Molecular pathogenicity of 1-nonadecene and L-lactic acid, unique metabolites in radicular cysts and periapical granulomas.

Recently, 1-nonadecene and L-lactic acid were identified as unique metabolites in radicular cysts and periapical granuloma, respectively. However, the biological roles of these metabolites were unknown. Therefore, we aimed to investigate the inflammatory and mesenchymal-epithelial transition (MET) effects of 1-nonadecene, and the inflammatory and collagen precipitation effects of L-lactic acid on both periodontal ligament fibroblasts (PdLFs) and peripheral blood mononuclear cells (PBMCs). PdLFs and PBMCs were treated with 1-nonadecene and L-lactic acid. Cytokines' expression was measured using quantitative real-time polymerase chain reaction (qRT-PCR). E-cadherin, N-cadherin, and macrophage polarization markers were measured using flow cytometry. The collagen, matrix metalloproteinase (MMP)-1, and released cytokines were measured using collagen assay, western blot, and Luminex assay, respectively. In PdLFs, 1-nonadecene enhances inflammation through the upregulation of some inflammatory cytokines including IL-1ß, IL-6, IL-12A, monocyte chemoattractant protein (MCP)-1, and platelet-derived growth factor (PDGF) α. 1-Nonadecene also induced MET through the upregulation of E-cadherin and the downregulation of N-cadherin in PdLFs. 1-Nonadecene polarized macrophages to a pro-inflammatory phenotype and suppressed their cytokines' release. L-lactic acid exerted a differential impact on the inflammation and proliferation markers. Intriguingly, L-lactic acid induced fibrosis-like effects by enhancing collagen synthesis, while inhibiting MMP-1 release in PdLFs. These results provide a deeper understanding of 1-nonadecene and L-lactic acid's roles in modulating the microenvironment of the periapical area. Consequently, further clinical investigation can be employed for target therapy.

Tertiary Lymphoid Structures Are Related to Inflammatory Progression and Bone Loss in Human Apical Periodontitis.

INTRODUCTION: Bone loss is strongly associated with the immunologic milieu in apical periodontitis (AP). Tertiary lymphoid structures (TLSs) are organized lymphoid cell aggregates that form in nonlymphoid tissues under persistent inflammatory circumstances. To date, there has been no relevant report of TLSs in periapical lesions. This work aimed to investigate the formation and potential function of TLSs in AP. METHODS: Tissues from human apical lesions (n = 61) and healthy oral mucosa (n = 5) were collected. Immunohistochemistry and multiplex immunofluorescence were used to detect the formation of TLSs. Correlation analyses were performed between clinical variables and TLSs. In addition, immunohistochemistry was used to evaluate the expression of interleukin-1 beta, interleukin-6, receptor activator of nuclear factor kappa-B ligand, and macrophage subsets in the apical lesions. RESULTS: Periapical granulomas (n = 24) and cysts (n = 37) were identified by histologic evaluation. TLSs, composed of B-cell and T-cell clusters, developed in periapical granulomas and radicular cysts. The CXC-chemokine ligand 13, its receptor CXC-chemokine receptor 5, follicular dendritic cells, and high endothelial venules were localized in TLSs. The quantity and size of TLSs were positively associated with bone loss in AP. Moreover, proinflammatory cytokines and macrophage subsets were also substantially elevated in TLS regions of apical lesions. CONCLUSIONS: The formation of TLSs in periapical granulomas and cysts was closely associated with persistent immune responses and bone loss in apical lesions. TLSs provide an updated insight into the complicated immune response process in AP.

Immunohistochemical evaluation of cyclooxygenase-2 and mast cell density in periapical lesions.

