periodontal diseases ( pd ) are chronic inflammatory disorders encompassing destructive 
 and nondestructive diseases of the periodontal supporting tissues of the teeth . aggressive periodontitis is characterized by severe and rapid loss of periodontal 
 attachment often commencing at or after puberty . even though the 
 diagnosis and classification of pd is essentially based on clinical parameters , new 
 auxiliary diagnostic tools based on the analysis of saliva and gingival crevicular fluid 
 have been studied and developed . in 
 particular , saliva has been extensively studied in relation to pd since it can be easily 
 collected and analyzed . saliva is necessary for the maintenance of oral health , and the unique properties of 
 this fluid are derived in large part from the proteins that are present . in the oral 
 cavity , salivary proteins participate in formation of the acquired enamel pellicle , 
 occur in the biofilm covering oral surfaces , initiate digestion , promote agglutination 
 and clearance of bacteria , and protect oral tissues against noxious compounds produced 
 by various microorganisms . in particular , mucin glycoprotein-2 ( mg2 ) was reported to be present in pellicle formed 
 on cementum surfaces , to exhibit 
 candidacidal activity , and to kill 
 the periodontal pathogen aggregatibacter 
 actinomycetemcomitans . salivary lactoferrin removes free iron in fluids decreasing the 
 availability of this metal to microbes . in addition , lactoferrin exhibits antibacterial , antimycotic , 
 antiviral , and anti - inflammatory activity . thus , mg2 and lactoferrin are active components of the innate 
 immune system in the oral environment . several studies reported that the levels of distinct salivary proteins are altered in 
 individuals with pd . therefore , the present study was 
 undertaken to investigate and compare the pattern of secretion and the expression of mg2 
 and lactoferrin in individuals with aggressive periodontitis , chronic periodontitis and 
 without periodontitis . during a 12-month period , individuals that were referred to the authors ' department 
 for treatment and volunteered to participate in this study were screened . exclusion 
 criteria for the study included : pregnancy ; use of antibiotics within the past 3 
 months ; individuals with systemic debilitating diseases or conditions that could 
 affect periodontal status ( e.g. diabetes ) ; smokers and former smokers ; recent trauma . additionally , individuals that had aggressive or chronic periodontitis and had 
 received periodontal treatment within the previous year were excluded in order to 
 observe the expression of the salivary glycoproteins mg2 and lactoferrin in the 
 inflammatory context of periodontitis . individuals meeting inclusion criteria received a complete periodontal exam that 
 included assessment of probing depth , probing attachment level and recession , 
 evaluation of bleeding and/or suppuration on probing , radiographic examination and 
 complete anamnesis . probing depths were measured at six sites per 
 tooth ( mesio- , mid- , disto - vestibular / palatal or lingual ) . the specific inclusion 
 criteria used to differentiate the 3 groups were based on previous 
 publications including : aggressive periodontitis group ( apg ) : ( i ) localized aggressive periodontitis - 
 periodontal damage being localized to permanent first molars and incisors ; 
 generalized aggressive periodontitis : ( ii ) generalized interproximal attachment loss 
 affecting at least 3 permanent teeth other than the permanent first molars and 
 incisors ; ( iii ) clinical attachment loss ( cal ) 5 mm ; probing depths 6 mm . 
