Agnieszka Kozubska1, Janina Grzegorczyk2, Magdalena Konieczka3, *Joanna Szczepańska4
Analysis of the level of non-specific and specific immunity parameters in saliva of children with osteogenesis imperfecta and study of relationships between selected proteins, disease symptoms and sociodemographic factors
Analiza stężeń parametrów odporności nieswoistej i swoistej w ślinie dzieci z osteogenesis imperfecta oraz badanie zależności pomiędzy wybranymi białkami a objawami choroby i czynnikami socjodemograficznymi
1Doctoral studies, Department of Pediatric Dentistry, Medical University in Łódź, Poland
Head of Department: Professor Joanna Szczepańska, MD, PhD
2Immunology and Allergology Clinic, Medical University in Łódź, Poland
Head of Clinic: Professor Marek Kowalski, MD, PhD
3Department of Microbiology and Laboratory Medical Immunology, Medical University in Łódź, Poland
Head of Department: Dorota Pastuszak-Lewandoska, MD, PhD
4Department of Pediatric Dentistry, Medical University in Łódź, Poland
Head of Department: Professor Joanna Szczepańska, MD, PhD
Wstęp. Osteogenesis imperfecta towarzyszą charakterystyczne objawy ze strony układu stomatognatycznego, m.in. dentinogenesis imperfecta (DGI). Do czynników modyfikujących proces próchnicowy zaliczamy nawyki higieniczne pacjenta, dietę oraz działanie śliny i jej składowych. Obecne w ślinie o działaniu antybakteryjnym oraz immunomodulującym są elementy odporności nieswoistej – defensyny hBD-1, hBD-2, katelicydyna LL-37, lizozym oraz odporności swoistej – wydzielnicza immunoglobulina A.
Cel. Analiza stężeń defensyn hBD1 i hBD2, katelicydyny LL-37, lizozymu oraz sIgA w ślinie dzieci z osteogenesis imperfecta oraz w grupie porównawczej.
Materiał i metody. W latach 2015-1018 zbadano 62 pacjentów z wrodzoną łamliwością kości, od 30 z nich oraz 30 pacjentów z grupy porównawczej pobrano próbkę śliny niestymulowanej. Pomiar poziomu badanych czynników przeprowadzono, posługując się testami immunoenzymatycznymi Elisa. Przeprowadzono analizę statystyczną wyników badań laboratoryjnych. Różnice/zależności istotne statystycznie ustalono przy poziomie istotności p<0,05.
Wyniki. Zawartość badanych białek w ślinie nie różniła się istotnie statystycznie w obu badanych grupach. Stwierdzono istotne statystycznie dodatnie korelacje pomiędzy: wiekiem a stężeniem sIgA w ślinie badanych pacjentów; wiekiem a stężeniem hBD-1 i hBD-2 w ślinie dzieci z grupy porównawczej; wiekiem a stężeniem sIgA w ślinie pacjentów z grupy badanej i porównawczej. Dla grupy porównawczej dostrzeżono istotne statystycznie różnice w stężeniu sIgA w ślinie pomiędzy dziewczynkami i chłopcami.
Wnioski. Wyniki badań laboratoryjnych śliny pod kątem stężeń badanych białek wskazują na konieczność dalszych badań laboratoryjnych, które mogłyby wyjaśnić niskie poziomy próchnicy u dzieci z OI. Wzrost stężenia sIgA w ślinie wraz z wiekiem pacjentów (z OI i z grupy porównawczej) może świadczyć o dojrzewaniu układu odporności swoistej w miarę upływu lat lub może odzwierciedlać wpływ na ten parametr środowiska zewnętrznego.
Introduction. Osteogenesis imperfecta is characterized by dental symptoms, including dentinogenesis imperfecta (DGI). Carious process can be modified by factors such as patient’s hygiene habits, diet, saliva and its components. Saliva’s elements of non-specific immunity with antibacterial and immunomodulatory action are hBD1, hBD2 defensins, cathelicidin LL-37, lysozyme and specific immunity – secretory immunoglobulin A.
