Dental caries: Progress in microbiology and immunology

Journal o f Infection (1981) 3, 107-133

SPECIAL REVIEW

Dental caries: progress in microbiology and immunology Crispian Scully Immunology Unit, Department of Oral Medicine and Pathology, University of Glasgow, Glasgow Dental Hospital and School, 378 Sauchiehall Street, Glasgow Summary Dental caries is the most prevalent infectious disease affecting man. Considerable evidence implicates Streptococcus mutans as a major causal microorganism although other bacteria are clearly involved in at least some circumstances. Dietary factors, especially the pattern of sucrose intake, and host factors are important. Normal salivation is essential to the host defences against caries and immunological factors are important. Immunisation of rodents and primates has been successful in protecting against caries and the search for antigens of Str. mutans that are protective and free of possible toxic effects is showing promise. Preventive dentistry already has a large armamentarium capable of preventing and controlling caries. Dietary restriction of sucrose and the use of fluorides, especially systemic fluoridation, are capable of producing a major reduction in caries. However, in spite of the success and safety of fluoridation this major advance, introduced first over 35 years ago, still reaches only a minority of the population. In view of the negative public reaction to fluoridation and the current concern involving vaccination against more life-threatening disease, the future of immunisation against caries would appear to depend on many factors other than scientific feasibility.

Introduction

Dental caries is the localised destruction of tooth tissue caused mainly by demineralisation commencing at the external surface of the tooth. Caries is the most prevalent infectious disease affecting m o d e r n man (Scherp, 1971). Dental caries is now recognised to result from the interaction of microbial, dietary and host factors (Keyes, 1962). Certain sites on the teeth are especially prone to carious attack and these sites (fissures, pits, adjacent to the gingiva and the contact points between the teeth) are also frequently covered by a dense and complex microbial m a s s - - t h e dental plaque. This plaque consists of many different microbial species whose distribution varies .not only between species, individuals of the same species, and at different sites within the oral cavity, but also from one site to another on the same tooth 0163-4453/81/020107+27 $01.00/0

©1981 The British Society for the Study of Infection

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(Gibbons and Van Houte, 1973). Demineralisation of the tooth is caused by acids liberated during the fermentation of dietary carbohydrates---especially sucrose (Gustaffson, Quensel, Lambe, Lundqvist, Graham, Bonow and Krasse, 1954; Keyes, 1968). Evidence that host factors are involved in the pathogenesis of caries includes the effects on caries of acquired changes in salivary flow rate and composition (Frank, Herdly and Phillippe, 1965) and genetic differences in predisposition to caries (Peri and Wagner, 1977). Prevention of dental caries is already quite feasible and is most readily achieved on an individual basis by reducing the amount of sucrose ingested and decreasing the frequency of ingestion. The mechanical removal of plaque (e.g. by toothbrushing) is desirable mainly as a means of preventing periodontal disease as it is mechanically impossible to clean completely those sites most prone to caries. Chemotherapeutic measures are also of some value but the other single most effective measure in the prophylaxis of caries is fluoridation. Optimal results are achieved with systemic fluoridation which can result in at least a 50 per cent reduction in the incidence of caries. Topical fluoride applications are also of value but the protection falls short of that achieved by systemic fluoridation (Murray, 1976). Unfortunately, for a number of ethical and other reasons, fluoridation has not received widespread acceptance. The potential of immunisation against dental caries has however, been recognised and this review attempts to summarise the progress towards that end. A microbial aetiology in dental caries

It was almost a century ago that acid production was demonstrated in carious dentine and it was reported that salivary bacteria fermented carbohydrates to produce sufficient acid to decalcify teeth (Miller, 1890). Following these early observations there began a prolonged controversy as to the causal microbial agents. Lactobacilli routinely could be isolated from carious lesions (Goadby, 1910; Kligler, 1915a) and it was shown that caries could be produced by incubating teeth in a broth of lactobacilli (McIntosh, James and Lazarus-Barlow, 1922; Bunting and Palmerlee, 1925; Bunting, Nickerson and Hard, 1926; Jay and Voorhees, 1927; Bunting, Nickerson, Hard and Crowley, 1928; Enright, Friesell and Trescher, 1932). However, in 1924 Clarke, working at St. Mary's Hospital, London, reported the isolation from the deeper layers of carious lesions of a streptococcus which assumed a rod shape in an environment of low pH and was therefore classified Streptococcus mutans (Clarke, 1924). Interest in Str. mutans and other viridans streptococci waned however, until Bibby (1939) reported that streptococci were the most common organisms in plaque and caries was induced in rats using a viridans type of streptococcus (Hammond and Tunnicliff, 1940). The involvement of bacteria in the aetiology of caries was further supported by the observations that antibiotics which suppressed the oral flora prevented caries in rats (McClure and Hewitt, 1946) and

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inhibited caries in man (Littleton and White, 1964; Handelman, Mills and Hawes, 1966). The bacterial aetiology of caries was confirmed by demonstrating that gnotobiotic rats remained free of caries when fed on a known cariogenic diet. (Orland, Blayney, Harrison, Reyniers, Trexler, Wagner, Gordon and Luckey, 1954). However, if these animals were infected with enterococci (Str. faecalis) together with either a proteolytic aerobic rod or an anaerobic pleomorphic rod, they then developed caries (Orland, Blayner, Harrison, Reyniers, Trexler, Ervin, Gordon and Wagner, 1955). The infectious nature of dental caries

Keyes (1960) showed that 'caries-resistant' Syrian albino hamsters, which failed to develop caries when maintained on a high sucrose diet, developed caries if they were caged with 'caries-active' hamsters or if they were orally inoculated with the faecal flora from these animals. It was also observed that suppression of the penicillin-sensitive oral flora of Osborne-Mendel rat dams during the suckling period only, resulted in greatly reduced caries in the pups (Keyes, 1960). Caries was, therefore, established as an infectious and transmissible disease. The role of Streptococcus mutans in dental caries

Following the preliminary evidence for the involvement of streptococci in caries, Fitzgerald and Keyes (1960) isolated a specific streptococcus from rodent caries which could produce caries if introduced into gnotobiotic rats (Fitzgerald, Jordan and Stanley, 1960). This work was supported by the induction of caries in animals using streptococci from human caries (Zinner, Aran, Jablon, Brust and Saslaw, 1964; Zinner, Jablon, Aran and Saslaw, 1965; Gibbons, Berman, Knoettner and Kapsimalis, 1966). The responsible streptococcus was soon found to be identical with Str. mutans which has now been implicated as a major organism in the aefiology of experimental dental caries in a number of animals including rats (Fitzgerald, Jordan and Stanley, 1960; Gibbons, Berman, Knoettner and Kapsimalis, 1966), hamsters (Zinner, Aran, Jablon, Brust and Saslaw, 1964; Zinner, Jablon, Aran and Saslaw, 1965; Krasse, 1966), gerbils (Fitzgerald and Fitzgerald, 1966), irus monkeys (Bowen, 1969) and rhesus monkeys (Lehner, Challacombe and Caldwell, 1975a, b). Examination of the distribution and numbers of different streptococci in the oral cavity has revealed that the dominant organism in human dental plaque is also Str. mutans (Carlsson, 1967). Higher numbers of cariogenic streptococci were found in humans with active caries, and children who developed caries of smooth surfaces during the subsequent year had a higher number of plaque streptococci at the time of initial examination than did those who failed to develop caries (Carlsson, 1967). These findings have

