Immunological profile of teeth with inflammatory periapical disease from chronic liver disease patients.

AIM: To evaluate the mRNA expression levels of the cytokines interferon-γ, tumour necrosis factor-α, interleukin (IL)-1ß, IL-10, IL-6, VEGF, and AGT and the chemokine CCL2/MCP-1 in periapical interstitial fluid associated with root canal infections before and after the reduction of the bacterial load using a cleaning procedure. METHODOLOGY: The case group included 11 patients with chronic liver disease, and the control group included 11 healthy patients. Clinical samples were taken from teeth with pulp necrosis. After cleaning and drying the canal, three paper points were introduced into the root canal and passed through the root apex (2 mm) into the periapical tissues for 1 min. The samples were collected immediately after root canal cleaning and 7 days later to characterize those gene expression levels using real-time PCR. The data were subjected to the Shapiro-Wilk and the Wilcoxon tests. RESULTS: In the control group, significantly increased expression of the pro-inflammatory cytokines IFN-γ and TNF-α was observed in teeth with restrained bacterial loads (day 7) (P < 0.05). Similarly, increased TNF-α expression was found on day 7 in the liver group (P < 0.05). No differences were observed in the expression levels of the IL-1ß, IL-10 and, IL-6, MCP-1/CCL-2 and VEGF between the first collection (day 0) and second collection (day 7), over time in either group. CONCLUSION: Chronic liver disease patients exhibited sufficient immunologic ability showing relatively similar expression levels of cytokines, chemokines and angiogenic factors in periapical samples compared with the responses from no-chronic liver disease patients. The outcomes of this study suggest that liver impairment did not compromise the periapical immune response.

Methyl mercaptan and hydrogen sulfide products stimulate proinflammatory cytokines in patients with necrotic pulp tissue and endodontically treated teeth.

Bacterial infections of the residual dentin or infected pulp tissue are responsible for most cases of endodontic treatment failures. Persisting microorganisms in necrotic pulp tissue produce sulphur components such as methyl mercaptan and hydrogen sulfide as well as thioether derivatives. Although there is emerging evidence that these sulphur compounds stimulate immune cells and induce the inflammatory cascade, the immunological mechanisms of local and systemic inflammation have not been described. In this retrospective study we evaluated the ex-vivo immune response of peripheral blood mononuclear cells to sulphur compounds in 53 patients with clinical or radiologic endodontic treatment failure, 20 patients with clinical discomfort or radiological findings without previous endodontic treatment and a control group of 31 patients who had received successful endodontic treatment at least five years previously. Patients with endodontic abnormalities showed significantly higher ex-vivo sulphur compound-stimulated interferon-gamma (IFN-γ) and interleukin-10 (IL-10) levels as compared to the control group. The association between ex-vivo-stimulated cytokines and endodontically derived sulphur compounds was further substantiated by the fact that the number of IFN-γ and/or IL-10-positive patients decreased significantly 3-8 months after re-treatment of the root canal or tooth extraction. Furthermore, serum tumor necrosis factor-alpha (TNF-α) levels were higher in patients than in controls, and at the same time, the TNFA -308 G/A polymorphism was associated with endodontic treatment failure in our study population. We conclude that a cellular immune response to sulphur compounds contributes to the inflammatory process observed in relation to endodontic treatment failures.

Immunological profile of periapical endodontic infections from HIV- and HIV+ patients.

