Antigenicity of Primary Endodontic Infection against Macrophages by the Levels of PGE 2 Production Frederico C. Martinho, DDS, MSc,* Wanderson Miguel Maia Chiesa, DDS, MSc,* Fabio R.M. Leite, DDS, MSc, PhD, † Joni A. Cirelli, DDS, MSc, ‡ and Brenda P.F.A. Gomes, DDS, MSc* Abstract Introduction: Root canal contents are potent stimuli for proinflammatory cytokines involved in apical periodonti- tis. 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 nigres- cens (13/21), Fusobacterium nucleatum (6/21), and Porphyromonas endodontalis (6/21) were the most frequently observed species. A positive associ- ation was found between F. nucleatum and P. endo- dontalis (P < .05). A correlation was found between the number of gram-negative bacterial species and the levels of endotoxins, as well as PGE 2 (P < .05). Higher levels of endotoxin were detected in teeth with exuda- tion, whereas elevated levels of PGE 2 were found in teeth with tenderness to percussion and pain on palpa- tion. 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 secre- tion. (J Endod 2011;37:602–607) Key Words Bacteria, endotoxin, macrophages, prostaglandin E2, root canal P rimary endodontic infection is a polymicrobial infection caused predominantly by gram-negative anaerobic bacteria (1). A restricted group of bacteria, especially Prevotella, Porphyromonas spp, and Fusobacterium nucleatum, is considered path- ogenically important for odontogenic infection (2–6). Each of the previously described gram-negative bacterial species possesses lipo- polysaccharide (LPS, endotoxin) as a cell wall constituent. LPS released during bacterial disintegration, multiplication, and death (1) can egress into periradicular tissue (7), being potent stimuli against different cells (8–12) and leading to periapical inflammatory responses and bone destruction (8). The inflammatory tissue present in periapical lesions is populated predominantly by macrophage (13). Gram-negative bacterial LPSs are one of the mainly potent stimuli for macrophages cells in the release of PGE 2 (14–16). PGE 2 is implicated in most of the inflammatory and destructive changes that occur in apical lesions, such as vasodilatation, increasing vascular permeability, collagen degradation, and bone resorption (17). The possible role of PGE 2 in the pathogenesis of apical periodontitis has been provided in endodontic literature (14, 15, 17–20). Clinical investigations had elucidated the strong correlation between higher levels of endotoxins in root canals with spontaneous pain (21–23) and clinical symptomatology such as pain on palpation, tenderness to percussion, and exudation (22–25). PGE 2 has been reported in inflamed human dental pulp (20), exudation (19), and periapical tissue (18). However, studies correlating all these aspects have not yet been provided in endodontic literature. Therefore, the aim of this clinical study was to investigate the presence of target gram-negative bacterial species and the levels of endotoxin in primary endodontic infection with apical periodontitis and to determine their antigenicity against macrophages through the levels of PGE 2 , evaluating their rela- tionship with clinical findings. Materials and Methods Patient Selection Twenty-one patients needing endodontic treatment who attended the Piracicaba Dental School, S ~ ao Paulo, Brazil, were included in this research. The age of the patients ranged from 13 to 73 years old. Samples were collected from 21 root canals with pulp necrosis, all showing radiographic evidence of apical periodontitis. The selected teeth showed absence of periodontal pockets more than 4 mm in depth. A detailed dental history was obtained from each patient. Those who had received antibiotic treatment during the last 3 months or who had any general disease were excluded. The Human Research Ethics Committee of the Piracicaba From the *Department of Restorative Dentistry, Endodontic Division, Piracicaba Dental School, State University of Campinas, Piracicaba, S~ ao Paulo, Brazil; † Department of Semiology and Clinics, Periodontic Division, Dental School, Federal University of Pelotas, Pelotas, Brazil; and ‡ Department of Diagnosis and Oral Surgery, Periodontic Division, Araraquara Dental School, State University of S~ ao Paulo, S~ ao Paulo, Brazil. Supported by the Brazilian agencies FAPESP (07/58518-4; 08/58299-3; 08/ 57954-8, 08/57551-0) and CNPq (3470820/2006-3; 471631/2008-6; 302575/2009-0). Address requests for reprints to Dr Brenda P.F.A. Gomes, Department of Restorative Dentistry, Endodontic Division, Piracicaba Dental School, State University of Campinas-UNICAMP, Av Limeira 901, Bairro Areiao, Piracicaba, S~ ao Paulo, Brazil, CEP 13414-903. E-mail address: bpgomes@fop.unicamp.br 0099-2399/$ - see front matter Copyright ª 2011 American Association of Endodontists. doi:10.1016/j.joen.2010.12.005 Clinical Research 602 Martinho et al. JOE — Volume 37, Number 5, May 2011