Salivary profile in diabetic patients: biochemical and immunological evaluation

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1 DOI /s BMC Reserch Notes RESEARCH ARTICLE Open Access Slivry profile in dibetic ptients: biochemicl nd immunologicl evlution Monic Virgini Viegs Lim Argão, João de Jesus de Oliveir Junior, Márci Cristin Gonçlves Mciel, Lucilene Amorim Silv, Flávi Rquel Fernndes do Nscimento nd Rosne Nssr Meireles Guerr * Abstrct Bckground: The im of this study ws to evlute the biochemicl nd immunologicl chrcteristics of sliv from dibetic ptients compred to non-dibetic dults. Methods: Eighty-eight dibetic dults nd 39 non-dibetic dults (control) were included in the study. Glucose, ure, clcium, totl protein nd mylse were determined by colorimetric method. The levels of secretory IgA nd the IgA nti-streptococcus mutns nd nti-insulin IgA ntibodies were mesured by enzyme-linked immunosorbent ssy (ELISA). Cries sttus ws evluted using the DMFT index. Results: Glucose, ure, clcium, nti-s. mutns IgA, totl IgA, nd nti-insulin IgA were significntly higher in dibetic ptients, wheres totl protein nd mylse levels were lower in these ptients. There ws no positive correltion between blood nd slivry glucose levels in either group. Dibetic ptients hd higher DMFT index. Conclusions: The present study showed for the first time tht IgA levels in dibetic ptients sliv, shows correltion with systemic biochemicl prmeters. Thus the sliv is n useful tool to follow the systemic helth sttus in these ptients. Keywords: Sliv, Composition, Dibetes mellitus, IgA, Glucose, Ure, Clcium Bckground Dibetes mellitus is public helth problem since, in ddition to importnt socil repercussions. It is chronic disese tht ffects growing number of individuls from different countries which re t different stges of economic nd socil development [1]. Dibetes is clssified into four clinicl types: type 1 dibetes which results from β-cell destruction nd usully leds to bsolute insulin deficiency; type 2 dibetes which results from progressive insulin secretory defect ssocited with insulin resistnce; other specific types of dibetes due to other cuses, nd gesttionl dibetes mellitus [2]. Systemic diseses such s dibetes compromise slivry glnd function nd consequently influence the quntity nd qulity of sliv produced. Altertions in the chemicl components nd physicl properties of sliv *Correspondence: roguerr@globo.com Lbortory of Immunophysiology, Cmpus Universitário do Bcng, Universidde Federl do Mrnhão (UFMA), 1966 Centro, São Luís, MA 65080, Brzil cn serve s dignostic prmeters nd sliv testing my therefore be dded to the rsenl of complementry tests [3]. Sliv hs severl dvntges over serum, including its simple nd noninvsive collection nd esy storge, trnsport nd hndling [4]. The wide vriety of components found in sliv permits its use for dignosis, prognosis nd monitoring of humn diseses [5], such s hereditry or congenitl diseses [6], utoimmune diseses, crdiovsculr diseses, infections [7], cncer [8], dibetes [9], cries [10], nd periodontl disese [11], mong others. Differences in sliv production nd composition hve been observed previously between dibetic nd non-dibetic subjects [12, 13]. Some uthors hve shown vrible correltion rtes between serologicl nd sliv dt [9, 12, 14]. However, despite this pprent controversil the sliv seems to be importnt nd useful to evlute physiologic ltertions. The im of the present study ws to evlute the biochemicl nd immunologicl profile of sliv in dibetic 2016 Lim-Argão et l. This rticle is distributed under the terms of the Cretive Commons Attribution 4.0 Interntionl License ( which permits unrestricted use, distribution, nd reproduction in ny medium, provided you give pproprite credit to the originl uthor(s) nd the source, provide link to the Cretive Commons license, nd indicte if chnges were mde. The Cretive Commons Public Domin Dediction wiver ( org/publicdomin/zero/1.0/) pplies to the dt mde vilble in this rticle, unless otherwise stted.