AIM: To evaluate CD4(+) CD28(+) and CD8(+) T-cell genes and the gene expression of IFN-γ, TNF-α, IL-1-ß, IL-17A, IL-10, CCL-2/MCP-1, CCL-4, CCL-5 (RANTES), CXCR4, CCR5 and RANKL from cells in the periapical interstitial fluid from root canal infections in healthy patients (HIV-) and HIV-positive individuals (HIV+). METHODOLOGY: Subjects included 20 HIV- and 23 HIV+ patients referred to the School of Dentistry at the Universidade Federal de Minas Gerais (Belo Horizonte, MG, Brazil). Almost all HIV+ patients were undergoing highly active antiretroviral therapy (HAART). Clinical samples were taken from teeth with pulp necrosis, and no patients had acute periapical symptoms at the time of the appointments. After cleaning and drying, 3 paper points were introduced into the root canal, passing passively through the root apex (2 mm) into the periapical tissues for 1 min. The samples were collected immediately after root canal cleaning and 7 days later (restrained root canal bacterial load) to characterize those gene expressions using real-time PCR. RESULTS: Significantly higher levels of CD4(+) CD28(+) and CD8(+) T cells in teeth with restrained bacterial loads (second collection) compared with the first collection were observed in both HIV- and HIV+ samples. In HIV- patients, an increase in IL-10 and CXCR4 expression was demonstrated as well as a decrease in pro-inflammatory cytokines such as RANKL, IFN-γ, IL1-ß and CCL5. However, in HIV+ patients an increase in cytokines IFN-γ, IL-1-ß, TNF-α and IL-17A, and chemokines CCL-2, CXCR4 and CCR5 were observed. The chemokine CCL-5 was not detected in HIV+ individuals. CONCLUSIONS: These findings suggest that after reducing the root canal bacterial load in HIV- individuals an anti-inflammatory response is generated whilst in HIV+ patients a pro-inflammatory response is sustained in the periapical area.

Influence of genetic regulatory effects modified by environmental immune activation on periapical disease.

The objective of this study was to compare the periradicular responses in endodontic infections among members of two populations: an urban Brazilian population and a non-mixed indigenous population. Samples were collected immediately and 7 days after the cleaning and shaping procedures (after reducing the intracanal microbial load) in an attempt to characterize the expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-9, interferon (IFN)-γ, IL-17, IL-10, and the chemokines CXCR4, CCL2/monocyte chemotactic protein (MCP)-1, and CCR6. The endogenous cytokine and chemokine expression levels were analyzed using real-time PCR. Only the urban population showed a significant increase in TNF-α, CCL2/MCP-1, CXCR4, and CCR6 expression following the cleaning and shaping of the root canal system. The IFN-γ levels were increased at the 2nd collection (p < 0.05) in the indigenous population. In turn, a significant increase in IL-10 and IL-17 expression (p < 0.05) was observed after the cleaning and shaping procedures (2nd collection) in both populations. No significant differences in the IL-1ß, IL-9, and CCL4 expression levels were observed between the 1st and 2nd collections in both populations. The results demonstrate a cytokine and chemokine expression profile that is specific to each analyzed population. However, immune modulation mediated by IL-10 began on the 7th day after the beginning of the endodontic treatment in both populations.

Cytokines and chemokines associated with Treg/Th17 response in chronic inflammatory periapical disease.

Cytokines and chemokines have a fundamental role in the maintenance of inflammation and bone response, which culminate in the development of chronic periapical lesions. Regulatory (Treg) and Th17 cytokines play a key role in regulating the immune response involved in this process. The aim of this study was to investigate the role of Treg and Th17 cells in chronic inflammatory periapical disease, by comparing the expression of the immunoregulatory mediators TGF-ß, IL-10, CCL4, and the proinflammatory IL-17 and CCL20 in the periapical tissue of teeth with pulp necrosis, with and without associated chronic lesions. Eighty-six periapical tissue samples were obtained from human teeth. The samples were divided into three groups: pulp necrosis with a periapical lesion (n=26); pulp necrosis without a periapical lesion (n=30), and control (n=30). All samples were submitted to histopathological analysis and cytokine and chemokine measurement through ELISA. Statistical analyses were done with Kruskal-Wallis and Mann-Whitney tests and Spearman correlation. The group with pulp necrosis and a periapical lesion showed a higher expression of CCL4 and TGF-ß in comparison with pulp necrosis without a lesion. CCL20 was higher in the group with a periapical lesion when compared to the control. In all groups there was a weak positive correlation between IL-17/CCL20, IL-10/CCL4, and IL-17/TGF-ß. Both types of cytokines, pro-inflammatory and immunoregulatory, occur simultaneously in periapical tissue. However, a rise in immunosuppressive cytokines and chemokines (CCL4 and TGF-ß) in periapical lesions suggests a role of these cytokines in stable periapical disease.

