Comparison of cathelicidine, alpha 1 defensin, histatin 1, and statherin levels in saliva between children with early childhood caries and caries-free children (case–control study)

Comparison of cathelicidine, alpha 1 defensin, histatin 1, and statherin levels in saliva between children with early childhood caries and caries-free children (case–control study) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Comparison of cathelicidine, alpha 1 defensin, histatin 1, and statherin levels in saliva between children with early childhood caries and caries-free children (case–control study) Vlasta Merglova, Jindra Windrichova, Lucie Baborska, Martina Pestova, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6905254/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Background Early childhood caries (ECC) is a chronic, microbial, carbohydrate-driven multifactorial disease. The composition of saliva, quantity, pH buffering capacity, presence of microorganisms, and amounts of peptides and proteins play important roles in the development and progression of dental caries. Salivary peptides and proteins are crucial for the formation of acquired enamel pellicles, protection against infections, and maintenance of calcium homeostasis. This study aimed to investigate the salivary levels of antimicrobial peptides (cathelicidine, alpha 1 defensin, histatin 1, and statherin) in children with early childhood caries and caries-free children. Methods This case‒control study involved 82 children (mean age 5.18 years) from the Pediatric Dentistry Department in Pilsen, Czech Republic. Children were enrolled on the basis of medical history and clinical examination and then divided into a case group (children with ECC) and a control group (caries-free children). Dental caries was recorded according to the WHO criteria, and the dmf index was calculated. Stimulated saliva was collected, and selected antimicrobial peptides were assessed via magnetic bead-based fluorescence assay kits following the manufacturer's instructions (Cloud-Clone Corp.). The data were statistically analyzed via a version without a multivariate regression model and a version with a multivariate regression model adjusted for age and sex. Graphical comparisons of antimicrobial peptide levels between groups were performed via box plots, with all tests performed at a significance level of 5%. Results Significant differences in age were found between the case group (mean 5.5 years) and the control group (mean 4.56 years), with no significant differences in sex. Compared with caries-free children, children with ECC (mean dmf index of 7.2) had higher levels of cathelicidin and statherin and lower levels of alpha defensin and histatin 1 in stimulated saliva. However, these differences were not statistically significant. Conclusions The results of the present study did not confirm statistically significant differences in the levels of selected antimicrobial peptides in the saliva, yet this issue deserves further attention. Early childhood caries saliva antimicrobial peptides biomarkers defensins histatins cathelicidins statherins Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Early childhood caries (ECC) is defined as the presence of one or more carious lesions (either noncavitated or cavitated) on any primary tooth, as well as any filling or tooth lost due to caries and its complications in children under six years of age [ 1 , 2 ]. Dental caries in early childhood, similar to dental caries in general, is a chronic, microbial, carbohydrate-driven multifactorial disease that arises from an imbalance between the processes of demineralization and remineralization on the surfaces of hard dental tissues. Dental caries shares common risk factors with other noncommunicable diseases, where excessive carbohydrate consumption plays a significant role in their etiology, such as cardiovascular disease, diabetes, and obesity [ 1 ]. Current methods for preventing ECC emphasize the early detection of risk factors, enabling the initiation of preventive measures before the first signs of disease manifest [ 3 ]. To determine the risk of developing dental caries, much attention has been given to saliva, not only in terms of its quantity, pH, buffering capacity and presence of microorganisms but also in terms of the peptides and proteins contained within it [ 4 , 5 ]. Saliva is readily available, can be obtained noninvasively, and contains many biomolecules, including those found in serum, that are considered useful for diagnosing and monitoring disease progression. In addition to salivary gland products, saliva also contains gingival sulcus and mucosal transudate fluids, products of bacterial metabolism, components of nasal and pharyngeal secretions, food debris, loose epithelial cells, blood cells and microorganisms. It consists of 98–99% water, in which inorganic substances (hydrogen, potassium, calcium, magnesium, bicarbonates, phosphates and fluorides) and low- and high-molecular-weight organic compounds, including proteins, peptides and enzymes, are dissolved [ 6 ]. Saliva performs a number of physiological functions, such as digestion; aids in food swallowing; lubricates tissues; protects hard dental tissues; and has antimicrobial, antiviral and antifungal functions. The composition of saliva responds not only to oral diseases but also to general diseases and their therapy; therefore, saliva is considered a suitable source of biomarkers for oral diseases and many general diseases [ 7 , 8 ]. Salivary peptides and proteins are essential for the proper function of saliva. The oral cavity is a gateway for a number of microorganisms, and it is the site where hard dental tissues are in contact with an aggressive environment. The role of peptides and proteins is important in the initial phase of digestion, in the formation of the acquired enamel pellicle, in providing protection against various infectious influences, and in maintaining calcium homeostasis, which is required for enamel remineralization processes [ 9 ]. Antimicrobial peptides are an essential part of innate immunity, forming the first line of defense against microbial colonization of the oral cavity and the development of infections. Most antimicrobial peptides act against gram-negative and gram-positive bacteria, fungi and viruses [ 10 , 11 ]. Tehy is produced by epithelial tissues, phagocytes, the parotid gland, gingival tissue, and lateral tongue tissue. In the oral cavity, they can be found in saliva, gingival epithelium, and gingival sulcus fluid. Salivary antimicrobial peptides protect hard dental tissues from caries and the oral mucosa. They work in conjunction with other salivary antimicrobial components to enhance their effects on oral microorganisms, stimulate the immune system, increase the production of IgA and IgC immunoglobulins, and prevent biofilm formation. Salivary antimicrobial peptides are classified on the basis of amino acid composition, structure, and size, with major groups including defensins, histatins, cathelicidins, and statherins. The aim of the current study was to compare the salivary levels of the most important antimicrobial peptides related to dental caries development in children with ECC and in caries-free children. Ethical consideration Ethical approval for the investigation and trial registration were obtained from the Research Ethics Committee of the Medical Faculty in Pilsen, Charles University, Czech Republic (No 367/2020), and the study was conducted in accordance with the Helsinki Declaration of 1975, as revised in 2013 [ 12 ]. Prior to the study, the legal guardians of all the children provided informed consent for their children to participate. Materials and methods Study participants All the children were recruited from the Pediatric Dentistry Department (Department of Stomatology) at the University Hospital in Pilsen, Czech Republic. The study group included Caucasian children of both genders. Children were recruited from the Pediatric Dentistry Department during the study period (2023–2024) and gradually included in the project over a 12-month period as they came in for examination. The main inclusion criteria were children between the ages of 3 and 7 years and self-reported middle socioeconomic class. The case group included children reporting ECC in their primary dentition with one or more decayed, missing, or filled teeth in any primary tooth. The control group included children without decayed teeth in their primary dentition, without fillings and without a history of primary tooth extractions due to caries. The exclusion criteria for both groups were the presence of systemic disease, uncooperative behavior during examination, disability, and age over 7 years. Clinical evaluation The children´s medical history was obtained through interviews with legal guardians. The dental examinations were conducted by one experienced examiner under standardized conditions at the Pediatric Dentistry Department via a sterile dental mirror, a dental probe and artificial light. Dental caries was recorded according to the WHO criteria [ 13 ]. All tooth surfaces were visually examined. The dental probe was used only to remove debris and food particles to improve visibility. Caries was recorded as present when a lesion in a pit or fissure or on a smooth tooth surface had an unmistakable cavity, undermined enamel or a detectably softened floor or wall. The dmf score was calculated by adding the number of primary teeth affected by caries, missing due to caries, and teeth with fillings. Saliva collection and analysis Compared with