Immunological Activities of Streptococcus mutans KCTC3065 Polysaccharide

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(1)Journal of Dental Hygiene Science Vol. 1, No. 1, pp. 1~6 (2001). Streptococcus mutans KCTC3065 ~~ ' W. ò {. †. fÂ& ~*" Immunological Activities of Streptococcus mutans KCTC3065 Polysaccharide Man-Deuk Han Dept. of Dental Hygiene, Kimcheon College, Kimcheon, Gyeongbuk 740-704, Korea. "#4 #453 53""$5 The oral micro-organism Streptococcus mutans synthesizes extracellular glucosyltransferase(GTFs) which polymerize the glucose moiety of sucrose into glucan polymers. These glucan was known to cause dental caries. The study was designed to evaluate immunoactivities of glucan obtained from the cell wall and extracellular polysaccharides of S. mutans. The anticomplementary activities showed 37.9% at cell wall polysaccharides(CWP) and 21.9% at extracellular polysaccharides(ECP). In examination of macrophage activation, murine macrophage cell line RAW 264.7 cells activated by CWP significantly elevated formation of nitric oxide, but not significantly formated that of ECP. Cell wall polysaccharideactivated RAW 264.7 cells secrete 24.6 µM(p<0.01) of nitric oxide(NO) after 24hr incubation at 37oC. However, ECPactivated macrophages release 4.4 µM of NO. In indirect cytotoxicity assay using ECP and CWP-activated RAW 264.7 culture supernatants, the cytotoxicity against KB cell line(oral epithelial carcinoma) was dependent on amount of nitric oxide production in RAW 264.7 cell culture. The results indicate that S. mutans polysaccharides have different macrophage activation and may play a role for oral hygiene. ,FZ XP XPSE SETT. Streptococcus mutans, Extracellular polysaccharide, Macrophage activation, Anticomplementary activity,. Cytotoxicity. B. ' BRMbº OK-432& ®b, º z º» B ÷Ï R z^ Bö ÆÒWj < ®º bî ôf & Úr . b W~º BRMb VËj ¾æÚº ~º Corynebacterium parvum~ Wª , BCG 5 Norcardia rubra~ ^ã Wª 5 ~~. ~ ® . ß® ~~ (13)-β-D-glucan ~º *Ò ªçöB zj ~ò~º ÒÏ~º º»B β-(13), β-(14), β-(1 6)glucanj "º Ïb ~º PSK, β-(13)~ Ïö β-(16)glucan ªæB schizophyllan, Ò Lentinan ® . *Ò ÒÏ> ®º zº»B & z^ö F' ßW 'Ú ¦·Ï ôf >, ?f BRMf ?"ö ¦·Ïj ~ ¾æÚæ pbB «· j ÛB~º ·Ïj ¾æÚV r^ . ~~ «· ·Ï V*f " &^¾ Úêf &NB ê¢ Wz~ «·Wj ¾æÞ . ß® BRM W ~ WzB &^º «·ZÒ¶(tumor necrosis factor, TNF), interleukin-1(IL-1), interleukin-6(IL-6)" ?f cytokine" nitric oxide(NO) " ?f &Ò>w 7* Öb~ Wj / ê . bîf ?"ö «· Î"¢ ¾æÚæò, / W"Ãöê &~º ·W ® . Hirose f ~"î~ ®º ÷²º nitric oxidef cytokine ªj>Ú ^. . ;~ Úêº ÚW" ^W ê ªB . Ú W f ~f¾ æb¦V ªjB &®Öö ~ Úæ , ^W f " ~f b¦V. ; W.(polymorphonucler leukocyte), ª2, " ?f W.ö ~ Úê . 6 · ;Ú çÒ ^f ÷öW ^~ Ë" .V B÷";j ÛB~º ^' ·Ïj >¯ . ß® Ú~ ªjW Ú(secretory IgA)º ÖW streptococci~ >ÏÚf >w~¾, glucosyltransferasej ÛB~ glucanb Fê>º j & W ~j¦Oj ÛB~º ©b C&r . V¢B ;ö çÒ~º ^ ~ · öf ; W. 5 &^ (macrophage)~ Wz¢ /ê~ ·Ï j Fê~ ¢N~ ' ";j ¢bÎ . ß® êç ³~ ^ ãf ;zB(BRM, biological response modifier) ÒWj ¾æÚº ©b >î . êç ³~ &. 6). 7). 1). 8). 9). 5). 2). 5). 3). 10). 11). 4-5). †. Corresponding author Tel: 054-420-4161 Fax: 054-420-4060 E-mail: [email protected]. 12). .