Granulocyte colony-stimulating factor induced reduction in pulpal necrosis.

AIM: To examine the effect of recombinant granulocyte colony-stimulating factor (G-CSF) on the number and function of neutrophils and on the histopathology of pulpal inflammation in normal and neutropenic rats. METHODOLOGY: The effect of G-CSF on changes in pulpal tissue was investigated at 2, 4, 7, and 10 days after pulpal exposure of the mandibular first molar of normal rats and of those with methotrexate-induced neutropenia. The area of pulpal necrosis was measured. The neutrophil count in peripheral blood was determined, and their phagocytosis and chemotactic reaction were also examined. Statistical significance was examined by use of the two way analysis of variance. RESULTS: In untreated rats, G-CSF significantly (P < 0.05) increased the number of peripheral neutrophils and their chemotactic reaction, but did not affect pulpal inflammation. In methotrexate-induced neutropenic rats, the phagocytosis and migration of neutrophils reduced, and the area of pulpal necrosis enlarged. After the G-CSF injection, the decreases in neutrophil count and their functions significantly (P < 0.05) reversed, and the enlargement of pulpal necrosis inhibited. CONCLUSIONS: These findings indicate that G-CSF prevented the reduction in neutrophil function and reduced the pulpal necrosis observed in the neutropenic rats, and suggest that neutrophils defend against bacterial invasion in pulpal tissue.

Role of IL-2 and helper T-lymphocytes in limiting periapical pathosis.

The purpose of this study was to examine the role of IL-2 and helper T-lymphocytes in the development of periradicular lesions in rats. In control animals, periradicular lesions developed within 28 days following pulpal infection. Immunologically, some anti IL-2 and anti CD4-reactive helper T-lymphocytes infiltrated the periapical tissue at 14 days, and their numbers increased at 28 days. In experimental animals, tacrolimus (FK506) was injected every day to inhibit the IL-2 production by helper T-cells. Histologically, the pulpal necrosis and periradicular inflammation in tacrolimus-treated rats were more severe than those in the control rats. Furthermore, the areas of pulpal necrosis and periradicular lesion in the immunosuppressed rat were significantly greater than those in the normal ones. The numbers of IL-2- and CD4-positive cells in the lesion of the experimental rats were statistically lower than those of the control ones. These results show that the decrease in IL-2 might have promoted the development of periradicular lesions.

Reduction of infection-stimulated periapical bone resorption by the biological response modifier PGG glucan.

Pulpal and periodontal diseases are bacterial infections which result in local connective tissue and bone destruction. Effective host resistance to these infections is primarily mediated by neutrophils and other phagocytic cells. PGG glucan (poly-beta 1-6-glucotriosyl-beta 1-3-glucopyranose glucan) is a biological response modifier which stimulates the production of neutrophils and upregulates their phagocytic and bactericidal activity. In the present studies, the effect of PGG glucan on infection-stimulated alveolar bone resorption was tested in an in vivo model. Periapical bone resorption was induced in Sprague-Dawley rats by surgical pulp exposure and subsequent infection from the oral environment. Animals were administered PGG glucan (0.5 mg/kg) or saline (control) subcutaneously the day before and on days 2, 4, 6, 9, 11, 13, 16, and 18 following the pulp exposure procedure. PGG glucan enhanced the number of circulating neutrophils and monocytes and increased neutrophil phagocytic activity approximately two-fold. PGG glucan-treated animals had significantly less infection-stimulated periapical bone resorption than control animals, as determined radiographically (-48.0%; p < 0.001) and by histomorphometry (-40.8% and -42.4% for first and second molars, respectively; p < 0.001). PGG glucan-treated animals also had less soft tissue destruction, as indicated by decreased pulpal necrosis. Only 3.3% of the first molar pulps from PGG glucan-treated animals exhibited complete necrosis, as compared with 40.6% of pulps from controls. Finally, PGG glucan had no effect on either PTH- or IL-1-stimulated bone resorption in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

IL-1 alpha and TNF-alpha expression in early periapical lesions of normal and immunodeficient mice.

