Saliva; An Oral Microbial Modulating Agent

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Saliva: An Oral Microbial

Modulating Agentby

Ajeigbe Yekeen Abiola

IN PARTIAL FULFILMMENT OF THE AWARD OF

THE REQUIREMENTS FOR THE AWARD OF

BACHELOR OR SCIENCE B.Sc. DEGREE INMICROBIOLOGY, UNIVERSITY OF ILORIN,

KWARA STATE, NIGERIA.


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Table of ContentsTable 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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Chapter 1: Introduction

The mouth is the gateway of the body to the external world and represents one of the most bio-

logically complex and significant sites in the body. Saliva (also referred to as spit, spittle, drool

orslobber) is a watery, sometimes frothy fluid found in the oral cavity ofHomo 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 electro-

lytes: 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 ap-

proximately 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 above-

mentioned 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 car-


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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 (Jen-

sen 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 exoge-

nous nutrients are limiting. Bacteria can also produce glycan hydrolases that cleave sugar resi-

dues 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 sub-

stances can be excreted from the body, including mercury, lead, potassium iodide, bromide,

morphine, ethyl alcohol, and certain antibiotics such as penicillin, streptomycin, and chlortetra-

cycline (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 sialo-

chemistry 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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Fig 1: The location of salivary glands in the oral cavity

Source Britannica 2003

1.1 Salivary Glands

Saliva is secreted by three (3) major

salivary glands namely parotid, sub-

mandibular and sub-lingual (Fig 1)

and these secrete about 90% of the

saliva in the mouth. Hundreds of mi-

nor salivary glands also secrete saliva

and these accounts for the remaining

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 con-

tains 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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names imply, serous secretions contain more water than the viscous saliva produced by mucous

glands (Jensen et al., 2003: Pedersen et al., 2002).

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 differ-

ent surfaces for microbial colonization. The constant production of saliva and intermittent provi-

sion 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

Fig 2: Gland contribution of unstimulated salivary flow

Source: Patricia et al., 2008


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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 com-

mensal microorganism is defined as one that lives on or within a host but does not cause any ap-

parent 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 mi-

crobes 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 Aggregatibacteractinomycetemcomitans, while Actinomyces is-

raelii can cause oral cysts. Overt pathogens are organisms that usually cause disease when pre-

sent, 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 (Meningo-

cocccus) andHaemophilus 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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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, implanti-

tis 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 Description Microorganisms implicated

Caries Decay (loss) of tooth enamel (dental caries)

or dentin (dentinal caries), or root dentin (root

caries)

Streptococcus, Lactobacillus,

Actinomyces (root caries)

Gingivitis Redness and swelling (inflammation) of the

gingival tissues (gums)

Actinomyces, Fusobacterium,

Bacteroides, Prevotella

Periodontitis Inflammation and either rapid (aggressive,

either generalized or localized) or slower

(chronic) destruction of the tissues supporting

the tooth

Aggregatibacter (localized),

Porphyromonas, Treponema,

Tannerella, Fusobacterium,

Prevotella

Implantitis Infection and destruction of tissues surround-

ing a dental titanium implant

Staphylococcus, Pseudomo-

nas, Porphyromonas,

Prevotella

Pulpitis Infection of the pulp, inflammation around Fusobacterium, Dialister,


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the apex of the root, leading to abscess for-

mation (periapical granuloma)

Peptostreptococcus, Porphy-

romonas

Halitosis Oral malodor Fusobacterium, Porphyromo-

nas, Prevotella, Treponema,

Eubacterium

Pharyngitis Redness and inflammation of the pharynx. Group A Streptococcus, Neis-

seria, Haemophilus, Coxsack-

ie A virus

Tonsillitis Infection and inflammation of the tonsils. Group A Streptococcus, Hae-

mophilus

Leukoplakia White patches on the buccal mucosal epithe-

lium or tongue

Candida, human papilloma

virus (HPV)

Stomatitis Reddening and inflammation of the oral mu-

cosa

Candida albicans, Candida

tropicalis, other Candida spe-

cies.