 generalized chronic periodontitis group ( cpg ) : ( i ) more than 30% of sites involved ; 
 moderate : cal 3 - 4 mm ; probing depths 4 - 6 mm ; severe : cal 5 mm ; probing depths 6 mm . each group had 5 individuals ( 2 males and 3 females ) with age ranges of 19 - 28 years 
 for apg , 36 - 50 years for cpg , 21 - 50 years for cg and as expected individuals from apg exhibited a 
 lower age range . this study was independently reviewed and approved by the local 
 research ethics committee , informed consent was obtained from all individuals prior 
 to their participation and subjects ' rights were protected at all times . non - stimulated and stimulated submandibular and sublingual saliva ( smsl ) was 
 collected from subjects between 10:00 a.m. and 11:00 a.m. subjects were asked to 
 refrain from eating or drinking two h prior to collection . subjects rinsed with 
 water , and smsl samples were collected with a custom - fitted device as described 
 previously . the first 4 min of non - stimulated smsl were discarded in order to avoid possible 
 interference of the device dead volume . four consecutive two - minute samples collected 
 in separate 1.5 ml centrifuge tubes and subsequently four consecutive two - minute 
 samples of stimulated smsl were collected in separate 15 ml falcon screw cap tubes . gustatory stimulation of stimulated samples was induced by placing lemon fruit 
 flavored candies on the tongue . tubes of non - stimulated smsl were labeled t1 , t2 , t3 
 and t4 . all samples were 
 kept on ice during the collection procedure , the flow rate from each subject was 
 recorded and samples were frozen at -20c until used . smsl samples were thawed , and for each subject , equal volumes ( 50 l ) of 
 non - stimulated and stimulated smsl were lyophilized , taken up in 15 l of sample 
 buffer , heated at 95c for 5 min and subjected to sodium dodecylsulfate polyacrylamide 
 gel electrophoresis ( sds - page ) on 7.5% gels . proteins in gels were electrophoretically transferred to nitrocellulose membranes 
 ( protran , schleicher and schuell , keene , nh , usa ) in 25 mm tris - hcl , ph 8.3 , 
 containing 192 mm glycine and 20% methanol at 100 volts for 1 h at room temperature . briefly , blots were equilibrated in 10 mm tris - hcl , ph 8.0 containing 
 150 mm nacl and 0.1% tween-20 ( tbst ) for 5 min and blocked with 5% non - fat , dried 
 milk in tbst overnight at room temperature . after blocking , the specificity of the anti - mg2 antibody has been 
 demonstrated in previous studies , and this antibody was diluted 
 1:1000 . antibodies were diluted in 1% milk / tbst and incubated for 1 h at room 
 temperature . after washing , blots were incubated with the second antibody , which was 
 anti - rabbit igg coupled to alkaline phosphatase ( promega , madison , wi , usa ) diluted 
 1:7,500 in 1% milk / tbst for 1 h at room temperature . blots were washed three times in 
 tbst for 5 min and color development was carried out with 
 5-bromo-4-chloro-3-indolyl - phosphate ( bcip ) and nitro blue tetrazolium ( nbt ) 
 according to the manufacturer 's ( promega ) instructions . comparison of the effect of non - stimulated and stimulated conditions on smsl flow 
 rate was tested for statistical significance ( =0.05 ) by a two - way repeated - measures 
 analysis of variance ( anova ; post hoc analysis - fisher 's exact 
 test ) . the statistical analysis was performed using statview 4.5 ( abacus concepts 
 inc . , berkeley , ca , usa ) . during a 12-month period , individuals that were referred to the authors ' department 
 for treatment and volunteered to participate in this study were screened . exclusion 
 criteria for the study included : pregnancy ; use of antibiotics within the past 3 
 months ; individuals with systemic debilitating diseases or conditions that could 
 affect periodontal status ( e.g. diabetes ) ; smokers and former smokers ; recent trauma . additionally , individuals that had aggressive or chronic periodontitis and had 
 received periodontal treatment within the previous year were excluded in order to 
 observe the expression of the salivary glycoproteins mg2 and lactoferrin in the 
 inflammatory context of periodontitis . individuals meeting inclusion criteria received a complete periodontal exam that 
 included assessment of probing depth , probing attachment level and recession , 
 evaluation of bleeding and/or suppuration on probing , radiographic examination and 
 complete anamnesis . probing depths were measured at six sites per 
 tooth ( mesio- , mid- , disto - vestibular / palatal or lingual ) . the specific inclusion 
 criteria used to differentiate the 3 groups were based on previous 
 publications including : aggressive periodontitis group ( apg ) : ( i ) localized aggressive periodontitis - 
 periodontal damage being localized to permanent first molars and incisors ; 
 generalized aggressive periodontitis : ( ii ) generalized interproximal attachment loss 
 affecting at least 3 permanent teeth other than the permanent first molars and 
 incisors ; ( iii ) clinical attachment loss ( cal ) 5 mm ; probing depths 6 mm . 