T-helper and B-lymphocytes may contribute to mechanisms that result in bone-resorptive cytokine production in periapical lesion. Mice with severe combined immunodeficiency (scid) lack functional B- and T-cell immunity. The purpose of this study was to investigate the progression of pulp necrosis and the histomorphometric features of periapical lesions in scid vs. normal mice. The expression of the bone-resorptive cytokines IL-1 alpha and TNF-alpha was also investigated. Sixteen five-week-old homozygous scid mice and 14 normal BALB/cJ mice were used. The pulps of mandibular first molars were exposed for 1, 2, 3, or 4 weeks. Blocks of tissue containing the mandibular teeth and supporting structures were processed for both light microscopic examination and immunohistochemical staining for IL-1 alpha dna TNF-alpha. Central sections were randomized, their images were blindly digitized into a computer, and the areas of the lesions surrounding the distal root apices were measured. The cells that stained positively for the cytokines in the same area of adjacent sections were counted. Pulp necrosis progressed at similar rates in teeth from both strains. A progressive and significant increase in the periapical lesion size in both strains was observed. The scid mice lesions were significantly smaller than the controls at only the three-week period. There was heavy cytokine staining in periapical lesions from both strains, especially in areas that contained a mixed inflammatory infiltrate or fibroblasts. The number of positively staining cells was proportional to the lesion size. Therefore, pulpal and periapical pathosis were independent of the presence of functional T- and B-cells in this model.

Determination and relationship of C-reactive protein in human dental pulps and in serum.

C-reactive protein (CRP), an acute phase protein synthesized by the liver, increases in serum as much as 3000 times above its normal level in response to acute inflammation. The purpose of this study was to determine whether CRP levels in dental pulps could be correlated with the histological disease status of the pulp and with systemic blood levels of CRP. Inflamed and necrotic pulps were extirpated during routine endodontic therapy. Normal pulps were removed from extracted, intact third molars. One half of each pulp specimen was placed in formalin for histological study; the other half was frozen for immunological study. A serum sample was obtained from each patient at the end of the dental visit. CRP levels were determined by the enzyme-linked immunosorbent assay. Pulps were categorized histologically as normal, inflamed, inflamed/necrotic, or necrotic. The correlation between CRP levels of pulp and serum was not significant. CRP levels of normal pulps differed significantly only from inflamed pulps (p less than 0.05, Dunnett). This increase in CRP appears to be a local phenomenon resulting from the interaction of CRP with various inflammatory mediators in the pulp.

Evaluating IL-2 levels in human pulp tissue.

In murine and human CD4+ T cell populations, there are three subpopulations of T helper cell types. Hahn et al. demonstrated the ratio of CD4/ CD8 + cells significantly increases in inflamed dental pulps compared with normal pulps. Elevated levels of interleukin (IL)-2 have been detected in inflamed dental pulps and the level of IL-2 could be used as a marker for inflammation. In this study, levels of IL-2 were evaluated by using a human IL-2 cytokine assay kit on 80 samples of freshly extracted human pulp tissue. Applying standard diagnostic procedures, the tissue samples were clinically categorized into one of three experimental groups. The results demonstrated that there were no significant differences between the concentrations of IL-2 in any of the experimental groups. Our findings are different from results reported previously. Further investigation is warranted to determine if a correlation exists between the concentration of IL-2 or other interleukins and the degree of inflammation present in the dental pulp.

The serum immunoglobulins level in maxillofacial bacterial infections.

The IgG, IgA and IgM levels in the serum of 23 patients with maxillofacial bacterial infections were determined. The high IgG level noted on admission further increased in the serum of 14 patients with acute inflammation and decreased in the serum of 10 patients with a chronic course. The level of IgM in the serum of patients with acute inflammation was higher than in the patients with chronic inflammation and decreased in both groups on the 7th day of treatment. The IgA level was high on admission in both groups and fell on the 7th day of treatment. Our results suggest that analysis of the serum level of IgG, IgA and IgM may be of diagnostic and prognostic value in the treatment of maxillofacial bacterial inflammation.

