Eligibility criteria for study participants.
The presence of periodontal pathogens in periodontally healthy children is often overlooked or ignored. Since house dust mite allergy often appears among children with chronic gum disease, it is important to understand the role of lipopolysaccharide—a major immunodominant antigen of Porphyromonas gingivalis—in stimulating atopic inflammatory markers of allergies. The aim of the present study is to investigate whether any correlation exists between salivary lipopolysaccharide of Porphyromonas gingivalis with circulatory Immunoglobulin-E and Immunoglobulin-G4 in periodontally healthy children with house dust mite allergy. We did an analytic observational study between January and December 2017. We recruited 20 periodontally healthy children (aged 6–16 years) from pediatric allergy-immunology clinic at Dr. Soetomo General Hospital (Indonesia). Lipopolysaccharide of Porphyromonas gingivalis was obtained from salivary secretion, while Immunoglobulin-E and Immunoglobulin-G4 were obtained from venous puncture simultaneously. Immunoglobulin analyses were performed by direct-sandwich ELISA, and lipopolysaccharide analyses were performed by limulus amebocyte lysate (LAL) assay. The average of salivary lipopolysaccharide was 7.21 ± 3.06 μg/ml, Immunoglobulin-E was 114.44 ± 26.19 pg/ml, and Immunoglobulin-G4 was 31.02 ± 9.09 ng/ml. There was a strong correlation between salivary lipopolysaccharide and Immunoglobulin-E (r2 = 0.695, p < 0.001), and a very strong correlation between salivary lipopolysaccharide and Immunoglobulin-G4 (r2 = 0.796, p < 0.001). Conclusion: data revealed significant correlations between salivary lipopolysaccharide of Porphyromonas gingivalis with circulatory Immunoglobulin-E and Immunoglobulin-G4 in periodontally healthy children with house dust mite allergy.
- periodontal pathogen
- Porphyromonas gingivalis
Given the complexity of community-based health care systems in the era of national insurance and high rate of poverty in Indonesia, it is not surprising that limited health literacy is common especially in rural areas . Numerous studies show a link between limited parental health literacy with poor oral health among their child [2, 3, 4]. Parents’ health literacy is often positively correlated with the frequency of dental visits and their knowledge/understanding of preventive measures in terms of oral or gingival diseases . Thus, problems in the mouth range from bacterial biofilm, dental caries, chronic gingivitis, and locally aggressive gingivitis often reported in children whose parents have limited health literacy .
The presence of bacterial biofilm has an association with the presence of periodontal pathogen, which may lead to a periodontal disease when left untreated or undertreated . Bacterial biofilm is often found among children with limited health literacy . When left untreated or undertreated, these periodontal pathogens can spread below the gum line and may develop into gingivitis and periodontitis in the future . Gingivitis is a reversible dental plaque-induced inflammation limited to the gingiva , while periodontitis is usually accompanied by gingivitis but involves irreversible destruction of the supporting tissues .
While many people believe that periodontal pathogen is an adult issue, nowadays this kind of pathogen is also a pediatric issue, since its presence is often reported in children aged 6–16 years [11, 12]. Even though the origin and transmission of these pathogens are not fully understood, few researches have linked them with salivary transmission. Saliva is the most probable vehicle for person-to-person transmission of periodontal pathogens, such as
Evidence suggests that the composition of the oral microbiome differs between children with and without allergy , and disruption of the bacterial biofilm in children leads to allergic responses following allergen challenge in subjects not previously sensitized to the allergen . It is likely that the greatest concentration of
The aim of the present study is to investigate whether any correlation exists between salivary lipopolysaccharide of
2.1 Ethical considerations
This is the grant number for ethics approval (20/Panke.KKE/I/2017). A written informed consent was obtained from every parent to be included in the study. This study was approved by Institutional Review Boards of Dr. Soetomo General Hospital Ethics Committee for Health Research at Surabaya, January 20, 2017. Results will be disseminated through peer-reviewed publications within 1 year after experiment has been finished.
