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    Quantification Of Endotoxins In Infected Root Canals And Acute Apical Abscess Exudates: Monitoring The Effectiveness Of Root Canal Procedures In The Reduction Of Endotoxins

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    Introduction This clinical study was conducted to measure the endotoxin levels in infected root canals (RCs) and exudates related to acute apical abscesses (AAAs). In addition, the effectiveness of RC procedures in reducing the endotoxin levels in RCs was monitored. Methods Paired samples of infected RCs and exudates from AAAs were collected from 10 subjects by using paper points. RCs samples were collected before (RCS1) and after chemomechanical preparation (CMP) (RCS2), after 17% EDTA (RCS3), and after 30 days of intracanal medication (Ca[OH]2 + chlorhexidine) (RCS4). A turbidimetric kinetic limulus amebocyte lysate assay was used for the measurement of endotoxins. Results Endotoxins were detected in 100% of the baseline samples of AAAs and RCs (RCS1) with median values of 175 EU/mL and 41.5 EU/mL, respectively (P <.05). After CMP (RCS2), endotoxins were reduced to a median value of 0.54 EU/mL (P <.05). Subsequent irrigation with EDTA (RCS3) failed to present a significant effectiveness in reducing the endotoxin levels (median= 0.37 EU/mL) (P =.07). However, intracanal medication for 30 days (RCS4) reduced endotoxins to median values of 0.03 EU/mL (P <.01). Conclusions The present study revealed a strong association between the high levels of endotoxins found in AAAs and RCs collected from the same tooth. Moreover, the effectiveness of CMP in reducing the endotoxin levels from RCs in acute endodontic infection was improved by the use of RC medication. Copyright © 2014 American Association of Endodontists.402177181Siqueira, Jr.J.F., Rocas, I.N., Microbiology and treatment of acute apical abscesses (2013) Clin Microbiol Rev, 26, pp. 255-273Rocas, I.N., Siqueira, Jr.J.F., Identification of bacteria enduring endodontic treatment procedures by a combined reverse transcriptase-polymerase chain reaction and reverse-capture checkerboard approach (2010) J Endod, 36, pp. 45-52Montagner, F., Jacinto, R.C., Signoretti, F.G., Treponema species detected in infected root canals and acute apical abscess exudates (2010) J Endod, 36, pp. 1796-1799Montagner, F., Jacinto, R.C., Signoretti, F.G., Clustering behavior in microbial communities from acute endodontic infections (2012) J Endod, 38, pp. 158-162Gomes, B.P., Martinho, F.C., Vianna, M.E., Comparison of 2.5% sodium hypochlorite and 2% chlorhexidine gel on oral bacterial lipopolysaccharide reduction from primarily infected root canals (2009) J Endod, 35, pp. 1350-1353Gomes, B.P., Endo, M.S., Martinho, F.C., Comparison of endotoxin levels found in primary and secondary endodontic infections (2012) J Endod, 38, pp. 1082-1086Martinho, F.C., Chiesa, W.M., Leite, F.R., 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 (2010) J Endod, 36, pp. 1467-1474Martinho, F.C., Chiesa, W.M., Leite, F.R., Antigenicity of primary endodontic infection against macrophages by the levels of PGE(2) production (2011) J Endod, 37, pp. 602-607Pitts, D.L., Williams, B.L., Morton, Jr.T.H., Investigation of the role of endotoxin in periapical inflammation (1982) J Endod, 8, pp. 10-18Hong, C.Y., Lin, S.K., Kok, S.H., The role of lipopolysaccharide in infectious bone resorption of periapical lesion (2004) J Oral Pathol Med, 33, pp. 162-169Garcia De Aquino, S., Manzolli Leite, F.R., Stach-Machado, D.R., Signaling pathways associated with the expression of inflammatory mediators activated during