(2) . Journal of Dental Hygiene Science. 6"ö & OÚ¢ >¯~ "Ã ê¯ö Ö;' j ~º ©b ~& . V¢B ~jÖÃö &~º Streptococcus mutans& W~º ~º BRMb Ú êö ÚÊ 'Ëj ~ºæ jº& ® . æ.ræ S. mutans& W~º ~ö & ' 5 ~jÖ V*f ô Úrb¾ , ; ÚöB ÚÊ ' Wj ¾æÚº º ~ Úææ p~ . V ¢B º S. mutans~ ~¢ Ï~ &^ ¦V nitric oxide W;êf Úê Wj rj, Wz B &^ V·j Ï~ ;çbz ^"ö & ^ ëWÎ"¢ rj¶ . ?f º ;Ú êç W~º ~& ;ç' ;~ãöB~ ·Ï ö ò jî¢ ~"" 5 ~"î~ &~º &^ö ÚÊ Î"¢ ~ºæ rjV * º>, §'bº ; î~ .Oj * £b BBö wÏF > ®j ©b Î ê . 13,14). Òò5O» 1.. þÒò. þö ÒÏ ~º S. mutans KCTC3065öB º ÂÁ;B ©j ÒÏ~& . BRMb ÒÏB & ~ β-glucan f Sigma ÒöB «~& . z^" Væ DMEM(Dulbecco's modified eagle's medium) 5 RPMI 1640 f fetal calf serumf GIBCO(Grand Island, NY, USA) Ò ¦V «®, streptomycin 5 penicillin, NaNO , mitomycin C(from Streptomyces caespitosus) f Sigma Ò(St. Louis, MO, USA) ¦V «~& . 13). . 2. 2.. þÿb 5 «·^. 3.. Ú 5 ^ãW ~~ ªÒ. ÚWj G;j * ÒÏB ÿbf guinea pig â G»ÖöB «~& . ;çbz« KB cellf ^" f¯, RAW 264.7 &^"º ö7&v ~"&b¦V ª·Aj ÒÏ~& . þö ÒÏ S. mutans ~~ ºÂÁB O»f. r" ? . V·b¦V ^W ~(ECP)f ^ã W ~(CWP)~ ºÂ O»f ~ O» j æ;~. ¯~& (Fig. 1). ^W ~~ ºÂ 5 ªÒº r" ? ¯~& . 3¢* V·B 100 ml~ Új 6,000Üg öB öªÒ ê, >³B *bö 20 ml~ salinej I ;~² z Ú >^ r çVf ÿ¢ b öªÒ ~& . f ?f >^O»b 3² >~ áÚê Î ç[ö TCA¢ « ³ê 2.5%& >ê Î&~& . ê ç[ö 70%~ ³ê& >ê öêR(99.9%)j &~ ïËöB ~!ÿn O~~& . O~B Ïf öªÒ (6,000Üg) b ÚW ~ ª³j *V . *B. ~ ª³f Ã~>ö CÎ ê dialysis membrane bag(M.W cut off: 10,000)ö IÚ vº böB 5¢ ÿn RCV . «'b RCB ª³ f 6,000ÜgöB ö ªÒ~ ÿÖb ÚW ~¢ ªÒ~& . 14). 'JH Procedure for purification method of S. mutans KCTC 3065 polysaccharides.. ^ãW ~~ ºÂ";f r" ? . B ÚW. ~¢ áV * 1N ö ªÒ¢ ~ áÚê *bö 100 ml~ Ã~>¢ &~ 3N~ ³ê& >ê NaOH¢ & ~ 12*ÿn v>V . v>B Ïf *çN *ãj Ï~ ^ã~ 2ê¢ { êö acetic acid¢ &~ 7z r, öªÒ~ cell debris¢ B~& . ç[f 3Vï(v/v)~ öêRj &~ ~!ÿn O~B ~¢ *Î ê, ³öªÒV(Beckman XL-90) öªÒ (15,000Üg)~ ^ã Wªj ªÒ~& . öªÒ ê áÚ ê *bf ²ï~ Ã~>¢ & r dialysis membrane bagö IÚ vº böB 5¢ ÿn RCV . RCB ª³. f ÿÖ~ ^ãW ~ ª³j áî .. Ú W. 4.. Ú W(anticomplementary activity)f Yamada ~ O»j æ;~ G;~& . 