ARTÍCULOS ORIGINALES
Differential reactivity of salivary igA and igG against streptococcus mutans proteins in humans with different caries experience
Soledad I. Gómez, Lorenza M. Jaramillo, Gloria C. Moreno, Nelly S. Roa, Adriana Rodríguez
Center for Dental Research, School of Dentistry, Pontificia Universidad Javeriana. Bogotá Colombia.
CORRESPONDENCE Dr. Adriana Rodriguez Pontificia Universidad Javeriana, Facultad de Odontologia, Centro de Investigaciones Odontologicas. Carrera 7 #40-62, edificio 26. Bogota, D.C., Colombia. arodrig@javeriana.edu.co
ABSTRACT
Dental caries is an infectious disease which still constitutes a public health concern. It begins at an early age and is caused mainly Streptococcus mutans (S. mutans). The aim of this study was to characterize the salivary humor immune response to S. mutans proteins in patients with caries, with history of caries and without caries, in order to determine which S. mutans proteins participate in the immunological response in subjects with different caries experience. Saliva was collected by spontaneous salivation for 5 minutes from 60 subjects aged 18 to 30 years, classified according to their caries experience as: without caries (Group I), with active caries (Group II) and with history of caries (Group III). The antigens derived from S. mutans by sonication were recognized by salivary IgA and IgG by Western Blot. The results showed that all the individuals studied recognized S. mutans proteins with molecular weights in the range of 8 to 191 kDa, with similar recognition profiles for salivary IgA and IgG. Subjects without caries recognized the 29 kDa protein, also known as S. mutans Antigen A, via salivary IgA, differing from patients with caries and history of caries, who recognized it via IgG. The protective response against S. mutans is mediated by IgA. To conclude, a differential response to the 29 kDa protein between study individuals may be indicative of resistance to dental caries and may have a protective role in the induction of IgA antibodies against dental caries, as found in the group without caries, in contrast to subjects with active caries and history of caries.
Key words: Streptococcus mutans; Saliva; Immunoglobulin A; Immunoglobulin G; Dental caries; Immunoblotting.
RESUMEN
Reactividad diferencial de igA e igG salivales contra proteinas de streptococcus mutans en humanos con diferentes estadios de caries dental
La caries dental es una enfermedad infecciosa que continua siendo un problema de salud publica, inicia a temprana edad y es causada principalmente por Streptococcus mutans (S. mutans). El objetivo de este estudio fue caracterizar la respuesta inmune humoral salival, ante las proteinas de S. mutans, en pacientes con caries, historia de caries e individuos libres de caries, para asi establecer que proteinas de S. mutans participan en la respuesta inmunologica en los diferentes estadios de caries. La saliva de 60 individuos entre 18 y 30 anos de edad, clasificados de acuerdo al estado de caries: libres de caries (grupo I), caries activa (grupo II) e historia de caries (grupo III), se colecto por salivacion espontanea durante 5 minutos. Los antigenos derivados de S. mutans por sonicacion, fueron reconocidos por IgA e IgG salivales por Western Blot. Los resultados mostraron que todos los individuos estudiados reconocen las proteinas de S. mutans en el rango de 8 a 191 kDa de peso molecular con perfiles de reconocimiento similares para IgA e IgG salival. Se encontro que los sujetos libres de caries reconocen por IgA salival la proteina de 29 kDa, tambien llamada Antigeno A de S. mutans, de manera diferente que los pacientes con caries e historia de caries quienes reconocieron la proteina via IgG. La respuesta protectora frente a S. mutans es mediada por IgA. En conclusion, una respuesta diferencial a la proteina de 29 kDa entre los individuos estudiados, puede ser indicativo de resistencia a la caries dental y tener un papel protector en la induccion de anticuerpos IgA frente a la caries dental, como se encontro en el grupo libre de caries, a diferencia de los sujetos con historia de caries y caries activa.
Palabras clave: Streptococcus mutans; Saliva; Inmunoglobulina A; Inmunoglobulina G; Caries Dental; Inmunodeteccion.