AIM: Periapical granuloma (PG) and cyst (PC) are formed as a protective response consequent to pulpal infection leaching through the apical foramen and lateral canals. Various inflammatory mediators like mast cells and cyclooxygenase (COX)-2 are involved in this intricate process. This pilot study aimed to evaluate and compare the immunoexpression of tryptase and COX-2 in periapical granuloma and periapical cyst, and also correlate them with intensity of inflammatory infiltrate and thickness of cystic epithelial lining. METHODOLOGY: An observational and cross-sectional study was conducted on paraffin-embedded tissue sections of 50 PGs and 50 PCs submitted for morphological and immunohistochemical analysis using anti-tryptase and anti-COX-2 antibodies. The mean number of mast cells (total, granulated and degranulated), mean COX-2 expression and inflammatory score was calculated. The data obtained were analysed using Mann Whitney U, Student's T, Chi-square and Spearman correlation test (p < .05). RESULTS: The inflammatory score, total mast cells and COX-2 expression were similar in PGs and PCs (p = .352, .339 and .352) however, the degranulated mast cells were highly significant in PC while granulated mast cells were highly significant in PG respectively (p < .001 in both). Although a non-significant correlation existed between COX-2 and total mast cells in both groups but, total mast cells were significantly correlated with epithelial thickness in PC (p = .029). CONCLUSIONS: Mast cells and cyclooxygenase-2 proved to be independent inflammatory markers in periapical lesions. Further studies should be planned on mast cell and COX-2 inhibitors as treatment modalities of periapical lesions.

The Paradigm of the Inflammatory Radicular Cyst: Biological Aspects to be Considered.

Inflammatory radicular cysts (IRCs) are chronic lesions that follow the development of periapical granulomas (PGs). IRCs result from multiple inflammatory reactions led initially by several pro-inflammatory interleukins and growth factors that provoke the proliferation of epithelial cells derived from epithelial cell rests of Malassez present in the granulomatous tissue, followed by cyst formation and growth processes. Multiple theories have been proposed to help explain the molecular process involved in the development of the IRC from a PG. However, although multiple studies have demonstrated the presence of epithelial cells in most PGs, it is still not fully understood why not all PGs turn into IRCs, even though both are stages of the same inflammatory phenomenon and receive the same antigenic stimulus. Histopathological examination is currently the diagnostic gold standard for differentiating IRCs from PGs. Although multiple studies have evaluated the accuracy of non-invasive or minimally invasive methods in assessing the histopathological nature of the AP before the intervention, these studies' results are still controversial. This narrative review addresses the biological insights into the complex molecular mechanisms of IRC formation and its histopathological features. In addition, the relevant inflammatory molecular mediators for IRC development and the accuracy of non-invasive or minimally invasive diagnostic approaches are summarised. (EEJ-2022-03-041).

The expression of cyclic GMP-AMP synthase in human apical periodontitis: A laboratory investigation.

AIM: As a key DNA sensor, cyclic GMP-AMP synthase (cGAS) has emerged as a major mediator of innate immunity and inflammation. Human apical periodontitis has yet to be studied for the presence of cGAS. This investigation was conducted to determine if cGAS is involved in the pathological process of human apical periodontitis. METHODOLOGY: Sixty four human periapical lesions, comprising 20 periapical granulomas and 44 radicular cysts, were employed in this investigation. Healthy gingiva (n = 6), dental pulp (n = 3), and apical papilla (n = 3) were used as control samples. The expression of cGAS in the periapical tissues was discovered using immunohistochemical staining. mRNA-Sequencing and qRT-PCR were utilized to determine the differentially expressed genes (DEGs) associated with DNA-sensing signalling in periapical lesions compared to the healthy control. Immunofluorescence labelling was used to identify the co-expression of cGAS, interleukin-1ß, and interleukin-18. RESULTS: A significantly greater expression level of cGAS was discovered in the periapical lesions, with no significant difference between radicular cysts and periapical granulomas. mRNA-Sequencing analysis and qRT-PCR identified differentially expressed mRNA, such as cGAS and its downstream DEGs, between periapical lesions and healthy control tissues. Immunofluorescence labelling further revealed that cGAS, interleukin-1, and interleukin-18 were co-localized. CONCLUSIONS: These observations imply that along with the synthesis of interleukin-1 and interleukin-18, cGAS may be involved in the aetiology of apical periodontitis.

Elevated Foxo3a and Fas-ligand expression in human periapical granulomas as a potential treatment target.

