human saliva , a complex secretion that contains a mixture of inorganic and organic molecules , plays an essential role in the maintenance of oral health [ 13 ] .
the tooth surface is protected by a film of salivary mucins and proline - rich glycoprotein .
human saliva has been shown to contain at least 2 structurally and functionally distinct populations of secreted mucins : the high molecular weight ( mr>10 da ) polymeric , gel - forming population , muc5b and the lower molecular weight ( mr 1.21.510 da ) non - polymerizing population muc7 [ 510 ] .
the secreted salivary mucins , muc5b and muc7 , provide lubrication , protect oral tissue surfaces , and modulate oral microorganisms .
secreted mucins are considered the first line of defense for epithelial tissues since they act as physical barriers between extracellular milieu and the mucosal surface .
although they are components of the non - immune oral host defense system , they are only transient components of saliva and are swallowed soon after secretion .
it has been shown that the major and minor salivary glands , as well as oral epithelial cells , express the membrane - bound mucin muc1 .
muc1 is ubiquitously expressed in the epithelial cells lining the respiratory , reproductive , and gastrointestinal tracts .
this mucin remains on the cell surface after secretion and is primarily involved in the protection of epithelial surfaces , acting as the second line of defense , and possibly in the mediation of intracellular signal processing .
the core protein of this glycoprotein is translated as a single polypeptide that is proteolytic cleaved into 2 subunits in endoplasmic reticulum .
the 2 fragments form a stable but noncovalent heterodimeric complex that remains associated during its post - translational processing and transport to the cell surface .
a portion of the membrane - associated muc1 can be shed , presumably by the second proteolytic cleavage of the cytodomain . in this way
previous studies have reported that muc1 is a critical element of the mucosal barrier to infection [ 1820 ] , and up - regulation of muc1 follows infection with a bacterial pathogen .
it has also been suggested that the protective effects of muc1 may be especially important in the oral cavity , where epithelial surfaces are constantly exposed to a variety of both pathogenic and commensal microbes .
in fact , up - regulation of muc1 expression in oral epithelial cells has been reported to result from porphyromonas gingivalis infection or increases in pro - inflammatory cytokines such as il-1 , il-6 , and tnf- .
there is also evidence that secreted mucin muc2 and muc5ac genes , which are not expressed in oral tissues , are up - regulated by pro - inflammatory mediators such as interleukin-1 beta and tnf alpha [ 22 , 23 ] .
therefore , there is a possibility that expression of membrane ( muc1 ) and secreted mucins ( muc5b and muc7 ) in the oral mucosal epithelial cells is affected by changes in pro - inflammatory cytokine levels in saliva .
the importance of salivary secreted mucins ( muc5b and muc7 ) has been the focus of much research in the last 2 decades .
the aim of this study was to compare salivary muc5b , muc7 , and muc1 content in saliva of young people with dental caries .
the adolescents ( age 18 years ) from a high school where students have a high prevalence of dental caries were chosen and served as the experimental group .
the high caries rate of adolescents was examined using the caries intensity index dmf ( decay / missing / filled ; d+m+f / number of the examined ) .
eight adolescents with dmf=3 served as a control group and 27 adolescents with dmf>11 were the research group .
examination of patients dentition with regard to caries was carried out in a school surgery , in artificial light , using basic diagnostic instruments .
the depth of gingival pouches and gingival hemorrhage were examined with a periodontal probe in individual sites in the teeth .
additionally , dental plaque and lesions on the mucus membrane of the oral cavity were evaluated .
saliva from all subjects was collected and the levels of mucins were recorded . the research protocol was approved by the committee for ethics and supervision on human research , medical university of biaystok , with informed consent from the patients .
samples from the subjects were collected between 9:00 and 11:00 a.m. all subjects abstained from eating and drinking for 2 h. unstimulated whole saliva was collected for 10 min by a spitting method .
saliva samples were homogenized and clarified by centrifugation at 10.000  g for 15 min at 4c .
the high sensitivity assay kits ( uscnk ) were used to determine the levels of muc5b and muc7 in the saliva samples .
