Relationship between the salivary concentrations of proteinase-3 and interleukin-8 and severe early childhood caries.

BACKGROUND: Severe early childhood caries (S-ECC) is a multifactorial transmissible infectious disease continuing to affect infants and toddlers worldwide. Saliva plays a modulatory role in the pathogenesis of dental caries. OBJECTIVES: The present study aimed to assess the salivary levels of proteinase-3 (PR3) and interleukin-8 (IL-8) as pro-inflammatory cytokines related to the function of neutrophils in association with S-ECC and its treatment. MATERIAL AND METHODS: Fifty children aged 36-60 months were recruited (25 caries-free controls and 25 S-ECC patients). Saliva sampling was performed in all participants. In the S-ECC group, sampling was repeated 6-8 weeks after restorative treatment. The salivary concentrations of PR3 and IL-8 were determined using the enzyme-linked immunosorbent assay (ELISA). The χ2 test, Fisher's exact test, the independent t test, and the paired t test were applied at p < 0.05. RESULTS: The baseline salivary concentrations of PR3 and IL-8 in the S-ECC group were significantly higher than in the caries-free group (p < 0.001). A significant reduction occurred in the levels of these cytokines following restorative treatment in the S-ECC group (p < 0.001), although they were still significantly higher than in the caries-free group (p < 0.05). CONCLUSIONS: The salivary levels of PR3 and IL-8 were significantly affected by the presence of dental caries in children, implying their potential efficiency as non-invasive indicators in the determination of the caries risk and treatment effectiveness.

Relationship between the salivary concentration of matrix metalloproteinases 8 and 20 and severe early childhood caries.

BACKGROUND: Dental caries is initiated through mineral dissolution by bacterial acids and collagen degradation by endogenous proteolytic enzymes, mainly collagenolytic matrix metalloproteinases (MMPs). OBJECTIVES: The present research aimed to evaluate the relationship between severe early childhood caries (S-ECC) and salivary MMP-8 and MMP-20 concentrations. MATERIAL AND METHODS: Fifty children aged 36-60 months were assigned to either the caries-free (control) group or the S-ECC group. Standard clinical examinations were performed, and approx. 1 mL of expectorated unstimulated whole saliva was collected from all participants. In the S-ECC group, the sampling was repeated 3 months after restorative treatment. All samples were analyzed for the salivary concentrations of MMP-8 and MMP-20, using the enzyme-linked immunosorbent assay (ELISA). Statistical analysis employed the t test, the Mann-Whitney U test, the χ2 test, Fisher's exact test, and the paired samples t test. The level of significance was set at 0.05. RESULTS: At baseline, the subjects in the S-ECC group presented with significantly elevated levels of MMP-8 as compared to the control group. However, the salivary concentration of MMP-20 did not exhibit a significant difference between the 2 groups. A significant reduction occurred in the levels of MMP-8 and MMP-20 3 months after restorative treatment in the S-ECC group. CONCLUSIONS: The salivary levels of MMP-8 and MMP-20 were significantly affected by dental restorative treatment in children. Furthermore, MMP-8 was observed to be a better indicator of the dental caries status than MMP-20.

Effects of Lipopolysaccharide from Porphyromonas gingivalis and Escherichia coli on Gene Expression Levels of Toll-like Receptors and Inflammatory Cytokines in Human Dental Pulp Stem Cells.