OBJECTIVE: Periapical granuloma is a common periodontitis type involving chronic inflammation; however, the efficacy of current therapies is limited. Its molecular pathogenesis also remains obscure. Forkhead box transcription factor class o3a (Foxo3a) and Fas-ligand (FasL) are associated with chronic inflammation. Therefore, in this study, we aimed to clarify the roles of Foxo3a and FasL in periapical granuloma pathophysiology. SUBJECTS AND METHODS: Periapical lesions were obtained from patients during endodontic surgery and tooth extraction; those diagnosed with periapical granulomas using haematoxylin and eosin staining were further analysed. Immunohistochemical analysis was performed for Foxo3a and FasL, and real-time polymerase chain reaction was performed for FOXO3A, FASL and interleukin (IL)-1ß. Healthy gingival tissues were also examined as controls. RESULTS: Neutrophils, lymphocytes and plasma cells in the periapical granulomas, but not healthy tissues, expressed Foxo3a. Dual-colour immunofluorescence imaging revealed Foxo3a and FasL co-expression in leukocytes. FOXO3A, FASL and IL-1ß mRNA levels in healthy gingival tissues were significantly lower than those in the periapical granulomas. Additionally, FOXO3A and IL-1ß expressions were negatively correlated. CONCLUSIONS: Phosphorylated Foxo3a may reduce IL-1ß release by inhibiting apoptosis through FasL in periapical periodontitis and prevent exacerbation. Thus, Foxo3a is a potential therapeutic agent for periapical periodontitis.

Release of Matrix Metalloproteinases by Macrophages in Radicular Cysts and Residual Radicular Cysts.

INTRODUCTION: Radicular cysts (RCs) and residual radicular cysts (RRCs) are the sequelae of dental caries and that leads to proliferation of epithelial rests of Malassez in periapical tissues. OBJECTIVES: The aim was to evaluate the relationship between Langerhans cells, macrophages, matrix metalloproteinases (MMP-9, MMP-13), and tumor necrosis factor-alpha (TNF-α) in the capsule and lining epithelium of cystic lesions. MATERIALS AND METHODS: Twenty RCs and 20 RRCs were submitted to immunohistochemical analysis with anti-CD68, anti-CD1a, anti-MMP-9, anti-MMP-13, and anti-TNF-α antibodies. The Mann-Whitney test and the Spearman correlation test were used for analysis of the data (P<0.05). RESULTS: The immunoexpression of MMP-13 and CD68 was significantly higher in RCs when compared with RRCs (P=0.011 and 0.012, respectively). The presence of an intense inflammatory infiltrate was significantly correlated with the immunoexpression of CD68 in RCs (P=0.025). Expression of CD68 showed a significant positive correlation with MMP-13 (P=0.015). A moderate correlation was observed between MMP-9 and MMP-13 (P=0.010). TNF-α expression was more common in RCs (P=0.001). CD1a was more frequently expressed in atrophic epithelium (P=0.041) and was significantly correlated with TNF-α (P=0.014). CONCLUSION: Langerhans cells induce a greater release of TNF-α which, in turn, is responsible for the stimulation of M1 macrophages. Higher immunoexpression of MMP-13 and MMP-9 is observed in the early stages of RCs compared with RRCs. Therefore, the toxins of microorganisms present in highly inflamed RCs are the main factors triggering a proinflammatory immune response and greater cystic expansion in the early stages of these lesions.

Comparative Metabolomics Reveals the Microenvironment of Common T-Helper Cells and Differential Immune Cells Linked to Unique Periapical Lesions.