 generalized chronic periodontitis group ( cpg ) : ( i ) more than 30% of sites involved ; 
 moderate : cal 3 - 4 mm ; probing depths 4 - 6 mm ; severe : cal 5 mm ; probing depths 6 mm . each group had 5 individuals ( 2 males and 3 females ) with age ranges of 19 - 28 years 
 for apg , 36 - 50 years for cpg , 21 - 50 years for cg and as expected individuals from apg exhibited a 
 lower age range . this study was independently reviewed and approved by the local 
 research ethics committee , informed consent was obtained from all individuals prior 
 to their participation and subjects ' rights were protected at all times . non - stimulated and stimulated submandibular and sublingual saliva ( smsl ) was 
 collected from subjects between 10:00 a.m. and 11:00 a.m. subjects were asked to 
 refrain from eating or drinking two h prior to collection . subjects rinsed with 
 water , and smsl samples were collected with a custom - fitted device as described 
 previously . the first 4 min of non - stimulated smsl were discarded in order to avoid possible 
 interference of the device dead volume . four consecutive two - minute samples collected 
 in separate 1.5 ml centrifuge tubes and subsequently four consecutive two - minute 
 samples of stimulated smsl were collected in separate 15 ml falcon screw cap tubes . gustatory stimulation of stimulated samples was induced by placing lemon fruit 
 flavored candies on the tongue . tubes of non - stimulated smsl were labeled t1 , t2 , t3 
 and t4 . all samples were 
 kept on ice during the collection procedure , the flow rate from each subject was 
 recorded and samples were frozen at -20c until used . smsl samples were thawed , and for each subject , equal volumes ( 50 l ) of 
 non - stimulated and stimulated smsl were lyophilized , taken up in 15 l of sample 
 buffer , heated at 95c for 5 min and subjected to sodium dodecylsulfate polyacrylamide 
 gel electrophoresis ( sds - page ) on 7.5% gels . proteins in gels were electrophoretically transferred to nitrocellulose membranes 
 ( protran , schleicher and schuell , keene , nh , usa ) in 25 mm tris - hcl , ph 8.3 , 
 containing 192 mm glycine and 20% methanol at 100 volts for 1 h at room temperature . briefly , blots were equilibrated in 10 mm tris - hcl , ph 8.0 containing 
 150 mm nacl and 0.1% tween-20 ( tbst ) for 5 min and blocked with 5% non - fat , dried 
 milk in tbst overnight at room temperature . after blocking , the specificity of the anti - mg2 antibody has been 
 demonstrated in previous studies , and this antibody was diluted 
 1:1000 . the anti - lactoferrin antibody ( sigma , st . antibodies were diluted in 1% milk / tbst and incubated for 1 h at room 
 temperature . after washing , blots were incubated with the second antibody , which was 
 anti - rabbit igg coupled to alkaline phosphatase ( promega , madison , wi , usa ) diluted 
 1:7,500 in 1% milk / tbst for 1 h at room temperature . blots were washed three times in 
 tbst for 5 min and color development was carried out with 
 5-bromo-4-chloro-3-indolyl - phosphate ( bcip ) and nitro blue tetrazolium ( nbt ) 
 according to the manufacturer 's ( promega ) instructions . comparison of the effect of non - stimulated and stimulated conditions on smsl flow 
 rate was tested for statistical significance ( =0.05 ) by a two - way repeated - measures 
 analysis of variance ( anova ; post hoc analysis - fisher 's exact 
 test ) . as expected , the observed flow rate values obtained at rest ( t1-t4 ) were lower than 
 stimulated ( t5-t8 ) conditions in all groups . the mean value for flow rates under 
 non - stimulated conditions was 0.460.18 ml / min for apg , 0.300.20 for cpg and 0.470.31 
 for cg . under stimulated conditions , it was 1.790.92 ml / min for apg , 1.470.38 ml / min 
 for cpg and 1.870.99 ml / min for cg . stimulation significantly raised salivary volumes 
 in all groups ( apg - p=0.01 ; cpg - p=0.03 ; cg - p=0.01 ) ; no significant time effect 
 was observed for non - stimulated or stimulated conditions , and the observed flow rates 
 under both conditions did not differ among groups . mg2 immunoreactive bands from apg , cpg and cg exhibited a weak immunoreactive signal 
 under non - stimulated conditions whereas upon gustatory stimulation , the intensity of 
 the immunoreactive signal increased ( figure 
 1a ) . blots probed with anti - lactoferrin antibodies revealed a difference in 
 banding pattern among groups ( figure 1b ) ; 
 individuals from apg and cpg had an increase in expression upon gustatory 
 stimulation , whereas individuals from cg exhibited a decrease . a - representative western 
 blots of mg2 pattern observed in all individuals from each group . b - 
 representative western blots of lactoferrin pattern observed in all individuals 
