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Aim: Apical periodontitis is an inflammatory disorder triggered by an immune response to bacterial infection, leading to the periapical tissue damage and alveolar resorption. However, the underlying mechanisms driving this process remain elusive, due to the complex and interconnected immune microenvironment within the local lesion site. In this study, the influence of Nlrp3 inflammasome-mediated immune response on the apical periodontitis was investigated., Methodology: RNA sequencing, immunohistochemistry and ELISA assay were performed to investigate the activation of Nlrp3 inflammasome signalling pathways in the human periapical tissues, including radicular cysts, periapical granulomas and healthy oral mucosa. A mouse model of apical periodontitis was established to study the role of Nlrp3 knockout in periapical bone resorption and Treg cell stability, and the underlying mechanism was explored through in vitro experiments. In vivo Treg cell adoptive transfer was performed to investigate the effects of Treg cells on the progression of apical periodontitis., Results: Our findings find that the hyperactivated Nlrp3 inflammasome is present in human periapical lesions and plays a vital role in the immune-related periapical bone loss. Using a mouse model of apical periodontitis, we observe that Nlrp3 deficiency is resistant to bone resorption. This protection was accompanied by elevated generation and infiltration of local Treg cells that displayed a notable ability to suppress RANKL-dependent osteoclast differentiation. In terms of the mechanism of action, Nlrp3 deficiency directly inhibits the osteoclast differentiation and bone loss through JNK/MAPK and NF-κB pathways. In addition, Nlrp3 induces pyroptosis in the stem cells from apical papilla (SCAPs), and the subsequent release of cytokines affects the stability of Treg cell in periapical lesions, leading indirectly to enhanced bone resorption. In turn, adoptive transfer of both Nlrp3-deficient and wild-type Treg cells effectively prevent the bone erosion during apical periodontitis., Conclusions: Together, our data identify that the Nlrp3 inflammasome modulates the Treg cell stability and osteoclastogenesis in the periapical inflammatory microenvironment, thus determining the progression of bone erosion., (© 2024 British Endodontic Society. Published by John Wiley & Sons Ltd.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Altaie, Venkatachalam, Samaranayake, Soliman and Hamoudi.)

Introduction: Dental cysts can be of inflammatory (radicular cysts) or noninflammatory (dentigerous cysts) origin. Apical periodontitis is a necrosis of the pulp and infection of the root canal causing the development of apical granulomas or radicular cysts. The immunology of granuloma and cyst formation is important because modern root filling materials are immunologically active and can contribute to the resolution of apical granulomas. In contrast, radicular cysts often require apicectomy. A better understanding of the pathophysiology of inflammation and bone resorption in apical periodontitis could be the basis for developing new root filling materials with superior immunomodulatory properties., Methods: Forty-one apical granulomas, 23 radicular cysts, and 23 dentigerous cysts were analyzed in this study. A tissue microarray of the 87 consecutive specimens was created, and human leukocyte antigen-DR isotype (HLA-DR)-, CD83-, receptor activator of nuclear factor kappa B ligand-, macrophage colony-stimulating factor (MCSF)-, galectin-3 (Gal3)-, CD4-, and CD8-positive cells were detected by immunohistochemistry. Tissue microarrays were digitized, and the expression of markers was quantitatively assessed., Results: HLA-DR, CD83, MCSF, and Gal3 expression was significantly (P < .05) higher in radicular cysts compared with apical granulomas. HLA-DR, CD83, MCSF, receptor activator of nuclear factor kappa B ligand, and Gal3 expression in dentigerous cysts was significantly (P < .05) lower than in both periapical lesions (apical granulomas and radicular cysts). CD4 and CD8 infiltration was not statistically different between apical granulomas and radicular cysts. Dentigerous cysts showed a significantly (P < .05) lower T-cell infiltration than apical periodontitis. The CD4/CD8 ratio was not significantly different between the analyzed groups., Conclusions: The development of radicular cysts in apical periodontitis is associated with an increased expression of myeloid inflammatory markers and bone resorption parameters. Antigen-presenting cells and myeloid cells might be more relevant for the pathogenesis of apical periodontitis than T cells. Increased inflammation might promote the formation of radicular cysts and more pronounced bone resorption., (Copyright © 2019 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Objectives: Apical periodontitis can appear clinically as apical granulomas or radicular cysts. There is evidence that immunologic factors are involved in the pathogenesis of both pathologies. In contrast to radicular cysts, the dentigerous cysts have a developmental origin. Macrophage polarization (M1 vs M2) is a main regulator of tissue homeostasis and differentiation. There are no studies comparing macrophage polarization in apical granulomas, radicular cysts, and dentigerous cysts., Materials and Methods: Forty-one apical granulomas, 23 radicular cysts, and 23 dentigerous cysts were analyzed in this study. A tissue microarray (TMA) of the 87 consecutive specimens was created, and CD68-, CD11c-, CD163-, and MRC1-positive macrophages were detected by immunohistochemical methods. TMAs were digitized, and the expression of macrophage markers was quantitatively assessed., Results: Radicular cysts are characterized by M1 polarization of macrophages while apical granulomas show a significantly higher degree of M2 polarization. Dentigerous cysts have a significantly lower M1 polarization than both analyzed periapical lesions (apical granulomas and radicular cysts) and accordingly, a significantly higher M2 polarization than radicular cysts. Macrophage cell density in dentigerous cysts is significantly lower than in the periapical lesions., Conclusions: The development of apical periodontitis towards apical granulomas or radicular cysts might be directed by macrophage polarization. Radicular cyst formation is associated with an increased M1 polarization of infiltrating macrophages. In contrast to radicular cysts, dentigerous cysts are characterized by a low macrophage infiltration and a high degree of M2 polarization, possibly reflecting their developmental rather than inflammatory origin., Clinical Relevance: As M1 polarization of macrophages is triggered by bacterial antigens, these results underline the need for sufficient bacterial clearance during endodontic treatment to prevent a possible M1 macrophage-derived stimulus for radicular cyst formation.

The aim of this study was to compare the number of CD57+ natural killer (NK) cells and CD8+ T lymphocytes between periapical granulomas (PGs) and radicular cysts (RCs). Twenty-fives cases of PGs and 25 of RCs were submitted to histological analysis and immunohistochemistry using anti-CD57 and anti-CD8 biomarkers. Positive cells were counted in 10 fields (400× magnification) and the median value was calculated for each case. Statistical tests were used to evaluate differences in the number of CD57+ NK cells and CD8+ T lymphocytes according to type of lesion, intensity of the infiltrate and thickness of the lining epithelium. The number of CD57+ NK cells and CD8+ T lymphocytes was higher in PGs than in RCs (p = 0.129 and p = 0.541, respectively). Comparison of the number of CD57+ NK cells in atrophic and hyperplastic epithelium revealed a larger number of cells in the atrophic epithelium (p = 0.042). A larger number of CD57+ NK cells and CD8+ T lymphocytes were observed in grade III infiltrates compared to grade I/II (p = 0.145 and p = 0.725, respectively). CD8+ T lymphocytes were more prevalent than CD57+ NK cells in most cases when PGs and RCs were analyzed separately or in combination (p < 0.0001). CD57+ NK cells and CD8+ T lymphocytes play a key role in antiviral defense and the presence of these cells supports evidence suggesting the participation of these microorganisms in the pathogenesis of PGs and RCs. The response mediated by CD8+ T lymphocytes was more frequent, indicating greater participation of the adaptive immunity in these chronic lesions.

