adolescent rats. A severe lower thoracic (vertebral T10) spinal cord injury was generated by weight drop (10 g×50 mm). Severely injured and body weight-matched uninjured male Sprague-Dawley rats were studied. At 3 and 5 days post-injury, we performed histological analysis of the distal femoral metaphysis, TUNEL assay, immunohistochemistry, real-time PCR, and western blot analysis compared to uninjured controls. We observed severe hindlimb functional deficits typical of this model. We detected uncoupled remodeling with increased osteoclast activity in the absence of osteoblast activity. We detected osteoblast, osteocyte, and chondrocyte apoptosis with suppressed osteoblast and chondrocyte proliferation and growth plate arrest due to spinal cord injury. We also detected altered gene expression in both whole bone extracts and bone marrow monocytes following spinal cord injury. We conclude that spinal cord injury results in altered gene expression of key regulators of osteoblast and chondrocyte activity. This leads to premature cellular apoptosis, suppressed cellular proliferation, growth plate arrest, and uncoupled bone remodeling in sublesional bone with unopposed osteoclastic resorption.

Tetracycline antibiotics, including doxycycli\e (DOX), have been used to treat bone resorptive diseases, partially because of their activity to suppress osteoclastogenesis induced by receptor activator of nuclear factor kappa B ligand (RANKL). However, their precise inhibitory mechanism remains unclear. Therefore, the present study examined the effect of Dox on osteoclastogenesis signaling induced by RANKL, both in vitro and in vivo. Although Dox inhibited RANKL-induced osteoclastogenesis and down-modulated the mRNA expression of functional osteoclast markers, including tartrate-resistant acid phosphatase (TRAP) and cathepsin K, Dox neither affected RANKL-induced MAPKs phosphorylation nor NFATc1 gene expression in RAW264.7 murine monocytic cells. Gelatin zymography and Western blot analyses showed that Dox down-regulated the enzyme activity of RANKL-induced MMP-9, but without affecting its protein expression. Furthermore, MMP-9 enzyme inhibitor also attenuated both RANKL-induced osteoclastogenesis and up-regulation of TRAP and cathepsin K mRNA expression, indicating that MMP-9 enzyme action is engaged in the promotion of RANKL-induced osteoclastogenesis. Finally, Dox treatment abrogated RANKL-induced osteoclastogenesis and TRAP activity in mouse calvaria along with the suppression of MMP9 enzyme activity, again without affecting the expression of MMP9 protein. These findings suggested that Dox inhibits RANKL-induced osteoclastogenesis by its inhibitory effect on MMP-9 enzyme activity independent of the MAPK-NFATc1 signaling cascade., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background and Objective: 18β-Glycyrrhetinic acid (GA) is a natural anti-inflammatory compound derived from licorice root extract (Glycyrrhiza glabra). The effect of GA on experimental periodontitis and its mechanism of action were determined in the present study., Material and Methods: Periodontitis was induced by oral infection with Porphyromonas gingivalis W83 in interleukin-10-deficient mice. The effect of GA, which was delivered by subcutaneous injections in either prophylactic or therapeutic regimens, on alveolar bone loss and gingival gene expressions was determined on day 42 after initial infection. The effect of GA on lipopolysaccharide (LPS)-stimulated macrophages, T cell proliferation and osteoclastogenesis was also examined in vitro., Results: 18β-Glycyrrhetinic acid administered either prophylactically or therapeutically resulted in a dramatic reduction of infection-induced bone loss in interleukin-10-deficient mice, which are highly disease susceptible. Although GA has been reported to exert its anti-inflammatory activity via downregulation of 11β-hydroxysteroid dehydrogenase-2 (HSD2), which converts active glucocorticoids to their inactive forms, GA did not reduce HSD2 gene expression in gingival tissue. Rather, in glucocorticoid-free conditions, GA potently inhibited LPS-stimulated proinflammatory cytokine production and RANKL-stimulated osteoclastogenesis, both of which are dependent on nuclear factor-κB. Furthermore, GA suppressed LPS- and RANKL-stimulated phosphorylation of nuclear factor-κB p105 in vitro., Conclusion: These findings indicate that GA inhibits periodontitis by inactivation of nuclear factor-κB in an interleukin-10- and glucocorticoid-independent fashion., (© 2010 John Wiley & Sons A/S.)

