archives of oral biology 52 (2007) 10881096

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Age-dependent deciency in saliva and salivary antibodies secretion in Downs syndrome

S. Chaushu a,*, G. Chaushu b, M. Zigmond c, E. Yefenof d, A. Stabholz e, J. Shapira f, J. Merrick g, G. Bachrach c
Department of Orthodontics, Hebrew University-Hadassah School of Dental Medicine, P.O. Box 12272, Jerusalem 91120, Israel b Department of Oral and Maxillofacial Surgery, The Maurice and Gabriela Goldschleger School of Dental Medicine, Tel Aviv University, Tel Aviv, Israel c Institute of Dental Sciences, Hebrew University-Hadassah School of Dental Medicine, Jerusalem, Israel d Lautenberg Center of Immunology, Hebrew University-Hadassah School of Medicine, Jerusalem, Israel e Department of Periodontics, Hebrew University-Hadassah School of Dental Medicine, Jerusalem, Israel f Department of Pediatric Dentistry, Hebrew University-Hadassah School of Dental Medicine, Jerusalem, Israel g National Institute of Child Health and Human Development, Ofce of the Medical Director, Division for Mental Retardation, Ministry of Social Affairs, Israel
a

article info
Article history: Accepted 8 June 2007 Keywords: Downs syndrome Saliva Antibodies Ageing Mucosal immune system

abstract
Objective: Downs syndrome (DS) individuals suffer from an increased susceptibility to infections. Here, we assessed age-related changes in the salivary-specic humoral immunity of DS subjects. Design: Parotid and whole saliva were collected from a young group of DS (YDS, n = 30, 23.3 4 years), an older group of DS individuals (ODS, n = 10, 51.9 8 years) and compared to two age-matched groups of healthy volunteersa young group (YC, n = 29, 22.8 5 years) and an older group (OC, n = 10, 48.4 9 years). The levels of total IgA, and specic antibodies to three common oral pathogens (Porphyromonas gingivalis, Actinobacillus (Aggregatibacter) actinomycetemcomitans and Streptococcus mutans) were analysed. Results: The limited increases in IgA concentrations could not compensate the dramatic reduction in the salivary ow rate observed in DS individuals. Therefore, the median secretion rates of the specic antibodies in whole and parotid saliva were 7077% and 3460% (respectively) lower in YDS individuals as compared to YC and farther 77100% and 7588% (respectively) lower in ODS compared to YDS. In contrast, the antibody secretion rates were similar for parotid saliva, or even increased for whole saliva of OC, compared with YC. Consequently, a dramatic cumulative extreme reduction (>92%) in the bacterial specic salivary antibodies differentiated the adult DS individuals from to their age-matched controls. Conclusions: Our results indicate a severe immunodeciency in the secretion rate of the specic salivary IgA response of in DS individuals which intensies with age. # 2007 Elsevier Ltd. All rights reserved.

* Corresponding author. Fax: +972 2 6427613. E-mail address: drchaushu@gmail.com (S. Chaushu). 00039969/$ see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2007.06.002

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1.

Introduction

Human saliva has been suggested to play an important role in oral health, especially in protection against noxious compounds produced by microorganisms. There is no agreement as to the actual role of saliva in the pathogenesis of dental caries and periodontal disease. It appears that no single salivary agent is far more important than the other. Nevertheless, data suggests that inhibition of the virulence factors may protect the host. Novel strategies for inducing safe mucosal immunotherapy for these diseases have been proposed. Although the clinical efcacy of such approaches is still unsatisfactory, emerging technologies are expected to improve the effectiveness of such procedures in the near future.1,2 Studies carried out on healthy subjects found an age related decrease in the amount of whole saliva.3 In contrast, no correlation was found between age, the amount of parotid saliva4,5 and the ability to launch an antibody response.68 A reduced salivary ow rate9 and an increased prevalence of mucosally derived infections in the elderly6 are frequently found as side effects of systemic disorders and medication intake. Downs syndrome (DS) is a genetic disorder resulting from a trisomy of chromosome 21 that leads to multiple abnormalities. DS patients are susceptible to infectious, malignant, and autoimmune diseases.10 For unknown reasons,11,12 the most abundant infections in DS are observed in the mucosalgastrointestinal and respiratory systems.10 Their prevalence and severity increase with age.13 DS individuals have an increased prevalence of periodontal disease, which develops at early age and is rapid and extensive,1417 while the incidence of carious lesions is contrastingly low.18,19 The present investigation was undertaken to examine agerelated changes in salivary ow rate and specic antibody responses to bacterial antigens commonly expressed in periodontal diseases and dental caries, in whole and parotid saliva of DS individuals.