Background: Chronic periapical lesions (CPLs) are common lesions of the oral cavity and are the result of caries, tooth fracture, iatrogenic causes, or factors causing contamination and pulp necrosis. Inflammatory cells participate in the expansion of CPLs by releasing factors that stimulate or inhibit osteolytic activity. The objective of this study was to investigate the participation of RANKL, TNF-α, cathepsin K, IL-33, and OPG in the development of radicular cysts (RCs) and periapical granulomas (PGs)., Methods: Paraffin-embedded sections of 30 RCs and 22 PGs were submitted to immunohistochemistry., Results: Immunoexpression of the proteins studied was observed in the epithelium and capsule of RCs, as well as in connective tissue of PGs. The expression of the osteoclastogenic factors studied differed significantly in RCs and PGs (P < .001), with lower expression of OPG in RCs. In PGs, the lowest expression was observed for cathepsin K. Comparison of the 2 lesions showed a similar participation of RANKL and IL33, while a significant difference was observed for OPG (P < .001), TNF-α (P = .002), and cathepsin K (P = .016). No association of the expression of the proteins with lesions size was observed., Conclusions: This study demonstrated the participation of RANKL, TNF-α, IL-33, cathepsin K, and OPG in the development of RCs and PGs, with emphasis on the highest immunoreactivity of cathepsin in RCs and TNF-α and OPG in PGs. OPG possibly determines the slower growth of PGs compared to RCs., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The objective of this study was to evaluate the expression of matrix metalloproteinase 9 (MMP-9) and transforming growth factor beta (TGF-β1) in periapical lesion samples correlated with the intensity of the inflammatory infiltrate and thickness of the epithelial lining. Forty-five cases of periapical lesions (23 periapical granulomas and 22 radicular cysts) were subjected to morphological and immunohistochemical analyses using anti-MMP-9 and anti-TGF-β1 antibodies. The data were analyzed using the following tests: non-parametric Mann-Whitney, chi-square, Fisher's exact test and Spearman's correlation test (P<0.05). Analysis of inflammatory infiltrate revealed that 78% of periapical granulomas presented infiltrate grade III, in contrast with 32% of radicular cysts (P<0.001). Morphological evaluation of the epithelial thickness in radicular cysts revealed the presence of atrophic epithelium in 86% of the cysts. The immunostaining of MMP-9 was score 2 in 67% of the granulomas and 77% of the cysts. Both lesions were predominantly score 1 for TGF-β1. Significant differences were confirmed between the expression scores of TGF-β1 and MMP-9 in periapical granulomas (p = 0.004) and in radicular cysts (p < 0.001). Expression of TGF-β1 was different for periapical granulomas and radicular cysts. This immunoregulatory cytokine seems more representative in asymptomatic lesions. The extracellular matrix remodeling process dependent on MMP-9 seems to be similar for both periapical granulomas and radicular cysts. TGF-β1 and MMP-9 may play an important role in the maintenance of periapical lesions.

The objective of this study was to observe the distribution of macrophages (MPs) expressing transforming growth factor beta-1 (TGF-β1) in tissue samples from patients with different human chronic periapical diseases. In this study, samples were collected from 75 volunteers, who were divided into three groups according to classified standards, namely, healthy control (N = 25), periapical granuloma (N = 25), and periapical cyst (N = 25). The samples were fixed in 10% buffered formalin for more than 48 h, dehydrated, embedded, and stained with hematoxylin and eosin for histopathology. Double immunofluorescence was conducted to analyze the expression of TGF-β-CD14 double-positive MPs in periapical tissues. The number of double-positive cells (cells/mm 2 ) were significantly higher in the chronic periapical disease tissues (P < 0.01) compared to that in the control tissue; in addition, the density of TGF-β1-CD14 double positive cells was significantly higher in the periapical cyst group than in the periapical granuloma group (P < 0.01). The number of TGF-β1 expressing macrophages varied with human chronic periapical diseases. The TGF-β1-CD14 double-positive cells might play an important role in the pathology of human chronic periapical diseases.

Introduction: This study tested the hypothesis that the inflammatory cell profile (CD3-, CD4-, CD8-, CD20-, and CD68-positive cells) and the expression of immunologic markers (tumor necrosis factor α, interferon-γ, interleukin-6, and interleukin-18) in chronic apical periodontitis are the same between non-HIV-infected patients and HIV-infected patients undergoing highly active antiretroviral therapy (HAART)., Methods: Thirty-four surgically excised chronic apical periodontitis lesions were sampled from 34 patients (17 HIV-infected and 17 non-HIV-infected). The lesions were extracted from teeth with no previous endodontic treatment. All HIV-infected patients were undergoing HAART. The specimens were submitted to histopathologic and immunohistochemical analyses by using an optical microscope. Immunoexpression was graded into 2 levels, focal to weak and moderate to strong. The χ(2), Fisher exact, and Mann-Whitney tests were used to analyze all significant differences between groups., Results: Periapical cysts represented 70.6% and 52.9% of the lesions in the HIV-infected and non-HIV-infected groups, respectively; however, no statistically significant difference was observed (P = .481). There were no statistically significant differences between groups for the inflammatory cell profile and for any of the immunologic markers (P > .05)., Conclusions: There are no statistically significant differences of the cellular profile and expression of immunologic markers in chronic apical periodontitis between non-HIV-infected patients and HIV-infected patients undergoing HAART., (Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Aim: To evaluate and compare the immunoexpression of tryptase in samples of periapical granulomas (PGs) and radicular cysts (RCs) correlating it with the type of lesion, localization, intensity of the inflammatory infiltrate and thickness of the cystic epithelial lining, in order to gain insight into the phlogistic role of these cells in the lesions studied., Methodology: Twenty-five PGs and twenty-five RCs obtained from human teeth without endodontic treatment were submitted to morphological and immunohistochemical analysis using anti-tryptase antibody. Mast cells were identified and counted in three regions: intra-epithelial, central/superficial and deep portions. The data were analysed using the Mann-Whitney U-test (P < 0.05)., Results: In comparison with RCs, PGs exhibited higher immunoexpression of tryptase-positive mast cells located in both central/superficial and deep regions (P < 0.001 and P < 0.001, respectively). When considering the total number of mast cells and disregarding the location, the number of tryptase-positive mast cells increased gradually from RCs to PGs (P < 0.001). Lesions with inflammatory infiltrate grade III had greater number of tryptase-positive mast cells located in both central/superficial and deep regions than lesions with inflammatory infiltrates grade II (P = 0.045 and P = 0.025). When the location was ignored, the lesions with inflammatory infiltrate grade III also exhibited higher immunostaining of tryptase-positive mast cells (P = 0.01)., Conclusions: Tryptase-positive mast cells were present in chronic periapical lesions in a larger number in periapical granulomas than in radicular cysts, in both central/superficial and deep regions., (© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Introduction: Interleukin (IL)-17 expression has been detected in apical periodontitis lesions, but its role in the disease process remains unclear. The present study compared the expression of IL-17 in periradicular cysts and granulomas and evaluated the association of this cytokine with clinical and radiographic findings., Methods: Apical periodontitis lesions (18 cysts and 20 granulomas) were obtained from 38 patients subjected to periradicular surgery. Some clinical, radiographic, and cone-beam computed tomographic features were recorded. Silanized slides containing paraffin sections were used for the immunohistochemical reactions using anti-IL-17 antibody. Image analysis was performed using an optical microscope, and each sample was divided into 5 high-power fields, which were evaluated for the expression of IL-17 in the epithelium and connective tissues. Results were evaluated for correlations with the lesion size and the occurrence of symptoms and sinus tract., Results: Expression of IL-17 was significantly higher in cysts than in granulomas (P = .02). Among the periradicular cysts, a thin epithelium showed significantly increased labeling for IL-17 when compared with a hyperplastic epithelium (P = .003). IL-17 expression was usually associated with focal accumulations of polymorphonuclear leukocytes. No association of IL-17 expression with symptoms, sinus tract, or lesion size was observed (P > .05)., Conclusions: The present study reinforces the notion that IL-17 may take part in the pathogenesis of apical periodontitis lesions. A role in the exacerbation of chronic inflammation and cyst formation is suspected. Further studies are required to shed light on the specific functions of IL-17 in periradicular inflammatory processes., (Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Unlabelled: 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., Methods: The 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., Results: The 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)., Conclusion: There 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.

