日本睡眠歯科学会口腔内装置診療ガイドライン作成委員会の活動報告

Oral appliance therapy was approved by national health insurance in Japan in 2004 and oral appliances（OAs）have since been widely used in the treatment of obstructive sleep apnea（OSA）. We herein described the process of making clinical practice guidelines by the task force of the Japanese Academy of Dental Sleep Medicine as a work report. In Japan, OAs are covered by national health insurance. In consideration of the balance between medical treatment fees and the price of technical materials, we used a single-piece（monoblock）OA that advanced the mandible forward and limited mouth opening in OSA patients in Japan. The Japanese Academy of Dental Sleep Medicine（JADSM）focused on OAs frequently used for the treatment of OSA in Japan, and considered an evaluation of their effects to benecessary. Clinical practice guidelines were developed using the Grading of Recommendations, Assessment, Development, and Evaluation（GRADE）system. We recommend OAs that advanced the mandible forward and limited mouth opening for patients with OSA.However, CPAP should be used by patients for whom it has been indicated. OAs are desirable for those who cannot use CPAP（GRADE 1B, strong recommendation/quality of evidence, “Moderate quality”）. The long-term effects and side effects, OSA severity, and comorbidities of OA therapy were not examined, which represented a limitation to the present study. In future studies, the Japanese Academy of Dental Sleep Medicine plan to update clinical practice guidelines for oral appliances used in OSA

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<p><i>C</i>. <i>ochracea</i> ATCC27872 (A) and TmAI2 (B) were incubated anaerobically on coverglasses in 12-well polystyrene plates for 48 h. Representative images of the biofilms are shown at the indicated magnifications. Arrowheads indicate partially vacant spaces.</p

Quantitative analysis of the biofilm structure of <i>C</i>. <i>ochracea</i>.

<p>The height (A) and % volume occupied by <i>C</i>. <i>ochracea</i> in total volume (B) are shown. The height was evaluated by using Zen 2009 software and the volume by use of IMARIS software. Data are presented as means ± SD of OD₄₀₅. The valuation was performed at six points. *, <i>P</i> < 0.05 compared to wild type.</p

Growth curves of the <i>C</i>. <i>ochracea</i> wild-type and TmAI2 strains.

<p><i>C</i>. <i>ochracea</i> ATCC27872 and TmAI2 were cultured in TS broth at 37°C, under anaerobic conditions, and their growth in 1.0 x TS broth (A) and 0.5 × TS broth (B) was measured as OD₆₆₀. Data are means ± SD (n = 3).</p

Adherence of <i>C</i>. <i>ochracea</i> to polystyrene plates.

<p><i>C</i>. <i>ochracea</i> ATCC27872 and TmAI2 were incubated in 96-well polystyrene plates for 1 h. Adherence was measured at OD₄₀₅ with an ELISA-based assay. Data are presented as means ± SD (n = 12).</p

Schematic of inactivation and complementation of <i>luxS</i>.

<p>(A) Inactivation of <i>luxS by</i> using an <i>ermFermAM</i> cassette. The sequences that flanked the 5′- and 3′- ends of <i>luxS</i> (Coch_1216) were amplified with primers Capno1 and 2, and primers Capno3 and 4, respectively. The <i>ermFermAM</i> cassette was inserted between the amplified fragments and cloned. The plasmid was linearized and introduced into <i>C</i>. <i>ochracea</i> ATCC 27872 by electroporation. The resultant <i>luxS</i>∷<i>ermFermAM</i> strain was named TmAI2. (B) Inactivation of <i>luxS by</i> using <i>tetQ</i>. The <i>tetQ</i> fragment was inserted between the sequences that flanked the 5′- and 3′- ends of <i>luxS</i> by using the PCR-based overlap extension method and self-ligattion of the fragment, and then amplified in <i>E</i>. <i>coli</i>. The resultant plasmid was linearized and introduced into <i>C</i>. <i>ochracea</i> ATCC 27872 by electroporation. The resultant <i>luxS</i>∷<i>tetQ</i> strain was named LKT7. (C) Complementation of <i>luxS</i>. An <i>ermF-luxS</i> fragment was constructed by using the PCR-based overlap extension method to express both genes under control of the <i>ermF</i> promoter, and the fragment was cloned into the center of the <i>tetQ</i> gene in pAITQ. The resultant plasmid was linearized and introduced into <i>C</i>. <i>ochracea</i> LKT7 by electroporation. The resultant <i>tet</i>∷<i>ermF-luxS</i> strain was named luxS-C3.</p

AI-2 production from <i>C</i>. <i>ochracea</i>.