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since been confirmed by several workers (Schamschula and Charlton, 1971; Englander and Jordan, 1972). Streptococcus mutans appears to colonise teeth in a highly localised manner. It is more frequently isolated from areas recognised clinically as susceptible to caries (Ikeda and Sandham, 1971; Gibbons, de Paola, Spinell and Skobe, 1974; Shklair, Keene and Cullen, 1974), initially colonising pits and fissures (Ikeda and Sandham, 1971) but then increasing on smooth surfaces, especially at the contact areas (Ikeda, Sandham and Bradley, 1973). Higher numbers of Str. mutans were isolated from the plaque of carious than of non-carious tooth surfaces (de Stoppelaar, Von Houte and Backer-Dirks, 1969; Littleton, Kakehashi and Fitzgerald, 1970; Shklair, Keene and Simonson, 1972) and high numbers were associated with early carious lesions (Duchin and Van Houte, 1978). Of the seven serotypes of Str. mutans recognised (Bratthall, 1970; Perch, Kjems and Ravn, 1974) it is the serotype c that is most prevalent in caries in most Western populations. Streptococcus mutans is not however, invariably isolated from individual surfaces prior to the initiation of caries, and relatively few studies have examined the relation between Str. rnutans in plaque and the development of caries over a period of time. Caries has also occurred in some subjects in whom Str. mutans could not be detected (Ikeda, Sandham and Bradley, 1973; Mikkelsen and Poulsen, 1976; Swenson, Liljemark and Schuman, 1976). It appears likely that different microorganisms may be involved in the carious process at different sites on the tooth and that once a carious cavity is produced significant changes may occur in the composition of the plaque flora (Loesche and Syed, 1973). A wide range of microorganisms may be isolated from carious lesions and since several are cariogenic for experimental anim.als (Fitzgerald, Jordan and Stanley, 1960; Fitzgerald, Jordan and Archard, 1966) this might raise problems in immunisation. The role of these other microorganisms is therefore summarised below. Streptococci and other Streptococcus mutans A variety of streptococci other than Str. mutans are present in plaque (Carls-

son, 1968; Colman and Williams, 1972; Hardie and Bowden, 1976). Enterococci occur in only small numbers in plaque (Williams, Forbes, Blau and Eickenberg, 1950; Bahn, Shklair, Mazzarella and Calandra, 1960) and very small numbers ofStr, faecalis are found (Gold, Jordan and Van Houte, 1975). Enterococci can initiate caries in gnotobiotic animals, but Str. faecalis appears to be less cariogenic than Str. mutans (Drucker and Green, 1979). Streptococcus sanguis is universally present in plaque (Carlsson, 1967). It is less cariogenic in rats than is Str. mutans (Drucker and Green, 1979) and it is not cariogenic for hamsters (Krasse and Carlsson, 1970), monkeys (Bowen, 1968; Caldwell, Challacombe and Lehner, 1977) or man (de Stoppelaar, Van Houte and Backer-Dirks, 1969). Indeed in the latter study Str. sanguis showed an inverse relationship with the presence of Str. mutans and with caries.

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Streptococcus mitior (mitis) also is a prominent coloniser of plaque, is cariogenic in rodents (Gibbons and Banghart, 1968a) but is less cariogenic than Str. mutans (Drucker and Green, 1979). Streptococcus salivarius constitutes only a small percentage of the plaque flora (Gibbons, Kapsimalis and Socransky, 1964; Gibbons, Socransky, de Aranjo and Van Houte, 1964) and although it is cariogenic for rodents (Gibbons and Banghart, 1968a; Zinner and Jablon, 1968; Kelstrup and Gibbons, 1970; Krasse and Carlsson, 1970; Drucker and Green, 1979) it is rarely isolated from human caries (Loesche and Syed, 1973). Streptococcus milleri (anginosus) is only slightly less cariogenic for rodents than is Str. mutans (Drucker and Green, 1979). Lactobacilli

The association of Lactobacillus acidophilus with human caries has been confirmed by many groups (McIntosh, James and Lazarus-Barlow, 1922; Bunting and Palmerlee, 1925; Bunting, Nickerson and Hard, 1926; Jay and Voorhees, 1927) and recent studies have demonstrated an association of L. casei with caries in the rat (Fitzgerald, Jordan and Archard, 1966), monkey (Bowen, 1965) and man (Carlsson, Grahnen and Jonsson, 1975). The role of lactobacilli has been investigated by Ikeda, Sandham and Bradley (1973) who found that the onset of caries in children was preceded by a rise in the number of Str. mutans but often occurred in the absence of lactobacilli. Furthermore, although caries was associated with a rise in plaque lactobacilli in some instances, the lactobacilli became a sizeable proportion of the flora only after the initiation of caries. Lactobacilli appear therefore, to play at least a secondary role in the aetiology of caries. Other species

Actinomyces viscosus and A. naeslundii are found in significant numbers in

human plaque (Collins, Gerencser and Slack, 1973). Although in general actinomyces species appear incapable of creating a pH low enough for demineralisation (Larje and Frostell, 1968), A. naeslundii can produce root caries in gnotobiotic rats (Socransky, Hubersak and Propas, 1970) and A. viscosus may be isolated from advanced caries (Loesche and Syed, 1973), especially from caries of the root cement (Jordan and Hammond, 1972; Syed, Loesche, Pape and Grenier, 1975). The evidence suggests therefore, that bacteria other than Str. mutans may contribute to the carious process (Fitzgerald, 1976), but nevertheless Str. mutans does appear to have a major aetiological role. Cariogenic attributes of Streptococcus mutans

The capacity ofStr, mutans to initiate dental caries depends on the ability to release organic acids and to produce adhesive extracellular polysaccharides (Gibbons and Van Houte, 1975a, b). Streptococcus mutans also appears to

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have the capacity to store fermentable substrates (de Stoppelaar, Konig, Plasschaert and Van der Hoeven, 1971). Frequent intake of sucrose fosters colonisation of plaque by Str. m u t a n s (Krasse, 1965a, b; Krasse, Edwardson, Svensson and Trell, 1967), plaque formation (Krasse, 1965a, b; Rolla, 1976) and the production of caries, especially on smooth surfaces (Fitzgerald and Keyes, 1960; Krasse, 1965b; Krasse, Edwardson, Svensson and Trell, 1967). Fermentation of the glucose and fructose moieties of sucrose by Str. m u t a n s results in the production of lactic and other organic acids which appear to be responsible for the demineralisation of tooth substance (Jordan, 1965; Keyes, 1968; Tanzer, Krickevsky and Keyes, 1969; Tanzer, 1972). The ability ofStr, rnutans to colonise tooth surfaces and to develop plaque is related to the synthesis of adhesive extracellular polymers (Jordan and Keyes, 1966; Gibbons and Banghart, 1967; Newbrun, 1972). Glucose or other carbohydrates will promote the growth of Str. m u t a n s but not its adherence, since the polymers are synthesised exclusively from sucrose (Fitzgerald and Jordan, 1968; Gibbons, 1968; Gibbons and Fitzgerald, 1969). Streptococcus m u t a n s produces constitutive enzymes known as dextransucrases, sucrose-6-glucosyltransferases or glucosyltransferases (GTase, GTF. EC.2.4.1.5.) which hydrolyse sucrose to synthesise a variety of high molecular weight levans and dextrans (Gibbons and Nygaard, 1968; Robrish, Reid and Krichevsky, 1972; McCabe and Smith, 1973). The dextrans (glucans) are made up of otl-6 linked glucose moieties with variable portions of ot1-3 glucosidic linkages either as inter-residue or interchain linkages (Lewicki, Long and Edwards, 1971; Ebisu, Misaki, Kato and Kotani, 1974). Water-soluble glucans possess mainly a l - 6 links whereas a 1-3 links characterise the water-insoluble glucans (Freedman, Birkhed and Granath, 1978). Glucans are responsible for the adherence of Str. m u t a n s to hard surfaces (McCabe, Keyes and Howell, 1967; Gibbons and Nygaard, 1968; Olson, Bleiweis and Small, 1972; Mukasa and Slade, 1973, 1974a, b) and are involved in bacterial agglutination and adherence during plaque formation (Gibbons and Banghart, 1967; Guggenheim and Schroeder, 1967; Gibbons and Fitzgerald, 1969; Mukasa and Slade, 1973, 1974a, b; Kuramitsu, 1974). The importance of glucans in adherence is emphasised by the ability of the enzyme dextranase to cause disintegration of plaque deposits (Fitzgerald, Keyes, Stoudt and Spinell, 1968; Fitzgerald, Spinell and Stoudt, 1968) and by the inability of mutants of Str. m u t a n s that cannot produce glucans, but are still acidogenic, to adhere to surfaces or to initiate caries (Tanzer, Freedman, Fitzgerald and Larson, 1974). The water-insoluble glucans (mutans) appear to be most important in the adherence of Str. m u t a n s (Guggenheim and Schroeder, 1967; Gibbons and Nygaard, 1968; Olson, Bleiweis and Small, 1972; Mukasa and Slade, 1973, 1974a; Freedman and Tanzer, 1974; Genco, Evans and Taubman, 1974; Montville, Cooney and Sinskey, 1977). The importance of mutans in cariogenicity has been emphasised by Michalek, Shiota, Ikeda, Navia and McGhee (1975) who found that mutant strains of Str. m u t a n s that synthes-