Actinomycosis Hard swelling (cyst) within the gums Actinomyces israelii

Cold sores Surface (superficial) red, dry lesions close to

the lips

Herpes smplex virus (HSV)

Source: Lamont and Jenkinson, 2010.


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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:

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

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


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glucose, sodium, chloride, and urea) and its capacity to provide the dissolution of substances al-

lows 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 tis-

sues 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 antimicro-

bial 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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Mastication, speech, and deglutition are also aided by the lubricant effects of the proteins (mu-

cins) 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 res-

idues 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).

Fig 4: Illustration of salivary seromucosal covering

Source: Patricia et al., 2008


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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 avail-

ability 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, sali-

vary 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 (cit-

rate), and to macromolecules (statherin, histidine-rich peptides, and proline-rich proteins). A


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special case of the combination of calcium is its strong link with -amylase, where it acts as a co-

factor 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 den-

tal 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 im-

portant, 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 William-

son, 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 con-

centration 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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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 op-timization 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 in-

creasing 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 H 2PO4

-. This acid-base

pair has a pKa value in the range 6.8-7.2, which has a maximum buffering capacity that is rela-

tively 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 concentra-

tions 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;

Edgaret al., 2004; Jaffe et al., 1986; Ertugrul et al., 2003).


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Ammonia, a product of urea and amino acid metabolism, is potentially cytotoxic to gingival tis-

sues. It is an important factor in the initiation of gingivitis because it may increase the permeabil-

ity 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 en-

zyme -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 co-

agulation 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 sub-

mandibular glands (Cate, 1998).


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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 secre-

tory immunoglobulin A (S-IgA) etc.

Sugars in their free form are present in total stimulated and unstimulated saliva at a mean con-

centration 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, par-

ticularly 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 func-

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

Constituent Whole Saliva (mg/100ml)

Resting State Stimulated State

Protein 220 280

IgA 19

IgG 1

IgM Less than 1

Amylase 38

Lysozyme 22 11

Albumin tr tr

Sodium 15 60

Calcium 6 6

Magnesium Less than 1 Less than 1

Phosphate 17 12

Bicarbonate 31 200

* Note: tr =trace amounts

Source: Marsh and Martin, 2009


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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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Carbonic anhydrase I (CAI) is one out of ten CA isoenzymes that have been identified in hu-

mans. 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 occur-

rence in living systems and its role in several physiological processes (Tashian 1989). This en-

zyme 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 Lind-

skog 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 im-

portant processes such as CO2 transport as HCO3- acid-base homeostasis, ion transport, for-

mation of aqueous humour and gastric juice, and syntheses of urea, glucose and fatty acids (Ma-

ren, 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 concen-

tration 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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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 sal-

ivary 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 proteas-

es and up-regulating cytokines. Von Ebner gland protein is another cysteine protease inhibitor

(Lamont and Jenkinson, 2010)

Fig 5: Suggested model for the function of CA VI on the dental surface.

Source: Saliva and Dental Caries, 2000


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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 activi-

ty (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 lipo-

polysaccharides of the external membranes of Gram-negative bacteria and are potent inhibitors

ofCandida 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 for-

mation and inhibition of histamine release by the mastocytes, suggesting a role in oral inflamma-

tion (Schenkels and Veerman, 1995).

At least 12 histatins are present in saliva, resulting from truncations or proteolysis of the genet-

ically distinct histatins 1 and 3. Histatin-5 (the N-terminal 24 amino acids of histatin-3) is a ma-

jor 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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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 struc-

ture 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 sys-

tems against microbial agents invading the organism mostly via mucosal tissues. Lactoferrin af-


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fects the growth and proliferation of a variety of infectious agents including both Gram-positive

and negative bacteria, viruses, protozoa, or fungi (Kirkpatricket 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. Lac-

toferrin 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 de-

mands such asLactobacillus sp. orBifidobacterium 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 as-

partic acid); while an arginine chain is responsible for binding the carbonate ion (Baker, 1994;

Ward et al., 1996).


