Human Periapical Granulomas Research Articles

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

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. 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)-1β. Healthy gingival tissues were also examined as controls. 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-1β mRNA levels in healthy gingival tissues were significantly lower than those in the periapical granulomas. Additionally, FOXO3A and IL-1β expressions were negatively correlated. Phosphorylated Foxo3a may reduce IL-1β release by inhibiting apoptosis through FasL in periapical periodontitis and prevent exacerbation. Thus, Foxo3a is a potential therapeutic agent for periapical periodontitis.

Role of S100A4 in the Pathogenesis of Human Periapical Granulomas.

S100A4 expression is associated with the pathology of chronic inflammatory diseases. In this study, we investigated the role of S100A4 and four inflammatory mediators (IL-1β, IκB, IL-10, and TNF-α) in human periapical granulomas (PGs). S100A4 expression in PGs obtained by apicoectomy was examined by immunohistochemistry. Further, the expression of S100A4 and four inflammatory mediators was compared between PGs and healthy gingival tissues (HGTs) using real-time PCR. In the PGs, S100A4 was found to be expressed in endothelial cells and fibroblasts. Furthermore, real-time PCR revealed that the expression of S100A4 and IL-1β in PGs was significantly higher than that in HGTs. Although a correlation between the expression of S100A4 and IκB or IL-10 was not detected, a positive correlation between the expression of S100A4 and IL-1β or TNF-α was observed. The expression of S100A4 correlates with the pathogenesis of PGs.

Epstein-Barr virus reactivation by persistent apical periodontal pathogens.

To assess whether Epstein-Barr virus (EBV) reactivation is triggered by persistent apical periodontitis-related microbes using in vitro and ex vivo methodologies. Surgically removed human periapical granulomas (n=50) and healthy gingival tissues (n=10) were analysed to determine the presence of EBV and seven persistent apical periodontitis-related microbes. In addition, real-time polymerase chain reaction was used to detect the mRNA expression of BZLF-1, an immediate-early gene of EBV. Expression of latent membrane protein (LMP)-1 and ZEBRA, an early lytic protein of EBV encoded by BZLF-1, was also examined using triple-colour immunofluorescence staining. n-Butyric acid produced by the microbes was quantified, and luciferase assays were performed in association with bacterial lysates. In addition, Daudi cells were cultured with bacterial lysates, and the expression levels of BZLF-1 mRNA and ZEBRA protein were determined. EBV DNA and BZLF-1 mRNA were detected in 47 out of 50 periapical granulomas, but not in healthy gingival tissues. The EBV DNA copy number and the number of Fusobacterium nucleatum were significantly positively correlated with BZLF-1 expression in periapical granulomas. The number of Prevotella intermedia was slightly correlated with BZLF-1 expression; however, the other microbes were not. CD79a-positive B cells in periapical granulomas, but not those in healthy gingival tissues, expressed both LMP-1 and ZEBRA. n-Butyric acid production was the highest in F. nucleatum and the lowest in P. intermedia. Enterococcus faecalis, Candida albicans and the other tested microbes did not produce n-butyric acid. An F. nucleatum lysate exhibited significantly increased BZLF-1-luciferase activity in the same manner of commercial butyric acid, whereas P. intermedia did not. F. nucleatum also induced the expression of BZLF-1 mRNA and ZEBRA protein by Daudi cells, indicating that EBV reactivation was induced. Among the persistent apical periodontitis-related bacteriathat were tested, F. nucleatum most strongly reactivated latent EBV, whereas E. faecalis and C. albicans as well as the other microbes did not.

