Effect of lipopolysaccharide derived from surabaya isolates of Actinobacillus actinomycetemcomitans on alveolar bone destruction

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1 Veterinry World, EISSN: Aville t RESEARCH ARTICLE Open Aess Effet of lipopolyshride derived from sury isoltes of Atinoillus tinomyetemomitns on lveolr one destrution Rini Devijnti Ridwn 1, Sidrningsih 1, Tuti Kusumningsih 1 nd Shermn Slim 2 1. Deprtment of Orl Biology, Fulty of Dentl Mediine, Universits Airlngg, Sury, Indonesi; 2. Deprtment of Prosthodonti, Fulty of Dentl Mediine, Universits Airlngg, Sury, Indonesi. Corresponding uthor: Rini Devijnti Ridwn, e-mil: rini-d-r@fkg.unir..id Co-uthors: Sidrningsih: sidrningsih@fkg.unir..id, TK: tuti-k@fkg.unir..id, SS: shermn-s@fkg.unir..id Reeived: , Aepted: , Pulished online: doi: /vetworld How to ite this rtile: Ridwn RD, Sidrningsih, Kusumningsih T, Slim S (2018) Effet of lipopolyshride derived from sury isoltes of Atinoillus tinomyetemomitns on lveolr one destrution, Veterinry World, 11(2): Astrt Bkground: Atinoillus tinomyetemomitns lipopolyshride (LPS) hs high virulene ftor. It interts with serum protein through reeptors on the epithelil ell surfe, therey inresing oth interleukin (IL)-1β, nd IL-6 whih results in dmge to periodontl tissue. Aim: The im of the study ws to identify nd evlute the effet of LPS derived from lol isoltes (A. tinomyetemomitns) on the destrution of lveolr one y mens of severl iomrkers, inluding; the numer of osteolsts nd osteolsts, the expression of IL-6, mtrix metllopeptidse 1 (MMP-1), nd reeptor tivtor of nuler ftor kpp-β lignd (RANKL). Mterils nd Methods: The isoltion of LPS from A. tinomyetemomitns ws lulted using phenol, while purifition ws performed using Sephdex C-18 olumn hromtogrphy. 40 Wistr rts were divided into four groups of 10. Eh tretment ws divided into two groups whih were 0.9% NCl nd LPS indued for 7 nd 14 dys, respetively. Gingivl nd lveolr ones were further introdued into the indution re, followed y the mesuring of osteolst nd osteolst with hemtoxylin-eosin stining, IL-6, MMP-1 nd RANKL expression with immunohistohemil. Results: Redued numers of osteolsts t the 7 th nd 14 th dy of tretment were deteted, while those of osteolsts inresed. There ws n inresed expression of IL-6, MMP-1, nd RANKL in the 7 th nd 14 th -dy tretment group. Tretment of LPS from A. tinomyetemomitns over 7 nd 14 dys resulted in dmge to periodontl tissue nd lveolr one in Wistr rts. Conlusion: LPS of A. tinomyetemomitns dministrtion for 7 nd 14 dys uses periodontl nd lveolr tissue destrution in Wistr rts. Keywords: Atinoillus tinomyetemomitns lol isolte, lipopolyshride, interleukineil-6, mtrix metllopeptidse-1, reeptor tivtor of nuler ftor kpp-β, reeptor tivtor of nuler ftor kpp-β lignd. Introdution Lipopolyshride (LPS) is omponent of A. tinomyetemomitns forming its ell wll s virulene ftor. LPS interts with serum protein through reeptors on the epithelil ell surfe [1]. The high onentrtion of LPS inreses interleukin (IL) - 1β nd IL-6 whih, in turn, results in the destrution of periodontl tissue due to ytokine response in the epithelium, neutrophil, firolst, nd monoyte. The proess ours through host stimultion nd plys n importnt role in tissue dmge y stimulting the mrophge to relese (IL-1), IL-1β, nd tumor nerosis ftor (TNF). These pro-inflmmtory ytokines re responsile for using one dmge [2]. The LPS interferes with the homeostsis of ollgen metolism through ollgen phgoytosis through Copyright: Ridwn, et l. Open Aess. This rtile is distriuted under the terms of the Cretive Commons Attriution 4.0 Interntionl Liense ( y/4.0/), whih permits