unstimulated saliva samples, stimulated saliva was used for the current research because of its ease of collection in young children and its time-saving nature. Saliva was collected via a commercial product, Salivette® (Sarstedt, Germany). To collect saliva, it is important to follow the recommended procedure [ 14 ]. Collection should be performed via a cellulose cotton swab at least 30 minutes after the consumption of food and drinks to avoid contamination. Smoking and the consumption of caffeinated beverages before collection are also advised. Contamination can occur from cosmetics or blood, especially in patients with gingivitis, periodontitis, or recent dental hygiene procedures. To make it easier to obtain a sufficient saliva sample in children, it is advisable to drink at least 200 ml of fluid an hour before collection; for adults, an amount of fluid of 250–500 ml is recommended. To collect stimulated saliva, the patient gently chews a cellulose swab for 60 seconds; during chewing, the stimulated saliva is absorbed into the swab. The samples of stimulated saliva were immediately transported to the laboratory and centrifuged, the supernatant was collected, and aliquots of 350 µl were stored in the biobank at -80°C until processing. The determination of selected antimicrobial peptides was performed via magnetic bead-based fluorescence assay kits according to the manufacturer's instructions (Cloud-Clone Corp., USA). These kits apply multiplex sandwich enzyme immunoassays based on xMAP® technology for in vitro quantitative measurement of CAMP, DEF alfa 1, HTN1, and STATH in human serum, plasma, tissue homogenates, cell lysates, cell culture supernatants, and other biological fluids. Detection was performed with a MAGPIX instrument (Luminex Corporation, USA). The xMAP® Technology and MAGPIX system uses color-coded magnetic microspheres coated with antibodies to perform multiplex quantitative sandwich immunoanalysis of proteins in a variety of sample matrices. The amount of each salivary antimicrobial peptide is given in ng/ml of saliva. Statistical analysis The data obtained were statistically analyzed via STATISTICA 14 software (StatSoft, USA). Both the version without a multivariate regression model and the version with a multivariate regression model were used for statistical processing. Continuous variables are presented as the mean ± standard deviation (SD). Two-sample t tests and multivariate regression models adjusted for age and sex were used to compare the monitoring parameters cathelicidin (CAMP), alpha defensin (DEF alfa1), histatin 1 (HTN1), and statherin (STATH) between the case and control groups. Graphical comparisons of parameters between groups were conducted via box plots; all tests were performed at a significance level of 5%. The box plots display the median (box: 25th /75th percentiles), and the whiskers show the minimum/maximum levels of salivary antimicrobial peptides in the case and control groups. Outliers that significantly differ from the rest of the dataset are presented as individual points beyond the whiskers on the box plot. Results A total of 82 participants were enrolled in the study. The case group was composed of 55 children (32 boys and 23 girls) and the control group comprised 27 children (10 boys and 17 girls). The demographic and clinical characteristics (age, sex and dmf index) of all participants are presented in Table 1 . The children of the case group have a mean age 5.49 years and mean dmf index of 7.2, and the children of the control group has a mean age 4.56 years and dnf index zero. Table 1 Descriptive statistics of sex, age and dmf index in children of the case and control groups gender dmf index number male female mean age ± SD (years) mean median max. min. SD Case group 55 32 (58.18%) 23 (41.82%) 5.4909 ± 0.9598 7.2 7 19 1 4.45 Control group 27 10 (37,04%) 17 (62.96%) 4.5556 ± 0.89156 0 0 0 0 0 Total 82 42 (51.21%) 40 (48,79%) 5.1829 ± 1.0319 SD – Standard Deviation Comparison of variables between the case and control groups The results of the p-values of the individual two-sample tests revealed that there was a statistically significant difference between the case and control groups only for the variable age, and the observed variables CAMP, DEF alfa1, HTN1, and STATH did not differ significantly between the case and control groups (Table 2 ). Table 2 Comparison of salivary antimicrobial peptide concentration and age between the case and control groups of children Variable Case group (mean concentration) Control group (mean concentration) t- value p SD case group SD control group CAMP 179.7973 136.3122 1.104079 0.272869 198.4513 93.08503 Def alfa1 0.8138 0.8763 -0.643233 0.521912 0.4055 0.42910 HTN1 3.8011 4.0193 -0.394905 0.693964 2.6359 1.60530 STATH 22.4573 23.4674 -0.233092 0.816285 17.8401 19.63323 age 5.4909 4.5556 4.242808 0.000059* 0.9598 0.89156 SD – Standard Deviation P < 0.005 statistically significant Comparison of variables in the case group The results of the p values of the individual two-sample tests revealed that in the case group, the observed variables CAMP, DEF alfa1, HTN1, and STATH did not differ between the sexes.. Comparison of variables in the control group The resulting p values of the individual two-sample tests revealed that in the control group, the observed variables CAMP, DEF alfa1, HTN1, and STATH did not differ between the sexes. Multivariate regression model A multivariate regression model was used to assess differences in the observed parameters CAMP, DEF alfa1, HTN1, and STATH between groups, adjusting for the age and sex of the children. The results of the individual regression models revealed that there was no statistically significant difference between the case and control groups for any of the observed parameters (CAMP, DEF alfa1, HTN1, and STATH) after adjustment for age and sex). Graphical comparisons of the parameters between the groups were performed via box plots. Children with ECC (case group) had higher mean CAMP levels in saliva than did children with intact dentition (control group). However, the difference was not statistically significant (p = 0.41) (Fig. 1 ). The findings were different for the mean values of DEF alfal and HTN1. Decreased values of these antimicrobial proteins were detected in the saliva of the children in the case group. The difference was not statistically significant (p = 0.78 resp. 0.93) (Figs. 2 , 3 ). With respect to STATH, a similar situation to CAMP was found. Children with ECC (case group) had higher mean salivary levels of this antimicrobial peptide than did the children in the control group. The difference between the case and control groups of children was again statistically nonsignificant (p = 0.63) (Fig. 4 ). Discussion Dental caries in early childhood is one of the most widespread diseases of childhood and can cause many complications for the child and significantly reduce the quality of life of the child and the whole family. ECC is a multifactorial disease involving dental microbial plaque, dietary sugars, the quality of hard dental tissues, the amount and composition of saliva and likely other risk factors that have not yet been fully investigated. These factors include proteins present in saliva, which are potential biomarkers of dental caries in early childhood [ 15 – 17 ]. The importance of salivary peptides and proteins in relation to dental caries and in determining the risk of dental caries has been addressed by a number of authors [ 18 – 22 ], who have not reached unequivocal conclusions in their studies. Among the cathelicidins, the human cationic antimicrobial peptide hCAP18, which cleaves serine proteases to the antimicrobial peptide LL-37, is found in the oral cavity and respiratory tract of humans. These molecules cause damage to the cell membranes of microorganisms. LL-37 is characterized by high activity against Streptococcus mutans (SM), Streptococcus sanguis , Streptococcus mitis , Fusobacterium nucleatum , Aggregatibacter actinomycetemcomitans , and Capnocytophaga sputigena [ 11 ]. Some authors [ 23 – 25 ] suggest that reduced levels of antimicrobial peptides, especially LL-37, in saliva may be associated with the occurrence of dental caries. The relationships between dental caries in children with a mean age of 8.7 years and salivary LL-37 concentrations were investigated by Davidopoulou et al. [ 25 ]. They reported that children with intact dentition had significantly higher LL-37 concentrations than did children with high caries activity. The results of this study suggest that the antimicrobial peptide LL-37 is present in saliva in early childhood, but its concentration is low. The concentration of this antimicrobial peptide in saliva increases with age and reaches a maximum in late adolescence. Almondi et al. [ 26 ] came to similar conclusions and hypothesized a protective role of salivary LL-37 in caries development. The authors reported low levels of salivary LL-37 and high levels of SM and Streptococcus sobrinus in saliva samples from children with ECC. Different conclusions were reached by Colombo et al. [ 27 ], who monitored the levels of LL-37, beta defensin 2, beta defensin 3 and histatin 5 in children with ECC. They reported no relationship between the presence of ECC and the levels of these antimicrobial peptides. The antimicrobial peptide LL-37 was evaluated in the current study for both the case group (caries active) and the control group (caries free). The results revealed that the salivary levels of LL-37 in caries-active children were greater than those in those without