the course of two models of experimental periodontitis (2009) Life Sci, 84, pp. 745-754Schein, B., Schilder, H., Endotoxin content in endodontically involved teeth (1975) J Endod, 1, pp. 19-21Horiba, N., Maekawa, Y., Abe, Y., Cytotoxicity against various cell lines of lipopolysaccharides purified from Bacteroides, Fusobacterium, and Veillonella isolated from infected root canals (1989) J Endod, 15, pp. 530-534Martinho, F.C., Chiesa, W.M., Leite, F.R., Correlation between clinical/radiographic features and inflammatory cytokine networks produced by macrophages stimulated with endodontic content (2012) J Endod, 38, pp. 740-745Ricucci, D., Siqueira, Jr.J.F., Fate of the tissue in lateral canals and apical ramifications in response to pathologic conditions and treatment procedures (2010) J Endod, 36, pp. 1-15Martinho, F.C., Chiesa, W.M., Marinho, A.C., Clinical investigation of the efficacy of chemomechanical preparation with rotary nickel-titanium files for removal of endotoxin from primarily infected root canals (2010) J Endod, 36, pp. 1766-1769Martinho, F.C., Chiesa, W.M., Zaia, A.A., Comparison of endotoxin levels in previous studies on primary endodontic infections (2011) J Endod, 37, pp. 163-167Martinho, F.C., Gomes, B.P., Quantification of endotoxins and cultivable bacteria in root canal infection before and after chemomechanical preparation with 2.5% sodium hypochlorite (2008) J Endod, 34, pp. 268-272Oliveira, L.D., Carvalho, C.A., Carvalho, A.S., Efficacy of endodontic treatment for endotoxin reduction in primarily infected root canals and evaluation of cytotoxic effects (2012) J Endod, 38, pp. 1053-1057Xavier, A.C.C., Martinho, F.C., Chung, A., One-visit versus two-visit root canal treatment: Effectiveness in the removal of endotoxins and cultivable bacteria (2013) J Endod, 39, pp. 959-964Jacinto, R.C., Gomes, B.P., Shah, H.N., Quantification of endotoxins in necrotic root canals from symptomatic and asymptomatic teeth (2005) J Med Microbiol, 54, pp. 777-783Csako, G., Elin, R.J., Hochstein, H.D., Physical and biological properties of U.S. Standard endotoxin EC after exposure to ionizing radiation (1983) Infect Immun, 41, pp. 190-196Vianna, M.E., Zilio, D.M., Ferraz, C.C., Concentration of hydrogen ions in several calcium hydroxide pastes over different periods of time (2009) Braz Dent J, 20, pp. 382-388Rôças, I.N., Baumgartner, J.C., Xia, T., Prevalence of selected bacterial named species and uncultivated phylotypes in endodontic abscesses from two geographic locations (2006) J Endod, 32, pp. 1135-1138Buck, R.A., Cai, J., Eleazer, P.D., Detoxification of endotoxin by endodontic irrigants and calcium hydroxide (2001) J Endod, 27, pp. 325-327Buttler, T.K., Crawford, J.J., The detoxifying effect of varying concentrations of sodium hypochlorite on endotoxins (1982) J Endod, 8, pp. 59-66Tanomaru Filho, M., Leonardo, M.R., Da Silva, L.A., Effect of irrigating solution and calcium hydroxide root canal dressing on the repair of apical and periapical tissues of teeth with periapical lesion (2002) J Endod, 28, pp. 295-299Wilson, M., Susceptibility of oral bacterial biofilms to antimicrobial agents (1996) J Med Microbiol, 44, pp. 79-87Berutti, E., Marini, R., Angeretti, A., Penetration ability of different irrigants into dentinal tubules (1997) J Endod, 23, pp. 725-727Petsch, D., Anspach, F.B., Endotoxin removal from protein solutions (2000) J Biotechnol, 76, pp. 97-119Burton, A.J., Carter, H.E., Purification and characterization of the lipid a component of the lipopolysaccharides from Escherichia coli (1964) Biochemistry, 3, pp. 411-418Vianna, M.E., Horz, H.P., Conrads, G., Comparative analysis of endodontic pathogens using checkerboard hybridization in relation to culture (2008) Oral Microbiol Immunol, 23, pp. 282-290Endo, M.S., Zaia, A.A., Ferraz, C.C.R., Quantitative and qualitative analysis of microorganisms in root-filled teeth with persistent infection: Monitoring of the 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    Clinical Investigation Of Bacterial Species And Endotoxin In Endodontic Infection And Evaluation Of Root Canal Content Activity Against Macrophages By Cytokine Production