Periapical abscesses, radicular cysts, and periapical granulomas are the most frequently identified pathological lesions in the alveolar bone. While little is known about the initiation and progression of these conditions, the metabolic environment and the related immunological behaviors were examined for the first time to model the development of each pathological condition. Metabolites were extracted from each lesion and profiled using gas chromatography-mass spectrometry in comparison with healthy pulp tissue. The metabolites were clustered and linked to their related immune cell fractions. Clusters I and J in the periapical abscess upregulated the expression of MMP-9, IL-8, CYP4F3, and VEGF, while clusters L and M were related to lipophagy and apoptosis in radicular cyst, and cluster P in periapical granuloma, which contains L-(+)-lactic acid and ethylene glycol, was related to granuloma formation. Oleic acid, 17-octadecynoic acid, 1-nonadecene, and L-(+)-lactic acid were significantly the highest unique metabolites in healthy pulp tissue, periapical abscess, radicular cyst, and periapical granuloma, respectively. The correlated enriched metabolic pathways were identified, and the related active genes were predicted. Glutamatergic synapse (16-20),-hydroxyeicosatetraenoic acids, lipophagy, and retinoid X receptor coupled with vitamin D receptor were the most significantly enriched pathways in healthy control, abscess, cyst, and granuloma, respectively. Compared with the healthy control, significant upregulation in the gene expression of CYP4F3, VEGF, IL-8, TLR2 (P < 0.0001), and MMP-9 (P < 0.001) was found in the abscesses. While IL-12A was significantly upregulated in cysts (P < 0.01), IL-17A represents the highest significantly upregulated gene in granulomas (P < 0.0001). From the predicted active genes, CIBERSORT suggested the presence of natural killer cells, dendritic cells, pro-inflammatory M1 macrophages, and anti-inflammatory M2 macrophages in different proportions. In addition, the single nucleotide polymorphisms related to IL-10, IL-12A, and IL-17D genes were shown to be associated with periapical lesions and other oral lesions. Collectively, the unique metabolism and related immune response shape up an environment that initiates and maintains the existence and progression of these oral lesions, suggesting an important role in diagnosis and effective targeted therapy.

Overexpression of Cyclophilin A in Human Periapical Lesions.

INTRODUCTION: Cyclophilin A (CypA) is a cytosolic protein involved in multiple biological functions, such as inflammation, tissue remodeling, tumorigenesis, and vascular diseases. Human periapical lesions are induced by bacterial infections. However, the expression of CypA in human periapical lesions remains unclear. This study aimed to investigate the presence of CypA in human periapical lesions and the possible association of CypA with angiogenesis, inflammatory cell infiltration, and alveolar bone degradation during inflammatory development. METHODS: Fifty-eight human periapical tissues, including periapical granulomas (PGs, n = 28), radicular cysts (RCs, n = 24), and healthy control tissues (control group, n = 6) were collected. Samples were fixed and analyzed. CypA expression was detected and analyzed by immunohistochemistry in different cross sections. Double immunofluorescence was assessed to colocalize CypA with CD34, CypA with matrix metalloproteinase 9 (MMP-9), and CD147 with MMP-9. RESULTS: CypA was significantly overexpressed in the RC and PG groups compared with the control group (P < .05), but the difference between the RC and PG groups was insignificant (P > .05). CypA-positive cells were mainly lymphocytes, endothelial cells, epithelial cells, and plasma cells. The double-labeling analysis of CypA with CD34 suggested that CypA expression was associated with angiogenesis during periapical lesions. MMP-9 colocalized with both CypA and CD147 indicated that CypA may colocalize with CD147 and may be associated with the degradation of soft and hard tissues around human periapical lesions. CONCLUSIONS: CypA may be involved in the development of periapical lesions with an increase in inflammatory cell infiltration, angiogenesis acceleration, and alveolar bone degradation.

Vasoactive Intestinal Peptide Immunoregulatory Role at the Periapex: Associative and Mechanistic Evidences from Human and Experimental Periapical Lesions.

INTRODUCTION: The balance between the host proinflammatory immune response and the counteracting anti-inflammatory and reparative responses supposedly determine the outcome of periapical lesions. In this scenario, the vasoactive intestinal peptide (VIP) may exert a protective role because of its prominent immunoregulatory capacity. In this study, we investigated (in a cause-and-effect manner) the potential involvement of VIP in the development of human and experimental periapical lesions. METHODS: Periapical granulomas (n = 124) and control samples (n = 48) were comparatively assessed for VIP and multiple immunologic/activity marker expression through real-time polymerase chain reaction. Experimental periapical lesions (C57Bl/6 wild-type mice) were evaluated regarding endogenous VIP expression correlation with lesion development and the effect of recombinant VIP therapy in lesion outcome. CCR4KO and IL4KO strains and anti-glucocorticoid-induced TNFR-related protein inhibition were used to test the involvement of Treg and Th2 cells in VIP-mediated effects. RESULTS: VIP expression was more prevalent in periapical granulomas than in controls, presenting a positive association with immunoregulatory factors and an inverse association/correlation with proinflammatory mediators and the receptor activator of nuclear factor kappa B ligand/osteoprotegerin ratio. Endogenous VIP expression up-regulation was temporally associated with lesion immunoregulation and a decline of bone loss. VIP therapy in mice prompted the arrest of lesion development, being associated with an anti-inflammatory and proreparative response that limits the proinflammatory, Th1, Th17, and osteoclastogenic response in the periapex. The VIP protective effect was dependent of Treg migration and activity and independent of interleukin 4. CONCLUSIONS: Our results show that VIP overexpression in human and experimental periapical lesions is associated with lesion inactivity and that VIP therapy results in the attenuation of experimental lesion progression associated with the immunosuppressive response involving Treg cells.

microRNA-146a and Hey2 form a mutual negative feedback loop to regulate the inflammatory response in chronic apical periodontitis.