Aim. Analysis of levels of hBD1 and hBD2 defensins, cathelicidin LL-37, lysozyme and sIgA in saliva of children with osteogenesis imperfecta and in a comparative group of healthy children.
Material and methods. In the years 2015-2018, 62 individuals with brittle bone disease were examined, samples of non-stimulated saliva were collected from 30 patients and from 30 subjects of the comparative group. Levels of the examined parameters were measured using ELISA immunoassays tests. Statistical analysis of laboratory test results was carried out. Statistically significant differences/relationships were established at the significance level of p < 0.05.
Results. The concentration of the studied proteins in saliva did not differ significantly between the groups. There were statistically significant positive correlations between: age and sIgA concentration in saliva of the examined patients; age and concentration of hBD1 and hBD2 in saliva of children from the comparative group; age and sIgA concentration in saliva of patients from the study and comparative groups. For the comparative group, statistically significant differences in sIgA concentrations in saliva between girls and boys were observed.
Conclusions. Laboratory test results of hBD1 and hBD2 defensins, cathelicidin LL-37, lysozyme and sIgA levels in saliva indicate that there is a need for further laboratory tests, which could explain the low levels of caries index in children with osteogenesis imperfecta. The increased levels of sIgA in saliva with growing age of the patients (with OI and healthy children) may indicate that specific immunity system matures with age or may reflect the impact of external environment on this parameter.
Osteogenesis imperfecta (OI) is a hereditary disorder of the connective tissue caused by mutations in genes encoding collagen type I – COL1A1 and COL1A2, or in a small percentage in genes encoding proteins involved in collagen biosynthesis. Most often it is inherited in an autosomal dominant pattern. The phenotype of the disease ranges from mild to severe and even lethal. It is characterized by reduced mass and density of the bones, increased brittleness, which leads to repeated fractures even due to minor injuries and deformations of the long bones (1-3). Patients with osteogenesis imperfecta are also characterized by short stature, laxity of the joints, they may experience hearing impairment, blue sclerae and dental abnormalities. Characteristic features of their stomatognathic system include dentinogenesis imperfecta type I (DGI), malocclusion, lack of permanent tooth germs, ectopic eruption of permanent teeth (4, 5).
Tooth decay is a local, post-eruption, pathological process of external origin that causes decalcification of enamel, decay of tooth’s hard tissues and leads to tissue loss (WHO). It develops when components such as bacterial biofilm, carbohydrates supplied with food and the host’s susceptibility coexist in the appropriate period of time (6). The caries process can be modified by factors such as hygienic habits of the patient, diet and action of saliva and its components.
Elements with antibacterial and immunomodulatory properties present in saliva include: hBD1 defensins, hBD2, cathelicidin LL-37, lysozyme (non-specific immunity), and secretory immunoglobulin A (specific immunity). Defensins and cathelicidin LL-37 belong to antimicrobial peptides, they have bactericidal effect on Gram-negative and Gram-positive bacteria, they neutralize toxins, furthermore they have antiviral, antiparasitic and antifungal properties. Moreover, they exhibit a number of immunomodulatory functions, such as induction and inhibition of inflammatory process, influence on cell differentiation and chemotaxis. Cathelicidin LL-37, acting on the FPRL1 receptor, activates fDCs (follicular dendritic cells) to produce CXCL13 chemokine, which stimulates B lymphocytes through the CXCR5 receptor, further increasing production of BAFF activating factor. In this way, it increases proliferation of B lymphocytes and secretion of immunoglobulins, including immunoglobulin A. Secreted IgA prevents adhesion of microbes to mucosal epithelial cells, and by agglutinating removes them from the oral cavity, neutralizes microbial toxins, enzymes and viruses. Lysozyme is an enzyme that has bactericidal effect on Gram-positive and Gram-negative bacteria (7-10).