2 Pge 2 of 7 ptients, considering the totl production of IgA, nd the concentrtion specific IgA nti-insulin nd nti-streptococcus mutns, s dditionl biomrkers in sliv to stte the differences between dibetic nd non dibetics ptients. Methods Popultion The smple size ws clculted bsed on the prevlence of dibetes in Mrnhão (MS Hiperdi [15]), with confidence intervl of 95 %. Dt were collected systemticlly during 6 month. The smple consisted of 142 subjects (93 dibetics nd 49 controls). The popultion ws divided into two groups ge-sex mtched: DIABETIC nd NON DIABETIC. The dibetic group comprises ptients with dibetes type I nd Type II, since there re no sttistic differences mong them, with previous dignosis of more thn 4 yers. This study used convenience smple bsed on the following inclusion criteri: ge older thn 18 yers, previous dignosis of dibetes mellitus, nd prticiption in the Dibetes Progrm of Presidente Dutr University Hospitl, São Luís, Mrnhão, Brzil. Only 88 dibetic ptients, who met the inclusion criteri, hd sufficient slivry flow for nlysis nd effectively greed to prticipte in the study. The following criteri were used for inclusion in the control group: ge older thn 18 yers, no systemic disese or dibetes, nd no history of mediction use. Only 39 non-dibetic dults, who met the inclusion criteri, effectively prticipted in the study. Sociodemogrphic dt were obtined by semi-structured interview. All subjects received informtion regrding the objective nd procedures of the study nd only prticipted in the dt collection fter signing free informed consent form. The study protocol ws pproved by the Ethics Committee of Universidde Federl do Mrnhão (Protocol No ). Collection of sliv smples Unstimulted whole sliv ws collected, in fsting, from dibetic nd non-dibetic subjects over period of 5 min. After rinsing their mouth with filtered wter, the subjects were sked to spit sliv into sterile flsk. The flsks were seled immeditely fter smple collection nd refrigerted t 4 C before trnsport to the lbortory. The sliv smples were centrifuged (10,000 rpm, 10 min) to reduce slivry debris nd viscosity. Glucose nd totl protein concentrtions were determined immeditely fter centrifugtion. The other prmeters were evluted lter in superntnt stored t 20 C. Biochemicl nlysis Glucose, totl protein, ure, clcium nd mylse concentrtions were determined by colorimetric method. The nlyses were performed using utomted procedures on n Architect C8000 pprtus (Abbott ) for glucose, mylse, totl protein nd clcium. Individul smples were tested in triplicte. Determintion of IgA levels Slivry totl IgA, nti-s. mutns IgA nd nti-insulin IgA ntibodies were determined by enzyme-linked immunosorbent ssy (ELISA) s described previously [11]. Briefly, nti-humn IgA (Sigm, St. Louis, USA) ws diluted to concentrtion of 50 µg/ml in crbontebicrbonte buffer (ph 9.6) nd dsorbed onto ELISA strips in flt-bottom wells (NUNC, Roskilde, Denmrk), followed by overnight incubtion t 4 C. The wells were then wshed five times with 0.15 M phosphte-buffered sline (PBS; ph 7.2) contining 0.05 % Tween 20 (Promeg, Mdison, USA; PBS-T) nd incubted in PBS contining 1 % bovine serum lbumin (Sigm) for 1 h t 37 C. The wells were wshed five times nd 100 µl of tenfold dilutions (1:100 to 1:1000) of sliv smples in PBS ws dded. After 30 min t 37 C nd further wshing, 100 µl lkline phosphtse-lbeled got ntihumn IgA (Sigm; diluted 1:1000 in PBS-T) ws dded to the wells nd the strips were incubted for 2 h t 37 C. The strips were then wshed five times with PBS-T. The color ws developed by dding 100 µl of solution of ρ-nitrophenylphosphte (Sigm) nd incubting the strips for 30 min in the drk. The rection ws