Chronic apical periodontitis (CAP) is defined as chronic inflammation of the dental pulp and root canal system. Porphyromonas endodontalis lipopolysaccharide ( P. endodontalis LPS) plays an important role in inducing an inflammatory response in CAP. microRNA-146a (miR-146a) is a key regulator of inflammation and is induced by LPS. Hairy and enhancer-of-split related with YRPW motif 2 (Hey2) has been confirmed to be induced by the Notch signaling pathway, which is involved in tooth development, pulp regeneration, and repair after injury. Our study aimed to investigate the functional role of miR-146a via the targeting of Hey2 in CAP as well as the underlying mechanism. Compared with 13 healthy controls, miR-146a and Hey2 expressions were significantly higher in 20 patients with CAP. In addition, miR-146a, Hey2, interleukin (IL)-6, IL-1ß, and tumor necrosis factor (TNF)-α expressions were significantly increased in MC3T3-E1 cells stimulated with different concentrations (0-20 µg/mL) of P. endodontalis LPS for different amounts of time (0-48 hours). Moreover, miR-146a, which acts as an anti-inflammatory mediator, negatively regulated the expression of IL-6, IL-1ß, and TNF-α, and Hey2 was confirmed as a target gene of miR-146a by a luciferase reporter assay. Hey2 also negatively regulated miR-146a, IL-6, IL-1ß, and TNF-α expressions, and P. endodontalis LPS strongly induced Hey2 recruitment to the IL-6 promoter (-400 ~ -200 bp). These findings suggest that miR-146a and Hey2 form a mutual negative feedback regulatory loop, demonstrating a novel mechanism that regulates inflammatory responses in CAP.

Immunohistochemical Analysis of Cyclooxygenase-2 and Tumor Necrosis Factor Alpha in Periapical Lesions.

INTRODUCTION: The purpose of this study was to evaluate the expression of cyclooxygenase-2 (COX-2) and tumor necrosis factor alpha (TNF-α) in periapical granuloma (PG) and radicular cyst (RC) samples and to correlate it with the type of lesion, the intensity of the inflammatory infiltrate, and the thickness of the epithelial lining. METHODS: A total of 51 cases of periapical lesions (25 PGs and 26 RCs) were subjected to morphologic analysis and immunohistochemical study. The anti-COX-2 and anti-TNF-α antibodies were applied using the immunoperoxidase technique. Data were analyzed by the Mann-Whitney test, Pearson chi-square test, Fisher exact test, and Spearman correlation. RESULTS: Analysis of the inflammatory infiltrate revealed that 80% of PGs exhibited a grade III infiltrate as opposed to a 19% rate in RCs (P < .001). Morphologic evaluation of the epithelial thickness of RCs revealed the presence of atrophic epithelium in 73% of cases. The majority of PGs had a score of 1 for COX-2 immunoexpression (n = 14, 54%) and a score of 2 for TNF-α expression (n = 16, 64%), whereas in cases of RCs a score of 1 was more prevalent for COX-2 and TNF-α expression (n = 17, 65%). Significant differences in the expression scores of COX-2 and TNF-α were detected in periapical lesions (P < .001). CONCLUSIONS: Based on these findings, we emphasize that RCs and PGs have a similar expression of inflammatory mediators (COX-2 and TNF-α) although the secretion of TNF-α by macrophages and of COX-2 by several cells was higher in PGs, indicating a greater inflammatory response in these lesions.

Expression of the Forkhead box transcription factor Foxo3a in human periapical granulomas.