2.2 Research design and setting
This is an analytic observational study with cross-sectional study design to assess the correlation between salivary lipopolysaccharide of
2.3 Sample size estimation
The sample size is based on a substantial meaningful change in IgE level observed in the group . Korn et al.  stated that ELISA is able to assay for free IgE in a concentration range of 1–2000 pg/ml from peripheral blood samples with a substantial meaningful change of 0.5 pg/ml and the expected standard deviation of IgE concentration is assumed to be 0.01 pg/ml based on findings by Korn . For this randomized controlled trial design, the formula is:
All parameters were assumed as follows: mean change of IgE in intervention group = 10 pg/ml; mean change of IgE in control group = 0 pg/ml; α = 0.05; β = 0.20; δ = 10 pg/ml; s = 7; and s2 = 49.
We calculated and found the value of N = 17.71. This estimate requires 18 patients, to obtain 80% statistical power with 5% significance level for an independent samples and paired t-test. Due to the nature of this study being a pilot study, we aimed to recruit a minimal of 22 subjects in order to compensate for an estimate of 20% drop outs.
Study participants are recruited by direct invitation and pamphlets from pediatric allergy-immunology clinic at Dr. Soetomo General Hospital (Indonesia). Data collectors reviewed the clinic appointment schedules to identify subjects with HDM allergy and met with subjects and their parents or families after their physician visit for a screening to assess their eligibility. Patients were enrolled after considering various inclusion and exclusion criteria such as mentioned in (Table 1). By total sampling, 32 subjects were obtained from this hospital. They were prescreened from January 31 to February 28, 2017 at pediatric allergy-immunology clinic at Dr. Soetomo General Hospital (Indonesia). Based on the inclusion and exclusion criteria mentioned in (Table 1), 22 subjects were eligible to participate in the study. Informed consent is given as a voluntary agreement to participate in this research, out of which, 20 parents agreed and 2 parents declined to participate in the study.
|Inclusion Criteria||Exclusion Criteria|
|Children aged 6–16 years at any gender and ethnic||Any sign of allergic diseases (asthma, hay fever, food allergy, eczema) within 1 month before observation|
|Have been diagnosed with HDM allergies by positive skin-prick test||Taking any antihistamines or steroid within 1 month before observation|
|Periodontally healthy children without any sign of chronic gingival diseases||Experienced dental scaling and root planning within 6 months before observation|
|Positive culture of ||The presence of low-grade fever due to infections rather than gingivitis/periodontitis|
|Understand and able to cooperate to the research protocol||Recent blood disorders or congenital abnormalities|
|Parents or legal representatives have signed the written consent in accordance with our institutional policies||Any medical conditions that may be harmful to be involved in this study, in which phlebotomy is contra-indicated|
2.5 Data collection
Data were collected by taking venous puncture for measuring total serum IgE and IgG4 from subjects’ blood samples and taking saliva for measuring lipopolysaccharide of
We used a simple sample-processing method for PCR to detect
2.6 Statistical analysis
The obtained data were tabulated and analyzed using Statistical Package of Social Science (SPSS version 17, IBM, New York, USA). First, univariable linear regression procedures were conducted to examine associations between circulatory Ig-E and all determinants. In advance, dummy variables were created for all categorical determinants. Second, a multiple linear regression analysis with a stepwise exclusion method was conducted with all continuous and dummy variables. Determinants that seemed relevant for prediction of activation were kept in the model. Statistical significance was set at
A total of 20 periodontally healthy children accepted to participate in this study; 8 were males and 12 were females. The average age was 10.52 ± 2.78 years (range, 6–16 years). The patients’ general characteristics are as shown in Table 2.
3.1 Correlation between salivary LPS-Pg and Ig-E levels
Analysis of correlation between salivary LPS-Pg and Ig-E levels showed a strong correlation (r2 = 0.695;
3.2 Correlation between salivary LPS-Pg and Ig-G4 levels
Analysis of correlation between salivary LPS-Pg and Ig-G4 levels showed a very strong correlation (r2 = 0.796;
3.3 Results of univariate logistic regression analysis
As shown in Table 3, univariate logistic regression analysis uses gender, age, height, weight, BMI, family size, insurance status, number of colony-forming unit (CFU) of Pg, level of salivary LPS-Pg, level of circulatory Ig-G4 as the independent variables, and circulatory Ig-E as the dependent variable. Data revealed that number of colony-forming units (CFU) of Pg, level of salivary LPS-Pg, and level of circulatory Ig-G4 were associated with circulatory Ig-E (
|Age (years)||10.52 (2.78)||6||16|
|Height (cm)||140.28 (14.99)||108.50||162.00|
|Weight (kg)||36.12 (7.96)||23.00||51.00|
|BMI (kg/m2)||18.24 (1.92)||15.27||21.82|
|Family size (n)||4.70 (1.38)||3||8|
|Insurance status (n)|
|CFU-Pg (×105 CFU/ml)||9.93 (8.34)||1.08||29.45|
|LPS-Pg (μg/ml)||7.21 (3.06)||1.96||10.76|
|Ig-E (pg/ml)||114.44 (26.20)||77.69||141.41|
|Ig-G4 (ng/ml)||31.02 (9.09)||15.96||40.96|
3.4 Results of multivariable regression analysis
Multivariable regression analysis was performed using indexes which were demonstrated to be related to circulatory Ig-E in univariate analysis. The results showed that only level of salivary LPS-Pg and level of circulatory Ig-G4 remained a risk factor for circulatory Ig-E (Table 4).