unrestrited use, distriution, nd reprodution in ny medium, provided you give pproprite redit to the originl uthor(s) nd the soure, provide link to the Cretive Commons liense, nd indite if hnges were mde. The Cretive Commons Puli Domin Dedition wiver ( retiveommons.org/pulidomin/zero/1.0/) pplies to the dt mde ville in this rtile, unless otherwise stted. firolst. A study y Shddox et l. [3], found tht LPS stimultes the synthesis of osteolsti IL-1β, TNFα, IL-6, nd reeptor NF-кB lignd (RANKL). The virulene potentil of the A. tinomyetemomitns might e different. Speifi Polyshride Antigen (SPA), serotype of A. tinomyetemomitns is responsile for the resistne mehnism to phgoytosis nd kills polimorfonuler leukoyte in humns [4]. Tkhshi et l. [4] reported tht the SPA of serotype nd indues IL-1 relese y murine s mrophge whih is lower thn the SPA of A. tinomyetemomitns serotype. Menwhile, Ptil et l. [5] demonstrted the higher sensitivity of vrious ntimiroils in other serotypes ompred to tht of A. tinomyetemomitns. Some kinds of yests serete toxins nd these types lled killer yests. These yests n inhiit the growth of other yest strins nd lso hve ntimiroil tivities inhiiting growth of teri [6,7]. The ims of this study were to determine the effet of LPS in lol isolte of A. tinomyetemomitns, s hrterized y severl mrkers of dmged lveolr, through the mesuring of osteolst nd osteolst, s well s IL-6, Mtrix Metllopeptidse 1 (MMP-1) nd RANKL expression. Veterinry World, EISSN:

2 Mterils nd Methods Ethil pprovl This study reeived n ethil lerne pprovl letter relting to humn sujets from the Ethis Reserh Committee of Fulty of Dentl Mediine, Universits Airlngg, with numer 29/KKEPK/ FKG/III/2015. The reserh onstituted Anlytil Oservtionl involving the use of n Experimentl Lortory nd ross-setionl method. A. tinomyetemomitns lol isolte preprtion A. tinomyetemomitns ws otined from ptients with ggressive periodontitis nd further suultured in Luri Berthni (Merk KgA, Drmstdt, Germny) for 2-3 dys to otin its morphology (visulized in 2 pltes). An A. tinomyetemomitns ulture of 200 µl ws required for four niml model groups (40 rts in totl); therefore, 10 ml ulture ws stored t density of 10 8 ells every dy for period of 14 dys. 200 µl LPS of A. tinomyetemomitns ws produed ontining 200 µg/ml proteins to indue sulus of the mxill molr of Wistr rts over 7 th dy to 14 th dy. LPS ws isolted nd purified following Westphl nd Jnn s [6] phenoli-sed method. The resulting purified rude LPS nd isolte were susequently gel-filtrted in Sephdex C-18 (Sigm Aldrih, Drmstdt, Germny) t room temperture with disggregtion uffer s solvent (0.05 M Tris-HCl ph 9, M EDTA, nd 0.3 deoxyholte) (Sigm-Aldrih, Drmstdt, Germny). Animl model study preprtion The niml model used onsisted of Wistr rts divided into four groups eh ontining 10 rodents. Group 1 onstituted ontrol group, indued with NCl 0.9% for 7 dys Group 2 reeived tretment with LPS for 7 dys. Group 3 ws indued with NCl 0.9% for 14 dys, while Group 4 underwent tretment with LPS for 14 dys [4,5]. Tretment ws injeted into the sulus of the first mxillry molr (M1) following Dumistresu s method [8,9]. Eh group ws divided into two sugroups, one treted for 7 dys, the other for 14 dys. Injetions of 200 µg of LPS, with protein level of 200 µg/ml, nd density of 108 were dministered for t lest 7 dys to otin signifint ggressive periodontitis symptoms [9]. Gingivl nd lveolr ones were susequently exmined into the indution re, followed y the mesuring of osteolst nd osteolst with hemtoxylin-eosin (HE) stining, nd IL-6, MMP-1, nd RANKL expression with n immunohistohemil mrker kit (Merk KgA, Drmstdt, Germny). At the next