INTRODUCTION
Dental caries is the local destruction of hard tissues
in the tooth by acid products from bacterial
fermentation of carbohydrates. It is usually chronic,
site-specific, multifactorial, dynamic, and results in
physiological imbalance between the mineral
portion of the tooth and bacterial plaque, when the
reduction in pH leads to loss of minerals over time.
It is an infectious disease which is produced not by
a single microorganism but by many, and it can be
stopped at any point in time 1- 6.
Dental caries is an epidemiological problem. The
latest National Oral Health Study (Estudio Nacional
de Salud Bucal, ENSAB III) conducted by
Colombia's Ministry of Health, reports that "caries
history in permanent dentition occurs in 19.9% of
seven-year olds and 71.9% of twelve-year-olds. The
percentage increases during adolescence to 89.5%.
At thirty-five years of age, the DFMT index (D:
decayed, F: filled, M: missing, T: teeth) is 15" 7.
Given the infectious nature of caries, there is
worldwide interest in developing a vaccine to prevent
it. This vaccine would focus on neutralizing the
virulence factors of S. mutans, the most widely
studied causal agent, which participate in the
pathophysiological mechanisms of dental caries, such
as adherence to teeth and the production of insoluble
glucans, by inducing a response of specific salivary
antibodies of the type secretory immunoglobulin A
(sIgA) and serum immunoglobulins A and G (IgG),
the latter as part of the salivary component through
blood extravasation through the gingival fluid 8-10.
Research groups such as Chia, Lehner and Naspitz
conducted studies with the aim of characterizing the
humoral immune response to different S. mutans
proteins, in order to correlate them with the absence or
presence of caries. It is deduced from the results that
there is a high frequency of infection, suggesting that
the immune response against S. mutans may be non
protective. Only a small percentage of the population is
unaffected by dental caries and it has not been possible
to determine a factor that would explain this natural
resistance. The differences in immune response to S.
mutans between individuals who suffer from the disease
and the small percentage who do not, are unclear.
Under the hypothesis that these differences that
maintain or keeps an individual caries free, depends
on that the humoral immune response in without caries
individuals, is directed against particular antigens of
S. mutans that neutralize its pathophysiological
mechanisms and despite of being present in oral
cavity, it does not cause the disease11-26, the aim of this
study was to characterize the specific salivary IgAand
IgG-mediated response against S. mutans proteins
in patients with caries, with history of caries, and
without caries.
Finding S. mutans antigens that induce a protective
response against dental caries in naturally sensitized
humans would contribute additional strategies for
mass protection of the population through a vaccine
designed with those antigens.
MATERIALS AND METHODS
Population and Sample
The study included 60 subjects aged 18 to 30 years,
with no systemic and oral pathology other than
dental caries, who visited the clinics at the School
of Dentistry at Pontificia Universidad Javeriana.
After a review of the clinical history and an oral
examination, they were classified into 3 groups
according to DFMT index (D: decayed, F: filled, M:
missing, T: teeth), with 20 subjects per group, as
follows: (I) without caries: DFMT equal to zero; (II)
with active caries: DFMT greater than zero and D
equal to 1 or more, and (III) with history of caries:
DFMT greater than zero, D equal to zero and F equal
to 1 or more, who received treatment for caries at
least 6 months prior to being included in the study.
After subjects had signed informed consent, saliva
samples (5 ml) were taken by spontaneous
salivation. The project was approved by the Ethics
and Research Committee of the School of Dentistry
at Pontificia Universidad Javeriana (CIEFOUJ).
S. mutans colony-forming units
count from saliva
To determine the number of colony-forming units
(CFU) per ml, we used a quantitative method for
which a series of saliva dilutions were made in saline
solution (1:10, 1:100, 1:1000). Aliquots (50 ml) of
each dilution were plated on mitis salivarius agar
(Becton Dickinson) and incubated for 48 hours in
microaerophilic conditions.