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ised increased amounts of mutans exhibited increased adherence and greater cariogenicity than a wild type of Str. mutans. Mutants which synthesised but failed to bind glucan, failed to adhere to hard surfaces in vitro and produced 50 per cent fewer caries in mono-infected rodents in spite of remaining acidogenic (de Stoppelaar, Konig, Plasschaert and Van der Hoeven, 1971; Tanzer, Freedman, Fitzgerald and Larson, 1974). Immunisation against dental caries

The recognition that caries is an infectious disease prompted the hypothesis that there might be a host response which could mediate resistance to caries. The concept that prophylaxis against caries could be achieved by vaccination was first suggested over half a century ago (Ross, Krasnow and Samet, 1927). Preliminary but somewhat futile attempts to vaccinate humans were carried out using lactobacilli (Jay, Crowley and Bunting, 1932; Jay, Crowley, Hadley and Bunting, 1933; Canby and Bernier, 1942; Williams, 1944). There appears to have been no further work on the vaccination of man subsequent to these reports, but it is of interest to note a report showing significantly less caries in children vaccinated with Mycobacterium tuberculosis as compared with non-vaccinated children (Cuppini, Borea, Steganini and Capuzi, 1969). It is possible that this apparent beneficial effect may have been due to non-specific stimulation of immunity. Studies in rodents

There have been several attempts to induce protection against dental caries in experimental animals by the injection of live, heat-killed or formalinised bacteria, or bacterial products. Early studies on the active immunisation of animals against caries were however, largely unsuccessful. Jay, Crowley, Hadley and Bunting (1933) and Rosebury, Foley, Greenberg and Pollack (1934) immunised rats subcutaneously with lactobacilli but reported no protection against caries. Fitzgerald and Keyes (1962) reported that the subcutaneous immunisation of hamsters with a vaccine of whole cells of Str. mutans was unsuccessful but Wagner (1966, 1967) successfully protected gnotobiotic rats by the use of an intramuscular injection of whole cells of Str. faecalis in Freund's complete adjuvant (FCA). The protected rats had higher titres of serum and salivary agglutinins to Str. faecalis and also fewer Str. faecalis in saliva, suggesting that specific antibody was produced against the cariogenic bacterium and was secreted into the serum and saliva (Wagner, 1967). In spite of the early success, immunisation of rats has not invariably been effective in protecting against caries. Guggenheim, Muhlemann, Regolati and Schmid (1970) for example, on immunising rats intravenously with whole viable cells of Str. mutans were unsuccessful in protecting against caries. Tanzer, Hageage and Larson (1970) were also unsuccessful using subcutaneous immunisation of rats with formalinised whole cells of Str. mutans. This group later reported partial success in protecting rats against

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smooth surface and fissure caries by subcutaneous immunisation with the same preparation ofStr, mutans but administered in the region of the major salivary glands (Tanzer, Hageage and Larson, 1973). Immunisation induced a rise in titre of both serum and salivary agglutinins, but there was no reduction in the numbers of Str. mutans isolated. Moreover, the protection against caries was highly variable, with even an increase in caries in some animals. Partial success in immunisation against caries was also achieved by Gaffar, Marcussen, Ruffner and Kestenbaum (1970) in hamsters whose oral flora had been suppressed with antibiotics. This group used as the infecting and immunising organism a cariogenic bacterium Streptococcus SS2 (ATCC 27006) related to, but not identical with Str. salivarius. This microorganism produces caries in both rats and hamsters (Gibbons and Banghart, 1968; Gaffar, Marcussen, Ruffner and Kestenbaum, 1970). Intramuscular injections of hamsters with formalinised whole cells of Str. SS2 in FCA resulted in almost a 70 per cent reduction of caries in some animals, associated with haemagglutinating antibodies in both the serum and saliva (Gaffar, Marcussen, Schlissel and Volpe, 1971). Gaffar (1976) however, failed to induce protection against caries using killed whole cells of Str. mutans. The initial success in inducing protection against caries in rats by the subcutaneous immunisation with formalised whole cells ofStr, mutans in the region of the major salivary glands (Tanzer, Hageage and Larson, 1973) was followed by a series of reports in which a similar procedure was employed with the object of inducing salivary rather than serum antibodies (Taubman, 1973; Taubman and Smith, 1974, 1977). Immunisation of rats with whole cells of Str. mutans in FCA induced agglutinins in whole saliva (Taubman, 1973), a significant reduction in caries, and a reduction in the numbers of Str. rnutans. The salivary antibodies were mainly of the IgA class. Such a procedure however, also induced serum antibody titres up to 1 in 3000 although the class of antibodies was not determined (Taubman, 1973). The protection against caries was attributed to the induction of salivary antibodies but the presence of serum antibodies at high titres (and their possible relevance in protection) appears to have been neglected. Deposition of an antigen into the parenchyma of a secretory gland produces both an IgA and IgG antibody response and indeed IgG is usually the more prevalent class (Hurlimann and Lichoa, 1976). Olson, Bleiweis and Small (1972) showed that injection of Str. mutans directly into the submandibular glands was followed by an increase in serum IgG antibody detected by agglutination, but no antibody activity was detected in the saliva. Protection that has been attributed to a salivary IgA response may therefore have been related in part to a serum response, or to salivary antibodies other than those of the IgA class (p. 121). Studies in primates

The first successful immunisation of primates against caries was reported by Bowen (1969). Three Macaca irus (fascicularis) monkeys were immunised intravenously with a vaccine of live whole cells of Str. mutans, and a rise in