10 ml þ&ö 150 µl~ GVB jÏÏ(gelatin veronal buffered saline: 0.15 mM CaCl , 0.5 mM MgCl , 1.8 mM sodium barbital, 3.1 mM barbituric acid, 141 mM NaCl 5 0.1% gelatin, pH 7.4)" 250 µg/ml ³ê~ ' ò 50 µl¢ & r, Vö 50 µl (100U/ml)~ guinea pig serumj complement &~ 37 CöB 30ª* >wÎ ê, 4.75 ml~ GVB jÏÏj &~ Ú~ « ³ê¢ 1 unit/ml ;~& . ;B Ú bbj 1.0 unit, 1.2 unit 5 1.6 unit >² ' þ& ö ª" r, anti-sheep hemolysin(2MHU/ml)" ÿï~ SRBC(5Ü10 cells/ml)¢ b~ NöB 30ª* 6· ê, 6·B SRBC 2 ml¢ þ&ö Î&~ « 5 ml& > ê GVB jÏÏb ;~& . ''~ þ&j ¾ b Î ê 37 CöB 60ª* >wV . >wj 7æÊV *~ 70 µl~ 0.5M EDTA Ïj &~ b ê, 400 15). 2+. 2. 2. o. 2+. 8. 2+. o.

(3) Immunological Activities of Streptococcus mutans KCTC3065 Polysaccharide. ÜgöB 5ª* ö ªÒ~ ç[~ 7ê¢ 541 nmöB G;~& . ÚWf & &j C Ú Ï.(50% of total complement hemolysis, TCH )~ &æN(Inhibition of TCH , ITCH ) ¾æÚî . 50. 50. 50. . æ¢ B~ F 0.25% trypsin" 0.02% EDTA Ïj &~ V· flaskj 37 CöB 3-5ª* O~~& . Vö V æ¢ &~ ^¢ >³ r 15 ml~ tubeö IÚ öª Ò(1000 rpm)~ Væ¢ B ê, V· flaskö I F Væ¢ &~ ¦FÎ ê 5% CO ~öB 37 C 48* V·~& . V·B KB cellf >³~ F RPMI Væ 2Ü10 cells/ml ; r 96 wellö ª"~& . ª"B wellö 50 µg/ml 5 10 µg/ml~ S. mutans ~, ECPf CWP Ò WzB RAW 264.7 cell V·bj & ~& . r & £bº mitomycinj 1 µg/ml >ê &~& . WzB RAW 264.7 cell~ V·j ;çbFz ^" KBö 'Ï ê ^ëWf MTT O» b G;~ & . ¯ 96 wellö V·B ^ ç[ ªÒ ' wellö 10 µl MTT solution(5 mg/ml)j IÚB 37 CöB 4* O ~~& . V·j ªÒ 100 µl DMSO(100%)j IÚB debris& j*® j rræ microplate reader(Ceres UV900 Hbi, USA)öB 5ª* shaking ê 540 nmöB 7ê¢ G;~& . o. o. 2. ITCH50 (%) = TCH50 of control-TCH50 treated with sample TCH50 of control. Ü100. ~ G;. 5. Nitric oxide(NO) RAW 264.7 NO , , 10% heat inactivated fetal bovine serum(FBS), 40 mM , (100 µg/ml) (100U/ml) 0.37% NaHCO3 16) . DMEM ECP CWP RAW 264.7 S. mutans . RAW 264.7 Colstar 6 petri-dish 2 10 5% CO2 37oC . RAW 264.7 37oC DMEM , . RAW 264.7 tryphan blue 95% . hemocytometer 2 106 cells/ml 37oC 24 well 1 ml 90 5% CO2 . 