It has been reported that Forkhead box transcription factor class O3a (Foxo3a) is expressed in rheumatoid arthritis, a chronic inflammatory condition accompanied by bone resorption, and plays a role in its pathology. However, it has remained unclear whether Foxo3a is involved in the pathogenesis of periapical granulomas. The present study was performed to compare the expression of Foxo3a in periapical granulomas and healthy gingival tissues. Samples were obtained surgically from patients, and subjected to hematoxylin-eosin staining for histopathologic diagnosis. Two-color immunofluorescence staining was also performed using antibodies against Foxo3a and markers for three types of inflammatory cells: neutrophils, T lymphocytes, and B lymphocytes. This revealed that Foxo3a was expressed in all three cell types in periapical granulomas but not in healthy gingival tissues. Foxo3a was expressed in 82.1%, 78.3%, and 77.5% of neutrophils, T lymphocytes, and B lymphocytes, respectively, and statistical analysis using the Kruskal-Wallis test followed by the Steel-Dwass test showed no significant difference of Foxo3a expression among the three cell types. Our results suggest that Foxo3a transcription factors may be involved in the pathogenesis of periapical granulomas.

Expression of silent information regulator 2 homolog 1 (SIRT1) in periapical granulomas.

Silent information regulator 2 homolog 1 (SIRT1) inhibits oxidative injury and has anti-inflammatory effects. SIRT1 may be involved in healing of periapical periodontitis; however, SIRT1 expression in periapical periodontitis lesions has not been investigated. This study evaluated SIRT1 expression and a marker of oxidative stress-8-hydroxy-2'-deoxyguanosine (8-OHdG)-in periapical granulomas. First, we used real-time polymerase chain reaction to determine whether U-937 monocytes express SIRT1. U-937 cells treated with the SIRT1 activator resveratrol exhibited the highest SIRT1 mRNA level after 6-h incubation. By contrast, treating cells with the SIRT1 inhibitor sirtinol returned SIRT1 expression level to that of the control. In addition, immunocytochemical analysis using cytospin specimens showed that U-937 cells co-expressed SIRT1 and Ki-67. Dual-color immunofluorescence imaging showed that round cells in periapical granulomas co-expressed SIRT1 and 8-OHdG; however, neither was expressed in healthy gingival tissues. The number of 8-OHdG-expressing cells was significantly greater than the number of SIRT1-expressing cells. Our findings suggest that macrophages express SIRT1 and that wound healing in periapical granulomas is enhanced by a SIRT1-mediated reduction in the level of oxidative stress.

Increased interleukin 1α and interleukin 1ß expression is involved in the progression of periapical lesions in primary teeth.

BACKGROUND: Interleukin 1 (IL-1) is involved in bone resorption. However, the role of IL-1 in periapical lesions characterized by periapical bone destruction in primary teeth has not yet been fully elucidated. This study aimed to detect the distribution and expression of IL-1 in periapical lesions in primary teeth and assess the relationship between the cytokines and the degree of inflammatory cell infiltration. METHODS: A total of 106 chronic periapical lesions in primary teeth were harvested. Haematoxylin and eosin (H&E) staining was used to determine the histological type and the inflammatory cell infiltration grade (mild, moderate, and severe), and immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) were used to detect the distribution and expression of IL-1α and IL-1ß. RESULTS: Of the 106 chronic periapical lesion samples, there were 85 cases of periapical granuloma, accounting for 80.19% of the total samples, and 21 cases of radicular cysts, accounting for 19.81%; no cases of abscess were detected. Immunohistochemistry results showed that both IL-1α and IL-1ß were expressed in periapical granulomas and cysts. ELISA results showed that IL-1α and IL-1ß levels were higher in the periapical granuloma group than in the radicular cyst and normal control groups (P < 0.05). In the periapical granuloma group, IL-1α and IL-1ß were detected at higher levels in the severe inflammatory cell infiltration subgroup than in the mild-inflammatory cell infiltration subgroup (P < 0.05), and IL-1ß expression was also higher in the moderate inflammatory cell infiltration subgroup than in the mild inflammatory cell infiltration subgroup (P < 0.01). A significant positive correlation was observed between the protein expression levels of IL-1α and IL-1ß and the inflammation grade in periapical granulomas from primary teeth (P < 0.05). CONCLUSION: Expression levels of the cytokines IL-1α and IL-1ß in periapical granulomas from primary teeth increased with increasing inflammatory severity and appeared to be a contributing factor to the progression of periapical lesions.