Introduction: Cysts and periapical granulomas are inflammatory reactions that develop in response to periapical infection by microbial species in dental root canal. It is known that toll-like receptors (TLRs) are pathogen recognition molecules and that galectins are lectins that can be associated with the inflammatory process, stimulating or inhibiting the immune system. The objective of this study was to evaluate the in situ expression of TLRs and galectins in radicular cysts and periapical granulomas., Methods: We analyzed 62 cases (30 radicular cysts, 27 periapical granulomas, and 5 control cases). Indirect immunohistochemistry was used to evaluate the expression of TLRs (TRL-2 and TLR-4) and galectins (Gal-3 and Gal-9)., Results: The expression of Gal-3 and Gal-9 was significantly higher in periapical granulomas and radicular cysts than in the control group. Similarly, both Gal-3 and Gal-9 were expressed significantly more in periapical granulomas than in radicular cysts. The expression of TLR-2 was significantly higher in periapical granulomas and radicular cysts than in the control group, and it was also significantly higher in radicular cysts with sinus tract than in the cases without sinus tract. Furthermore, the expression of TLR-4 was significantly higher in the cases of periapical granulomas with sinus tract than in the cases without sinus tract., Conclusions: Gal-3/Gal-9 and TLR-2/TLR-4 expression in the periapical granulomas and radicular cysts is associated with reactive periapical inflammation. Pathobiology of periapical disease is a very complex interplay of many bioactive molecules involved in immunoinflammatory responses. Up-regulation of these bioactive molecules might be an important modulator of inflammatory periapical lesions., (Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: CXC ligand 12/stromal-derived factor-1 (CXCL12/SDF-1) is a pleiotropic chemokine that regulates the influx of a wide range of leukocytes. The aim of this study was to characterize CXCL12/SDF-1 in apical lesions (ALs) of endodontic origin, with special emphasis in associated immune cell populations., Methods: In this case-control study, 29 individuals with chronic apical periodontitis and 21 healthy volunteers were enrolled. ALs and healthy periodontal ligament samples were obtained for tissue homogenization, immune Western blotting, and enzyme-linked immunosorbent assay to determine CXCL12/SDF-1 forms and levels. Anatomopathologic diagnosis, immunostaining for CXCL12/SDF-1, CD117-CXCL12/SDF-1, and toluidine blue were also performed to identify tissue and cell localization. Finally, a set of tissue samples were digested and analyzed by flow cytometry to identify CXCL12/SDF-1 in different immune cell populations. Data were analyzed with Stata v11 and WinDi 2.9 software, and significance was considered if P < .05., Results: CXCL12/SDF-1 was predominantly identified as monomers; levels of CXCL12/SDF-1 were significantly higher in ALs compared with controls, and it was primarily localized to inflammatory infiltrates. Expression of CXCL12/SDF-1 was colocalized to mast cells in tissue sections. Furthermore, CD117(+) mast cells were the second most frequent infiltrating cells and the main CXCL12/SDF-1 expressing cells, followed by CD4(+) lymphocytes, monocytes/macrophages, neutrophils, and dendritic cells., Conclusions: ALs of endodontic origin demonstrated higher levels of CXCL12/SDF-1 compared with controls. CXCL12/SDF-1 was identified in immune cell populations, whereas mast cells represented the major CXCL12/SDF-1 expressing cells, suggesting that this chemokine might play a central role in apical tissue destruction, most probably inducing persistent recruitment of immune cells, particularly of mast cells., (Copyright © 2013 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: Different cell types and cytokines have been identified as contributors to the formation of periapical lesions. In this perspective, this study aimed to evaluate the immunoexpression of interleukin (IL)-17, transforming growth factor (TGF)-β1, and the forkhead box P3 (FoxP3) in periapical lesions, correlating them with the type of lesion, the intensity of the inflammatory infiltrate, and the thickness of the cystic epithelial lining., Methods: Twenty periapical granulomas (PGs), 20 radicular cysts (RCs), and 20 residual radicular cysts (RRCs) were submitted to immunohistochemical analysis using anti-IL-17, anti-TGF-β1, and anti-FoxP3 antibodies., Results: In comparison with PGs and RCs, RRCs exhibited a lower immunoexpression of IL-17 and TGF-β1 (P = .021 and P < .001, respectively). The number of FoxP3+ cells increased in this order: RRCs, RCs, and PGs (P < .001). In comparison with lesions with inflammatory infiltrates grades I and II, lesions with inflammatory infiltrate grade III exhibited a higher number of FoxP3+ cells (P = .002). Similarly, in comparison with lesions with inflammatory infiltrates grades II and III, lesions with inflammatory infiltrate grade I showed a tendency for a lower expression of IL-17 and TGF-β1 (P = .085 and P = .051, respectively). For all groups, there was a positive correlation between the immunoexpressions of IL-17 and TGF-β1 (P < .05). Positive correlations between the number of FoxP3+ cells and the immunoexpressions of IL-17 and TGF-β1 (P < .05) were found only in PGs., Conclusions: Th17 and Treg cells seem to interact at the site of injury, suggesting the involvement of proinflammatory and immunoregulatory cytokines in the pathogenesis of periapical lesions., (Copyright © 2013 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: Chronic dental periapical lesions result from chronic inflammation of periapical tissues caused by continuous antigenic stimulation from infected root canals. Recent findings have suggested that T helper (Th) 1 and Th2-like cytokines are important in the pathogenesis of chronic periapical inflammatory diseases. However, the mechanisms regulating these immunoinflammatory pathways have not been fully elucidated. Thus, the aim of this study was to evaluate interleukin (IL)-4, IL-12, and interferon γ (IFN-γ) protein levels in human radicular cysts and periapical granulomas., Methods: Archived samples of cysts (n = 52) and granulomas (n = 27) were sectioned and submitted to immunohistochemistry to evaluate the tissue expression of IL-4, IL-12, and IFN-γ. The data were analyzed using the Mann-Whitney U test (P < .05)., Results: An increased expression of IFN-γ was observed in radicular cysts. IL-4 expression was stronger in periapical granulomas than in radicular cysts. IL-12 was not detected in any of the samples., Conclusions: Our study showed that IFN-γ protein levels are increased in radicular cysts, whereas IL-4 expression is stronger in samples of periapical granulomas. Further studies are necessary to elucidate the signaling pathways mediated by these cytokines and to facilitate the development of more effective periapical disease management strategies., (Copyright © 2013 American Association of Endodontists. All rights reserved.)