stopped with 1 N NOH (Sigm) nd the opticl density ws mesured t wvelength of 405 nm. Clinicl exmintion The cries sttus of the ptients ws evluted using the DMFT (decyed, missing nd filled permnent teeth) index nd ws performed by single exminer. Dt on orl hygiene, ccess to dentl cre, nd slivry flow were lso collected. Sttisticl nlysis Quntittive vribles were compred between the two groups by the Student t test. The correltion between dibetic nd non-dibetic ptients ws evluted using Person s liner correltion coefficient. A receiver operting chrcteristic (ROC) curve ws constructed for the dignostic test. This nlysis considered glucose, totl protein, ure, nti-insulin Ig, nd mylse concentrtions. The test ws defined s positive when ltertions in t lest four prmeters were observed. The sensitivity of the test ws 88 %, specificity ws 90 %, nd dignostic

3 Pge 3 of 7 ccurcy ws 89 %. Ctegoricl vribles were compred by Fisher s exct test. The level of significnce for rejection of the null hypothesis ws set t p 0.05 in ll tests. All nlyses were performed using the GrphPd Prism 5.0 progrm. Results The control group consisted of 39 cliniclly helthy subjects with men (±SD) ge of 23 ± 6 yers (rnge: yers). The men (±SD) ge of the dibetic ptients ws 52 ± 18 yers (rnge: yers). Fifty-seven of the 88 dibetic ptients were femles nd 31 were mles, nd 59 were using insulin. The time since dignosis of dibetes ws 11 ± 8 yers. Blood glucose levels were 261 ± 131 mg/dl. The dt obtined for dibetic ptients were compred to control (non-dibetic) group of 39 subjects, including 22 femles nd 17 mles. The men (±SD) blood glucose levels were 92 ± 9 mg/dl in this group. Tble 1 shows the slivry biochemicl prmeters nd Fig. 1 illustrtes the immunologicl prmeters. Glucose, ure, clcium, nti-s.mutns IgA, totl IgA nd ntiinsulin IgA were significntly higher in dibetic ptients (p < 0.05). On the other hnd, totl protein nd mylse concentrtions were significntly lower in dibetic ptients (p < 0.05). No positive correltion ws observed between blood nd slivry glucose levels in either group, lthough glucose levels were higher in dibetic ptients thn in the control group (Tble 1). A negtive correltion ws found between slivry glucose concentrtion nd nti-insulin IgA. The dibetic ptients were divided into two subgroups ccording to the use or not of insulin. The results showed no significnt differences between dibetes I or dibetes II ptients. For this reson, the slivry dt were evluted together s dibetic ptients (Tble 2). A ROC curve ws constructed to vlidte the slivry prmeters tht could be used for dignostic testing. Glucose, totl protein, ure, nti-insulin IgA, mylse Tble 1 Slivry prmeters of dibetic nd non-dibetic ptients Prmeter Control Dibetic p vlue Glucose (mg/dl) 3 ± 0.03 b 11 ± Ure (mg/dl) 17 ± 1 27 ± Clcium (mg/dl) 2 ± ± Totl protein (mg/dl) 2 ± ± Amylse (AU/dL) 37 ± ± Sliv smples obtined from 39 non-dibetic (control) subjects nd 88 dibetic ptients of both genders were evluted b Results re reported s the men ± stndrd devition (X ± SD) Fig. 1 Slivry concentrtion of totl IgA, nti-mutns (Streptococcus mutns) IgA nd nti-insulin IgA in dibetic ptients nd helthy controls. The results re reported s the men ± stndrd devition (X ± SD) of 88 dibetic ptients nd 39 controls. (*) Significnt when p < 0.05 compred to the control group Tble 2 Slivry composition of dibetic ptients who used insulin or not Prmeter Use of insulin p vlue Yes Glucose 12 ± 3 b 9 ± 3 b 0.51 Ure 28 ± 1 25 ± Clcium 2 ± ± Amylse 0.3 ± ± Totl protein 37 ± 1 37 ± Anti-mutns IgA 21 ± 1 20 ± Totl IgA 35 ± 1 36 ± Anti-insulin IgA 28 ± 2 27 ± Sliv smples obtined from 88 dibetic ptients of both genders were evluted b Results re reported s the men ± stndrd devition (X ± SD) nd clcium concentrtion were included in the nlysis. Only clcium ws excluded fter ROC nlysis since it exhibited n re of 0.5, i.e., this prmeter ws not sttisticlly significnt (Tble 3). Tble 3 Are under the ROC curve obtined for slivry prmeters of dibetic ptients Prmeter Are under the ROC curve Sliv smples obtined from 88 dibetic ptients of both genders were evluted No Stndrd error p vlue Glucose < Totl protein < Amylse < Anti-insulin IgA < Ure < Clcium