Heat Shock 70 Protein Genes and Genetic Susceptibility to Apical Periodontitis

IntroductionHeat shock proteins (HSPs) protect cells under adverse conditions such as infection, inflammation, and disease. The differential expression of HSPs in human periapical granulomas suggests a potential role for these proteins in periapical lesion development, which may contribute to different clinical outcomes. Therefore, we hypothesized that polymorphisms in HSP genes leading to perturbed gene expression and protein function may contribute to an individual's susceptibility to periapical lesion development. MethodsSubjects with deep carious lesions with or without periapical lesions (≥3 mm) were recruited at the University of Texas School of Dentistry at Houston and at the University of Pittsburgh. Genomic DNA samples of 400 patients were sorted into 2 groups: 183 cases with deep carious lesions and periapical lesions (cases) and 217 cases with deep carious lesions but without periapical lesions (controls). Eight single nucleotide polymorphisms (SNPs) in HSPA4, HSPA6, HSPA1L, HSPA4L, and HSPA9 genes were selected for genotyping. Genotypes were generated by end point analysis by using Taqman chemistry in a real-time polymerase chain reaction assay. Allele and genotype frequencies were compared among cases and controls by using χ2 and Fisher exact tests as implemented in PLINK v.1.07. In silico analysis of SNP function was performed by using Polymorphism Phenotyping V2 and MirSNP software. ResultsOverall, SNPs in HSPA1L and HSPA6 showed significant allelic association with cases of deep caries and periapical lesions (P < .05). We also observed altered transmission of HSPA1L SNP haplotypes (P = .03). In silico analysis of HSPA1L rs2075800 function showed that this SNP results in a glutamine-to-lysine substitution at position 602 of the protein and might affect the stability and function of the final protein. ConclusionsVariations in HSPA1L and HSPA6 may be associated with periapical lesion formation in individuals with untreated deep carious lesions. Future studies could help predict host susceptibility to developing apical periodontitis.

Expression and immunohistochemical localization of leptin in human periapical granulomas.

BackgroundLeptin, initially described as an adipocyte-derived hormone to regulate weight control, is expressed in normal and inflamed human dental pulp, being up-regulated during pulp experimental inflammation. Leptin receptor (LER) has been identified in human periapical granulomas. The aim of this study was to analyze and characterize the expression of leptin in human periapical granulomas.Material and MethodsFifteen periapical inflammatory lesions were obtained from extracted human teeth and teeth which underwent periapical surgery. After their morphological categorization as periapical granulomas and gradation of the inflammatory infiltrate, they were examined by immunohistochemistry using human leptin policlonal antibodies. Leptin mRNA expression was also determined by quantitative real-time PCR (qRT-PCR) and the amount of leptin protein was analyzed by immunoblot.ResultsAll periapical lesions exhibited the characteristic of chronic granulomatous inflammatory process with inflammatory infiltrate grade III. Leptin+ cells were detected in 13 periapical granulomas (86.6%). The median number of Leptin+ cells in periapical granulomas was 1.70 (0.00-7.4). Amongst the inflammatory cells in the periapical granulomas, only macrophages were reactive to leptin antibodies. Western blot analysis revealed the presence in all samples of a protein with apparent molecular weight of approximately 16 kDa, corresponding to the estimated molecular weights of leptin. The expression of leptin mRNA was confirmed by qRT-PCR analysis and the size of the amplified fragment (296 bp for leptin and 194 bp for cyclophilin) was assessed by agarose gel electrophoresis.ConclusionsFor the first time, it has been demonstrated that human periapical granuloma expresses the adipokine leptin. Key words: Apical granuloma, dental pulp, endodontics, leptin, leptin receptor, immune system, immunohistochemistry, periapical inflammatory response.

Analysis of Multiple Cytokine Polymorphisms in Individuals with Untreated Deep Carious Lesions Reveals IL1B (rs1143643) as a Susceptibility Factor for Periapical Lesion Development