Introduction: The development of periapical granulomas is dependent on the host response and involves Th1, Th2, Th17, and Treg-related cytokines. The discovery of new Th9 and Th22 subsets, with important immunomodulatory roles mediated by interleukin (IL)-9 and IL-22, respectively, emphasizes the need for reevaluation of current cytokine paradigms in context of periapical lesions. We investigated the expression of IL-9 and IL-22 in active and stable human granulomas and throughout experimental lesion development in mice., Methods: Periapical granulomas (N = 83) and control specimens (N = 24) were evaluated regarding the expression of IL-9 and IL-22 via real-time polymerase chain reaction. Experimental periapical lesions were induced in mice (pulp exposure and bacterial inoculation) and the lesions evolution correlation with IL-9 and IL-22 expression kinetics was evaluated., Results: IL-9 and IL-22 mRNA expression was higher in periapical lesions than in control samples; higher levels of IL-9 and IL-22 were observed in inactive than in active lesions. In the experimental lesions model, increasing levels of IL-9 and IL-22 mRNA were detected in the lesions, and inverse correlations were found between IL-9 and IL-22 and the increase of lesion area in the different time point intervals., Conclusions: Our results suggest that Th9 and Th22 pathways may contribute to human and experimental periapical lesion stability., (Copyright © 2013 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Aim: To determine the association of functional single nucleotide polymorphisms in genes of the pro-inflammatory cytokines tumour necrosis factor-α, interleukin-1β, interleukin-8 and interleukin-12B with the development of two clinical forms of apical periodontitis (AP): acute suppurative and chronic nonsuppurative., Methodology: The study included 120 patients from Bucaramanga City, Colombia, 63 diagnosed with acute suppurative AP (ASAP) and 57 diagnosed with chronic nonsuppurative AP (CNAP). Genotyping for IL1B +3954 (rs1143634), IL8 / CXCL8 -251 (rs4073), IL12B +1188 (rs3212227) and TNFA -308 (rs1800629) was performed by the PCR-restriction fragment length polymorphisms method. The statistical analysis was performed using STATA 10.0 and PLINK V1.07 software., Results: Significant differences in the distribution of IL8 / CXCL8 -251 A allele (P adjusted = 0.041; OR adjusted = 0.41, CI adjusted = 0.31-0.97) and IL8 / CXCL -251 TT genotype (P adjusted = 0.04; OR adjusted = 2.24, CI adjusted = 1.04-4.84) were observed comparing patients diagnosed with ASAP and CNAP. No association was observed in genotype and allele distribution for other genetic polymorphisms analysed., Conclusion: This study provides molecular epidemiological evidence that suggests in the present cohort that IL8 / CXCL8 -251 T allele, which is associated with higher production of IL8/CXCL8, is also associated with a higher risk of developing acute suppurative form of AP, whereas IL8 / CXCL8 -251 A allele, which is associated with lower production of IL8/CXCL8, is associated with chronic nonsuppurative form of AP. This suggests a pivotal role for IL-8/CXCL8 in periapical disease because of its ability to induce chemotaxis and modulating the directed migration of neutrophils to the site of inflammation in response to microbial infection of pulp., (© 2012 International Endodontic Journal.)

Introduction: Because herpesviruses might be etiologically involved in periapical pathosis of endodontic origin, this study aimed to determine the occurrence of human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), and the expression of mRNA transcripts of tumor necrosis factor (TNF)-α, γ-interferon (IFN), interleukin (IL)-1β, IL-6, IL-12, and IL-10 in periapical granulomatous lesions collected in conjunction with apicoectomy., Methods: A total of 9 symptomatic and 6 asymptomatic teeth with periapical lesions were studied. Periapical samples were collected in conjunction with apicoectomy, which was being performed because of radiographic evidence of incomplete periapical healing after conventional root canal therapy. By using established polymerase chain reaction primers and procedures, polymerase chain reaction assays were used to identify herpesvirus and cytokine gene expression., Results: The difference in occurrence of HCMV, EBV, and cytokines between symptomatic and asymptomatic periapical lesions was statistically significant: HCMV (P = .048), EBV (P = .002), IFN (P = .001), IL-1 (P = .012), IL-6 (P = .026), IL-10 (P = .026), IL-12 (P = .012), and TNF (P < .001) (Mann-Whitney U test). There was a significant correlation between EBV, HCMV, and TNF, γ-IFN, IL-1, and IL-12 in symptomatic periapical lesions (Spearman test)., Conclusions: The present findings provide evidence of a putative role of HCMV and EBV in the pathogenesis of symptomatic periapical pathosis. The release of tissue-destructive cytokines might be of pathogenetic significance., (Copyright © 2012 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Aim: To investigate the expression of TLR2 in refractory periapical lesions., Methodology: Refractory periapical lesion biopsies were histopathologically and clinically categorized into asymptomatic periapical granuloma (n=10), symptomatic periapical granuloma (n=10) or periapical cyst (n=10) and prepared for immunohistochemical staining using antibodies to TLR2, CD3 and CD19 or staining with methyl green pyronin. Sections were viewed under light microscopy and the presence or absence of the target cells was correlated with the histopathological and clinical data. Additionally, TLR2 expression was quantified by counting TLR(+) cells., Results: Various mononuclear inflammatory cells in the bacteria-induced periapical lesions were reactive to TLR2 antibody, with many showing morphological similarities to lymphocytes and plasma cells. Lymphocytes were the most numerous cells in the inflammatory infiltrate. In refractory periapical granuloma, CD3(+) T cells were more numerous, whereas in periapical cysts, CD19(+) B cells were more numerous. There was a statistically significant (P<0.05) higher expression of TLR2 in symptomatic periapical granuloma than asymptomatic periapical granuloma or periapical cyst., Conclusion: The presence of TLR-expressing cells in periapical granulomas and cysts provides further evidence that periapical cysts are likely to be sustained by the immune system via reaction to bacterial antigens., (© 2011 International Endodontic Journal.)

Aim: To study the expression of monocyte chemotactic protein-3 (MCP-3, also known as chemokine CCL-7) in tissue from apical lesions (AL) and to associate MCP-3 expression with symptomatic or asymptomatic apical periodontitis., Methodology: To determine the expression of MCP-3 in AL, biopsies obtained during tooth extraction procedures were fixed, subjected to routine processing and diagnosed as apical granuloma (AG) (n = 7) or radicular cyst (RC) (n = 5). As controls, apical periodontal ligament (PDL) specimens from healthy premolars extracted for orthodontics reasons were included (n = 7). All specimens were immunostained for MCP-3 and examined under a light microscope. In addition, homogenates from AL (n = 14) and healthy PDL samples (n = 7) were studied through immunowestern blot. Finally, periapical exudates samples were collected from root canals of teeth having diagnosis of symptomatic (n = 14) and asymptomatic apical periodontitis (n = 14) during routine endodontic treatments and analysed by immunowestern blot and densitometry., Results:   MCP-3 was detected in AG and RC and localized mainly to inflammatory leucocytes, whereas no expression was observed in healthy PDLs. MCP-3 was also detected in periapical exudate, and its levels were significantly higher in symptomatic than in asymptomatic apical periodontitis., Conclusions: MCP-3 was expressed in AL and its levels associated with clinical symptoms. MCP-3 might play a role in disease pathogenesis, possibly by stimulating mononuclear chemotaxis., (© 2010 International Endodontic Journal.)

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]-beta), chemokines (macrophage inflammatory protein [MIP]-1beta 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-beta (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., (Copyright 2010 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Background: Periapical lesions are a host response that involves immune reaction to prevent dissemination of bacteria from an infected root canal. The purpose of this study was to evaluate the levels of nitric oxide (NO), IL-4, TGF-beta, tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) in chronic periapical lesions and to determine their possible association with clinical and radiographic parameters., Methods: Seventeen human radicular cysts and 30 periapical granulomas were used in this study. Cytokines and NO were assessed by enzyme-linked immunosorbent assay and by the Griess reaction respectively confirmed by immunohistochemical., Results: TNF-alpha and IFN-gamma were detected in 10% of granulomas and in 41.2% and 70% of radicular cysts. IL-4 was reactive in 24% of cysts, and TGF-beta was positive in all samples. Patients with tenderness showed significantly higher levels of IFN-gamma and IL-4 (P < 0.05). Swelling was associated with high levels of TNF-alpha, IFN-gamma, and IL-4 (P < 0.05). Lesions presenting bone resorption were associated with high levels of NO (P < 0.05)., Conclusions: Periapical granulomas display a regulatory environment characterized by high TGF-beta and low inflammatory cytokine levels, while radicular cysts has mist Th1 and Th2 inflammatory reaction with the presence of IFN-gamma, TNF-alpha, and IL-4.