4 Pge 4 of 7 Tble 4 Index of decyed, missing nd filled permnent teeth (DMFT) in dibetic ptients Index Dibetic (n = 88) Control (n = 39) p vlue D 4 ± ± 0.2 <0.001 M 16 ± ± 0.2 <0.001 F 1 ± ± 0.4 <0.001 DMFT b 20 ± 1 5 ± 0.5 <0.001 b Results re reported s the men ± stndrd error of the men DMFT decyed, missing, filled permnent teeth Tble 5 Orl hygiene, ccess to dentl cre nd dry mouth senstion in dibetic ptients nd controls Dibetic f (%) Control f (%) p vlue b Number of tooth brushing < Totl Use of dentl floss Yes < No Totl Lst dentl visit 1 yer < yers Totl Dry mouth senstion Yes 63 5 < No Totl f (%), frequency expressed s percentge b Fisher s exct test Clinicl exmintion showed high DMFT index in dibetic ptients (Tble 4). Furthermore, compred to the control group, these ptients hd poor orl hygiene nd less ccess to dentl cre nd exhibited xerostomi signs such dry mouth (Tble 5). Discussion Whole sliv is mixture of slivry glnd secretions contining substnces derived from gingivl creviculr fluid, desqumted epithelil cells, food rests, microorgnisms, nd products derived from microbil metbolism [16]. Since stimultion ffects both the production of sliv nd the concentrtion of some of its components [3, 17], in the present study unstimulted whole sliv ws used to evlute orl ltertions ssocited with dibetes in dult ptients. A significnt increse in the slivry concentrtion of glucose, clcium nd ure ws observed in dibetic ptients. Similr results of clcium concentrtion hve been reported previously [18]. Furthermore, the increses were similr to those reported by other investigtors tht evluted biochemicl prmeters in the sliv of dibetic ptients [12]. Chnges in slivry composition hve been suggested to ffect the development, symptoms nd severity of mny orl diseses, prticulrly in dibetic ptients [9]. No correltion between slivry nd blood glucose levels ws observed in dibetic ptients despite higher slivry glucose concentrtion in this group. Slivry nd blood levels of glucose my be considered independent vribles s previously described for dults [12 14] nd children with dibetes [19]. According to Juryst et l. [13], slivry glucose concentrtion mesured either in unstimulted or stimulted sliv is lwys higher in dibetic ptients thn in control subjects, nd this high concentrtion exerts potentilly unfvorble effects on the orl helth of these ptients. These results gree with the present findings nd confirm the wek ssocition between blood glucose levels nd glucose concentrtion or excretion in sliv, t lest in individul cses [14]. Totl mylse nd protein were significntly lower in dibetic ptients. Amylse ws evluted s mrker of metbolic nd hormonl chnges in slivry glnd products. Yvuzyilmz et l. [20] lso reported lower levels of mylse in dibetic ptients. In contrst, slivry totl protein concentrtion differed from tht reported in studies evluting this prmeter fter freezing nd storge of the smple [21]. This difference might be explined by the fct tht in the present study the ssy ws performed immeditely fter collection nd centrifugtion of the sliv smples to prevent endogenous proteolytic ctivity [4]. Evlution of immunologicl chnges in sliv showed higher totl IgA