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    Introduction: This study investigated the presence of different Gram-negative bacterial species and the levels of endotoxins found in primary endodontic infection (PEI), determining their stimulation ability against macrophages through the levels of interleukin (IL)-1, IL-6, IL-10, and tumor necrosis factor alpha (TNF-α), and evaluated their relationship with clinical and radiographic findings.Material and methods: Samples were taken from 21 root canals with primary endodontic infection with apical periodontitis (PEIAP). Molecular techniques were used for bacterial detection. Limulus amebocyte lysate assay was used to measure endotoxins. Pro-inflammatory cytokines were measured by ELISA assay.Results: All samples were positive for bacterial DNA (21/21). Prevotella nigrescens (57.2 %) was the most frequent species. Higher levels of endotoxins were found in teeth with pain on palpation and exudation (all p < 0.05). Positive correlations were found between endotoxins and the levels of TNF-α and IL-1β, whereas a negative correlation was found between endotoxin and the amount of IL-10 (p < 0.05). Endotoxin levels were found to be a risk factor for exudation and increased the number of Gram-negative bacterial species for the presence of a larger area of bone destruction (all p < 0.05).Conclusion: A wide variety of Gram-negative bacterial species are involved in primary endodontic infection, with participation of different Treponema species. Thus, the levels of endotoxins and the number of Gram-negative bacteria species present in root canals were considered risk factors for the severity of endodontic infection.Clinical relevance: The present study revealed that Gram-negative bacterial species and endotoxins play an important role in the development of signs/symptoms and the severity of bone destruction, this knowledge is essential for the establishment of an effective therapy.18920952102Siqueira, J.F., Jr., Rocas, I.N., Microbiology and treatment of acute apical abscesses (2013) Clin Microbiol Rev, 26, pp. 255-273. , PID: 23554416Martinho, F.C., Chiesa, W.M., Leite, F.R., Cirelli, J.A., Gomes, B.P., 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 (2010) J Endod, 36, pp. 1467-1474. , PID: 20728711Montagner, F., Jacinto, R.C., Signoretti, F.G., Gomes, B.P., Treponema species detected in infected root canals and acute apical abscess exudates (2010) J Endod, 36, pp. 1796-1799. , PID: 20951290Rocas, I.N., Siqueira, J.F., Jr., Identification of bacteria enduring endodontic treatment procedures by a combined reverse transcriptase-polymerase chain reaction and reverse-capture checkerboard approach (2010) J Endod, 36, pp. 45-52. , PID: 20003934Rocas, I.N., Siqueira, J.F., Jr., Andrade, A.F., Uzeda, M., Oral treponemes in primary root canal infections as detected by nested PCR (2003) Int Endod J, 36, pp. 20-26. , PID: 12656510Gomes, B.P., Lilley, J.D., Drucker, D.B., Associations of endodontic symptoms and signs with particular combinations of specific bacteria (1996) Int Endod J, 29, pp. 69-75. , PID: 9206427Siqueira, J.F., Jr., Rocas, I.N., PCR-based