Objective: The aim of this study was to qualitatively and semi-quantitatively analyze mast cells in periapical lesions., Materials and Methods: Biopsy specimens of 96 periapical lesions were stained with hematoxylin-eosin, histochemical Giemsa and immunohistochemical CD 117 (C kit) antibody. Mast cell count below 100 mast cells on 1000 fields of high power magnification was noted as 'negative', 101-400 as 'mild', 401-800 cells as 'moderate', and over 800 cells as 'severe'., Results: Mast cells are found in 68 (70.8%) lesions. The presence of mast cells was greater in cysts than in granulomas (P < 0.0028). There was no difference in semi-quantitative expression of CD 117 in granulomas and cysts (P > 0.05). Mast cells were placed in both: inflammatory infiltrate and in fibroblastic areas of periapical lesions, and their presence was most frequently mild to moderate., Conclusions: The findings of present study could suggest a role of mast cells in regulation of cellular immune mechanisms in periapical lesions, balancing between alterative and reparatory processes in inflamed periapical tissue.

Introduction: Periapical chronic lesion formation involves activation of the immune response and alveolar bone resorption around the tooth apex. However, the overall roles of T helper type 1 (Th1), Th2, and T-regulatory cell (Treg) responses and osteoclast regulatory factors in periapical cysts and granulomas have not been fully determined. This study aimed to investigate whether different forms of apical periodontitis, namely cysts and granulomas, show different balances of Th1, Th2 regulators, Treg markers, and factors involved in osteoclast chemotaxis and activation., Methods: Gene expression of these factors was assessed using quantitative real-time polymerase chain reaction, in samples obtained from healthy gingiva (n = 8), periapical granulomas (n = 20), and cysts (n = 10)., Results: Periapical cysts exhibited a greater expression of GATA-3, while a greater expression of T-bet, Foxp3, and interleukin-10 (IL-10) was seen in granulomas. The expression of interferon-gamma, IL-4, and transforming growth factor-beta was similar in both lesions. Regarding osteoclastic factors, while the expression of SDF-1alpha/CXCL12 and CCR1 was higher in cysts, the expression of RANKL was significantly higher in granulomas. Both lesions exhibited similar expression of CXCR4, CKbeta8/CCL23, and osteoprotegerin, which were significantly higher than in control., Conclusion: Our results showed a predominance of osteoclast activity in granulomas that was correlated with the Th1 response. The concomitant expression of Treg cell markers suggests a possible suppression of the Th1 response in granulomas. On the other hand, in cysts the Th2 activity is augmented. The mechanisms of periradicular lesion development are still not fully understood but the imbalance of immune and osteoclastic cell activity in cysts and granulomas seems to be critically regulated by Treg cells.

Introduction: Radicular cysts (RCs) are periapical lesions resulting in jaw bone destruction. The inflammatory dental periapical granuloma (PG) is considered to be the origin of RC formation; however the mechanism of RC development remains unclear., Methods: Cell suspension from the surgically extirpated tissue of 27 RCs and 25 PGs was obtained. Bacteriological analysis of the PG tissue samples was performed in order to define two major groups of PG according to the prevailing causative bacterial infection: the streptococcal PG (PG-S, n=10) and the anaerobe PG (PG-A, n=9) group. The inflammatory response of tissue infiltrating lymphocytes was assessed by following T lymphocyte activation (HLA-DR expression) as well as interferon gamma (IFN-gamma) and interleukin 4 (IL-4) production which were evaluated by the flow cytometry., Results: In comparison to RC both types of PG contained a higher proportion of activated T cells (HLA-DR) and lower proportion of IL-4 producing cells. PG-A tissue contained increased percentage of CD3 cells and increased percentage of T helper 1 (Th1) cells in comparison with PG-S. In RC the IFN-gamma production is higher than in streptococcal PG-S but similar as in PG-A., Discussion: Tissue infiltration by Th2 cells and IL-4 production is likely to play an etiopathogenic role in RC formation.

In this study, we examined the role of nitric oxide (NO) in controlling vascular integrity mediated by vascular endothelial (VE)-cadherin in chronic inflammation. Periapical granulomas were analysed for the expression of inducible NO synthase (iNOS) and VE-cadherin, and more iNOS expression than VE-cadherin was shown. Human umbilical vein endothelial cells (HUVECs) were stimulated with proinflammatory cytokines and lipopolysaccharide extracted from Porphyromonas gingivalis and it induced iNOS expression, whereas it reduced VE-cadherin expression, compared with negative controls. On the other hand, pre-incubation with 1400W, an iNOS-specific inhibitor, markedly reduced iNOS expression in stimulated HUVECs and restored VE-cadherin expression to its control level, suggesting that vascular integrity was modulated in conjunction with the reduction of NO. Immunocytochemistry confirmed the functional role of NO in cultured HUVEC monolayers with or without 1400W. These data are consistent with a hypothesis suggesting that NO could attenuate VE-cadherin-mediated vascular integrity in human chronic inflammation.

Inflammatory cytokines contribute to periapical tissue destruction. Their activity is potentially regulated by suppressors of cytokine signaling (SOCS), which downregulate signal transduction as part of an inhibitory feedback loop. We investigated the expression of the cytokines tumor necrosis factor alpha (TNF-alpha); interleukin (IL)-10 and RANKL; and SOCS-1, -2, and -3 by real-time polymerase chain reaction in 57 periapical granulomas and 38 healthy periapical tissues. Periapical granulomas exhibited significantly higher SOCS-1, -2, and -3, TNF-alpha, IL-10, and RANKL messenger RNA levels when compared with healthy controls. Significant positive correlations were found between SOCS1 and IL-10 and between SOCS3 and IL-10. Significant inverse correlations were observed between SOCS1 and TNF-alpha, SOCS1 and RANKL, and SOCS3 and TNF-alpha. Increased SOCS-1, -2, and -3 messenger RNA levels in periapical granulomas may be related to the downregulation of inflammatory cytokines in these lesions; therefore, SOCS molecules may play a role in the dynamics of periapical granulomas development.

This study evaluated the presence and density of natural killer (NK) cells as well as collagen density in chronic apical periodontitis lesions and tried to find any correlations with concomitant herpesvirus infection or histopathological status of the lesion. Surgical specimens of chronic apical periodontitis lesions were surveyed for the presence and density of NK cells by immunohistochemical analysis. Collagen density in these lesions was quantified by means of histochemistry. All specimens were positive for the presence of CD57-positive cells. Topographically, CD57-positive cells were found singly or forming clusters in the granulomatous tissue, as well as subjacent and within the cystic epithelium. No significant differences in the density of CD57-positive cells were found between nonepithelialized and epithelialized lesions or between herpesvirus-positive and herpesvirus-negative lesions. Significant differences were found in volumetric density of collagen when comparing nonepithelialized and epithelialized lesions, with the latter demonstrating higher values. When no distinction of lesion type was made, there was no significant difference in collagen density between herpesvirus-positive and herpesvirus-negative lesions. When comparing the collagen density in herpesvirus-positive and herpesvirus-negative specimens from the same lesion type, a significant difference was found in nonepithelialized lesions, with herpesvirus-positive lesions showing lower values. The presence of CD57-positive cells in all chronic apical periodontitis specimens may indicate that activated NK cells play a role in the pathogenesis of this disease, possibly by participating in innate immunity events involved in the control of virus infection. Collagen density may vary in function of the type of lesion and presence of herpesvirus infection.