Background: NHAoc/NHA2 is highly and selectively expressed in osteoclasts and plays a role(s) in normal osteoclast differentiation, apoptosis and bone resorptive function in vitro. Extensive mutational analysis of a bacterial homologue, NhaA, has revealed a number of amino acid residues essential for its activity. Some of these residues are evolutionarily conserved and have been shown to be essential not only for activity of NhaA in bacteria, but also of NHAoc/NHA2 in eukaryotes., Methods: The salt-sensitive Saccharomyces cerevisiae strain BW31a was used for heterologous expression of mutants of NHAoc/NHA2. Membrane expression of NHAoc/NHA2 was confirmed by confocal microscopy. Intracellular concentration of Na+ (a measure of Na+ antiporter activity) was estimated by atomic absorption spectroscopy. The growth phenotypes of cells expressing NHAoc/NHA2 mutants were studied on YNB agar supplemented with NaCl and by growth curves in YNB broth., Results: Mutations in amino acid residues V161 and F357 reduced the ability of transfected BW31a cells to remove intracellular sodium and to grow in NaCl-containing medium. Yeast expressing the double mutant F357 F437 cannot grow in 0.4M NaCl, suggesting that these residues are also essential for antiporter activity., Conclusions: Evolutionarily conserved amino acids are required for full antiporter function., General Significance: Mutations in these amino acid residues may impact NHAoc activity and therefore osteoclast function in vitro and in vivo., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The central role of reactive oxygen species (ROS) in osteoclast differentiation and in bone homeostasis prompted us to characterize the redox regulatory system of osteoclasts. In this report, we describe the expression and functional characterization of PAMM, a CXXC motif-containing peroxiredoxin 2-like protein expressed in bone marrow monocytes on stimulation with M-CSF and RANKL. Expression of wild-type (but not C to G mutants of the CXXC domain) PAMM in HEK293 cells results in an increased GSH/GSSG ratio, indicating a shift toward a more reduced environment. Expression of PAMM in RAW264.7 monocytes protected cells from hydrogen peroxide-induced oxidative stress, indicating that PAMM regulates cellular redox status. RANKL stimulation of RAW 264.7 cells caused a decrease in the GSH/GSSG ratio (reflecting a complementary increase in ROS). In addition, RANKL-induced osteoclast formation requires phosphorylation and translocation of NF-kappaB and c-Jun. In stably transfected RAW 264.7 cells, PAMM overexpression prevented the reduction of GSH/GSSG induced by RANKL. Concurrently, PAMM expression completely abolished RANKL-induced p100 NF-kappaB and c-Jun activation, as well as osteoclast formation. We conclude that PAMM is a redox regulatory protein that modulates osteoclast differentiation in vitro. PAMM expression may affect bone resorption in vivo and help to maintain bone mass.

Periodontal disease is a chronic inflammatory disease in the oral cavity, which culminates in alveolar bone loss. Porphyromonas gingivalis is a consensus periodontal pathogen that has been implicated in adult forms of periodontitis. We previously demonstrated that IL-10-deficient mice exhibit a hyperinflammatory phenotype and are highly susceptible to P. gingivalis-induced periodontitis, indicating an important anti-inflammatory effect of IL-10 in suppressing bone loss. In this study, we analyzed the pathway(s) by which IL-10 deficiency leads to severe P. gingivalis-induced periodontitis. Because Stat3 is essential in IL-10 signaling, immune cell-specific Stat3-deficient mice were subjected to P. gingivalis infection to identify the key IL-10-responsive cells in preventing periodontitis. Myeloid cell-specific Stat3-deficient mice exhibited increased periodontal bone loss (p < 0.001), whereas T cell- and B cell-specific Stat3 mice were resistant, suggesting that macrophages (MP) and/or polymorphonuclear leukocytes are the key target cells normally suppressed by IL-10. Myeloid cell-specific Stat3-deficient mice exhibited elevated gingival CD40L gene expression in vivo compared with wild-type controls (p < 0.01), and Stat3-deficient MPs exhibited vigorous P. gingivalis-stimulated IL-12 production in vitro and induced elevated Ag-specific T cell proliferation compared with wild-type MPs (p < 0.01). Of importance, both IL-12p40/IL-10 and T cell/IL-10 double-deficient mice were resistant to P. gingivalis-induced periodontitis, demonstrating roles for both IL-12p40 and T cells in pathogenesis in a hyperinflammatory model of disease. These data demonstrate that P. gingivalis-induced periodontitis in IL-10-deficient mice is dependent upon IL-12p40-mediated proinflammatory T cell responses.