Type I of DGI, present in OI, is characterized by changes in tooth color, ranging from brown to opalescent blue or gray. There is often enamel loss as a result of abnormal dentin structure, exposure of dentin and its rapid abrasion to the level of gingiva, which could enhance the carious process (11).
Analysis of hBD1 and hBD2 defensins, cathelicidin LL-37, lysozyme and sIgA concentrations in saliva of children with osteogenesis imperfecta and in the comparative group.
Material and methods
In 2015-2018, 62 patients with OI, from Department of Paediatrics, Newborn Pathology and Metabolic Diseases of Bones of the Central Clinical Hospital – University Center of Paediatrics in Łódź were examined and subjected to interview and the physical examination, dmft/DMFT index was also calculated. The p/P component included changes with code 3 according to ICDAS II, when a localized lesion in the form of opaque or discolored enamel is visible, with no visible signs of dentin involvement, located in the place of stagnation of plaque and with codes 4, 5 and 6 as carious lesions with different advancement affecting the dentin. In 30 of them and in 30 patients of the Department of Paediatric Dentistry of Medical University of Łódź (the comparative group), laboratory tests of saliva were performed. The study included children aged from 3 to 16 years, whose legal guardian/parent consented to the planned procedures, moreover the study group included individuals with osteogenesis imperfecta, and the comparative group included children not affected by this disorder.
The laboratory part was performed at the Department of Microbiology and Laboratory Medical Immunology, Medical University of Łódź. In order to perform statistical analysis of the laboratory tests results, the patients were divided four times: by gender (girls vs boys); by presence or absence of dentinogenesis imperfecta; by severity of osteogenesis imperfecta (mild vs severe) and by presence or absence of OI (the comparative group) (tab. 1).
Tab. 1. Distribution of patients for statistical analysis
|Division of patients||Average age||Gender||Dentinogenesis imperfecta||Severity of the disease||Presence of OI|
|Girls||Boys||DGI||Without DGI||Mild||Severe||OI||Without OI|
|Number of people in the group||Study group||8 years and 6 months||15||15||13||17||19||11||30||–|
|Comparative group ||8 years and 7 months||16||14||–||–||–||–||–||30|
Thirty patients with osteogenesis imperfecta, in the morning hours, after breakfast, had a sample of unstimulated saliva in the amount of at least 2 cm3 collected into round-bottomed polypropylene tubes. The children spat out saliva directly into the test tube or, in case of difficulties, it was collected from the bottom of the oral cavity with a sterile syringe with simultaneous massage of the sublingual and submandibular areas. The procedure lasted about 5 minutes and depended on the degree of patient cooperation. The same procedure was performed, also in the morning, in the comparative group of 30 children who were patients of the Department of Paediatric Dentistry of Medical University of Łódź.
Within an hour after collection, the saliva samples were transported in a portable refrigerator at 5°C to the Department of Microbiology and Laboratory Medical Immunology, Medical University of Łódź. After proper preparation in accordance with instructions attached to diagnostic tests, the samples were stored in freezers at -70°C until tests were performed. The samples were defrosted before testing. The saliva samples required additional preparation by centrifugation – 3000 rpm for 10 minutes at 19°C, then the supernatant was poured into Eppendorf tubes. In order to measure levels of the studied agents, Elisa enzyme immunoassay tests were used. For analysis the following tests were used: 1) defensins (hBD1 and hBD2) – ELISA Kit for Beta-defensin 1 test and ELISA Kit for Beta-defensin 4A test from ElAab Science, Wuhan, China; 2) Cathelicidin LL-37 (CAMP) – ELISA Kit for Cathelicidin Antimicrobial Peptide (CAMP) test from Cloud-Clone Corp, San Jose, USA; 3) lysozyme – Lysozyme ELISA test from Immunodiagnostic, Hameenlinna, Finland; 4) secretory immunoglobulin A (sIgA) – Demeditec Diagnostics Secretory IgA test, Demeditec, Kiel, Germany. Altogether 300 determinations were made for 60 saliva samples. Spectrophotometric measurement of absorbance (OD) was performed with a Multiskan GO device (Thermo Scientific). Concentrations of agents was calculated on the basis of calibration curves. The results for hBD1 were given in pg/ml, for sIgA in μg/ml, for hBD2, CAMP and lysozyme in ng/ml.