the microtiter plates provided in the kits were pre - coated with a monoclonal antibody specific to muc5b or muc7 . standards and samples were added to the appropriate microtiter plate wells with a biotin - conjugated polyclonal antibody preparation specific for muc5b or muc7 and were incubated for 1 h at 37c . after washing away any unbound substances , avidin conjugated to horseradish peroxidase was added to each microplate well and incubated .
after another aspiration and washing step , a tmb substrate solution was added to each well .
the enzyme - substrate reaction was terminated by the addition of a sulfuric acid solution and the color change was measured at a wavelength of 450 nm .
the assay was performed in duplicate and the concentration of muc5b and muc7 in the samples was then determined by comparing the o.d . of the samples to the standard curve .
the range of the standard curve was 0.62540 ng / ml for muc5b and 0.31220 ng / ml for muc7 .
the minimum detectable dose of human muc5b was generally less than 0.247 ng / ml and muc7 was less than 0.312 ng / ml . assay of muc1 was done according to a previously published protocol .
samples of human saliva were diluted in pbs to protein concentration 10 g / ml and aliquots ( 50 l ) were coated onto microtiter plates ( nunc f96 ; maxisorp , roskilde , denmark ) at room temperature overnight .
the wells were washed 3 times ( 100 l ) with pbs , ph 7.4 , 0.05% tween ( pbs - t ; washing buffer ) between all ensuing steps .
unbound sites were blocked with 100 l of 1% blocking reagent for elisa ( roche diagnostics , mannheim , germany ) for 1 h. the wells were then incubated for 2 h with 100 l of anti - muc1 monoclonal antibody ( anti - ema , clone gp1.4 ; sigma , st louis , mo , usa ) diluted in pbs - t/1% bsa ( sigma , st louis , mo , usa ) 1:500 ( v / v ) .
next , plates were incubated ( 1 h ) with 100 l of secondary antibody , horseradish peroxidase conjugated anti - mouse igg ( sigma , st louis , mo , usa ) diluted in pbs - t/1% bsa 1:1500 ( v / v ) .
plates were washed 4 times in pbs and the colored reaction was developed by incubating with 2.2-azino - bis(3-ethylbenzthiazoline-6-sulfonic acid ) ( abts )  liquid substrate for horseradish peroxidase ( sigma , st louis , mo , usa ) .
absorbance at 405 nm was measured after 3045 min in an elisa reader . as controls , wells with no saliva
the assay was performed in triplicate for saliva and the results are presented as the mean difference between the readings in optical density units ( od ) in experimental wells and controls .
since the data were not normally distributed , the mann - whitney nonparametric u test for unrelated results of muc5b and muc7 was used to compare differences between the groups .
correlations between mucins level and dmf , gi , and pi values in dental caries subjects were assessed by spearman s coefficients ( level of significance , p<0.05 ) .
the adolescents ( age 18 years ) from a high school where students have a high prevalence of dental caries were chosen and served as the experimental group .
the high caries rate of adolescents was examined using the caries intensity index dmf ( decay / missing / filled ; d+m+f / number of the examined ) .
eight adolescents with dmf=3 served as a control group and 27 adolescents with dmf>11 were the research group .
examination of patients dentition with regard to caries was carried out in a school surgery , in artificial light , using basic diagnostic instruments .
the depth of gingival pouches and gingival hemorrhage were examined with a periodontal probe in individual sites in the teeth .
additionally , dental plaque and lesions on the mucus membrane of the oral cavity were evaluated .
saliva from all subjects was collected and the levels of mucins were recorded . the research protocol was approved by the committee for ethics and supervision on human research , medical university of biaystok , with informed consent from the patients .
samples from the subjects were collected between 9:00 and 11:00 a.m. all subjects abstained from eating and drinking for 2 h. unstimulated whole saliva was collected for 10 min by a spitting method .
saliva samples were homogenized and clarified by centrifugation at 10.000  g for 15 min at 4c .
the high sensitivity assay kits ( uscnk ) were used to determine the levels of muc5b and muc7 in the saliva samples .