immunohistohemil exmintion on glss ojet with 50% glyerin dropped ove the glss ojet nd viewed the results with n Eletron Mirosope (Automted Eletron Mirosope, Olympus, USA). Dt in norml distriution were nlyzed using Shpiro-Wilk test (p>0.05). Sttistil nlysis Dt homogeneity ws nlyzed using ANOVA (p<0.05). Sttistil nlysis ws effeted y mens of Sttistil Pkge for the Soil Sienes (SPSS) 17.0 softwre for windows 8.1 y SPSS In., Chigo, United Sttes. Results Mesurement of osteolst nd osteolst The mesurement of osteolst from lveolr ones on dy 7 showed dt were normlly distriuted nd homogenous with signifint vlue of p>0.05. ANOVA nlysis showed there ws onsiderle differene on dy 7 in the osteolst etween the tretment group 16.5±3.84 nd the ontrol group 5±0.82 (p<0.05). Mesurement of osteolst from lveolr ones rried out on dy 14 showed ws normlly distriuted nd homogenous p>0.05. ANOVA nlysis showed there ws mrked differene in osteolst etween the tretment group 22.6±2.41 on dy 14 nd tht of the ontrol group 5.8±1.48 (p<0.05). The mesurement of osteolst from lveolr ones on dy 7 showed dt to e normlly distriuted nd homogenous p ANOVA nlysis showed there ws no stle ontrst in osteolst etween the tretment group on dy 7 nd the ontrol group (p<0.05). The mesurement of osteolst from lveolr ones on dy 14 onfirmed tht dt were normlly distriuted nd homogenous with signifint vlue of p ANOVA nlysis showed there ws onsiderle differene in osteolst etween the tretment group on dy 14 nd the ontrol group (p<0.05). A grphi of the verge lveolr osteolst on dy 7 showed 15 ells in the ontrol group nd 7.4 ells in the tretment group. The mesurement of osteolst from gingiv tissue on dy 7 onfirmed there to e 5 ells in the ontrol group nd 16.5 ells in the tretment group. Osteolst mesurements tken from the lveolr on dy 14 showed tht osteolst represented 22.6 ells in the ontrol group, ut only 4.5 ells in the tretment group. The mesurement of osteolst-derived from gingiv tissue on dy 14 estlished the presene of 5.8 ells in the ontrol group, in ontrst to the 22.6 ells in the tretment group. The verge presene of osteolst nd osteolst in the lveolr one n e seen in Figure-1, while the results of HE stining of gingiv tissue re shown in Figure-2. Expression of IL-6, MMP-1, nd RANKL The expression of IL-6 in gingivl tissue during dy 7 showed dt were normlly distriuted nd homogenous (p>0.05). ANOVA nlysis reveled signifint differene of IL-6 expression etween the dy 7 tretment group 15.8±0.79 nd the ontrol group 4.7±1.16 (p<0.05). The expression of IL-6 from gingivl tissue undergoing dy 14 tretment showed the dt to e normlly distriuted nd homogenous (p>0.05). ANOVA nlysis negted ny signifint differene of IL-6 expression etween Veterinry World, EISSN:

3 the dy 14 tretment group 2.1±0.74 nd the ontrol group 1.9±0.74 (p<0.05). The verge IL-6 expression in gingivl tissue is shown in Figure-3, while the result of IHC stining of Wistr rt gingiv fetures in Figure-4. The dt of MMP-1 expression in gingiv in dy 7 tretment were normlly distriuted nd homogenous (p>0.05). ANOVA nlysis onfirmed onsiderle differene in IL-6 expression etween the dy 7 tretment 15.3±0.67 nd ontrol groups 6.1±0.99 (p<0.05). The dt of MMP-1 expression in gingiv during dy 14 tretment were normlly distriuted nd homogenous (p>0.05). ANOVA nlysis onfirmed signifint differene in IL-6 expression etween the dy 14 tretment group 5.9±0.74 nd the ontrol group 3.9±0.74 (p<0.05). The verge MMP-1 expression in gingivl tissue is shown in Figure-5, with the result of IHC stining of Wistr rt gingiv ontined in Figure-6. The dt of RANKL expression in gingiv fter tretment lsting 7 dys ws normlly