caries. This difference was not statistically significant (p = 0.27). These results are consistent with the findings of Nireeksha et al. [ 24 ]. The relationship between LL–37 and ECC is currently unclear [ 18 ]. Defensins are short cationic peptides with low molecular weights and 6–8 cysteine residues that form 3–4 intramolecular disulfide bonds. They have the ability to act against all types of gram-positive and gram-negative bacteria, fungi and viruses [ 9 – 11 ]. They are categorized into alpha defensins and beta defensins on the basis of structural variances and tissue distributions. Alpha defensins are formed in neutrophils and salivary gland duct cells and are present in gingival sulcus fluid and tissues affected by inflammation. Beta defensins (hBD-1, hBD-2, and hBD-3) are produced by oral epithelial cells and are found in gingival sulcus fluid and saliva [ 6 ]. The mechanisms of action of defensins on pathogens are not yet fully understood, but defensins are likely to disrupt bacterial cell walls. Tao et al. [ 19 ] investigated the correlation between the antimicrobial peptide alpha defensin and dental caries. They reported significantly higher levels of alpha defensin in children aged 11–15 years without dental caries than in a control group of children with dental caries of the same age. Low levels of alpha defensin in the saliva of schoolchildren may be associated with the presence of dental caries, as described by Dale et al. [ 28 ], who also reported low levels of alpha defensin in patients with dental caries. Ribeiro et al. [ 29 ] reported that alpha and beta defensins decrease the incidence of ECC. However, studies using more precise methods of salivary protein determination have not shown significant differences in the levels of alpha and beta defensins in the saliva of children with ECC and those with intact dentition [ 27 , 30 , 31 , 32 ]. Similarly, this study revealed no statistically significant difference in the amount of alpha 1 defensin in the saliva of children with ECC and those without caries (p = 0.52). On the basis of available studies, systematic reviews, and the current study, defensins cannot be considered reliable for determining the risk of ECC [ 27 , 30 – 33 ]. Histatins are a group of cationic antimicrobial peptides with low molecular weights and high contents of the amino acid histidine. The main representatives are histatins 1, 3 and 5, which make up 1–1.5% of all low-molecular-weight salivary proteins. The levels of histatins in saliva change with age. Research [ 33 ] has shown that the concentration of histatin 1 is significantly greater in children aged 3–5 years than in older children. These peptides are produced by the cells of the parotid and submandibular salivary glands and are known for their antimicrobial effects against SM and Porphyromonas gingivalis [ 34 ]. Histatins work by integrating into the cell membrane of bacteria and increasing membrane permeability, leading to bacterial destruction [ 35 ]. They also inhibit the growth of Candida fungi [ 10 ] and have the ability to form complexes with metal ions, making them inaccessible to enzymes and releasing reactive oxygen species that affect bacterial cellular structures [ 30 , 35 ]. Histatins also have an affinity for the enamel surface and play a significant role in the formation of the enamel pellicle, limiting bacterial colonization on tooth surfaces. Additionally, they suppress the spontaneous growth of hydroxyapatite crystals in saliva containing a supersaturated solution of calcium phosphate [ 35 ]. A study by Sun et al. [ 36 ] investigated the salivary peptidome in children aged 3–5 years with severe ECC. Severe ECC is an aggressive form of ECC characterized by specific criteria, such as caries on smooth tooth surfaces in children under 3 years of age, caries on upper frontal teeth and specific dmf index values at different ages. Children aged 3–5 years with severe ECC were found to have lower histatin 1 levels before treatment than after treatment. Histatin 1 could serve as a biomarker for severe ECC on the basis of previous studies and systematic reviews [ 32 , 33 , 37 ]. Another study by Vitorino et al. [ 38 ] revealed a strong correlation between the absence of dental caries and high levels of histatin 1, statherin, and proline-rich proteins (PRPs). In the present study, higher amounts of histatin 1 were detected in the caries-free children, although the difference between the case and control groups was not statistically significant (p = 0.69). Statherins are low-molecular-weight acidic peptides composed of 43 amino acids. They have the ability to bind to the hydroxyapatite of enamel, inhibit the spontaneous precipitation of calcium and phosphate salts from supersaturated saliva, and inhibit the growth of hydroxyapatite crystals [ 6 ]. Statherins are considered precursors of the acquired pellicle because of their strong affinity for hydroxyapatite. They allow remineralization of enamel and cause aggregation of bacteria, thereby reducing their ability to adhere to hard and soft tissues of the oral cavity. The relationship between statherin and ECC is also under investigation. Vitorino et al. [ 38 ] reported an association between elevated levels of PRPs, histatin 1, and statherin in children without caries. Increased amounts of salivary statherin were detected in the caries-free children in the present study, but the difference between the children with ECC and the caries-free children was not statistically significant (p = 0.81). The suitability of statherin as a biomarker of ECC requires further validation in clinical studies. The results of the present study did not confirm the expected differences in the amount of selected antimicrobial peptides in the saliva of children with ECC and children without dental caries. The results of the current study are partially in accordance with the findings of other authors [ 24 , 27 , 30 – 32 , 38 ], but the differences in the levels of antimicrobial peptides between the case and control groups of children are not statistically significant. An explanation for this may be that the saliva used for analysis in the present study was stimulated saliva, whereas the available studies used unstimulated saliva. Saliva collection might affect proteomic biomarkers. The major salivary glands are more involved in the production of stimulated saliva, and this saliva has different levels of specific ions, proteins, and water than unstimulated saliva. Unstimulated saliva represents an equilibrated condition, with less impact on the major salivary glands [ 39 ]. Stimulated saliva seems to be less suitable for the determination of salivary biomarkers of ECC. Conclusion Salivary antimicrobial peptide (CAMP, DEF alfa1, HTN1 and STATH) levels were detected in the stimulated saliva of children with ECC and caries-free children. Children with ECC had higher levels of CAMP and STATH than did caries-free children, whereas the situation was reversed for DEF alfa1 and HTN1. Children with ECC had lower amounts of these antimicrobial peptides in stimulated saliva, but these differences were not statistically significant. The study partially confirmed the results of other authors. ECC is a multifactorial disease with various main risk factors, such as poor dietary habits, poor oral hygiene, sociodemographic factors, and factors related to the composition of oral bacteria. In addition to oral hygiene, nutrition plays an important role in the development of dental caries in childhood. When determining the risk of ECC, these risk factors should be focused on and easily identified through interviews with the child's guardians and during the clinical examination, followed by evaluation of salivary proteins. Limitations of the study Limitations of the study include inconsistencies in the age of the children in the groups, with the control group being younger than the case group. The study also did not evaluate oral hygiene practices or nutritional habits, relying solely on stimulated saliva to determine the levels of antimicrobial peptides. Abbreviations ECC – early childhood caries WHO – World Health Organization dmf index – decayed, missing and filled teeth CAMP – cathelicidin DEF alfa1 – alpha defensin hBD – 1 - beta defensin 1 hBD – 2 - beta defensin 2 hBD – 3 – beta defensin 3 HTN1 – histatin 1 STATH – statherin PRPs – proline-rich proteins SD - standard deviation hCAP18 – human cationic antimicrobial peptide SM – Streptococcus mutans ng/ml – nanograms per millilitre m1 - microlitre Declarations Ethics approval and consent to participate Ethical approval for the investigation and trial registration were obtained from the Research Ethics Committee of Medical Faculty in Pilsen, Charles University, Czech Republic (No 367/2020). Before the study, the legal guardians of all children provided the informed consent for their children to participate in the study. Consent for publication Not applicable Availability of data and materials The datasets analysed during the current study are available from the corresponding author on reasonable request. Competing interest The authors declare that they have no competing interests. Funding This research was supported by a grant from the Ministry of Health of the Czech Republic – Conceptual Development of Research Organization Faculty Hospital in Pilsen – FNPI, 00669806. Author´s contributions VM designed the study JW performed the meassurment of peptides LB collected clinical samples MP performed the saliva processing MR analyzed the data The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. References Pitts NB, Baez RJ, Diaz- Guillory C, Donly KJ, Feldens CA, McGrath C et al. Early Childhood Caries: Bangkok Declaration IAPD. Int J Paediatr Dent. 2019;29(3):384‐6. doi: 10.1111/ipd.12490. Ramalingam L, Messer LB. 