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serum agglutinating antibodies to Str. mutans dextran and GTF was associated with a significant reduction in caries of the deciduous teeth (Bowen, 1969). The same group (Bowen, Cohen, Cole and Colman, 1975) protected irus monkeys against caries in permanent teeth by the use of a vaccine of live whole cells of Str. mutans given intravenously, or cell wall preparations injected submucosally. Cohen, Colman and Russell (1979) have confirmed that the protection against caries extends to at least nine years after immunisation, and this group have also induced protection using a vaccine of formalinised whole cells of Str. mutans in aluminium hydroxide given subcutaneously and possibly with cell walls administered submucosally without adjuvant. Lehner, Challacombe and Caldwell (1975a, b) used a vaccine of heatkilled Str. mutans in Freund's incomplete adjuvant (FIA) to induce protection against smooth surface caries in deciduous teeth of rhesus monkeys (Macaca mulatta). Subcutaneous or submucosal immunisation was associated with a rise in serum complement-fixing antibodies to cell wall antigens and to a hydroxyapatite extract of Str. mutans culture supernatants. Effective immunisation was achieved later using a vaccine of formalinised Str. mutans in FIA (Lehner, Challacombe, Wilton and Caldwell, 1976). In neither study (Lehner, Challacombe and Caldwell, 1975a, b, c) was there a significant rise in salivary haemagglutinating IgA; antibodies were serum IgG antibodies. The permanent dentition is also protected by such immunisation (Lehner, Caldwell and Challacombe, 1977) and there is a correlation between the number of cavities and the colony forming units of Str. mutans isolated from cervical plaque (Caldwell, Challacombe and Lehner, 1977). It should be emphasised that in a number of these studies (Lehner, Challacombe and Caldwell, 1975 a,b,c; Caldwell, Challacombe and Lehner, 1977; Lehner, Caldwell and Challacombe, 1977) there was little difference in salivary haemagglutinating IgA antibody titres in immunised as compared with control monkeys. Evans, Emmings and Genco (1975) however, immunised irus monkeys subcutaneously with formalinised whole cells of Str. mutans in the region of the major salivary glands, followed by direct instillation into the parotid ducts, and produced a marked reduction in the number of tooth sites infected and in the numbers of Str. mutans isolated from infected sites. Immunised monkeys did not show protection against a non cross-reacting strain, implicating the involvement of specific antibodies (Emmings, Evans and Genco, 1976). The reduction inStr, mutans correlated with salivary secretory IgA antibodies to Str. mutans but the rise in salivary antibodies was accompanied by a rise in titre of serum IgG, IgM and IgA antibodies after subcutaneous injection or ductal instillation (Evans, Genco and Emmings, 1976). Schick, Klimek, Weimann and Zwisler (1978) immunised irus monkeys submucosally with cell walls of Str. mutans without adjuvant and confirmed that a reduction in caries in immunised animals correlated with a rise in serum, rather than salivary, IgG antibodies. It would appear therefore, that protection against caries is associated in some instances with a

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salivary antibody and in others with a serum antibody response; this is discussed below (p. 121). Oral immunisation studies

Salivary IgA antibodies appear to be associated with protection against caries in at least some species. Oral administration of killed Str. rnutans to gnotobiotic rats has induced solely a secretory IgA antibody response (Taubman and Smith, 1973; Michalek, McGhee, Mestecky, Arnold and Bozzo, 1976) with no detectable serum or salivary IgG, or salivary IgM antibody response (Michalek, McGhee, Mestecky, Arnold and Bozzo, 1976). Such an immunisation procedure has induced significant protection against caries and a reduction of Str. m u t a n s in plaque (Michalek, McGhee and Babb, 1978). The oral administration of killed Str. m u t a n s to man (McGhee, Mestecky, Arnold, Michalek, Prince and Babb, 1978; Mestecky, McGhee, Arnold, Michalek, Prince and Babb, 1978) or monkey (Challacombe and Lehner, 1980) has elicited mainly a secretory IgA antibody response but these studies are too preliminary to assess with respect to caries protection. In contrast, oral administration of live Str. m u t a n s to monkeys has induced mainly a serum (Ciardi, Bowen, Reilly, Hsu, Gomez, Kuzmiak-Jones and Cole, 1978) or a serum and salivary (Challacombe and Lehner, 1980) antibody response. Passive immunisation studies

Early passive immunisation experiments were unsuccessful in protecting animals against caries. Fitzgerald and Keyes (1962) attempted to immunise hamsters passively using rabbit hyperimmune antisera against cariogenic streptococci. In a brief study lasting only two months, there appeared to be no serum antibodies in the recipient animals at the termination of the study and no protection against caries. Sweeney, Shaw and Childs (1966), who gave y-globulin from the serum of caries-resistant rats subcutaneously to nonresistant animals also failed to induced protection against caries, but the antibody titres in donors or recipients were not reported. Caries immunity has however, been passively derived by rat pups suckling dams that have been previously immunised intravenously with Str. m u t a n s whole cells (McGhee, Michalek, Navia and Narkates, 1976). The colostrum of the dams contained antibodies of both IgA and IgG classes, which were transferred to the pups and conferred immunity (Michalek, Rahman and McGhee, 1975; McGhee, Michalek and Ghanta, 1976; Michalek and McGhee, 1977). Passive immunity to caries has also been transferred to rat pups by the oral administration of dried milk from cows immunised with Str. m u t a n s (Michalek, McGhee, Arnold and Mestecky, 1978). Successful passive transfer of immunity to caries has also been reported in monkeys using serum immunoglobulins from immunised animals (Lehner, Russell, Challacombe, Scully and Hawkes, 1978; Lehner, Russell, Wilton, Challacombe, Scully and Hawkes, 1978).

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Antigens used in immunisation

The antigens used in most of the immunisation studies discussed above have been whole cells o f S t r , m u t a n s which elicit a wide variety of responses. Little is known about the nature of the protective antibodies other than in a few instances, of their classes or of the general associations of rising titres with increased protection. Antigens suggested as the possible candidates for stimulation of protective immunity include glucosyltransferase (Evans and Genco, 1973), cell-associated glucans (Genco, Emmings, Evans and Apicella, 1976), glucan receptors (McCabe and Smith, 1976), serotype specific polysaccharide antigens (Linzer and Slade, 1974; Iacono, Taubman, Smith and Levine, 1975), lipoteichoic acid (Bleiweis, Taylor, Deepak, Brown and Wetherell, 1976) and several cell wall protein antigens (Russell and Lehner, 1978; M.W. Russell, 1979; R.R.B. Russell, 1979a, b, 1980; Russell, Bergmeier, Zanders and Lehner, 1980). Since glucosyltransferase (GTF) activity and the adherence of Str. m u t a n s are involved in the pathogenesis of caries, and protective immunisation schedules using antigens other than GTF often elicit serum and/or salivary antibodies to GTF (Genco, Evans and Taubman, 1974) or to fractions containing GTF activity (Bowen, 1969; Emmings, Evans and Genco, 1975; Lehner, Challacombe and Caldwell, 1975a, b), several groups have attempted to induce protection against caries by immunising with preparations of GTF. Immunisation does not invariably result in an antibody response to GTF. Ciardi, Bowen, Reilly, Hsu, Gomez, Kuzmiak-Jones and Cole (1978) failed to elicit a rise in titre of serum antibodies to GTF by immunising monkeys submucosally with formalinised Str. m u t a n s , although they did elicit a rise in antibody titre using live organisms. Bahn, Pinter, Quillman and Hayashi (1969) successfully protected rats against caries by the intraperitoneal injection of crude GTF from Str. m u t a n s in FIA and protection was associated in some animals with a rise in serum inhibitory antibodies to GTF (Hayashi, Shklair and Bahn, 1972). Crude GTF from Str. m u t a n s given intraperitoneally to hamsters also induced partial protection against caries (Gaffar, 1976) and a rise in titres of serum and salivary haemagglutinating inhibitory antibodies against GTF (Gaffar, Marcussen, Schlissel and Volpe, 1971). GTF from Str. m u t a n s in FIA injected into the salivary gland region of rats and hamsters induced a salivary antibody response and protection against caries comparable with that obtained by immunising with whole cells of Str. m u t a n s (Smith, Taubman, and Ebersole, 1976a, b; Taubman and Smith, 1977). However, in contrast to these successes using GTF in rodents, Guggenheim, Muhlemann, Regolati and Schmid (1970) were unsuccessful in protecting rats against caries by intravenous immunisation with crude GTF from Str. rnutans. In these animals, in spite of a rise in titre of serum antibodies, there was even a slight increase in caries above that of controls. Immunisation of monkeys with GTF preparations from Str. m u t a n s has also given variable results. Bowen, Cohen, Cole and Colman (1975) immunised