BACKGROUND: Periodontal diseases originate from a group of oral inflammatory infections initiated by oral pathogens. Among these pathogens, Gram-negative bacteria such as p. gingivalis play a major role in chronic periodontitis. P. gingivalis harbours lipopolysaccharide (LPS) which enables it to attach to TLR2. OBJECTIVES: Evaluating the effects of P. gingivalis and E. coli LPS on the gene expression of TLRs and inflammatory cytokines in human dental pulp stem cells (hDPSCs). METHODS: We evaluated the expression level of TLR2, TLR4, IL-6, IL-10, and 1L-18 in hDPSCs treated with 1µg/mL of P. gingivalis lipopolysaccharide and E. coli LPS at three different exposure times using Real-time RT-PCR. RESULT: The test group treated with P. gingivalis LPS showed a high level of TLR4 expression in 24 hours exposure period and the lowest expression in 48 hours of exposure time. In the case of IL-10, the lowest expression was in the 24 hours exposure period. Although in the E.coli LPS treated group, IL-10 showed the highest expression in 24 and lowest in 48 hours exposure period. Moreover, IL-18 in P. gingivalis LPS treated group showed a significant difference between 6, 24, and 48-time periods of exposure, but not in the E. coli LPS treated group. CONCLUSION: Both types of LPS stimulate inflammation through TLR4 expression. P. gingivalis LPS performs more potentially than E. coli in terms of stimulating inflammation at the first 24 hours of exposure. Nevertheless, our study confirmed that increasing P. gingivalis and/or the E.coli LPS exposure time, despite acting as an inflammatory stimulator, apparently showed anti-inflammatory properties.

Evaluating the effect of LPS from periodontal pathogenic bacteria on the expression of senescence-related genes in human dental pulp stem cells.

The human dental pulp stem cells (hDPSCs) are one of the readily available sources of multipotent mesenchymal stem cells (MSCs) and can be considered as a type of tool cells for cell-based therapies. However, the main limitation in the clinical use of these cells is DPSC senescence, which can be induced by lipopolysaccharide (LPS) of oral pathogenic bacteria. Up to now, far little attention has been paid to exploring the molecular mechanisms of senescence in DPSCs. So, the current study aimed to investigate the underlying molecular mechanism of senescence in hDPSCs stimulated with Porphyromonas gingivalis (P. gingivalis) and Escherichia coli (E. coli)-derived LPSs, by evaluating both mRNA and protein expression of four important senescence-related genes, including TP53, CDKN1A, CDKN2A and SIRT1. To this purpose, hDPSCs were stimulated with different LPSs for 6, 24 and 48 h and then the gene expression was evaluated using quantitative real-time polymerase chain reaction (qPCR) and western blotting. Following stimulation with P. gingivalis and E. coli-derived LPSs, the relative mRNA and protein expression of all genes were significantly up-regulated in a time-dependent manner, as compared with unstimulated hDPSCs. Moreover, the hDPSCs stimulated with P. gingivalis LPS for 6 and 24 h had the highest mRNA expression of CDKN1A and SIRT1, respectively (p < 0.0001), whereas the highest mRNA expression of CDKN2A and TP53 was seen in hDPSCs stimulated with E. coli LPS for 48 h (p < 0.0001). In summary, because DPSCs have been reported to have therapeutic potential for several cell-based therapies, targeting molecular mechanisms aiming at preventing DPSC senescence could be considered a valuable strategy.

Comparison of Gene Expression of Different Isoforms of Osteopontin in Symptomatic Irreversible Pulpitis of Human Dental Pulp.

Introduction: Osteopontin (OPN), plays an important role in immune system modulation. OPN can activate osteoclasts, thus causing resorption of bone. In addition, it might have a protective function against polymicrobial endodontic infections. Since different isoforms of OPN might have diverse roles, the aim of the present study was to compare gene expression of different isoforms of osteopontin in symptomatic irreversible pulpitis and normal pulps of the human dental pulp. Materials and Methods: Pulps were taken from 20 teeth with symptomatic irreversible pulpitis as the case group and from 20 intact premolars scheduled for extraction as the control group. After RNA extraction and synthesis of complementary DNA (cDNA), quantitative real-time polymerase chain reaction (PCR) was used for the evaluation of gene expression of OPN, OPN2 and OPN3. The Mann-Whitney U, t and Chi-square tests were used to analyze differences between the groups. Results: Mean values of OPN, OPN2 and OPN3 in normal pulps were 0.695±0.295, 0.656±0.298 and 0.816±0.422, respectively. Mean values of OPN, OPN2 and OPN3 in symptomatic irreversible pulpitis were 2.52±1.82, 1.99±0.899 and 1.816±0.954, respectively. Unlike OPN and OPN2, OPN3 exhibited significantly higher expression in normal pulps (P<0.05). Conclusion: The results of the present case- control study showed that some variants of OPN are upregulated during pulpitis and it might be due to their prominent modulatory roles in dental pulps.