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Ribeiro TR, Dria KJ, de Carvalho CB, Monteiro AJ, Fonteles MC,de Moraes Cavalho K et al. Salivary peptide profile and its association with early childhood caries. Int J Paediatr Dent. 2013;23(3):225-34. doi: 10.1111/j.1365-263X.2012.01258.x Malcolm J, Sherriff A, Lappin DF, Ramage G, Conway DI, Macpherson LMD. Salivary antimicrobial proteins associate with age-related changes in streptococcal composition in dental plaque. Mol Oral Microbiol.2014;29(6):284-93. doi: 10.1111/omi.12058 Toomarian L, Sattari M, Hashemi N, Tadayon N, Baghban AA. Comparison of neutrophil apoptosis, alpha-defensins and calprotectin in children with and without severe early childhood caries. Iranian J Immunol. 2011;8(1):11-9. Jurczak A, Košcielniak D, Papiež M, Vyhouskkaya P, Krzyšciak W. A study of β- defensin – 2 and histatin – 5 as diagnostiic marker of early childhood caries progression. Biol Res.2015;48:61. doi: 10.1186/s40659-015-0050-7. Hemadi AS, Huang R, Zhou Y, Zou J.Salivary proteins and microbiota as biomarkers for early childhood caries risk assessment. Int J Oral Sci. 2017;9(11): e1. doi: 10.1038/ijos.2017.35. Gusman H, Travis J, Helmerhorst EJ, Potempa J, Troxler RF, Oppenheim FG. Salivary Histatin 5 Is an Inhibitor of Both Host and Bacterial enzymes Implicated in Periodontal Disease. Infect Immun. 2001;69(3):1402-8. doi: 10.1128/IAI69.3.1402.2001. Melino S, Di Nardo CSP, Sarkar B. Histatins: salivary peptides with copper (II) – and zinc (II) – binding motifs. FEBS J. 2013;281(3):657-72. doi: 10.1111/febs.12612. Sun X, Huang X, Tan X, Si Y, Wang X, Chen F et al. Salivary peptidome profiling for diagnosis of severe early childhood caries. J Transl Med. 2006;14(1):240. doi:10.1186/s12967-016-0996-4. Ao S, Sun X, Shi X, Huang X, Chen F, Zheng S . Longitudinal investigation of salivary proteomic profiles in the development of early childhood caries. J Dent.2017;61:21-7. doi: 10.1016/j.dent.2017.04006. Vitorino R, Lobo MJ, Duarte JR, Ferrer-Correia AJ, Domingues PM, Amado FM. The role of salivary peptides in dental caries. Biomed Chromatogr. 2005 Apr;19(3):214-22. doi: 10.1002/bmc.438. Ohshiro K, Rosenthal DI, Koomen JM, Streckfus CF, Chambers M, Kobayashi R. Preanalytic saliva processing affects proteomic results and biomarker screening of head and neck squamous carcinoma. Int J Oncol 2007;30(3):743–9. doi: 10.3892/ijo.30.3.743. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 07 Jul, 2025 Editor assigned by journal 05 Jul, 2025 Submission checks completed at journal 05 Jul, 2025 First submitted to journal 16 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6905254","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":471946349,"identity":"fd12f6cf-f07e-4a5e-a4ea-edb1959e90f7","order_by":0,"name":"Vlasta Merglova","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5klEQVRIiWNgGAWjYLCCByCCvRFIFRCrJQFE8Bw2YGAwIEmLRDKRWnTbex+/SKg4LG9w8zHb4wKDOjlzBubHH/BpMTtz3Mwi4cxhww23k9mNZxiwGVs2sBngtczsRhqbQWJbGuPM2fnHpHkMeBI3HOCBuBSnlvvPwFrsZ848zAbUIlEP0nIAvy1szA8S22wS+yWYQVoMEgwO8DA24PdLGhtDwhmb5H6eZDbpGQYJhhsOsxnj08FgdvwY84cPFRK2bexAhxVU1MkbHG/GH2JAwCYBYzEjkXgBM9xMIhSPglEwCkbBSAQAQ/pDpaYz5kcAAAAASUVORK5CYII=","orcid":"","institution":"University Hospital in Pilsen, Charles University","correspondingAuthor":true,"prefix":"","firstName":"Vlasta","middleName":"","lastName":"Merglova","suffix":""},{"id":471946350,"identity":"066b552c-5379-4550-938d-5c6a3b5deae5","order_by":1,"name":"Jindra Windrichova","email":"","orcid":"","institution":"University Hospital in Pilsen, Charles University","correspondingAuthor":false,"prefix":"","firstName":"Jindra","middleName":"","lastName":"Windrichova","suffix":""},{"id":471946351,"identity":"500072dd-e09f-460d-bb77-2b722741afde","order_by":2,"name":"Lucie Baborska","email":"","orcid":"","institution":"University Hospital in Pilsen, Charles University","correspondingAuthor":false,"prefix":"","firstName":"Lucie","middleName":"","lastName":"Baborska","suffix":""},{"id":471946352,"identity":"423bb027-de81-4b2f-ba38-81db3f617b73","order_by":3,"name":"Martina Pestova","email":"","orcid":"","institution":"University Hospital in Pilsen, Charles University","correspondingAuthor":false,"prefix":"","firstName":"Martina","middleName":"","lastName":"Pestova","suffix":""},{"id":471946353,"identity":"03039849-92b9-4911-b806-8747ebb8704b","order_by":4,"name":"Marian Rybar","email":"","orcid":"","institution":"MatStat","correspondingAuthor":false,"prefix":"","firstName":"Marian","middleName":"","lastName":"Rybar","suffix":""}],"badges":[],"createdAt":"2025-06-16 11:53:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6905254/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6905254/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84920844,"identity":"7172e2d6-fe9d-45c2-a9fd-609a32bf4f79","added_by":"auto","created_at":"2025-06-18 19:40:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":212814,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of salivary CAMP concentration between the case and control groups of children schowed no significant difference with p ˃ 0.05\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6905254/v1/74925c636630216167a70b72.png"},{"id":84921746,"identity":"57be44f7-1a5c-4a2e-8ebf-e9f0d90546bb","added_by":"auto","created_at":"2025-06-18 19:56:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":222186,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of salivary DEFalfa1 concentration between the case and control groups of children showed no significant difference with p ˃0.05\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6905254/v1/4deb0797872e0ab9a24a08d3.png"},{"id":84920847,"identity":"cb3c36b8-af38-4e58-ab0d-e54522d1a1f9","added_by":"auto","created_at":"2025-06-18 19:40:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":203923,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of salivary HTN1 concentration between he case and control groups of children showed no significant difference with p ˃0.05\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6905254/v1/ece2ff9ee01dc87cf4cc8ff0.png"},{"id":84920846,"identity":"05f2a7c7-2974-460c-b055-1fbd93de744b","added_by":"auto","created_at":"2025-06-18 19:40:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":217399,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of salivary STATH concentration between the case and control groups of children showed no significant difference s with p ˃0.05\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6905254/v1/2ca05ad531253116c5105642.png"},{"id":84922023,"identity":"fed23e42-0ff8-4016-8831-3892bb99cb7b","added_by":"auto","created_at":"2025-06-18 20:04:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1413189,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6905254/v1/e904bd1b-2fa6-4391-887e-4d05dc696782.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparison of cathelicidine, alpha 1 defensin, histatin 1, and statherin levels in saliva between children with early childhood caries and caries-free children (case–control study)","fulltext":[{"header":"Background","content":"\u003cp\u003eEarly childhood caries (ECC) is defined as the presence of one or more carious lesions (either noncavitated or cavitated) on any primary tooth, as well as any filling or tooth lost due to caries and its complications in children under six years of age [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Dental caries in early childhood, similar to dental caries in general, is a chronic, microbial, carbohydrate-driven multifactorial disease that arises from an imbalance between the processes of demineralization and remineralization on the surfaces of hard dental tissues. Dental caries shares common risk factors with other noncommunicable diseases, where excessive carbohydrate consumption plays a significant role in their etiology, such as cardiovascular disease, diabetes, and obesity [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Current methods for preventing ECC emphasize the early detection of risk factors, enabling the initiation of preventive measures before the first signs of disease manifest [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. To determine the risk of developing dental caries, much attention has been given to saliva, not only in terms of its quantity, pH, buffering capacity and presence of microorganisms but also in terms of the peptides and proteins contained within it [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Saliva is readily available, can be obtained noninvasively, and contains many biomolecules, including those found in serum, that are considered useful for diagnosing and monitoring disease progression. In addition to salivary gland products, saliva also contains gingival sulcus and mucosal transudate fluids, products of bacterial metabolism, components of nasal and pharyngeal secretions, food debris, loose epithelial cells, blood cells and microorganisms. It consists of 98\u0026ndash;99% water, in which inorganic substances (hydrogen, potassium, calcium, magnesium, bicarbonates, phosphates and fluorides) and low- and high-molecular-weight organic compounds, including proteins, peptides and enzymes, are dissolved [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Saliva performs a number of physiological functions, such as digestion; aids in food swallowing; lubricates