In previous studies we showed that biasing the immune response to Porphyromonas gingivalis antigens to the Th1 phenotype increases inflammatory bone resorption caused by this organism. Using a T cell screening strategy we identified eight P. gingivalis genes coding for proteins that appear to be involved in T-helper cell responses. In the present study, we characterized the protein encoded by the PG&#95;1841 gene and evaluated its relevance in the bone resorption caused by P. gingivalis because subcutaneous infection of mice with this organism resulted in the induction of Th1 biased response to the recombinant PG1841 antigen molecule. Using an immunization regime that strongly biases toward the Th1 phenotype followed by challenge with P. gingivalis in dental pulp tissue, we demonstrate that mice pre-immunized with rPG1841 developed severe bone loss compared with control immunized mice. Pre-immunization of mice with the antigen using a Th2 biasing regime resulted in no exacerbation of the disease. These results support the notion that selected antigens of P. gingivalis are involved in a biased Th1 host response that leads to the severe bone loss caused by this oral pathogen.

Objective: Individuals with spinal cord injury (SCI) develop a severe form of osteoporosis below the level of injury that is poorly understood. We conducted a preliminary investigation to assess whether circulating markers of bone turnover and circulating RANKL/OPG levels are related to the severity of SCI, aging, or to differences in mobility (i.e., walking or using a wheelchair)., Methods: Sixty-four caucasian men >or=1.6 years since injury selected based on locomotive mode provided blood samples and completed a health questionnaire at the VA Boston Healthcare System from 10/2003 to 6/2005. Plasma sRANKL, osteoprotegerin (OPG), osteocalcin and carboxyterminal telopeptide of type I collagen (CTx) levels were determined., Results: Increasing age was significantly associated with increased OPG and CTx. Injury severity was predictive of OPG levels, and adjusting for age, participants with cervical motor complete and ASIA C SCI (n=11) had significantly lower mean OPG (46.1 pg/ml) levels than others (63.4 pg/ml). Locomotive mode was not associated with differences in bone markers., Conclusions: Severe cervical spinal cord injury is associated with decreased circulating OPG levels placing these patients at risk for accelerated bone loss that appears unrelated to locomotive mode.

Significant progress has been made in studies of the mechanisms by which RANKL induces terminal osteoclast differentiation. However, many crucial details in the RANKL-evoked signaling pathway for osteoclast differentiation remain to be defined. We characterized genes specifically expressed in osteoclasts by differential screening of a human osteoclastoma cDNA library, and found that the regulator of G-protein signaling 10A (RGS10A), but not the RGS10B isoform, was specifically expressed in human osteoclasts. The expression of RGS10A is also induced by RANKL in osteoclast precursors and is prominently expressed in mouse osteoclast-like cells. RGS10A silencing by RNA interference blocked intracellular [Ca2+]i oscillations, the expression of NFAT2, and osteoclast terminal differentiation in both bone marrow cells and osteoclast precursor cell lines. Reintroduction of RGS10A rescued the impaired osteoclast differentiation. RGS10A silencing also resulted in premature osteoclast apoptosis. RGS10A silencing affected the RANKL-[Ca2+]i oscillation-NFAT2 signaling pathway but not other RANKL-induced responses. Our data demonstrate that target components of RGS10A are distinct from those of RGS12 in the RANKL signaling mechanism. Our results thus show the specificity of RGS10A as a key component in the RANKL-evoked signaling pathway for osteoclast differentiation, which may present a promising target for therapeutic intervention.

Osteoclastic bone resorption is a central mechanism in skeletal development, remodeling and pathology. RANKL is a mandatory factor controlling osteoclastogenesis; however, the underlying signaling pathways are only partially characterized. Using a screening array for the investigation of differential transcription factor activation, we identified activation of the Brn3 transcription factor family as a downstream event of RANKL signaling during terminal osteoclastogenesis. RANKL stimulation induces expression of Brn3a and b and maximal transcriptional activity of Brn3 family members concurrent with osteoclastic giant cell formation. Immunohistochemical analysis revealed both nuclear and cytoplasmic localization of Brn3a and b in mature osteoclasts. Functional inhibition of Brn3 transcription factors resulted in inhibition of pre-osteoclast fusion and reduction in bone resorbing activity of mature osteoclasts. Furthermore, we identified synaptotagmin-1, a regulator of membrane and vesicular fusion, as downstream target of Brn3 with a role in osteoclast function. We conclude that Brn-3 represents a novel molecular differentiation factor that controls osteoclast maturation and function, suggesting an important role in bone metabolism.