 from each group . t1t4 ( non - stimulated submandibular and sublingual 
 saliva - smsl ) and t5t8 ( stimulated submandibular and sublingual saliva - smsl ) . apg - aggressive periodontitis group ; cpg - generalized chronic periodontitis 
 group ; cg - control group comparison of expression among groups under non - stimulated conditions revealed that 
 cg exhibited the highest expression of mg2 and lactoferrin ( figures 2a and 2b ) . apg 
 showed the lowest levels of mg2 , and cpg of lactoferrin ( figures 2a and 2b ) . comparison of expression among groups under stimulated conditions showed that cg 
 exhibited the highest expression of mg2 , whereas apg the highest of lactoferrin 
 ( figures 2c and 2d ) . again , apg showed the lowest levels of mg2 , and cpg of 
 lactoferrin ( figures 2c and 2d ) . comparison of glycoproteins expression under non - stimulated ( a , b ) or 
 stimulated ( c , d ) conditions . a - mg2 western blot ; b - lactoferrin western blot ; 
 c - mg2 western blot ; d - lactoferrin western blot ; apgaggressive periodontitis 
 group ; cpg - generalized chronic periodontitis group ; cg - control group as expected , the observed flow rate values obtained at rest ( t1-t4 ) were lower than 
 stimulated ( t5-t8 ) conditions in all groups . the mean value for flow rates under 
 non - stimulated conditions was 0.460.18 ml / min for apg , 0.300.20 for cpg and 0.470.31 
 for cg . under stimulated conditions , it was 1.790.92 ml / min for apg , 1.470.38 ml / min 
 for cpg and 1.870.99 ml / min for cg . stimulation significantly raised salivary volumes 
 in all groups ( apg - p=0.01 ; cpg - p=0.03 ; cg - p=0.01 ) ; no significant time effect 
 was observed for non - stimulated or stimulated conditions , and the observed flow rates 
 under both conditions did not differ among groups . mg2 immunoreactive bands from apg , cpg and cg exhibited a weak immunoreactive signal 
 under non - stimulated conditions whereas upon gustatory stimulation , the intensity of 
 the immunoreactive signal increased ( figure 
 1a ) . blots probed with anti - lactoferrin antibodies revealed a difference in 
 banding pattern among groups ( figure 1b ) ; 
 individuals from apg and cpg had an increase in expression upon gustatory 
 stimulation , whereas individuals from cg exhibited a decrease . pattern of secretion of salivary glycoproteins . a - representative western 
 blots of mg2 pattern observed in all individuals from each group . b - 
 representative western blots of lactoferrin pattern observed in all individuals 
 from each group . t1t4 ( non - stimulated submandibular and sublingual 
 saliva - smsl ) and t5t8 ( stimulated submandibular and sublingual saliva - smsl ) . apg - aggressive periodontitis group ; cpg - generalized chronic periodontitis 
 group ; cg - control group comparison of expression among groups under non - stimulated conditions revealed that 
 cg exhibited the highest expression of mg2 and lactoferrin ( figures 2a and 2b ) . apg 
 showed the lowest levels of mg2 , and cpg of lactoferrin ( figures 2a and 2b ) . comparison of expression among groups under stimulated conditions showed that cg 
 exhibited the highest expression of mg2 , whereas apg the highest of lactoferrin 
 ( figures 2c and 2d ) . again , apg showed the lowest levels of mg2 , and cpg of 
 lactoferrin ( figures 2c and 2d ) . comparison of glycoproteins expression under non - stimulated ( a , b ) or 
 stimulated ( c , d ) conditions . a - mg2 western blot ; b - lactoferrin western blot ; 
 c - mg2 western blot ; d - lactoferrin western blot ; apgaggressive periodontitis 
 group ; cpg - generalized chronic periodontitis group ; cg - control group the infectious nature of the periodontitis is related to specific microbiota as well as 
 to the expression of particular inflammatory markers . additionally , initiation and progression of periodontal 
 infections is clearly modified by local and systemic conditions ( risk factors ) , such as 
 diabetes mellitus and cigarette smoking , as well as potentially important risk 
 indicators including stress . salivary proteins are not included in the category of risk indicators , but this concept 
 is being explored . investigation of salivary proteins in individuals with periodontitis may be useful to 
 enhance the knowledge of their role in this population . statistical analysis did not 
 reveal flow rate differences among groups , stimulated or non - stimulated . therefore , we 
 decided to analyze equal volumes ( 50 l ) of smsl by immunoblotting , instead of equal 
 amounts of protein . it has been previously reported that mg2 can agglutinate several pathogens from the oral 
 cavity , and in 
 addition , interact and kill the periodontal pathogen a. 