tissues; protects hard dental tissues; and has antimicrobial, antiviral and antifungal functions. The composition of saliva responds not only to oral diseases but also to general diseases and their therapy; therefore, saliva is considered a suitable source of biomarkers for oral diseases and many general diseases [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Salivary peptides and proteins are essential for the proper function of saliva. The oral cavity is a gateway for a number of microorganisms, and it is the site where hard dental tissues are in contact with an aggressive environment. The role of peptides and proteins is important in the initial phase of digestion, in the formation of the acquired enamel pellicle, in providing protection against various infectious influences, and in maintaining calcium homeostasis, which is required for enamel remineralization processes [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAntimicrobial peptides are an essential part of innate immunity, forming the first line of defense against microbial colonization of the oral cavity and the development of infections. Most antimicrobial peptides act against gram-negative and gram-positive bacteria, fungi and viruses [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Tehy is produced by epithelial tissues, phagocytes, the parotid gland, gingival tissue, and lateral tongue tissue. In the oral cavity, they can be found in saliva, gingival epithelium, and gingival sulcus fluid. Salivary antimicrobial peptides protect hard dental tissues from caries and the oral mucosa. They work in conjunction with other salivary antimicrobial components to enhance their effects on oral microorganisms, stimulate the immune system, increase the production of IgA and IgC immunoglobulins, and prevent biofilm formation. Salivary antimicrobial peptides are classified on the basis of amino acid composition, structure, and size, with major groups including defensins, histatins, cathelicidins, and statherins.\u003c/p\u003e \u003cp\u003eThe aim of the current study was to compare the salivary levels of the most important antimicrobial peptides related to dental caries development in children with ECC and in caries-free children.\u003c/p\u003e \u003cp\u003eEthical consideration\u003c/p\u003e \u003cp\u003eEthical approval for the investigation and trial registration were obtained from the Research Ethics Committee of the Medical Faculty in Pilsen, Charles University, Czech Republic (No 367/2020), and the study was conducted in accordance with the Helsinki Declaration of 1975, as revised in 2013 [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Prior to the study, the legal guardians of all the children provided informed consent for their children to participate.\u003c/p\u003e \u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eStudy participants\u003c/p\u003e \u003cp\u003eAll the children were recruited from the Pediatric Dentistry Department (Department of Stomatology) at the University Hospital in Pilsen, Czech Republic. The study group included Caucasian children of both genders. Children were recruited from the Pediatric Dentistry Department during the study period (2023\u0026ndash;2024) and gradually included in the project over a 12-month period as they came in for examination. The main inclusion criteria were children between the ages of 3 and 7 years and self-reported middle socioeconomic class. The case group included children reporting ECC in their primary dentition with one or more decayed, missing, or filled teeth in any primary tooth. The control group included children without decayed teeth in their primary dentition, without fillings and without a history of primary tooth extractions due to caries. The exclusion criteria for both groups were the presence of systemic disease, uncooperative behavior during examination, disability, and age over 7 years.\u003c/p\u003e \u003cp\u003eClinical evaluation\u003c/p\u003e \u003cp\u003eThe children\u0026acute;s medical history was obtained through interviews with legal guardians. The dental examinations were conducted by one experienced examiner under standardized conditions at the Pediatric Dentistry Department via a sterile dental mirror, a dental probe and artificial light. Dental caries was recorded according to the WHO criteria [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. All tooth surfaces were visually examined. The dental probe was used only to remove debris and food particles to improve visibility. Caries was recorded as present when a lesion in a pit or fissure or on a smooth tooth surface had an unmistakable cavity, undermined enamel or a detectably softened floor or wall. The dmf score was calculated by adding the number of primary teeth affected by caries, missing due to caries, and teeth with fillings.\u003c/p\u003e \u003cp\u003eSaliva collection and analysis\u003c/p\u003e \u003cp\u003eCompared with unstimulated saliva samples, stimulated saliva was used for the current research because of its ease of collection in young children and its time-saving nature. Saliva was collected via a commercial product, Salivette\u0026reg; (Sarstedt, Germany). To collect saliva, it is important to follow the recommended procedure [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Collection should be performed via a cellulose cotton swab at least 30 minutes after the consumption of food and drinks to avoid contamination. Smoking and the consumption of caffeinated beverages before collection are also advised. Contamination can occur from cosmetics or blood, especially in patients with gingivitis, periodontitis, or recent dental hygiene procedures. To make it easier to obtain a sufficient saliva sample in children, it is advisable to drink at least 200 ml of fluid an hour before collection; for adults, an amount of fluid of 250\u0026ndash;500 ml is recommended. To collect stimulated saliva, the patient gently chews a cellulose swab for 60 seconds; during chewing, the stimulated saliva is absorbed into the swab. The samples of stimulated saliva were immediately transported to the laboratory and centrifuged, the supernatant was collected, and aliquots of 350 \u0026micro;l were stored in the biobank at -80\u0026deg;C until processing. The determination of selected antimicrobial peptides was performed via magnetic bead-based fluorescence assay kits according to the manufacturer's instructions (Cloud-Clone Corp., USA). These kits apply multiplex sandwich enzyme immunoassays based on xMAP\u0026reg; technology for in vitro quantitative measurement of CAMP, DEF alfa 1, HTN1, and STATH in human serum, plasma, tissue homogenates, cell lysates, cell culture supernatants, and other biological fluids. Detection was performed with a MAGPIX instrument (Luminex Corporation, USA). The xMAP\u0026reg; Technology and MAGPIX system uses color-coded magnetic microspheres coated with antibodies to perform multiplex quantitative sandwich immunoanalysis of proteins in a variety of sample matrices. The amount of each salivary antimicrobial peptide is given in ng/ml of saliva.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe data obtained were statistically analyzed via STATISTICA 14 software (StatSoft, USA). Both the version without a multivariate regression model and the version with a multivariate regression model were used for statistical processing. Continuous variables are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Two-sample t tests and multivariate regression models adjusted for age and sex were used to compare the monitoring parameters cathelicidin (CAMP), alpha defensin (DEF alfa1), histatin 1 (HTN1), and statherin (STATH) between the case and control groups. Graphical comparisons of parameters between groups were conducted via box plots; all tests were performed at a significance level of 5%. The box plots display the median (box: 25th /75th percentiles), and the whiskers show the minimum/maximum levels of salivary antimicrobial peptides in the case and control groups. Outliers that significantly differ from the rest of the dataset are presented as individual points beyond the whiskers on the box plot.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eA total of 82 participants were enrolled in the study. The case group was composed of 55 children (32 boys and 23 girls) and the control group comprised 27 children (10 boys and 17 girls). The demographic and clinical characteristics (age, sex and dmf index) of all participants are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The children of the case group have a mean age 5.49 years and mean dmf index of 7.2, and the children of the control group has a mean age 4.56 years and dnf index zero.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDescriptive statistics of sex, age and dmf index in children of the case and control groups\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003egender\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c10\" namest=\"c6\"\u003e \u003cp\u003edmf index\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003enumber\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003emale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003efemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emean age\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (years)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003emean\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003emedian\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003emax.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003emin.