Hypercitrullination and anti-citrullinated protein antibodies in chronic apical periodontitis, a laboratory investigation. Does autoimmunity contribute to the pathogenesis?

AIM: The presence of Gram-negative anaerobic bacteria, in particular, Porphyromonas gingivalis (P. gingivalis) in periapical granulomas predicts the generation of citrullinated proteins in the lesion. Citrullination of proteins may lead to the formation of anti-citrullinated autoantibodies (ACPA-s) initiating the formation of an autoimmune loop which may contribute to the perpetuation of inflammatory reactions and tissue damage in chronic apical periodontitis. The objective of this study was to demonstrate the formation of citrullinated proteins in chronic apical periodontitis and whether they can act as autoantigens. METHODOLOGY: Twenty-five periapical granulomas (n = 25) were investigated in the study. Healthy periodontal tissue samples served as normal control tissue (n = 6). The peptidyl-citrulline level was determined with the dot blot method. ACPA levels were analysed using anti-citrullinated cyclic peptide (anti-CCP) EDIA kit. Differences between periapical granuloma and control samples were assessed using Mann-Whitney U tests. p Values <.05 were considered as statistically significant. RESULTS: Protein concentrations, peptidyl-citrulline levels and anti-CCP ratios were compared between periapical granuloma and healthy control groups. Multiple linear regression analysis revealed significant (p = .042) hypercitrullination in periapical granuloma samples. Moreover, there was a significant difference in the ACPA ratios between periapical granuloma (2.03 ± 0.30) and healthy control (0.63 ± 0.17) groups (p = .01). Seventeen of 25 periapical granuloma samples (17/25; 68%), whereas one out of six control samples (1/6; 17%) were shown to be positive for the presence of ACPA. CONCLUSIONS: This is the first study detecting the presence of citrullinated peptides and APCA in periapical granuloma, suggesting the contribution of autoimmune reactions in the pathogenesis and perpetuation of chronic apical periodontitis.

Apical periodontitis: preliminary assessment of microbiota by 16S rRNA high throughput amplicon target sequencing.

BACKGROUND: Apical periodontitis includes periapical granulomas and radicular cysts, which are histologically distinguished by the absence and the presence of an epithelial lining, respectively. The main cause of apical periodontitis is the bacterial colonization of the root canal space. This research aimed at assessing whether and how periapical granulomas and radicular cysts differ in terms of microbiota using high throughput amplicon target sequencing (HTS) techniques. METHODS: This study included 5 cases of Periapical Granulomas (PGs) and 5 cases of Radicular Cysts (RCs) selected on the base of histology out of 37 patients from January 2015 to February 2016. Complete medical history, panoramic radiograms (OPTs) and histologic records of each patient were assessed. Only lesions greater than 1 cm in diameter and developed in proximity to teeth with bad prognosis were included. The microbiota present in periapical granulomas and radicular cysts thus retrieved was finely characterized by pyrosequencing of the 16S rRNA genes. RESULTS: The core of OTUs shared between periapical granulomas and radicular cysts was dominated by the presence of facultative anaerobes taxa such as: Lactococcus lactis, Propionibacterium acnes, Staphylococcus warneri, Acinetobacter johnsonii and Gemellales. L. lactis, the main OTUs of the entire datasets, was associated with periapical granuloma samples. Consistently with literature, the anaerobic taxa detected were most abundant in radicular cyst samples. Indeed, a higher abundance of presumptive predicted metabolic pathways related to Lipopolysaccharide biosynthesis was found in radicular cyst samples. CONCLUSIONS: The present pilot study confirmed the different microbial characterization of the two main apical periodontitis types and shade light on the possible role of L. lactis in periapical granulomas.

Investigation of cultivable bacteria isolated from longstanding retreatment-resistant lesions of teeth with apical periodontitis.