ntibody concentrtion in dibetic ptients. These findings re probbly ssocited with the elevted levels of specific nti-s. mutns nd nti-insulin ntibody levels lso observed in these ptients. The determintion of chnges in whole sliv composition my be used to dignose possible vritions nd defective host immune responses, including the secretion of s-iga [22]. Similr results hve been reported in other studies evluting the slivry production of IgA ntibodies in dibetic ptients [20, 23]. According to Btist et l. [23], S. mutns counts re usully higher in dibetic ptients. Although the degree of infection of the ptients ws not quntified in this study, elevted nti-s. mutns ntibody titers might be relted to n incresed biovilbilityof bcteril ntigens. The locl response of specific ntibodies to certin plque bcteri such s S. mutns my identify sites with potentil risk of developing cries nd other orl

5 Pge 5 of 7 diseses even before onset of the first clinicl signs [24]. Streptococcusmutns is Grm-positive bcterium tht is potentilly ggressive to vrious tissues. Elevted nti-s. mutns IgA concentrtions my indicte tht the ptients re systemticlly exposed to this microorgnism, s well s higher cries risk [25], especilly if we consider the poor orl hygiene nd less ccess to dentl cre showed in the present study (Tble 5). In the present study high levels of nti-insulin IgA ntibodies were detected in sliv from dibetic ptients nd this result my explin the elevtion of glucose levels observed in dibetic ptients. The dibetes hyperglycemi Insulin is known to hve regultory role on glucose levels nd the nti-insulin ntibodies re frequently responsible to block the insulin ligtion to the respective receptor, incresing the biodisponibility of this hormone nd decresing its ction [26 28], resulting from defects in insulin secretion, insulin ction, or both [2], observed in dibetes. There is no previous report bout nti-insulin ntibodies in sliv from dibetic ptients in ssocition with other slivry profile. For this reson the results ltogether my highlight informtion bout the slivry biomrkers in dibetic ptients, since the detection of nti-insulin ntibodies re frequently ssocited to insulin ntibody-induced insulin resistnce nd cn be used s dignostic nd prognostic mrkers to dibetes, but lso to other utoimmune diseses [26, 27]. According Eisenbrth (2003) [28], specific ptterns of utontibody expression my predict risk nd progression to dibetes rnging from 20 % to >90 %, minly in dibetes type I. We propose detection of IgA nti-insulin in sliv s useful tool to predict dibetes risk erly, s well s, n importnt result to monitoring the progression of disese in dibetic ptients, with the sme ccurcy s tht obtined for serum nti-insulin ntibodies nd with less discomfort to the ptients during fluid collection. Considering lrge number of ptients were using injectble insulin, we investigted whether this tretment lters the vribles nlyzed. No significnt differences in slivry biochemicl or immunologicl prmeters were observed between non-insulin-dependent nd insulindependent dibetic ptients. Similr findings hve been reported by Juryst et l. [13] nd Yvuzyilmz et l. [20] who lso compred sliv of insulin-dependent nd noninsulin-dependent dibetic ptients. Biochemicl nd immunologicl ltertions in the sliv of dibetic ptients hve been described nd slivry chnges my serve s complementry prmeter for the dignosis of dibetes mellitus [3]. In the present study, the clinicl performnce of n individul test ws demonstrted by the ROC curve fter the vribles hd been combined nd were vlidted for dignostic use. The greter the cpcity of test to discriminte between dibetic nd non-dibetic subjects, the closer is