 actinomycetemcomitans . 
 in a previous study , the concentration of mg2 was determined in stimulated whole saliva 
 of subjects suffering from a. actinomycetemcomitans associated 
 pd . it has been reported that 
 mg2 output in the diseased subjects was decreased at least by a factor three in 
 comparison to periodontally healthy subjects and that this low concentration of mg2 
 suggested a decline in mucin defense and consequently , a higher susceptibility to oral 
 infections . in the present study , blotting analysis revealed that all groups showed an increase of 
 mg2 expression upon stimulation revealing a similar pattern ( figures 1a and 1b ) . a previous 
 study that analyzed the pattern of secretion of mg2 in samples from individuals without 
 periodontitis reported that pas 
 stained gels did not reveal differences in mg2 secretion patterns upon stimulation . 
 however , western blots indicated that mg2 expression was enhanced after stimulation ; a 
 result that is in agreement with the observed pattern in the present study for cg . it was also observed that individuals without periodontitis exhibited the highest 
 expression of this small salivary mucin under specific conditions ( figures 2a and 2c ) . the 
 reduced output of this salivary mucin in individuals with aggressive periodontitis 
 observed on blots ( figures 2a and 2c ) is similar to the one described . since in chronic periodontitis the 
 destruction of the periodontal ligament and loss of the adjacent supporting bone usually 
 displays a slow to moderate rate of progression , we hypothesize that the intermediate 
 expression of mg2 observed in individuals belonging to this group ( figures 2a and 2c ) could in 
 part contribute to decrease the disease progression rate in comparison to aggressive 
 periodontitis . salivary lactoferrin is a single chain iron - binding glycoprotein that binds ferric ions 
 and possesses antibacterial , antimycotic , antiviral and anti - inflammatory 
 properties . the impact of a surgical periodontal treatment in the 
 concentration of this glycoprotein in non - stimulated and stimulated whole saliva samples 
 from individuals with chronic periodontitis was analyzed previously . it was reported that the concentrations 
 decreased significantly in both non - stimulated and stimulated samples after the surgical 
 treatment , suggesting that lactoferrin may be suitable for monitoring periodontal 
 treatment results . another study evaluated lactoferrin levels from individuals with localized aggressive 
 periodontitis before and after periodontal therapy . among other samples , 
 paraffin - stimulated whole saliva was obtained from these individuals and from 
 periodontally healthy controls . differences between groups and with respect to 
 periodontal therapy were not observed in lactoferrin concentrations in whole 
 saliva . concentrations and output of lactoferrin were also determined in whole saliva samples 
 from subjects suffering from a. actinomycetemcomitans - associated 
 pd . the output of lactoferrin 
 was not significantly different in healthy and diseased subjects although a higher 
 iron - saturation of lactoferrin in subjects with the disease was described . lactoferrin expression was examined in whole and parotid saliva from individuals with 
 localized aggressive periodontitis and compared to age- , gender- , and race - matched 
 controls . it was reported that 
 whole saliva from the test group had higher levels of the glycoprotein although the 
 levels of bound iron were significantly reduced , suggesting that lactoferrin from 
 individuals with localized aggressive periodontitis might interfere in the disease 
 progression . in the present study , it was observed that individuals with or without periodontitis 
 exhibited a similar pattern of lactoferrin secretion ( figure 1b ) . comparison of expression among groups revealed that under 
 non - stimulated conditions individuals without periodontitis ( cg ) showed the highest 
 expression of lactoferrin ( figure 2b ) whereas 
 under stimulated conditions individuals with periodontitis ( apg and cpg ) exhibited a 
 more intense immunoreactive signal ( figure 
 2d ) . the higher expression of this glycoprotein in individuals with aggressive periodontitis 