Evaluation of NLRP3 and IL-18 Levels after Periodontal Therapy.

The progression of periodontitis depends on interactions between the periodontal pathogens and the host immune cytokines, including interleukin (IL)-1ß and IL-18. Production of IL-1ß is regulated by NOD-like receptors family pyrin domain containing 3 (NLRP3). This study aimed to evaluate the effect of periodontal treatment on the concentrations of IL-18 and NLRP3 in patients with chronic periodontitis. In this experimental study, 18 patients with chronic periodontitis and a mean age of 46.2±8.95 years, were included. The gingival crevicular fluid (GCF) was collected at the beginning of the study, 4 weeks after non-surgical (phase I), and 4 weeks after surgical periodontal treatment. The levels of NLRP3 and IL-18 were measured; using an enzyme-linked immunosorbent assay. Pearson correlation test was used to analyze the concentration of NLRP3 and IL-18 before and after the treatments with CAL and PD. There was a significant association between the level of NLRP3 and the mean values of PD and CAL before treatment. After each treatment phase, a significant decrease was observed in the NLRP3 level. There was no significant relationship between IL-18 and clinical parameters before and after periodontal treatments. Given the possible association between the level of NLRP3 and clinical parameters, we suggest it as a possible indicator of inflammation in chronic periodontitis and an index for evaluating the treatment outcome.

The expression levels of MicroRNA-146a, RANKL and OPG after non-surgical periodontal treatment.

OBJECTIVE: MicroRNA-146a (miR-146a) is a regulator of inflammatory response. Periodontitis is a disease with immune pathophysiology of the periodontium in which the inflammation results in the destruction of the soft tissues and alveolar bone. Therefore, the aim of this study was to investigate the expressions of miR-146a, OPG, and RANKL in diseased and healthy periodontal tissues to understand whether miR-146a expression level may associate with OPG and RANKL mRNA levels and OPG/RANKL ratio after non-surgical periodontal treatment. METHODS: The levels of miR-146a, RANKL, and OPG in gingival tissues from patients with generalized periodontitis stages II and III and grades A and B (n = 15, group A), patients with generalized periodontitis stages III and IV and grade C (n = 15, group B), and healthy individuals (n = 10) were determined by real-time PCR. The associations of miR-146a expression with OPG and RANKL levels were evaluated. RESULTS: The levels of miR-146a in two subgroups within periodontitis patients were significantly higher than healthy subjects (P < 0.0001). MiR-146a showed the increased level in group A of patients compared with group B (P < 0.05). Clinical parameters such as probing depth (PD) and clinical attachment loss (CAL) were significantly higher in patients than control group (P < 0.05). The levels of OPG and RANKL were increased in patients compared with healthy subjects, although the elevated levels were not statistically significant. MiR-146a was not associated with OPG and RANKL levels and OPG/RANKL ratio. CONCLUSIONS: The results of this study failed to show the associations of miR-146a level with OPG and RANKL levels and OPG/RANKL ratio in periodontitis after non-surgical periodontal treatment.

Study of the immunomodulatory effects of osteogenic differentiated human dental pulp stem cells.