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irus monkeys submucosally with a GTF preparation and reported an enhancement of caries. Subcutaneous immunisation of rhesus monkeys with crude GTF preparations also failed to induce protection against caries (Russell, Challacombe and Lehner, 1976) although it did elicit a rise in titre of serum GTF inhibitory antibodies (Bahn, Cummings and Hayashi, 1976; Russell, Challacombe and Lehner, 1976). Bahn's group however, induced a significant reduction in caries in monkeys immunised submucosally with GTF from Str. m u t a n s (Bahn, Shklair and Hayashi, 1977) and Schick, Klimek, Weimann and Zwisler (1978) submucosally immunised flus monkeys with' GTF from Str. m u t a n s in aluminium hydroxide inducing protection against caries in association with a rise in titre of serum antibodies. These preparations may however, have contained other antigens. Protective immunisation of monkeys is associated with a serum antibody response to cell wall and culture supernatant antigens of Str. m u t a n s (Lehner, Challacombe and Caldwell, 1975a, b, c; Cohen, Colman and Russell, 1979) and immunisation with cell walls of Str. m u t a n s induces protection against caries (Lehner, Challacombe and Caldwell, 1975b; Bowen, Cohen, Cole and Colman, 1975). However, immunisation with Str. m u t a n s cell walls treated with proteolytic enzymes has failed to induce protection, or has even increased caries (Colman and Cohen, 1979; Russell, Challacombe and Lehner, 1980) suggesting that a protein component of the cell wall is the immunogen responsi_ble for protective immunity. Protective immunisation may be associated with a rise in titre of serum antibodies to certain of these cell wall protein antigens (Colman and Cohen, 1979; Russell, Challacombe and Lehner, 1980) and immunisation of monkeys with selected antigens appears to induce protection against dental caries (Lehner, Russell and Caldwell, 1980). Problems in immunisation

Successful immunisation of a number of species with antigens of Str. m u t a n s has provided protection against the subsequent development o f S t r , m u t a n s induced caries, associated, especially in primates, with the induction of serum antibodies and with the induction of salivary antibodies in other species. The reduction in caries has been associated with fewer infecting organisms in several instances (Hayashi, Shklair and Bahn, 1972; Taubman and Smith, 1974; Bowen, Cohen, Cole and Colman, 1975; Evans, Emmings, and Genco, 1975; Emmings, Evans and Genco, 1976; Lehner, Challacombe and Caldwell, 1976; Caldwell, Challacombe and Lehner, 1977; Lehner, Caldwell and Challacombe, 1977). However, although it is now evident that immunisation of experimental animals against caries can be successfully carried out, a number of potential problems remain. The possibility of antigenic drift (Bratthall and Gibbons, 1975a, b) may be a theoretical rather than practical problem, since there is cross-reaction between several serotypes of Str. m u t a n s with respect to cell wall associated protein antigens (Russell and Lehner, 1978; R . R . B . Russell, 1979a, 1980), glucosyltransferases

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(R. R. B. Russell, 1979c) and opsonic antibodies (Scully and Lehner, 1978). It is also clear that immunisation of rodents (Smith, Taubman and Ebersole, 1976a,b; Michalek, McGhee, Arnold and Mestecky, 1978; Scully and Lehner, 1978; R. R. B. Russell, 1980) or monkeys (Scully and Lehner, 1978) with a single serotype of Str. m u t a n s may also elicit antibodies crossreacting with other serotypes. A further problem has been the necessity for the use of an adjuvant unacceptable for use in humans (such as Freund's complete or incomplete adjuvant) for the induction of immunity in many instances. Successful immunisation using aluminium hydroxide as adjuvant has however, now been achieved (Cohen, Colman and Russell, 1979; Lehner, Russell and Caldwell, 1980) and indeed an adjuvant may not be necessary (Cohen, Colman and Russell, 1979). A cautionary note has been struck by the demonstration in rabbit antisera to Str. m u t a n s of antibodies cross-reactive with human heart and skeletal muscle (Van de Rijn, Bleiweis and Zabriskie, 1976). Certain of the cell wall associated protein antigens appears to be cross-reactive with human heart tissue (Hughes, 1979; R. R. B. Russell, 1979b; Hughes, MacHardy, Sheppard and Woods, 1980; R. R. B. Russell, 1980). Antibodies cross-reactive with cardiac tissue may however, be of low avidity (Ferretti, Humphrey, Begay and Shea, 1979), and the presence of such serum antibodies does not necessarily imply subsequent host damage (Yang, Soprey, Wittner and Fox, 1977). No side-effects have been reported in monkeys immunised either with Str. m u t a n s cells (Cohen, Colman and Russell, 1979) or selected protein antigens (Lehner, Russell and Caldwell, 1980) and the protein antigen cross-reactive with heart tissue may not be the immunogen responsible for protection against caries (Colman and Cohen, 1979). This potential problem remains however, a source of some concern and much research activity. Ho~t effector mechanisms in the protection against dental caries

The mechanism(s) involved in immunological protection against caries have not been fully elucidated. Protection would appear to require one or more mechanisms that might reduce or control the number of cariogenic organisms at the sites of carious attack, or in some way block their cariogenicity. The lack of a blood supply directly at the site of dental p!aque accumulation does not preclude the involvement of specific antibodies in protection against bacterial colonisation, since antibody might enter the oral environment in the saliva (Tomasi and Bienenstock, 1968) or the gingival crevicular fluid (Brandtzaeg, 1965). Antibody-mediated protective mechanisms have been suggested to function by one of a number of mechanisms including: (1) inhibition of adherence by inhibiting GTF and thus preventing the formation of adhesive extracellular polysaccharides, or by blockade of cell surface binding sites for the GTF (Genco and Taubman, 1973) or for polymer (Mukasa and Slade, 1973, 1974; Olson, Guggenheim and Small,