identification of Treponema maltophilum, T amylovorum, T medium, and T lecithinolyticum in primary root canal infections (2003) Arch Oral Biol, 48, pp. 495-502. , PID: 12798152Metzger, Z., Macrophages in periapical lesions (2000) Endod Dent Traumatol, 16, pp. 1-8. , PID: 11202850Beutler, B., Cerami, A., The biology of cachectin/TNF—a primary mediator of the host response (1989) Annu Rev Immunol, 7, pp. 625-655. , PID: 2540776Keller, R., Fischer, W., Keist, R., Bassetti, S., Macrophage response to bacteria: induction of marked secretory and cellular activities by lipoteichoic acids (1992) Infect Immun, 60, pp. 3664-3672. , PID: 1500175Garcia de Aquino, S., Manzolli Leite, F.R., Stach-Machado, D.R., Francisco da Silva, J.A., Spolidorio, L.C., Rossa, C., Jr., Signaling pathways associated with the expression of inflammatory mediators activated during the course of two models of experimental periodontitis (2009) Life Sci, 84, pp. 745-754. , PID: 19285515Sousa, E.L., Martinho, F.C., Nascimento, G.G., Leite, F.R., Gomes, B.P., Quantification of endotoxins in infected root canals and acute apical abscess exudates: monitoring the effectiveness of root canal procedures in the reduction of endotoxins (2014) J Endod, 40 (2), pp. 177-181. , PID: 24461400Seymour, G.J., Gemmell, E., Cytokines in periodontal disease: where to from here? (2001) Acta Odontol Scand, 59, pp. 167-173. , PID: 11501886Martinho, F.C., Chiesa, W.M., Leite, F.R., Cirelli, J.A., Gomes, B.P., Correlation between clinical/radiographic features and inflammatory cytokine networks produced by macrophages stimulated with endodontic content (2012) J Endod, 38, pp. 740-745. , PID: 22595105Boyce, B.F., Li, P., Yao, Z., Zhang, Q., Badell, I.R., Schwarz, E.M., O’Keefe, R.J., Xing, L., TNF-alpha and pathologic bone resorption (2005) Keio J Med, 54, pp. 127-131. , PID: 16237274Huang, G.T., Do, M., Wingard, M., Park, J.S., Chugal, N., Effect of interleukin-6 deficiency on the formation of periapical lesions after pulp exposure in mice (2001) Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 92, pp. 83-88. , PID: 11458250Tinsley, J.H., South, S., Chiasson, V.L., Mitchell, B.M., Interleukin-10 reduces inflammation, endothelial dysfunction, and blood pressure in hypertensive pregnant rats (2010) Am J Physiol Regul Integr Comp Physiol, 298, pp. R713-R719. , PID: 20053959Leon, L.R., Kozak, W., Kluger, M.J., Role of IL-10 in inflammation. 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    Monitoring The Effectiveness Of Root Canal Procedures On Endotoxin Levels Found In Teeth With Chronic Apical Periodontitis

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    Objective: The aim of this study was to monitor the effectiveness of root canal procedures by using different irrigants and intracanal medication on endotoxin levels found in root canals of teeth with chronic apical periodontitis. Material and methods: Thirty root canals of teeth with pulpal necrosis associated with periapical lesions were selected and randomly divided into groups according to the irrigants used: GI - 2.5% NaOCl, GII - 2% chlorhexidine (CHX) gel, and GIII - saline solution (SS) (all, n=10). Samples were collected with sterile/apyrogenic paper points before (S1) and after root canal instrumentation (S2), after use of 17% ethylenediaminetetraacetic acid (EDTA) (S3), and after 30 days of intracanal medication (Ca(OH)2+SS) (S4). A turbidimetric kinetic Limulus Amebocyte Lysate assay was used for endotoxin measurement. Results: Endotoxins were detected in 100% of the root canals investigated (30/30), with a median value of 18.70 EU/mL. After