BACKGROUND AND AIMS: Dental pulp stem cells (DPSC) are promising tools in regenerative medicine due to their differentiation potential and immunomodulatory properties. However, it is not clearly known whether or not DPSCs maintain their immunosuppressive effects after differentiation. In the present study, we examined the immunomodulatory effects of osteogenic differentiated DPSCs (OD-DPSCs). METHODS: OD-DPSCs and undifferentiated DPSCs were co-cultured with allogenic PBMCs in different ratios and the proliferation of the PBMCs was measured. The concentration of IL-10, TGF-ß, PGE2, IL-6, and NO were then examined. Moreover, the expression of IDO, HLAG, and HGF genes were determined in undifferentiated and OD-DPSCs. FINDINGS: The results showed that OD-DPSCs could inhibit the proliferation of allogenic PBMCs. The levels of PGE2, IL-6, and TGF-ß anti-inflammatory cytokines increased after the co-culture. Moreover, the levels of NO increased during the differentiation process and the expression of IDO, HLAG, and HGF genes remained unchanged after osteogenic differentiation. SIGNIFICANCE: Although, there were some differences between the OD-DPSCs and undifferentiated DPSCs in terms of their cytokine and NO production, undifferentiated DPSCs maintained their immunomodulatory activities upon differentiation.

Evaluation of STAT1 and Wnt5a gene expression in gingival tissues of patients with periodontal disease.

Periodontal disease is a common chronic inflammatory disease of the oral cavity. This disease occurs as a consequence of uncontrolled inflammatory immune responses against periodontopathic bacteria. Several studies have documented the proinflammatory roles of the Signal Transducer and Activator of Transcription 1 (STAT1) and Wnt5a in inflammatory diseases. However, there has been no detailed investigation of STAT1 and Wnt5a genes expression in periodontal disease. So, we aimed to evaluate the expressions of STAT1 and Wnt5a in patients with chronic and aggressive periodontitis and determine their correlation with clinical parameters. Three groups of subjects were enrolled including control (20 healthy individuals), chronic (25 patients), and aggressive periodontitis patients (25 patients). The expressions of STAT1 and Wnt5a were evaluated in gingival tissue samples using a Real-time polymerase chain reactions assay. The expressions of STAT1 and Wnt5a were significantly upregulated in chronic and aggressive periodontitis compared with the healthy control. We also found that the expressions of STAT1 and Wnt5a increased in aggressive periodontitis compared with chronic periodontitis. In addition, there was the linear relationship between the expression of STAT1 and Wnt5a and the clinical parameters, including clinical attachment loss and periodontal pocket depth. A linear relationship between the expressions of Wnt5a and the clinical parameters was also identified. Taken together, our findings highlight the roles of STAT1 and Wnt5a in the pathogenesis of the periodontal inflammation, suggesting these molecules as valuable therapeutic targets.

Association of CD26/dipeptidyl peptidase IV mRNA level in peripheral blood mononuclear cells with disease activity and bone erosion in rheumatoid arthritis.

Dipeptidyl peptidase IV (DPP-IV, CD26) plays many roles in the pathogenesis of several autoimmune and inflammatory diseases. The current study evaluated the association of DPP-IV enzymatic activity and its gene expression with disease activity and bone erosion in rheumatoid arthritis. Blood samples were collected from 20 rheumatoid arthritis patients and 40 healthy volunteers. Patients were divided into four subgroups using DAS28 index. CD26 gene expression levels were analyzed in peripheral blood mononuclear cells by quantitative reverse transcription-polymerase chain reaction. Additionally, the enzymatic activity of this molecule in serum was determined using Gly-Pro-p-nitroanilide as substrate. Digital radiography was applied to obtain images for bone erosion assessment. No significant difference in serum DPP-IV activity level was seen between patients and controls (p = 0.140). However, patients exhibited an increase in CD26 mRNA expression (1.68 times) when compared to controls (p = 0.001). Moreover, a strong positive correlation between CD26 gene expression and DAS28 index as well as bone erosion in the hands was observed (r = 0.71, p = 0.002 and r = 0.61, p = 0.049, respectively). This study demonstrated that CD26 mRNA expression in rheumatoid arthritis patients is associated with disease activity and bone erosion, suggesting a potential role for this molecule in the immunopathology of rheumatoid arthritis and bone erosion.