Periodontal disease involves multi-bacterial infections accompanied by inflammatory bone resorption lesions. The abundant T and B lymphocyte infiltrates are the major sources of the osteoclast differentiation factor, receptor activator for nuclear factor-kappaB ligand (RANKL) which, in turn, contributes to the development of bone resorption in periodontal disease. In the present study, we found that the concentrations of RANKL and regulatory T cell (T(reg))-associated cytokine, interleukin (IL)-10, in the periodontal tissue homogenates were correlated negatively, whereas RANKL and proinflammatory cytokine, IL-1beta, showed positive correlation. Also, according to the fluorescent-immunohistochemistry, the frequency of forkhead box P3 (FoxP3)/CD25 double-positive cells was diminished strikingly in the bone resorption lesion of periodontal disease compared to healthy gingival tissue, while CD25 or FoxP3 single positive cells were still observed in lesions where abundant RANKL+ lymphocytes were present. Very importantly, few or no expressions of FoxP3 by the RANKL+ lymphocytes were observed in the diseased periodontal tissues. Finally, IL-10 suppressed both soluble RANKL (sRANKL) and membrane RANKL (mRANKL) expression by peripheral blood mononuclear cells (PBMC) activated in vitro in a bacterial antigen-specific manner. Taken together, these results suggested that FoxP3/CD25 double-positive T(reg) cells may play a role in the down-regulation of RANKL expression by activated lymphocytes in periodontal diseased tissues. This leads to the conclusion that the phenomenon of diminished CD25+FoxP3+ T(reg) cells appears to be associated with the increased RANKL+ T cells in the bone resorption lesion of periodontal disease.

The undesired movement of anchor teeth, and relapse of previously moved teeth, are major clinical problems in orthodontics. Dental implants are increasingly used to preserve anchorage, but these are costly and require invasive surgical procedures. An alternative strategy for maintaining anchorage may be the use of biological inhibitors of osteoclastic bone resorption. In the present study, we investigated the relative efficacy of pamidronate vs. osteoprotegerin (OPG) in inhibiting bone resorption and tooth movement, using a new orthodontic model in mice in which maxillary molars are moved for prolonged periods by near-constant, clinically relevant forces. Osteoclast influx to compression sites was initiated on day 3, was maximal on day 4, and persisted until at least day 12 after force application. Tooth movement paralleled osteoclast numbers. Minimal osteoclast apoptosis was observed, suggesting that recruitment, rather than programmed cell death, is a critical regulatory mechanism under conditions of constant force. Osteoclasts were reduced at compression sites by both OPG (95%) and pamidronate (70%); tooth movement was more dramatically inhibited by OPG (77% vs. 34%). Our findings indicate that constant orthodontic force regulates the recruitment, activation, and viability of osteoclasts, and that OPG could have clinical utility in preventing undesired tooth movement.

Objective: Interleukin-10 is an anti-inflammatory cytokine that reduces periapical bone loss, but its role in periodontal bone loss is unclear. In the present study, we tested the hypothesis that endogenous interleukin-10 is a potent suppressor of Porphyromonas gingivalis-induced alveolar bone loss in vivo., Methods: Interleukin-10 knockout (-/-) and wild-type mice were inoculated intraorally with P. gingivalis. Non-infected animals served as negative controls. Alveolar bone loss, gingival cytokine levels, and gingival gene expression were assessed using morphometric analysis, enzyme-linked immunosorbent assay (ELISA), and semiquantitative reverse transcription polymerase chain reaction (RT-PCR), respectively., Results: P. gingivalis-infected interleukin-10(-/-) mice exhibited severe alveolar bone loss compared to non-infected interleukin-10(-/-) and wild-type mice by day 42. Surprisingly, bone resorptive cytokines interleukin-1alpha and tumor necrosis factor alpha (TNF-alpha) were not up-regulated in gingival tissues by P. gingivalis-infection. Although interleukin-1beta was marginally increased, blockade of both interleukin-1 isoforms or interleukin-1 receptor type I with neutralizing antisera failed to reduce alveolar bone loss in interleukin-10(-/-) mice, indicating the operation of an interleukin-1-independent mechanism. No strong correlations between bone loss and other cytokines was observed, although interferon gamma (IFNgamma), interleukin-6, interleukin-4, and prostaglandin E2 were modestly up-regulated in infected interleukin-10(-/-) mice. P. gingivalis infection reduced the expression of cell markers in gingival tissue on days 7 and 14 in both interleukin-10(-/-) and wild-type animals, suggestive of bacteria-induced cytotoxicity or apoptosis. This was followed by up-regulated expression of receptor activator of nuclear factor kappa B ligand (RANKL) and CD40 ligand (CD40L on days 28 and 70 in infected interleukin-10(-/-) mice only., Conclusion: The interleukin-10(-/-) mouse is highly susceptible to bone loss induced by the periodontal pathogen P. gingivalis, which is mediated via an interleukin-1-independent pathway., ((c)Blackwell Munksgaard 2004)