the microtiter plates provided in the kits were pre - coated with a monoclonal antibody specific to muc5b or muc7 . standards and samples were added to the appropriate microtiter plate wells with a biotin - conjugated polyclonal antibody preparation specific for muc5b or muc7 and were incubated for 1 h at 37c . after washing away any unbound substances ,
avidin conjugated to horseradish peroxidase was added to each microplate well and incubated . after another aspiration and washing step ,
the enzyme - substrate reaction was terminated by the addition of a sulfuric acid solution and the color change was measured at a wavelength of 450 nm .
the assay was performed in duplicate and the concentration of muc5b and muc7 in the samples was then determined by comparing the o.d . of the samples to the standard curve .
the range of the standard curve was 0.62540 ng / ml for muc5b and 0.31220 ng / ml for muc7 .
the minimum detectable dose of human muc5b was generally less than 0.247 ng / ml and muc7 was less than 0.312 ng / ml .
samples of human saliva were diluted in pbs to protein concentration 10 g / ml and aliquots ( 50 l ) were coated onto microtiter plates ( nunc f96 ; maxisorp , roskilde , denmark ) at room temperature overnight . the wells were washed 3 times ( 100 l ) with pbs , ph 7.4 , 0.05% tween ( pbs - t ; washing buffer ) between all ensuing steps .
unbound sites were blocked with 100 l of 1% blocking reagent for elisa ( roche diagnostics , mannheim , germany ) for 1 h. the wells were then incubated for 2 h with 100 l of anti - muc1 monoclonal antibody ( anti - ema , clone gp1.4 ; sigma , st louis , mo , usa ) diluted in pbs - t/1% bsa ( sigma , st louis , mo , usa ) 1:500 ( v / v ) .
next , plates were incubated ( 1 h ) with 100 l of secondary antibody , horseradish peroxidase conjugated anti - mouse igg ( sigma , st louis , mo , usa ) diluted in pbs - t/1% bsa 1:1500 ( v / v ) .
plates were washed 4 times in pbs and the colored reaction was developed by incubating with 2.2-azino - bis(3-ethylbenzthiazoline-6-sulfonic acid ) ( abts )  liquid substrate for horseradish peroxidase ( sigma , st louis , mo , usa ) .
absorbance at 405 nm was measured after 3045 min in an elisa reader . as controls , wells with no saliva
the assay was performed in triplicate for saliva and the results are presented as the mean difference between the readings in optical density units ( od ) in experimental wells and controls .
since the data were not normally distributed , the mann - whitney nonparametric u test for unrelated results of muc5b and muc7 was used to compare differences between the groups .
correlations between mucins level and dmf , gi , and pi values in dental caries subjects were assessed by spearman s coefficients ( level of significance , p<0.05 ) . statistical analysis was carried out using the statistica 6.0 .
the index of intensity of caries / dental caries index - dmf was significantly higher ( p=0.002 ) in the experimental group compared to the control group ( figure 1a ) .
a mean value of gingival index 0.33 below the value of 1 indicates a mild form of gingivitis ( figure 1b ) .
gingivitis seems to be associated with dental plaque at the mean value of 0.53 pi plaque index / periodontal index ( figure 1c ) .
median of muc5b saliva concentration in research group with dmf>11 was 0.7 ng / ml of saliva ( 2575 : 0.0602.34 ) and was higher in comparison to patients with dmf=3 ( control group ) [ 0.05 ng / ml ( 2575 : 0.0500.78 ) ] .
the concentration of muc7 in saliva of patients with dmf>11 ranged from 0.06 ( 25 percentile ) to 0.42 ( 75 percentile ) with the median 0.13 and was lower in comparison to subjects with dmf=3 [ 0.17 ( 2575 : 0.060.32 ) ] .
the difference between groups was not statistically significant ( p=0.918 , table 1 ) due to variation between individuals in the level of muc7 .
figure 4 also shows the relative muc1 transmembrane mucin levels ( expressed as o.d . at 405 nm values ) in saliva of experimental subjects .
significantly higher muc1 level was found in subjects with dmf>11 ( research group ) in relation to subjects with dmf= 3 ( control group ) ( p=0.011 , table 1 ) .