The Expression of Interferon Regulatory Factor 8 in Human Periapical Lesions.

INTRODUCTION: Interferon regulatory factor 8 (IRF8) is a critical transcription factor in innate immune responses that regulates the development and function of myeloid cells. Human periapical lesions are caused by endodontic microbial infections. However, the presence of IRF8 in human periapical lesions remains elusive. This study aims to explore the expression of IRF8 in human periapical lesions and the possible association of IRF8 with macrophages, nuclear factor kappa B (NF-κB) signaling, and the autophagy process. METHODS: Thirty-nine human periapical tissues, including healthy control tissues (n = 15), radicular cysts (RCs, n = 11), and periapical granulomas (PG, n = 13), were examined. Tissues were fixed in paraformaldehyde and analyzed. The inflammatory infiltrates of lesions were evaluated by hematoxylin-eosin, and the expression of IRF8 was analyzed by immunohistochemistry. Double immunofluorescence assessment was performed to colocalize IRF8 with CD68, NF-κB p65, and LC3B. RESULTS: The expression of IRF8 was significantly higher in RCs and PGs than in the healthy control group, but no significant difference was found between RCs and PGs. There were significantly more IRF8-CD68 double-positive cells in RCs and PGs than in the healthy control group, but no significant difference was observed between RCs and PGs. Double-labeling analysis of IRF8 with NF-κB and LC3B indicated that IRF8 expression is associated with NF-κB signaling and the autophagy process during periapical lesions. CONCLUSIONS: IRF8 could be observed and might possibly be involved in macrophages in the development of periapical lesions.

Comparative Expression of CD34, Intercellular Adhesion Molecule-1, and Podoplanin and the Presence of Mast Cells in Periapical Granulomas, Cysts, and Residual Cysts.

INTRODUCTION: The aim of the present study was to compare the immunoexpression of CD34, intercellular adhesion molecule-1 (ICAM-1), and podoplanin and the presence of mast cells with clinical, demographic, radiologic, and histologic features from periapical granulomas, periapical cysts, and residual cysts. METHODS: Thirty-one lesions (5 granulomas, 15 periapical cysts, and 11 residual cysts) were selected. Histologic sections in silanized slides were used for the immunohistochemical reactions. The analysis of the images was performed by using an optical microscope, and data were analyzed with 5% significance (P < .05). RESULTS: Cysts presented atrophic and hyperplastic epithelium in 11 cases (35.5%) and 15 cases (48.8%), respectively (P > .05). The intensity of the inflammatory infiltrate was similar when comparing the 3 groups (P > .05). CD34 and podoplanin expression and the presence of mast cells were similar when comparing the 3 groups; ICAM-1 expression was more intense in granulomas than cysts (P < .05). There were no statistically significant differences associated with the expression of the evaluated markers according to the intensity of the inflammatory infiltrate. CONCLUSIONS: There were no differences in the expression of CD34 and podoplanin and in the presence of mast cells when the 3 groups were compared. ICAM-1 expression was more common in periapical granulomas.

Immunohistochemical Analysis of Galectins-1, -3, and -7 in Periapical Granulomas, Radicular Cysts, and Residual Radicular Cysts.

INTRODUCTION: Galectins play important roles in immunoinflammatory responses, but their participation in the development of periapical lesions remains unclear. This study aimed to evaluate the expressions of galectins-1, -3, and -7 in periapical lesions, correlating them with the intensity of the inflammatory infiltrate and the pattern of the cystic epithelium. METHODS: Twenty periapical granulomas (PGs), 20 radicular cysts (RCs), and 20 residual radicular cysts (RRCs) were submitted to immunohistochemistry using anti-galectin-1, -3, and -7 antibodies. The percentage of immunopositive cells in epithelial and connective tissues was determined. RESULTS: In connective tissue, PGs exhibited higher cytoplasmic/membrane expression of galectins-1 and -7 than RCs and RRCs (P < .05). There was higher nuclear expression of galectin-1 in PGs compared with RCs and RRCs (P < .05). The expression of galectins-1 and -7 in connective tissue was higher in lesions with grade III inflammation (P < .05). No significant differences in galectin-3 immunoexpression were observed for any of the parameters evaluated (P > .05). In the epithelial component, a higher nuclear expression of galectin-7 was detected in RRCs (P < .05), and a higher cytoplasmic/membrane expression of this protein was found in cysts with hyperplastic epithelium (P < .05). Positive correlations were observed between the nuclear and cytoplasmic/membrane expression of galectin-1 in connective tissue (P < .05) as well as between the nuclear and cytoplasmic/membrane expression of galectin-7 in epithelial tissue of cysts (P < .05). CONCLUSIONS: Galectins-1 and -7 may play important roles in the pathogenesis of PGs, RCs, and RRCs. On the other hand, the present results suggest only a minor involvement of galectin-3 in the development of these lesions.