For the immunological analysis, the remaining
saliva in each sample was clarified by centrifugation
(Eppendorf Centrifuge) at 10,000 rpm for 15
minutes and stored at –20° C (General Electric) until
it was used.
Preparation of S. mutans extracts
To prepare S. mutans extracts we employed a strain
from the American Type Culture Collection (ATCC,
#31989) grown in Todd Hewitt broth (Difco Bacto
Todd Hewitt Broth) supplemented with 1% glucose
(Carlo Erba) for 18 hours at 37°C, in microaerophilic
atmosphere.
The purity of the culture was tested by Gram stain
(Labsar). Bacterial culture density was adjusted to
tube 4 on the McFarland scale, corresponding to a
concentration of 1.2x109 cells/ml in phosphate buffer
solution (PBS: Sigma) – glycine (Pharmacia Biotech)
(0.5g/ml). Having previously standardized the
optimum conditions, the bacteria were suspended in
PBS-glycine supplemented with protease inhibitor
(Tris (0.1M) (Pharmacia Biotech), 10% n-propanol
(Merck), 2% EDTA, phenylmethanesulfonyl fluoride
(PMSF) 2 mM (Sigma) and subjected to 25 sonication
cycles (Fisher Scientific) at 20W for one minute, with
1-minute rests, in ice throughout the process.
The S. mutans extract was analyzed by SDS-PAGE,
and a Bradford test (Bradford Sigma reagent;
Human spectrophotometer) was used to quantify
proteins, obtaining a concentration of 2747 mg/ml.
Reactivity of salivary IgA and IgG
to S. mutans proteins
SDS-PAGE
To separate the proteins contained in S. mutans
extracts, we used the SDS-PAGE technique (Sodium
Dodecyl Sulfate Polyacrylamide Gel Electrophoresis)
on 10% polyacrylamide gels. The first well on each
gel served as a wide range molecular weight pattern
from 7.2 to 200 kDa (BIO-RAD). The other wells
were filled with 110 μg/well S. mutans extract.
Electrophoresis was done in tris-glycine buffer
(Pharmacia Biotech) at 150 mV (BIO-RAD. Mini
Protean Electrophoresis Cell) for approximately 1
hour. For the electrophoresis control, some gels were
stained with 0.25% Coomassie Blue (BIO-RAD) for
5 minutes.
The gels were transferred to a PVDF membrane (Pall
Life Sciences, BIO-RAD transfer chamber) for two
hours at 100 V and 150 mA. The transfer was
confirmed with 1% India ink (Pelikan) in PBS, and
the gels were stained with Coomasie blue (Sigma).
The membranes were rinsed for 5 minutes with TBS
buffer (Invitrogen), pH 7.5: Tris (10 mM), NaCl (150
mM) and Tween 20 (Sigma) (0.1%). After rinsing,
they were blocked with skimmed milk (Proleche) (5%
in TBS) for two hours under constant stirring
(Barnstead-Thermolyne magnetic stirrer), after which
the rinsing procedure was repeated. The membranes
were stored on filter paper at 4°C until they were used.
Immunoblotting
Prior to use, membranes were cut into 0.5 cm wide
strips and incubated with the saliva from each
subject diluted 1:5 in TBS for IgG and 1:12 for IgA
for two hours under constant stirring at room
temperature. After 5 rinses with TBS, they were
incubated for one hour with the secondary human
anti-IgA (Sigma) and anti -IgG (Sigma) antibodies
conjugated with peroxidase, diluted in TBS in a
proportion of 1:100,000 for IgG and 1:250,000 for
IgA, having been previously standardized.
Chemiluminescence
After rinsing, the membranes were placed on the
film (Light sensitive CL-Xposure Film, Pierce).
Chemiluminescent substrate (Luminol Supersignal
West Dura Extended Duration Substrate, Pierce)
was added, which had been previously prepared
following a five-minute protocol. The films were
developed by autoradiography and the molecular
weight for each protein in the extract, recognized
by each protein, was calculated by preparing a
calibration curve using the molecular weight
patterns and relative mobility of each protein.
