Host defense peptides are immediate responders of the innate immunity that express antimicrobial, immunoregulatory, and wound-healing activities. Neutrophils are a major source for oral host defense peptides, and phagocytosis by neutrophils is a major mechanism for bacterial clearance in the gingival tissue. Dysfunction of or reduction in the numbers of neutrophils or deficiency in the LL-37 host defense peptide was each previously linked with proliferation of oral Aggregatibacter actinomycetemcomitans which resulted in an aggressive periodontal disease. Surprisingly, A. actinomycetemcomitans shows resistance to high concentrations of LL-37. In this study, we demonstrated that submicrocidal concentrations of LL-37 inhibit biofilm formation by A. actinomycetemcomitans and act as opsonins and agglutinins that greatly enhance its clearance by neutrophils and macrophages. Improved uptake of A. actinomycetemcomitans by neutrophils was mediated by their opsonization with LL-37. Enhanced phagocytosis and killing of A. actinomycetemcomitans by murine macrophage-like RAW 264.7 cells were dependent on their preagglutination by LL-37. Although A. actinomycetemcomitans is resistant to the bactericidal effect of LL-37, our results offer a rationale for the epidemiological association between LL-37 deficiency and the expansion of oral A. actinomycetemcomitans and indicate a possible therapeutic use of cationic peptides for host defense

OBJECTIVE: Mycoplasma salivarium is a human oral potential pathogen that preferentially resides in dental plaques and gingival sulci. It has been suggested that this organism may play an etiological role in inflammatory processes in the oral cavity. The aim of this work was to determine whether M. salivarium possesses a potent oxidant scavenging capacity (OSC). DESIGN: The OSC of M. salivarium was quantified by a highly sensitive luminal-dependent chemiluminescence assay in the presence of cocktails that induced a constant flux of luminescence resulting from the generation of peroxide, hydroxyl radical (cocktail A) and NO, superoxide and peroxynitrites (cocktail B). RESULTS: M. salivarium markedly reduced oxidative stress by scavenging both free reactive oxygen and nitrogen species. The OSC of M. salivarium was much higher than that of other Mycoplasma species. Most of M. salivarium OSC was confined to the cytosolic fraction and was markedly increased in the presence of tannic acid, red blood cells or mucin. The cytosolic OSC of M. salivarium was heat stable and not affected by sodium azide or prolonged proteolysis. However, it was markedly decreased upon dialysis, suggesting that the major reducing activity is not enzymatic but rather, a low molecular weight compound(s). CONCLUSIONS: The ability of M. salivarium to scavenge oxidants may play a role in the survival and pathogenicity of this microorganism. The enhanced OSC of M. salivarium in the presence of tannic acid, red blood cells or mucin might have a significant importance to assess complex interactions with polyphenols from nutrients, salivary proteins and red blood cells extravasated from injured capillaries during infection and inflammation in oral tissues.