statistical analysis showed a positive correlation between muc1 levels and dmf in the research group ( table 2 ) .
dental caries , as well as obesity , is a growing epidemiological problem in adolescents .
there are many known predisposing factors for dental caries , especially injurious health behaviors and poor nutritional habits , which are based on a higher consumption of sweets and sweet beverages .
recently , links between production of pro - inflammatory cytokines and dental caries process were also indicated .
saliva contains many more components that participate in the protection of the oral tissues ( e.g. lysozyme , lactoferrin , lactoperoxidase , immunoglobulins , agglutinin , and mucins .
research results clearly indicate that salivary mucins perform a variety of functions essential to maintaining a stable oral defense . as a part of the enamel pellicle , mucins help initiate bacterial colonization by promoting the growth of benign commensal oral flora , forming a protective barrier and lubrication against excessive wear , providing a diffusion barrier against acid penetration , and limiting mineral egress from the tooth surface .
our results indicated that high concentration of secreted mucin muc5b predominates in people with severe caries . during caries development ,
a 14-fold increase in muc5b concentration was determined in adolescents with dmf>11 and muc7 mucin concentration was slightly decreased .
our data also demonstrate that muc1 levels are increased in saliva of patients with very severe caries .
our research also showed a statistically significant positive correlation between muc1 levels and dmf in a group with severe caries .
there have been no studies aimed at the comparison of membrane mucin muc1 levels in saliva of healthy subjects with those of people with caries . in a human study
, muc1 was detected as a component of the residual saliva that coats oral mucosal surfaces , meaning that muc1 may aid in the hydration of the oral surface and may interact with oral bacteria .
membrane - bound muc1 mucin may form a scaffold or structural framework upon which the gel - forming mucin muc5b assembles .
the resulting membrane - bound and secreted mucins complex would further strengthen the epithelial protective barrier and increase the lifetime of secreted mucins in the oral cavity .
the release of a muc1 ectodomain to the saliva may cause this mucin to become an inefficient  scaffold maintaining secreted mucins .
our study demonstrated the possible connections between dental caries disease and the concentration of mucins , especially muc1 and muc5b , in saliva .
the knowledge of functional properties of saliva , as well as those of its separate components , may permit a better assessment of dental caries susceptibility .
future research is essential to more completely characterize salivary components and their interactions and how these affect the caries process .
 backgroundhuman saliva , a complex secretion that contains a mixture of inorganic and organic molecules , plays an essential role in the maintenance of oral health . 
 mucins are the major macromolecular component of the secretion and are considered the first line of defense for epithelial tissues . 
 the aim of this study was to compare levels of mucins ( muc5b , muc7 , and muc1 ) in saliva of young subjects with dental caries.material/methodsall patients had dmf ( decay / missing / filled ) higher than value 0 . 
 eight subjects with dmf=3 ( control group ) and 27 adolescents with dmf > 11 ( research group ) were recruited for this study . 
 clinical evaluation procedures were oral examination , including tooth , periodontal , oral mucosal status , and collection of saliva samples . 
 saliva was collected for mucin assay . 
 enzyme - linked immunosorbent assay was used to quantitate muc5b , muc7 , and muc1.resultsour results indicate that adolescents with very high intensity of dental caries disease had increased levels of muc1 and muc5b . 
 the membrane mucin muc1 protein levels in the group with dmf>11 ( research group ) were higher compared to the group with dmf=3 ( control group ) , and the increase was statistically significant ( p=0.011 ) . 
 similarly , secreted mucin muc5b protein levels were higher ( p=0.06 ) in the group with dmf>11 ( research group ) . 
 although muc7 protein levels were slightly reduced in symptomatic subjects , the decrease was statistically insignificant ( p=0.918).conclusionsour data suggest links between the production of mucins , especially muc1 and muc5b in saliva , and dental caries disease . 
Background
Material and Methods
Study population
Salivary sample collection
Analysis of salivary MUC7 and MUC5B
Determination of membrane MUC1
Statistical analysis
Results
Discussion
Conclusions