Substantial numbers of dendritic cells have been detected in radicular granulomas. To test the hypothesis that local antigen presentation from dendritic cells to T-cells is involved critically in immunological responses within radicular granulomas, we compared characteristics of dendritic cells and macrophages by morphological and biological analyses. Under light microscopy, HLA-DR+ and CD68+ cells showed diverse profiles, including dendritic-shaped cells. Transmission electron microscopy revealed that HLA-DR+ dendritic cells, with long cytoplasmic processes and lacking distinct phagosomes, were concentrated in the lymphocyte-rich area. HLA-DR alpha-chain, CD83, and CD86 mRNAs from HLA-DR+ dendritic cells, and CD28 mRNA from CD28+ T-cells were up-regulated in lymphocyte-rich area. Scanning electron microscopy demonstrated that the density of gold particles on dendritic cells was higher than that on HLA-DR+ macrophages. These results suggest that dendritic cells in radicular granulomas are associated with local defense reactions as stronger antigen-presenting cells, as compared with macrophages.

Background/aim: It has been demonstrated that lymphocytes, plasma cells, macrophages and neutrophil granulocytes represent the predominant cells of the inflammatory lesion of the dental granulomas. Other cells, such as mast cells, eosinophils, dendritic cells comprise minor, but functionally important cell populations. Most of the data considering cells that take part in these processes have been derived from immunohistological studies. This study was undertaken with the aim to determine the phenotype profile of inflammatory cells of dental granulomas using immunohistochemical method in order to study the differences of their quantitative properties and distribution between symptomatic and asymptomatic lesions., Methods: The material for the analysis originated from 42 individuals with clinic and radiographic diagnosis of chronic periapical lesions. The tissue was take either during the periradicular surgery, or tooth extraction. Cryostat tissue sections were stained using the alkaline phosphatase-antialkaline phosphatase assay (APAAP). This method is highly valid and sensitive using a panel of specific monoclonal antibodies: CD3, CD4, CD8, CD19, CD38, CD14, CD1a, CD83, CD80, CD86, CD45 and CD123., Results: The composition of the cell population revealed that there was no homogenous and site-specific pattern of the distribution of inflammatory cells. The results of our investigation revealed that the majority of inflammatory cells comprised lymphocytes and plasma cells, followed by subpopulations CD4+, CD8+ and CD14+ cells. Much lower in number were CD80+, CD86+ and CD83+ and CD1a+ cells. There were no statistically significant differences in mean values of inflammatory cells number between symtomatic and asymptomatic lesions, with the exception of CD86+ cells, the number of which was statistically higher in symptomatic lesions., Conclusion: Inflamatory infiltrate cells in dental granulomas are dominated by T- and B-lymphocytes. It points out the complexity of immunopathogenic events in imitiating and progressing of dental granulomas that involve mechanisms of both cellular and humoral immunity. Regarding the quantitative presence of immunocompetent cells in symptomatic and asymptomatic lesions no statistically significant difference was determined unless in mature dendritic cells present in symptomatic lesions.

The Editorial Board of the Journal of Endodontics has developed a literature-based study guide of topical areas related to endodontics. This study guide is intended to give the reader a focused review of the essential endodontic literature and does not cite all possible articles related to each topic. Although citing all articles would be comprehensive, it would defeat the idea of a study guide. This section will cover potential immunological reactions, endotoxin, and cysts versus granulomas.

Objective: Dental granulomas (DGs) and radicular cysts (RCs) are chronic periapical lesions frequently involving the jaws. Langerhans cells (LCs) are dendritic cells responsible for the presentation of antigens to T lymphocytes. This study examined the expression of LCs in DG and RCs by immunohistochemical staining., Study Design: Eighteen cases of DGs and 26 cases of RCs were analyzed using anti-CD1a marker., Results: CD1a-labeled LCs were observed in 11.1% of DGs and in 69.2% of RCs, showing a significant correlation (P < 0.0001; Fisher's test). In DGs, LCs were only observed in granulation tissue, showing discrete immunostaining density. In RCs, LCs exhibited both a round and a dendritic shape in all epithelial layers. Although a correlation was observed between immunostaining density and epithelial thickness, as well as between immunostaining and inflammatory intensity, the differences were not significant in radicular cysts., Conclusion: Langerhans cells provide important insight into the immunopathogenesis of chronic periapical lesions.

We examined the presence of the receptor for advanced glycation end products (RAGE) in periapical granulomas and analyzed the interaction between RAGE and inducible nitric oxide synthase (iNOS) to elucidate inflammatory reaction mechanisms. Periapical lesions were surgically removed from 37 patients with chronic periapical periodontitis and halved. Paraffin sections were prepared from half of each lesion and stained with hematoxylin and eosin, whereas cryostat sections were prepared from the other half. Based on the histological evaluation, 33 of the lesions were diagnosed as periapical granulomas. These were examined by immunohistochemistry using serial cryostat sections probed with anti-human iNOS or RAGE antibodies. Macrophages, lymphocytes, and endothelial cells expressed RAGE and these cell types, in addition to plasma cells, exhibited anti-iNOS immunoreactivity. Serial cryostat sections demonstrated the infiltration of RAGE-expressing cells around iNOS-producing cells, suggesting that these molecules may be important in the tissue injury associated with periapical periodontitis.

Background: A connection of herpesvirus periapical infection with symptomatic and large-size periapical lesions has been recognized in adult patients, but no data exist about a possible involvement of herpesviruses in severe periapical pathosis in children. Herpesviruses have the potential to elicit potent bone resorption-inducing cytokines in mammalian cells., Aim: This study aimed to determine the occurrence of human cytomegalovirus and Epstein-Barr virus DNA, and mRNA transcripts of receptor activator of nuclear kappa B ligand (RANKL), osteoprotegerin, core binding factor alpha-1, colony stimulating factor-1, transforming growth factor-beta, and monocyte chemoattractant protein-1 in periapical symptomatic pathosis of deciduous teeth., Material and Methods: Twelve deciduous molar teeth from patients aged 2-8 years were extracted due to severe periapical infection, and granulomatous tissue adherent to the root tip of the extracted teeth was collected using a surgical knife. Non-diseased pulpal tissue, obtained from 12 teeth extracted for orthodontic reasons, served as negative control. Polymerase chain reaction assays were employed to identify herpesvirus DNA and cytokine gene expression, using established polymerase chain reaction primers and procedures., Results: Seven (58%) of the periapical lesions yielded human cytomegalovirus and eight (67%) Epstein-Barr virus. Only one (8%) periapical lesion showed neither human cytomegalovirus nor Epstein-Barr virus. In healthy pulpal tissue, one (8%) specimen demonstrated human cytomegalovirus and another (8%) specimen revealed Epstein-Barr virus. Of the cytokines examined, RANKL expression showed significantly higher occurrence in periapical pathosis than in healthy pulpal tissue (P < 0.040). No relationship was identified between the type of herpesvirus and cytokine expression in the periapical lesions studied., Conclusions: The present findings provide evidence of a putative role of human cytomegalovirus and Epstein-Barr virus in the pathogenesis of symptomatic periapical pathosis in deciduous teeth. Increased RANKL expression in periapical lesions may be of pathogenetic significance.

Background: Periapical lesions are thought to be the result of a local inflammatory response mediated by inflammatory cell infiltration and production of inflammatory mediators. Although chemokines are strongly implicated in the migration and activation of leukocytes in different inflammatory diseases and experimental models, little is known regarding the expression of chemokines and their receptors in human apical periodontitis., Objective and Methods: The objective of this study was to determine the expression of chemokines and their receptors by real-time polymerase chain reaction in samples obtained from healthy gingiva, periapical granulomas, and inflammatory periradicular cysts. The inflammatory infiltrate was characterized by immunohistochemistry., Results: Comparing cysts and granulomas, an increase in CD4+ and CD8+ cells was observed in granulomas, despite the similar numbers of CD45RO-positive cells detected in both lesions. The analysis of mRNA expression revealed increased levels of CCR1, CCR2, CCR3, CCR5, CXCR1, and CXCR3 in both types of lesion compared with controls. Cysts exhibited a higher expression of CCR3, CCR5, CXCR1, and CXCR3 compared to granulomas. A significantly higher expression of RANTES, IP-10, and MCP-1 was detected in cysts compared with controls or granulomas. The expression of interleukin-8, MIP-1alpha, and MIP-1beta was not different in the three experimental groups., Conclusions: The increase in Th1 type (CCR1, CCR5, and CXCR3) and Th2 type (CCR2 and CCR3) receptors in both periapical lesions suggests the concomitant occurrence of Th1 and Th2 responses. Furthermore, the prevalent expression of the receptors CCR3, CCR5, CXCR1, and CXCR3 and of the chemokines RANTES, IP-10, and MCP-1 in cysts may point to a role in the progression of granulomas to cysts.

