David Polak

Mouse model of experimental periodontitis induced by Porphyromonas gingivalis/Fusobacterium nucleatum infection: bone loss and host response

AIM To compare the effect of oral infection with Porphyromonas gingivalis or Fusobacterium nucleatum versus infection with both bacteria on mouse periodontal tissues, and to characterize the inflammatory response. MATERIALS AND METHODS Mice were orally infected with P. gingivalis, F. nucleatum or both. At 42 days post-infection, alveolar bone loss was quantified using micro-computerized tomography. Tumour necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1beta levels induced by the infection were quantified using the subcutaneous chamber model. RESULTS Mice orally infected with F. nucleatum/P. gingivalis exhibited significantly more bone loss compared with that of mono-infected and sham-infected mice. F. nucleatum/P. gingivalis infection also increased the levels of TNF-alpha and IL1beta compared with the levels found in the mono-infected groups. CONCLUSIONS Polymicrobial infection with P. gingivalis/F. nucleatum aggravates alveolar bone loss and induces a stronger inflammatory response compared with that observed upon infection with either bacterium alone. The results suggest that oral infection of mice with a mixture of P. gingivalis and F. nucleatum may be superior to mono-infection models of experimental periodontitis.

Direct Recognition of Fusobacterium nucleatum by the NK Cell Natural Cytotoxicity Receptor NKp46 Aggravates Periodontal Disease

Periodontitis is a common human chronic inflammatory disease that results in the destruction of the tooth attachment apparatus and tooth loss. Although infections with periopathogenic bacteria such as Porphyromonas gingivalis (P. gingivalis) and Fusobacterium nucleatum (F. nucleatum) are essential for inducing periodontitis, the nature and magnitude of the disease is determined by the hosts immune response. Here, we investigate the role played by the NK killer receptor NKp46 (NCR1 in mice), in the pathogenesis of periodontitis. Using an oral infection periodontitis model we demonstrate that following F. nucleatum infection no alveolar bone loss is observed in mice deficient for NCR1 expression, whereas around 20% bone loss is observed in wild type mice and in mice infected with P. gingivalis. By using subcutaneous chambers inoculated with F. nucleatum we demonstrate that immune cells, including NK cells, rapidly accumulate in the chambers and that this leads to a fast and transient, NCR1-dependant TNF-α secretion. We further show that both the mouse NCR1 and the human NKp46 bind directly to F. nucleatum and we demonstrate that this binding is sensitive to heat, to proteinase K and to pronase treatments. Finally, we show in vitro that the interaction of NK cells with F. nucleatum leads to an NCR1-dependent secretion of TNF-α. Thus, the present study provides the first evidence that NCR1 and NKp46 directly recognize a periodontal pathogen and that this interaction influences the outcome of F. nucleatum-mediated periodontitis.

Strain‐dependent activation of the mouse immune response is correlated with Porphyromonas gingivalis‐induced experimental periodontitis

AIMS To evaluate the effect of oral infection with three Porphyromonas gingivalis strains on alveolar bone loss (ABL) and its correlation with the mouse immune response. MATERIALS AND METHODS Mice were orally infected with P. gingivalis strains 381, 33277 and 53977. After 42 days, maxillae were analysed for ABL using micro-computed tomography and the serum for anti-P.gingivalis IgG1 and IgG2a levels. The cytokine response to P. gingivalis was tested using the subcutaneous chamber model. RESULTS The P. gingivalis 53977-infected group showed the highest ABL, which was significantly different from all other groups (p<0.001). In addition, the humoral response to P. gingivalis 53977 was significantly lower than the response to P. gingivalis 381 and 33277 (p< or =0.01). The IgG2a/IgG1 ratio was higher in the P. gingivalis 33277-infected group (1.6) compared with the P. gingivalis 381-infected group (0.51). Four days post-infection, interleukin (IL)-1beta levels remained significantly higher in the P. gingivalis 53977-infected group only (1198.2+/-260.0, p<0.05), while IL-4 levels remained significantly higher in the P. gingivalis 381-infected group (265.8+/-131.6, p<0.05). CONCLUSIONS The high levels of ABL induced by P. gingivalis 53977 were inversely correlated with the humoral response to this bacterium. In addition, ABL was correlated with an elevated pro-inflammatory response.