90 petri dish DMEM 950 µl well . RAW 264.7 ECP CWP DMEM 10 µg/ml 50 µg/ml 50 µl well . 10 IU/ml IFN-γ, 1 µg/ml lipopolysaccaharide(LPS from E. coli) . 24 well 5% CO2 o 37 C 24 . 24 RAW 264.7 nitric oxide . Ding 17) 100 µl Griess (1% sulfanilamide/ 0.1% N-(1-naphthyl)-ethylenediamine dihydrochloride/2.5% H3PO4) , ELISA reader − sodium nitrate(NaNO2) 540 nm . NO 2. ^¦V ¢ G;~V *B ^"º î ²ö &~ þö ÒÏ ^º &æ" ÊÞ?Æî 5 ¾î Ö Ò ~ j F VæöB ê&V·~& ?f Væö öB ªÒB f j Ï ^~ Wf r" ?. ^º ö Ü ~ ^& >ê ª"~ ~ ¢ /~ öB *V·~& V·B ^¢. þ* Fæ>º F Væ >^ r >³~& >³B ^º "ï ~ ~ ^Nj &î r þö Ï~& ^ >º ¢ Ï~ Ü ; ê ~öB ö O ª"~ ª* V·~& ª ê ö ¦O>æ pf ^º B ~ F Væ ¢ ''~ ö ª "~&. ^¢ Wz~V * f ¢ Væö f >ê B~ O ' ö Î&~& ·W&öº ~ ~ ¢ ~öB Î&~& ò& Î&B ''~ f * V·~&. * V·B ^¦V B>º ~ ;ïf ~ O»ö V¢ ¯~& *Û® J« ~ ~ V·" ÿï~ £ j D NöB O~ ê ¢ Ï öB G;~& ~ ³êº ¢ ³êê C~ 7ê¢ G;~ &Fj ·W~ áî . Nitric oxide~ Wïf ' òî 7þb 3² > ê Ûê ¾Ò~ ÖÂ~& .. ~ WzB RAW 264.7 &^ " V·ö ~ ;z ^ ëW ECPf CWP WzB RAW 264.7 ^~ V· &ÒÖ b ;çbFz ^" KB cellö & ^ëWj rj º ";f r" ? . ; z^ ëWj rjV * ÖF'b nitric oxide~ G;O»" ÿ¢~² ''~ ò RAW 264.7 ^¢ Wz~& . ;çbFz ^" KB cellf 10% heat inactivated fetal bovine serum(FBS)j & RPMI1640öB V·~& .. þö ÒÏF ^~ ê&V· O»f r" ? . ÖF V. 5. 18). o. 7.. Ûê ¾Ò. þ~ Ö"º 3²~ > þj Û áf >~¢ Ûê¾ Ò~ means Û S.E¢ ®, F~W~ {f Student's ttest ªC~& . Ö"5V. Ú W(anticomplementary activity) Okuda f ~ö ~ ÚÚ Úê Wj Û~ zW"~ ç&&ê& ® : ® . V¢B S. mutans KCTC3065 ~~ ÚW W ;ê¢ {~ V * Ú Wþj >¯~& . j V·~ ºÂ ª ÒB CWPf ECP ~~ ÚW ;êº Fig. 1" ?. . Ú Wf &~ total complement hemolytic (TCH 50)ö & &N(%) V~& . þ Ö" S. 1.. 19). 6. S. mutans. Anticomplementary activity of the polysaccharides obtained from S. mutans KCTC 3065. Each bar represents the mean S.E of three independent experiments done in duplicate. ECP; extracellular polysaccharide, CWP; cell wall polysaccharide from S. mutans. 'JH . Û.