Despite many studies on the topic, plasma cells found in human periapical chronic inflammatory lesions (granulomas) continue to present unresolved issues. In this study, we tried to assess quantitatively and qualitatively the nature of plasma cells of 4 human periapical granulomas. Samples were analyzed for relative amounts of IgG-, IgM-, IgA-, and IgE-positive plasma cells by immunohistochemistry, and for morphological changes by transmission electron microscopy (TEM). By immunohistochemistry, many plasma cells stained positively with anti-IgG and anti-IgM antibodies; fewer cells reacted with anti-IgE and anti-IgA. Russell Bodies, controversial aspects of plasma cell maturation, showed positive reactivity of the superficial layer only to antibodies against IgG and IgM. By TEM analysis, phenotypes of normal and dysfunctional plasma cells (Mott cells) were evident. Russell Bodies appeared as intra- or extracellular round vesicles, with an homogeneous internal core, and an external membrane, resembling rough endoplasmatic reticulum (RER). We can conclude that mucosal immune response is not the predominant type in the periapical lesions examined. Positive immunoreaction for IgG and IgM of Russell Bodies may be due to the residual RER membrane, whereas components of yet unidentified nature may occupy the internal core.

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.

Substance P is a neuropeptide believed to be a major mediator of neurogenic inflammation. The aim of our study was to evaluate whether substance P levels are elevated in the clinical biopsies collected from inflamed periradicular or control tissue. In this study, the presence of substance P was examined in infected human periradicular granulation tissue and control tissue. Sections from 19 periradicular granulomas and pulp tissues from two healthy control teeth were examined using the immunohistochemical method. Substance P-expressing neutrophils, macrophages, and plasma cells were found in both acute and chronic periradicular granulomas. In addition, we observed the presence of neutrophils expressing substance P without concurrent clinical symptoms of acute inflammation. Our results are consistent with the hypothesis that substance P may be released from neutrophils in the inflamed region, and thus, substance P may modulate clinical inflammatory response by release from either neuronal or immunocompetent cell populations.

Periapical granulomas are induced by bacterial infection of the dental pulp and result in destruction of the surrounding alveolar bone. In previous studies we have reported that the bone resorption in this model is primarily mediated by macrophage-expressed interleukin-1 (IL-1). The expression and activity of IL-1 is in turn modulated by a network of Th1 and Th2 regulatory cytokines. In the present study, the functional roles of the Th1 cytokine gamma interferon (IFN-gamma) and IFN-gamma-inducing cytokines IL-12 and IL-18 were determined in a murine model of periapical bone destruction. IL-12-/-, IL-18-/-, and IFN-gamma-/- mice were subjected to surgical pulp exposure and infection with a mixture of four endodontic pathogens, and bone destruction was determined by microcomputed tomography on day 21. The results indicated that all IL-12-/-, IL-18-/-, and IFN-gamma-/- mice had similar infection-stimulated bone resorption in vivo as wild-type control mice. Mice infused with recombinant IL-12 also had resorption similar to controls. IFN-gamma-/- mice exhibited significant elevations in IL-6, IL-10, IL-12, and tumor necrosis factor alpha in lesions compared to wild-type mice, but these modulations had no net effect on IL-1alpha levels. Recombinant IL-12, IL-18, and IFN-gamma individually failed to consistently modulate macrophage IL-1alpha production in vitro. We conclude that, at least individually, endogenous IL-12, IL-18, and IFN-gamma do not have a significant effect on the pathogenesis of infection-stimulated bone resorption in vivo, suggesting possible functional redundancy in proinflammatory pathways.

Oral epithelium may play a regulatory role in local immune responses when interacting with bacteria. The present study was undertaken to investigate the effects of selected bacterial pathogens found in periodontal and endodontic infections on oral epithelial cells. Expression of cell surface molecules (major histocompatibility complex (MHC) Class II, CD54, CD70, CD80 and CD86) and secretion of inflammatory cytokines (interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha) in response to selected bacterial challenge were examined on an immortalized oral epithelial cell line, HOK-18A and a skin epithelial cell line, HaCaT. Actinomyces viscosus, Actinomyces israelii, Fusobacterium nucleatum lipopolysaccharide (LPS) or primary human periradicular exudate from a granuloma were co-cultured with epithelial cells for 4 or 24 h. Subsequently, cell surface expression of MHC Class II, CD54, CD70, CD80 and CD86, along with pro-inflammatory cytokine levels were determined using flow cytometry, ELISA and RT-PCR. Results indicated that the selected oral bacteria have greater effects on oral versus skin epithelial cells. F. nucleatum increased MHC Class II and CD54 (ICAM-1) cell surface expression on HOK-18A and HaCaT cells. A. israelii also had enhancing effects on the expression of CD54 and MHC Class II. A. israelii and LPS induced a 2.8-fold (P < 0.001) and 4.4-fold (P < 0.005) TNF-alpha secretion, respectively, while F. nucleatum and LPS induced a 10-fold (P < 0.0004) and 6-fold (P < 0.01) IL-1beta secretion, respectively by HOK-18A. Interestingly, CD70, CD80, and CD86 were generally decreased upon bacteria and LPS challenge on HOK-18A. The effects of increased MHC Class II and decreased CD70 were also evident with challenge of human periradicular exudate on HOK-18A. The implications of the study are unique in that oral epithelial cells may play both activating and inhibitory roles in the host immune response towards infection by oral bacteria. We introduce a concept of 'dormancy' where the differential expression of key cell surface antigens on oral epithelial cells may keep the recruited immune effector cells in a state of unresponsiveness, thus contributing to the long term quiescent period observed in many periodontal and endodontic lesions.

Background: Substance P (SP), a potent proinflammatory peptide present in sensory neurons, is believed to be a major mediator of neurogenic inflammation. The aim of this study was to examine the localization and involvement of SP, mast cells and tumor necrosis factor-alpha (TNF-alpha)-positive cells in human periapical granulomas., Methods: Sections from seven periapical granulomas were stained using a variety of immunohistochemical methods., Results: SP-immunoreactive nerve fibers, mast cells and TNF-alpha-positive cells were found localized in the vicinity of blood vessels in all the samples of periapical granulomas. The vascular endothelial cells stained positively for E-selectin and intercellular adhesion molecule-1. SP, TNF-alpha-positive cells and E-selectin could not be detected in clinically healthy periodontal ligament, and served as a negative control., Conclusion: Our findings suggest that SP, mast cells, TNF-alpha-positive cells and E-selectin may modulate the pathogenesis of apical periodontitis and may be responsible for stimulating the formation of granuloma with the resorption of periapical bone.