IntroductionIt has been proposed that individual genetic predisposition may contribute to persistent apical periodontitis. Cytokines are associated with levels of inflammation and are involved in caries, pulpal, and periapical tissue destruction. We hypothesized that polymorphisms in cytokine genes may contribute to an individual's increased susceptibility to apical tissue destruction in response to deep carious lesions. MethodsSubjects with deep carious lesions with or without periapical lesions (≥3 mm) were recruited at the University of Pittsburgh, Pittsburgh, PA, and the University of Texas at Houston, Houston, TX. Genomic DNA samples of 316 patients were sorted into 2 groups: 136 cases with deep carious lesions and periapical lesions (cases) and 180 cases with deep carious lesions but no periapical lesions (controls). Nine single-nucleotide polymorphisms in IL1B, IL6, TNF, RANK, RANKL, and OPG genes were selected for genotyping. Genotypes were generated by end point analysis using TaqMan chemistry (Invitrogen, Carlsbad, CA) in a real-time polymerase chain reaction instrument. Allele and genotype frequencies were compared among cases and controls using the PLINK program (http://pngu.mgh.harvard.edu/purcell/plink/). Ninety-three human periapical granulomas and 24 healthy periodontal ligament tissues collected postoperatively were used for messenger RNA expression analyses of IL1B. ResultsA single-nucleotide polymorphism in IL1B (rs1143643) showed allelic (P = .02) and genotypic (P = .004) association with cases of deep caries and periapical lesions. We also observed altered transmission of IL1B marker haplotypes (P = .02) in these individuals. IL1B was highly expressed in granulomas (P < .001). ConclusionsVariations in IL1B may be associated with periapical lesion formation in individuals with untreated deep carious lesions. Future studies could help predict host susceptibility to developing periapical lesions.

FOXP3 DNA Methylation Levels as a Potential Biomarker in the Development of Periapical Lesions

IntroductionEpigenetic mechanisms, such as DNA methylation, can modify gene expression patterns without changing the DNA sequence, comprising a tool that cells use to lock genes in the “off” position. Variations in the methylation profile have been correlated to a variety of human diseases. Here, we hypothesize that DNA methylation in immune response–related genes may contribute to the development of periapical lesions. MethodsThe DNA methylation patterns of 22 immune response–related gene promoters were evaluated in 137 human periapical granulomas, 8 apical cysts, and 31 healthy gingival tissues from 2 independent cohorts using a pathway-specific real-time polymerase chain reaction array (EpiTect Methyl II; Qiagen Inc, Valencia, CA). Messenger RNA expression analysis by qualitative polymerase chain reaction was also performed. SABiosciences's hierarchical clustering and methylation (Qiagen, Valencia, CA) and Prism6 software (GraphPad Software, Inc, La Jolla, CA) were used for data analysis. ResultsFOXP3 gene promoter showed the highest level of methylation in both periapical granulomas and apical cysts (P < .001), and methylation levels were inversely correlated with FOXP3 messenger RNA expression in the lesions. Furthermore, FOXP3 expression was prevalent in inactive lesions and was positively correlated with interleukin-10 and transforming growth factor beta levels. ConclusionsOur results suggest that FOXP3 acts as a master switch governing the development and function of T-regulatory cells, whose functions include the inhibition of immune responses and temper inflammation. The observed differential methylation patterns of FOXP3 in periapical lesions may be crucial in determining its suppressive activity and may be involved in periapical lesion development.

Expression and immunohistochemical localization of leptin receptor in human periapical granuloma.

To investigate the expression and immunohistochemical localization of leptin receptor (LEPR) in human periapical granulomas. Periapical inflammatory lesions were obtained from extracted human teeth and teeth which underwent periapical surgery. After their histopathological categorization as periapical granulomas (n=20), they were examined by immunohistochemistry using human LEPR monoclonal antibodies. LEPR mRNA expression was also determined by quantitative real-time PCR (qRT-PCR), and the amount of LEPR protein was analysed by immunoblot. All granuloma samples expressed LEPR. Amongst inflammatory cells, only macrophages showed expression of LEPR. Western blot analysis revealed the presence in the samples of a protein with apparent molecular weight of ~120kDa, corresponding to the estimated molecular weight of LEPR. The qRT-PCR analysis demonstrated the expression of LEPR mRNA, corresponding the size of the amplified fragment (338 bp), assessed by agarose gel electrophoresis, to that of LEPR mRNA. Human periapical granulomas express LEPR. In periapical granulomas, only macrophages showed expression of LEPR. This finding suggests that leptin can play a role in inflammatory and immune periapical responses.