S2, significant median percentage reduction was observed in all groups, irrespective of the irrigant tested: 2.5% NaOCl (99.65%) (GI), 2% CHX (94.27%) (GII), and SS (96.79%) (GIII) (all p0.05). Intracanal medication for 30 days was able to significantly reduce residual endotoxins: 2.5% NaOCl (90%) (GI), 2% CHX (88.8%) (GII), and SS (85.7%) (GIII, p<0.05). No differences were found in the endotoxin reduction when comparing S2 and S4 treatment groups. Conclusion: Our findings demonstrated the effectiveness of the mechanical action of the instruments along with the flow and backflow of irrigant enduring root canal instrumentation for the endotoxin removal from root canals of teeth with chronic apical periodontitis. Moreover, the use of intracanal medication for 30 days contributed for an improvement of endotoxin reduction.226490495Berutti, E., Marini, R., Angeretti, A., Penetration ability of different irrigants into dentinal tubules (1997) J Endod, 23, pp. 725-727Buck, R.A., Cai, J., Eleazer, P.D., Staat, R.H., Hurst, H.E., Detoxification of endotoxin by endodontic irrigants and calcium hydroxide (2001) J Endod, 27, pp. 325-327Burton, A.J., Carter, H.E., Purification and characterization of the lipid A component of the lipopolysaccharides from Escherichia coli (1964) Biochemistry, 3, pp. 411-418Csako, G., Elin, R.J., Hochstein, H.D., Tsai, C.M., Physical and biological properties of U.S. Standard endotoxin EC after exposure to ionizing radiation (1983) Infect Immun, 41, pp. 190-196Gomes, B.P., Endo, M.S., Martinho FC Comparison of endotoxin levels found in primary and secondary endodontic infections (2012) J Endod, 38, pp. 1082-1086Gomes, B.P., Martinho, F.C., Vianna, M.E., Comparison of 2.5% sodium hypochlorite and 2% chlorhexidine gel on oral bacterial lipopolysaccharide reduction from primarily infected root canals (2009) J Endod, 35, pp. 1350-1353Hong, C.Y., Lin, S.K., Kok, Cheng, S., Lee, M.S., Wang, T.M., The role of lipopolysaccharide in infectious bone resorption of periapical lesion (2004) J Oral Pathol Med, 33, pp. 162-169Horiba, N., Maekawa, Y., Abe, Y., Ito, M., Matsumoto, T., Nakamura, H., Correlations between endotoxin and clinical symptoms or radiolucent areas in infected root canals (1991) Oral Surg Oral Med Oral Pathol, 71, pp. 492-495Jacinto, R.C., Gomes, B.P., Shah, H.N., Ferraz, C.C., Zaia, A.A., Souza-Filho, F.J., Quantification of endotoxins in necrotic root canals from symptomatic and asymptomatic teeth (2005) J Med Microbiol, 54, pp. 777-783Lüderitz, O., Tanamoto, K., Galanos, C., McKenzie, G.R., Brade, H., Zähringer, U., Lipopolysaccharides: Structural principles and biologic activities (1984) Rev Infect Dis, 4, pp. 428-433Marinho, A.C., Martinho, F.C., Zaia, A.A., Ferraz, C.C., Gomes, B.P., Influence of the apical enlargement size on the endotoxin level reduction of dental root canals (2012) J Appl Oral Sci, 20, pp. 661-666Martinho, F.C., Chiesa, W.M., Leite, F.R., Cirelli, J.A., Gomes, B.P., 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 (2010) J Endod, 36, pp. 1467-1474Martinho, F.C., Chiesa, W.M., Marinho, A., Zaia, A.A., Ferraz, C.C., Almeida, J.F., Clinical investigation of the efficacy of chemomechanical preparation with rotary nickel-titanium files for removal of endotoxin from primarily infected root canals (2010) J Endod, 36, pp. 1766-1769Martinho, F.C., Gomes, B.P., Quantification of endotoxins and cultivable bacteria in root canal infection before and after chemomechanical preparation with 2.5% sodium hypochlorite (2008) J Endod, 34, pp. 268-272Oliveira, L.D., Carvalho, C.A., Carvalho, A.S., Alves, J.S., Valera, M.C., Jorge, A.O., Efficacy of