Chemokines play an important role in immune and inflammatory responses by inducing migration and adhesion of leukocytes, and have also been reported to modulate osteoclast differentiation from hemopoietic precursor cells of the monocyte-macrophage lineage. In this study, we examined the effect of MIP-1 gamma, a C-C chemokine family member, on receptor activator of NF-kappa B ligand (RANKL)-stimulated osteoclast differentiation, survival, and activation. RANKL induced osteoclasts to dramatically increase production of MIP-1 gamma and to also express the MIP-1 gamma receptor CCR1, but had only minor effects on the related C-C chemokines MIP-1 alpha and RANTES. Neutralization of MIP-1 gamma with specific Ab reduced RANKL-stimulated osteoclast differentiation by 60-70%. Mature osteoclasts underwent apoptosis within 24 h after removal of RANKL, as shown by increased caspase 3 activity and DNA fragmentation. Apoptosis was reduced by the addition of exogenous MIP-1 gamma or RANKL, both of which increased NF-kappa B activation in osteoclasts. Neutralization studies showed that the prosurvival effect of RANKL was in part dependent on its ability to induce MIP-1 gamma. Finally, osteoclast activation for bone resorption was stimulated by MIP-1 gamma. Taken together, these results demonstrate that MIP-1 gamma plays an important role in the differentiation and survival of osteoclasts, most likely via an autocrine pathway.

Periapical granulomas are induced by bacterial infection of the dental pulp and result in destruction of the surrounding alveolar bone. In previous studies we have reported that the bone resorption in this model is primarily mediated by macrophage-expressed interleukin-1 (IL-1). The expression and activity of IL-1 is in turn modulated by a network of Th1 and Th2 regulatory cytokines. In the present study, the functional roles of the Th1 cytokine gamma interferon (IFN-gamma) and IFN-gamma-inducing cytokines IL-12 and IL-18 were determined in a murine model of periapical bone destruction. IL-12-/-, IL-18-/-, and IFN-gamma-/- mice were subjected to surgical pulp exposure and infection with a mixture of four endodontic pathogens, and bone destruction was determined by microcomputed tomography on day 21. The results indicated that all IL-12-/-, IL-18-/-, and IFN-gamma-/- mice had similar infection-stimulated bone resorption in vivo as wild-type control mice. Mice infused with recombinant IL-12 also had resorption similar to controls. IFN-gamma-/- mice exhibited significant elevations in IL-6, IL-10, IL-12, and tumor necrosis factor alpha in lesions compared to wild-type mice, but these modulations had no net effect on IL-1alpha levels. Recombinant IL-12, IL-18, and IFN-gamma individually failed to consistently modulate macrophage IL-1alpha production in vitro. We conclude that, at least individually, endogenous IL-12, IL-18, and IFN-gamma do not have a significant effect on the pathogenesis of infection-stimulated bone resorption in vivo, suggesting possible functional redundancy in proinflammatory pathways.