Simultaneous analysis of T helper subsets (Th1, Th2, Th9, Th17, Th22, Tfh, Tr1 and Tregs) markers expression in periapical lesions reveals multiple cytokine clusters accountable for lesions activity and inactivity status.

Previous studies demonstrate that the balance between pro- and anti-inflammatory mediators determines the stable or progressive nature of periapical granulomas by modulating the balance of the osteoclastogenic factor RANKL and its antagonist OPG. However, the cytokine networks operating in the development of periapical lesions are quite more complex than what the simple pro- versus anti-inflammatory mediators' paradigm suggests. Here we simultaneously investigated the patterns of Th1, Th2, Th9, Th17, Th22, Thf, Tr1 and Tregs cytokines/markers expression in human periapical granulomas.MethodsThe expression of TNF-α, IFN-γ, IL-17A, IL23, IL21, IL-33, IL-10, IL-4, IL-9, IL-22, FOXp3 markers (via RealTimePCR array) was accessed in active/progressive (N=40) versus inactive/stable (N=70) periapical granulomas (as determined by RANKL/OPG expression ratio), and also to compare these samples with a panel of control specimens (N=26). A cluster analysis of 13 cytokine levels was performed to examine possible clustering between the cytokines in a total of 110 granulomas. ResultsThe expression of all target cytokines was higher in the granulomas than in control samples. TNF-α, IFN-γ, IL-17A and IL-21 mRNA levels were significantly higher in active granulomas, while in inactive lesions the expression levels of IL-4, IL-9, IL-10, IL-22 and FOXp3 were higher than in active granulomas. Five clusters were identified in inactive lesion groups, being the variance in the expression levels of IL-17, IL-10, FOXp3, IFN-γ, IL-9, IL-33 and IL-4 statistically significant (KW p<0.05). Three clusters were identified in active lesions, being the variance in the expression levels of IL-22, IL-10, IFN-γ, IL-17, IL-33, FOXp3, IL-21 and RANKL statistically significant (KW p<0.05). ConclusionThere is a clear dichotomy in the profile of cytokine expression in inactive and active periapical lesions. While the widespread cytokine expression seems to be a feature of chronic lesions, hierarchical cluster analysis demonstrates the association of TNF-α, IL-21, IL-17 and IFN-γ with lesions activity, and the association of FOXP3, IL-10, IL-9, IL-4 and IL-22 with lesions inactivity.

Expression of Heat Shock Proteins in Periapical Granulomas

IntroductionCells from virtually all organisms respond to a variety of stresses by the rapid synthesis of a highly conserved set of polypeptides termed heat shock proteins (HSPs). HSPs protect cells under adverse conditions such as infection, inflammation, and disease. We hypothesize that endodontic infection might result in an imbalance in the expression of heat shock genes, accounting for different clinical outcomes in periapical lesions. MethodsWe analyzed the expression of 44 HSPs genes using a pathway-specific real-time polymerase chain reaction array in 93 human periapical granulomas and 24 healthy periodontal ligament tissues collected postoperatively. Observed variations in the expression of HSP genes were also analyzed based on the classification of periapical granulomas as active or inactive. In addition, U937 cells were differentiated into macrophages, infected with different concentrations of purified Escherichia coli lipopolysaccharide (LPS), and used as templates for the HSP gene array. Protein expression was assessed by immunohistochemistry. ResultsThe expression of HSP genes was significantly increased in granulomas compared with healthy periodontal ligament (P < .00001). Among the 44 HSP genes, DNAJC3, HSPA4, HSPA6, and HSPB1 showed the highest expression levels in both granulomas and LPS-treated macrophages. DNAJC3, HSPA6, and HSPB1 were highly expressed in active lesions, whereas HSPA4 expression was higher in inactive lesions (P < .005). Higher concentrations of LPS led to increased HSP expression in macrophages (P < .0001). Immunocytochemistry confirmed the expression and colocalization of HSPB1 and HSPA6 proteins in the cytoplasm of LPS-infected macrophages. ConclusionsThe observed differential expression patterns of HSPs in periapical granulomas and LPS-infected macrophages suggest that HSP genes and proteins are involved in periapical lesion development and may account for different clinical outcomes. Understanding the role of the heat shock response might provide additional insights into the process of periapical lesion development.