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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 lipopol-

ysacharide leads to impaired permeability and a higher sensitivity to lysozyme and other an-

timicrobial agents (Arnold et al., 1977; Yamauchi et al., 1993; Leitch and Willcox, 1998). Lipo-

polysacharide can be disposed of even without the direct contact of lactoferrin with the cell sur-

face (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 Anto-

nini, 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 bacte-

ricidal activity of neutrophils (Sanchez et al., 1992).In vitro lactoferrin is able to prevent Pseu-


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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 coloniza-

tion. 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 glycosamino-

glycans. 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 effec-

tive against the Herpes simplex virus (Fujihara and Hayashi, 1995; Marchetti et al., 1996), cyto-

megaloviruses (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 andEimeria 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 be-


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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 sub-

stances (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 inflamma-

tion and subsequent tissue damage caused by the release of pro-inflammatory cytokines and re-

active 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 secre-

tions 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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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 betweenN-acetylmuramic acid andN-acetylglucosamine in peptidoglycan (Fig-

ure 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 de-

stroy 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; Amer-

ongen 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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3.3.5 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 cata-

lyst for the oxidation of the salivary thiocyanate ion (SCN) by hydrogen peroxide into hypothi-

ocyanate (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 in-

hibits 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 oxi-

dative damage to the host soft tissues (Edgar, 1992; Humphrey and Williamson, 2001; Tenovuo

and Lagerlof, 1994; Amerongen and Veerman).

Fig 6: Action of lysozyme on Gram-positive bacterial peptidoglycan



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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 phos-

phate salts and the growth of hydroxyapatite crystals on the tooth surface, preventing the for-

mation 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 proline-

rich proteins is the capacity to selectively mediate bacterial adhesion to tooth surfaces (Edgar,

1992; Humphrey and Williamson, 2001; Tenovuo and Lagerlof, 1994; Amerongen and Veer-

man).

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 resi-

dues 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 small-

er mucin glycoprotein 2 (MG2). MG1 is encoded by the MUC5B gene and MG2 is encoded by

theMUC7gene. 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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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 ad-

jacent 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.

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).

Fig 7: Alignment of mucin chains



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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 nega-

tively 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 man-

ner 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). Sali-

vary agglutinin, a highly glycosylated protein frequently associated with other salivary proteins

Fig 8: Gel forming and membrane bound mucins in the oral cavity



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and with secretory IgA, is one of the main salivary components responsible for bacteria aggluti-

nation (Amerongen and Veerman, 2002).

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.

Fig 9: Depiction of mechanisms of bacterial agglutination by salivary mucins



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Secretory immunoglobulin A (IgA) is the largest immunologic component of saliva. It can neu-

tralize 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 (Humph-

rey 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 S-

IgA to the mucus lining of the epithelial surface and thus inhibits attachment and tissue penetra-

tion by viruses, bacteria, and their released antigens such as LPS, toxins and environmental anti-

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


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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 experi-

ments 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 host-

microbial 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 bacte-

ria 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 sali-

vary glands (Korsrud and Brandtzaeg, 1980). IgA secreting plasma cells predominate in the ma-

jor and minor salivary glands over plasma cells producing other Ig isotypes (Michalek and Chil-

ders, 1990). Polymeric IgA containing J chain, secreted by plasma cells, is specifically recog-

nized 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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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 trans-

location, disulfide bonds covalently link SC with polymeric IgA, which in turn stabilizes the

IgA-SC complex (Bradtzaeg, 1995).

Immunoactivity

Inhibition of bacterial adherence: The inhibition of bacterial adherence by SIgA is consid-ered one of the most important defense mechanisms against mucosal bacterial invasion. SIgA

interferes with bacterial adherence to host surfaces by preventing both nonspecific and stere-

ochemical 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 hydropho-

bic interactions between bacteria and host receptors (Marcotte and Lavoie, 1998). Free in sa-

liva, polymeric IgA effectively aggregates bacteria (Lamont and Jenkinson, 2010).

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


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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 bacte-

ria. 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 migra-

tion (Lamont and Jenkinson, 2010).