Periodontal disease is the consequence of a mixed Gram-negative infection in the gingival sulcus and has been associated with deficits in the neutrophil response. A novel, and heretofore untested, alternative approach to therapy is the use of biological-response modulators that enhance the neutrophil response. Poly-beta1-6-glucotriosyl-beta1-3-glucopyranose glucan (PGG-glucan) is an immunomodulator, derived from yeast, which specifically enhances neutrophil priming, phagocytosis and bacterial killing while failing to induce inflammatory cytokine expression. The hypothesis tested was that PGG-glucan could enhance host resistance to a Gram-negative periodontal pathogen, Porphyromonas gingivalis. Chambers were implanted subcutaneously in the dorsolumbar region of C57BL/6J mice and allowed to heal for 14 days. PGG-glucan was administered subcutaneously to one-half of the animals and saline to the other half. In the first set of experiments the chambers were inoculated with P. gingivalis (A7436) at 4 x 10 (6), 4 x 10 (7), and 4 x 10 (8) colony-forming units (CFU). In the second set of experiments the chambers were inoculated with 5 x 10 (8) CFU of either P. gingivalis or Streptococcus sanguis, a Gram-positive oral microbe that is not periodontopathic. Chambers were sampled over the following 2 weeks. The results demonstrated that: (1). bacterial CFU and neutrophils increased with increasing bacterial inoculum (P<0.02); (2). bacterial CFU were lower in the PGG-glucan-treated animals than in the saline controls (P<0.02); and (3). neutrophil counts were higher in the PGG-glucan-treated animals than in the saline controls (P<0.01). These results indicate that PGG-glucan significantly enhances neutrophil emigration and bacterial killing, thus decreasing the bacterial infection in this model system.

Objective: The present study was undertaken to determine the frequency and extent of apical root resorption associated with induced periradicular lesions in mice., Study Design: Bone and root resorption was quantified by using two- and three-dimensional micro-computed tomography (mu-CT) in the lower first molars of mice subjected to pulp exposure and infection., Results: mu-CT measurements showed significant apical resorption in exposed and infected teeth, resulting in an average distal root shortening of 12.7% (P <.001 vs unexposed). These findings were confirmed with three-dimensional reconstituted images that showed thinning and shortening of the distal root. Tartrate-resistant acid phosphatase clastic cells were associated with resorption lacunae on the cementum of root apices, as well as on bone at the periphery of the periradicular lesions. Brown and Brenn staining showed the presence of bacteria in dentinal tubules adjacent to resorbed cementum., Conclusions: Apical root resorption is a prominent and consistent finding associated with periradicular infection in the mouse. This species represents a convenient model for studying the pathogenesis of inflammatory root resorption in vivo.

Solubilization of bone mineral by osteoclasts depends on the formation of an acidic extracellular compartment through the action of a V-type ATPase. We previously cloned a gene encoding a putative osteoclast-specific proton pump subunit, termed OC-116 kDa, approved mouse Atp6i (ATPase, H+ transporting, [vacuolar proton pump] member I). The function of Atp6i as osteoclast-specific proton pump subunit was confirmed in our mouse knockout study. However, the transcription regulation of Atp6i remains largely unknown. In this study, the gene encoding mouse Atp6i and the promoter have been isolated and completely sequenced. In addition, the temporal and spatial expressions of Atp6i have been characterized. Intrachromosomal mapping studies revealed that the gene contains 20 exons and 19 introns spanning approximately 11 kilobases (kb) of genomic DNA. Alignment of the mouse Atp6i gene exon sequence and predicted amino acid sequence to that of the human reveals a strong homology at both the nucleotide (82%) and the amino acid (80%) levels. Primer extension assay indicates that there is one transcription start site at 48 base pairs (bp) upstream of the initiator Met codon. Analysis of 4 kb of the putative promoter region indicates that this gene lacks canonical TATA and CAAT boxes and contains multiple putative transcription regulatory elements. Northern blot analysis of RNAs from a number of mouse tissues reveals that Atp6i is expressed predominantly in osteoclasts, and this predominant expression was confirmed by reverse-transcription polymerase chain reaction (RT-PCR) assay and immunohistochemical analysis. Whole-mount in situ hybridization shows that Atp6i expression is detected initially in the headfold region and posterior region in the somite stage of mouse embryonic development (E8.5) and becomes progressively restricted to anterior regions and the limb bud by E9.5. The expression level of Atp6i is largely reduced after E10.5. This is the first report of the characterizations of Atp6i gene, its promoter, and its gene expression patterns during mouse development. This study may provide valuable insights into the function of Atp6i, its osteoclast-selective expression, regulation, and the molecular mechanisms responsible for osteoclast activation.