INTRODUCTION: The objective of this research was to investigate the presence of viable bacteria in tissue samples from persistent apical lesions and to correlate the microbiological findings with the histopathological diagnosis of the lesion. METHODS: Twenty persistent apical lesions associated with well-performed endodontic retreatment were collected. Tissue samples were processed through culture techniques including serial dilution, plating, aerobic and anaerobic incubation, and biochemical tests for microbial identification followed by histopathological diagnosis. RESULTS: Cysts were more frequently diagnosed (13/20). Strict anaerobic species predominated in both cysts (80.4% of the species detected) and granulomas (65% of the species detected). Viable gram-positive bacteria were frequently recovered from apical lesions (cysts = 70.6%, granulomas = 84.4%). Gemella morbillorum and Propionibacterium acnes were the most frequently recovered species from cysts and granulomas, respectively. At least 1 gram-positive bacterial species was present in almost every sample (cysts = 12/13, granulomas = 7/7). No significant correlation was found between histologic findings and bacterial species. CONCLUSIONS: In conclusion, although cysts were more frequent than granulomas in cases of failure of endodontic retreatment, bacteria were isolated from both types of lesions, with a predominance of gram-positive species, suggesting that these species can survive outside the root canal and might be related to the persistence of the pathological process even after accurate endodontic retreatment.

Bacterial intensity and localization in primary molars with caries disease.

AIM: The aim was to assess the characteristics and outcomes of infections affecting the structures of carious primary molars. MATERIALS AND METHODS: Forty primary molars were used and classified according to the following clinical situation: With profound caries lesion, with bone loss at the furcation region, with perforation of the pulp chamber floor, and residual roots. The teeth were demineralized, cut, and stained with both haematoxylin-eosin and Brown and Brenn staining techniques. Assessment was performed using optical microscopy. RESULTS: Statistical analysis of the data by means of the Chi-square test suggests that there was a significant relationship (P<0.001) between the intensity and localization of infection and the level of destruction of dental structures. A significant difference was also observed in the intensity and localization of infection between the groups regarding crown, furca, and root (P<0.001). CONCLUSION: More intense and profound the infection, more severe is the dental destruction. The groups of residual roots showed the most severe bacterial infection compared to other groups.

Biofilms and apical periodontitis: study of prevalence and association with clinical and histopathologic findings.

INTRODUCTION: This study evaluated the prevalence of bacterial biofilms in untreated and treated root canals of teeth evincing apical periodontitis. The associations of biofilms with clinical conditions, radiographic size, and the histopathologic type of apical periodontitis were also investigated. METHODS: The material comprised biopsy specimens from 106 (64 untreated and 42 treated) roots of teeth with apical periodontitis. Specimens were obtained by apical surgery or extraction and were processed for histopathologic and histobacteriologic techniques. RESULTS: Bacteria were found in all but one specimen. Overall, intraradicular biofilm arrangements were observed in the apical segment of 77% of the root canals (untreated canals: 80%; treated canals: 74%). Biofilms were also seen covering the walls of ramifications and isthmuses. Bacterial biofilms were visualized in 62% and 82% of the root canals of teeth with small and large radiographic lesions, respectively. All canals with very large lesions harbored intraradicular biofilms. Biofilms were significantly associated with epithelialized lesions (cysts and epithelialized granulomas or abscesses) (p < 0.001). The overall prevalence of biofilms in cysts, abscesses, and granulomas was 95%, 83%, and 69.5%, respectively. No correlation was found between biofilms and clinical symptoms or sinus tract presence (p > 0.05). Extraradicular biofilms were observed in only 6% of the cases. CONCLUSIONS: The overall findings are consistent with acceptable criteria to include apical periodontitis in the set of biofilm-induced diseases. Biofilm morphologic structure varied from case to case and no unique pattern for endodontic infections was identified. Biofilms are more likely to be present in association with longstanding pathologic processes, including large lesions and cysts.

Nonsurgical root canal therapy of large cyst-like inflammatory periapical lesions and inflammatory apical cysts.

It is a general belief that large cyst-like periapical lesions and apical true cysts caused by root canal infection are less likely to heal after nonsurgical root canal therapy. Nevertheless, there is no direct evidence to support this assumption. A large cyst-like periapical lesion or an apical true cyst is formed within an area of apical periodontitis and cannot form by itself. Therefore, both large cyst-like periapical lesions and apical true cysts are of inflammatory and not of neoplastic origin. Apical periodontitis lesions, regardless of whether they are granulomas, abscesses, or cysts, fail to heal after nonsurgical root canal therapy for the same reason, intraradicular and/or extraradicular infection. If the microbial etiology of large cyst-like periapical lesions and inflammatory apical true cysts in the root canal is removed by nonsurgical root canal therapy, the lesions might regress by the mechanism of apoptosis in a manner similar to the resolution of inflammatory apical pocket cysts. To achieve satisfactory periapical wound healing, surgical removal of an apical true cyst must include elimination of root canal infection.

T lymphocyte activation and cytokine expression in periapical granulomas and radicular cysts.

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.