microbial culture. The total amount collected over a 5-min period was registered, and the salivary ow rates (ml/min) were calculated. Parotid saliva samples were collected using a parotid salivary gland cup. Salivary secretion was stimulated using 100 ml of 2% citric acid applied to the tongue every 15 s over a period of 1020 min. It has been shown that ow rates obtained during the initial 2-min interval are closely similar to ow rates of subsequently produced saliva.20 Accordingly, saliva ow rate was measured from the initiation of the gustatory stimulation and the salivary ow rates (ml/min) were recorded. The saliva samples were centrifuged at 12,000 g for 10 min at room temperature and kept frozen at 70 8C until assayed. All participants provided an informed consent (in the case of Downs syndrome patients by a parent or guardian) to a protocol that was approved by the institutional ethical committee.

2.1.

Bacterial culture

Porphyromonas gingivalis ATCC 33277 was cultured in Wilkins Chalgren Anaerobe Broth (Oxoid, Basingstoke, Hampshire, England). Actinobacillus (Aggregatibacter) actinomycetemcomitans ATCC 29523 (serotype a), was grown in TSBV medium.21 Both species were grown in an anaerobic chamber (Coy Laboratory Products, Ann Arbor, MI) under an atmosphere of 85% N2, 10% H2, 5% CO2 at 37 8C. Streptococcus mutans ATCC 27351 was grown in Brain Heart Infusion medium (Difco, Massachusetts, USA), in an aerobic environment supplemented with 5% CO2. Bacterial purity was determined by phase contrast microscopy and Gram staining. The presence of these three pathogens in the oral ora of Downs syndrome patients has been previously demonstrated.22,23

2.2.

Bacterial antigen preparation

2.

Materials and methods

Parotid and whole saliva were collected from a group of 39 healthy volunteers (mean age 29 13 years, 25 females and 14 males), and 40 DS patients (mean age 30.4 13.6 years, 16 females and 24 males), between the hours of 9.00 a.m. and 12.00 a.m. Twenty-nine healthy volunteers and 30 DS patients were younger than 30 years (mean age 23 5, 19 female and 10 male volunteers and mean age 23.3 4 years, 13 female and 17 male DS), and were referred to as young controls-YC and young DS-YDS. Ten healthy volunteers and 10 DS patients were older than 30 years (mean age 46 12, 6 female and 4 male volunteers, and 51.9 8 years, 3 female and 7 male DS and were referred to as older controls-OC and older DS-ODS). The clinical and radiographic periodontal status of the young DS and controls has been previously described.17 The periodontal status of the older DS could not be accurately assessed since 9 out of 10 individuals were partially or totally (7) edentulous. Whole saliva samples (unstimulated) were collected with the aid of test tubes used in the collection of sputum for

Late logarithmic phase bacterial cultures (300 ml) were harvested by centrifugation for 10 min at 3500 g. Bacterial pellets were re-suspended in 1.2 ml carbonatebicarbonate buffer (15 mM Na2CO3, 35 mM NaHCO3, 0.02% NaN3, pH 9.6), supplemented with antiprotease cocktail (SigmaAldrich, Rehovot, Israel). Cell suspensions were transferred into 2 ml microfuge tubes, in which one-third of the volume contains glass beads ($16 m, SigmaAldrich, Rehovot, Israel). Cells were disrupted using the Fast Prep cell disruptor (Bio 101-Savant Instruments, Inc., New York, USA) at a speed of 6 m/s for 30 s and then incubated on ice for 1 min. This procedure was repeated ve times. Suspensions were centrifuged 10 min at 12,000 g and supernatants were collected. Protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad, USA).

2.3.