Analysis of information
The data obtained were expressed as percentages of
individuals in each group that recognize each S.
mutans protein band. Comparison between groups
was done using the Chi-square test, and significance
was established at values of *p<0.05.
RESULTS
The increase in colony forming units
is related to degree of caries
Data reported in the literature 27 show that the
number of S. mutans colony-forming units was
lower in subjects without caries than in those with
caries in 3- to 5-year-old children. In our study, the
number of S. mutans CFU in saliva of adult
individuals was measured to determine whether this
reflect the caries experience in these groups. The
group with active caries (Group II) had a
significantly higher number of CFU (p=0.001) than
the other two groups (history of caries (Group III)
and caries-free (Group I)). The latter two had low
counts with no difference between them (Fig. 1).
Fig. 1: Microbiological analysis. The graph shows interquartile distribution (median and range) of the colony-forming units
(CFU) obtained from plating saliva on mitis salivarius agar, for
subjects from each study group. The dots represent extreme
values with respect to the median.
Salivary IgA response to S. mutans proteins
in different caries experience
Analysis of the results from the 60 subjects included
in this study, shows that 29 different S. mutans
proteins are recognized by IgA, with molecular
weights ranging from 8 to 191 kDa (Table 1, Fig. 2).
On average, each individual recognized 8 proteins,
in a range of 5 to 12. The response with highest
diversity was observed in an individual with active
caries and in an individual without caries. Table 1
shows the different percentages of salivary IgA
response to the different S. mutans proteins.
Table 1: Percentage of individuals with different caries
experience that recognize S. mutans proteins
via salivary IgA and IgG. Proteins separated
by molecular weight.
Fig. 2: Response of salivary IgA to S. mutans proteins. Image
showing chemiluminescence of 5 samples per group, revealing
recognition by salivary IgA of different S. mutans proteins of
individuals in the three study groups. Lines 1-5 Group I (active
caries); lines 6-10 Group II (history of caries) and lines 11-15
Group III (without caries). Line M is the pattern of molecular
weight used. The S. mutans extracts obtained by sonication
were subjected to electrophoresis to separate proteins,
transferred to PVDF membranes and incubated with saliva
from the study subjects. Human anti-IgA conjugated with
peroxidase was used for immunoblotting. It was developed by
chemiluminescence.
Patients in Group I have the highest diversity in recognition (average 9 proteins of different molecular weights), followed by Group II (8 different proteins), and Group III (7 proteins). Comparative analysis between groups shows no significant difference in diversity in recognition among the three groups (p>0.05). The individual with the highest recognition (12 different proteins) belonged to the caries-free group. Table 1 describes the most immunogenic antigens for each study group. Chi-square analysis of the number of individuals who recognize S. mutans proteins of different molecular weights via IgA showed significant differences for some proteins between groups. Only the recognition of the 29 kDa protein showed differences among all three groups (p=0.015). Highest recognition occurred in individuals without caries, followed by those with active caries, and lastly those with caries history (Fig. 3). Recognition of 191 kDa and 91 kDa proteins is significantly lower in the active caries group than in the group without caries, but similar between the group with caries history and the group without caries. The 125 and 10 kDa antigens are recognized by fewer individuals in the group with caries history than in the other two groups (p= 0.036) (Fig. 4).
Fig. 3: Response of salivary IgG to S. mutans proteins. Image showing chemiluminescence of 5 samples per group, revealing
recognition by salivary IgG of different S. mutans proteins of
individuals in the three study groups. Lines 1-5 Group I (active
caries); lines 6-10 Group II (history of caries) and lines 11-15
Group III (without caries). Line M is the pattern of molecular
weight used. The S. mutans extracts obtained by sonication were
subjected to electrophoresis to separate proteins, transferred to
PVDF membranes and incubated with saliva from the study
subjects. Human anti-IgG conjugated with peroxidase was used
for immunoblotting. It was developed by chemiluminescence.