 is in agreement with a previous report , but is in contrast to others . the differences in 
 these two studies from the present could be at least in part explained by variations in 
 samples that were analyzed ( i.e. smsl versus whole saliva ) since lactoferrin derived 
 from the gingival crevicular fluid will be present in samples from whole saliva . the increase in the amount of lactoferrin in stimulated samples from smsl of individuals 
 with generalized chronic periodontitis ( figure 2d ) 
 found in the present study is in contrast to another report in which the levels of lactoferrin in samples from 
 non - stimulated whole saliva were higher than in samples of stimulated whole saliva . again , the differences in this study from the present could also be in part explained by 
 smsl versus whole saliva . exclusion of individuals that had aggressive or chronic periodontitis and received 
 periodontal treatment during the last year had a direct impact in our sample size , since 
 it was difficult to select individuals that met this specific criterion . nevertheless , 
 this is the first study that compares the expression of salivary proteins among 
 individuals with aggressive and chronic periodontitis at the same time , and that 
 evaluates the influence of stimulation over time in the expression of mg2 and 
 lactoferrin in individuals with periodontitis . analysis of these salivary constituents 
 appears to have potential to be used as markers for aggressive or chronic periodontitis , 
 and since both mg2 and lactoferrin possess antimicrobial activity , specific domains 
 could be considered as possible candidates for the development of new drugs that in 
 principle could be of benefit to individuals with periodontitis . the biological plausibility of the differences observed in smsl physiology in our study 
 could be derived by periodontal bacterial components triggering the host - immune 
 response , and causing inflammation and activation of pro - inflammatory mediators . it has 
 been described that these molecules traveling via blood to other organs and tissues 
 might influence a variety of mechanisms . it is important to mention that the observed results show that 
 future studies should purposely collect non - stimulated and/or stimulated saliva samples 
 since protein expression under these circumstances varies . the reduced expression of 
 these glycoproteins observed in apg and cpg particularly under non - stimulated 
 conditions , decrease the oral cavity innate defense mechanisms provided by these 
 salivary components in these individuals . it remains unclear to what extent the reduced output of mg2 and lactoferrin might impact 
 the etiopathogenesis of aggressive and chronic periodontitis . however , the data suggest 
 an active role of these glycoproteins in the innate immune regulation of periodontal 
 bacterial colonization and disease progression .
objectives the aim of this study was to compare the pattern of secretion and the expression 
 of mucin glycoprotein-2 ( mg2 ) and lactoferrin in individuals with or without 
 periodontitis . 
 material and methods five individuals with aggressive periodontitis ( apg ) , 5 with generalized chronic 
 periodontitis ( cpg ) and 5 without periodontitis ( cg ) were enrolled after informed 
 consent . non - stimulated and stimulated submandibular and sublingual 
 saliva was 
 collected and samples analyzed by western blot probed with specific antibodies . 
 results stimulated and non - stimulated salivary flow rates did not differ among groups . 
 western blot analysis revealed that stimulation led to : an increase in mg2 
 expression in all groups , and to lactoferrin expression in apg and cpg . in 
 non - stimulated saliva 
 , cg exhibited the highest expression of both glycoproteins . 
 in stimulated saliva 
 , cg exhibited the highest expression of mg2 , whereas apg the 
 highest of lactoferrin . 
 conclusions the pattern of secretion of mg2 and lactoferrin in health and disease is complex . 
 although the present study analyzed samples from a limited number of participants , 
 the reduced expression of mg2 and lactoferrin in apg and cpg under non - stimulated 
 condition , the predominant circumstance of salivary secretion during the day , 
 suggests that these salivary constituents may play a role in the etiopathogenesis 
 of these diseases .