Interleukin-17 and interleukin-23 levels in gingival crevicular fluid of patients with chronic and aggressive periodontitis.

INTRODUCTION: Interleukin-17 is a pro-inflammatory cytokine with a wide range of protective and destructive effects in periodontitis. The role of IL-23 is stabilisation and expansion of Th-17. The aim of this study was to assess whether patients with aggressive and chronic periodontitis exhibit different gingival crevicular fluid (GCF) concentrations of IL-17 and IL-23 compared with clinically healthy subjects. MATERIAL AND METHODS: GCF samples were obtained from 32 patients: 10 with chronic periodontitis (CP), 12 with aggressive periodontitis (AgP), and 10 healthy controls (HC). IL-23 and IL-17 concentrations were measured using enzyme-linked immunosorbent assay (ELISA). Comparison of study groups was performed with ANOVA and Tukey HSD tests. Spearman's correlation coefficient was used to assess correlations between the variables. RESULTS: IL-17 concentration was significantly higher in the healthy group compared to the AgP and CP groups (p < 0.001), but there were no significant differences between the CP and AgP groups. IL-23 levels in the healthy group were significantly higher than that in the AgP group (p < 0.001). Cytokine concentrations did not correlate significantly with probing depths and clinical attachment levels. CONCLUSIONS: Gingival crevicular fluid concentrations of IL-17 and IL-23 were significantly higher in the healthy group compared to periodontitis groups.

Effect of Periodontal Treatment on the Crevicular Level of High-mobility Group Box 1 and Soluble Triggering Receptor Expressed on Myeloid Cells 1 in Patients with Chronic Periodontitis.

The present study aimed to compare the levels of high-mobility group box 1(HMGB1) and soluble triggering receptor expressed on myeloid cells (sTREM1) in the gingival crevicular fluid (GCF). This cross-sectional cohort trial investigated two groups of 22 eligible chronic periodontitis and 22 periodontally healthy individuals (student volunteers) both before and after the periodontal treatment.  GCF was collected from the deepest pockets with clinical attachment loss≥3 mm. Both groups received oral hygiene instructions, and scaling and root planning were performed in the test group. Enzyme-linked immunosorbent assay kit (ELISA) was used to measure the levels of HMGB1 and sTREM1 in GCF samples collected before and 1 month after non-surgical periodontal treatment. The results showed that HMGB1 levels were significantly higher in the chronic periodontitis patients than those of the healthy individuals before treatment (p<0.02) and decreased significantly after periodontal treatment, which reduced gingival inflammation. Furthermore, the levels of sTREM1 marker were significantly higher in periodontitis patients before (p<0.001) and 1 month after treatment than in healthy individuals (p<0.003) although its crevicular levels decreased after periodontal therapy in periodontitis group. The higher levels of sTREM1 and HMGB1 cytokines in GCF of periodontitis patients and the significant decrease after the introduction of the periodontal treatment underlines the importance of HMGB1 and sTREM1 in pathogenesis of periodontitis.

Relationships between High-mobility Group Protein B1 and Triggering Receptor Expressed on Myeloid Cells Concentrations in Gingival Crevicular Fluid and Chronic Periodontitis.