Expression Analysis of Wound Healing Genes in Human Periapical Granulomas of Progressive and Stable Nature

Introduction Wound healing process involves the activation of extracellular matrix components, remodeling enzymes, cellular adhesion molecules, growth factors, cytokines and chemokines genes. However, the molecular patterns underlying the healing process at the periapical environment remain unclear. Here we hypothesized that endodontic infection might result in an imbalance in the expression of wound healing genes involved in the pathogenesis of periapical lesions. Furthermore, we suggest that differential expression of wound healing markers in active and latent granulomas could account for different clinical outcomes for such lesions. Methods Study samples consisted of 93 periapical granulomas collected after endodontic surgeries and 24 healthy periodontal ligament tissues collected from premolars extracted for orthodontic purposes as control samples. Of these, 10 periapical granulomas and 5 healthy periapical tissues were used for expression analysis of 84 wound healing genes by using a pathway-specific real-time polymerase chain reaction array. The remaining 83 granulomas and all 24 control specimens were used to validate the obtained array data by real-time polymerase chain reaction. Observed variations in expression of wound healing genes were analyzed according to the classification of periapical granulomas as active/progressive versus inactive/stable (as determined by receptor activator for nuclear factor kappa B ligand/osteoprotegerin expression ratio). Results We observed a marked increase of 5-fold or greater in SERPINE1, TIMP1, COL1A1, COL5A1, VTN, CTGF, FGF7, TGFB1, TNF, CXCL11, ITGA4, and ITGA5 genes in the periapical granulomas when compared with control samples. SERPINE1, TIMP1, COL1A1, TGFB1, and ITGA4 mRNA expression was significantly higher in inactive compared with active periapical granulomas ( P < .001), whereas TNF and CXCL11 mRNA expression was higher in active lesions ( P < .001). Conclusions The identification of novel gene targets that curb the progression status of periapical lesions might contribute to a more accurate diagnosis and lead to treatment modalities more conducive to endodontic success.

Receptor for advanced glycation end products (RAGE)-expressing endothelial cells co-express AGE and S100 in human periapical granulomas

Objective The engagement of the receptor for advanced glycation end products (RAGE) by AGE or S100 perturbs homeostatic mechanisms and provides a basis for cellular dysfunction in pathological situations. To assess the mechanism of vascular immune reactions in chronic periapical periodontitis, we analysed co-expression of RAGE and AGE or S100 in periapical granulomas. Methods Surgically removed periapical lesions, which had been diagnosed as chronic periodontitis, were inspected histologically using paraffin-embedded sections stained with haematoxylin and eosin. Cryostat sections of the tissues, which were identified histologically as periapical granulomas, were then examined by double immunohistochemistry using polyclonal antibodies raised against human CD34 and monoclonal antibodies specific for human RAGE, AGE or S100. Dual-colour immunofluorescence image analysis was also performed to assess the co-expression of RAGE and AGE or RAGE and S100 by endothelial cells. Results Marked expression of RAGE, AGE, and S100 by CD34 + endothelial cells was noted. Dual-colour immunofluorescence image analysis revealed that the RAGE-expressing endothelial cells co-expressed AGE and S100; however, the number of RAGE–AGE-expressing endothelial cells was significantly higher than that of RAGE–S100-expressing endothelial cells. Conclusions Co-expression of RAGE and AGE by endothelial cells in periapical granulomas is more relevant than that of RAGE and S100. The possible engagement of RAGE and AGE may trigger cellular activation and mediate tissue injury.