<p>AI-2 levels are expressed as the ratio of the <i>V</i>. <i>herveyi</i> bioluminescence intensity after addition of culture supernatant to the intensity obtained after addition of TS broth alone. 27872; <i>C</i>. <i>ochracea</i> ATCC 27872 (wild type), TmAI2; <i>C</i>. <i>ochracea</i> TmAI2. *, <i>P</i> < 0.05 compared with TmAI2.</p

Effect of extrinsic AI-2 on biofilm formation by the <i>luxS</i>-deficient mutant and wild-type strain.

<p>(A) Assessment of biofilm formation by mutant and wild-type strains by using a two-compartment system. <i>C</i>. <i>ochracea</i> ATCC 27872 and TmAI2 were inoculated into TS broth in the indicated compartments (upper or lower well) in the wells of a 12-well polystyrene plate, and were then incubated for 48 h. Biofilm formation by the strain in the lower compartment of each well was stained with crystal violet staining. After rinsing and ethanol extraction, the mass of biofilm was quantified as OD₅₉₅ of extracted stain. Data are presented as means ± SD (n = 10). *, <i>P</i> < 0.05 compared with the biofilm formation by <i>C</i>. <i>ochracea</i> ATCC 27872 in the lower compartment with <i>C</i>. <i>ochracea</i> ATCC 27872 in the upper compartment. (B) Effect of mixed-culture on biofilm formation by mutant and wild-type strains. <i>C</i>. <i>ochracea</i> ATCC 27872 and TmAI2 were inoculated into the wells of a 96-well plate, incubated for 48 h, and biofilm formation was stained with crystal violet staining. After rinsing and ethanol extraction, the mass of biofilm was quantified as OD₅₉₅ of extracted stain. The data are presented as means ± SD (n = 21). * <i>P</i> < 0.05 compared to <i>C</i>. <i>ochracea</i> ATCC 27872 alone. (C) Effect of wild-type culture supernatant of the wild-type strain on biofilm formation by <i>C</i>. <i>ochrachea</i>. <i>C</i>. <i>ochracea</i> ATCC 27872 or TmAI2 cells were inoculated into the wells of a 96-well plate, and culture supernatant of <i>C</i>. <i>ochracea</i> ATCC 27872 was added. The plates were incubated for 48 h anaerobically, and then biofilm formation was stained with crystal violet staining. After rinsing and ethanol extraction, the mass of biofilm was quantified as OD₅₉₅ of extracted stain. Data are presented as means ± SD (n = 6). * <i>P</i> < 0.05 compared with <i>C</i>. <i>ochracea</i> ATCC 27872 alone. (D) Effect of <i>luxS</i> mutant culture supernatant on biofilm formation by <i>C</i>. <i>ochrachea</i>. One hundred microliters of <i>C</i>. <i>ochracea</i> ATCC 27872 or TmAI2 were inoculated into the wells of a 96-well polystyrene plate, and culture supernatant of <i>C</i>. <i>ochracea</i> TmAI2 was added. Then the plates were incubated for 48 h anaerobically, and biofilm formation was stained with crystal violet staining. After rinsing and ethanol extraction, the mass of biofilm was quantified as OD₅₉₅ of extracted stain. Data are presented as means ± SD (n = 6). * <i>P</i> < 0.05 compared with <i>C</i>. <i>ochracea</i> ATCC 27872 alone.</p

Effect of <i>luxS</i> complementation on biofilm formation by <i>C</i>. <i>ochracea</i>.

<p><i>C</i>. <i>ochracea</i> ATCC27872, LKT7 (<i>luxS</i>∷<i>tetQ</i>), and luxS-C3 (<i>luxS</i> complemented strain) were incubated in TS broth for 48 h under anaerobic conditions. Biofilm formation was then assayed by means of crystal violet staining. Data are presented as means ± SD (n = 6) *, <i>P</i> < 0.05 compared with wild type, § <i>P</i> < 0.05 compared with the LKT7.</p

Biofilm formation by <i>C</i>. <i>ochracea</i> wild type and TmAI2.

<p><i>C</i>. <i>ochracea</i> ATCC27872 and TmAI2 were incubated in (A) 1.0 x TS broth for 24 h, (B) 1.0 x TS broth for 48 h, (C) 0.5 × TS broth for 24 h, or (D) 0.5 × TS broth for 48 h, in a 12-well plate under anaerobic conditions. Biofilm formation was then assayed by crystal violet staining. Data are presented as means ± SD (n = 10). *, <i>P</i> < 0.05 compared with wild type.</p