Quantication of salivary Ig

Pathogen specic IgA and total IgA were determined by enzyme-linked immunosorbent assay (ELISA). Briey, 96-well polystyrene plates (Nunc, Roskilde, Denmark) were coated with the bacterial A. actinomycetemcomitans, P. gingivalis, and

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S. mutans antigens (0.05 mg/well for whole saliva antibodies and 2, 0.1, and 0.1 mg/well, respectively, for parotid saliva antibodies) or rabbit anti-human Ig antibodies (SigmaAldrich, Rehovot, Israel) (for measuring total IgA) diluted 1:800 in carbonatebicarbonate buffer, for 1 h of at 37 8C. The plates were washed six times with PBST (phosphate buffered saline, 0.05% Tween 20) and blocked with 1% PBSTBSA (PBST, 1% bovine serum albumin) at 4 8C overnight. Whole saliva dilutions (1:800 for total IgA, 1:8 for specic IgA) and parotid saliva dilutions (1:160 for total IgA, 1:4 for specic IgA) were placed in triplicate wells and incubated for 2 h at 37 8C. The plates were washed six times, and incubated for 1 h with 1:500 diluted alkaline phosphatase conjugated rabbit anti-human IgA specic for a chains (SigmaAldrich, Rehovot, Israel). The plates were washed again (six times) and 100 ml p-nitrophenylphosphate (SigmaAldrich, Rehovot, Israel) dissolved in diethanolamine pH 9.8 (1 mg/ml) was added. Plates were incubated at 37 8C. Colour development was stopped by adding sodium hydroxide 3 M and optical density at 405 nm was determined by an automated ELISA reader (Dynatech, Chantilly, VA, USA). The mean value of the triplicates and the background of each saliva sample (without antigen) were determined. A pool of whole saliva (pooled from four healthy volunteers) was used as standard to obtain relative antibody activity (ELISA units = EU). The concentrations of specic reactive antibodies were calculated in ELISA units (EU/ml), by comparing the antibody activity of the saliva pool with the test specimen. The antigen-reactive IgA secretion rates (EU/min) were calculated by multiplying antibody titers with salivary ow.

3.
3.1. 3.1.1.

Results
Whole saliva Whole saliva ow rate (Fig. 1A)

The median resting whole saliva ow rate in the entire control group was 0.55 ml/min (range 0.051.64 ml/min) compared to 0.05 ml/min (range 00.41 ml/min) in the entire DS group. Thus, DS individuals had a 90% reduction in the secretion of whole saliva. When examined by age, no statistically signicant differences were found in the whole saliva ow rates of YC versus OC individuals (median 0.5 ml/min, range 0.051.64 ml/min, and median 0.75 ml/min, range 0.061.47 ml/min, respectively). In contrast, the median whole saliva ow rate was 0.11 ml/min (range 00.41 ml/min) in YDS, compared to 0.02 ml/min (range 00.08 ml/min) in ODS subjects. These results indicate an 80% reduction of whole saliva ow rate in YDS versus YC, and a further 80% reduction in ODS versus YDS individuals. This adds up to a 97% decrease in ODS ow rate compared to that in older controls.

3.1.2.

Whole saliva antibody concentrations (Fig. 2)

The most prominent changes were found in the median concentrations of total IgA and specic IgA antibodies in the OC group, which were signicantly increased

3.1.3.

Whole saliva antibody secretion rates (Fig. 3)

2.4.

Statistical analysis

The differences were evaluated, analysed and compared using nonparametric rank tests (Wilcoxon and Exact Wilcoxon tests). All p values given are based on two-tailed tests and p < 0.05 was the criterion of signicance. Differences were evaluated, analysed and compared between the YDS and the YC, YC and OC, YDS and ODS, and between OC and ODS.

The median secretion rate (the product of concentration and ow rate, expressed as EU/min) of total IgA in whole saliva was 85% lower in the entire group of DS versus the controls, anti-P. gingivalis was 84% lower, anti-A. actinomycetemcomitans 78% lower, and anti-S. mutans IgA was 85% lower. When grouped by age, the median secretion rate of total IgA in whole saliva was 70% lower in YDS individuals as compared to YC, anti-P. gingivalis was 75% lower, anti-A. actinomycetemcomitans was 70% lower and anti-S. mutans IgA was 77% lower. The ODS subgroup had 85% lower total IgA secretion than in YDS, 77% lower anti-P. gingivalis and 80% lower anti-S. mutans.