A potential role for the silent information regulator 2 homologue 1 (SIRT1) in periapical periodontitis.

AIM: To investigate the role played by silent information regulator 2 homologue 1 (SIRT1) during angiogenesis of periapical periodontitis. METHODOLOGY: Periapical granulomas were subjected to dual-colour immunofluorescence imaging and real-time polymerase chain reactions assaying the expression levels of SIRT1, vascular endothelial growth factor (VEGF) and VE-cadherin. The association between Ki-67 and SIRT1 expression was also examined. Human umbilical vein endothelial cells (HUVECs) were treated with a combination of lipopolysaccharide and resveratrol (a SIRT1 activator) or sirtinol (a SIRT1 inhibitor); and the levels of mRNAs encoding SIRT1, VEGF and VE-cadherin were determined. HUVEC tube formation was assayed in the presence of resveratrol or sirtinol. The Mann-Whitney U-test or the Tukey-Kramer test was used for statistical analysis. RESULTS: Ki-67-expressing cells, including endothelial cells, lay adjacent to SIRT1-expressing cells in periapical granulomas. In addition, SIRT1-expressing cells were detected adjacent to VEGF-expressing cells and VEGF- or VE-cadherin-expressing endothelial cells. SIRT1, VEGF and VE-cadherin mRNA expression levels in periapical granulomas were significantly higher (P = 0.0054, 0.0090 and 0.0090, respectively) than those in healthy gingival tissues. HUVECs treated with resveratrol exhibited significantly higher expression of mRNAs encoding SIRT1, VEGF and VE-cadherin (P = 0.0019, 0.00005 and 0.0045, respectively) compared with controls, but sirtinol inhibited such expression. Resveratrol caused HUVECs to form tube-like structures, whilst sirtinol inhibited this process. CONCLUSIONS: These findings suggest that SIRT1 may stimulate angiogenesis in periapical granulomas by triggering the proliferation of endothelial cells and inducing VEGF and VE-cadherin expression.

Inflammatory cell expression of Toll-like receptor-2 (TLR2) within refractory periapical granuloma.

Background: Toll-like receptor-2 (TLR2) is highly important within the immune system. Characterization of the expression of TLR2 within inflammatory cells in periapical lesions could help in diagnosis and management of refractory cases. The aim of the study is identification of Toll-like receptor (TLR2) through immunohistochemical and immunofluroscence expression in inflammatory cells within refractory periapical granuloma cases. Methods: Eight cases of refractory periapical granuloma were selected out of 772 cases. Histological examination and immunohistochemical staining with polyclonal rabbit antihuman TLR2, monoclonal mouse antihuman CD38, CD68 and CD83 primary antibodies, as well as immunofluorescence staining with goat anti-rabbit TLR2, donkey anti-mouse CD38, CD68 and CD83 primary antibodies was conducted. Positive controls, negative controls and experimental sections with no primary antibody were included in the study. Qualitative analysis and double immunofluorescence technique was used to characterize the TLR + cells. Results: In periapical granuloma, lymphocytes (CD38 cells) expressed the most amount of TLR reactivity followed by macrophages (CD68 cells), and odontogenic epithelial cells. Neutrophils, red blood cells (RBCs) and collagen ground substance were negative to TLR2.  Conclusion: TLR2 was highly expressed by lymphocytes and plasma cells indicative of their major role in the inflammatory process and antigen recognition in refractory periapical granuloma. Dendritic cells expressing TLR2 were low in number suggesting a minor role in sustaining these lesions.