Despite existing literature pointing to a potential role of lipopolysaccharide in modulating allergic reactions, it remains a relatively underresearched subject matter. Notably, the majority of existing research has only investigated the interactions between periodontitis and clinical symptoms of allergies . In this research, we had observed a direct correlation between lipopolysaccharide of
Lipopolysaccharide is a gram-negative endotoxin, ubiquitous in the environment and can therefore exacerbate allergic responses . Existing literature has demonstrated the pathogenic role of
As a unique endotoxin, lipopolysaccharide-driven inflammatory responses can induce a more pronounced pro-inflammatory cytokine response . At very low concentration, lipopolysaccharide may induce atopic inflammatory responses by Th-1 shifting into Th-2, which is more potent to stimulate antibodies production . Lipopolysaccharide binds to Toll-like receptor (TLR) 4 and greatly enhances the response of TLR4-transfected cells . Thus, damage from lipopolysaccharide extends beyond the exhaustion of host innate immunity .
Lipopolysaccharide activates macrophage via the TLR4/NF-κB pathway . In turn, TLR4 activation increases SOCS3 mRNA expression . Since SOCS3 is an inducible endogenous negative regulator of JAK/STAT pathway , therefore administration of lipopolysaccharide in a model of experimental periodontal disease will be correlated with dynamics of the atopic inflammatory reaction . Meanwhile, given the unique lipopolysaccharide-induced atopic inflammatory responses and lipopolysaccharide-triggered mast cell derived, we can predict that lipopolysaccharide of this periodontal pathogen may stimulate the level of circulatory Ig-E and Ig-G4, even in the healthy populations [36, 37].
In line with the hypothesis, our present study confirms the existence of a significant correlation between salivary lipopolysaccharide of
This study had several limitations. Firstly, this study had small sample size, limiting the generalizability of the results. Perhaps more observations with longer periods would have been statistically significant with a larger sample size. Secondly, the cross-sectional approach of this current study cannot draw any conclusions concerning direct relationships. Given the correlational nature of the analysis, we cannot clarify whether salivary lipopolysaccharide of
In conclusion, salivary lipopolysaccharide of
The authors thank all subjects and their parents for participating in this study. The authors also thank laboratory assistants and pediatric dentistry nurses at Dr. Soetomo General Hospital (Surabaya, Indonesia) for their technical support in this study.
Conflict of interests
All authors declare no conflict of interests in this study.
Availability of data and materials
Most of the experimental data acquired/analyzed during this study have been included in this published version. Information on rest of the data is available from the corresponding author on reasonable request.
Consent for publication
Ethics approval and consent to participate
This is the grant number for ethics approval (20/Panke.KKE/I/2017). A written informed consent was obtained from every parent to be included in the study. This study was approved by Institutional Review Boards of Dr. Soetomo General Hospital Ethics Committee for Health Research at Surabaya, January 20, 2017. Informed consent was validated by the Institutional Review Boards of Dr. Soetomo General Hospital Ethics Committee for Health Research. All parents provided written informed consent to participate and were free to decline.
SCN designed the study and helped to draft the manuscript. RAN analyzed the data and initially wrote the manuscript. AE participated in the biochemical analysis. YDS participated in the preparation of the materials and carried out the experimental studies. HU participated in the figures preparation and analysis of data. SK, UT, and SP analyzed the data and critically revised the manuscript. All authors read and approved the final manuscript.
This study was supported partly by Faculty of Dental Medicine Universitas Airlangga Health Research Projects. The funding agencies had no role in data collection, analysis, interpretation, or the decision to submit the results.