Fig. 1 Box plots of salivary flow rate in Downs syndrome individuals (DS), controls (C), young Downs syndrome individuals (YDS), older Downs syndrome individuals (ODS), young controls (YC) and older controls (OC). (A) Whole saliva and (B) parotid saliva. Upper and lower limits of boxes represent 75th and 25th percentiles, respectively. Whisker caps represent 95th and 5th percentiles. The medians are indicated by horizontal lines. Symbols outside box represent outlier (o) or extreme values (*). Closed circles indicate significance: p < 0.05 and p < 0.01.

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Fig. 2 Box plots of IgA concentration (EU/ml) in whole saliva of controls (C), Downs syndrome individuals (DS), young controls (YC), young Downs syndrome individuals (YDS), older controls (OC), and older Downs syndrome individuals (ODS). (A) Total IgA, (B) IgA anti-Porphyromonas gingivalis, (C) IgA anti-Actinobacillus (Aggregatibacter) actinomycetemcomitans, and (D) IgA anti-Streptococcus mutans. Upper and lower limits of boxes represent 75th and 25th percentiles, respectively. Whisker caps represent 95th and 5th percentiles. The medians are indicated by horizontal lines. Symbols outside box represent outlier (o) or extreme values (*). Closed circles indicate significance: p < 0.05 and p < 0.01.

Anti-A. actinomycetemcomitans antibodies were hardly detected in whole saliva of ODS individuals. In contrast, the median secretion rates of total IgA and the specic antibodies in whole saliva of OC were signicantly higher than in YC. Hence, the cumulative reduction in the secretion rates in ODS compared to the age-matched OC reached 98%, and was highly signicant.

did not affect the salivary ow rate of OC, who surprisingly had a slightly higher ow rate than the YC. As a result, the marked difference between the ODS and OC was further accentuated, and ODS had a 90% reduction of parotid salivary secretion when compared to their age-matched controls.

3.2.2.

Parotid saliva antibody concentrations (Fig. 4)

3.2. 3.2.1.

Parotid saliva Parotid saliva ow rate (Fig. 1B)

The median stimulated parotid saliva ow rate in the entire control group was 0.25 ml/min (range 0.101.40 ml/min). The median parotid saliva ow rate in DS individuals was 0.09 ml/ min (range 00.65 ml/min). Thus, DS individuals had a 64% reduction in the secretion of parotid saliva. When divided into age subgroups, the median stimulated parotid salivary ow rate in YDS was 0.11 ml/min (range 0.04 0.65 ml/min) in comparison to 0.22 ml/min (range 0.100.86 ml/ min) in YC. This accounts for a 50% reduction of parotid ow rate in YDS versus YC. The median parotid salivary ow rate showed a further 60% reduction in ODS (0.04 ml/min, range 00.14 ml/min) compared to YDS individuals. In contrast, age

The median total IgA concentration was higher by 19% in the entire DS group versus the controls. When divided by age, the median total IgA was 20% higher in YDS versus YC, but 73% lower in ODS in comparison to YDS. In contrast, in OC the total IgA concentration was similar to YC. No signicant differences were found in the bacterial specic salivary IgA concentrations between the two age-related DS and control subgroups.

3.2.3.

Parotid saliva antibody secretion rates (Fig. 5)

The calculated median secretion rate of total IgA in parotid saliva was 60% lower in the entire DS versus the entire control group, anti-P. gingivalis was 54% lower, anti-A. actinomycetemcomitans 50% lower and anti-S. mutans IgA was 66% lower. When grouped by age, the median secretion rate of total IgA in parotid saliva of YDS was 35% lower than that of the YC.

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Fig. 3 Box plots of IgA secretion rate (EU/min) in whole saliva of controls (C), Downs syndrome individuals (DS), young controls (YC), young Downs syndrome individuals (YDS), older controls (OC), and older Downs syndrome individuals (ODS). (A) Total IgA, (B) IgA anti-P. gingivalis, (C) IgA anti-A. actinomycetemcomitans, and (D) IgA anti-S. mutans. Upper and lower limits of boxes represent 75th and 25th percentiles, respectively. Whisker caps represent 95th and 5th percentiles. The medians are indicated by horizontal lines. Symbols outside box represent outlier (o) or extreme values (*). Closed circles indicate significance: p < 0.05 and p < 0.01.