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eSD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCase group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32 (58.18%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23 (41.82%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.4909\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9598\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e4.45\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10 (37,04%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17 (62.96%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.5556\u0026thinsp;\u0026plusmn;\u0026thinsp;0.89156\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e42 (51.21%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e40 (48,79%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.1829\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0319\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"10\"\u003eSD \u0026ndash; Standard Deviation\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eComparison of variables between the case and control groups\u003c/p\u003e \u003cp\u003eThe results of the p-values of the individual two-sample tests revealed that there was a statistically significant difference between the case and control groups only for the variable age, and the observed variables CAMP, DEF alfa1, HTN1, and STATH did not differ significantly between the case and control groups (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of salivary antimicrobial peptide concentration and age between the case and control groups of children\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCase group\u003c/p\u003e \u003cp\u003e(mean concentration)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eControl group (mean concentration)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003et- value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSD case group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSD control group\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCAMP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e179.7973\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e136.3122\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.104079\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.272869\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e198.4513\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e93.08503\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDef alfa1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.8138\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.8763\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-0.643233\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.521912\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.4055\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.42910\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHTN1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.8011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.0193\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-0.394905\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.693964\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2.6359\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.60530\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSTATH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e22.4573\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e23.4674\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-0.233092\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.816285\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e17.8401\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e19.63323\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.4909\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.5556\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.242808\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.000059*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.9598\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.89156\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eSD \u0026ndash; Standard Deviation\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.005 statistically significant\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eComparison of variables in the case group\u003c/p\u003e \u003cp\u003eThe results of the p values of the individual two-sample tests revealed that in the case group, the observed variables CAMP, DEF alfa1, HTN1, and STATH did not differ between the sexes..\u003c/p\u003e \u003cp\u003eComparison of variables in the control group\u003c/p\u003e \u003cp\u003eThe resulting p values of the individual two-sample tests revealed that in the control group, the observed variables CAMP, DEF alfa1, HTN1, and STATH did not differ between the sexes.\u003c/p\u003e \u003cp\u003eMultivariate regression model\u003c/p\u003e \u003cp\u003eA multivariate regression model was used to assess differences in the observed parameters CAMP, DEF alfa1, HTN1, and STATH between groups, adjusting for the age and sex of the children. The results of the individual regression models revealed that there was no statistically significant difference between the case and control groups for any of the observed parameters (CAMP, DEF alfa1, HTN1, and STATH) after adjustment for age and sex). Graphical comparisons of the parameters between the groups were performed via box plots.\u003c/p\u003e \u003cp\u003eChildren with ECC (case group) had higher mean CAMP levels in saliva than did children with intact dentition (control group). However, the difference was not statistically significant (p\u0026thinsp;=\u0026thinsp;0.41) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The findings were different for the mean values of DEF alfal and HTN1. Decreased values of these antimicrobial proteins were detected in the saliva of the children in the case group. The difference was not statistically significant (p\u0026thinsp;=\u0026thinsp;0.78 resp. 0.93) (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). With respect to STATH, a similar situation to CAMP was found. Children with ECC (case group) had higher mean salivary levels of this antimicrobial peptide than did the children in the control group. The difference between the case and control groups of children was again statistically nonsignificant (p\u0026thinsp;=\u0026thinsp;0.63) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e "},{"header":"Discussion","content":"\u003cp\u003eDental caries in early childhood is one of the most widespread diseases of childhood and can cause many complications for the child and significantly reduce the quality of life of the child and the whole family. ECC is a multifactorial disease involving dental microbial plaque, dietary sugars, the quality of hard dental tissues, the amount and composition of saliva and likely other risk factors that have not yet been fully investigated. These factors include proteins present in saliva, which are potential biomarkers of dental caries in early childhood [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The importance of salivary peptides and proteins in relation to dental caries and in determining the risk of dental caries has been addressed by a number of authors [\u003cspan additionalcitationids=\"CR19 CR20 CR21\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], who have not reached unequivocal conclusions in their studies.\u003c/p\u003e \u003cp\u003eAmong the cathelicidins, the human cationic antimicrobial peptide hCAP18, which cleaves serine proteases to the antimicrobial peptide LL-37, is found in the oral cavity and respiratory tract of humans. These molecules cause damage to the cell membranes of microorganisms. LL-37 is characterized by high activity against \u003cem\u003eStreptococcus mutans\u003c/em\u003e (SM), \u003cem\u003eStreptococcus sanguis\u003c/em\u003e, \u003cem\u003eStreptococcus mitis\u003c/em\u003e, \u003cem\u003eFusobacterium nucleatum\u003c/em\u003e, \u003cem\u003eAggregatibacter actinomycetemcomitans\u003c/em\u003e, and \u003cem\u003eCapnocytophaga sputigena\u003c/em\u003e [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSome authors [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] suggest that reduced levels of antimicrobial peptides, especially LL-37, in saliva may be associated with the occurrence of dental caries. The relationships between dental caries in children with a mean age of 8.7 years and salivary LL-37 concentrations were investigated by Davidopoulou et al. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. They reported that children with intact dentition had significantly higher LL-37 concentrations than did children with high caries activity. The results of this study suggest that the antimicrobial peptide LL-37 is present in saliva in early childhood, but its concentration is low. The concentration of this antimicrobial peptide in saliva increases with age and reaches a maximum in late adolescence. Almondi et al. [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] came to similar conclusions and hypothesized a protective role of salivary LL-37 in caries development. The authors reported low levels of salivary LL-37 and high levels of SM and \u003cem\u003eStreptococcus sobrinus\u003c/em\u003e in saliva samples from children with ECC. Different conclusions were reached by Colombo et al. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], who monitored the levels of LL-37, beta defensin 2, beta defensin 3 and histatin 5 in children with ECC. They reported no relationship between the presence of ECC and the levels of these antimicrobial peptides. The antimicrobial peptide LL-37 was evaluated in the current study for both the case group (caries active) and the control group (caries free). The results revealed that the salivary levels of LL-37 in caries-active children were greater than those in those without caries. This difference was not statistically significant (p\u0026thinsp;=\u0026thinsp;0.27). These results are consistent with the findings of Nireeksha et al. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The relationship between LL\u0026ndash;37 and ECC is currently unclear [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDefensins are short cationic peptides with low molecular weights and 6\u0026ndash;8 cysteine residues that form 3\u0026ndash;4 intramolecular disulfide bonds. They have the ability to act against all types of gram-positive and gram-negative bacteria, fungi and viruses [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. They are categorized into alpha defensins and beta defensins on the basis of structural variances and tissue distributions. Alpha defensins are formed in neutrophils and salivary gland duct cells and are present in gingival sulcus fluid and tissues affected by inflammation. Beta defensins (hBD-1, hBD-2, and hBD-3) are produced by oral epithelial cells and are found in gingival sulcus fluid and saliva [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The mechanisms of action of defensins on pathogens are not yet fully understood, but defensins are likely to disrupt bacterial cell walls.\u003c/p\u003e \u003cp\u003eTao et al. [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] investigated the correlation between the antimicrobial peptide alpha defensin and dental caries. They reported significantly higher levels of alpha defensin in children aged 11\u0026ndash;15 years without dental caries than in a control group of children with dental caries of the same age. Low levels of alpha defensin in the saliva of schoolchildren may be associated with the presence of dental caries, as described by Dale et al. [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], who also reported low levels of alpha defensin in patients with dental caries. Ribeiro et al. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] reported that alpha and beta defensins decrease the incidence of ECC. However, studies using more precise methods of salivary protein determination have not shown significant differences in the levels of alpha and beta defensins in the saliva of children with ECC and those with intact dentition [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Similarly, this study revealed no statistically significant difference in the amount of alpha 1 defensin in the saliva of children with ECC and those without caries (p\u0026thinsp;=\u0026thinsp;0.52). On the basis of available studies, systematic reviews, and the current study, defensins cannot be considered reliable for determining the risk of ECC [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan additionalcitationids=\"CR31 CR32\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHistatins are a group of cationic antimicrobial peptides with low molecular weights and high contents of the amino acid histidine. The main representatives are histatins 1, 3 and 5, which make up 1\u0026ndash;1.5% of all low-molecular-weight salivary proteins. The levels of histatins in saliva change with age. Research [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] has shown that the concentration of histatin 1 is significantly greater in children aged 3\u0026ndash;5 years than in older children. These peptides are produced by the cells of the parotid and submandibular salivary glands and are known for their antimicrobial effects against SM and \u003cem\u003ePorphyromonas gingivalis\u003c/em\u003e [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Histatins work by integrating into the cell membrane of bacteria and increasing membrane permeability, leading to bacterial destruction [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. They also inhibit the growth of Candida fungi [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] and have the ability to form complexes with metal ions, making them inaccessible to enzymes and releasing reactive oxygen species that affect bacterial cellular structures [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Histatins also have an affinity for the enamel surface and play a significant role in the formation of the enamel pellicle, limiting bacterial colonization on tooth surfaces. Additionally, they suppress the spontaneous growth of hydroxyapatite crystals in saliva containing a supersaturated solution of calcium phosphate [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA study by Sun et al. [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] investigated the salivary peptidome in children aged 3\u0026ndash;5 years with severe ECC. Severe ECC is an aggressive form of ECC characterized by specific criteria, such as caries on smooth tooth surfaces in children under 3 years of age, caries on upper frontal teeth and specific dmf index values at different ages. Children aged 3\u0026ndash;5 years with severe ECC were found to have lower histatin 1 levels before treatment than after treatment. Histatin 1 could serve as a biomarker for severe ECC on the basis of previous studies and systematic reviews [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Another study by Vitorino et al. [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] revealed a strong correlation between the absence of dental caries and high levels of histatin 1, statherin, and proline-rich proteins (PRPs). In the present study, higher amounts of histatin 1 were detected in the caries-free children, although the difference between the case and control groups was not statistically significant (p\u0026thinsp;=\u0026thinsp;0.69).\u003c/p\u003e \u003cp\u003eStatherins are low-molecular-weight acidic peptides composed of 43 amino acids. They have the ability to bind to the hydroxyapatite of enamel, inhibit the spontaneous precipitation of calcium and phosphate salts from supersaturated saliva, and inhibit the growth of hydroxyapatite crystals [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Statherins are considered precursors of the acquired pellicle because of their strong affinity for hydroxyapatite. They allow remineralization of enamel and cause aggregation of bacteria, thereby reducing their ability to adhere to hard and soft tissues of the oral cavity.\u003c/p\u003e \u003cp\u003eThe relationship between statherin and ECC is also under investigation. Vitorino et al. [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] reported an association between elevated levels of PRPs, histatin 1, and statherin in children without caries. Increased amounts of salivary statherin were detected in the caries-free children in the present study, but the difference between the children with ECC and the caries-free children was not statistically significant (p\u0026thinsp;=\u0026thinsp;0.81). The suitability of statherin as a biomarker of ECC requires further validation in clinical studies.\u003c/p\u003e \u003cp\u003eThe results of the present study did not confirm the expected differences in the amount of selected antimicrobial peptides in the saliva of children with ECC and children without dental caries. The results of the current study are partially in accordance with the findings of other authors [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], but the differences in the levels of antimicrobial peptides between the case and control groups of children are not statistically significant. An explanation for this may be that the saliva used for analysis in the present study was stimulated saliva, whereas the available studies used unstimulated saliva. Saliva collection might affect proteomic biomarkers. The major salivary glands are more involved in the production of stimulated saliva, and this saliva has different levels of specific ions, proteins, and water than unstimulated saliva. Unstimulated saliva represents an equilibrated condition, with less impact on the major salivary glands [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Stimulated saliva seems to be less suitable for the determination of salivary biomarkers of ECC.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eSalivary antimicrobial peptide (CAMP, DEF alfa1, HTN1 and STATH) levels were detected in the stimulated saliva of children with ECC and caries-free children. Children with ECC had higher levels of CAMP and STATH than did caries-free children, whereas the situation was reversed for DEF alfa1 and HTN1. Children with ECC had lower amounts of these antimicrobial peptides in stimulated saliva, but these differences were not statistically significant. The study partially confirmed the results of other authors. ECC is a multifactorial disease with various main risk factors, such as poor dietary habits, poor oral hygiene, sociodemographic factors, and factors related to the composition of oral bacteria. In addition to oral hygiene, nutrition plays an important role in the development of dental caries in childhood. When determining the risk of ECC, these risk factors should be focused on and easily identified through interviews with the child's guardians and during the clinical examination, followed by evaluation of salivary proteins.\u003c/p\u003e \u003cp\u003eLimitations of the study\u003c/p\u003e \u003cp\u003eLimitations of the study include inconsistencies in the age of the children in the groups, with the control group being younger than the case group. The study also did not evaluate oral hygiene practices or nutritional habits, relying solely on stimulated saliva to determine the levels of antimicrobial peptides.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eECC \u0026ndash; early childhood caries\u003c/p\u003e\n\u003cp\u003eWHO \u0026ndash; World Health Organization\u003c/p\u003e\n\u003cp\u003edmf index \u0026ndash; decayed, missing and filled teeth\u003c/p\u003e\n\u003cp\u003eCAMP \u0026ndash; cathelicidin\u003c/p\u003e\n\u003cp\u003eDEF alfa1 \u0026ndash; alpha defensin\u003c/p\u003e\n\u003cp\u003ehBD \u0026ndash; 1 \u0026nbsp;- beta defensin 1\u003c/p\u003e\n\u003cp\u003ehBD \u0026ndash; 2 \u0026nbsp;- beta defensin 2\u003c/p\u003e\n\u003cp\u003ehBD \u0026ndash; 3 \u0026ndash; beta defensin 3\u003c/p\u003e\n\u003cp\u003eHTN1 \u0026ndash; histatin 1\u003c/p\u003e\n\u003cp\u003eSTATH \u0026ndash; statherin\u003c/p\u003e\n\u003cp\u003ePRPs \u0026ndash; proline-rich proteins\u003c/p\u003e\n\u003cp\u003eSD - standard deviation\u003c/p\u003e\n\u003cp\u003ehCAP18 \u0026ndash; human cationic antimicrobial peptide\u003c/p\u003e\n\u003cp\u003eSM \u0026ndash; \u003cem\u003eStreptococcus mutans\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eng/ml \u0026ndash; nanograms per millilitre\u003c/p\u003e\n\u003cp\u003em1 - microlitre\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eEthical approval for the investigation and trial registration were obtained from the Research Ethics Committee of Medical Faculty in Pilsen, Charles University, Czech Republic (No 367/2020). Before the study, the legal guardians of all children provided the informed consent for their children to participate in the study. \u003c/p\u003e\n\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\n\u003cp\u003eAvailability of data and materials\u003c/p\u003e\n\u003cp\u003eThe datasets analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\n\u003cp\u003eCompeting interest\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis research was supported by a grant from the Ministry of Health of the Czech Republic \u0026ndash; Conceptual Development of Research Organization Faculty Hospital in Pilsen \u0026ndash; FNPI, 00669806.\u003c/p\u003e\n\n\u003cp\u003eAuthor\u0026acute;s contributions\u003c/p\u003e\n\u003cp\u003eVM designed the study\u003c/p\u003e\n\u003cp\u003eJW performed the meassurment of peptides\u003c/p\u003e\n\u003cp\u003eLB collected clinical samples\u003c/p\u003e\n\u003cp\u003eMP performed the saliva processing\u003c/p\u003e\n\u003cp\u003eMR analyzed the data\u003c/p\u003e\n\u003cp\u003eThe manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePitts NB, Baez RJ, Diaz- Guillory C, Donly KJ, Feldens CA, McGrath C et al. Early Childhood Caries: Bangkok Declaration IAPD. Int J Paediatr Dent. 2019;29(3):384‐6. doi: 10.1111/ipd.12490.\u003c/li\u003e\n\u003cli\u003eRamalingam L, Messer LB. Early childhood caries: an update. Singapore Dent J. 2004; 26(1):21-9.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003e.\u003c/strong\u003e Young DA, Feartherstone JDB. Caries management by risk assessment. Community Dent Oral Epidemiol. 20013;41(1):e53-e63. doi: 10.1111/cdoe.12031.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003e. \u003c/strong\u003eMej\u0026agrave;re I, Axelsson S, Dahl\u0026eacute;n G, Espelid I, Norlund S, Tran\u0026aelig;s S et al. Caries Risk assessment. A systematic review. 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Biol Res.2015;48:61. doi: 10.1186/s40659-015-0050-7.\u003c/li\u003e\n\u003cli\u003eHemadi AS, Huang R, Zhou Y, Zou J.Salivary proteins and microbiota as biomarkers for early childhood caries risk assessment. Int J Oral Sci. 2017;9(11): e1. doi: 10.1038/ijos.2017.35.\u003c/li\u003e\n\u003cli\u003eGusman H, Travis J, Helmerhorst EJ, Potempa J, Troxler RF, Oppenheim FG. Salivary Histatin 5 Is an Inhibitor of Both Host and Bacterial enzymes Implicated in Periodontal Disease. Infect Immun. 2001;69(3):1402-8. doi: 10.1128/IAI69.3.1402.2001.\u003c/li\u003e\n\u003cli\u003eMelino S, Di Nardo CSP, Sarkar B. Histatins: salivary peptides with copper (II) \u0026ndash; and zinc (II) \u0026ndash; binding motifs. FEBS J. 2013;281(3):657-72. doi: 10.1111/febs.12612.\u003c/li\u003e\n\u003cli\u003eSun X, Huang X, Tan X, Si Y, Wang X, Chen F et al. Salivary peptidome profiling for diagnosis of severe early childhood caries. J Transl Med. 2006;14(1):240. doi:10.1186/s12967-016-0996-4.\u003c/li\u003e\n\u003cli\u003eAo S, Sun X, Shi X, Huang X, Chen F, Zheng S\u003cstrong\u003e.\u003c/strong\u003e Longitudinal investigation of salivary proteomic profiles in the development of early childhood caries. J Dent.2017;61:21-7. doi: 10.1016/j.dent.2017.04006.\u003c/li\u003e\n\u003cli\u003eVitorino R, Lobo MJ, Duarte JR, Ferrer-Correia AJ, Domingues PM, Amado FM. The role of salivary peptides in dental caries. Biomed Chromatogr. 2005 Apr;19(3):214-22. doi: 10.1002/bmc.438. \u003c/li\u003e\n\u003cli\u003eOhshiro K, Rosenthal DI, Koomen JM, Streckfus CF, Chambers M, Kobayashi R. Preanalytic saliva processing affects proteomic results and biomarker screening of head and neck squamous carcinoma. \u003cem\u003eInt J Oncol \u003c/em\u003e2007;30(3):743\u0026ndash;9. doi: 10.3892/ijo.30.3.743. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-oral-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ohea","sideBox":"Learn more about [BMC Oral Health](http://bmcoralhealth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ohea/default.aspx","title":"BMC Oral Health","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Early childhood caries, saliva, antimicrobial peptides, biomarkers, defensins, histatins, cathelicidins, statherins","lastPublishedDoi":"10.21203/rs.3.rs-6905254/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6905254/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eEarly childhood caries (ECC) is a chronic, microbial, carbohydrate-driven multifactorial disease. The composition of saliva, quantity, pH buffering capacity, presence of microorganisms, and amounts of peptides and proteins play important roles in the development and progression of dental caries. Salivary peptides and proteins are crucial for the formation of acquired enamel pellicles, protection against infections, and maintenance of calcium homeostasis. This study aimed to investigate the salivary levels of antimicrobial peptides (cathelicidine, alpha 1 defensin, histatin 1, and statherin) in children with early childhood caries and caries-free children.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThis case‒control study involved 82 children (mean age 5.18 years) from the Pediatric Dentistry Department in Pilsen, Czech Republic. Children were enrolled on the basis of medical history and clinical examination and then divided into a case group (children with ECC) and a control group (caries-free children). Dental caries was recorded according to the WHO criteria, and the dmf index was calculated. Stimulated saliva was collected, and selected antimicrobial peptides were assessed via magnetic bead-based fluorescence assay kits following the manufacturer's instructions (Cloud-Clone Corp.). The data were statistically analyzed via a version without a multivariate regression model and a version with a multivariate regression model adjusted for age and sex. Graphical comparisons of antimicrobial peptide levels between groups were performed via box plots, with all tests performed at a significance level of 5%.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eSignificant differences in age were found between the case group (mean 5.5 years) and the control group (mean 4.56 years), with no significant differences in sex. Compared with caries-free children, children with ECC (mean dmf index of 7.2) had higher levels of cathelicidin and statherin and lower levels of alpha defensin and histatin 1 in stimulated saliva. However, these differences were not statistically significant.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe results of the present study did not confirm statistically significant differences in the levels of selected antimicrobial peptides in the saliva, yet this issue deserves further attention.\u003c/p\u003e","manuscriptTitle":"Comparison of cathelicidine, alpha 1 defensin, histatin 1, and statherin levels in saliva between children with early childhood caries and caries-free children (case–control study)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-18 19:40:11","doi":"10.21203/rs.3.rs-6905254/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-07T14:53:40+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-05T10:44:38+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-05T10:42:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Oral Health","date":"2025-06-16T11:38:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-oral-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ohea","sideBox":"Learn more about [BMC Oral Health](http://bmcoralhealth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ohea/default.aspx","title":"BMC Oral Health","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"aaade6f2-4eb1-4b6c-9842-64dbb9e5df0d","owner":[],"postedDate":"June 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-12-15T12:23:48+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-18 19:40:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6905254","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6905254","identity":"rs-6905254","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}