One of the inflammatory mediators which is secreted by inflammatory cells is high-mobility group protein B1 (HMGB1). Interaction of HMGB1 and toll-like receptors (TLRs) leads to increased production of inflammatory cytokines. On the other hand, it was shown that triggering receptor expressed on myeloid cells (TREM-1) also can be activated by TLRs, and its soluble form (sTREM-1) can be formed by cleaving of membrane-bound form of TREM-1 proteinases. Since there is not enough knowledge about the precise role of HMGB1 and sTREM-1 in periodontal diseases, the aim of this study was to evaluate the concentration of HMGB1 and sTREM-1 in gingival crevicular fluid (GCF) samples of patients with chronic periodontitis. Gingival crevicular fluid (GCF) samples were obtained from a total of 24 individuals with clinically healthy gingiva and 24 patients with moderate to severe chronic periodontitis. For collecting GCF samples, periopapers were placed at the entrance of the crevice and left in position for 30 seconds. Then, they were stored at -80°C. Enzyme-linked immunosorbent assay (ELISA) was used for measuring the concentration of HMGB1 and sTREM-1 in GCF samples. The concentration of HMGB1 (p<0.001) and sTREM-1 (p<0.017), was significantly higher in chronic periodontitis group. In addition, there was a significant positive correlation between HMGB1 and sTREM-1 concentration in chronic periodontitis group (p<0.05). We also found significant positive correlation between PD (Pocket depth) and the concentration of HMGB1 (p<0.001) and sTREM-1 (p<0.015). It is concluded that both HMGB1 and sTREM-1 are released during the inflammatory response of periodontal tissues and they can promote inflammatory process, which leads to tissue destruction.

Effect of platelet-rich plasma concentrations on the proliferation of periodontal cells: An in vitro study.

OBJECTIVE: The purpose of this study was to evaluate the effect of different concentrations of platelet-rich plasma (PRP) on the proliferation of undifferentiated periodontal ligament (PDL) fibroblasts. MATERIALS AND METHODS: The undifferentiated PDL fibroblasts were obtained from two healthy human premolar teeth and cultured in Dulbecco's modified Eagle's medium. Cell wells were divided into five groups. Experimental groups received 0.1%, 5%, or 50% PRP; the positive and negative control groups were cultured in fetal bovine serum (FBS) 12% and in a medium without FBS 12%, respectively. The plates were incubated at 37°C for 1, 2, 3, 4, and 7 days. PDL cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide proliferation assay. Statistical analysis of the data was accomplished using repeated measure ANOVA and Tukey's test. P < 0.05 was considered statistically significant. RESULTS: The 5% PRP had the greatest effect on undifferentiated fibroblast proliferation, which was significant on the 3rd day. There was no significant difference between 0.1% PRP and positive control during the first 3 days. The group with 50% PRP presented significantly lower proliferation, compared to other experimental and control groups. CONCLUSIONS: It may be concluded that the growth-stimulating effect of PRP is dose dependent with the best results in low concentrations.

Evaluation of MicroRNA-146a and Its Targets in Gingival Tissues of Patients With Chronic Periodontitis.

BACKGROUND: MicroRNAs (miRNAs) are a group of small non-coding RNAs that play an important role in the regulation of gene expression. miRNA-146a (miR-146a), a member of the miR-146 family, is involved in the control of inflammation. Periodontitis is a set of chronic inflammatory disorders of the tissues surrounding the teeth that lead to the breakdown of alveolar bone and tooth loss. In this study, expression levels of miR-146a and its targets, including tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6, are evaluated in human patients with chronic periodontitis (CP). METHODS: The study population consisted of 10 healthy controls and 20 individuals with CP. For each participant, clinical parameters including probing depth and clinical attachment level were measured, and a gingival tissue sample was collected. Levels of miR-146a, TNF-α, IL-1ß, and IL-6 were quantified using real-time polymerase chain reaction. RESULTS: Levels of miR-146a were significantly higher in patients with CP (P <0.001). There was a positive correlation between levels of miR-146a and clinical parameters (P <0.05). Elevated miR-146a was accompanied by a significant reduction in TNF-α and IL-6 (P <0.001). CONCLUSIONS: Patients with CP had higher levels of miR-146a than healthy individuals, accompanied by reduced levels of TNF-α and IL-6. A positive relationship between miR-146a levels and clinical parameters suggests a pathophysiologic role of miR-146a in CP.

Relationship of Salivary Lactoferrin and Lysozyme Concentrations with Early Childhood Caries.