the curve to the left corner of the grph, with n re under the curve close to 1 [29]. The sequence of individul vribles tht best discriminte dibetic ptients ws glucose (re of 0.99), totl protein (re of 0.98), mylse (re of 0.95), nti-insulin IgA (re of 0.83), nd ure (re of 0.80). The ccurcy of the dignostic test combining these prmeters ws 89 %. A similr ccurcy (90 %) hs been reported by Lopez et l. [19] who used sliv for the dignosis of dibetes in children considering clcium, ure, totl sugrs, glucose nd totl protein concentrtion s done in the present study for dult ptients. The ssocition of dentl cries with dibetes is less cler. In this study, dibetic ptients hd higher DMFT index thn control subjects. The verge number of teeth lost ws 16 nd the verge number of decyed teeth ws 3.7, which were responsible for the high DMFT index in these ptients. The dry mouth senstion nd incresed slivry glucose levels observed here my lter the effectiveness of slivry protection ginst cries [10]. Other fctors such s poor orl hygiene nd deficient regulr cre of these ptients cn ggrvte pre-existing diseses. Dt from clinicl evlution my explining the high level of IgA nti-s.mutns, s low slivry flow, nd high DMFT index re frequently ssocited to n increse in bcteril counts nd the risk of infection mong dibetic ptients. Its importnt to emphsizes the ge differences between the control group (23 ± 6, rnge: yers) nd dibetic ptients (52 ± 18), s well the rnge intr group (rnge: yers), due to the inclusion criteri Wht cn lso influence the differences found between the two groups. Dibetic ptients with reduced slivry flow hve n incresed risk of enmel hypominerliztion nd cries formtion [30]. A decrese in slivry flow cn cuse orl ltertions such s n increse in the concentrtion of mucin nd glucose. Prolifertion of pthogenic microorgnisms my lso occur. The consequences of chronic hyperglycemi include tongue ltertions, periodontl disese, tooth deminerliztion, nd n incresed infection risk [31]. The erly detection nd tretment of hyperglycemi nd hyposlivtion my provide useful strtegy to prevent the dentl complictions of dibetes nd to promote orl helth in this popultion [32]. Slivry composition in dibetes type 1 nd 2 cn be correlted to n incresed risk of periodontl disese [33, 34]. Crd et l. [16], evluting type 2 dibetic ptients, found symptoms of periodontl disese ssocited with tooth decy nd tooth loss in ll ptients. Jones et l. [35] demonstrted n incresed cries risk

6 Pge 6 of 7 in 457 ptients with dibetes mellitus. Their study included ptients with type 1 nd type 2 dibetes, similr to the present study. There ws positive correltion between DMFT nd totl IgA nd negtive correltion between DMFT nd tooth brushing in dibetic ptients. Similr results hve been reported by Smith nd Tubmn [36] for dult ptients. In the group with cries, the uthors observed significnt positive correltion between plque index nd specific IgA. The dignostic vlue of sliv hs been demonstrted in series of studies showing the ccurcy nd specificity of this body fluid for the dignosis of diseses [3 5, 7 9, 12, 13, 17, 19, 20, 37]. The ese of collection of sliv smples nd the lower risk of contmintion of helthcre workers during smple hndling re importnt dvntges tht support the use of this fluid s dignostic tool. Furthermore, studies compring the results of sliv nlysis with other relible scientific methods, such s blood mesurements, hve demonstrted tht the method is relible nd its ppliction is therefore vlid. There is no previous report bout the detection of ntiinsulin in sliv of dibetic ptients nd its reltion to severl biochemicl prmeters. The present study ws the first to demonstrte tht combintion