Fig. 4: IgA recognition of S. mutans proteins in study groups.
Behavior of recognition frequencies by IgA of Streptococcus
mutans proteins of different molecular weights, which showed
some significant differences among the three study groups. The
signals obtained by chemiluminescence were taken as proteins
recognized by salivary IgA, and the molecular weights of each
peptide in the extract recognized was calculated by preparing
a calibration curve using molecular weight patterns as a
reference and the relative mobility of each protein. Frequencies
between groups was compared by Chi-square and a value
*p<0-05 was considered significant.
Salivary IgG response to S. mutans proteins
in different caries experience
The IgG from the 60 individuals included in this
study recognized 21 different S. mutans proteins
ranging in molecular weight from 10 to 191 kDa
(Table 1, Fig. 3). On average, each individual
recognized 6 proteins, in a range of 2 to 12 proteins.
The response with highest diversity was observed
in an individual with active caries. Table 1 shows
the percentages of specific salivary IgG-mediated
response to S. mutans proteins.
On average, the patients from the group with active
caries recognized the greatest diversity of proteins
of different molecular weights (8 proteins), followed
by the group with caries history (6) and group
without caries (6). Similarly to the observations for
IgA, no difference was found among groups
regarding the diversity recognized via IgG. However,
patients who recognized the highest number of
different proteins belong to the group with active
caries (18) and the group with history of caries (12).
Table 1 shows the most immunogenic antigens for
each group.
Statistical analysis by Chi-square of the number of
patients whose IgG recognizes S. mutans proteins
of different molecular weights showed significant
differences between groups for proteins of
molecular weights 17kDa (p= 0.01376), 36 KDa
(p= 0.0179) and 43 KDa (p= 0.0029), which were
recognized in greater numbers by patients from the
group with active caries than by individuals without
caries. The 29 kDa protein was recognized less by
subjects without caries (p=0.09), and the group with
history of caries did not recognize the 10 KDa
antigen (p= 0.01720) (Fig. 5).
Fig. 5: IgG recognition of S. mutans proteins in study groups. Behavior of recognition frequencies by IgG of Streptococcus
mutans proteins of different molecular weights, which showed
some significant differences among the three study groups. The
signals obtained by chemiluminescence were taken as proteins
recognized by salivary IgG, and the molecular weights of each
peptide in the extract recognized was calculated by preparing
a calibration curve using molecular weight patterns as a
reference and the relative mobility of each protein. Frequencies
were compared between groups by Chi-square and a value
*p<0-05 was considered significant.
Analysis of simultaneous IgA and IgG
recognition of S. mutans 29 kDa protein
An analysis of the recognition behavior of proteins
with significant differences between groups showed
a pattern in the recognition of the 29 kDa protein
by the two types of IgA and IgG antibodies
simultaneously, which differed significantly among
the three groups (p=0.00001).
Of the 14 caries-free individuals that recognized the
29 kDa protein via IgA, only 3 recognized it
simultaneously via IgG, in contrast to the active
caries group, where most of the individuals that
recognized the protein via IgA (12) also did so via
IgG (8) (p=0.0001716) (Fig. 6).
Of the 6 individuals with caries history who
recognized the 29 kDa protein via IgA, 4 also
recognized it via IgG. The simultaneous expression
in this group differs significantly from the group
without caries (p=0.0002994) (Fig. 6).
There was no significant difference in the
simultaneous expression of IgA and IgG against the
29 kDa S. mutans protein between the group that
has the disease and the group that has had the
disease (p=0.7709) (Fig. 6).
Fig. 6: Simultaneous expression of
IgA and IgG against the 29 kDa S.
mutans protein. Shows the number
of individuals in each study group
that recognized 29 kDa S. mutans
antigen by the IgA type antibody
(whole bar); the black section on
each bar represents simultaneous
recognition in these individuals via
IgG, and gray section shows
individuals that did not recognize
it via IgG. There are significant
differences among the three groups
(p=0.00001), when the number of
individuals recognizing the S.
mutans 29 kDa protein via IgA and
IgG is compared by Chi-square.