Cytokines and chemokines associated with Treg/Th17 response in chronic inflammatory periapical disease

Abstract Cytokines and chemokines have a fundamental role in the maintenance of inflammation and bone response, which culminate in the development of chronic periapical lesions. Regulatory (Treg) and Th17 cytokines play a key role in regulating the immune response involved in this process. The aim of this study was to investigate the role of Treg and Th17 cells in chronic inflammatory periapical disease, by comparing the expression of the immunoregulatory mediators TGF-β, IL-10, CCL4, and the proinflammatory IL-17 and CCL20 in the periapical tissue of teeth with pulp necrosis, with and without associated chronic lesions. Eighty-six periapical tissue samples were obtained from human teeth. The samples were divided into three groups: pulp necrosis with a periapical lesion (n=26); pulp necrosis without a periapical lesion (n=30), and control (n=30). All samples were submitted to histopathological analysis and cytokine and chemokine measurement through ELISA. Statistical analyses were done with Kruskal-Wallis and Mann-Whitney tests and Spearman correlation. The group with pulp necrosis and a periapical lesion showed a higher expression of CCL4 and TGF-β in comparison with pulp necrosis without a lesion. CCL20 was higher in the group with a periapical lesion when compared to the control. In all groups there was a weak positive correlation between IL-17/CCL20, IL-10/CCL4, and IL-17/TGF-β. Both types of cytokines, pro-inflammatory and immunoregulatory, occur simultaneously in periapical tissue. However, a rise in immunosuppressive cytokines and chemokines (CCL4 and TGF-β) in periapical lesions suggests a role of these cytokines in stable periapical disease.

Signaling pathways activation by primary endodontic infectious contents and production of inflammatory mediators.

INTRODUCTION: This study investigated the bacterial community involved in primary endodontic diseases, evaluated its ability to activate the macrophage Toll-like receptor 4 receptor through p38 mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) signaling pathways, and determined the levels of endotoxins and interleukins (interleukin [IL]-6 and -10) produced by endodontic content-stimulated macrophages. METHODS: Samples were taken from 21 root canals by using sterile/apyrogenic paper points. Raw 264.7 macrophages were stimulated with root canal contents. Checkerboard DNA-DNA hybridization was used for bacterial analysis and the limulus amebocyte lysate assay for endotoxin measurement; p38 MAPK and NF-κB activation was determined by Western blot analysis. IL-6 and IL-10 were measured using the enzyme-linked immunosorbent assay. RESULTS: Bacteria and endotoxins were detected in 100% of the samples (21/21). The most frequently observed species were Parvimonas micra (16/21, 76%), Fusobacterium nucleatum ssp. nucleatum (15/21, 71%), and Porphyromonas endodontalis (14/21, 66%). Correlations were found between endotoxins and IL-6 and IL-10 (P < .05); p38 phosphorylation had a peak at 60 minutes, and NF-κB was quickly activated after 10 minutes of stimulation. CONCLUSIONS: It was concluded that the complex bacterial community was shown to be a potent activator of TLR-4 determined by the p38 MAPK and NF-κB signaling pathways, culminating in a high antigenicity against macrophages through the levels of IL-6 and IL-10, all significantly affected by endotoxin levels.

The impact of chlorhexidine-based endodontic treatment on periapical cytokine expression in teeth.

INTRODUCTION: Root canal treatment typically involves cleaning and shaping procedures followed by treatment with antibacterial endodontic dressing between appointments and, ultimately, 3-dimensional,hermetic filling. Chlorhexidine (CHX) is effective as an irrigation solution and is used as an endodontic dressing. The aim of this study was to examine the influence of CHX on periapical cytokine expression. METHODS: Expression levels of the cytokines interferon γ, tumor necrosis factor α, interleukin (IL)-1ß, IL-17A, IL-10, and the chemokine monocyte chemoattractant protein-1 (CCL2/MCP-1) were assayed by real-time polymerase chain reaction immediately after root canal cleaning and 15 days later. RESULTS: Messenger RNA expression of IL-1ß, interferon γ, IL-10, and CCL2/MCP-1 was increased on day 15 in teeth without endodontic dressing. No statistical change was observed in the messenger RNA expression of cytokines when comparing sampling times for teeth that received endodontic dressing. CONCLUSIONS: The results show that CHX application between appointments prevented the increase of both proinflammatory and immunoregulatory cytokines 15 days after the dental procedure.