endodontic treatment for endotoxin reduction in primarily infected root canals and evaluation of cytotoxic effects (2012) J Endod, 38, pp. 1053-1057Oliveira, L.D., Jorge, A.O., Carvalho, C.A., Koga-Ito, C.Y., Valera, M.C., In vitro effects of endodontics irrigants on endotoxins in root canals (2007) Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 104, pp. 135-142Ozdemir, H.O., Buzoglu, H.D., Calt, S., Cehreli, Z.C., Varol, E., Temel, A., Chemical and ultramorphologic effects of ethylenediaminetetraacetic acid and sodium hypochlorite in young and old root canal dentin (2012) J Endod, 38, pp. 204-20818- Paredes-Vieyra, J., Enriquez, F.J., Success rate of single- versus two-visit root canal treatment of teeth with apical periodontitis: A randomized controlled trial (2012) J Endod, 38, pp. 1164-1169Pérez-Heredia, M., Ferrer-Luque, C.M., González-Rodríguez, M.P., Martín-Peinado, F.J., González-López, S., Decalcifying effect of 15% EDTA, 15% citric acid, 5% phosphoric acid and 2.5% sodium hypochlorite on root canal dentine (2008) Intendod J, 41, pp. 418-423Petsch, D., Anspach, F.B., Endotoxin removal from protein solutions (2000) J Biotech, 76, pp. 97-119Pitts, D.L., Williams, B.L., Morton, T.H., Jr., Investigation of the role of endotoxin in periapical inflammation (1982) J Endod, 8, pp. 10-18Schein, B., Schilder, H., Endotoxin content in endodontically involved teeth (2006) J Endod, 32, pp. 293-295Silva, L.A., Leonardo, M.R., Assed, S., Tanomaru-Filho, M., Histological study of the effect of some irrigating solutions on bacterial endotoxin in dogs (2004) Braz Dent J, 15, pp. 109-114Silva, L.A., Nelson-Filho, P., Leonardo, M.R., Rossi, M.A., Pansani, C.A., Effect of calcium hydroxide on bacterial endotoxin in vivo (2002) J Endod, 28, pp. 94-9825- Sousa, E.L., Martinho, F.C., Nascimento, G.G., Leite, F.R., Gomes, B.P., Quantification of endotoxins in infected root canals and acute apical abscess exudates: Monitoring the effectiveness of root canal procedures in the reduction of endotoxins (2014) J Endod, 40, pp. 177-181Tanomaru, J.M., Leonardo, M.R., Tanomaru Filho, M., Bonetti Filho, L., Silva, L.A., Effect of different irrigation solutions and calcium hydroxide on bacterial LPS (2003) Int Endod J, 36, pp. 733-739Thaler, A., Ebert, J., Petschelt, A., Pelka, M., Influence of tooth age and root section on root dentine dye penetration (2008) Int Endod J, 41, pp. 1115-1122Vera, J., Siqueira, J.F., Jr., Ricucci, D., Loghin, S., Fernández, N., Flores, B., One- versus two-visit endodontic treatment of teeth with apical periodontitis: A histobacteriologic study (2012) J Endod, 38, pp. 1040-105229- Vianna, M.E., Horz, H.P., Conrads, G., Zaia, A.A., Souza-Filho, F.J., Gomes, B.P., Effect of root canal procedures on endotoxins and endodontic pathogens (2007) Oral Microbiol Immunol, 22, pp. 411-418Xavier, A.C., Martinho, F.C., Chung, A., Oliveira, L.D., Jorge, A.O., 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    Antigenicity Of Primary Endodontic Infection Against Macrophages By The Levels Of Pge2 Production

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    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 PGE2, 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&amp;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 &lt; .05). A correlation was found between the number of gram-negative bacterial species and the levels of endotoxins, such as PGE2 (P &lt; .05). Higher levels of endotoxin were detected in teeth with exudation, whereas elevated levels of PGE2 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 PGE2 secretion. 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