Background and aims. Lysozyme and lactoferrin are salivary proteins which play an important role in innate defense mechanisms against bacteria. This study investigated the association of salivary lysozyme and lactoferrin concentrations with early childhood caries (ECC). Materials and methods. This study was carried out on 42 healthy children (age range, 36 to 71 months), of whom 21 were caries free (CF) and 21 had ECC. Disposable needle-less syringes were used to collect unstimulated saliva from buccal and labial vestibules. Fifteen children who had ECC were treated completely and their saliva was collected in the same way for the second time, three months after treatment. Lysozyme and lactoferrin concentrations were measured and recorded by the ELISA method. The intergroup comparisons were carried out using chi-square, Student's t-test and Wilcoxon signed ranked test. A P-value less than 0.05 was considered as statistically significant. Results. The mean concentration of lysozyme was significantly higher in CF group compared with that of ECC group (P = 0.04). Although the mean concentration of lactoferrin in ECC group was higher in comparison with ECC group, the difference was not statistically significant (P = 0.06). After dental treatment, the mean concentrations of lysozyme and lactoferrin did not change in comparison with their concentrations before treatment. Conclusion. ECC may have a relationship with lower concentrations of unstimulated salivary lactoferrin and lysozyme and reduced amounts of these two salivary proteins may be a risk factor for dental caries in children.

Correlation between salivary Toll like receptor-2 concentration and early childhood caries.

BACKGROUND: Early childhood caries (ECC) is a common health problem in developing countries. Basic knowledge about etiology and pathogenesis of ECC plays an important role in its prevention. OBJECTIVE: To determine the relationship between salivary TLR-2 concentration and early childhood caries formation. METHODS: Twenty-Eight children with ages ranging from 36 to 71 months (15 in ECC group and 13 in caries free group) were chosen based on inclusion criteria. Their saliva was aspirated in the volumes of 1-2 ml. Resampling was done for 8 subjects of ECC group 3 months after dental restoration. TLR-2 concentration was measured using ELISA. RESULTS: Mean concentrations of TLR-2 in ECC and caries free group were 2.12 and 1.42 ng/ml, respectively. The difference between concentrations was statistically significant (p=0.008). Three months after treatment in 8 ECC, the mean concentration of TLR-2 (0.925 ng/ml) significantly decreased compared to the original concentration in ECC (p<0.001) and caries free groups (p<0.001). CONCLUSION: Elevated concentration of TLR-2 in ECC group compared to caries free group and its decrease after treatment point to the participation of innate immune system and specially TLR-2 in the pathogenesis of early childhood caries.

Recombinant Leishmania tarentolae encoding the HPV type 16 E7 gene in tumor mice model.

BACKGROUND: Cervical cancer, the third most prevalent cause of cancer in women worldwide, is associated with HPVs. The critical role of E7 protein in HPV-related malignancies has designated it as a strong contender for generating vaccines against HPV. MATERIALS & METHODS: In this study, we developed a novel live vaccine using recombinant Leishmania tarentolae expressing E7-green fluorescent protein (GFP) fusion protein for the protection of mice against HPV-associated tumors. In order to transfect L. tarentolae with E7-GFP fusion construct, pLEXSY-neo2 system was applied. Followed by PCR, fluorescence imaging and fluorescence-activated cell sorting analysis, integration of E7-GFP gene into parasites genome was confirmed. A comparative study of six groups of C57BL/6 mice was performed to analyze antigen-specific humoral and cellular immune responses against E7 encoding live and DNA vaccines. Furthermore, the anti-tumor protective effect of L. tarentolae-E7-GFP was compared to other vaccination strategies, namely pcDNA-E7 as the DNA vaccine and pcDNA-E7/L. tarentolae-E7-GFP as the prime-boost regimen. RESULTS: We found that E7-GFP expressing recombinant L. tarentolae induces significant levels of IgG2a and IFN-γ, while there is no significant IL-5 production compared with that of other strategies and control groups before and after challenge with TC-1 tumor cells. It is noteworthy that the designed live vaccine showed the best protection and minimum tumor size among all groups against TC-1-induced tumors. CONCLUSION: Overall, the results obtained revealed that the E7-GFP recombinant L. tarentolae could be a potential live vaccine for induction of immune responses in vivo.