Objective: Distribution and density of CD1a-labeled Langerhans cells (LCs) were examined in human dental periapical inflammatory lesions, and compared with inflammatory cell infiltration or epithelial cell proliferation., Materials and Methods: Eighty three periapical lesions (26 apical granulomas (AGs), 8 epitheliated granulomas (EGs), 34 radicular cysts (RCs), 15 residual radicular cysts (RRCs)) were collected. As control, specimens of periodontal ligaments including Malassez epithelial rests were curetted from 21 teeth. LC densities were measured and various degrees of inflammatory cell infiltration were examined immunohistochemically. Labeling indices for the cellular proliferation markers Ki-67 antigen and DNA topoisomerase II alpha were calculated in the epithelial components., Results: LCs were found in all periapical lesions but not in Malassez epithelial rests. LCs were more abundant in epithelial components than in subepithelial layers. Intraepithelial LCs were more frequent in RCs than in RRCs, whereas subepithelial LCs were less frequent in RRCs than in AGs and EGs. T lymphocytes consistently outnumbered macrophages, plasma cells and B lymphocytes. The range of the CD4/CD8-positive cell ratio differed according to the lesions. Increased LC density was associated with the severity of CD3-positive cell infiltration. Ki-67- and Topo II-LI showed various degrees of epithelial immunoreactivity. There was a significant correlation between LC density and proliferative potential of the epithelium in periapical lesions., Conclusion: These findings suggested that LCs appeared to be associated with T lymphocyte infiltration and proliferative potential of the epithelial tissue in periapical lesions.

The infiltration of leukocytes into inflammation sites such as observed in human periapical granulomas is considered to be mediated by chemotactic factors. In this study, we examined the presence of chemokine- and chemokine receptor-positive cells in samples obtained from human subjects by means of immunohistochemical methods. Macrophage chemotactic protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1alpha, MIP-1beta and IFN-inducible protein 10 (IP-10)-producing cells were present in periapical granulomas. In addition, chemokine receptor CCR3-, CCR5-, and CXCR3-positive cells were also present. In contrast, no factor expression was observed in clinically healthy periodontal ligament, serving as a negative control. Our findings suggest that these chemokines are responsible for modulating the process of disease, such as human apical periodontitis., (Copyright 2001 Academic Press.)

In the periapex, the interaction among inflammatory cells and microorganisms and their products results both in specific and nonspecific immune responses. Many studies have reported quantitative analysis of the immunocompetent cells in periapical lesions, but the exact ratio of these cells in cysts and granulomas remains unknown. In the present study, we undertook a quantitative analysis of CD68+ cells and CD8+ T cells in human periapical granulomas and cysts. Immunoperoxidase staining revealed that CD68+ cells were present in both lesions, with no statistically significant difference, mainly distributed in the inner portion of the lesion, where the inflammation site is more active. On the other hand, CD8+ lymphocytes were more numerous in cysts. Thus, it appears that CD8+ T cells may play a more important role in a later phase of periapical lesion progression, probably exerting regulatory or cytotoxic functions in cellular immune response, which may lead to the stabilization of these lesions.

To study mediators associated with the progression of disease and the process of bone regeneration in human apical periodontitis, we examined samples of periapical granulation tissues and regeneration tissues obtained from five patients by use of immunohistochemical methods. Periapical granulation tissues were found to contain a large number of CD4-positive T cells and CD68-positive monocytes/macrophages (CD4: 35.2%, CD68: 32.7%). The CD4-positive T cells and CD68-positive monocytes/macrophages were predominantly present in regeneration tissues (CD4: 62.1%, CD68: 16.0%). In these the percentages of CD4-positive T cells were higher as compared with periapical granulation tissues (from 35.2% to 62.1%). In periapical granulation tissues, CD4-positive T cells stained positively for interferon-gamma (IFN-gamma) and negatively for interleukin-4 (IL-4). In regeneration tissues, IL-4-producing cells could be detected. However, IFN-gamma-producing cells could not be detected. These results suggest that IFN-gamma and IL-4 may modulate the pathogenesis of infectious disease and the process of bone regeneration in local inflammation sites such as human apical periodontitis., (Copyright 2001 Academic Press.)

Immunoglobulin synthesizing plasma cells for IgG were quantitated in 20 periapical granulomas and 20 periapical cysts, using unlabelled antibody peroxidase-antiperoxidase complex method. Result showed that immunoglobulin G producing plasma cells were predominant in periapical cyst as compared with periapical granuloma. A statistical significant relation was observed between these two lesions.

Objective: Proinflammatory cytokines are involved in the pathogenesis of periapical lesions. The purpose of this study was to evaluate the presence of the cytokines tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta(1) (TGF-beta(1)) in periapical pathosis and to determine their relationship to the size of the lesions., Study Design: One tooth from each of 25 patients was root-end resected, and the periapical lesion was collected. The amounts of TNF-alpha and TGF-beta(1) were assessed by enzyme-linked immunosorbent assay., Results: TGF-beta(1) was detected in 21 of 25 lesions. In samples with scar tissue, no TGF-beta(1) activity was detected. A statistically significant correlation was found between TGF-beta(1) per milligram of tissue and the diameter of the lesions. TNF-alpha was detected in only 2 samples., Conclusions: TGF-beta(1) was present in periapical granulomas and cysts but not in lesions with scar tissue. The correlation between the amount of TGF-beta(1) per milligram of tissue and the size of the lesion was significant.

Apical periodontitis is initiated primarily by the mixed microflora of infected root canals. Continuous flow of bacteria and their products through the apical foramen induces influx, activation and coordinated interaction of immune-inflammatory cells within the periapical area. Successful mobilization of host defense mechanisms prevents abundant extraradicular bacterial invasion. However, anti-infective effector mechanisms are not restricted to killing the invading microorganisms but also destroy normal tissue components and induce bone absorption, resulting ultimately in the loss of the affected teeth. Moreover, autocrine and paracrine loops of stimulation may lead to the perpetuation of the local inflammatory lesion and may also alter the function of remote tissues and organs. This review attempts to summarize current knowledge about the pathogenic mechanism of apical periodontitis, focusing on the formation of a special granulation tissue that effectively fights bacteria originated from the infected pulp chamber and, by exerting this protective function, also contributes to harmful local and distant events. The dynamic equilibrium between defensive and destructive mechanisms may provide a pathobiological basis for better understanding of clinical signs and symptoms of various forms of apical periodontitis lesions and influence treatment strategy and practice.

The aims of this study were to investigate the expression of pro-inflammatory, anti-inflammatory and immune-related cytokines present in periapical lesions. We investigated the expression of cytokines: namely interleukins IL-2, IL-4, IL-6, IL-10 and interferon-gamma (IFN-gamma) in formalin-fixed, paraffin-embedded sections of periapical granulation tissue. The study samples were biopsies from 24 patients with periapical lesions: 12 with periapical granulomas and 12 patients with radicular cysts. Immunohistochemistry was also performed on tonsillar tissue which served as a control. We utilised a set of specific monoclonal antibodies and polyclonal monospecific antibodies to detect cells that expressed the different cytokines within the tissues. We also considered the nature of the periapical immune response by investigation of the T-helper 1 (Th-1) and T-helper 2 (Th-2) lymphocyte subsets using their cytokine profile, i.e., Th-1: IL-2 and IFN-gamma and Th-2: IL-4, IL-5 and IL-6. Only a few cells were weakly positive for the IL-2 protein in each of the tissue sections. Cells that expressed IL-4 or IL-6 were far more numerous than cells that expressed either IL-2 or IFN-gamma. Thus, we demonstrated a greater number of Th-2 cells in periapical lesions. This relative ratio of the T-cell subsets underlines the importance of the anti-inflammatory mechanisms taking place in the diseased tissue manifested by the wide array of IL-10-expressing cells: B cells, T suppressor cells (CD8 (+)) and tissue macrophages. The numbers of inflammatory cells expressing the anti-inflammatory molecules far outnumbered the cells that expressed pro-inflammatory cytokines. Thus, the downregulation of the inflammatory response and the predominant Th-2 or humoral immune response in periapical periodontitis may be important features that dictate the outcome of the disease process in the periapical lesion.