Toll-like receptor 2 knockout mice showed increased periapical lesion size and osteoclast number.

INTRODUCTION: The aim of this study was to characterize the formation and progression of experimentally induced periapical lesions in TLR2 knockout (TLR2 KO) mice. METHODS: Periapical lesions were induced in molars of 28 wild type (WT) and 27 TLR2 KO mice. After 7, 21, and 42 days, the animals were euthanized, and the mandibles were subjected to histotechnical processing. Hematoxylin-eosin-stained sections were examined under conventional light microscopy for the description of pulpal, apical, and periapical features and under fluorescence microscopy for the determination of the periapical lesion size. The subsequent sections were evaluated by tartrate resistant acid phosphatase histoenzymology (osteoclasts), Brown and Brenn staining (bacteria), and immunohistochemistry (RANK, RANKL, and OPG). Data were analyzed by the Mann-Whitney U and Kruskal-Wallis tests (α = 0.05). RESULTS: The WT group showed significant differences (P < .05) in the periapical lesion size and the osteoclast number between 7 and 42 days and between 21 and 42 days. In the TLR2 KO group, significant differences (P < .05) in the periapical lesion size and the osteoclast number were found between 7 days and the other periods. There was a significant difference (P < .05) between the 2 types of animal regarding the periapical lesion size, which was larger in the TLR2 KO animals. No significant differences (P > .05) were found between WT and TLR2 KO mice related to the pulpal, apical, and periapical features; bacteria localization; and immunohistochemical results (except for RANK expression). CONCLUSIONS: TLR2 KO animals developed larger periapical lesions with a greater number of osteoclasts, indicating the important role of this receptor in the host's immune and inflammatory response to root canal and periradicular infection.

T-lymphocyte and cytokine expression in human inflammatory periapical lesions.

INTRODUCTION: Lymphocytes, among many cells, express different sets of cytokines, chemokines, and receptors, which are considered important mediators of periapical immune response to infection. METHODS: The aim of this study was to evaluate the mRNA expression of CD4(+)CD28(+) and CD8(+) T genes and the gene expression of interferon-γ, tumor necrosis factor-α, interleukin (IL)-1ß, IL-17A, IL-10, CCL2/MCP-1, CCL4, CCL5, CXCR4, CCR5, and receptor activator for nuclear factor kappa B ligand (RANKL) in periapical interstitial fluid from human root canal infections. The samples were collected immediately after root canal cleaning and 7 days later (restrained root canal bacterial load) to characterize those gene expressions. RESULTS: Real-time polymerase chain reaction demonstrated significantly higher levels of CD4(+)CD28(+) and CD8(+) T-cell markers in the former root canal condition and an increase of IL-10 and CXCR4, followed by a decrease of proinflammatory cytokines such as RANKL, interferon-γ, IL-1ß, and CCL5. CONCLUSIONS: Analyses of T-lymphocyte and cytokine expression in periapical area were able to show that distinct root canal conditions might play regulatory roles in controlling local immune/inflammatory processes.

Inflammatory myofibroblastic tumor of the maxillary sinus related with pulp necrosis of maxillary teeth: case report.