Saliva has become a central topic of research in many scientific categories. It is involved in mastication, lubrication, buffering action, maintenance of tooth integrity, physicochemical and antimicrobial defense, immunization, wound healing, taste, and early digestion (Amerongen and Veerman, 2002; Fábián et al, 2008). It is also important in biofilm formation on tooth surfaces, bacterial adhesion, serves as an important source for genetic and forensic profiles and maintains mucosal integrity of the oral and upper gastrointestinal mucosal surfaces. The oral cavity can be considered a ‘bio-reactor’ (Gorelik et al, 2008; Kanner et al, 2012) where, on a daily basis, multiple interactions occur among salivary electrolytes, thousands of different proteins including the glycoprotein mucin, plasma-derived albumin, immunoglobulins, digestive enzymes such as alpha amylase as well as with substantial amounts of polyphenols from nutrients. However, saliva also contains potentially toxic H2O2 generated by oral streptococci, salivary lactoperoxidase generates bactericidal and cytocidal thiocyanate anion (SCN−) and activated phagocytes release a series of toxic oxidants (Grisham and Ryan, 1990; Nagler et al, 2002; Halliwell and Gutteridge, 2007). This implies that saliva and the oral structures may be constantly exposed to oxidative stresses. Over the evolution, saliva has evolved the protective low molecular weight antioxidants (LMWA) uric acid, ascorbate, reduced glutathione, and plasma albumin possessing antioxidant activity, is delivered to saliva via the crevicular fluid (Sculley and Langley-Evans, 2002; Liskmann et al, 2007; Ginsburg et al, 2012). However, saliva may also contain red blood cells extravasated either following tooth brushing, use of tooth picks, during orthodontic treatment or in oral pathologies. Erythrocytes had been proposed to serve not only as transporters of oxygen and removers of CO2 but also as ‘sinks’ for reactive oxygen species (ROS) and as protectors of other cells against oxidant toxicity (Richards et al, 1998; Koren et al, 2009, 2010; Ginsburg et al, 2012). It was also proposed that quantifications of antioxidants be performed in whole blood but not exclusively in plasma (Ginsburg et al, 2011b). Today, the main justification to present a ‘letter to the editor’ on salivary functions, stems from a series of recent novel observations, which shed a new insight on the interactions of salivary proteins with polyphenols from nutrients and with blood cells and how such interactions might affect the redox status and the integrity of the oral cavity. The following are the main highlights: Microbial and red blood cells acquired enhanced oxidant-scavenging abilities (OSA) by avidly binding to their surfaces a large assortment of antioxidant polyphenols from nutrients. Such complexes acted in synergy with the antioxidants in whole unstimulated saliva to decompose ROS (Koren et al, 2009, 2010; Ginsburg et al, 2011a). Many of the polyphenols in aqueous beverages (e.g. red wine, tea, coffee, cocoa, cinnamon, cranberries, pomegranate etc.) might not exist in a full soluble state, and therefore not available as effective antioxidants. However, this shortcoming could be overcome by simply mixing the various agents either with fresh un-stimulated saliva (Ginsburg et al, 2012) or with mucin and albumin, which all serve as ‘solubilizers’ of lipophilic agents to render polyphenols more available as efficient antioxidants. Polyphenols in plants and fruit beverages can strongly adhere to the huge surface area of the oral cavity are retained there for long periods and this, despite a normal salivary flow. This suggested that bound polyphenols could act as a ‘slow release apparatus’ helping to maintain a proper redox status and probably also the defense against oxidative stresses (Ginsburg et al, 2012). The OSA in the oral cavity is a sum result of the synergistic interactions among antioxidants in saliva, crevicular fluid, antioxidant polyphenols from nutrients, blood elements, and paradoxically perhaps, also the indigenous catalase-positive colonizing microbial flora (Ginsburg et al, 2011b). Figure 1 represents one example, of many studied, showing a synergistic OSA resulting from the interactions among saliva, whole blood and the tea major polyphenol epigallocatechin gallate (EGCG). However, one has also to take into consideration that the presence in the oral cavity of excessive amounts of heme proteins which occur in periodontal pathologies, may also act as a ‘double-edged sword’ by supplying excessive amounts of Fe+2, instrumental in the generation, via the Fenton reaction, of the highly toxic hydroxyl radical. Antioxidant polyphenols might under certain conditions, also act as pro-oxidants and as signaling molecules which can generate pro-inflammatory agents (Rahman et al, 2006; Halliwell and Gutteridge, 2007; Halliwell, 2008). Figure 1. Luminol-dependent chemiluminescence patterns (Ginsburg et al, 2004) induced by combinations among unstimulated fasting saliva, whole blood, and epigallocatechin gallate (EGCG). The various agents and combinations among them were added to test tubes containing 800 μl of Hanks balanced salt solution (HBSS). This was followed by the addition of a ‘cocktail’ comprised of luminol (10 μM), sodium selenite (1 mM), H2O2 (1 mM) and CoCl2.6H2O (10 μM) which induced a rapid light wave due to H2O2 and hydroxyl radical. Note that sub inhibitory amounts of saliva and of EGCG acted in synergy with whole blood to significantly quench luminescence (n = 5) Because of the very low bioavailability of polyphenols resulting from intensive metabolism in the liver and by the microbial flora of the gastrointestinal tract and due to a strict regulatory mechanism (Ginsburg et al, 2011b) only micromolar amounts of antioxidant polyphenols capable of protecting LDL from oxidation, eventually manage to reach plasma. It was therefore recently proposed that polyphenols from nutrients exert their beneficial effect as antioxidants mainly in the oral cavity and in the stomach (Gorelik et al, 2008; Kanner et al, 2012) but to a much lesser extent in plasma. Accordingly, polyphenols in red wine, coffee, tea, and in other beverages which undergo solubilization by salivary proteins, may now neutralize advanced lipid oxidation end-products generated in the stomach during the metabolism of fatty acids (Gorelik et al, 2008; Kanner et al, 2012) and thus able to prevent the oxidation of LDL in the circulation. It was therefore proposed that consumption of fatty foods be always accompanied by a simultaneous consumption of fruit beverages rich in antioxidant polyphenols. Further research is however needed to assess the role of salivary antioxidants, polyphenols from nutrients, blood elements, and those antioxidants associated with microbial flora as potential players in the homeostasis and in the complex milieu of the oral cavity in health and in disease states.

Saliva has become a central research issue in oral physiology and pathology. Over the evolution, the oral cavity has evolved the antioxidants uric acid, ascorbate reduced glutathione, plasma-derived albumin and antioxidants polyphenols from nutrients that are delivered to the oral cavity. However, blood cells extravasated from injured capillaries in gingival pathologies, or following tooth brushing and use of tooth picks, may attenuate the toxic activities of H2O2 generated by oral streptococci and by oxidants generated by activated phagocytes. Employing a highly sensitive luminol-dependent chemiluminescence, the DPPH radical and XTT assays to quantify oxidant-scavenging abilities (OSA), we show that saliva can strongly decompose both oxygen and nitrogen species. However, lipophilic antioxidant polyphenols in plants, which are poorly soluble in water and therefore not fully available as effective antioxidants, can nevertheless be solubilized either by small amounts of ethanol, whole saliva or also by salivary albumin and mucin. Plant-derived polyphenols can also act in collaboration with whole saliva, human red blood cells, platelets, and also with catalase-positive microorganisms to decompose reactive oxygen species (ROS). Furthermore, polyphenols from nutrient can avidly adhere to mucosal surfaces, are retained there for long periods and may function as a “slow- release devises” capable of affecting the redox status in the oral cavity. The OSA of saliva is due to the sum result of low molecular weight antioxidants, albumin, polyphenols from nutrients, blood elements and microbial antioxidants. Taken together, saliva and its antioxidants are considered regulators of the redox status in the oral cavity under physiological and pathological conditions.