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1974); (2) interference with carbohydrate transport mechanisms in the cariogenic organisms (Gibbons, 1972; Tanzer, Brown and Mclnemey, 1973); (3) opsonisation of cariogenic organisms followed by the phagocytosis and killing by phagocytic cells (Lehner, Challacombe and Caldwell, 1976; Scully, 1979). Saliva is clearly of great importance in the control of the oral flora and protection against caries, since caries may become rampant if the salivary flow is reduced in a number of conditions including salivary gland aplasia, Sjogren's syndrome, or irradiation involving the major salivary glands (Zaus and Teuscher, 1940; Frank, Herdly and Philippe, 1965; Llory, Dammron and Frank, 1971; Daly and Drane, 1974). However, the situation is compli: cated by a marked alteration in the dietary habits of patients with severe xerostomia, with an increased frequency in ingestion of soft cariogenic foods (Brown, Dreizen, Rider and Johnston, 1976) and studies in healthy subjects have produced little evidence of a major effect of differences in salivary flow rate upon the incidence of caries (Mandel, 1974). Saliva has long been recognised as possessing antibacterial activity. Mechanical cleansing may well be important, but non-specific antibacterial mechanisms include lysozyme (Mandel and Zengo, 1973), peroxidases (Dogon, Kerr and Amdur, 1962), glucanases (Staat, Gawronski and Schachtele, 1973), lactoferrin (Masson, Heremans and Dive, 1966) bacteriocins (Hamada and Ooshima, 1975) and glycoprotein aggregating factors (Hay, Gibbons and Spinell, 1971; Kashket and Guilmette, 1978). Apart from the many possible non-immunological anti-bacterial mechanisms, the presence of y-globulins in whole (Ellison, Mashimo and Mandel, 1960) and parotid saliva (Mandel and Ellison, 1961) suggested that saliva may contain antibacterial mechanisms of an immunological nature. IgA is the major salivary immunoglobulin, but IgG and IgM (Brandtzaeg, 1965; Brandtzaeg, Fjellanger and Gjeruldsen, 1970), IgD (Sewell, Matthews, Flack and Jefferis, 1979) and IgE (Salmon, 1970) are also found. At least some of the salivary IgG and IgM enters mixed saliva from the plasma via the gingival crevice (Brandtzaeg, Fjellanger and Gjeruldsen, 1970; Holmberg and Killander, 1971; Challacombe, Russell, Hawkes, Bergmeier and Lehner, 1978). Saliva contains secretory IgA (slgA) naturally-occurring antibodies to oral bacteria (Evans and Mergenhagen, 1965; Kraus and Konno, 1965) including antibodies to Str. rnutans (Challacombe, Guggenheim and Lehner, 1973; Bratthall and Gibbons, 1975b; Arnold, Mestecky and McGhee, 1976). Such IgA antibodies can bind to oral bacteria (Brandtzaeg, Fjellanger and Geruldsen, 1968; Klein, Guinard and Frank, 1974; Nisengard and Jarrett, 1976) and may confer protection by inhibition of bacterial adherence (Williams and Gibbons, 1972). In vitro studies have suggested that antibodies might inhibit the adherence of Str. mutans (Genco, Evans and Catlin, 1970; Hamada and Slade, 1976). sIgA appears unlikely to be protective by a bactericidal effect, as it does not fix complement (Colten and Bienenstock, 1974) and IgA appears not to be opsonic (Quie, Messner and

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Williams, 1968) but may inhibit phagocytosis (Van Epps, Reed and Williams, 1978) including the phagocytosis of Str. m u t a n s (Scully, 1979). IgA-mediated inhibition of bacterial adherence appears not however, to be the sole mechanism involved in the immunological protection against caries. As discussed above, in the rodent at least, salivary IgG often rises in association with the rise in salivary IgA that accompanies immun~sation (McGhee, Michalek and Ghanta, 1976) and may be responsible for some of the protection attributed to IgA. Furthermore, many of the immunisation schedules that have induced salivary 'protective' antibodies in rodents have also induced serum antibodies (Hayashi, Shklair and Bahn, 1972; Tanzer, Hageage and Larson, 1973; Taubman and Smith, 1974; McGhee, Michalek, Webb, Navia, Rahman and Legler, 1975), although as discussed above this has not invariably been the case. In contrast to the results in rodents, immunisation of monkeys induces a rise mainly in serum antibodies whether the immunisation is given subcutaneously (Lehner, Challacombe and Caldwell, 1975, 1976) or submucosally (Bowen, Cohen, Cole and Colman, 1975; Lehner, Challacombe and Caldwell, 1975). Although a salivary antibody response has been reported in some studies using immunisation into the salivary gland or duct (Evans, Emmings and Genco, 1975) this also is usually associated with a rise in serum antibody titres. The serum antibodies are mainly IgG complement-fixing antibodies to cell wall and other antigens (Lehner, Challacombe and Caldwell, 1975) and are opsonic for Str. m u t a n s (Scully and Lehner, 1979b). T cell-B cell cooperation appears to be critical to the formation of such antibodies (Lamb, Kontiainen andLehner, 1979, 1980) and an early rise in serum antibodies to Str. m u t a n s appears to confer protection against caries (Lehner, ChaUacombe and Caldwell, 1976; Peri and Wagner, 1977; Lehner, Russell, Scully, Challacombe and Caldwell, 1979). The sequence of production of antibodies of the different immunoglobulin classes appears to be important in determining the degree of protection achieved (Lehner, Russell, Wilton, Challacombe, Scully and Hawkes, 1978) and an early rise in IgG antibodies to Str. m u t a n s confers protection (Lehner, Russell, Scully, Challacombe and Caldwell, 1979). Passive transfer experiments in monkeys also indicate that the IgG class of serum antibodies confers protection (Lehner, Russell, Challacombe, Scully and Hawkes, 1978) and it may be that other antibody classes are non-protective, or impair protection (Lehner, Russell, Wilton, Challacombe, Scully and Hawkes, 1978). In view of the evidence for the involvement of serum antibodies in protection against caries in immunised monkeys it was suggested that one mechanism involved in the protection might be the opsonisation and phagocytosis of the cariogenic bacteria mediated by serum antibodies and phagocytic cells (Lehner, Challacombe and Caldwell, 1976). This concept has been supported by the finding that immunisation is associated within six weeks with a significant rise in serum opsonic activity for Str. m u t a n s (ScuUy and Lehner, 1979a) which resides in the IgG and IgM fractions (Scully and Lehner,

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1979b) and can be passively transferred (Scully, 1979). It is of great interest that serum opsonic antibodies to Str. mutans are induced only by immunisation with antigens of Str. mutans that elicit protection against caries (Scully, Russell and Lehner, 1980). Furthermore, opsonic antibodies induced by immunisation with the most prevalent serotype of Str. mutans (serotype c) are cross-reactive in opsonisation with several other serotypes (Scully and Lehner, 1978), suggesting that protection against a range of serotypes may be possible. Although the teeth erupt into an avascular site, plasma components still gain access to the enamel surface of the teeth since there is a continual flow of fluid from the plasma into the crevice that lies between the tooth and the gingiva (the gingival crevice) (Weinstein and Mandel, 1964). The crevicular fluid exerts a small mechanical rinsing effect (Brill, 1962) which can eliminate some micro-organisms (Green and Kass, 1970), and contains nonimmunological defence mechanisms such as lysozyme ,(Brandtzaeg and Mann, 1964). It was however, apparent from early studies that the crevicular fluid originated from plasma (Brill, 1959) and subsequent studies have demonstrated the presence in this fluid of many plasma components including IgG, IgA and IgM (Brandtzaeg, 1965; Holmberg and Killander, 1971; Shillitoe and Lehner, 1972) and various complement components (Attstrom, Laurel, Lahsson and Sjoholm, 1975; Courts, Boackle, Fudenberg and Silverman, 1977; Holmberg and Killander, 1971; Shillitoe and Lehner, 1972). Identification of C3 and the C3 proactivator in their converted forms suggests that complement has been activated in vivo (Attstrom, Laurel, Lahsson and Sjoholm, 1975). Most of the crevicular fluid immunoglobulins originate in the plasma rather than by local synthesis (Challacombe, Russell and Hawkes, 1978; Challacombe, Russell, Hawkes, Bergmeier and Lehner, 1978) and opsonic antibodies to Str. mutans, presumably derived from plasma, have been detected in crevicular fluid (Scully, 1980a, b). Gingival crevicular fluid also contains many leucocytes (Sharry and Krasse, 1960; Egelberg, 1963) of which over 90 per cent are polymorphonuclear leucocytes (Egelberg, 1963; Attstrom, 1970, 1971; Schiott and Loe, 1970; Skapski and Lehner, 1976; Wilton, Renggli and Lehner, 1976). Indeed, the gingival crevice is the main site through which leucocytes enter the oral cavity (Attstrom and Egelberg, 1970; Scully and Challacombe, 1979) and the estimation that over 6 per cent of the total blood leucocytes enter the oral cavity daily (Noguchi, 1972) suggests that there is a large migration of cells through the gingival crevice. There is evidence that the crevicular polymorphonuclear leucocytes are functional phagocytes for Str. mutans (Scully, 1980a, b). All the immune components necessary for the opsonisation of Str. mutans (i.e. leucocytes; opsonic antibodies; complement) appear therefore to be present in the gingival crevice. Early observations that leucocytes from saliva could also phagocytose- a number of bacteria including Str. viridans (Orban and Weinmann, 1939a, b), have been followed by brief reports of the phagocytosis of other