of immunologicl nd biochemicl prmeters s useful dt to monitor dibetic ptients, since significnt differences were observed in the sliv composition of dibetic ptients, The ltertions detected indicting strong reltionship between our findings nd the systemic helth sttus of the ptient. Moreover, some slivry prmeters were found to be useful to clssify n dult s dibetic. This difference in slivry composition thus suggests the use of sliv s n lterntive fluid to monitor ptients with dibetes mellitus, considering the increse of dibetes not only in Brzil but lso in other countries nd the possibility to find new forms of dignosis to design prevention strtegies. Abbrevitions IgA: immunoglobulin A; s-iga: secretory IgA; ELISA: enzyme-linked immunosorbent ssy; DMFT index: decyed, missing nd filled permnent teeth index; ROC curve: receiver operting chrcteristic; X ± SD: men ± stndrd devition. Authors contributions MVVL-A, JJOJr, MCGM nd LAM: hve been involved in cquisition of dt, nlysis nd interprettion of dt; drfting the mnuscript nd importnt intellectul content. FRFN: Hve been involved in nlysis nd interprettion of dt; drfting the mnuscript nd revising it criticlly for importnt intellectul content. RNMG: Ws responsible to the conception, design, cquisition of dt, nlysis, interprettion of dt; drfting the mnuscript nd intellectul content s the leder of this reserch group. All uthors red nd pproved the finl mnuscript. Acknowledgements To Mrnhão Stte Foundtion (FAPEMA)-EditlPPSUS, Federl University of Mrnhão (UFMA) nd Ntionl Council for Scientific nd Technologicl Development (CNPq) for the finncil support nd to CNPq for the reserch fellowship of RNMG nd FRFN. Competing interests The uthors declre tht they hve no competing interests. Received: 6 November 2015 Accepted: 22 Jnury 2016 References 1. Wild S, Roglick G, Green A, Sicree R, King H. Globl prevlence of dibetics: estimtes for the yer 2000 nd projections for Dibetes Cre. 2004;27: Americn Dibetes Assocition. Stndrds of medicl cre in dibetes. Dibetes Cre. 2012;35(Suppl 1):S Mour SAB, Medeiros AMC, Cost FRH, Mores PH, Oliveir filho SA. Vlor dignóstico d sliv em doençs oris e sistêmics: um revisão d litertur. Pesq Brs Odontop Clin Integr. 2007;7: Bigler LR, Streckfus CF, Dubinsky WP. Slivry biomrkers for detection of mlignnt tumors tht re remote from orl cvity. Clin Lb Med. 2009;29: Oliveir Júnior JJ. Guerr RNM. Biomrcdores imunológicos d sliv. RevCiencSude (São Luís). 2010;12: Grody WW. Cystic fibrosis: moleculr dignosis, popultion screening, nd public policy. Arch Pthol Lb Med. 1999;123: Pink R, Simek J, Vondrkov J, Fber E, Michl P, Pzder J, et l. Sliv s dignostic medium. Biomed Pp Med FcUnivPlcky Olomouc Czech Repub. 2009;153: Guerr RNM, Oliveir-Junior JJ, Mouchrek-Filho JCE, Liberio AS, Lim MVV, Pim DBS, Brito CXL, Mendonç C, Nscimento FRF, Pereir ALA. Slivry evlution of peditric ptients with cncer, before nd fter ntineoplstic tretment. J Orl Pthol Med. 2012;41: Negrto CA, Trzi O. Buccl ltertions in dibetes mellitus. Dibetol Metbolic Syndr. 2010;2: Miko S, Smbrus SJ, Shfin S, et l. Dentl cries nd dolescents with type 1 dibetes. Br Dent J. 2010;208: Brnco-de-Almeid LS, Alves CMC, Lopes FF, Pereir AFV, Guerr RNM, Pereir ALA. Slivry IgA nd periodontl tretment needs in dibetic ptients. Brz Orl Res. 2011;25: Crd C, Mosquer-Llored N, Slom L. Gomez De Ferrris ME, Peydró A. Structurlndfunctionlslivrydisorders in type 2 dibeticptients. Med Orl Pthol Orl. Cir Bucl. 2006;11: Juryst C, Bulur N, Oguzhn B, Stmn I, Yilmz TM, Mlisse WJ, Sener A. Slivry glucose concentrtion nd excretion in norml nd dibetic subjects. J Biomed Biotech. 