For each tested parameter in saliva of the study group and the comparative group, the following measures of distribution were determined: arithmetic mean, median, range (min-max) and standard deviation. The Shapiro-Wilk and U Mann-Whitney tests were used for statistical analysis, and the Spearman’s rank correlation factor was used to assess a connection between two measurable features. Statistically significant differences/correlations were established at the significance level of p < 0.05. The results of laboratory tests were statistically analyzed by GRETL and Excel programs.
The values of the dmft/DMFT index for patients with OI are presented in table 2. In patients with dentinogenesis imperfecta, no signs of active caries were found (tab. 2). The dmft/DMFT rates were low in primary dentition, and they equaled for 3-year-old children: 0.14; for 5-year-old children: 0; for 7-year-old children: 0.2; in adolescents with permanent teeth: for 12-year-olds: 0; for children aged 15: 4.67.
Tab. 2. Value of dmft/DMFT index in children with OI compared to epidemiological data
|Age||dmft/DMFT in patients with OI||Epidemiological data (19)|
|Total (patients with DGI and without DGI)|| Patients with DGI|
| ||Primary dentition|
|3 years old||0.14||0||2.4 (2015)|
|5 years old||0||0||4.7 (2016)|
|7 years old||0.25||0||5.61 (2016)|
| ||Permanent dentition|
|12 years old||0||–||3.75 (2016)|
|15 years old||4.67||–||5.75 (2015)|
The mean value of sIgA in saliva was slightly higher in the comparative group (188.24 μg/mL) than in the study group (185.43 μg/mL). Mean values of hBD1 and hBD2 defensins were at a higher level in the study group than in the comparative group and equaled respectively: for hBD1 – 184.91 and 149.69 pg/mL and for hBD2 1.0962 and 0.98891 ng/mL. For cathelicidin LL-37, the mean value was higher in the study group – 3381.8 ng/mL, similarly, the mean value of lysozyme was higher in the study group – 2782.4 ng/mL. The obtained differences were not statistically significant (tab. 3).
Tab. 3. Measurements of distribution of the studied parameters in saliva in the study and comparative groups
| ||Study/comparative group||Arithmetic mean||Median||Minimum||Maximum||Standard deviation|
The Shapiro-Wilk test was used to check the normal distribution of a random variable. It was shown that the studied feature (proteins content in saliva) did not have normal distribution. Hence, the Mann-Whitney U test was used to determine whether the protein content in saliva in these two groups (the OI group and the comparative group) did not differ. The content (median) of the studied proteins in saliva – sIgA, hBD1, hBD2, CAMP and lysozyme did not differ statistically in both groups.
Relationship between age and the studied parameters
A positive correlation was found between age and concentration of sIgA in saliva of the studied patients (R Spearman = 0.544, p = 0.002), i.e. the concentration of sIgA in saliva increased with age. A similarly statistically significant positive correlation was observed between the age of children in the comparative group and the concentration of hBD1 defensin (R Spearman = 0.502, p = 0.005) and hBD2 defensin (R Spearman = 0.421, p = 0.02), i.e. the concentration in saliva increases with age for both hBD1 and hBD2. The results considered simultaneously for the study group and the comparative group showed a statistically significant positive correlation only between age and the concentration of sIgA in saliva (R Spearman = 0.365, p = 0.004) (tab. 4).
Tab. 4. Analysis of relationships between the studied parameters in saliva and age of the subjects
|Correlation between age and:||n||Rank correlation coefficient by Spearman||Statistical significance p|
|Study and comparative groups|
Relationship between the number of broken bones and the studied parameters
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