Effective Immunity to Dental Caries: Studies of Active and Passive Immunity to Streptococcus mutans in Malnourished Rats JERRY R. MC GHEE, SUZANNE M. MICHALEK, JUAN M. NA VIA, and ANNIE JO NARKA TES Department of Microbiology, and Institute of Dental Research, University of Alabama in Birmingham, Birmingham, Alabama 35294, USA

It is generally recognized that protein malnutrition of varying degrees of severity afflicts most of the world's population. Furthermore, it is becoming clear that malnutrition and infection interact synergistically in the host since malnutrition predisposes the host to infection, whereas infection often intensifies existing malnutrition.1 During the past 50 years, several studies have been directed toward understanding how nutritional deficiency contributes to disease susceptibility. Studies with both man and experimental animals have demonstrated a lowering of both humoral and cell-mediated immunity.2-5 These observations could be partially explained by a lack of good quality protein during any of the critical stages of development which can bring about irreversible changes in tissues, including lymphoid organs, that are experiencing their period of maximum growth.6 For example, in man, the teeth, particularly the enamel, begin to form in utero and continue to develop during the first 20 years of life and this is the period when the tooth is most susceptible to caries attack. Recent studies by Menaker and Navia7 have shown that specific protein malnutrition imposed during the early development of rats results in increased caries susceptibility when compared with normally nourished rats. Dental caries is an infectious disease of This investigation was supported by a grant from the John A. Hartford Foundation, Inc.; a grant from the World Health Organization; Grant IN-66L from the American Cancer Society; and Grants DE 03041-04, DE 02670, and DE 04217-01 from the National Institute of Dental Research, National Institutes of Health, Bethesda, Md.

multifactorial origin that includes factors such as host susceptibility, diet containing fermentable carbohydrate, and cariogenic microorganisms.8 Each of these factors must be considered in any attempt to induce immunity to dental caries. In recent years, certain oral streptococci, notably Streptococcus mutans, have been considered as the principal etiologic agents of dental caries.9-12 S mutans produces the enzyme, dextransucrase, which uses sucrose as a substrate to synthesize high and low molecular weight glucans.13-15 Studies have suggested that these glucans are important determinants of virulence since they facilitate S mutans adherence to hard surfaces and contribute to plaque formation.14,16-21 In addition, fermentation of glucose and fructose moieties of sucrose by S mutans results in production of lactic acid, which is involved in the demineralization of the tooth surface, thus initiating the carious lesion.12,22,23 Studies with both germfree and conventional rats have shown that rats locally injected with S mutans in the salivary gland region and challenged with the homologous bacterium can elicit a protective immune response against dental caries.24-28 However, to determine whether specific immunity to dental caries can be effected in conditions of protein malnutrition, it is necessary to evaluate the nature of the immune response to S mutans in a nutritionally compromised host. Our initial studies were directed toward measurement of immunoglobulin (Ig) levels in rat serum, milk, and saliva from both protein-malnourished and normal rats and their offspring.29-30

C206 Downloaded from jdr.sagepub.com at FUDAN UNIV LIB on May 9, 2015 For personal use only. No other uses without permission.

Vol 55 1976

EFFECTIVE IMMUNITY TO DENTAL CARIES

Materials and Methods Sprague-Dawley derived COBS/CD rats were used in all studies. Timed-pregnant females were obtaineda and separated into two groups. One group was offered a low protein diet, no. 408, containing 8% protein and the other group was fed diet no. 425 that contains a nutritionally adequate level of 25% protein.31 To ensure that caries that developed in rat offspring resulted from S mu tans infection, a method was developed to suppress the indigenous microbial flora of rat dams and their offspring. This method consists of dietary supplementation of antibiotics shown to be effective against the total flora.32 The flora of each dam was determined between days -21 and -14 of gestation by obtaining oral swabs and fecal samples and culturing these on blood, Mitis-Salivarius (MS), and Rogosa agar plates. Antibiotic sensitivity disks were applied to a duplicate set of plates in order to determine the antibiotics that are effective against the indigenous flora. Beginning on clay -7 of gestation, dams were fed either diet no. 408 or 425 supplemented with three antibiotics shown to be effective on in vitro suppression of the oral flora. This method has been shown to be very effective in suppression of the microbial flora in rats.32 After one week of antibiotic treatment, 80% of the rats were bacterial-suppressed and 95% were suppressed after two weeks of treatment. Oral swabs were taken from each dam and three pups from each litter at weekly intervals throughout the experiment and sampled to ensure continual microbial suppression. For determination of Ig levels in milk, serum and saliva of both protein-malnourished and normally nourished rats, the following procedures were used. Oxytocinstimulated milk was collected from each dam at two- to five-day intervals throughout lactation. Rat pups were sequentially bled from day of birth, before nursing, and at five-day intervals thereafter until they were killed. Pilocarpine-stimulated whole saliva was collected from individual rat pups 20 to 45 days of age at approximately five-day intervals. Immunoglobulins in colostrum, milk, serum, and saliva were quantitated by radial immunodiffusion using purified rat IgA, a Charles River Laboratories, Wilmington, Mass.