Prevalence and clonal analysis of Porphyromonas gingivalis in primary endodontic infections.

This study investigated the prevalence of Porphyromonas gingivalis in 62 teeth with primary endodontic infections by using a species-specific 16S rRNA gene-based nested polymerase chain reaction assay. P. gingivalis isolates recovered from 2 infected root canals were also analyzed for clonal diversity by using arbitrarily primed PCR. Overall, P. gingivalis was found in 48% of the samples. This species was specifically detected in 36% of canals of teeth with chronic apical periodontitis, in 46% of the cases of acute apical periodontitis, and in 67% of acute apical abscesses. P. gingivalis was significantly more frequent in abscess aspirates than in canals of teeth with chronic apical periodontitis (P < .05). Typing of colonies retrieved from 2 infected canals revealed 2 clones per individual. These findings confirmed that P. gingivalis can be an important endodontic pathogen, mostly associated with acute abscesses, and demonstrated that different clonal types of this species can colonize the root canal in the same individual.

Submandibular tuberculous lymphadenitis after endodontic treatment of the mandibular first premolar tooth: report of a case.

Cervical tuberculous lymphadenitis (scrofula) is an infectious granulomatous disease that requires a precise diagnosis. The differential diagnosis involves mainly the pathologic conditions involving the regional lymph nodes and the submandibulary salivary glands. Although tuberculous lesions generally develop secondary to pulmonary disease, clinical manifestations are occasionally seen with no evidence of involvement of the lungs. In this report, a case of tuberculous submandibular lymphadenitis developing after endodontic treatment of the mandibular first premolar tooth is described.

Commensal oral bacteria antigens prime human dendritic cells to induce Th1, Th2 or Treg differentiation.

In various immunopathologic conditions, bacterial flora induce an immune response which results in inflammatory manifestations, e.g. periapical granuloma. Dendritic cells provide the main orchestration of specific immune responses. The aim of our study was to test the capacity of distinct oral bacterial antigens (prepared from Streptococcus mitis, Propionibacterium acnes, and Bacteroides spp.) to prime human dendritic cells for stimulation of the T-lymphocyte response. To assess the T-lymphocyte response, the expression of CD25, CD69, intracellular interferon gamma (cIFN-gamma), and intracellular interleukin 4 (cIL-4) was determined. Dendritic cells were prepared from leukocyte buffy coat from healthy blood donors. Monocytes were stimulated with IL-4 and GM-CSF and dendritic cells activated with bacterial lysates. Cell suspensions contained up to 90% dendritic cells, which represented 2-12% of the initial number of mononuclear cells. Lymphocyte subsets that developed in lymphocyte cultures after 1 week of stimulation were analyzed by flow cytometry. Dendritic cells, primed with antigens of Bacteroides fragilis have shown significantly higher activation and expression of intercellular IFN-gamma by T lymphocytes compared to negative controls. The dendritic cells primed with antigens of P. acnes had no effect on T-lymphocyte activation or cytokine production; instead they induced differentiation of T lymphocytes into CD25bright cells (regulatory T cells) with a potentially inhibitory effect on immune response. Dendritic cells primed with antigens of S. mitis induced increased expression of cIL-4. We conclude that commensal oral bacteria antigens prepared from B. fragilis, S. mitis, and P. acnes prime human dendritic cells to induce Th1, Th2, and T(reg) differentiation, respectively. This may advance our understanding of immunopathologic manifestations in the oral cavity and offer new possibilities for redirecting immune responses in mucosal vaccination.

Herpesviral-bacterial coinfection in periapical pathosis.

Two members of the herpesvirus family, human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV), seem to be important putative pathogens of human periodontitis and symptomatic periapical lesions, causing pathosis either by inducing immunosuppression with a subsequent risk of aggressive bacterial infections or by infecting of periodontal cells directly. This study aimed to relate periapical occurrence of HCMV, EBV, and herpes simplex virus active infections to clinical characteristics of periapical lesions and periapical bacterial flora. Microbial samples were collected from 34 periapical lesions in conjunction with periapical surgery. Part of the periapical specimen was frozen for virologic examination, and another part was transferred to anaerobic transport medium for bacteriologic examination. RNA was isolated by means of a guanidinium isothiocyanate-acid phenol procedure, and cDNA was produced using herpesvirus-specific primers and reverse-transcription polymerase chain reaction amplification. Bacteriologic examination was performed according to established anaerobic culture methods. Of the 34 periapical lesions studied, 20 showed both HCMV and EBV, seven showed only HCMV, one showed only EBV, and six showed neither HCMV nor EBV. Herpes simplex virus was detected in two lesions. Higher occurrence of herpesvirus was detected in large versus small periapical lesions (p < 0.001) and in symptomatic versus asymptomatic periapical lesions (p < 0.001). A total of 18 microbial groups and an average of 2.1 to 3.0 bacterial groups were isolated from various categories of periapical lesions. The important finding of this study was that most teeth with necrotic pulp and periapical lesions harbored herpesviruses in periapical granulomatous tissue. Herpesvirus species in cooperation with endodontopathic bacteria may play major roles in the etiopathogenesis of aggressive types of periapical pathosis in humans.