Inflammatory myofibroblastic tumor (IMT) is a benign lesion composed of myofibroblasts accompanied by varying numbers of inflammatory cells. Various pathogenetic factors have been proposed, but the etiology of most IMTs remains unknown. This article presents a case of IMT occurring in the left maxillary sinus. A 24-year-old man complained of throbbing pain in the maxillary left molars and swelling of the left cheek. His maxillary left second molar was diagnosed as pulp necrosis and root canal treatment performed. After that, his symptoms continued and he was referred to the Department of Otolaryngology. Computerized tomography disclosed compact soft tissue masses in the left maxillary sinus with obstruction of maxillary ostium. Under general anesthesia, the lesions were fully excised. Histopathologically, the lesions were composed of plump or spindled myofibroblasts. Cells were immunoreactive for smooth muscle actin and ß-catenin, and were negative for ALK1, CD34, and EMA. The diagnosis was IMT of left maxillary sinus. Although it is very rare, IMT should be included as a differential diagnosis in patients with compact masses in maxillary sinus.

Antigenicity of primary endodontic infection against macrophages by the levels of PGE(2) production.

INTRODUCTION: Root canal contents are potent stimuli for proinflammatory cytokines involved in apical periodontitis. This study investigated target gram-negative bacterial species and endotoxins in primary endodontic infection with apical periodontitis, determined their antigenicity against macrophages through the levels of PGE(2), and evaluated their relationship with clinical findings. METHODS: Samples were taken from 21 root canals with primary infection and apical periodontitis by using paper points. Polymerase chain reaction (16S rDNA) was used for bacterial detection and limulus amebocyte lysate assay for endotoxin measurement. Levels of prostaglandin E2 (PGE(2)) were measured by enzyme-linked immunosorbent assay (Duoset Kit; R&D, Minneapolis, MN). RESULTS: Prevotella nigrescens (13/21), Fusobacterium nucleatum (6/21), and Porphyromonas endodontalis (6/21) were the most frequently observed species. A positive association was found between F. nucleatum and P. endodontalis (P < .05). A correlation was found between the number of gram-negative bacterial species and the levels of endotoxins, such as PGE(2) (P < .05). Higher levels of endotoxin were detected in teeth with exudation, whereas elevated levels of PGE(2) were found in teeth with tenderness to percussion and pain on palpation. CONCLUSIONS: Our findings imply an additive effect between the number of gram-negative bacterial species involved in endodontic infection regarding the induction of proinflammatory cytokine by macrophage cells. Moreover, teeth with clinical symptomatology were related to higher levels of endotoxins and PGE(2) secretion.

Antigenic activity of bacterial endodontic contents from primary root canal infection with periapical lesions against macrophage in the release of interleukin-1beta and tumor necrosis factor alpha.

INTRODUCTION: Periradicular tissue chronic stimulation by endotoxin may cause apical periodontitis. This study investigated the microbial profile and the levels of endotoxin found in primary root canal infection with apical periodontitis, determined their antigenicity against macrophages through the levels of interleukin (IL)-1beta and tumor necrosis factor alpha (TNF-alpha), and evaluated their relationship with clinical and radiographic findings. METHODS: Samples were taken from 21 root canals with primary endodontic infection and apical periodontitis with paper points. PCR technique (16S rDNA) was used for the detection of the target bacteria. Limulus Amebocyte Lysate (LAL) was used to measure endotoxin. The amounts of IL-1ss/ TNF-alpha in macrophages supernatants were measured by enzyme-linked immunosorbent assay--Duoset-kit (ELISA). RESULTS: Prevotella nigrescens (13/21), Porphyromonas endodontalis (6/21), and Treponema socranskii (6/21) were the most frequently detected gram-negative bacterial species. The presence of the sinus tract (2/21) was related to the detection of Filifactor alocis (p < 0.05), whereas a tooth with a radiolucent area > or = 2 mm was related to the detection of Treponema denticola. A correlation was found between the number of gram-negative bacteria and the levels of IL-1beta/TNF-alpha (p < 0.05). Increased levels of endotoxin were followed by TNF-alpha release (p < 0.05). Higher levels of IL-1beta (p < 0.05) and endotoxin contents were related to the larger size of the radiolucent area. CONCLUSION: The antigenicity of the endodontic contents is not only related to the amount of endotoxin found in the root canal but also to the number of different species of gram-negative bacteria involved in the infection. Moreover, a larger size (> or = 2 mm) of the radiolucent area was related to IL-1beta and endotoxin.