distriuted nd homogenous (p>0.05). ANOVA nlysis showed there ws signifint differene in RANKL expression etween the dy 7 tretment group 15±2.5 nd the ontrol group 10.1±2.5 (p<0.05). Dt relting to RANKL expression in gingiv fter 14-dy tretment ws normlly distriuted nd homogenous (p>0.05). ANOVA nlysis showed there to e signifint differene in RANKL expression etween the dy 14 tretment group 18.8±1.03 nd the ontrol group 15±1.2 (p<0.05). The verge RANKL expression in gingiv is shown in Figure-7 with the result of IHC stining of Wistr rt gingiv ontined in Figure-8. Disussion The highest onentrtion of osteolsts in the lveolr one ws found in the dy 14 ontrol group nd differed signifintly ompred to tht of the tretment group. These results ourred due to the sene of ggressive periodontitis whih, in ontrst, ws found to e present in the LPS tretment group. Osteolst plys n importnt role in minerliztion nd hydroxyptite preipittion y regulting lium nd phosphte levels during the omposition of hydroxyptite. Osteolst produes lkline phosphtse in dequte mounts in the plsm memrne responsile for one minerliztion. Osteolst lso produes ytokines suh s olony stimulting Figure-3. Averge interleukin-6 expression in gingivl tissue. Figure-1: Averge levels of osteolst nd osteolst in tretment on dys 7 nd 14 in lveolr one. Figure-2: Hemtoxilin-Eosin stining of gingiv tissue. Control (); with 7-dy indution of lipopolyshride (LPS) (); Group with 7-dy indution of LPS (). The lk rrow indites osteolst, while the yellow shows the lotion of osteolst. Figure-4: Expression of interleukin-6 in gingiv of Wistr rt. Control (); Atinoillus tinomyetemomitns lipopolyshride (LPS) (dy 7) (); A. tinomy etemomitns LPS (dy 14) (), indited y the rown regions in the nuleus (lk rrow). Ativted expression of interleukin -6 ws shown s rown in ytoplsm of gingiv epithelium (lk rrow) t 400 mgnifition. Veterinry World, EISSN:

4 Figure-5. Averge mtrix metllopeptidse-1 expression in gingiv. Figure-7: Averge reeptor tivtor of nuler ftor kpp-β lignd expression in lveolr. Figure-6: Mtrix metllopeptidse-1 (MMP-1) expression in gingiv of Wistr rt. Control (); tretment of lipopolyshride (LPS) (7-dy) (); tretment of LPS (14-dy) (), shown s rown in the nuleus (lk rrow). Ativted MMP-1 expression is highlighted s rown in ytoplsm of gingiv epithelium (lk rrow) t 400 mgnifition. ftor-1, RANKL, nd osteoprotegerin [8-10]. The highest onentrtion of lveolr osteolst ws found in the dy 14 LPS tretment group nd differed onsiderly ompred to tht of the ontrol group. The indution of LPS uses dhesion nd invsion in the host. LPS is mjor ftor in teri nd plys n importnt role in one resorption through osteolst stimultion in whih LPS tivtes osteolst to relese ftors ttrting nd tivting osteolst [11]. LPS n mke mrophges tivted, nd n lso trigger the synthesis o-f oth ytokines tht hve pro-inflmmtory tivities, suh s interleukin (IL)-1, IL-6, IL-8, nd TNF α, s well s nother ytokine, nmely, IL-10 serving s regultor [2,3,12]. LPS lso inhiits ollgen nd non-ollgen protein synthesis. Referring to study onduted y Tkhshi et l [4] nd Nir et l. [13] found tht the LPS of A. tinomyetemomitns uses one resorption in murine lvril y stimulting murine mrophge. A. tinomyetemomitns uses lveolr one destrution nd ntiody response in three rt strins; Fwn Hooded Hypersensitive, Dhl Slt Sensitive, nd Norwy Brown [14]. The proess of periodontitis psses through 4 phses: (1) Aumultion nd presene of teri Figure-8: Expression of reeptor tivtor of nuler ftor kpp-β lignd in lveolr of Wistr rt. Control (); Atinoillus tinomyetemomitns lipopolyshride (7 dys) (); A. tinomyetemomitns (14 dys) (), shown s rown in the