Epstein-Barr virus infection in chronically inflamed periapical granulomas.

Periapical granulomas are lesions around the apex of a tooth caused by a polymicrobial infection. Treatment with antibacterial agents is normally performed to eliminate bacteria from root canals; however, loss of the supporting alveolar bone is typically observed, and tooth extraction is often selected if root canal treatment does not work well. Therefore, bacteria and other microorganisms could be involved in this disease. To understand the pathogenesis of periapical granulomas more precisely, we focused on the association with Epstein-Barr virus (EBV) using surgically removed periapical granulomas (n = 32). EBV DNA was detected in 25 of 32 periapical granulomas (78.1%) by real-time PCR, and the median number of EBV DNA copies was approximately 8,688.01/µg total DNA. In contrast, EBV DNA was not detected in healthy gingival tissues (n = 10); the difference was statistically significant according to the Mann-Whitney U test (p = 0.0001). Paraffin sections were also analyzed by in situ hybridization to detect EBV-encoded small RNA (EBER)-expressing cells. EBER was detected in the cytoplasm and nuclei of B cells and plasma cells in six of nine periapical granulomas, but not in healthy gingival tissues. In addition, immunohistochemical analysis for latent membrane protein 1 (LMP-1) of EBV using serial tissue sections showed that LMP-1-expressing cells were localized to the same areas as EBER-expressing cells. These data suggest that B cells and plasma cells in inflamed granulomas are a major source of EBV infection, and that EBV could play a pivotal role in controlling immune cell responses in periapical granulomas.

Significance of human cytomegalovirus and Epstein-Barr virus in inducing cytokine expression in periapical lesions.

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)-1ß, 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.

Cytomegalovirus-infected inflammatory cells in dental periapical lesions.

INTRODUCTION: As cytomegalovirus may be etiologically involved in periapical pathosis of endodontic origin, this study aimed to determine the cellular source of periapical cytomegalovirus. METHODS: Periapical granulomatous tissue was collected from 15 extracted teeth with symptomatic periapical lesions. Multi-color flow cytometry was used to identify cytomegalovirus-infected cells. RESULTS: Cytomegalovirus infection was identified in 10 of the 15 (67%) study lesions, and in periapical monocytes/macrophages (40% of lesions) and T lymphocytes (54% of lesions), but not in periapical B lymphocytes. CONCLUSION: This study and previous polymerase chain reaction-based investigations show that cytomegalovirus is a frequent inhabitant of symptomatic periapical lesions.

Natural killer cells and alterations in collagen density: signs of periradicular herpesvirus infection?

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.

Herpesviruses in asymptomatic apical periodontitis lesions: an immunohistochemical approach.

INTRODUCTION: Human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) have been recently detected in samples from apical periodontitis lesions by means of molecular biology techniques and a role in the pathogenesis of this disease has been suggested. The present study was designed to survey asymptomatic primary apical periodontitis lesions for the presence of HCMV- and/or EBV-infected cells by means of immunohistochemistry. METHODS: Apical periodontitis lesions were obtained from 35 patients [26 human immunodeficiency virus (HIV) -seronegative patients and nine HIV-seropositive patients] after tooth extraction and subjected to immunohistochemical analysis using monoclonal antibodies specific for HCMV and EBV. RESULTS: Fifteen of the 35 apical periodontitis lesions were positive for the target herpesviruses. Overall, EBV was found in 31% of the samples and HCMV in 23%, with 14% of the lesions showing EBV and HCMV dual infection. No association was found between HCMV or EBV with any particular histopathological type of apical periodontitis (P > 0.05). HCMV was significantly more frequent in apical periodontitis lesions from HIV-positive patients (67%) than in lesions from HIV-negative patients (8%) (P = 0.001). EBV was detected in 44% of lesions from HIV-positive patients and in 27% of lesions from HIV-negative patients, but this difference was not significant (P = 0.91). CONCLUSION: Our results showed that cells infected by HCMV and EBV can be found in apical periodontitis lesions, with a higher prevalence in HIV-positive patients. The specific role that these viruses play in the pathogenesis of apical periodontitis remains to be described.

Human cytomegalovirus, Epstein-Barr virus and bone resorption-inducing cytokines in periapical lesions of deciduous teeth.

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.

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.

Polymerase chain reaction detection of human immunodeficiency virus DNA in human periradicular lesions.

Human immunodeficiency virus (HIV) has been previously reported to be present in the dental pulp of a patient with AIDS. The present report investigated the feasibility of polymerase chain reaction (PCR) amplification to detect the HIV proviral DNA in cells from periradicular lesions from an HIV-positive patient. The standard PCR amplification with 30 cycles and the nested PCR consisting of two 25-cycle amplifications were used. Samples from each reaction were separated by nondenaturing polyacrylamide gel electrophoresis and stained with ethidium bromide for visualization. Gels were electroblotted to nylon membranes, which were then fixed, denatured, and dried. Membranes were hybridized to specific radioactive oligonucleotide probes and placed next to Kodak XAR film for visualization of the HIV-specific bands. No evidence of HIV-specific reaction was observed in cells (negative control) or in two periradicular lesions from two HIV-negative patients. The ethidium bromide strains revealed that PCR amplification of DNA extracts from two lesions from the HIV-positive patient yielded PCR bands (with both primer pairs) which corresponded to HIV-specific bands of the expected size.