T-Helper Cell Type 17/Regulatory T-Cell Immunoregulatory Balance in Human Radicular Cysts and Periapical Granulomas

Introduction Cysts and granulomas are chronic periapical lesions mediated by a set of inflammatory mediators that develop to contain a periapical infection. This study analyzed the nature of the inflammatory infiltrate, presence of mast cells, and in situ expression of cytokines (interleukin [IL]-17 and transforming growth factor [TGF]-ß), chemokines (macrophage inflammatory protein [MIP]-1ß and monocyte chemotactic protein [MCP]-1), and nuclear transcription factor (FoxP3) in human periapical granulomas and cysts compared with a control group. Methods Fifty-five lesions (25 periapical cysts, 25 periapical granulomas, and 5 controls) were analyzed. The type of inflammatory infiltrate was evaluated by hematoxylin-eosin staining, and the presence of mast cells was analyzed by toluidine blue staining. Indirect immunohistochemistry was used to evaluate the expression of cytokines, chemokines, and FoxP3. Results The inflammatory infiltrate mainly consisted of mononuclear cells. In cysts, mononuclear infiltrates were significantly more frequent than mixed (polymorphonuclear/mononuclear) infiltrates ( P = .04). Mixed inflammatory infiltrates were significantly more frequent in patients with sinus tract ( P = .0001). The number of mast cells was significantly higher in granulomas than in cystic lesions ( P = .02). A significant difference in the expression of IL-17 ( P = .001) and TGF-ß ( P = .003) was observed between cysts and granulomas and the control group. Significantly higher IL-17 levels were also observed in cases of patients with sinus tract ( P = .03). Conclusions We observed that chronic periapical lesions might experience a reagudization process that is correlated with an increased leukocyte infiltration, with the predominance of neutrophils attracted by a chemokine milieu, as well as the increased presence of IL-17.

Th17/Treg immunoregulatory balance in human periapical lesions (47.5)

Abstract Cysts and granulomas are chronic periapical lesions mediated by a set of inflammatory mediators that develop in order to contain a periapical infection. This study analyzed the inflammatory infiltrate, mast cells and in situ expression of cytokines, chemokines and FoxP3 in human periapical granulomas and cysts compared to controls. Fifty-five lesions (25 cysts, 25 granulomas and 5 controls) were analyzed. The type of inflammatory infiltrate was evaluated by HE staining and the presence of mast cells was analyzed by toluidine blue staining. Indirect immunohistochemistry was used to evaluate the expression of cytokines, chemokines and FoxP3. The inflammatory infiltrate mainly consisted of mononuclear cells. In cysts, mononuclear infiltrates were significantly more frequent than mixed (polymorphonuclear/mononuclear) infiltrates (p=0.04). Mixed inflammatory infiltrates were significantly more frequent in patients presenting fistulae (p=0.0001). The number of mast cells was significantly higher in granulomas than in cystic lesions (p=0.02). A significant difference in the expression of IL-17 (p=0.001) and TGF-β (p=0.003) was observed between cysts and granulomas and the control group. Significantly higher IL-17 levels were also observed in cases of patients with fistulae (p=0.03). Mast cells, TGF-β and IL-17 probably participate in the modulation of the inflammatory process in established periapical lesions, especially in granulomas.

High Matrix Metalloproteinase Activity Is a Hallmark of Periapical Granulomas

Introduction The inability to distinguish periapical cysts from granulomas before performing root canal treatment leads to uncertainty in treatment outcomes because cysts have lower healing rates. Searching for differential expression of molecules within cysts or granulomas could provide information with regard to the identity of the lesion or suggest mechanistic differences that may form the basis for future therapeutic intervention. Thus, we investigated whether granulomas and cysts exhibit differential expression of extracellular matrix (ECM) molecules. Methods Human periapical granulomas, periapical cysts, and healthy periodontal ligament tissues were used to investigate the differential expression of ECM molecules by microarray analysis. Because matrix metalloproteinases (MMP) showed the highest differential expression in the microarray analysis, MMPs were further examined by in situ zymography and immunohistochemistry. Data were analyzed by using one-way analysis of variance followed by the Tukey test. Results We observed that cysts and granulomas differentially expressed several ECM molecules, especially those from the MMP family. Compared with cysts, granulomas exhibited higher MMP enzymatic activity in areas stained for MMP-9. These areas were composed of polymorphonuclear cells (PMNs) in contrast to cysts. Similarly, MMP-13 was expressed by a greater number of cells in granulomas compared with cysts. Conclusion Our findings indicate that high enzymatic MMP activity in PMNs together with MMP-9 and MMP-13 stained cells could be a molecular signature of granulomas unlike periapical cysts.