DISCUSSION
This study was designed to determine whether
individuals with and without caries recognize S.
mutans antigens differently, which might explain
natural resistance. It looks at one group of persons
free from the disease and another group of diseased
patients, in turn divided into two groups, considering
that persons with active carious lesions have higher levels of S. mutans than those who have had the
disease but no longer have lesions (group with caries
history), even though they might have the same
susceptibility, as reported in the consensus in the
literature about the association between caries and
S. mutans 28.
The profile of S. mutans proteins recognized by
salivary IgA and IgG was established for each patient.
Salivary IgA represents local immunity, while IgG
represents systemic immunity but has an influence on
the response in the oral cavity because it is present in
whole saliva through the gingival fluid 29.
There is controversy in the literature about the
salivary IgA and IgG response regarding the
number and type of S. mutans proteins recognized.
For example, Chia et al. report that the proteins
most often recognized by salivary IgA and serum
IgG are those having molecular weight 60 to 63 kDa
30, whereas in our study, this molecular weight range
was not recognized by salivary antibodies in most
of the study subjects. The most immunogenic
proteins for both classes of antibodies reported here
were those with molecular weights 91, 45, 32 and
29 kDa. The 191 kDa protein induced an IgA
response, while the 43, 38, 36 and 32 kDa proteins
primarily activated an IgG response.
Chia et al.30 report that a protein similar in size to
the 191 kDa protein was recognized by IgG in most
patients, but not by IgA, as it was in our study. This
antigen is particularly relevant due to its proximity
to the molecular weight reported for the protein
PAc, also known as antigen I/II (Ag I/II),31 which is
one of the primary S. mutans virulence factors.
Other S. mutans proteins are immunogenic for
different population groups; for IgA, the proteins of
approximately 92 kDa,30 170 and 190 KDa 32-34 (which may c
orrespond to glycosyltransferases
(GTFs) and Ag I/II, respectively) and for IgG, the
39 and 97 kDa proteins 27.
In addition to different studies reporting diversity
in recognition, the molecular weights of some of
the most immunogenic proteins are different
according to the populations studied, possibly due
to genetic influence of the Major Histocompatibility
Complex (MHC)35-40 and other factors such as diet,
oral hygiene and exposure to fluoride 32-34.
The comparative analysis of results between groups
showed that all three groups recognize a similar
number of proteins; however, the response differs
according to the experience of disease. For IgA,
recognition is similar in caries-free individuals and
patients with caries history, while it differs in patients
with active caries. In contrast, IgG recognition is
similar in patients with caries history and active
caries, and different in caries-free individuals, whose
response is lower in most cases.
Within this comparison it was found that only the
29kDa protein is significantly recognized by IgA in
individuals without caries compared to subjects with
caries history. In contrast, subjects without caries
tend to respond less via IgG to the same protein,
compared to the other two groups, which respond
similarly. The 29 kDa protein has been described as
S. mutans antigen A since 1979 41 and has been a
candidate for a vaccine antigen after being tested in
monkeys, because it induced significantly high
levels of serum IgG 42, but the quantity of IgA
induced has not been assessed.
This study assessed simultaneous recognition by
IgA and IgG of S. mutans 29 kDa protein, and found
that there are significantly more individuals who
recognize it via IgG in the groups which had or have
the disease, although most individuals who
recognize it via IgA belong to the group without
caries. Taking into account that a lower quantity of
S. mutans was found in caries-free individuals, it
suggests that the quantity of specific IgA against
the 29 kDa protein and low amount of IgG protected
them at the local level.