Anti-P. gingivalis parotid IgA secretion rate was 39% lower, anti-S. mutans was 60% lower and anti-A. actinomycetemcomitans parotid IgA secretion rate was 34% lower than in controls. The secretion rate of total IgA in ODS was 77% lower than in YDS, anti-P. gingivalis was 85% lower, anti-S. mutans was 75% lower, and anti-A. actinomycetemcomitans 88% lower. In contrast, the median secretion rate of total IgA in parotid saliva of OC was slightly higher, albeit not statistically signicant, than in YC ( p = 0.06), while the specic antibodies secretion rates were similar. Therefore, the differences between the secretion rates in ODS versus the age-matched OC were again emphasized and highly statistically signicant.

3.3.

Gender

No statistically signicant differences were found between males and females pertaining to all the evaluated parameters.

4.

Discussion

There is no consensus concerning the role of the secretory immune system in the development of periodontal disease.

Studies supporting a protective role showed inhibition of bacterial colonization by salivary IgA,24 a high risk of periodontal disease in individuals with low levels of secretory IgA and specic antibodies2527 and an increased frequency of tooth loss in immunodecient subjects.28 In contrast, other studies found no differences in the periodontal status29 or even less gingival inammation in immunodecient patients.30,31 Colonization of tooth surfaces with S. mutans is mandatory for the initiation of dental caries.32 A protective role of salivary IgA antibodies against the development of dental caries has been attributed to their ability to inhibit sucrose independent or sucrose dependent streptoccocal accumulation on tooth surfaces.33 Animal models demonstrated an increase in the specic salivary IgA antibody response against cariogenic bacteria and a reduced number of caries lesions following immunization with anti-caries vaccines.34 Our analysis indicated that DS patients display a signicant reduction in their salivary ow, both resting whole and stimulated parotid, compared to controls (Fig. 1). While some reports are consistent with our ndings,3539 others found no differences.4042 Tylenda et al.43 reported that submandibular gland uid secretion capacity is maintained with ageing in healthy

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Fig. 4 Box plots of IgA concentration (EU/ml) in parotid saliva of controls (C), Downs syndrome individuals (DS), young controls (YC), young Downs syndrome individuals (YDS), older controls (OC), and older Downs syndrome individuals (ODS). (A) Total IgA, (B) IgA anti-P. gingivalis, (C) IgA anti-A. actinomycetemcomitans, and (D) IgA anti-S. mutans. Upper and lower limits of boxes represent 75th and 25th percentiles, respectively. Whisker caps represent 95th and 5th percentiles. The medians are indicated by horizontal lines. Symbols outside box represent outlier (o) or extreme values (*). Closed circles indicate significance: p < 0.05 and p < 0.01.

individuals. By contrast, Percival et al.3 demonstrated a signicant decrease in the secretion rates of unstimulated whole saliva in relation to age. In the present study age had no effect on the secretion rate of whole saliva in healthy individuals. However, a large (80%) age-related salivary ow rate reduction in unstimulated whole saliva of DS individuals was observed (Fig. 1A). Data on stimulated parotid salivary ow rate in healthy individuals revealed no signicant age-related uctuations.4 6,44,45 In the present study stimulated parotid salivary ow rates in older healthy controls, were similar to, or even higher than those in younger healthy controls suggesting that age per se, is not a predisposing factor for hypofunction of the parotid gland. In contrast, stimulated parotid saliva of ODS individuals showed a 60% age-related reduction compared to that of YDS, and a 90% reduction compared to their age-matched controls (Fig. 1B). This result concurs with our previous report.38 The age-related dramatic reduction in both resting whole and stimulated parotid saliva may contribute to the compromised oral health seen in such patients.17,46 The relative importance of salivary Ig secretion rates as opposed to concentrations has been a matter of discussion in the past.47 The diminished output of salivary IgA, rather than