C207

TABLE I CONCENTRATION OF IGA IN COLOSTRUM AND MILK FROM RAT DAMS FED 25% OR 8% PROTEIN DIETS Group Day of Lactation

1 2 4 6 10 15

20

25% Protein

8% Protein

1.20

1.15 0.18 1.04 0.28 1.85 0.39 0.70 0.14

0.21

1.26 0.31 1.42 0.34 0.75 0.16 0.71±0.08 0.65 0.07 0.55 ± 0.05

0.74±0.12 0.70 0.09 0.61 ± 0.03

* Milligrams of IgA/ml sample ± standard error. Each value is the mean of six to eight dams.

IgG22, and IgM as standards and anti-rat a, y, and ,u antiserums.29 Results and Discussion Table I shows that levels of IgA in colostrum and milk were not significantly different in dams fed either diet no. 408 or 425. From days 0 to 4, the level increased in both groups; however, by day 6, the IgA in milk in both groups of dams had decreased to one half the level observed in early milk and remained relatively constant throughout the remainder of lactation. It should be noted that dams fed diet no. 408 produced only 25 to 50% of the quantity of milk as dams fed a normal protein regimen. However, the level of IgG2a in milk from malnourished rats was significantly lower than the level in normal rats througlhout the entire period of lactation (Fig 1). This Ig increased in both groups between days 0 and 6 and remained constant throughout the remainder of lactation. No IgM could 2.0 Q

.~10

.PA

I..

0

10

15

DAYS(Lact.Iion)

20

FIG 1.-Levels of IgG2. in rat colostrum and milk during lactation: open circles, dams fed 25% protein diet; solid circles, dams fed 8% protein diet.

Downloaded from jdr.sagepub.com at FUDAN UNIV LIB on May 9, 2015 For personal use only. No other uses without permission.

C208

J Dent Res Special Issue C

MCGHEE ET AL

AGE(DNy.)

FIG 2.-Levels of serum IgG2. in rat offspring: open circles, pups from dams fed 25% protein diet; solid circles, pups from dams fed 8% protein diet.

be detected in colostrum or milk from either malnourished or normal rats at any interval tested. As shown in Figure 2, the level of serum IgG2a in malnourished rat offspring was significantly lower than the level observed in normally nourished rats at every interval tested. Both groups of rats demonstrated increasing levels of IgG25 during lactation and reached a maximum between 15 and 20 days of age in both malnourished (3.96 mg/ml) and normal (6.36 mg/ml) pups. After weaning, both groups of rats showed a rapid decrease in IgG2 levels until day 35. These results suggest that the initial increase in the level of this Ig was due to passive transfer. By 45 days of age, this serum Ig had increased; however, the level in malnourished rats was still approximately half that observed in normal rats. No differences were observed in the levels of IgA or IgM

in the serum of malnourished or normal rats. Salivary IgA was first observed in 25-dayold malnourished and normal rats and no significant differences could be discerned in the level of this Ig between the two groups (Table 2). However, salivary IgG2. was observed in both groups by day 19. The level of this Ig remained constant throughout the entire test period and no significant differences were observed. No salivary IgM could be detected at any interval tested. These results suggest that malnourished and normal rat dams passively transfer IgG2a to their offspring since the level of this Ig in the serum of rat pups increased during suckling and decreased after weaning. Furthermore, these results demonstrate that proteinmalnourished rat dams have approximately twofold less IgG2a in their milk and their offspring have approximately twofold less IgG2, in their serum when compared to normally nourished rat dams and their offspring. In a second series of experiments, the effect of local injection of S mutans in the submandibular region on protection against S mutans-induced dental caries was studied.28 The experimental design used in this series of experiments is shown in Figure 3. Beginning on day -7 of gestation, rat dams were fed either diet no. 408 or 425 supplemented with cephalothin, ampicillin, and carbenicillin (1 gm of each drug per kilogram diet), the antibiotics shown to effectively suppress the rats' oral flora. Rat dams and litters on each dietary regimen were subsequently separated into three groups. At 14 days of age, all rats received subcutaneous, bilateral

TABLE 2 IMMUNOGLOBULIN* LEVELS IN SALIVA OF RATS FROM DAMS FED DIET No. 408 OR 425 IgA

Group

19 days

No. 425 No. 408

25 days

30 days

35 days

45 days

Effective immunity to dental caries: studies of active and passive immunity to Streptococcus mutans in malnourished rats.

Effective Immunity to Dental Caries: Studies of Active and Passive Immunity to Streptococcus mutans in Malnourished Rats JERRY R. MC GHEE, SUZANNE M...
590KB Sizes 0 Downloads 0 Views