Periapical bone resorption occurs following infection of the dental pulp and is mediated mainly by IL-1alpha in the murine model. The production and activity of IL-1alpha is modulated by a network of regulatory cytokines, including those produced by Th1 (pro-inflammatory) and Th2 (anti-inflammatory) subset T cells. This study was designed to assess the functional role of the Th2-type cytokines IL-4 and IL-10 in infection-stimulated bone resorption in vivo. The dental pulps of the first molars were exposed and infected with a mixture of four common endodontic pathogens, and bone destruction was determined by micro-computed tomography at sacrifice on day 21. The results demonstrate that IL-10(-/-) mice had significantly greater infection-stimulated bone resorption in vivo compared with wild-type mice (p < 0.001), whereas IL-4(-/-) exhibited no increased resorption. IL-10(-/-) had markedly elevated IL-1alpha production within periapical inflammatory tissues (>10-fold) compared with wild type (p < 0.01), whereas IL-4(-/-) exhibited decreased IL-1alpha production (p < 0.05). IL-10 also suppressed IL-1alpha production by macrophages in a dose-dependent fashion in vitro, whereas IL-4 had weak and variable effects. We conclude that IL-10, but not IL-4, is an important endogenous suppressor of infection-stimulated bone resorption in vivo, likely acting via inhibition of IL-1alpha expression.

An association has been reported between polymorphisms in the genes encoding IL-1alpha (-889) and IL-1beta (+3953) (periodontitis susceptibility trait, PST), and an increased severity of periodontitis (18). The IL-1beta polymorphism was reported to correlate with increased IL-1beta expression by monocytes in response to bacterial stimulants. In the present study, we determined if PST positive subjects with periodontitis exhibit elevated production of IL-1beta, compared to PST negative periodontitis patients. Peripheral blood monocytes were obtained from 10 PST+ and 10 PST- age- and disease-balanced subjects with adult forms of periodontitis. Monocytes were cultured with a panel of bacterial stimulants, including Escherichia coli and Porphyromonas gingivalis LPS, and whole formalinized periodontal pathogens P. gingivalis, Bacteroides forsythus and Prevotella intermedia, and health-associated organisms Veillonella parvula and Streptococcus sanguis. Our results demonstrate that monocytes from PST+ and PST- patients showed no significant differences in IL-1beta production in response to any stimulant tested. In addition, the periodontal pathogens P. gingivalis, B. forsythus and P. intermedia failed to stimulate higher IL-1beta responses compared to health-associated species V. parvula and S. sanguis. A marked interindividual variation in production of IL-1beta was seen, with high, low and intermediate responders present in both PST+ and PST- groups. We conclude that genetic loci other than the PST polymorphisms are also important regulators of monocyte IL-1 responses.

Solubilization of bone mineral by osteoclasts depends on the formation of an acidic extracellular compartment through the action of a V-proton pump that has not yet been characterized at the molecular level. We previously cloned a gene (Atp6i, for V-proton pump, H+ transporting (vacuolar proton pump) member I) encoding a putative osteoclast-specific proton pump subunit, termed OC-116kD (ref. 4). Here we show that targeted disruption of Atp6i in mice results in severe osteopetrosis. Atp6i-/- osteoclast-like cells (OCLs) lose the function of extracellular acidification, but retain intracellular lysosomal proton pump activity. The pH in Atp6i-/- liver lysosomes and proton transport in microsomes of Atp6i-/- kidney are identical to that in wild-type mice. Atp6i-/- mice exhibit a normal acid-base balance in blood and urine. Our results demonstrate that Atp6i is unique and necessary for osteoclast-mediated extracellular acidification.

Infections of the dental pulp commonly result in infraosseus inflammation and bone destruction. However, the role of phagocytic leucocytes in the pathogenesis of pulpal infections has been uncertain. In this work we used P/E-/- selectin-deficient mice, which lack rolling adhesion of leucocytes to endothelium and mimic the human syndrome, leucocyte adhesion deficiency II (LAD-II), to test the hypothesis that phagocytic leucocytes protect against pulpal infection and subsequent periapical infraosseus bone resorption. P/E-/- mice and P/E+/+ wild-type controls were subjected to surgical pulp exposure, and both groups were infected with a mixture of pulpal pathogens including Prevotella intermedia, Fusobacterium nucleatum, Peptostreptococcus micros and Streptococcus intermedius. Animals were killed after 20 days, and the extent of infraosseus bone destruction was quantified by histomorphometry. In two separate experiments, P/E-/- mice had significantly greater bone resorption than P/E+/+ controls. The increased bone destruction correlated with a twofold decrease in polymorphonuclear (PMN) infiltration into periapical inflammatory tissues of P/E-/- mice. P/E-/- mice had higher tissue levels of the bone resorptive cytokine, interleukin (IL)-1alpha. Tissue levels of IL-2, IL-4, IL-10, tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) were all higher in P/E-/- mice, but the increases were not statistically significant. Only IL-12 was higher in P/E+/+ mice, possibly reflecting a greater number of infiltrating monocytes in wild-type mice. These findings demonstrate that phagocytic leucocytes are protective in this model, and suggest that elevated expression of inflammatory cytokines is responsible for the observed bone destruction.