(4) Journal of Dental Hygiene Science. . ^W ~(ECP)¢ 60 µg/mlj R~&j r Ú WÎ"º 21.3Û2.1% îb, ^ãW ~ (CWP)º 37.9Û2.1%~ Ú Wj ¾æÚî . ?f Ö"º & β-glucan 20.9Û1.4%~ Ú Wj ¾ æÞ © ECPº 0.4%, CWPº 17%~ Ú W Î" & Ö>~² ¾æÒ . ß® CWPº ECP Ú W Ö>~& . mutans. &^~ nitric oxide(NO) WË f ~ bî f &^¢ Wz~ Væ Úö ¢ W~º ©b rJrb , Asai f öB ºÂB zW .B OK-432& &^~ NO synthase B*j Fê~ º ©b : ® . öBº Öö S. mutans KCTC3065¦V ºÂB ~ ª³ &^¢ WB, NO Wj Fê~ºæ {~& . S. mutans KCTC3065¢ V·~, V·ÒÚ~ ^W. ~¢ RAW 264.7 ^ V·ö 10 µg/ml 6º 50 µg/ml O R~&j r Fig. 3" ? & NO~ Wj /êV . ¯, RAW 264.7 ^ö ¶bj R~æ pf rW&öBº 4.5Û16 µM~ NOj W~&b¾, ECP. ~ 10 µg/ml¢ R öBº 4.4Û1.5 µM, CWPº 24.8 Û2.8 µM~ NO¢ '' W~ &^~ W Ö>~&. . 6 50 µg/mlj R ^öBº ECP~ ãÖ 20.1Û 1.5 µM, CWPº 44.4 µM~ NOj '' W~& . r · W &b Ï IFN-γ~ ãÖ 10IU/mlj R~&j r 14.4Û2.1 µM, E. coli~ LPS¢ 1 µg/mlj R ^ö Bº 38.8Û3.1 µM~ NO¢ W~& . Ú ~ &Ú NO~ Wj &Ë ô FêÎ ©f 50 µg/ml~ CWP¢ R VæB 44.4 µM~ NO¢ W 2.. IFN-γ LPS NO2− Streptococcus sp. RAW 264.7 murine. 20,21). 22). V . ~ ö ~~ CWPº S. mutans KCTC 3065~ ^ãW ~B £ 70 kDa¢ " peak ~º ª¶W, C ê>zbf 48%, Wî 43.5% W> îb, gas chromatography ªC Ö", W~ jN glucosef galactose& 62.2f 38.9% Úê ~î. . &^ö & ÒW ~~ NO W /ê Î "º CWPf ?f j>ÏW ~& >ÏW ECP Ö >~& . ?f Ö"º ~& &^¢ ÎN'b ¶ ~V *Bº öW~ V²& ôf ª¶ j &ÏW bîö ~ &^~ W /ê>º ©b Îê . 13). 3.. ;çbz ^" KB cellö & ^ëW Î". b¾ b Öbö & ?"~ >w "; 7 .V >wf ö~ epitopeö & ß' >wb · ~æò, >>>º >wf jß' >wb >w~ ö bî öò jî¢ b 6º z^¢ ÚÊ º V* ¢ÚÂ . ?f >w &Ú &^& Wz¢ Fê~º bîº LPSf ?f ^~ ^ã Wª, Ò IFN-γf ?f cytokine &' . . Decker f WzB &^º ^ ªjW «·ZÒ¶(TNFα)¢ W~, º z^ö ^ëWj ¾æÚ, &^ ~ L-jVò ~W &ÒÖb nitric oxide(NO)¢ W~ º ©b ~& . NOº * Ò~º 7* &Òb î z^~ Æ~Òj ^ê¢ N º Hibbs ~ B ê ôf & Ú rb, Asai f P815 « ·^"¢ ÚÊV *Bº 80 µM~ NO ³ê& ºB. ~& . öB S. mutans~ ~ WzB &^~ V ·b ;z ^"ö & ^ëWj {~& . ¯ zWj ¦ï~V * S. mutans ~¢ RAW263.7 & ^"ö R~, V· 10%¢ ;z ^" KB cellö 'Ï~& . ECP~ ãÖ 50 µgb R ³êöBº 18.5%~ ^ëWj &b¾, 10 µg~ ³êöBº 5.6%~ ^ëWj ¾æÚî . >ö CWPº 50 µg/ml RB ³ êöB 34.4%, 10 µg/ml~ ³êöB 12.7%~ ^ëWj ¾ æÚî . & £b zB mitomycin~ ãÖ, 1 µg/mlö B 55.3%~ ^ëWj ¾æÚîb, interferon-γf LPS WzB VæöBº 10% Ú~ ^ëWj ¾æÚî . «'b " r B þ nitric oxide~ WË Ö >~&~ CWP& KB cellö & ^ëWj ¸² ¾æÚî. . ?f Ö"º Higuchi &^~ ^ëW V* ö & öB «·ZÒ¶f nitric oxideº «·^ö & "º ^ëW ¶ ·Ï º þ' Ö"f FÒ ~² ¾æÒ . S. mutans~ ~ö ~ &^& Wz>º z' V*bº Leif Morrison B © ¾" B â*, T â* 5 &^öº LPS-binding protein Ò~, β-glucan, òJ®, ê >ÏÚ & ®Ú, bîö ~ &^º Wz>º ©¾" S. mutans~ glucanö & RAW 264.7 ^º >ÏÚ& Ò ~º ©b Îê . " Nature æö B Brownf Gordon~ ¢^ö ~~ &^f Væ W. öº zWj ¾æÚº β-glucanö &~ Ú >ÏÚ 3(com23). 24). 25). 26). 27). 