In vitro biocompatibility of endodontic sealers incorporating antibacterial nanoparticles

The main cause of endodontic disease is bacteria. Disinfection is presently achieved by cleaning the root canal system prior to obturation. Following setting, root canal filling is devoid of any antibacterial effect. Endodontic sealers with antimicrobial properties yet biocompatible may enhance root canal therapy. For this purpose, quaternized polyethylenimine nanoparticles which are antibacterial polymers, biocompatible, nonvolatile, and stable may be used. The aim of the present study was to examine the impact of added QPEI on the cytotoxicity of AH Plus, Epiphany, and GuttaFlow endodontic sealers. The effect of these sealers on the proliferation of RAW264.7 macrophage and L-929 fibroblast cell lines and on the production of TNFa from macrophages was examined. Cell vitality was evaluated using a colorimetric XTT assay. The presence of cytokines was determined by two-site ELISA. Results show that QPEI at 1% concentration does not impair the basic properties of the examined sealers in bothmacrophages and fibroblast cell lines. Incorporation of 1% QPEI into the sealers did not impair their biocompatibility. QPEI is a potential clinical candidate to improve antibacterial activity of sealers without increasing cytotoxicity

Vaccination of mice with Porphyromonas gingivalis or Fusobacterium nucleatum modulates the inflammatory response, but fails to prevent experimental periodontitis

AIM To assess the potential of using vaccination with Porphyromonas gingivalis or Fusobacterium nucleatum, in modulating local subcutaneous inflammatory response and alveolar bone loss following coinfection with both bacteria. MATERIALS AND METHODS Mice were immunized against either P. gingivalis or F. nucleatum. The cytokine response to mixed infection with P. gingivalis and F. nucleatum was evaluated using the subcutaneous chamber model. The alveolar bone loss induced by oral mixed infection was evaluated by micro-CT using the experimental periodontitis model. Serum levels of specific antibodies were determined by ELISA. RESULTS Vaccination with either bacterium produced a specific humoral response before infection. Animals immunized against either bacteria following a mixed infection with P. gingivalis and F. nucleatum, showed decreased TNFalpha (but not IL-1beta) levels as compared with non-immunized animals. However, the vaccination did not change the level of mixed infection-induced alveolar bone loss when compared with non-immunized animals. Six weeks following the oral mixed infection, specific antibody titres remained high. Furthermore, specific antibodies against the non-immunized bacterium were present at high levels. CONCLUSIONS While vaccination produced specific antibodies and suppressed the inflammatory response, it failed to prevent or reduce the progression of experimental periodontitis induced by mixed infection with P. gingivalis and F. nucleatum.

An update on the evidence for pathogenic mechanisms that may link periodontitis and diabetes

AIM To provide an update of the review by Taylor (Journal of Clinical Periodontology, 2013, 40, S113) regarding the scientific evidence of the biological association between periodontitis and diabetes. METHODS Literature searches were performed using MeSH terms, keywords and title words and were published between 2012 and November 2016. All publications were screened for their relevance. The data from the articles were extracted and summarized in tables and a narrative review. RESULTS Small-scale molecular periodontal microbiome studies indicate a possible association between altered glucose metabolism in pre-diabetes and diabetes and changes in the periodontal microbiome, with no evidence for casual relationships. Clinical and animal studies found elevated gingival levels of IL1-β, TNF-α, IL-6, RANKL/OPG and oxygen metabolites in poorly controlled diabetes. In addition, individuals with diabetes and periodontitis exhibit high levels of circulating TNF-α, CRP and mediators of oxidative stress, and successful periodontal treatment reduces their levels. CONCLUSIONS The elevated pro-inflammatory factors in the gingiva of patients with poorly controlled diabetes suggest a biological pathway that may aggravate periodontitis. Some evidence suggests that the systemic inflammatory burden in periodontitis has the potential to affect diabetes control, but no studies addressed the impact of successful periodontal therapy on the pathophysiological mechanisms involved in systemic complications of diabetes.