Examination on Candida spp. in refractory periapical granulomas.

AIM: To examine the occurrence of Candida spp. in refractory periapical granulomas. METHODOLOGY: One hundred and three surgically removed periapical granulomas were subjected to molecular analysis for the occurrence of Candida albicans. DNA was extracted from the samples using a modified phenol/chloroform/isoamyl alcohol method and was subjected to polymerase chain reaction (PCR) with OPA-03 and repetitive sequence (GACA)4 primers. The PCR products were separated in agarose gel electrophoresis, stained with ethidium bromide, visualized using UV light and the sequences were analysed. Samples indicating possible occurrence of Candida were further investigated by histological and immunohistological methods. Periodic acid-Shiff staining (PAS) was used to detect yeast cells and hyphae, and specific monoclonal antibodies to recognize high molecular mass mannoproteins present in the C. albicans cell wall. DNA extraction was controlled by running PCR using beta-actin primers (a housekeeping gene). C. albicans CCUG19915, C. tropicalis ATCC750, C. krusei ATCC6258, C. guilliermondii ATCC6260 and C. glabrata CCUG32725 served as positive controls in PCR. A tissue preparation of chronic atrophic candidosis in oral buccal mucosa served as a positive control for histological and immunohistological examinations. RESULTS: Polymerase chain reaction with beta-actin primers indicated successful DNA extraction in 68 out of 103 samples. The majority of the samples (50) were negative whereas 18 of the samples showed PCR products indicating possible occurrence of Candida spp. PAS-staining and immunohistological examination of these samples were, however, negative. Further analysis of the PCR products revealed sequences not typical for Candida spp. CONCLUSIONS: Candida spp. do not seem to occur in periapical granuloma.

A new bacterial species associated with failed endodontic treatment: identification and description of Actinomyces radicidentis.

OBJECTIVE: This report describes 2 endodontic patients who had persistent signs and symptoms after conventional root canal treatment. The aim of this study was to determine what microorganisms were present in the root canals of the teeth with failed endodontic therapy. STUDY DESIGN: After removal of the root fillings, the canals were sampled by advanced microbiological techniques and the isolates were characterized by various tests. RESULTS: Bacteria, which grew in pure cultures, were isolated in each case. The bacteria were similar to each other and were classified as Actinomyces on the basis of phylogenic and phenotypic evidence. The bacteria were different from others within the genus, thus warranting designation as a new species, Actinomyces radicidentis. CONCLUSIONS: The 2 cases of endodontic failure were infected with A radicidentis, a new Actinomyces species. This bacterium joins a restricted group of other microorganisms that have been associated with failure of root canal treatment.

Tumor necrosis factor-alpha and transforming growth factor-beta1 in chronic periapical 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.

Cytotoxic T lymphocytes versus streptococcal colonization in periapical granulomas.

Seventeen dental periapical lesions were investigated to study bacterial colonization. Periapical lesions, obtained after apicotomy, were also enzymatically desegregated to quantitatively analyze lymphocyte subpopulations by flow cytometry. Fourteen samples yielded a positive bacterial growth when homogenized and cultured. We isolated enough lymphocytes to make flow cytometric analysis from 12 samples. A significant increase in interleukin-2 receptor and ICAM-1 molecule expression on T cells was found, compared with peripheral blood lymphocytes. Furthermore, a decreased expression of interleukin-2 receptors and HLA DR molecules on CD8+ T cells was found in granulomas predominantly colonized by Streptococcus spp., compared with lesions predominantly colonized by strict anaerobes.

Microbiological status of root-filled teeth with apical periodontitis.