28). Ü. Nitric oxide formation of RAW 264.7 macrophage activated by S. mutans KCTC3065. RAW 264.7 cells(2 106 cell/ml) were cultured for 24hrs in DMEM with 10 µg/ml and 50 µg/ml of polysaccharides. The supernatant were collected and assayed for nitrite production. Each bar represents the mean S.E of three independent experiments done in duplicate. Con; control, ECP; extracellular polysaccharide, CWP; cell wall polysaccharide from S. mutans, IFN-γ; interferon-γ, LPS; lipopolysaccharide. 'JH . Û.

(5) Immunological Activities of Streptococcus mutans KCTC3065 Polysaccharide. . b R ³êöBº 18.5%~ ^ëWj &b, 10 µg~ ³êöBº 4.5%~ ^ëWj ¾æÚî . > ö CWPº 50 µg/ml RB ³êöB 34.4%, 10 µg/ ml~ ³êöB 12.7%~ ^ëWj ¾æÚî . 4. CWPº ECP Ú W" RAW 264.7 ^¦ V nitric oxide~ W ô~b, KB ^"ö & *7' ^ëWöê ¸² ¾æÚî . ç" ?f Ö" j S. mutans& W~º ~ f ~jÖÃj ¢bÊº *W ~¾, Ú~ &^ W 5 Ú W " ?f ";j Û /W"Ã> wö &~, ~"÷ 5 ;z B j ÛB~º 3'' j > ®º ©b ÒòB . Indirect cytotoxicity effect of S. mutans polysaccharide against KB cell line, oral epithelial carcinoma. The RAW 264.7 macrophage(2 106) were cultured for 24hrs containing 50 µg/ ml and 10 µg/ml of polysaccharides from S. mutans KCTC3065. The cultured medium supernatant of RAW 264.7 cell was collected and added 10%(v/v) of the supernatant to the culture medium of KB cell line(2 105 cells/ml in RPMI). After culture for 48hrs, the cytotoxicity against KB cells was assayed by MTT. Each bar represents the mean S.E of three independent experiments done in duplicate. The significance of differences as compared with the control. *: P<0.01. MMC; mitomycin, ECP; extracellular polysaccharide, CWP; cell wall polysaccharide from S. mutans, IFN-γ ; interferon-γ, LPS; lipopolysaccharide. 'JH . Ü. Ü. Û. " dectin-1¢º >ÏÚ& Ò~ W z>º ©b C&r . ß® Hirose ö ~~, ~"î~ ~ "º ÷ö Porphyromonas gingivalisf Actinobacillus actinomycetemcomitansº ²rWb < ®º Úë²(LPS)ö ~ ~"" ê¯ &^¢ Wz~ cytokine(IL-1α, IL-8, IL-10)f þ ï~ nitric oxide ~ OÂ~º ©b >î . Ö" S. mutans j LPSf ?f Úë²¢ W~æ pæò, W~º ÚW 6º ÚW ~ &^¢ Wz ~ nitric oxide~ OÂ~& . V¢B S. mutans~ ~ º j ~jÖÃ Bö Ö;' j ~¾, Nz &^ Wö ~ ;z ö & z·Ï" ~f 5 ~" "Ã>wö 3''b ·Ï &ËW ® . plement receptor 3). 29). 12). º. £. S. mutans KCTC3065~ ÚöB W>º ~ &Ú ^W ~~ &æ Ò' W Î"º r" ? . 1. Ú Wf ^W ~ ª³(ECP) 21.3% îb, ^ãW ª³ ~(CWP)º 37.9% î . 2. &^ RAW 264.7 ^" V·ö 10 µg/ml O R ~&j r, ECP ~~ ãÖ 4.4 µM~ NO¢ W Î >, CWPº 24.6 µM~ NO¢ W~ &^ ~ NO WW ECP Ö>~& . 3. S. mutans ~ WzB &^ V·~ ;z ^"ö & indirect cytotoxicityº ECP~ ãÖ, 50 µg. ^^ò 1. Garside P, Mowart AM: Oral tolerance. Semin Immunol 13(3): 177-185, 2001. 