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RESEARCH REPORT |
1 Department of Orthodontics, Hebrew University-Hadassah School of Dental Medicine, founded by the Alpha Omega Fraternity, PO Box 12272, Jerusalem 91120, Israel;
2 Lautenberg Center of Immunology, Hebrew University-Hadassah School of Medicine, Jerusalem, Israel;
3 Department of Pediatric Dentistry, Hebrew University-Hadassah School of Dental Medicine;
4 Department of Oral and Maxillofacial Surgery, The Chaim Sheba Medical Center, Tel Hashomer, Israel;
*corresponding author, drshaush{at}netvision.net.il
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: saliva • Ig • Down Syndrome • MIS
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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The reasons for an increased susceptibility to infections in DS subjects are uncertain. Over the past 20 years, a large body of research focusing on abnormalities in the systemic immune system of DS was performed, demonstrating selective cell-mediated immunodeficiencies, defective neutrophil polymorphonuclear leukocyte chemotaxis, impaired antibody response to specific pathogens, low T-cell lymphocyte counts, and immature subsets of T-lymphocytes (Ugazio, 1981; Reuland-Bosma and Van Dijk, 1986; Lockitch et al., 1987; Morinushi et al., 1997).
The highest incidence of infections in DS is observed in the mucosal-gastrointestinal and respiratory systems (Desai, 1997), which comprise the largest area of mucous membrane through which micro-organisms interact with the immune system. The mucosal immune system (MIS) provides specific immune defense at mucosal surfaces. Secretory IgA (SIgA) is the dominant antibody isotype in human external secretions (Brandtzaeg, 1998). The MIS is not necessarily in concert with systemic immunity (Waldman and Ganguly, 1974; Mowat and Viney, 1997) and constitutes a first natural barrier against invading pathogens (Brandtzaeg et al., 1999).
Salivary sialometry and sialochemistry are expanding fields of interest used by an increasing number of researchers, to gain a deeper understanding of oral physiology and pathology. The literature yields only occasional and contradictory reports of the use of sialometry and sialochemistry to explain the role of saliva in the oral health and well-being of DS subjects. The few studies performed over the past four decades, in an attempt to quantify salivary secretion, reported either decreased or normal flow rates (Winer et al., 1965; Coburn et al., 1967; Cutress, 1972; Jara et al., 1991).
A review of the literature reveals sporadic studies concerned with the function of the DS MIS (Epstein and Baumgarten, 1991; Barr-Agholme et al., 1998). We therefore undertook to assess the levels of salivary immunoglobulins (Ig) in a group of DS individuals and its bearing on their susceptibility to mucosal surface infections.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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. All those with hypothyroidism were receiving treatment with replacement therapy (Eltroxin, GlaxoSmithKline, Pittsburgh, PA, USA; 50-200 mg/day), and their thyroid hormone levels were well-controlled.
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All participants provided informed consent (in the case of DS subjects, a parent or guardian provided the consent) to a protocol that was reviewed and approved by the institutional ethics committee.
Saliva Collection
Saliva samples were collected by means of a parotid salivary gland cup. Salivary secretion was stimulated by means of 100 µL of 2% citric acid applied to the tongue every 15 sec over a period of 4 to 40 min. The length of the saliva collection varied with the rate of saliva flow and subject cooperation. It has been shown that flow rates obtained during the initial two-minute interval are similar to flow rates of saliva produced subsequently (Baum et al., 1985). Accordingly, saliva flow rate was measured from the initiation of the gustatory stimulation. Saliva samples were collected between 9:00 a.m. and 12:00 Noon, and the salivary flow rate was expressed in milliliters/minute/gland.
Quantification of Salivary Ig
Total salivary IgM, IgG, and IgA were determined by enzyme-linked immunosorbent assay (ELISA), with 96-well polystyrene plates (Nunc, Roskilde, Denmark) coated with rabbit anti-human Ig antibodies (BioMakor, Kiryat Weizmann, Rehovot, Israel). At least 4 salivary dilutions were placed in triplicate wells and incubated for 2 hrs at 37°C. The plates were washed 6 times before alkaline-phosphatase-conjugated rabbit anti-human 
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, or µ heavy chains (Sigma-Aldrich, Rehovot, Israel) were added. After further incubation for 1 hr at 37°C, the plates were washed again, and p-nitrophenylphosphate was added to the wells. Color development was stopped after 30 min at 37°C by the addition of sodium hydroxide, and the optical density at 405 nm was determined in an automated ELISA reader (Dynatech, Chantilly, VA, USA). Human myeloma proteins, from the pooled Hadassah Hospital stock, were used as standards, which were calibrated in a commercial agar immunodiffusion assay against reference proteins provided by the manufacturer (Behring Werke, Marburg, Germany). The median value of the triplicates was used. Parallelism between the standard curves based on serum myeloma proteins and the unknown salivary samples tested at different dilutions was a prerequisite for the determination of salivary Ig concentrations.