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bacteria (Rovelstad, 1960; Eichel and Lisanti, 1964) and latex beads (Kenney, Kraal, Saxe and Jones, 1977). It would appear that leucocytes which enter the gingival crevice may retain viability for sufficient time for these cells to carry out phagocytic activity (Scully, 1980a, b) but the activity of salivary leucocytes remains to be substantiated. Opsonisation of Str. mutans therefore, appears to be possible in the gingival crevice where it may mediate immunological protection against dental caries in the rhesus monkey. However, it is possible that serum antibodies might afford protection against caries by an additional or alternative mechanism, and it is likely that salivary antibodies are protective in certain circumstances. References Arnold, R. R., Mestecky, J. and McGhee, J. R. (1976). Naturally occurring secretory immunoglobulin A antibodies to Streptococcus mutans in human colostrum and saliva. Infection and Immunity, 14, 355. Attstrom, R. (1970). Presence of leucocytes in crevices of healthy and chronically inflamed gingivae. Journal of Periodontal Research, 5, 42. Attstrom, R. (1971). Studies of neutrophil polymorphonuclear leucocytes at the dentogingival junction in gingival health and disease. Journal of Periodontal Research, Supplement 8, 7. Attstrom, R. and Egelberg, J. (1970). Emigration of blood neutrophiis and monocytes into the gingival crevice Journal of Periodontal Research, 5, 48. Attstrom, R., Laurel, A., Lahsson, U. and Sjoholm, A. (1975). Complement factors in gingival crevice material from healthy and inflamed gingiva in humans. Journal of Periodontal Research, 10, 19. Bahn, A. N., Cummings, S. and Hayashi, J. A. (1976). Inhibition of glucan and levan synthesis and neuraminidase activity of oral streptococci by monkey antiserum. Journal of Dental Research, 55, C134. Bahn, A. N., Pinter, J. K., Quillman, P. D. and Hayashi, J. A. (1969). Immunisation with enzymes against caries in the rat. International Association for Dental Research, Abstract, 64. Bahn, A. N., Shklair, I. L. and Hayashi, J. A. (1977). Immunization with dextransucrases, levansucrases and glycosidic hydrolases from oral streptococci. Journal of Dental Research, 56, 1586. Bahn, A. N., Shklair, I. L., Mazzarella, M. and Calandra, J. C. (1960). Incidence of oral group D streptococci. Journal of Dental Research, 39, 686. Bibby, B. G. (1939). Oral bacteriology: basic considerations bearing on disease and therapy. Journal of National Dental Association, 26, 629. Bleiweis, A. S., Taylor, M. C., Deerpak, S., Brown, T. A. and Wetherell, S. R. (1976). Comparative chemical compositions of cell walls of Streptococcus mutans. Journal of Dental Research, 55, Special Issue A, A103. Bowen, W. H. (1965). A bacteriological study of experimental dental caries in monkeys. International Dental Journal, 15, 12. Bowen, W. H. (1968). Dental caries in monkeys. Advances in Oral Biology, 3, 185. Bowen, W. H. (1969). A vaccine against dental caries. A pilot experiment with monkeys (Macaca mulatta). British Dental Journal, 126, 159. Bowen, W. H., Cohen, B., Cole, M. F. and Colman, G. (1975). Immunisation against dental caries. British Dental Journal, 139, 45. Brandtzaeg, P. (1965). Immunochemical comparison of proteins in human gingival pocket fluid, serum and saliva. Archives of Oral Biology, 10, 795. Brandtzaeg, P., Fjellanger, I. and Gjeruldsen, S. T. (1968). Adsorption of immunoglobulin A on to oral bacteria in vivo. Journal of Bacteriology, 96, 242. Brandtzaeg, P., Fjellanger, I. and Gjeruldsen, S. T. (1970). Human secretory immunoglobulins, I. Salivary secretions from individuals with normal or low levels of serum immunoglobulins. Scandinavian Journal of Haematology, Supplement 12, 1. Brandtzaeg, P. and Mann, W. V. (1964). A comparative study of the lysozyme activity of human gingival pocket fluid, serum and saliva. Acta Odontologica Scandinavica, 22, 441. Bratthall, D. (1970). Demonstration of five serological groups of streptococcal strains resembling Streptococcus mutans. Odontologisk Revy , 21, 143.

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BratthaU, D. and Gibbons, R. J. (1975a). Antigenic variation of Streptococcus mutans colonising gnotobiotic rats. Infection and Immunity, 12, 1231. Bratthall, D. and Gibbons, R. (1975b). Changing agglutination activities of salivary immunoglobulin A preparations against oral streptococci. Infection and Immunology, 11, 603. Brill, N. (1959). Influence of capillary permeability on flow of tissue fluid into gingival pockets. Acta Odontologica Scandinavica, 17, 23. Brill, N. (1962). Acta Odontologica Scandinavica, 20, Supplement 32, 1. Brown, L R., Dreizen, S., Rider, L. J. and Johnston, D. A. (1976). The effect of radiation-induced xerostomia on saliva and serum lysozyme and immunoglobulin levels. Oral Surgery, 41, 83. Bunting, R. W. and Palmerlee, F. (1925). The role of Bacillus acidophilus in dental caries. Journal ofthe American Dental Association, 12, 38-1. Bunting, R. W., Nickerson, G. and Hard, D. C. (1926). Further studies of the relation of Bacillus acidophilus to dental caries. Dental Cosmos, 68, 931. Bunting, R. W., Nickerson, G., Hard, D. G. and Crowley, M. (1928). Further studies of the relation of Bacillus acidophilus to dental caries. Dental Cosmos, 70, 1. Caldwe!l, J., Challacombe, S. J. and Lehner, T. (1977). A sequential bacteriological and serological investigation of rhesus monkeys immunised against dental caries with Streptococcus mutans. Journal of Medical Microbiology, 10, 213. Canby, C. P. and Bernier, J. L. (1942). Bacteriologic and immunologic studies in dental caries: a preliminary report. Journal o f the American Dental Association, 29, 606. Carlsson, J. (1967). Presence of various types of non-haemolytic streptococci in dental plaque and in other sites of the oral cavity in man. Odontological Revy, 18, 55. Carlsson, J. (1968). A numerical taxonomic study of human oral streptococci. Odontological Revy, 19, 137. Carlsson, J., Grahnen, H. and Jonsson, G. (1975). Lactobacilli and streptococci in the mouth of children. Caries Research, 9, 333. Challacombe, S. J., Guggenheim, B. and Lehner, T. (1973). Antibodies to an extract of Streptococcus mutans, containing glucosyltransferase activity, related to dental caries in man. Archives of Oral Biology, 18, 657. Challacombe, S. J. and Lehner, T. (1980). Salivary antibody responses in rhesus monkeys immunised with Streptococcus mutans by the oral, submucosal or subcutaneous routes. Archives o f Oral Biology, 24, 917. ChaUacombe, S. J., Russell, M. W. and Hawkes, J. E. (1978). Passage of intact IgG from plasma to the oral cavity via crevicular fluid. Clinical Experimental Immunology, 34, 417. Challacombe, S. J., Russell, M. W., Hawkes, J. E., Bergmeier, L. A. and Lehner, T. (1978). Passage of immunoglobulins from plasma to the oral cavity in rhesus monkeys. Immunology, 35, 923. Ciardi, J. E., Bowen, W. H., Reilly, J. A., Hsu, S. D., Gomez, I., Kuzmiak-Jones, H. and Cole, M. F. (1978). Antigens of Streptococcus mutans implicated in virulence production of antibodies.Advances in Experimental Biology and Medicine, 107, 281. Clarke, J. K. (1924). On the bacterial factor in the aetiology of dental caries. British Journal of Experimental Pathology, 5, 141. Cohen, B., Colman, G. and Russell, R. R. B. (1979). Immunisation against dental caries: further studies. British Dental Journal, 147, 9. Collins, P. A., Gerencser, M. A. and Slack, J. M. (1973). Numeration and identification of actinomycetaceae in human dental calculus using the fluorescent antibody technique. Archives of Oral Biology, 18, 145. Colman, G. and Cohen, B. Immunisation of monkeys (Macaca fascicularis) with Streptococcus mutans. In Pathogenic Streptococci (Parker, M. T., Ed.), Reedbooks, Chertsey (1979), 214-215. Colman, G. and Williams, R. E. O. Taxonomy of some human viridans streptococci. In Streptococci and Streptococcal Diseases (Wannamaker, L. W. and Matsen, J. M., Eds), Academic Press, New York (1~72), pp. 281-299. Colten, H. R. and Bienenstock J. Lack of C3 activation through dassical~or alternative pathways by human secretory IgA anti-blood group A antibody. In The Immunoglobulin A System (Mestecky, J. and Lawton, A. R., Eds), Plenum Press, New York (1974), pp. 305-308. Cuppini, A., Borea, G., Steganini, F. and Capuzzi, P. (1969). I1 vaccino VDS e profilassi della carie dentaria: ricerche clinico statisticlie. Mondo Odontostomat, U, 453. Curtis, F. J., Boackle, R. J., Fudenberg, H. H. and Silverman, M. S. (1977). Detection of functional complement components in gingival crevicular fluid from humans with periodontal disease. Journal o f Dental Research, 56, 327.