2. Nakahara H, Sato EF, Ishisaka R, Kanno T, Yoshioka T, Yasuda T, Inoue M, Utsumi K: Biochemical properties of human oral polymorphonuclear leukocyte. Free Radic Res 28(5): 485-495, 1998. 3. Jespersgard C, Hajishengallis G, Greenway TE, Smith DJ, Russell MW, Michalek SM: Functional and immunogenic characterization of two cloned regions of Streptococcus mutans glucosyltransferase I. Infect Immun 67(2): 810-816, 1999. 4. Regan DR, Cohen PL, Cromatie WJ, Schwab JH: Immunosuppresive macrophage induced by arthropathic peptidoglycan-polysaccharide polymers from bacterial cell wall. Clin Exp Immunol 74(3): 365370, 1988. 5. Franz, G: Polysaccharides in pharmacy: current applications and future concepts. Planta Med 55: 493-497, 1989. 6. Ujiie T: Chemoimmunotherapy of L1210 leukemia with adriamycin, cyclophosphamide, and OK-432, and their effects on the generation of antitumor immunity. Jpn J Cancer Res 78(7): 737-747, 1987. 7. Purnell D, Bartlet G, Kreider J, Biro T: Corynebacterium parvum and cytophosphamide as combination treatment for a murine mammary adenocarcinoma. Cancer Res 37: 1137-1140, 1977. 8. Yamaura Y, Sakatani M, Ogura T, Azuma I: Adjuvant immunotheraphy of lung cancer with BCG cell-wall skeleton (BCG-CWS). Cancer 43: 1314-1319, 1979. 9. Kagawa K, Yamashita T, Tsubura E, Yamamura Y: Inhibition of pulmonary metastasis by Norcardia rubra cell wall skeleton, with special reference to macrophage activation. Cancer Res 44: 665670, 1984. 10. Franz G: Polysaccharides in pharmacy: current applications and future concepts. Planta Med 55: 493-497, 1989. 11. Suzuki I, Hashimoto K, Okikawa S, Sato K, Osawa M, Yadomae T: Antitumor and immunomodulating activities of a β-glucan obtained from liquid-cultured Grifola flondosa. Chem Pharm Bull 37: 4010-4013, 1980. 12. Hirose M, Ishihara K, Saito A, Nakagawa T, Yamada S, Okuda K: Expression of cytokines and inducible nitric oxide in inflamed gingival tissue. J Periodontol 72(5): 590-597, 2001. 13. Wiater A, Choma A, Szczodrak J: Insoluble glucans synthesized by cariogenic streptococci: a structural study. J Basic Microbiol 39(4): 265-273, 1999. 14. Han MD, Lee JW, Ra SJ, Lee ES, Jeon ES: Chemical properties of Streptococcus mutans KCTC 3065 polysaccharide purified by fraction. J Korean Acad Dent Health 24(3): 259-270, 2000. 15. Yamada H, Kiyohara H, Cyon JC, Kojima Y, Kumazawa Y, Otsuka: Studies on polysaccharides from Angelica acutiloba. Planta Med 50: 163-167, 1984. 16. Kwon NS, Steuhr DS, Nathan CF: Inhibition of tumor cell ribonucleotide reductase by macrophage-derived nitric oxide. J.

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