Products of periodontopathic bacteria exert immunomodulatory effects on various lymphoid cell populations, some of which have been implicated in the pathogenesis of periodontitis. It has recently been suggested that some of these bacterial products may possess superantigenic (SAg) activity. SAg bind simultaneously to the V beta chain of T cell receptors and to class II major histocompatibility complex molecules, thereby activating as many as 35% of T cells to proliferate and produce cytokines. In order to examine this question, the proliferation of splenic and thymic T cells from immunologically naive, 3-6-wk-old Balb/c (H-2d), C57BL/6 (H-2b) and C3H/HeJ (H-2k) mice was assessed in response to sonic extracts of periodontopathogens. Laboratory and/or reference strains of a.o. Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia and Prevotella nigrescens were used as stimulants. Staphylococcal enterotoxin B (SEB), a known superantigen, was utilized as a positive control. Unfractionated spleen cells responded to several of the tested preparations of the different bacteria, as well as to SEB, Con A and Escherichia coli LPS. Thymocytes responded to Con A and SEB, but not to LPS or to any sonic extract. Spleen cells depleted of B cells by panning responded to SEB and Con A, but not to LPS and showed a reduced response to sonicates. The residual response of B cell-depleted spleen cells was reduced essentially to background by treatment with anti-Thy 1.2 + C'. Similar results were obtained in the presence of 5% added mitomycin-treated antigen presenting cells, indicating that these cells were not limiting. These results demonstrate that extracts of periodontopathic bacteria do not stimulate murine T cells in a manner consistent with superantigenic activation.

Bacterial products have served as important immunological tools to study lymphocyte activation. The lipopolysaccharides of the Gram-negative bacteria are well known to be potent activators of B lymphocytes. Several Gram-positive bacteria produce exotoxins that are superantigens for T cells. In the present study, we demonstrate that the Gram-positive bacteria Clostridium botulinum C and D produce a high molecular weight mitogen (Cb mitogen) that is a potent activator of murine B lymphocytes. The Cb mitogen was discovered as a consequence of our attempt to investigate a possible superantigen activity present in the botulinum exotoxins. We observed initially that mouse spleen cells were strongly stimulated to proliferate by culture supernatants of C. botulinum C and D. However, the characterization of the responding cell ruled out superantigen because only the B lymphocytes were stimulated to proliferate and to secrete immunoglobulins, and they did so independent of T cell help. In addition, the molecular characterization of the Cb mitogen demonstrated that the purified botulinum toxin was devoid of mitogenic activity. In contrast, the fractionation of the culture supernatant of C. botulinum C in an FPLC Superose 12 column indicated that the Cb mitogen was present in the void volume of the column (MW > or = 300 kDa) which had no toxigenic activity. However, the fractions containing molecules of 150 kDa were highly toxic for mice and had no mitogenic activity. The possibility that LPS was present as a contaminant in the Cb mitogen preparations was excluded because spleen cells from the LPS non-responder C3H/HeJ mice responded well to the Cb mitogen, and the antibiotic polymyxin B, which is an inhibitor of LPS, had no effect on the Cb-mitogen activity. However, an anti-lipoteichoic acid monoclonal antibody (3-1 mAb) inhibited to a great extent the proliferation of spleen cells induced by the Cb mitogen but had no effect on the LPS or concanavalin A stimulation of these cells. Moreover, the Cb mitogen was specifically adsorbed and eluted from a protein G Sepharose column to which the anti-lipoteichoic acid 3-1 mAb had been conjugated. These results support the view that lipoteichoic acid is a selective B cell mitogen.