Antibacterial activity of bone cement containing quaternary ammonium polyethyleneimine nanoparticles

Sir, A bone cement mantle is commonly used in orthopaedic surgery, and creates an ideal environment for bacterial growth. Bacterial infection following joint replacement surgery is a catastrophic complication. It is estimated that infections occur in up to about 2% of primary hip and knee replacements, whereas revision surgery carries a 2–3-fold higher risk. The most common bacteria isolated from infected joints are Grampositive cocci (Staphylococcus epidermidis, Staphylococcus aureus and Streptococcus spp.) followed by Gram-negative bacteria and, less frequently, mixed and fungal infections. Addition of antibiotics to the cement has been advocated, especially in revision surgery. However, several studies describe the decreasing potency of common antibiotics in treating the infecting organisms and a parallel rise in the prevalence of resistant strains. As the implant itself lacks a blood supply, it is inherently susceptible to colonization by bacteria, which are less likely to be eradicated than in viable tissue. Therefore, efforts are focused on prevention of implant colonization. However, attempts to protect the implants using an antibiotic coating as well as the addition of antibiotics to the cement in the cemented implant have not proved successful. A novel strategy involves the use of antibacterial molecules that are bound to the implant or to the surrounding cement and are not released but remain functional for long periods of time. Quaternary amine residues proved to have excellent antibacterial properties. – 7 The purpose of this study was to modify a commonly used bone cement to obtain a safe and long-lasting antibacterial effect using quaternary ammonium polyethyleneimine (QPEI) nanoparticles. QPEI nanoparticles were synthesized as previously described. The tested materials were prepared by adding the synthesized powder to clinically available bone cement [SimplexTM P Bone Cement: 75% methyl methacrylate–styrene copolymer, 15% polymethylmethacrylate (PMMA) plus 10% barium; Stryker, Kalamazoo, MI, USA]. QPEI nanoparticles were added at 0%, 1%, 2% or 3% (w/w) to the bone cement and homogeneously mixed according to the manufacturer’s instructions. An antimicrobial effect against S. aureus ATCC 8325-4 and Enterococcus faecalis (a clinical isolate from the Maurice and Gabriela Goldschleger School of Dental Medicine at Tel Aviv University, Israel) was tested using the direct contact test (DCT) and agar diffusion test (ADT). Biocompatibility was tested on human primary polymorphonuclear cells as previously described. Cell viability was measured using the XTT assay (Biological Industries) and levels of tumour necrosis factor-a (TNF-a) in the supernatant were measured using an ELISA kit (Biolegend, San Diego, CA, USA). Additionally, physical properties of the cements were evaluated. Testing was performed using a Controlled Teststore 25 Tons MTS Device (Minneapolis, MN, USA) and results were analysed using MPT software (Multi-Purpose Testware 793.10, MTS System Corporation, Eden Prairie, MN, USA). Strain (1) and Young’s modulus (E) were calculated for each specimen. A strong antibacterial effect after an ageing period of 4 weeks was evident (P,0.05) in all the bone cement samples in which the QPEI nanoparticles were incorporated compared with bone cement samples with no additives, which showed no antibacterial effect. The DCT showed significant antibacterial activity against both bacteria for at least 4 weeks (Figure 1); the ADT revealed no inhibition halo in the agar plates for both tested bacteria, indicating that the nanoparticles are retained in the PMMA and do not diffuse into the agar. Moreover, QPEI nanoparticles did not change the biocompatibility properties of PMMA. Addition of the nanoparticles at all tested percentages didnot result inasignificant change in cell viability compared with that of the bone cement group; incorporation of 0%, 1%, 2% and 3% QPEI Research letters

Protective potential of non-dialyzable material fraction of cranberry juice on the virulence of P. gingivalis and F. nucleatum mixed infection.