Statistical Analysis
Information gathered was statistically analyzed by Kruskal-Wallis and Wilcoxon rank-sum tests. These non-parametric rank tests were used since the sample size was relatively small and not of normal distribution. All p values given are based on two-tailed tests, and p < 0.05 was the criterion of significance. Differences were evaluated, analyzed, and compared between the entire group of Down patients and the control group.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). No statistically significant differences were found between the median salivary IgA concentration in the study group and that in the controls (Fig. 2A), while the median salivary IgG and IgM concentrations were statistically significantly higher by 25% (p = 0.05) and 340% (p < 0.001), respectively (Figs. 2B, 2C). Since the secretory dynamics of parotid Ig is dependent on the secretion flow rate, the salivary rate (the product of concentration and flow rate, expressed as µg/min/gland) is a better measure of parotid Ig output than its absolute concentration. The median secretion rates of IgA and IgG were diminished by 83% (p < 0.001) and 75% (p = 0.05), respectively, when compared with those of the controls, while the median secretion rate of IgM was not statistically significantly lower (Fig. 3). Generally, the Ig levels reported in our study are compatible with those reported previously (Brandtzaeg et al., 1970; Ben-Aryeh et al., 1986; Challacombe et al., 1995).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Young DS patients have high rates of infections, malignancies, and autoimmune diseases (Desai, 1997), which may be seen in non-DS subjects at a much older age. Histological aberrations of the thymus as well as abnormal distribution of T-cell populations suggest that the majority of the immunological disabilities may be ascribed to an abnormal thymic physiology. The impairment in both B- and T-cell function is expressed in abnormal antibody and Ig production. In DS children younger than 6 yrs, the level of serum Ig is similar to that of healthy controls (Nespoli et al., 1993). After the age of 5 yrs, hypergammaglobulinemia of the IgA and IgG type is found in the serum (Burgio et al., 1975), as well as an abnormal distribution of IgG subclasses (Avanzini et al., 1988). Vaccine-induced response to hepatitis B antigen in DS patients is a mere 22% of the response in controls. IgM levels decrease during adolescence and are lower than normal in the majority of DS adults. Based on these findings, it has been inferred that while the primary immune defect is in the cellular compartment, both cellular and humoral immunity in DS subjects undergoes precocious aging (Nespoli et al., 1993).
The present study focuses on the use of saliva as a model for monitoring mucosal immunocompetence in DS subjects by monitoring the level of salivary total Ig. The use of saliva satisfied the criteria for: (i) a non-invasive sample collection procedure and (ii) a secretion that reflects a central compartment of the MIS (oral cavity) and could be processed by simple immunological diagnostic means. A drawback in the use of saliva for the evaluation of the function of MIS is that it reflects merely the result following recruitment of mucosal B-lymphocytes to the salivary glands. In addition, fluctuations in flow rate may influence Ig concentrations in individual results. Another difficulty that should be taken into consideration when the level of IgG in parotid saliva is measured is the fact that IgG does not reflect secretory immunity but rather a passive diffusion from systemic immunity. Therefore, additional factors influencing the parotid gland, such as chronic parotitis, may be responsible for fluctuations in IgG secretion rate. Salivary Ig level is flow-rate-dependent. Generally, Ig levels decrease in response to stimulation of saliva secretion. These impediments can be overcome by the measurement of albumin or total protein concentrations for adjustment of flow rate when salivary Ig concentrations are evaluated (Gleeson et al., 1995).
The use of saliva as a corollary of MIS has an additional important consideration. Whole saliva is more accessible than a specific gland for collection and therefore is preferred in many laboratories. It has certain limitations, however: Whole saliva is an admixture of secretions from the major and minor salivary glands, with food debris, bacteria, shed cells, and leukocytes. Therefore, while whole saliva may be suitable for qualitative studies, the use of a specific gland, as a source of pure and unadulterated secretion, is much more suitable for quantitative studies (Mandel, 1980).
Several studies have demonstrated that freezing and thawing significantly reduces the detection of Ig in whole saliva, while their effects on parotid salivary Ig are minimal. Inter-individual differences in salivary Ig levels in stimulated parotid saliva are minimal (Brandtzaeg, 1971; Ben-Aryeh et al., 1986). It was for these reasons that parotid saliva was chosen to be tested in the present investigation.
Immunological aberrations are more prevalent in older age groups of DS patients (Nespoli et al., 1993). It was therefore considered important in the present study to focus on a homogenous age group of DS subjects and age-matched controls. The use of a non-homogenous group would have yielded inconclusive results (Lockitch et al., 1987).