its absolute concentration, has been associated with a high incidence of recurrent mucosal infections in DS individuals,39 and also suggested as an etiologic factor for the oral health problems seen in Sjogrens syndrome patients.48 The median whole and parotid salivary total IgA concentration in the YDS group was slightly higher than that of the controls, while no changes were demonstrated in the specic antibody response (Figs. 2 and 4). These rather limited increases in IgA level could not compensate the dramatic reduction in ow rate rendering the YDS individuals highly immunodecient in both total and specic salivary IgA response (Figs. 3 and 5). The signicant lower levels of salivary antibodies in the present YDS group were accompanied by an increase in the prevalence, extent and severity of periodontitis.17 These results are in agreement with the early colonization with periodontal pathogens previously described in DS individuals.49,50 The salivary immunodeciency was further accentuated in the ODS group in which the ow rate was severely diminished and the concentrations were either similar or lower compared to YDS individuals. In contrast, adult healthy individuals had similar levels of total and specic IgA antibodies as younger controls (Figs. 3 and 5).

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Fig. 5 Box plots of IgA secretion rate (EU/min) in parotid saliva of controls (C), Downs syndrome individuals (DS), young controls (YC), young Downs syndrome individuals (YDS), older controls (OC), and older Downs syndrome individuals (ODS). (A) Total IgA, (B) IgA anti-P. gingivalis, (C) IgA anti-A. actinomycetemcomitans, and (D) IgA anti-S. mutans. Upper and lower limits of boxes represent 75th and 25th percentiles, respectively. Whisker caps represent 95th and 5th percentiles. The medians are indicated by horizontal lines. Symbols outside box represent outlier (o) or extreme values (*). Closed circles indicate significance: p < 0.05, and p < 0.01.

The results of the present study support previous studies, which found unimpaired or even higher IgA levels in parotid and whole saliva of the elderly.8,51,52 The literature is much scarcer in studies on age-related changes in bacterial specic salivary antibodies. However, a study by Percival et al.6 also found an age related increase in anti-S. mutans, A. actinomycetemcomitans, and Escherichia coli salivary IgA, suggesting that the ability to mount IgA responses is not impaired with ageing of healthy individuals. Reuland-Bosma et al.53 reported lower prevalence of P. gingivalis in older DS subjects, which seems to contradict the severe IgA deciency reported here. A possible explanation for this apparent discrepancy is that the severe loss of the dentition characteristic of DS at this age, results in the elimination of periodontal pathogens from the oral cavity.53,54 Our data suggests that the secretory immunity in the oral cavity of DS subjects is characterized by a severe immunodeciency. This immunodeciency is more severe in whole saliva than in parotid saliva, and is extremely exacerbated with age. It might be speculated that such differences between whole and parotid saliva arise from removal of some of the secreted antibodies in whole saliva by binding to bacterial

antigens from the oral cavity. However, such a hypothesis awaits evidence in future studies. Recently, we analysed the secretion rates of the antibacterial protein LL-37 in whole saliva of DS individuals.55 Lack in LL-37 was correlated with severe periodontitis in individuals with the Morbus Kostman genetic disorder.56,57 In contrast to the reduced levels of salivary-specic antibodies, the secretion rate of LL-37 was found to be normal and not inuenced by the decrease in salivary ow rate.55 The combined results of our previous and present studies suggest that normal salivary LL-37 levels are not sufcient to protect against periodontal disease in DS patients, when accompanied with deciencies in the oral mucosal acquired immunity (specic antibodies). Caries associated gram-positive oral bacteria were found to be more susceptible to human cationic antimicrobial peptides (including LL-37) compared to the primarily gram-negative oral pathogens associated with periodontitis.58 These ndings support our previous hypothesis that normal amounts of cationic antimicrobial peptides in saliva of DS individuals might protect against caries, while a deciency in their acquired immunity (specic antibodies) accounts for their high prevalence of periodontitis.

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Acknowledgements
This investigation was supported by the Dr. Izador I. Cabakoff Research Endowment Fund. We thank Dr. Ilan Feldberg, Chief Dental Consultant, Division for Mental Retardation, Ministry of Social Affairs and the staff in the Dental Clinic of Elwyn Center for the Disabled, especially Mrs. Ofra Saadon and Dr. Moshe Einhorn for their cooperation in this study. We are also grateful to Prof. Adrian Becker for his support and editorial assistance.
18.

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