Distribution of <i>P</i>. <i>gingivalis</i>, <i>T</i>. <i>denticola</i>, <i>T</i>. <i>forsythia</i>, <i>F</i>. <i>nucleatum</i> genomic DNA in ApoE^null mouse tissue by PCR.

<p>Hematogenous dissemination and invasion of four periodontal pathogens in systemic organs were detected by presence of bacterial genomic DNA by PCR.</p><p>Distribution of <i>P</i>. <i>gingivalis</i>, <i>T</i>. <i>denticola</i>, <i>T</i>. <i>forsythia</i>, <i>F</i>. <i>nucleatum</i> genomic DNA in ApoE^null mouse tissue by PCR.</p

Polybacterial infection-induced alteration of serum inflammatory markers in ApoE^null mice.

<p>Sera from polybacterial-infected mice (n = 6) and sham-infected mice (n = 6) at both 12 and 24 weeks was analyzed as described in Materials and Methods.</p><p>Polybacterial infection-induced alteration of serum inflammatory markers in ApoE^null mice.</p

Polybacterial infection elicits a distinct splenic T cell response.

<p>Representative images of the gating scheme are shown in panels 2 and 3, with panel 2 showing the lymphocyte gate, panel 3 showing the CD3⁺ CD4⁺ lymphocyte gate, and panel 4 showing the histogram of CD3⁺ CD4⁺ Receptor⁺ lymphocytes. (A). CD3⁺ CD4⁺ IFNγR⁺ cells indicate Th1 response. (B). CD3⁺ CD4⁺ IL4R⁺ cells indicate Th2 response. (C). CD3⁺ CD4⁺ IL17R⁺ cells indicate Th17 response. Poly—polybacterial infection. ** p < 0.01.</p

Viable bacteria were recovered from infected mouse heart and aorta.

<p>(A). FISH reveals cluster of coccobacilli morphotype of <i>P</i>. <i>gingivalis</i> in mouse aortic arch at 12 weeks, indicated by white arrow heads. (B). Enlarged inset in D showing clusters of <i>P</i>. <i>gingivalis</i> coccobacilli morphotype indicated by white arrow heads. (C). No bacteria were detected in sham-infected mouse aortic tissues. Scale bar is 10 micrometers.</p

Polymicrobial infection-induced altered expression of atherosclerosis related genes in ApoE^null mouse.

<p>Twenty-four week polybacterial-infected and sham-infected aortic tissue samples were processed and analyzed as described in Methods.</p><p>Polymicrobial infection-induced altered expression of atherosclerosis related genes in ApoE^null mouse.</p

Polybacterial infection significantly increased atherosclerotic plaque growth in the ascending aorta and the aortic root.

<p>(A). Total plaque area of infected mice was elevated at both 12- and 24 weeks of infection, but was significant compared to controls at 24 weeks of infection (p < 0.05). (B). Intimal/medial layer thickness ratio of infected mice was increased relative to sham-infected mice at both 12- and 24 weeks of infection, although not significant (p = 0.1599 and 0.1339). (C). CD3⁺ T cell counts were significantly higher in 24 week–infected mice than sham-infected mice. (D). H & E stained aortic section illustrates large plaques in infected mice. (E). H & E stained aortic section showing small plaques in sham-infected mice. (F). Aortic Sections demonstrating numerous CD3⁺ T cells in 24-week polybacterial-infected mouse (20X magnification). (G). Aortic Sections demonstrating numerous CD3⁺ T cells in 24-week polybacterial-infected mouse (200X magnification). (H). Aortic section from sham-infected mice devoid of infiltrating CD3⁺ T cells. Black arrow heads indicate atherosclerotic plaque. Red arrows indicate infiltrated CD3⁺ T cells. L—artery lumen, I—intimal layer, M—medial layer, A—adventitial layer. * p < 0.05.</p

Polybacterial-infection induced bacterial-specific humoral immune response and alveolar bone resorption.

<p>(A). Serum IgG antibody response to the four bacteria used in periodontal infection at 12 and 24 weeks. (B). Serum IgM antibody response to the four bacteria used in periodontal infection at 12 and 24 weeks. Representative left maxilla lingual view of polybacterial-infected and sham-infected mice at both 12- and 24 weeks with area of bone resorption outlined from alveolar bone crest to cementoenamel junction. (C). Total alveolar bone resorption is significantly greater than controls at 24 weeks of polybacterial infection. (D). Representative left maxilla lingual view of polybacterial-infected and sham-infected mice at both 12- and 24 weeks with area of bone resorption outlined from alveolar bone crest to cementoenamel junction. Poly—polybacterial infection, Con—sham-infected control, <i>Pg/Td/Tf/Fn</i>–polybacterial infection <i>Pg</i>–<i>P</i>. <i>gingivalis</i>, <i>Td</i>–<i>T</i>. <i>denticola</i>, <i>Tf</i>–<i>T</i>. <i>forsythia</i>, <i>Fn</i>–<i>F</i>. <i>nucleatum</i>. * p < 0.05, ** p < 0.01, *** p < 0.001.</p

Polybacterial infection enhances atherogenic potential of infected ApoE^-/- mice.

<p>(A). Total serum cholesterol and total serum triglycerides are significantly elevated in infected mice. (B). Chylomicrons and VLDL are significantly elevated in infected mice. (C). Acute inflammatory marker SAA is not significantly elevated in infected mice. (D). Oxidized LDL is significantly elevated in infected mice. (E). Serum nitric oxide is significantly decreased in infected mice. Chol—cholesterol, Trigly—triglycerides, CM—chylomicrons, VLDL—very low-density lipoprotein, LDL—low density lipoprotein, HDL—high density lipoprotein, Cn—control, Ply—poly infection, <i>Pg</i>–<i>P</i>. <i>gingivalis</i> infection, <i>Td</i>–<i>T</i>. <i>denticola infection</i>, <i>Tf</i>–<i>T</i>. <i>forsythia</i> infection, <i>Fn</i>–<i>F</i>. <i>nucleatum</i> infection, OxyLDL—oxidized LDL. n = 6, * p < 0.05, ** p < 0.01.</p

Oral infection schedule and periodontal disease parameters.

<p>(A) Oral infection schedule. (B) Twelve-week infection-induced horizontal alveolar bone resorption was statistically significant on the maxilla palatal and mandible lingual sides. (C) Twenty-four-week infection-induced horizontal ABR was statistically significant on the maxilla palatal, maxilla buccal, and mandible lingual sides. (D) Representative left maxilla lingual view images of horizontal ABR measurements, outlining the area between the cemento-enamel junction (CEJ) and alveolar bone crest (ABC) of molars 1, 2, and 3 of 12- and 24 week <i>F</i>. <i>nucleatum</i>-infected and sham-infected mice. (E) Representative images of gingival histology of 12- and 24 week-infected and sham-infected mice demonstrating minimal gingival inflammation. * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001.</p

Histological analysis of rat periodontal tissue.

<p>Histological analysis of rat jaws (H&E staining). Each molar surface was evaluated for apical migration of JE and the length of migration along the cementum was measured in μm. Vertical bone resorption was also examined by measuring the length in μm from the CEJ to ABC. Polymorphonuclear neutrophils and blood vessels along the interdental area were counted in 50 μm² sections. Asterisks indicate the following <i>P</i>-values when compared to controls *<i>P</i><0.05, ** <i>P</i><0.01.</p