Differential Patterns of Receptor Activator of Nuclear Factor Kappa B Ligand/Osteoprotegerin Expression in Human Periapical Granulomas: Possible Association with Progressive or Stable Nature of the Lesions

Receptor activator of nuclear factor kappa B ligand (RANKL) and osteoprotegerin (OPG) are expressed in apical periodontitis, suggesting a role for these molecules during lesion development. However, the profiles of RANKL/OPG expression in periapical lesions remain unknown. In this study we investigated the patterns of RANKL and OPG mRNA expression by real-time polymerase chain reaction in human periapical granulomas (N = 44) and compared them with sites presenting characteristic bone resorbing activity: healthy (n = 14) and orthodontically stretched and compressed periodontal ligament (n = 26), healthy gingiva (n = 24), chronic gingivitis (n = 32), and chronic periodontitis (n = 34) samples. Both RANKL and OPG mRNA expression was higher in periapical granulomas when compared with healthy periodontal ligament. Distinct patterns of RANKL and OPG expression ratio were found in the granulomas and in different physiologic and pathologic conditions, with characteristic bone resorption activity potentially being indicative of the stable or progressive nature of the lesions. Lesions with radiographic image smaller than 5 mm showed higher RANKL/OPG expression than images greater than 5 mm. Periapical granulomas presented heterogeneous patterns of RANKL and OPG expression, ranging from samples with RANKL/OPG ratio similar to that seen in sites with minimal or absent bone resorption to samples with RANKL/OPG expression pattern comparable with active bone resorption sites.

Receptor activator NFκB-ligand and osteoprotegerin protein expression in human periapical cysts and granulomas

The purpose of this study was to determine the expression of receptor activator of NFkappaB ligand (RANKL) and osteoprotegerin (OPG) associated with bone destruction in periapical cysts and granulomas. Forty human dental chronic periapical lesions were collected after periapical surgery. The lesions collected were fixed in 10% buffered formalin and histologically processed. At least 2 sections of each specimen were stained with hematoxylin and eosin for microscopic diagnosis. After that, 10 human periapical granulomas and 10 cysts were selected for immunohistochemical analysis for RANKL, OPG, and CD68+. Polymorphonuclear neutrophils, macrophages, endothelial cells, and lymphocytes were stained for RANKL and OPG in both lesions. Epithelial cells were also stained for RANKL and OPG in periapical cysts. Quantitative analysis was conducted and the results were expressed as a ratio of the number of immunostained cells over the total number of cells in the field (n = 100). The ratio of RANKL+/total cells was higher than OPG+/total cells in periapical granulomas (0.553 +/- 0.153 and 0.483 +/- 0.189, respectively; P < .0012; paired t test) and in cysts (0.519 +/- 0.09 and 0.339 +/- 0.117, respectively; P < .0001; paired t test). The ratios of OPG+/total cells (P < .0001; paired t test) and RANKL+/total cells (P < .0322; paired t test) were greater in granulomas than in cysts. However, the ratio RANKL+/OPG+ in granulomas (1.336 +/- 0.723) and cysts (1.404 +/- 0.385) was not significantly different. The ratio of CD68+/total cells was significantly higher in granulomas (0.381 +/- 0.040) than in cysts (0.307 +/- 0.068) (P < .0001; unpaired t test with Welch correction). Taking into account the limitations of the experimental approach employed, our findings indicate the presence of RANKL and OPG in cysts and granulomas, strongly suggesting the involvement of these gene products in the development of periapical lesions.