2009;2009: Sores MSM, Btist-Filho MMV, Pimentel MJ, Pssos IA, Küstner EC. Determintion of slivry glucose in helthy dults.med Orl Pthol Orl. Cir Bucl. 2009;14: MS Ministério d Súde do Brsil-HIPERDIA: cpturedon August site: Kufmn E, Lmster IB. The dignostic ppliction of sliv. A review. Crit Rev Orl Biol. 2002;13: Mndel ID. The dignostic uses of sliv. J Orl Pthol Med. 1990;19: Monteiro AMD, Arújo RPC, Gomes Filho IS. Dibetes Mellitus tipo 2 e Doenç Periodontl. RGO. 2002;50: Lopez ME, Colloc ME, Páez RG, Schllmch JN, Koss MA, Chervongur A. Slivry chrcteristics of dibetic children. Brz Dent J. 2003;14: Yvuzyilmz E, Yumr O, Akdognli T, Ymlik N, Ozer N, Ersoy F, Yeni YI. The ltertions of whole sliv constituents in ptients with dibetes mellitus. Aust Dent J. 1996;41: Flegl KM, Ezzti TM, Hrris MI, Hynes SA, Jurez RZ, Knowler WC. Prevlence of dibetes in Mexicn Americns, Cubns, nd Puerto Ricns from the Hispnic helth nd nutrition survey, Dibetes Cre. 1991;14:

7 Pge 7 of Bchrch G, Muster Z, Rz I, Chushu G, Stbholz A, Nussbum G, et l. Assessing the levels of immunoglobulins in the sliv of dibetic individuls with periodontitis using checkerbord immunodetection. Orl Dis. 2008;14: Btist JE, Btist CEM, MonteiroNeto V, Guerr RNM. Colonizção por Streptococcusmutns e dosgens de nticorpos ntiinsulin n sliv de dibéticos. Rev CiêncSúde (São Luís). 2004;6: Brtthll D, et l. Immunoglobulin A rection to orl streptococci in sliv of subjects with different combintions of cries nd levels of mutns streptococci. Orl Microbiol Immunol. 1997;12: Kog-Ito CY, Mrtins CAP, Blducci I, Jorge AOC. Correltion mong mutns streptococci counts, dentl cries, nd IgA to Streptococcus mutns in sliv. Brz Orl Res. 2004;18: Yu L, Robles DT, Abiru N, Kur P, Rewers M, Kelemen K, Eisenbrth GS. Erly expression of nti-insulin uto-ntibodies of humns nd the NOD mouse: evidence erly determintion of subsequent dibetes. Proc Ntl Acd Sci. 2000;97: Kmrdt T, Mitchison NA. Advnces in immunology: tolernce nd utoimmunity. New Engl J Med. 2001;344: Eisenbrth G. Insulin utoimmunity: imumunogenetics/immunopthogenesis of type 1A dibetes. Ann NY Acd Sci. 2003;1005: Mrtinez EZ, Louzd-Neto F, Pereir BB. A curv de ROC pr testes dignósticos. Cdernos de Súde Coletiv. 2003;11: Moore PA, Guggenheimer J, Etzel KR, et l. Type 1 dibetes mellitus, xerostomi, nd slivry flow rtes. Orl Surg Orl Med Orl Pthol Orl RdiolEndod. 2001;92: Vernillo AT. Dentl considertions for the tretment of ptients with dibetes mellitus. J Am Dent Assoc. 2003;134:24S 33S. 32. Yeh CK, Hrris SE, Mohn S, Horn D, Fjrdo M, Chun YHP, Jorgensen J, McDougll M, Abboud-Wener S. Hyperglycemi nd xerostomi re key determinnts of tooth decy in type 1 dibetic mice. Lb Invest. 2012;92: Ben-Aryeh H, Serouy R, Knter Y, Szrgel R, Lufer D. Orl helth nd slivry composition in dibetic ptients. J Dibetes Complictions. 1993;7: Arriet J, Brtolomé B, Jiménez E, Svedr P, Arriet F. Problems bucodentlesen pcientes con dibetes mellitus. Med Orl. 2003;8: Jones RB, McCllum RM, Ky EJ, Kirkin V, McDonld E. Orl helth nd orl helth behviour in popultion of dibetic clinic ttenders. Community Dent Orl Epidemiol. 1992;20: Smith DJ, Tubmn MA. Ontogeny of immunity to orl microbiot in humns. Crit Rev Orl Biol Med. 1992;3: Lwrence HP. Slivry mrkers of systemic disese: noninvsive dignostic of disese nd monitoring of generl helth. J Cn Dent Assoc. 2002;68: Submit your next mnuscript to BioMed Centrl nd we will help you t every step: We ccept pre-submission inquiries Our selector tool helps you to find the most relevnt journl We provide round the clock customer support Convenient online submission Thorough peer review Inclusion in PubMed nd ll mjor indexing services Mximum visibility for your reserch Submit your mnuscript t