DS subjects showed a significant reduction in their salivary flow. While some published reports are consistent with our findings (Coburn et al., 1967; Cutress, 1972), others (Winer et al., 1965; Jara et al., 1991) found no difference in saliva flow between DS subjects and controls. Since Ig levels are flow-dependent, a salivary source whose flow rate is steady over years should allow for an accurate evaluation of immunological changes without being affected by age. In this regard, parotid saliva provides a reliable source for such analysis, since it was demonstrated that its stimulated flow rate in healthy donors remains constant over time (Baum, 1981; Heft and Baum, 1984).
A superficial evaluation of the data may suggest that the null hypothesis of the present study was incorrect. The median salivary IgA concentration in the study group was similar to that of the controls, while the median salivary IgG and IgM concentrations were higher by 25% and 340%, respectively. These findings are compatible with previously described Ig levels in whole saliva of DS subjects (Barr-Agholme et al., 1998). In the aforementioned study, however, no adjustment for flow rate was made. The only adjustment performed was the proportion of IgG1-IgG4 expressed as a percentage of total IgG. When this was done, the relative level of IgG1 was significantly higher in DS subjects.
To the best of our knowledge, the present study is unique in measuring parotid salivary Ig levels in DS subjects. The importance of flow rate adjustment in the evaluation of salivary Ig concentrations has been addressed in a study that compared children suffering from recurrent colds and influenza with an age-matched control group of healthy children. Salivary IgA concentrations did not differ significantly between the groups. However, following adjustment of albumin, the level of SIgA was significantly lower in recurrently ill children, demonstrating a relationship between a compromised local immune response and recurrent upper respiratory tract infections (Drummond and Hewson-Bower, 1997).
It has been previously suggested (Brandtzaeg et al., 1970) that the secretion rate (the product of concentration and flow rate, expressed as µg/min/gland) is a better measure of an individual's parotid Ig output than its concentration. This suggestion was adopted in the present study, since immune defense at mucosal surfaces is dependent on the available Ig output that confronts a potential pathogen more than on its absolute concentration. The median secretion rates of IgA and IgG were found to be diminished by more than 75% when compared with those of the controls, while the median secretion rate of IgM was not significantly different. According to these results, secretory immunity in the oral cavities of DS subjects is characterized by severe immunodeficiency. This may provide a partial explanation for the high rate of recurrent infections in the respiratory and gastro-intestinal tracks notable in DS patients.
Given the relative scarcity of similar studies concerning the MIS in DS subjects, the results of the present investigation could be compared only with serologic findings. Several investigations of the immunological response to various vaccines ought to be addressed in the present discussion. Reactogenicity and immunogenicity of an acellular pertussis vaccine in 24 DS subjects and in 10 normal infants have been evaluated elsewhere (Li Volti et al., 1996). The vaccine administration elicited protective levels of antibodies in all DS subjects, although the geometric mean titers of IgG antibodies against Bordetella pertussis in these subjects were significantly lower than in normal controls. DS subjects vaccinated with hepatitis B demonstrated a significantly lower IgG1 response to the vaccine than did the controls (Avanzini et al., 1988). Peripheral blood leukocytes from DS individuals were evaluated for antibody responses to a bacterial antigen (tetanus toxoid) and to viral antigens (influenza A/Bangkok and B/Singapore) and were found to be significantly decreased in DS patients. Although no difference was observed between the DS and control groups in the percentages of total T- and B-lymphocytes, a decreased level of CD4+ T-cells and an increased level of CD8+ T-cells were noted (Philip et al., 1986). Antibody response to pneumococcal vaccine was lower in DS than in controls (Nurmi et al., 1982).
Immunodeficiency provides a unique model for evaluation of the significance of the MIS. Subjects with IgA deficiency are often asymptomatic, but if symptoms exist, they usually appear as increased frequency of respiratory infections, pneumonia, intestinal infections, and autoimmune disease (Koskinen, 1996). Salivary IgA is, in general, significantly lower in infection-prone children than in healthy controls (Lehtonen et al., 1987). It has been suggested that IgA-deficient subjects, who are compensated by a significant increase in IgM, are less infection-prone than those in whom no IgM compensation is recorded (Mellander et al., 1986).
The above studies, as well as our own, suggest that DS patients might have immunodeficiency in the humoral mucosal immune response. Since no compensatory increase in the total salivary IgM output has been reported in the present study, this could provide a partial explanation for the high ratio of recurrent infections in target organs of the MIS in DS subjects.
Future studies are required to provide additional evidence in support of this hyopthesis. Factors such as age, institutionalization-vs.-home environment, the comparison with the serological immune status, and correlation to oral diseases may shed further light on the role of the MIS in chronic recurrent infections in DS patients.
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ACKNOWLEDGMENTS |
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Received April 25, 2001; Last revision February 19, 2002; Accepted February 28, 2002
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REFERENCES |
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