The present study examined the microbiological status of 100 root-filled teeth with radiographically verified apical periodontitis--the pathology (P) group--and of 20 teeth without signs of periapical pathosis--the technical (T) group. In the P group 117 strains of bacteria were recovered in 68 teeth. In most of the cases examined one or two strains were found. Facultative anaerobic species predominated among these isolates (69% of identified strains). Growth was classified as 'sparse' or 'very sparse' in 53%, and as 'heavy' or 'very heavy' in 42%. Enterococci were the most frequently isolated genera, showing 'heavy' or 'very heavy' growth in 25 out of 32 cases (78%). In 11 teeth of the T group no bacteria were recovered, whilst the remaining nine yielded 13 microbial strains. Eight of these grew 'very sparsely'. It is concluded that the microflora of the obturated canal differs from that found normally in the untreated necrotic dental pulp, quantitatively as well as qualitatively. Nonsurgical retreatment strategies should be reconsidered.

Microorganisms in closed periapical lesions.

The purpose of this study was to investigate the microorganisms of strictly selected closed periapical lesions associated with both refractory endodontic therapy and pulpal calcification. Definitive criteria were established that assured complete clinical isolation of the periapical lesion from the oral and periodontal environment. A total of 13 criteria-referenced lesions were selected from 70 patients with endodontic surgical indications. A well controlled culturing method was used in all cases and samples were taken by one clinician at three separate sites during each surgery. Samples taken at the surgical window and within the body of the lesion served as controls, whilst a third sample was taken at the apex. In all 13 cases, samples taken from the apex yielded microorganisms comprising 63.6% obligate anaerobes and 36.4% facultative anaerobes. Prevalence of the isolated species was 31.8% for Actinomyces sp., 22.7% Propionibacterium sp., 18.2% Streptococcus sp., 13.6% Staphlyococcus sp., 4.6% Porphyromonas gingivalis, 4.6% Peptostreptococcus micros and 4.6% Gram-negative enterics. The results of this investigation indicate that closed periapical lesions associated with calcified teeth or those resistant to root canal treatment harbour bacteria. The inability to eradicate all root canal microorganisms during root canal treatment, along with anatomical factors, may allow further bacterial colonization of the root apex and surrounding periapical tissues, and consequently prevent healing.

Identification and antibiotic sensitivity of bacteria isolated from periapical lesions.

Periradicular tissues from 28 refractory endodontic cases requiring surgical intervention were submitted for histological diagnosis and microbiological culture. Bacteria isolated from these lesions were identified and then tested for their antibiotic sensitivity to a panel of common antibiotics. The periapical tissue specimens of 22 out of 28 lesions (79%) contained microorganisms. Of the 22 cases showing positive growth cultures, 15 were polymicrobial and 7 were single species isolates. Fifty-three different species were recovered: 29 anaerobes, 19 facultative anaerobes, and 5 aerobes. Microbes were observed under light microscopy in only one case. The most common organisms isolated were Propionibacterium acnes, Staphylococcus epidermidis, Streptococcus intermedius, Wolinella recta, Fusobacterium species, and Clostridium species. Antibiotic susceptibility results showed no clear cut evidence of significant antibiotic resistance among the species tested. The results of this study seem to corroborate earlier studies regarding the microbial population of periapical lesions refractory to nonsurgical endodontics.

Periradicular curettage.

Periradicular curettage is a part of the treatment procedure of periradicular surgery. Its main purpose is to remove pathological periradicular tissues for visibility and accessibility to facilitate the treatment of the apical root canal system, or sometimes for the removal of harmful foreign materials present in the periradicular area. Inflammatory periradicular lesions (granuloma and cysts) are the responses of the periradicular tissues to irritants from the root canal and not from the periradicular area unless medicaments and/or filling materials have been forced through the apical foramina or perforations into the periodontium. Histologically, the inflammatory periradicular lesion is similar to healing granulation tissue, which is composed of cells which have natural and specific immunological defence capability and cooperate by means of cytokines to amplify the protective mechanisms of the host. Accordingly, it is not necessary to completely curette out all the inflamed periradicular tissues during surgery, since this granulation-like tissue will be incorporated into the new granulation tissue as part of the healing process. To control the source of irritants in the root canal is far more important than to remove all periradicular tissues affected by the irritants. The successful removal of all irritants from the root canal system results in resolution of pulpally induced periradicular lesions. In the case where the periradicular lesion is caused by endodontic instruments or cytotoxic filling materials placed in the periradicular tissues, removal of these foreign objects is required for resolution of the lesion.