nuleus (lk rrow) t mgnifition of 400. in gingivl sulus (oloniztion); (2) teril invsion of the epithelium nd gingiv; (3) host response stimultion, quired nd innte (inflmmtion) immune response tivtion; nd (4) destrution of onnetive tissue tthment on dentl enmel nd one using irreversile dmge [1]. The presene of LPS dereses osteolst. LPS genertes stimultion on RANKL whih further inds to RANK, stimulting tumor nerosis reeptor-ssoited ftors-6 for osteolst progenitor tivtion leding to osteolst differentition, tivtion nd n inresed numer of osteolsts. This, in turn, uses lveolr one destrution whih n serve s mrker if suh destrution hs ourred [15]. IL-6 expression in gingiv during dy 7 tretment showed n inrese nd mrked differene ompred to the ontrol, inditing the presene of LPS in periodontl tissue. LPS stimultes pro-inflmmtory ytokine tivtion, suh s IL-6, stimulting one resorption, nd inhiiting one formtion leding to periodontl nd lveolr dmge [2,16]. Veterinry World, EISSN:

5 The expression of MMP-1 in the dy 7 nd dy 14 tretment groups showed signifint ontrst ompred to tht of the ontrol group. The indution of LPS A. tinomyetemomitns uses pro-inflmmtory ytokine stimultion. IL-8 produed y monoyte, kertinoyte, endothelil, nd firolst ell stimultes the relese of MMPs y neutrophil. One suh MMP ws MMP-1 whih is potentil ollgense-1 nd plys role in the degrdtion of onnetive tissue in the inflmed re. The high expression of MMP-1 provokes periodontl nd lveolr dmge in ordne with the study onduted y Clesson et l. [17] tht onfirmed A. tinomyetemomitns to e strong stimulnt to MMP-8 relese, minly due to Leukother eing one of its 15 virulene ftors. The expression of RANKL in lveolr ones tends to inrese nd shows signifint differene in the dy 7 nd dy 14 tretment groups ompred to the ontrol group. The result onfirms LPS-l s n ntigeni omponent rising host immune response nd further stimulting pro-inflmmtory ytokines, espeilly TNF-α nd IL-1. TNF-α nd IL-1 stimulte RANKL whih inrese osteolst. The high expression of osteolst ugments osteolin tht n led to lveolr one resorption. A previous study onduted y Bullon et l. [18], posited tht the inresed numer of osteolins ws mrker of one formtion inhiition. An elevtion osteolin level in serum ws ssoited with the rte of one destrution. Studies involving nimls onfirmed the role of osteolin in lveolr one resorption. Conlusion LPS indues IL-6 expression, osteolstogenesis, nd one resorption. Injetion of porphyromons gingivlis from IL-6-removed rts uses redued level of one loss. This implies tht IL-6 ontriutes to teri progressivity whih promotes lveolr dmge. RANKL regultes the pthologil nd physiologil onditions of one resorption. During the pthologil inflmmtion phse of the one disese, RANKL expression ws found to e present in B ell, T ell, nd monoyte. Ativtion of T-ell nd B-ells re ellulr soures of RANKL in one resorption in gingiv during inflmmtion. These results support the onlusion tht LPS of A. tinomyetemomitns dministrtion over dy 7 nd dy 14 uses periodontl nd lveolr dmge in Wistr rts. Authors Contriutions Coneption nd design of the study: RDR, TK Aquisition of dt:s RDR, Sidrningsih, Anlysis nd/or interprettion of the dt: RDR, SS. Drfting nd revising the mnusript ritilly for importnt intelletul ontent: RDR, TK nd SS. All uthors red nd pproved the finl mnusript. Aknowledgments The uthors would like to thnk the Fulty of Dentl Mediine. Reserh grnt is funded y Diretorte of Reserh nd Community Servie, Diretorte Generl of Strengthening Reserh nd Development of Reserh, Tehnology nd Higher Edution Ministry: Letter of Appointment Agreement of Reserh Progrm numer: 018/SP2H/LT/DRPM/ II/2016, Ferury 17, 2016 Competing Interests The uthors delre tht they hve no ompeting interests. Referenes 1. Luo, Q., Yng, X., Yu, S., Shi, H., Wng, K., Xio, L., Zhu, G., Sun, C., Li, T., Li, D. nd Zhng, X. (2017) Struturl sis for lipopolyshride extrtion y ABC trnsporter LptB2FG. Nt. Strut. Mol. Biol., 24: Tessro, F.H., Ayl, T.S., Nolso, E.L., Bell, L.M. nd Mrtins, J.O. (2017) Insulin influenes LPS-indued TNF-α nd IL-6 relese through distint pthwys in mouse mrophges from different omprtments. Cell Physiol. Biohem., 42: Shddox, L.M., Gonçlves, P.F., Vovk, A., Allin, N., Hung, H., Hou, W., Aukhil I. nd Wllet, S.M. (2013) LPSindued inflmmtory response fter therpy of ggressive periodontitis. J. Dent. Res., 92: Tkhshi, N., Koyshi, M., Tkki, T., Tkno, K., Miyt, M., Okmtsu, Y., Hsegw, K., Nishihr, T. nd Ymmoto, M. (2008) Atinoillus tinomyetemomitns lipopolyshride stimultes ollgen pgoytosis y humn gingivl firolst. Orl Miroiol. Immunol., 23: Ptil, C., Ross, C. Jr. nd Kirkwood, K.L. (2006) Atinoillus tinomyetemomitns lipopolyshride indues interleukin-6 expression through multiple mitogen-tivted protein kinse pthwys in periodontl ligment firolsts. Orl Miroiol. Immunol., 21: Jnus, M.M., Crielrd, W., Volgennt, C.M.C., vn der Veen, M.H., Brndt, B.W. nd Krom, B.P. (2017) Cndid lins lters the teril miroiome of erly in vitro orl iofilms. J. Orl Miroiol., 9: Younis, G., Awd, A., Dwod, R.E. nd Nehl, E. (2017) Antimiroil tivity of yests ginst some pthogeni teri. Vet. World, 10: Westermn, R.B., He, Y., Keen, J.E., Littledike, E.T. nd Kwng, J. (1997) Prodution nd hrteriztion of monolonl ntiodies speifi for the lipopolyshride of Esherihi oli O157. J. Clin. Miroiol., 35: Dumistresu, A.L. (2006) Histologil omprison of periodontl inflmmtory hnges in two models of experimentl periodontitis the rt. A pilot study. Timisor Med. J., 56: Li, S.D., Chen, Y.B., Qiu, L.G. nd Qin, M.Q. (2017) G-CSF indiretly indues poptosis of osteolsts during hemtopoieti stem ell moiliztion. Clin. Trnsl. Si., 10: Strålerg, F., Kssem, A., Ksprzykowski, F., Arhmson, M., Gru, A., Lindholm, C. nd Lerner, U.H. (2017) Inhiition of lipopolyshride-indued osteolst formtion nd one resorption in vitro nd in vivo y ysteine proteinse inhiitors. J. Leuko. Biol., 101: Nirwn, I., Rhmdi, P. nd Rinti, D. (2017) Potentil of pomegrnte fruit extrt (Puni grntum Linn.) to inrese vsulr endothelil growth ftor nd pltelet-derived growth ftor expressions on the post-tooth extrtion wound of Cvi Coy. Vet. World, 10: Nir, S.P., Meghji, S., Wilson, M., Reddi, K., White, P. nd Henderson, B. (1996) Bterilly indued one destrution: Mehnisms nd misoneptions. Infet. Immun., 64(7): Veterinry World, EISSN:

6 14. Shreiner, H., Mrkowitz, K., Mirylkr, M., Moore, D., Diehl, S. nd Fine, D.H. (2010) Aggregtiter tinomyetemomitns-indued one loss nd ntiody response in three rt strin. J. Periodontol., 82: Clesson, R., Johnsson, A., Beliskis, G., Hänstrőm, L. nd Klfs, S. (2002) Relese nd tivtion of mtrix metlloproteinse 8 from humn neutrophils triggered y theleukotoxin of Atinoillus tinomyetemomitns. J. 15. Grves, D.T., Otes, T. nd Grlet, G.P. (2011) Review of Periodontl. Res., 37: osteoimmunology nd the host response in endodonti nd periodontl lesion. J. Orl Miroiol., 3: Bullon, P., Chndler, L., Segur Ege, J.J., Cno, R.P. nd Shuquillo, A.M. (2007) Osteolin in serum, sliv nd 16. Nield-Gehrig, J.S. nd Willmnn, D.E. (2007) Foundtion of Periodontis for the Dentl Hygienist. Lippinott Willims & Wilkins, Phildelphi, PA. gingivl reviulr fluid: Their reltion with periodontl tretment outome in postmenopusl women. Med. Orl. Ptol. Orl Cir. Bul., 12(3): E193-E197. ******** Veterinry World, EISSN: