Saliva: An Oral Microbial Modulating Agent

by Ajeigbe Yekeen Abiola

IN PARTIAL FULFILMMENT OF THE AWARD OF THE REQUIREMENTS FOR THE AWARD OF BACHELOR OR SCIENCE B.Sc. DEGREE IN MICROBIOLOGY, UNIVERSITY OF ILORIN, KWARA STATE, NIGERIA.

Table of Contents
Table of Contents .......................................................................................................................................... 2 Chapter 1: Introduction ................................................................................................................................. 4 1.1 Salivary Glands .................................................................................................................................... 6 1.2 Oral Microbiota & General Health ...................................................................................................... 7 Chapter 2: General Functions of Saliva ...................................................................................................... 11 2.1 Taste .............................................................................................................................................. 11 2.2 Physical-Mechanical Activities (Lubrication, Dilution, Flushing and Cleaning Of Oral Cavity) 12 2.3 Modulation of the oral microbiota ................................................................................................ 14 2.4 Maintaining the integrity of the tooth enamel ............................................................................... 14 2.5 Buffer for the oral cavity ............................................................................................................... 15 2.6 Digestion ....................................................................................................................................... 17 2.7 Tissue Repair ................................................................................................................................ 17 Chapter 3: Major Components of the Salivary Pellicle .............................................................................. 18 3.0 Albumin ........................................................................................................................................ 20 3.1 Digestive Components of the Salivary Pellicle................................................................................. 20 3.1.1 Amylase ..................................................................................................................................... 20 3.2 Buffering Components of the Salivary Pellicle ................................................................................ 20 3.2.1 Carbonic anhydrase (CA)........................................................................................................... 20 3.3 Immunoactive/Immunologic Components of the Salivary Pellicle .................................................. 22 3.3.1 Cystatins..................................................................................................................................... 22 3.3.2 Histatins ..................................................................................................................................... 23 3.3.3 Lactoferrin.................................................................................................................................. 24 3.3.4 Lysozyme ................................................................................................................................... 28 3.3.5 Peroxidase .................................................................................................................................. 30 3.3.6 Proline-rich Proteins (PRPs) ...................................................................................................... 31

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3.3.7 Mucin-glycoprotein 1 and 2 (MG 1 & 2) ................................................................................... 31 3.3.8 Secretory Immunoglobulin A (SIgA)......................................................................................... 34 3.3.9 Statherin ..................................................................................................................................... 40 3.4 Summary of Immunoactive Components of the Saliva .................................................................... 40 Chapter 4: Inactivation of salivary defenses ............................................................................................... 42 4.1 Salivary Flow and Factors Affecting It ............................................................................................. 42 4.2 Effects of Reduced Salivary Flow .................................................................................................... 43 4.3 Saliva and Dental Caries Formation .................................................................................................. 45 References ................................................................................................................................................... 48

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4 Chapter 1: Introduction The mouth is the gateway of the body to the external world and represents one of the most biologically complex and significant sites in the body. Saliva (also referred to as spit, spittle, drool or slobber) is a watery, sometimes frothy fluid found in the oral cavity of Homo sapiens. Whole saliva is a dilute, viscous solution. Around 0.5 to 1.5 liters of saliva are secreted into the mouth each day. Saliva is hypotonic, with an average pH of around 6.7. Saliva contains both organic compounds (23 g/l protein, notably the enzyme amylase), and inorganic compounds including the electrolytes: bicarbonate, chloride, potassium and sodium (Lamont and Jenkinson, 2010). In a healthy mouth, the mean volume of saliva ranges from approximately 1.07mL before swallowing to approximately 0.77mL after swallowing (Dawes, 2004), total daily flow of saliva ranges from 500 mL to 1.5L (Jensen et al., 2003: Humphrey and Williamson, 2003). While much is known about the digestive properties of saliva, the other roles of saliva and especially its role as part of the immune system has not been widely publicized or acknowledged despite quite a lot of research in this field. Human saliva not only lubricates the oral tissues, making oral functions such as speaking, eating, and swallowing possible, but also protects teeth and oral mucosal surfaces in different ways. The lubricating and antimicrobial functions of saliva are maintained mainly by resting saliva. Stimulation of saliva results in a flushing effect and the clearance of oral debris and noxious agents. However, the protective functions of saliva are not limited to the abovementioned functions (Jensen et al., 2003: Humphrey and Williamson, 2003). Recent studies have revealed a large number of functions, mediated by both the inorganic and organic components of saliva that should be considered in assessments of the effects of human saliva on dental caries. Some of these studies have introduced a new approach to dental carPage | 4

5 ies from being a bacterially induced multifactorial disease to a disease which may also be influenced by inherited salivary factors. Such genetically regulated salivary components may influence both the colonization and the clearance of micro-organisms from the oral cavity (Jensen et al., 2003: Humphrey and Williamson, 2003). Saliva can provide growth nutrients for bacteria. Various bacteria produce proteases that degrade salivary proteins into peptides and amino acids, which can be used by the bacteria when exogenous nutrients are limiting. Bacteria can also produce glycan hydrolases that cleave sugar residues from the salivary glycoproteins, so that the sugars can be used for bacterial growth. Again, through co-evolution the bacteria that readily colonize and grow in saliva are mostly harmless and may help exclude pathogens (Lamont and Jenkinson, 2010). The secretion of saliva also provides a mechanism whereby certain organic and inorganic substances can be excreted from the body, including mercury, lead, potassium iodide, bromide, morphine, ethyl alcohol, and certain antibiotics such as penicillin, streptomycin, and chlortetracycline (Jensen et al., 2003: Humphrey and Williamson, 2003). At present, due to its role in the oral cavity, saliva represents an increasingly useful auxiliary means of diagnosis (Mahmud, 2003). Many researchers have made use of sialometry and sialochemistry to diagnose systemic illnesses, monitoring general health, and as an indicator of risk for diseases creating a close relation between oral and systemic health (Gonzales and Sanchez, 2003). However, since several factors can influence salivary secretion and composition a strictly standardized collection must be made so the above-mentioned exams are able to reflect the real functioning of the salivary glands and serve as an efficient means for monitoring health (Mahmud, 2003).

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1.1 Salivary Glands

Saliva is secreted by three (3) major salivary glands namely parotid, submandibular and sub-lingual (Fig 1) and these secrete about 90% of the saliva in the mouth. Hundreds of minor salivary glands also secrete saliva and these accounts for the remaining
Fig 1: The location of salivary glands in the oral cavity Source; Britannica, 2003

10% of saliva in the oral cavity. Minor salivary glands include the lingual, la-

bial, buccal, palatine and glossopalatine (Pedersen et al., 2002: Humphrey and Williamson, 2001: Cassolato and Turnbull, 2001). The parotid ducts/gland is located in the cheeks and supplies a fluid containing bicarbonate and phosphate ions, agglutinins (glycoproteins), -amylase, proline-rich proteins and other proteins & glycoproteins. S-IgA is also produced in the parotid ducts by plasma cells that localize there from the bone marrows (Lamont and Jenkinson, 2010). The sub-mandibular gland produce between 60-70% of the saliva in the oral cavity which contains mucous and serum derived (serous) components. The sub-lingual glands located anterior to the sub-mandibular glands produce mainly mucous secretions. Salivary secretions are classified as serous (primarily from the parotid glands), mucous (from the minor salivary glands), or mixed (from the submandibular and sublingual glands). As their

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7 names imply, serous secretions contain more water than the viscous saliva produced by mucous glands (Jensen et al., 2003: Pedersen et al., 2002).

Fig 2: Gland contribution of unstimulated salivary flow Source: Patricia et al., 2008

1.2 Oral Microbiota & General Health

The oral cavity is the most complex and the most accessible microbial ecosystem of the human body. The teeth, gingivae (gums), tongue, throat and buccal mucosa (cheeks) all provide different surfaces for microbial colonization. The constant production of saliva and intermittent provision of sugars and amino acids from ingested food provides nutrients for microbial growth. The human oral cavity is home to about 700 identified species of bacteria. This number will probably turn out to be closer to 1000 in the future, when all taxa and phyla have been recorded (Patricia et al., 2008). It is also home to at least 30 species of fungi (mainly of the genus Candida), several species of protozoa (which graze on the bacteria for food), and various intracellular viruses. Generally, in a single subject it is usual to find between 2050 species of bacteria at healthy oral

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8 sites. At diseased sites there is a tendency for higher numbers of different species to be present, perhaps 200 or more (Lamont and Jenkinson, 2010). The organisms present in the oral cavity are a mixture of commensals and pathogens. A commensal microorganism is defined as one that lives on or within a host but does not cause any apparent disease. However, this terminology may be misleading, as many commensal bacteria can, under certain conditions, be associated with human disease. Subjects whose immune systems are not working optimally, i.e. immunocompromised, are especially susceptible to infections by microbes that are commensal in healthy individuals. For these reasons, commensals are nowadays often referred to as opportunistic pathogens. Many of the cultivated bacteria present in the mouth probably contribute to oral diseases to a greater or lesser extent, because these diseases are almost always associated with polymicrobial infections. Monospecies infections are rare; however, localized aggressive periodontitis (LAP) is predominantly associated with Aggregatibacter actinomycetemcomitans, while Actinomyces israelii can cause oral cysts. Overt pathogens are organisms that usually cause disease when present, unless the host has protective immunity. There are very few organisms in the oral cavity and nasopharynx that can be considered overt pathogens. Streptococcus pyogenes (Group A Streptococcus), Streptococcus pneumoniae (Pneumococcus), Neisseria meningitidis (Meningococccus) and Haemophilus influenzae all reside within the nasopharynx and have the potential to cause life-threatening diseases. It is important to note, however, that even in such cases these bacteria may also be carried by subjects with no overt signs of disease. This is termed the carrier state (Lamont and Jenkinson, 2010).

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9 Almost every member of the human population is afflicted at some stage of their lives with an oral disease. The incidence of dental caries has declined generally in the developed world, due in part to fluoride in the water supply, in toothpaste, or taken in tablet form. However there are many groups within locales such as in Sub Saharan Africa that are still seriously afflicted with caries. Polymicrobial infections of the gingivae and sub-gingival regions (periodontitis, implantitis and pulpitis) are major conditions requiring clinical intervention. These diseases impose a significant burden on the health care system of such places (Lamont and Jenkinson, 2010). Table 1: Important Oral Diseases, their manifestations and microorganisms implicated Disease Caries Description Microorganisms implicated

Decay (loss) of tooth enamel (dental caries) Streptococcus, Lactobacillus, or dentin (dentinal caries), or root dentin (root Actinomyces (root caries) caries)

Gingivitis

Redness and swelling (inflammation) of the Actinomyces, Fusobacterium, gingival tissues (gums) Bacteroides, Prevotella Aggregatibacter (localized),

Periodontitis

Inflammation and either rapid (aggressive, either generalized or localized) or slower (chronic) destruction of the tissues supporting the tooth

Porphyromonas, Treponema, Tannerella, Prevotella PseudomoFusobacterium,

Implantitis

Infection and destruction of tissues surround- Staphylococcus, ing a dental titanium implant nas, Prevotella

Porphyromonas,

Pulpitis

Infection of the pulp, inflammation around Fusobacterium,

Dialister,

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1 0 the apex of the root, leading to abscess for- Peptostreptococcus, mation (periapical granuloma) Halitosis Oral malodor romonas Fusobacterium, Porphyromonas, Prevotella, Treponema, Eubacterium Pharyngitis Redness and inflammation of the pharynx. Group A Streptococcus, Neisseria, Haemophilus, Coxsackie A virus Tonsillitis Infection and inflammation of the tonsils. Group A Streptococcus, Haemophilus Leukoplakia White patches on the buccal mucosal epithe- Candida, human papilloma lium or tongue Stomatitis virus (HPV) Porphy-

Reddening and inflammation of the oral mu- Candida albicans, Candida cosa tropicalis, other Candida species.

Actinomycosis Cold sores

Hard swelling (cyst) within the gums

Actinomyces israelii

Surface (superficial) red, dry lesions close to Herpes smplex virus (HSV) the lips

Source: Lamont and Jenkinson, 2010.

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1 1 Chapter 2: General Functions of Saliva The saliva carries out a lot of activities in the oral cavity due to the actions of the components salivary pellicle. A summary of the functions of saliva with some of the active components are shown in Fig 3 below:

Abbreviations: Ca2+ = calcium; PRG = proline-rich glycoprotein; PRPs = proline-rich proteins; SLPI = secretory leukocyte protease inhibitor; VEGh = Von Ebner glands protein; Zn2+ = zinc

Fig 3: Summary of functions of saliva in oral cavity Source: Brosky, 2007

2.1 Taste The salivary flow initially formed inside the acini is isotonic with respect to plasma. However, as it runs through the network of ducts, it becomes hypotonic (Washington et al., 2000; Dawes et al., 2004; Turner and Sugiya, 2002; Constanzo, 2004). The hypotonicity of saliva (low levels of

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1 2 glucose, sodium, chloride, and urea) and its capacity to provide the dissolution of substances allows the gustatory buds to perceive different flavors. Gustin, a salivary protein, appears to be necessary for the growth and maturation of these buds (Berkovitz et al., 2002; Humphrey and Williamson, 2001; Cate, 1998; Stack and Papas, 2001). 2.2 Physical-Mechanical Activities (Lubrication, Dilution, Flushing and Cleaning Of Oral Cavity) Saliva is responsible for flushing the epithelial surfaces and for lubrication and protection of tissues and an adequate flow of saliva is essential for the maintenance of both hard and soft tissue integrity. Saliva forms a seromucosal covering that lubricates and protects the oral tissues against irritating agents (Stack and Papas, 2001; Nagler, 2004). Human saliva lubricates the oral tissues, making oral functions such as speaking, eating, and swallowing possible. The lubricating and antimicrobial functions of saliva are maintained mainly by resting saliva. Stimulation of saliva results in a flushing effect and the clearance of oral debris and noxious agents.

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1 3

Fig 4: Illustration of salivary seromucosal covering Source: Patricia et al., 2008

Mastication, speech, and deglutition are also aided by the lubricant effects of the proteins (mucins) present in saliva (Berkovitz et al., 2002; Edgar, 1992; Tenovuo 1994; Nagler 2004; Tabak et al., 2982; Schenkels et al., 1995; Amerongen and Veerman, 2002). In addition to diluting substances, its fluid consistency provides mechanical cleansing of the residues present in the mouth such as nonadherent bacteria and cellular and food debris. Salivary flow tends to eliminate excess carbohydrates, thus, limiting the availability of sugars to the biofilm microorganisms. The greater the salivary flow, the greater the cleaning and diluting capacity; therefore, if changes in health status cause a reduction in salivary flow, there would be a drastic alteration in the level of oral cleaning (Tenovuo and Lagerlof, 1994; Douglas, 2002; Cate, 1998; Stack and Papas, 2001; Nagler, 2004).

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1 4 2.3 Modulation of the oral microbiota Saliva contains anti-microbial components such as lysozymes, S-IgA, mucins, salivary histatins etc. which functions as part of an innate defense system which is non-specific and always active. This properties are further discussed under the components of saliva. 2.4 Maintaining the integrity of the tooth enamel Saliva plays a fundamental role in maintaining the physical-chemical integrity of tooth enamel by modulating remineralization and demineralization. The main factors controlling the stability of enamel hydroxyapatite are the active concentrations free of calcium, phosphate, and fluoride in solution and the salivary pH (Axelsson, 2000; Larsen and Bruun, 2001). The high concentrations of calcium and phosphate in saliva guarantee ionic exchanges directed towards the tooth surfaces that begin with tooth eruption resulting in post-eruptive maturation. Remineralization of a carious tooth before cavitation occurs is possible, mainly due to the availability of calcium and phosphate ions in saliva (Tenovuo and Lagerlof 2000; Cate, 1998; Stack and Papas, 2001). The concentration of salivary calcium varies with the salivary flow (Tenovuo and Lagerlof 2000; Axelsson, 2000) and is not affected by diet. However, diseases such as cystic fibrosis and some medications such as pilocarpine cause an increase in calcium levels. Depending on the pH, salivary calcium can be ionized or linked. Ionized calcium is important for establishing equilibrium between the calcium phosphates of enamel and its adjacent liquid. Non-ionized calcium can be linked to inorganic ions (inorganic phosphate, bicarbonate, fluoride), to small organic ions (citrate), and to macromolecules (statherin, histidine-rich peptides, and proline-rich proteins). A

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1 5 special case of the combination of calcium is its strong link with -amylase, where it acts as a cofactor necessary for the enzyme function (Tenovuo and Lagerlof 2000; Axelsson, 2000). Inorganic orthophosphate found in saliva consists of phosphoric acid (H3PO4) and primary (H2PO4-), secondary (HPO42-), and tertiary (PO43-) inorganic phosphate ions. The concentrations of these ions depend on salivary pH and vary in accordance with the salivary flow. As the flow increases, the total concentration of inorganic phosphate diminishes (Tenovuo and Lagerlof 2000; Axelsson, 2000). The most important biological function of this ion is to maintain the dental structure. The presence of fluoride in saliva, even at physiologically low levels, is decisive for the stability of dental minerals. Its concentration in total saliva is related to its consumption. It is dependent on the fluoride in the environment, especially in drinking water. Other sources are also important, such as dentifrices and other products used in caries prevention. The presence of fluoride ions in the liquid phase reduces mineral loss during a drop in biofilm pH, as these ions diminish the solubility of dental hydroxyapatite, making it more resistant to demineralization. It has also been demonstrated fluoride reduces the production of acids in biofilm (Humphrey and Williamson, 2001; Tenovuo and Lagerlof 2000; Axelsson, 2000; Larsen and Bruun, 2001). 2.5 Buffer for the oral cavity Oral pH is buffered to a small extent by saliva proteins and phosphate. The major influence on saliva pH however is bicarbonate ion which is a by-product of cell metabolism. Bicarbonate concentration increases in saliva as the flow rate rises and is due to the increased metabolic rate. This, in turn, raises the pH (more alkaline) of saliva.

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1 6 Saliva behaves as a buffer system to protect the mouth (Tenovuo and Lagerlof, 1994; Nagler 2004) as follows: It prevents colonization by potentially pathogenic microorganisms by denying them optimization of environmental conditions. Saliva buffers (neutralizes) and cleans the acids produced by acidogenic microorganisms, thus, preventing enamel demineralization (Cate, 1998). It is important to emphasize biofilm thickness, and the number of bacteria present determines the efficacy of salivary buffers (Humphrey and Williamson, 2004). Negatively loaded residues on the salivary proteins work as buffers. Sialin, a salivary peptide, plays an important role in increasing the biofilm pH after exposure to fermentable carbohydrates (Tenovuo and Lagerlf, 1994; Cate, 1998). The phosphate buffer is active in unstimulated saliva (Bardow et al., 2008). The mechanism for the phosphate buffer system is due to the ability of the secondary phosphate ion, HPO42-, to bind a hydrogen ion and form a primary phosphate ion H2PO4-. This acid-base pair has a pKa value in the range 6.8-7.2, which has a maximum buffering capacity that is relatively close to the salivary pH range; 6-8. Hence the phosphate buffer has the potential to be an effective buffer in the mouth. However, its effectiveness is limited due to insufficient concentrations of phosphate in the oral cavity. Urea is another buffer present in total salivary fluid which is a product of amino acid and protein catabolism that causes a rapid increase in biofilm pH by releasing ammonia and carbon dioxide when hydrolyzed by bacterial ureases (Edgar, 1992; Jenkins, 1978; Tenovuo and Lagerlf, 1994; Edgar et al., 2004; Jaffe et al., 1986; Ertugrul et al., 2003).

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1 7 Ammonia, a product of urea and amino acid metabolism, is potentially cytotoxic to gingival tissues. It is an important factor in the initiation of gingivitis because it may increase the permeability of the sulcular epithelium to other toxic or antigenic substances in addition to the formation of dental calculus (Macpherson and Dawes, 1991). 2.6 Digestion Saliva is responsible for the initial digestion of starch, favoring the formation of the food bolus (Cate, 1998; Constanzo, 2004). This action occurs mainly by the presence of the digestive enzyme -amylase (ptyalin) in the composition of the saliva. Its biological function is to divide the starch into maltose, maltotriose, and dextrins.This enzyme is considered to be a good indicator of properly functioning salivary glands (Enberg et al., 2001), contributing 40% to 50% of the total salivary protein produced by the glands. The greater part of this enzyme (80%) is synthesized in the parotids and the remainder in the submandibular glands. Its action is inactivated in the acid portions of the gastrointestinal tract and is consequently limited to the mouth (Edgar, 1992; Humphrey and Williamson, 2001; Tenovuo and Lagerlof, 1994; Douglas 2002; Schenkels et al., 1995). 2.7 Tissue Repair A tissue repair function is attributed to saliva since clinically the bleeding time of oral tissues appears to be shorter than other tissues. When saliva is experimentally mixed with blood, the coagulation time can be greatly accelerated (although the resulting clot is less solid than normal). Experimental studies in mice have shown wound contraction is significantly increased in the presence of saliva due to the epidermal growth factor it contains which is produced by the submandibular glands (Cate, 1998).

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1 8 Chapter 3: Major Components of the Salivary Pellicle Some components of the salivary pellicle includes; albumin, amylase, lysosome, lactoferrin, acidic proline-rich proteins, statherin, mucin-glycoprotein 1 and 2, carbonic anhydrase and secretory immunoglobulin A (S-IgA) etc. Sugars in their free form are present in total stimulated and unstimulated saliva at a mean concentration of 0.5 to 1 mg/100mL (Edgar, 1992; Jenkins, 1978). High concentrations of sugar in saliva mainly occur after the intake of food and drink (Edgar, 1992; Jenkins, 1978). It is known that there is a correlation between the glucose concentration in the blood and salivary fluid, particularly in diabetics, but because this is not always significant, saliva is not used as a means of monitoring blood sugar (Ben-Aryeh et al., 1988). The salivary components could be grouped into three (3) different groups based on their functions. These are: Digestive components, Buffering components and Immunoactive/immunologic components. Among the immunologic salivary components, there are enzymes (lysozyme, lactoferrin, and peroxidase), mucin glycoproteins, agglutinins, histatins, proline-rich proteins, statherins, S-IgA and cystatins (Axelsson, 2000; Schenkels, 1985).

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1 9
Table 2: The mean concentration (mg/100ml) of selected constituents of whole human saliva.

Constituent

Whole Saliva (mg/100ml)

Resting State Stimulated State 280

Protein IgA IgG IgM Amylase Lysozyme Albumin Sodium Calcium Magnesium Phosphate Bicarbonate * Note: tr = trace amounts

220 19 1 Less than 1 38 22 tr 15 6 Less than 1 17 31

11 tr 60 6 Less than 1 12 200

Source: Marsh and Martin, 2009

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2 0 3.0 Albumin Albumin is a major protein found in the body and is unique from other proteins in that it is not glycosylated. Albumin is water soluble, moderately soluble in concentrated salt solutions and can be denatured by heat (Meister-Green et al., 2010). 3.1 Digestive Components of the Salivary Pellicle 3.1.1 Amylase Amylase is an enzyme which initiates enzymatic hydrolysis in the mouth. Amylase is secreted in the parotid gland and converts starch into sugar. The action of amylase on substances causes the phenomenon where some food which contains little sugar but a lot of starch tastes sweet when in the mouth. Though three types of amylases have been found in nature namely -amylase, -amylase and amylase, only -amylase is found in the saliva. 3.2 Buffering Components of the Salivary Pellicle 3.2.1 Carbonic anhydrase (CA) Description and Functions: The carbonic anhydrases (or carbonate dehydratases) form a family of enzymes that catalyze the rapid interconversion of carbon dioxide and water to bicarbonate and protons (or vice versa), a reversible reaction that occurs rather slowly in the absence of a catalyst (Badger, 1994). CO2 + H2C HCO3- + H+

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2 1 Carbonic anhydrase I (CAI) is one out of ten CA isoenzymes that have been identified in humans. The isoform of carbonic anhydrase found in saliva is carbonic anhydrase VI (CA-VI) and it is coded by the CA6 gene (HGNC, 2012). Ever since its discovery by Meldrum and Roughton (1933), and Stadie and O'Brien (1933), the zinc enzyme carbonic anhydrase (CA) has been studied extensively because of its wide occurrence in living systems and its role in several physiological processes (Tashian 1989). This enzyme catalyzes the reversible hydration of carbon dioxide to bicarbonate with production of a proton (Meldrum and Roughton 1933; Coleman 1967; Lindskog et at 1984; Silverman and Lindskog 1988). It also catalyzes the hydrolysis of esters (Pocker and Stone, 1965; Tashain, et al., 1963, 1964), hydration of aldehydes (Pocker and Meany, 1965), and is associated with many important processes such as CO2 transport as HCO3- acid-base homeostasis, ion transport, formation of aqueous humour and gastric juice, and syntheses of urea, glucose and fatty acids (Maren, 1967, 1991; Coulson and Herbert, 1984; Tashian, 1989; Swenson 1991; Henry 1996). The physiological role of salivary CA VI has been clarified during recent years (Kivela et al, 1999a). Low salivary concentrations of CA VI appear to be associated with increased prevalence of caries and acid-peptic diseases (Kivela et al, 1999a). It was shown by Kivela and co-workers (1999b) that salivary CA VI correlates negatively with DMFT- values, especially in individuals with poor oral hygiene. In 1974, Szabo reported higher CA activity levels in caries-free children than in children with active caries. Since there is a positive correlation between CA VI concentration and salivary flow rate, and a negative correlation with the DMFT index, recent research suggests that salivary CA VI plays a role in protecting the teeth from caries (Kivela et al, 1999a, b).

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2 2

Fig 5: Suggested model for the function of CA VI on the dental surface. Source: Saliva and Dental Caries, 2000

Contrary to earlier predictions, CA VI does not seem to be directly involved in the regulation of actual salivary pH or buffer capacity, and no correlation has been found between salivary CA VI concentration and mutans streptococci or lactobacilli levels (Kivela et al, 1999b). CA VI has been reported to bind to the enamel pellicle and retain its enzymatic activity on the tooth surface (Fig. 4) (Leinonen et al, 1999). In the enamel pellicle, CA VI may catalyze the conversion of salivary bicarbonate and microbe-delivered hydrogen ions to carbon dioxide and water. 3.3 Immunoactive/Immunologic Components of the Salivary Pellicle 3.3.1 Cystatins Cystatins are cysteine rich peptides that inhibit bacterial cysteine proteases. Hence, cystatins are bacteriostatic in the oral cavity. Cystatins also regulate inflammation by inhibiting host proteases and up-regulating cytokines. Von Ebner gland protein is another cysteine protease inhibitor (Lamont and Jenkinson, 2010)

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2 3 The cystatins are related to acquired film formation and to hydroxyapatite crystal equilibrium. Due to its proteinase inhibiting properties, it is surmised they act in controlling proteolytic activity (Edgar, 1992; Humphrey and Williamson, 2001; Tenovuo and Lagerlof, 1994; Amerongen and Veerman; Blankenvoorde et al., 1996). 3.3.2 Histatins The histatins, a family of cationic histidine-rich peptides (Amerongen and Veerman, 2002) have antimicrobial activity against some strains of Streptococcus mutans (Mackay et al., 1984) and inhibit hemoagglutination of the periopathogen, Porphyromonas gingivallis (Murakami et al., 1992). They also kill Candida albicans (Lamont and Jenkinson, 2010). They neutralize the lipopolysaccharides of the external membranes of Gram-negative bacteria and are potent inhibitors of Candida albicans growth and development (Xu et al., 1991). Histatins have both fungicidal and bactericidal effects in the oral cavity. The bactericidal and fungicidal effects occur through the union of positively loaded histatins with the biological membranes resulting in the destruction of their architecture and altering their permeability. Other functions attributed to these peptides are: participation in acquired film formation and inhibition of histamine release by the mastocytes, suggesting a role in oral inflammation (Schenkels and Veerman, 1995). At least 12 histatins are present in saliva, resulting from truncations or proteolysis of the genetically distinct histatins 1 and 3. Histatin-5 (the N-terminal 24 amino acids of histatin-3) is a major salivary histatin and is very effective in killing yeast. Histatins bind to a Candida membrane receptor, and then the peptide is taken up by the cells. This result in arrest of the cell cycle and

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2 4 the cells lose ATP by efflux. Histatins can also regulate hydroxyapatite crystal growth, inhibit bacterial cysteine proteinases and prevent bacterial coaggregation (Lamont and Jenkinson, 2010). 3.3.3 Lactoferrin Description: Lactoferrin (formerly known as lactotransferrin) is a glycoprotein, and a member of a transferrin family, thus belonging to those proteins capable of binding and transferring iron (Fe3+) ions (Metz-Boutique et al., 1984). Lactoferrin is a glycoprotein with a molecular weight of about 80 kDa, which shows high affinity for iron. The molecular structure and amino acid sequence of human lactoferrin were discovered in 1984. Lactoferrin was then classified as a member of the transferrin family, due to its 60% sequence identity with serum transferrin (Metz-Boutique et al., 1984). Three different isoforms of lactoferrin have been isolated. Lactoferrin- is the iron binding form, but has no ribonuclease activity. On the other hand lactoferrin- and lactoferrin- demonstrate ribonuclease activity but they are not able to bind iron (Furmanski et al., 1989). There are three forms of lactoferrin according to its iron saturation: apolactoferrin (iron free), monoferric form (one ferric ion), and hololactoferrin (binds two Fe3+ ions). The tertiary structure in hololactoferrin and apolactoferrin is different (Jameson et al., 1998). Immunoactivity Lactoferrin is considered to be a part of the innate immune system. At the same time, lactoferrin also takes part in specific immune reactions, but in an indirect way (Legrand et al., 2005). Due to its strategic position on the mucosal surface lactoferrin represents one of the first defense systems against microbial agents invading the organism mostly via mucosal tissues. Lactoferrin afPage | 24

2 5 fects the growth and proliferation of a variety of infectious agents including both Gram-positive and negative bacteria, viruses, protozoa, or fungi (Kirkpatrick et al., 1971). Lactoferrin links to free iron in the saliva causing bactericidal or bacteriostatic effects on various microorganisms requiring iron for their survival such as the Streptococcus mutans group. Lactoferrin also provides fungicidal, antiviral, anti-inflammatory, and immunomodulatory functions (Edgar, 1992; Humphrey and Williamson, 2001; Tenovuo and Lagerlof, 1994; Amerongen and Veerman, 2002; Nikawa et al., 1993). Its ability to bind free iron, which is one of the elements essential for the growth of bacteria, is responsible for the bacteriostatic effect of lactoferrin (Arnold et al., 1980). A lack of iron inhibits the growth of iron-dependent bacteria such as E. coli (Brock, 1980). In contrast, lactoferrin may serve as iron donor and in this manner support the growth of some bacteria with lower iron demands such as Lactobacillus sp. or Bifidobacterium sp., but lacoferrin is generally considered as beneficial (Petschow et al., 1999; Sherman et al., 2004). Lactoferrins capability of binding iron is two times higher than that of transferrin, which can serve in some cases as donor of Fe3+ ions for lactoferrin. Two ferric ions can be bound by one lactoferrin molecule. One carbonate ion is always bound by lactoferrin concurrently with each ferric ion (Aisen and Liebman, 1972; Metz-Boutique et al., 1984; Baker, 1994). Although this bond is very strong and can resist pH values of as low as 4, its saturation does not exceed 10% in total (Mazurier and Spik, 1980). Four amino acid residues are most important for iron binding (histidine, twice tyrosine, and aspartic acid); while an arginine chain is responsible for binding the carbonate ion (Baker, 1994; Ward et al., 1996).
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2 6 The ability to keep iron bound even at low pH is important, especially at sites of infection and inflammation where, due to the metabolic activity of bacteria, the pH may fall under 4.5. In such a situation lactoferrin also binds iron released from transferrin, which prevents its further usage for bacterial proliferation (Valenti and Antonini, 2005). The bactericidal activity of lactoferrin is not iron-dependent and may be mediated through more than one pathway. Receptors for the N-terminal region of lactoferrin have been discovered on the surface of some microorganisms. The binding of lactoferrin to these receptors induces cell-death in Gram-negative bacteria due to a disruption in the cell wall. The subsequent release of lipopolysacharide leads to impaired permeability and a higher sensitivity to lysozyme and other antimicrobial agents (Arnold et al., 1977; Yamauchi et al., 1993; Leitch and Willcox, 1998). Lipopolysacharide can be disposed of even without the direct contact of lactoferrin with the cell surface (Rossi et al., 2002). Bactericidal activity affecting Gram-positive bacteria is mediated by electrostatic interactions between the negatively charged lipid layer and the positively charged lactoferrin surface that cause changes in the permeability of the membrane (Valenti and Antonini, 2005). It has been discovered that lactoferricin, a cationic peptide generated by the pepsin digestion of lactoferrin, has more potent bactericidal activity than the native protein. There are two forms known at present; lactoferricin H (derived from human lactoferrin) and lactoferricin B (of bovine origin) (Bellamy et al., 1992). As a result of the fusion of secondary granules with phagosomes, lactoferrin becomes an iron provider for the catalysis of free radical production and thereby increases the intracellular bactericidal activity of neutrophils (Sanchez et al., 1992). In vitro lactoferrin is able to prevent Pseu-

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2 7 domonas aeruginosa biofilm formation. The lack of iron in the environment forces bacteria to move. Therefore, they cannot adhere to surfaces (Singh et al., 2002). Lactoferrin may contribute to defense against the invasion of facultative intracellular bacteria into cells by binding both target cell membrane glycoaminoglycans and bacterial invasins, which prevents pathogen adhesion to target cells. This ability was first reported against enteroinvasive E. coli HB 101 and later also against Yersinia enterocolica, Yersinia pseudotuberculosis, Listeria monocytogenes, Streptococcus pyogenes, and Staphylococcus aureus (Valenti and Antonini, 2005). The proteolytic activity of lactoferrin is considered to inhibit the growth of some bacteria such as Shigella flexneri or enteropathogenic E.coli through degrading proteins necessary for colonization. However, this can be disabled by serine protease inhibitors (Orsi, 2004; Ward et al., 2005). Lactoferrin is capable of binding certain DNA and RNA viruses (Yi et al., 1997). Nevertheless, its main contribution to antiviral defense consists in its binding to cell membrane glycosaminoglycans. In this manner lactoferrin prevents viruses from entering cells and infection is stopped at an early stage (Ward et al., 2005). Such a mechanism has been demonstrated as being effective against the Herpes simplex virus (Fujihara and Hayashi, 1995; Marchetti et al., 1996), cytomegaloviruses (Andersen et al., 2001), and the human immunodeficiency virus (Harmsen et al., 1995), respectively. Lactoferrin acts against parasites in various ways. For example, the infectivity of Toxoplasma gondii and Eimeria stiedai sporozoites is reduced after their incubation with lactoferricin B. It is thought that lactoferricin breaches parasitic membrane integrity causing subsequent changes in interactions between the host and the parasite (Omata et al., 2001). The competition for iron bePage | 27

2 8 tween the parasite and lactoferrin is the basis of its antiparasitic activity against Pneumocystis carinii (Cirioni et al., 2000). In contrast, some parasites such as Tritrichomonas foetus are able to use lactoferrin as a donor of ferric ions (Tachezy et al., 1996) Besides iron lactoferrin is capable of binding a large amount of other compounds and substances such as lipopolysacharides, heparin, glycosaminoglycans, DNA, or other metal ions like Al3+, Ga3+, Mn3+, Co3+, Cu2+, Zn2+ etc. However, its affinity for these other ions is much lower. Apart from CO32, lactoferrin can bind a variety of other anions like oxalates, carboxylates, and others. In this way it is possible for lactoferrin to affect the metabolism and distribution of various substances (Baker, 1994). Thanks to its antimicrobial activity and capability of binding components of bacterial cell walls or their receptors, lactoferrin may prevent the development of inflammation and subsequent tissue damage caused by the release of pro-inflammatory cytokines and reactive oxygen species (Legrand et al., 2005). 3.3.4 Lysozyme Description and Synthesis: Lysozyme is a single chain polypeptide of 129 amino acids cross-linked with four disulfide bridges (Jolles, 1969) and is a basic protein found in most secretions, including saliva, where it is present in high concentrations. Salivary lysozyme originates from both the salivary gland secretions and from gingival crevicular fluid (Lamont and Jenkinson, 2010). Immunoactivity The activity of lysozyme is a function of both pH and ionic strength. The enzyme is active over a broad pH range (6.09.0). At pH 6.2, maximal activity is observed over a wider range of ionic strengths (0.020.100 M) than at pH 9.2 (0.010.06 M) (Davies et al., 1969)

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2 9 The natural substrate for lysozyme is the peptidoglycan layer of bacterial cell walls (Holtje, 1996). Hence, lysozyme hydrolyzes/digests the cell walls of Gram-positive bacteria by breaking the (1-4) bond between N-acetylmuramic acid and N-acetylglucosamine in peptidoglycan (Figure 6) and between N-acetyl-D-glucosamine residues in chitodextrin (Rupley, 1964; Holler, 1975). Gram-positive cells are quite susceptible to this hydrolysis as their cell walls have a high proportion of peptidoglycan. Gram negative bacteria are less susceptible due to the presence of an outer membrane and a lower proportion of peptidoglycan. (Sigma, 2012). Not surprisingly, many successful oral colonizers are relatively resistant to killing by lysozyme. Lysozyme being strongly cationic can activate the bacterial autolisines which are able to destroy bacterial cell wall components. Other antibacterial mechanisms have been proposed for this enzyme, such as inhibition of bacterial adherence by swallowing or expectoration (Edgar, 1992; Humphrey and Williamson, 2001; Tenovuo and Lagerlof, 1994; Schenkels et al., 1995; Amerongen and Veerman, 2002; Lamont and Jenkinson, 2010). In addition, lysozyme contains small amphipathic sequences in the C-terminal region that are capable of killing bacteria (Lamont and Jenkinson, 2010).

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3 0

Fig 6: Action of lysozyme on Gram-positive bacterial peptidoglycan Source: Lamont and Jenkinson, 2008.

3.3.5 Peroxidase Peroxidase in saliva is derived from the salivary glands and polymorphonuclear neutrophils (PMNs). Peroxidase or sialoperoxidase offers antimicrobial activity because it serves as a catalyst for the oxidation of the salivary thiocyanate ion (SCN) by hydrogen peroxide into hypothiocyanate (OSCN), a potent antibacterial substance which is produced by the aerobic metabolism of oral bacteria. At acid pH, OSCN becomes protonated (and uncharged) and readily passes through bacterial membranes. Hypothiocyanite oxidizes SH groups in bacterial enzymes and inhibits bacterial metabolism (Humphrey and Williamson, 2001; Tenovuo and Lagerlof, 1994). As a result of its consumption, proteins and cells are protected from the toxic and oxidant effects of hydrogen peroxide. Reduction of hydrogen peroxide to water by peroxidase also prevents oxidative damage to the host soft tissues (Edgar, 1992; Humphrey and Williamson, 2001; Tenovuo and Lagerlof, 1994; Amerongen and Veerman).
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3 1 3.3.6 Proline-rich Proteins (PRPs) These include acidic proline-rich proteins and proline-rich glycoproteins. Saliva contains acidic and basic proline-rich proteins (PRPs) which attach to oral streptococci. Basic PRPs inhibit HIV infectivity by binding to the gp120 regions of the virus (Lamont and Jenkinson, 2010). The proline-rich proteins and statherins inhibit the spontaneous precipitation of calcium phosphate salts and the growth of hydroxyapatite crystals on the tooth surface, preventing the formation of salivary and dental calculus. They favor oral structure lubrication, and it is probable both are important in the formation of acquired film. Another function proposed for the prolinerich proteins is the capacity to selectively mediate bacterial adhesion to tooth surfaces (Edgar, 1992; Humphrey and Williamson, 2001; Tenovuo and Lagerlof, 1994; Amerongen and Veerman). 3.3.7 Mucin-glycoprotein 1 and 2 (MG 1 & 2) Description and Synthesis: Mucins are present at all surfaces within the human body that are exposed to the environment. Mucins are composed of an amino acid chain backbone (polypeptide) with chains of sugar residues attached to the amino acids serine, threonine, or asparagine. Mucins are the basis of salivary function and are site specific. Salivary mucins are extremely large proteins carrying chains of sugar residues linked together. In saliva there are two main types of mucin, designated mucin glycoprotein 1 (MG1) and the smaller mucin glycoprotein 2 (MG2). MG1 is encoded by the MUC5B gene and MG2 is encoded by the MUC7 gene. Mucins are produced by all salivary glands except (or in very low amounts) by the parotid gland (Lamont and Jenkinson, 2010).

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3 2 Immunoactivity Mucins form complexes that are key to their functional properties. Charged carboxyl or amino groups present on amino acids can form ionic cross links with oppositely charged groups on adjacent polypeptide chains. Cysteine residues present in one chain can form disulfide bonds with cysteine residues in another chain. Both of these inter-chain interactions tend to align mucin chains with each other (Figure 5). Sialic acid residues, which are highly negatively charged, can then interact with sialic acid residues on aligned chains through bridging with Ca2+ ions.

Fig 7: Alignment of mucin chains Source: Lamont and Jenkinson, 2008.

The sialic acid residues are very important to mucin function, as they are to other systems in the human body. The negatively charged residues act to keep the mucin chains apart, allowing water molecules to become trapped between them. Mucin function depends upon this trapping of water molecules, thus determining the degree of hydration of the mucin gel. Mucin has high viscosity when it is hydrated and gel-like, and MG1 is the main gel-forming mucin in saliva (Figure 6).

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3 3

Fig 8: Gel forming and membrane bound mucins in the oral cavity Source: Lamont and Jenkinson, 2008.

The viscosity and elastic properties of saliva are attributed to the gel-forming mucins. These have multiple cysteine-rich domains that form disulfide bonds and thus generate multimeric complexes. The non-gel forming mucins provide a relatively close coating of epithelial cells, protecting the cell membrane from physical or biological damage. Some of them are membrane tethered (Figure 7). They lack the cysteine rich domains of the gel forming mucins (Lamont and Jenkinson, 2010). Since bacteria usually have a net negative surface charge, they tend not to bind directly to negatively charged mucins. Instead, they may interact through bridging reactions involving divalent cations such as Ca2+ ions (see Figure 7). However, many bacteria express cell surface proteins (lectins) that specifically recognize oligosaccharides present on the salivary mucins. In this manner salivary mucins are bound by multiple bacteria and agglutination results. MG2 is thought to be more important than MG1 for agglutinating bacteria in saliva. Agglutination of some oral streptococci is lost after removal of the terminal sialic acid of the oligosaccharide side chains, indicating that the bacterial lectin has sialic acid specificity (Lamont and Jenkinson, 2010). Salivary agglutinin, a highly glycosylated protein frequently associated with other salivary proteins
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3 4 and with secretory IgA, is one of the main salivary components responsible for bacteria agglutination (Amerongen and Veerman, 2002).

Fig 9: Depiction of mechanisms of bacterial agglutination by salivary mucins Source: Lamont and Jenkinson, 2008.

3.3.8 Secretory Immunoglobulin A (SIgA) Description: The major immunoglobulin in the salivary secretions is immunoglobulin A (IgA). This molecule is secreted as a complex with a linking chain by cells that are found close to the parotid gland. The secreted form of IgA is called secretory IgA (or S-IgA). It is found at all mucosal sites, such as the gastrointestinal tract, respiratory tract and urogenital tract, and it is also present in tears and breast milk (in addition to saliva). There are two isoforms, or subclasses, of IgA designated IgA1 and IgA2 and saliva contains approximately equal proportions of each.

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3 5

Fig 10: Schematic representation of SIgA. The wavy line represents the secretory component. Source: Marcotte and Lavoie, 1998.

Secretory immunoglobulin A (IgA) is the largest immunologic component of saliva. It can neutralize viruses, bacterial, and enzyme toxins. It serves as an antibody for bacterial antigens and is able to aggregate bacteria, inhibiting their adherence to oral tissues (Humphrey and Williamson, 2001: Axelsson, 2000; Cate, 1998; Schenkels et al., 1985). Other immunologic components, such as IgG and IgM, occur in less quantity and probably originate from gingival fluid (Humphrey and Williamson, 2001; Edgar, 1992). SIgA consists of at least two IgA monomers linked to a J chain and a secretory component. The J chain and secretory component are disulfide linked to the Fc region of the IgA molecule. Each IgA monomers consist of two a-heavy chains and two light chains linked covalently by disulfide bonds. The primary function of S-IgA is immune exclusion. S-IgA is glycosylated and this anchors SIgA to the mucus lining of the epithelial surface and thus inhibits attachment and tissue penetration by viruses, bacteria, and their released antigens such as LPS, toxins and environmental anti-

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3 6 gens. IgA antibodies do not activate complement, thus minimizing disruption of the epithelial barrier layer. Free in saliva, polymeric IgA effectively aggregates bacteria. SIgA is one of the principal factors preventing bacterial translocation, which can result in sepsis and death of the host. The classic view is that SIgA exerts its effect by aggregating bacteria, thereby mediating clearance of those bacteria from the gut (Williams and Gibbons, 1972) and preventing invasion of the body by the bacteria (Brandtzaeg, 1998; Brandtzaeg et al., 1985). This model of SIgA activity, termed immune exclusion, was originally based on in vitro experiments in which aggregation by SIgA was shown to decrease the ability of bacteria to adhere to cultured epithelial cells (Williams and Gibbons, 1972). As much as 2.5 g/day of SIgA is secreted into the lumen of the digestive tract, making it the most heavily produced protein in the body by weight (Conley and Delacroix, 1987) and suggesting its importance in maintaining normal hostmicrobial interactions (Everett et al., 2004). It is thought that the primary function of secretory IgA (SIgA) is, in conjunction with the mucus lining of the gut, to prevent translocation of bacteria across the epithelial barrier (Everett et al., 2004). Synthesis: Salivary IgA is produced by plasma cells that are located adjacent to the duct and acini of salivary glands (Korsrud and Brandtzaeg, 1980). IgA secreting plasma cells predominate in the major and minor salivary glands over plasma cells producing other Ig isotypes (Michalek and Childers, 1990). Polymeric IgA containing J chain, secreted by plasma cells, is specifically recognized by the PIgR located on the basolateral surface of the ductal and acinar cells (Tomasi, 1989). The polymeric IgA-PIgR complex is internalized into endocytic vesicles and transported to the apical surface of the epithelial cells. After fusion of the vesicles with the cell membrane,

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3 7 the PIgR is proteolytically cleaved, which releases a portion of PIgR, called SC, and polymeric IgA into the secretions as SIgA (Childers et al., 1989; Tomasi, 1989). During the external translocation, disulfide bonds covalently link SC with polymeric IgA, which in turn stabilizes the IgA-SC complex (Bradtzaeg, 1995).

Fig 11: Steps in the production and secretion of IgA at the mucosal surface

Immunoactivity Inhibition of bacterial adherence: The inhibition of bacterial adherence by SIgA is considered one of the most important defense mechanisms against mucosal bacterial invasion. SIgA interferes with bacterial adherence to host surfaces by preventing both nonspecific and stereochemical interactions. The binding of SIgA to adhesins can reduce the negative surface charge and the hydrophobicity of bacteria, thus limiting the potential for ionic and hydrophobic interactions between bacteria and host receptors (Marcotte and Lavoie, 1998). Free in saliva, polymeric IgA effectively aggregates bacteria (Lamont and Jenkinson, 2010).
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3 8 Toxin Neutralization: SIgA can neutralize toxins by blocking their binding to cell receptors (Marcotte and Lavoie, 1998). Glycans on S-IgA are also able to non-specifically trap bacteria. S-IgA interacts with mucins and so-called scavenger proteins such as gp340 present in saliva to generate heterotypic complexes that trap bacteria and stimulate macrophage migration (Lamont and Jenkinson, 2010). Viral Immunity: SIgA plays an important role in viral immunity because of its presence at the site of initial contact between virions and host cells. A protective effect of SIgA against respiratory and enteric viral infections has been demonstrated (Marcotte and Lavoie, 1998). Immune exclusion: Immune exclusion entails the prevention of bacterial movement across the mucosal barrier by a combination of a thick, flowing mucus barrier and the secretory immune system (Everett et al., 2004). One of the major functions of SIgA is to perform immune exclusion, which consists of limiting the penetration of antigenic materials through the mucosal epithelium. This involves the binding of SIgA antibodies with antigens, which facilitates their removal from mucosal surfaces (Marcotte and Lavoie, 1998). Immune exclusion is not considered to be promicrobial by any means. As stated by Brandtzaeg in 1998, The main purpose of the secretory antibody system is, in cooperation with innate mucosal defense mechanisms (Goldblum et al., 1996), to inhibit colonization and invasion of pathogens (Brandtzaeg, 1998). Thus, the relationship between SIgA and pathogenic bacteria is thought to be antagonistic. But what about the relationship between SIgA and the normal flora? It is known that SIgA, which is elicited in response to normal enteric bacteria (Shroff and Cebra, 1995; Shroff et al., 1995), also binds to the normal flora (Shroff and Cebra, 1995; Shroff et al., 1995; Orndoff et al.,, 2004). Thus, presumably, SIgA exercises a degree of immune exclusion toward the norPage | 38

3 9 mal flora, as well as toward pathogenic bacteria. However, given the known benefits of the normal flora (MacDonald and Pettersson, 2000; Harp et al., 1992), this conclusion is perhaps counterintuitive and, at the very least, gives pause for thought. The currently accepted paradigm is that SIgA-mediated aggregation of bacteria decreases adherence of those bacteria to epithelial surfaces (Williams and Gibbons, 1972).This theory stems from work by Williams and Gibbons (1972), who demonstrated that bacteria agglutinated by SIgA adhere to epithelial surfaces in vitro less well than do unagglutinated bacteria. Binding of Streptococcus sanguis (S. sanguis), S. mitis and two strains of S. salivarius to cultured human epithelial cells was inhibited by 68%, 63%, 68% and 76%, respectively, by the presence of SIgA at agglutinating concentrations. Thus, the study by Williams and Gibbons found a relatively simple and negative relationship between aggregation and adhesion using a particular set of conditions (Williams and Gibbons, 1972). Effects of SIgA Deficiency In past studies, Ig-deficient patients have been used as potential models for elucidating the role of host immunity in the control of diseases. Selective IgA deficiency is the most common primary immunodeficiency and can occur at a frequency within a population varying from 1:300 to 1:3,000 depending on the screened population (Liblau and Bach, 1992). Patients with antibody deficiencies show an increased susceptibility to microbial infections, more particularly in the intestinal and upper respiratory tracts (Amman and Hong, 1980; De Laat et al., 1991). Ig-deficient patients, especially those deficient in IgA, represent a useful model for studying the role of salivary IgA in oral health and diseases. If salivary IgA plays a major role in the maintenance of the homeostasis of the oral microbiota, Ig-deficient individuals should show an increased susceptibility to caries and periodontal diseases. It should be noted that Salivary IgA is part of a complex
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4 0 and interdependent immune defense system and hence its full effects and deficiencies are not fully researched or documented. Numerous studies have focused on the development of a vaccine that could induce salivary IgA antibodies and be effective in protecting against caries. 3.3.9 Statherin Statherin is a highly stable salivary protein of low molecular mass (5,380). A study of DNA from human-hamster somatic cell hybrids indicated that statherin is coded by a single-copy gene located in region 4q11-q13. Dentinogenesis imperfecta and perhaps juvenile periodontitis are coded in the same region. Statherin play an important role in the maintenance of oral health since, like the proline-rich proteins, it binds calcium and inhibits crystal growth. Statherin also inhibits spontaneous precipitation of calcium phosphate salts. Thus, statherin and PRPs may prevent build-up of harmful deposits in the salivary glands and on the tooth surfaces (Humphrey and Williamson, 2001). 3.4 Summary of Immunoactive Components of the Saliva
Table 3: Immunoactive Components in Saliva and their functions

Immunoactive Component Cystatin Histatin Lactoferrin Lysozyme

Function/Activity Bacteriostatic Bactericidal and fungicidal Iron sequestration Bactericidal (More effective on gram positive bacteria due to the peptidoglycan in their cell

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4 1 walls) Peroxidase Proline-Rich Proteins Mucin-glycoprotein 1 and 2 Bactericidal Bacteriostatic and bactericidal. Bactericidal, physical removal of microorganisms Secretory Immunoglobulin A Statherin Bacteriostatic, bactericidal, anti-viral Bacteriostatic Source: Anonymous

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4 2 Chapter 4: Inactivation of salivary defenses The oral microbiota has evolved to grow and survive in the human mouth despite the salivary defenses, and these organisms have devised means to resist IgA and salivary defenses. For example, some Streptococcus, Haemophilus and Neisseria species produce proteases that specifically cleave S-IgA1, disrupting functions such as complexing and clumping. S-IgA1 fragments may also promote bacterial adherence and accumulation. IgA2 has a deletion in the hinge region that renders it resistant to these proteases. Other bacteria produce glycan hydrolysases that cleave sugar chains from mucins. This causes changes in mucin properties making them much less efficient, both in binding bacteria and in lubrication. This adaptation could be a factor that allows Streptococcus mutans to be effective as a cariogenic agent (Lamont and Jenkinson, 2010). Also, some bacteria are able to adapt to lactoferrin activities by releasing siderophores (iron chelating compounds of bacterial origin) that compete with lactoferrin for Fe3+ ions (Crosa, 1989; Ratledge and Dover, 2000). Some other types of bacteria, including Neisseriaceae family, adapt to new conditions by expressing specific receptors capable of binding lactoferrin, and to cause changes in the tertiary structure of the lactoferrin molecule leading to iron dissociation (Schryvers et al., 1998; Ekins et al., 2004). Some parasites such as Tritrichomonas foetus are able to use lactoferrin as a donor of ferric ions (Tachezy et al., 1996). 4.1 Salivary Flow and Factors Affecting It Salivary flow varies in the stimulated (eg, chewing) and unstimulated states (Humphrey and Williamson, 2001). Stimulated flow contributes up to 90% of average daily saliva production, at a rate of between 0.2 and 7mL/min. In the stimulated state, the parotid glands contribute more than 50% of total salivary flow. In contrast, normal flow in the unstimulated state is more than 0.1 mL/min, with the submandibular glands contributing approximately 65% of total flow; the paPage | 42

4 3 rotid glands, 20%; and the sublingual glands, 7%8%. Certain drugs and therapies such as radiation therapy have been known to affect salivary flow. Emotional states such as anxiety, stress and fear also affect salivary flow as evidenced by the dry mouth phenomenon (Humphrey and Williamson, 2001). 4.2 Effects of Reduced Salivary Flow Xerostomia refers to dryness of the mouth caused by hyposalivation. Xerostomia disrupts the normal homeostasis of the oral cavity, leading to a range of oral and dental disorders. It also causes difficulty in speech, taste and eating (Brosky, 2007). Xerostomia can have a significant impact on patients health, well-being, and overall quality of life. Studies have documented speech difficulties (Rhodus et al., 1995), changes in taste sensation (Rose-Ped etal., 2002), swallowing difficulties and decreased dietary intake. The saliva is also reputed to have some anti-caryogenic properties. Probably the most important anti-caryogenic activity of saliva is the flushing and neutralizing effects, commonly referred to as "salivary clearance" or "oral clearance capacity" (Lagerlof and Oliveby, 1994). Hence, xerostomia patients are expected to be more susceptible to dental caries infections (Brown et al, 1978; Scully, 1986: Heintze et al., 1983) and indeed subjects with impaired saliva flow rate often show high caries incidence (Papas et al., 1993; Spak et al., 1994). Reduced salivary flow rate and the concomitant reduction of oral defense systems may also cause mucosal inflammations (Daniels et al., 1975; Van der Reijden et ah, 1996). Xerostomia is usually treated using topical therapies with lubricants such as mucin sprays, lozenges, humidifiers, gels, mouthwash and toothpaste; coating agents such as Gelchair and Magic

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4 4 Mouthwash; and finally saliva stimulants such as pastilles and sugar free gums and candies (Brosky, 2007). It must be emphasized, however, that no linear relationship exists among salivary secretion rate, caries activity, and DMFS/DMFT values (Birkhed and Heintze, 1989; Russell et al, 1990). Only weak or no association between saliva secretion rates and caries incidence has been shown (Mandel, 1987, 1989; Russell et al, 1991). Major and minor salivary gland secretion rates have also been assessed and correlated to the sensation and complaints of dry mouth (xerostomia), objectively reduced saliva secretion (hyposalivation), as well as to various oral health measures, and yet there is an unanswered question: How much saliva is enough? (Fox et al, 1987; Ship et al, 1991).

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4 5

4.3 Saliva and Dental Caries Formation

Dental caries, also known as tooth decay or a cavity, is an infection, usually bacterial in origin that causes demineralization of the hard tissues (enamel, dentin and cementum) and destruction of the organic matter of the tooth, usually by production of acid by hydrolysis of the food debris accumulated on the tooth surface.

Fig 8: Destruction of a tooth by cervical decay from dental caries (Root decay). Source: Wikipedia, 2012.

Caries is a unique multifactorial infectious disease. It is generally accepted, however, that saliva secretion and salivary components secreted in saliva are important for dental health. The final result, "caries to be or not to be", is a complex phenomenon involving internal defense factors, such as saliva, tooth surface morphology, general health, and nutritional and hormonal status, and a number of external factors-for example, diet, the microbial flora colonizing the teeth, oral hygiene, and fluoride availability (Lenander-Lumikar and Loimaranta, 2000).
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4 6

Fig 8: Factors affecting dental caries Source: Saliva and Dental Caries, 2000

The notion that dental caries in animals is an infectious, transmissible disease was first demonstrated by Keyes (1960). Since then, a group of phenotypically similar bacteria, collectively known as mutans streptococci, has been implicated as the principal bacterial component responsible for the initiation and the development of dental caries (Loesche, 1986). The tooth surface is unique among all body surfaces in two ways. First, it is a non-shedding hard surface, and, second, this surface is introduced into the human mouth during the first years of life. The earliest point at which the cariogenic mutans streptococci may become established is when the first teeth erupt. Solid surfaces are required for both streptococcal colonization and multiplication (Loesche, 1986). Once mutans streptococci become established, they are considered difficult to eliminate, and the caries process is made possible (Li and Caufield, 1995).
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4 7 The current concepts of dental caries focus on the fermentation of carbohydrates by cariogenicbacteria producing organic acids. Plaque bacteria produce a variety of end-products that may differ depending on the diet. When fermentable carbohydrates are present, the main organic acids produced are lactic, formic, and acetic acids (Geddes, 1975, 1981). These acids coincide with a pH drop in plaque, resulting in demineralization of the tooth (Loesche, 1986; Nyvad and Fejerskov, 1996) and creating an environment which is advantageous for further growth of Streptococcus mutans (Bradshaw et al., 1989; Dashper and Reynolds, 2000). Probably the most important caries-preventive functions of saliva are the flushing and neutralizing effects, commonly referred to as "salivary clearance" or "oral clearance capacity" (Lagerlof and Oliveby, 1994). In general, the higher the flow rate, the faster the clearance (Miura et al., 1991) and the higher the buffer capacity (Birkhed and Heintze, 1989). Reduced salivary flow rate and the concomitant reduction of oral defense systems may cause severe caries and mucosal inflammations (Daniels et al., 1975; Van der Reijden et ah, 1996). In conclusion, dental caries is probably the most common consequence of hyposalivation (Brown et al, 1978; Scully, 1986).

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