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Dally, T. E. find Drane, J. B. Seventh Annual Cancer Conference Proceedings. Luppincott, Philadelphia (1974), pp. 147-154. de Stoppelaar, J. D., Konig, K. G., Plasschaert, A. J. M. and Van der Hoeven, J. S. (1971). Decreased cariogenicity of a mutant of Streptococcus mutans. Archives o f Oral Biology, 16, 971. de Stoppelaar, J. D., Van Houte, J. and Backer-Dirks, O. (1969). The relationship between extracellular polysaccharide-producing streptococci and smooth surface caries in 13 year old children. Caries Research, 3, 190. Dogon, I. L., Kerr, A. C. and Amdur, B. H. (1962). Characterisation of an antibacterial factor in human parotid secretions, active against Lactobacillus casei. Archives o f Oral Biology, 7, 81. Drucker, D. B. and Green, R. M. Potential of streptococci for inducing dental caries in gnotobiotic rats. In Pathogenic Streptococci (Parker, M. T., Ed.), Reedbooks, Chertsey (1979), pp. 206--207. Duchin, S. and Van Houte, J. (1978). Relationship of Streptococcus mutans and lactobacilli to incipient smooth surface dental caries in man. Archives o f Oral Biology, 23, 779. Ebisu, S., Misaki, A., Kato, K. and Kotani, S. (1974). The structure of water-insoluble glucans of cariogenic Streptococcus mutans, formed in the absence and presence of dextranase. Carbohydrate Research, 38, 374. Edwardsson, S. (1974). Bacteriological studies on deep areas of carious dentine. Odontological Revy, 25, Supplement 32. Egelberg, J. (1963). Cellular elements in gingival pocket fluid. Acta Odontologica Scandinavica, 21, 283. Eichel, B. and Lisanti, V. F. (1964). Leucocyte metabolism in human saliva. Archives of Oral Biology, 9, 299. Ellison, S. A., Mashimo, P. A. and Mandel, I. D. (1960). Immunochemical studies of human saliva. The demonstration of serum proteins in whole and parotid saliva. Journal of Dental Research, 39, 892. Emmings, F. G., Evans, R. T. and Genco, R. S. (1975). Antibody response in parotid fluid and serum of Macaca fasc&ularis (irus) monkeys after localimmunisation with Streptococcus mutans. Infection and Immunity, 12, 281. Emmings, F. G., Evans, R. T. and Genco, R. J. (1976). Immunisation ofMacaca fascicularis (Macaca irus) monkeys with Streptococcus mutans: specificity of antibody responses in saliva. Journal o f Dental Research, 55C, C181. Englander, H. R. and Jordan, H. V. (1972). Relationship between Streptococcus mutans and smooth surface caries in the deciduous dentition. Journal o f Dental Research, 51, 1505. Enright, J. J., Friesell, H. E. and Trescher, M. O. (1932). Studies of the cause and nature of dental caries. Journal of Dental Research, 12, 759. Evans, R. T., Emmings, F. G. and Genco, R. J. (1975). Prevention of Streptococcus mutans infection of tooth surfaces by salivary antibody in irus monkeys (Macaca fascicularis ). Infection and Immunity, 12, 293. Evans, R. T. and Genco, R. J. (1973). Inhibition of glucosyltransferase activity by antisera to known serotypes of Streptococcus mutans. Infection and Immunity, 7, 237. Evans, R. T., Genco, R. J. and Emmings, F. G. (1976). Effects of antibodies on adherence and cell-associated glucan production by Streptococcus mutans cells. Journal o f Dental Research, C, C127. Evans, R. T. and Mergenhagen, S. E. (1965). Occurrence of natural antibacterial antibody in human parotid fluid. Proceedings o f the Society o f Experimental Biology and Medicine, 119, 815. Ferretti, J. J., Humphrey, M. W., Begay, M. R. and Shea, C. Two-dimensional immunoelectrophoresis of Streptococcus mutans antigens: immunological cross-reactions with mammalian tissue. In Pathogenic Streptococci (Parker, M. T., Ed.),, Reedbooks, Chertsey, (1979), pp. 224-225. Fitzgerald, R. J. The microbial ecology of plaque in relation to dental caries. In Microbial Aspects of Dental Caries (Stiles, H. M., Loesche, W. J. and O'Brien, T. C., Eds), Information Retrieval Inc., Washington (1976), pp. 849-858. Fitzgerald, D. B. and Fitzgerald, R. J. (1966). Induction of dental caries in gerbils. Archives of Oral Biology, 11, 139. Fitzgerald, R. J. and Jordan, H. V. Polysaccharide-produeing bacteria and caries. In Art and Science of Dental Caries Research (Harris, R. S., Ed.), Academic Press, New York (1968), pp. 79-861 Fitzgerald, R. J., Jordan, H. V. and Archard, H. O. (1966). Dental caries in gnotobiotic rats infected with a variety of Lactobacillus acidophilus. Archives o f Oral Biology, 11, 473. Fitzgerald, R. J., Jordan, H. V. and Stanley, H. R. (1960). Dental caries and gingival pathologic changes in the gnotobiotic rat. Journal of Dental Research, 39, 923.

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