RANKL in human periapical granuloma: possible involvement in periapical bone destruction

The cytokine receptor activator of nuclear factor kappaB-ligand (RANKL) has been involved in both the physiological and pathological regulation of osteoclast life span and bone metabolism. Periapical granuloma is a periradicular lesion characterized by periapical bone destruction. The aims of this study were to associate the RANKL mRNA levels to periapical granulomas using the real-time reverse transcriptase-polymerase chain reaction (RT-PCR) technique and to determine the specific cell involved in RANKL synthesis. In eight periapical granuloma and eight periodontal ligament samples from periodontally healthy volunteers, RANKL mRNA was detected by real-time RT-PCR. Expression of RANKL on infiltrate leukocytes was further investigated by flow cytometry in six periapical granulomas. Receptor activator of nuclear factor kappaB-ligand mRNA levels were higher in periapical granulomas than in healthy periodontal ligament as its RANKL mRNA cycle threshold (Ct) and DeltaCt were significantly lower than that of controls (33.07 +/- 1.24 vs 36.96 +/- 1.69 and 11.58 +/- 3.02 vs 15.60 +/- 3.31, respectively). A 16.2-fold (2.0-131.6) higher RANKL gene expression was detected in the granulomas compared with the control tissues. We determined by flow cytometry that lymphocytes were the predominant leukocyte cells (53.31%), and monocytes and dendritic cells were the main RANKL synthesizers in granuloma lesions. These data indicate that monocytes synthesized RANKL in periapical granulomas and suggest that RANKL is involved in bone loss associated with periapical lesions.

Co‐production of vascular endothelial cadherin and inducible nitric oxide synthase by endothelial cells in periapical granuloma

To clarify the mechanisms of inflammatory cell migration in human periapical granulomas by examining vascular endothelial (VE) cadherin and inducible nitric oxide synthase (iNOS)-producing cells. Periapical tissues were obtained from patients during endodontic surgery and were divided into two portions. After fixing the tissues with acetone or 4% paraformaldehyde in phosphate-buffered saline, 5-microm-thick paraffin or cryostat sections were prepared, respectively. The paraffin sections of the inflamed tissues were evaluated histologically with haematoxylin-eosin stains. Cryostat sections of the tissue, diagnosed as periapical granulomas, were then examined by either immunohistochemistry using anti-human VE-cadherin or iNOS antibodies (Abs) for the characterization of infiltrating cells. In addition, co-localization of VE-cadherin and iNOS production was also analysed by two-colour immunofluorescence image analysis. Endothelial cells were strongly stained with iNOS Abs. Macrophages, lymphocytes, polymorphonuclear leucocytes and fibroblasts also exhibited iNOS production. These iNOS-positive cells accumulated around the blood vessels. On the other hand, VE-cadherin production was exhibited in only endothelial cells. Two-colour immunofluorescence image analysis using VE-cadherin and iNOS Abs demonstrated that iNOS-producing endothelial cells also showed VE-cadherin production. Vascular endothelial-cadherin produced by endothelial cells could be regulated by iNOS-producing cells in periapical granulomas and might play a pivotal role in vascular permeability.

Presence OF CXCR3-Positive Cells and IFN-γ–Producing Cells in Human Periapical Granulomas

In an attempt to understand further the balance between the types of helper T (Th) cells in human apical periodontitis, we examined the difference in the expression of the chemokine receptor and cytokine in samples obtained from human subjects by means of immunohistochemical methods. Chemokine receptor CXCR3-positive cells and IFN-gamma-producing cells were found to be present in human periapical granulomas, whereas chemokine receptor CCR3-positive cells and IL-4-producing cells could not be detected. By contrast, no factor expression was observed in a clinically healthy periodontal ligament serving as a negative control. Our findings suggest that Th1 cells may play an important role in the pathological process of local inflammation such as apical periodontitis.