In contrast, among individuals who had or have the
disease, although there are fewer individuals with
specific IgA for the 29 kDa protein, most individuals
simultaneously recognized it via IgG. This may be
because higher numbers of CFUs were found in their
mouths than in caries-free individuals. The IgA
present in groups with caries or with history of caries
did not control the disease. This may be explained
from two points of view: (1) the response observed in
individuals with history of caries may be due to a
mechanism of tolerance because the microorganism
present in the oral cavity may be ingested in small
amounts during food intake, 43 which could in turn
explain why having had the disease is considered one
of the primary risk factors for having it again 44; and
(2) subjects with active caries who also had the
highest number of CFUs probably trigger a humoral
immune response due to the bacterial challenge, but
it is not as high and not enough to be effective.
A high percentage of individuals with active caries
recognized the 45 kDa protein primarily, as well as
the 125 kDa protein via IgA, and the 36, 43, 17 and
10 kDa proteins via IgG. These proteins have not been
described in the literature as dominant natural
antigens in terms of antigenicity and immunogenicity,
and may be related to the activity of the disease 30 and
correlated to the fact that caries-free individuals and
individuals with caries history reacted poorly to them.
This behavior reflects differences between the two
types of response: the local IgA-mediated response
and the systemic IgG-mediated response. In the
presence of large quantities of S. mutans, an initial
systemic IgG-mediated response is activated, which
subsequently changes to IgA in terms of predomi -
nance. The process that takes place to generate the
antibodies found in saliva is different for each type
of immunoglobulin 29; this is one of the few studies
reported in the literature describing the IgG
response to S. mutans in the oral cavity.
Even though deficient memory response to S. mutans
antigens has been shown,45 information gathered
from the literature and data from our study suggest
that the protective response may be mediated more
by IgA than by IgG. Our study found a greater
number of caries-free individuals who responded to
several S. mutans proteins which have been classified
in the literature as potential vaccine antigens,
including the 191 kDa, 29 kDa and 38 kDa proteins;
30 in contrast to the results reported by Smith et al.,
who did not find significant differences in the
pattern of response between patients with and
without S. mutans infection 15.
With regard to the antigen corresponding to the
PAc protein, Takahashi et al. claim that IgA type
antibodies against it play a part in protection against
colonization by S. mutans 31.A higher IgA response
has been shown in caries-free individuals than in
patients with active caries 39. In our study, the 191
kDa protein was similarly recognized by IgA in
caries-free individuals and in those with caries
history, but differently between individuals with
caries history and those with active caries. The
deviation of the response towards antigens which are
not relevant in the pathophysiology of the disease,
when there is a large number of microorganisms in
the environment, may be an S. mutans evasion
mechanism, in addition to the fact that it has been
shown that this protein is recognized by adults but
not by children, showing how the immune response
matures as the individual grows 27.
It is important to design studies that help clarify the
role of antibodies against the protein PAc in the
disease, considering that artificial induction of
maturation of the response to this antigen early in life
could provide protection against dental caries 46.
To date, it has not been possible to find antigens that
explain natural protection from the disease.
Differences might be observed by conducting
studies of specific antibody avidity and affinity.
Until these aspects are studied, the possibility of
finding an S. mutans antigen which will activate a
protective response should not be left aside.
Other topics that should be considered are S. mutans
evasion mechanisms that enable it to avoid or regulate
the specific immune response, and knowledge of
tolerance mechanisms that may be generated by the
microorganism and its constant ingestion. Future
strategies for controlling dental caries should aim to
control these mechanisms.
CONCLUSIONS
All the individuals studied have antibodies that recognize S. mutans proteins via salivary IgA and IgG, but the protective response against S. mutans seems to be more mediated by local IgA than by IgG. The 29 kDa protein, formerly known as Antigen A, was recognized mainly via IgA in individuals without caries, and simultaneously via IgG in individuals with caries history and active caries, showing an association between this protein in the protection of caries-free subjects, and tolerance and low response in subjects with caries history or active caries, respectively.
ACKNOWLEDGEMENTS
This study was financed by the Academic Vice-rector's Office of Pontificia Universidad Javeriana, project ID: 000935.
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