Viral Immunity: SIgA plays an important role in viral immunity because of its presence atthe 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 acrossthe mucosal barrier by a combination of a thick, flowing mucus barrier and the secretory im-

mune 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 mu-

cosal epithelium. This involves the binding of SIgA antibodies with antigens, which facili-

tates 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 patho-

genic 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; Shroffet al.,

1995), also binds to the normal flora (Shroff and Cebra, 1995; Shroffet al., 1995; Orndoffet

al.,, 2004). Thus, presumably, SIgA exercises a degree of immune exclusion toward the nor-


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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 theo-

ry stems from work by Williams and Gibbons (1972), who demonstrated that bacteria agglu-

tinated by SIgA adhere to epithelial surfaces in vitro less well than do unagglutinated bacte-

ria. Binding ofStreptococcussanguis (S. sanguis), S. mitis and two strains ofS. 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 Gib-

bons 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 prima-

ry 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 in-

testinal 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 sali-

vary 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 suscepti-

bility to caries and periodontal diseases. It should be noted that Salivary IgA is part of a complex


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and interdependent immune defense system and hence its full effects and deficiencies are not ful-

ly 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 lo-

cated in region 4q11-q13. Dentinogenesis imperfecta and perhaps juvenile periodontitis are cod-

ed in the same region.

Statherin play an important role in the maintenance of oral health since, like the proline-rich pro-

teins, it binds calcium and inhibits crystal growth. Statherin also inhibits spontaneous precipita-

tion of calcium phosphate salts. Thus, statherin and PRPs may prevent build-up of harmful de-

posits in the salivary glands and on the tooth surfaces (Humphrey and Williamson, 2001).

3.4 Summary of Immunoactive Components of the Saliva

Immunoactive Component Function/Activity

Cystatin Bacteriostatic

Histatin Bactericidal and fungicidal

Lactoferrin Iron sequestration

Lysozyme Bactericidal (More effective on gram positive

bacteria due to the peptidoglycan in their cell

Table 3: Immunoactive Components in Saliva and their functions


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walls)

Peroxidase Bactericidal

Proline-Rich Proteins Bacteriostatic and bactericidal.

Mucin-glycoprotein 1 and 2 Bactericidal, physical removal of microorgan-

isms

Secretory Immunoglobulin A Bacteriostatic, bactericidal, anti-viral

Statherin Bacteriostatic

Source: Anonymous


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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 ex-

ample, some Streptococcus, Haemophilus and Neisseria species produce proteases that specifi-

cally 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 effi-

cient, 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 che-

lating 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 (Schryv-

ers 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 Wil-

liamson, 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 pa-


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rotid glands, 20%; and the sublingual glands, 7%8%. Certain drugs and therapies such as radia-

tion 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 sensa-

tion (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, xerosto-

mia 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; Spaket 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, loz-

enges, humidifiers, gels, mouthwash and toothpaste; coating agents such as Gelchair and Magic


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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 etal, 1991). Major and minor salivary gland secretion rates have

also been assessed and correlated to the sensation and complaints of dry mouth (xerostomia), ob-

jectively 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 etal, 1987; Ship et

al, 1991).


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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.

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).

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

Source: Wikipedia, 2012.


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The notion that dental caries in animals is an infectious, transmissible disease was first demon-

strated by Keyes (1960). Since then, a group of phenotypically similar bacteria, collectively

known as mutans streptococci, has been implicated as the principal bacterial component respon-

sible 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 consid-

ered difficult to eliminate, and the caries process is made possible (Li and Caufield, 1995).

Fig 8: Factors affecting dental caries

Source: Saliva and Dental Caries, 2000


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The current concepts of dental caries focus on the fermentation of carbohydrates by cariogenic-

bacteria producing organic acids. Plaque bacteria produce a variety of end-products that may dif-

fer 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 Fejer-

skov, 1996) and creating an environment which is advantageous for further growth of Strepto-

coccus mutans (Bradshaw et al., 1989; Dashper and Reynolds, 2000).

Probably the most important caries-preventive functions of saliva are the flushing and neutraliz-

ing 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 in-

flammations (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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Saliva; An Oral Microbial Modulating Agent