BACKGROUND Periodontitis is a polymicrobial infectious disease. A novel potential chemical treatment modality may lie in bacterial anti-adhesive materials, such as cranberry juice fractions. The aim of this study is to explore the effect of high molecular weight cranberry constituent (non-dialyzable material [NDM]) on the virulence of a mixed infection with Porphyromonas gingivalis and Fusobacterium nucleatum in mice. METHODS In vitro, the anti-adhesive property of NDM was validated on epithelial cell culture, and inhibition of coaggregation was tested using a coaggregation assay. The in vivo effect was tested on the outcome of experimental periodontitis induced by a P. gingivalis and F. nucleatum mixed infection, and also on the local host response using the subcutaneous chamber model of infection. Phagocytosis was also tested on RAW macrophages by the use of fluorescent-labeled bacteria. RESULTS NDM was found to inhibit the adhesion of both species of bacteria onto epithelial cells and to inhibit coaggregation in a dose-dependent manner. NDM consumption by mice attenuated the severity of experimental periodontitis compared with a mixed infection without NDM treatment. In infected subcutaneous chambers, NDM alone reduced tumor necrosis factor-α (TNF-α) levels induced by the mixed infection. In vitro, NDM eliminated TNF-α expression by macrophages that were exposed to P. gingivalis and F. nucleatum, without impairing their viability. Furthermore, NDM increased the phagocytosis of P. gingivalis. CONCLUSIONS The results indicate that the use of NDM may hold potential protective and/or preventive modalities in periodontal disease. Underlying mechanisms for this trait may perhaps be the anti-adhesive properties of NDM or its potential effect on inflammation.

Virulence Mechanism of Bacteria in Mixed Infection: Attenuation of Cytokine Levels and Evasion of Polymorphonuclear Leukocyte Phagocytosis

BACKGROUND The objective of the present study is to evaluate the effect of bacterial viability on the virulence of mixed infection. METHODS Expression of pro- and anti-inflammatory cytokines (interleukin [IL]-1β and IL-10, respectively) was tested in vivo, following live versus heat-killed infection (mono or mixed), using the mouse chamber model of infection. Ex vivo, phagocytosis of fluorescently labeled bacteria was tested in primary mouse polymorphonuclear leukocytes by flow cytometry. RESULTS In monoinfection, heat-killed Porphyromonas gingivalis led to augmented levels of IL-1β 2 hours postinfection, whereas IL-10 levels remained unaffected. Phagocytosis of heat-killed P. gingivalis was reduced compared with that of the live P. gingivalis, whereas phagocytosis of heat-killed Fusobacterium nucleatum was augmented compared with that of live F. nucleatum. In mixed infection, both IL-1β and IL-10 levels were augmented 24 hours postinfection when the bacteria were heat-killed. Although the phagocytosis pattern of F. nucleatum in the mixed infection remained similar to that upon monoinfection, phagocytosis of P. gingivalis was reduced following mixed infection. CONCLUSIONS The inflammatory response to live mixed infection is attenuated with reduced phagocytosis, compared with that of heat-killed mixed infection. The lower response to live mixed infection could stem from a mechanism enabling the bacteria to evade the host response, thereby increasing bacterial survival.

Porphyromonas Gingivalis Capsule-Mediated Coaggregation as a Virulence Factor in Mixed Infection With Fusobacterium Nucleatum

BACKGROUND Porphyromonas gingivalis (Pg) capsule enables evasion from phagocytosis, invasion of keratinocytes, and bacterial survival. In mixed infection, the capsule also participates in coaggregation, which may lead to characteristic virulence not present in the monoinfection. The aim of this study is to evaluate the role of Pg capsule as a virulence factor in coaggregated mixed infection with Fusobacterium nucleatum (Fn). METHODS Mixed infections containing Fn and non-capsulated or capsulated strains of Pg were compared with the same infection with lactose as coaggregation inhibitor. Murine experimental periodontitis was used to assess disease severity. Primary polymorphonuclear leukocytes and keratinocytes were used to examine phagocytosis and bacterial invasion, respectively. RESULTS Mixed infection with capsulated Pg augmented alveolar bone loss compared with that of mixed infection with non-capsulated Pg. Addition of lactose led to attenuation of bone loss in the capsulated mixed infection and to intensification of bone loss in the non-capsulated mixed infection. In the latter mixed infection, Fn evaded phagocytosis, whereas in the capsulated mixed infection Pg displayed a greater capacity for invasion of keratinocytes. CONCLUSIONS Pg capsule was found to serve as a unique virulence factor in mixed infection with Fn. Capsule-dependent coaggregation led to augmented invasion of Pg and may be responsible for the severity of disease after mixed infection with Fn.