Receptor activator of nuclear factor kappa B ligand and osteoprotegerin levels in gingival crevicular fluid.

BACKGROUND: OSTEOCLASTOGENESIS IS COORDINATED BY THE INTERACTION OF THREE MEMBERS OF THE TUMOR NECROSIS FACTOR (TNF) SUPERFAMILY: Osteoprotegerin (OPG)/receptor activator of nuclear factor kappa B ligand (RANKL)/receptor activator of nuclear factor kappa B (RANK). The aim of this study was to investigate RANKL and OPG levels, and their relative ratio in gingival crevicular fluid (GCF) of patients with chronic and aggressive periodontitis, as well as healthy controls. MATERIALS AND METHODS: In this analytical study, GCF was obtained from healthy (n = 10), mild chronic periodontitis (n = 18), moderate chronic periodontitis (n = 18), severe chronic periodontitis (n = 20), and generalized aggressive periodontitis (n = 20) subjects. RANKL and OPG concentrations were measured by enzyme-linked immunosorbent assay. Statistical tests used were Kruskal-Wallis test, Mann-Whitney U rank sum test, and Spearman's rank correlation analysis. The level of statistical significance was set at P < 0.05. RESULTS: Mean RANKL concentration showed no statistically significant differences between groups (P = 0.58). There were also no significant differences between mean OPG concentration in the five groups (P = 0.0.56). Moreover, relative RANKL/OPG ratio did not reveal a significant difference between the three study group subjects: healthy, chronic periodontitis (mild, moderate, severe), and aggressive periodontitis (P = 0.41). There was statistically significant correlation between the concentration of sRANKL and Clinical Attachment Level (CAL) in moderate chronic periodontitis patients (R = 0.48, P = 0.04). There was also negative correlation between OPG concentration and CAL in moderate chronic periodontitis patients, although not significant (R = -0.13). CONCLUSION: RANKL was prominent in periodontitis sites, especially in moderate periodontitis patients, whereas OPG was not detectable in some diseased sites with bleeding on probing, supporting the role of these two molecules in the bone loss developed in this disease.

Immunomodulatory activities of chitin microparticles on Leishmania major-infected murine macrophages.

BACKGROUND: Chitin microparticles (CMPs) are found to be potent macrophage stimulators; however, their immunomodulatory effects on the parasite-infected macrophages have not yet been studied. To address this issue, we used a Leishmania major-infected murine macrophage model and characterized the regulatory effects of CMPs on the parasite-infected cells. METHODS: Mouse peritoneal macrophages were prepared and infected with L. major (MRHO/IR/1975/ER) standard strain. Following cell treatment with CMPs (500 µg/mL) for 48 h, percent of infected macrophages was determined by Giemsa staining and compared with untreated cells. To find the potential mechanisms of the activity of CMPs, TNF-α and accumulated nitrite in the culture supernatants of the treated and untreated cells were also measured by ELISA and colorimetric Griess assays, respectively. RESULTS: According to the obtained results, chitin microparticles reduced the ex vivo parasite infectivity by ∼12%. However, this inhibitory effect was not directly related to the increased biosynthesis and release of nitric oxide (NO) by macrophages. Instead, we observed a significant increase in the level of TNF-α secretion due to cell treatment with CMPs. Interestingly, this overexpression of TNF-α did not impair cell viability, suggesting the anti-apoptotic effects of the CMPs. CONCLUSIONS: These findings show that chitin microparticles have immunomodulatory effects on L. major-infected macrophages and further provide motivations for future studies on their in vivo effects.