Quantitative analysis of association between herpesviruses and bacterial pathogens in periodontitis

Background and Objective: The development of human periodontitis may depend upon cooperative interactions among herpesviruses, specific pathogenic bacteria and tissue‐destructive inflammatory mediators. This study sought to identify associations among human cytomegalovirus, Epstein–Barr virus and six putative periodontopathic bacteria in periodontitis lesions. Material and Methods: Fifteen periodontitis patients (nine with aggressive periodontitis and six with chronic periodontitis) and 15 periodontally normal subjects were included in the study. In each study subject, a microbiological sample was collected, using a curette, from the deepest periodontal probing depth of the dentition. A real‐time TaqMan® polymerase chain reaction assay was employed to determine the subgingival counts of human cytomegalovirus, Epstein–Barr virus, Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum and Campylobacter rectus. Statistical analysis was performed using the Student's t‐test, the Pearson correlation coefficient test and the single variable logistic regression test for odds ratio‐based risk calculation. Results: Human cytomegalovirus was detected in eight periodontitis lesions and in one normal periodontal site, Epstein–Barr virus was detected in nine periodontitis lesions and in two normal periodontal sites, and the study bacteria were detected in 6–15 periodontitis lesions and in 1–11 normal periodontal sites. Correlations were found between counts of human cytomegalovirus and Epstein–Barr virus, between counts of human cytomegalovirus and P. gingivalis, T. forsythia and C. rectus, and between counts of Epstein–Barr virus and P. gingivalis and T. forsythia. Human cytomegalovirus and Epstein–Barr virus counts were also positively associated with the level of periodontal attachment loss, probing pocket depth and gingival bleeding on probing. Conclusion: This study confirmed that periodontal human cytomegalovirus and Epstein–Barr virus are associated with major periodontopathic bacteria and with the severity of periodontal disease. The finding of abundant herpesviruses in periodontitis lesions redefines the pathogenic paradigm of the disease. Understanding the interplay between herpesviruses and specific bacterial species in the pathogenesis of periodontitis may form the basis for new approaches to preventing, reducing or delaying tissue breakdown from periodontal infections.     

Secreted adenosine triphosphate from Aggregatibacter actinomycetemcomitans triggers chemokine response

Extracellular ATP (eATP) is an important intercellular signaling molecule secreted by activated immune cells or released by damaged cells. In mammalian cells, a rapid increase of ATP concentration in the extracellular space sends a danger signal, which alerts the immune system of an impending danger, resulting in recruitment and priming of phagocytes. Recent studies show that bacteria also release ATP into the extracellular milieu, suggesting a potential role for eATP in host–microbe interactions. It is currently unknown if any oral bacteria release eATP. As eATP triggers and amplifies innate immunity and inflammation, we hypothesized that eATP secreted from periodontal bacteria may contribute to inflammation in periodontitis. The aims of this study were to determine if periodontal bacteria secrete ATP, and to determine the function of bacterially derived eATP as an inducer of inflammation. Our results showed that Aggregatibacter actinomycetemcomitans, but not Porphyromonas gingivalis, Prevotella intermedia, or Fusobacterium nucleatum, secreted ATP into the culture supernatant. Exposure of periodontal fibroblasts to filter sterilized culture supernatant of A. actinomycetemcomitans induced chemokine expression in an eATP‐dependent manner. This occurred independently of cyclic adenosine monophosphate and phospholipase C, suggesting that ionotrophic P2X receptor is involved in sensing of bacterial eATP. Silencing of P2X7 receptor in periodontal fibroblasts led to a significant reduction in bacterial eATP‐induced chemokine response. Furthermore, bacterial eATP served as a potent chemoattractant for neutrophils and monocytes. Collectively, our findings provide evidence for secreted ATP of A. actinomycetemcomitans as a novel virulence factor contributing to inflammation during periodontal disease.     

Macrophage subsets and osteoimmunology: tuning of the immunological recognition and effector systems that maintain alveolar bone

Chronic and aggressive periodontal diseases are characterized by the failure to resolve local inflammation against periodontopathogenic bacteria in the subgingival biofilm. Alveolar bone resorption is associated with altered innate and adaptive immune responses to periodontal pathogens. Macrophage‐derived cytokines, chemokines and growth factors, present in both destructive and reparative phases of periodontitis, are elevated in numerous animal and human studies. Macrophage polarization to either a predominantly pro‐inflammatory or anti‐inflammatory phenotype may be a critical target for monitoring disease activity, modulating immune responses to subgingival biofilms in patients at risk and reducing alveolar bone loss.     

In vivo and ex vivo actions of a novel P. gingivalis inhibitor on multi‐species biofilm,inflammatory response,and periodontal bone loss

Chronic periodontitis is one of the most common infectious inflammatory diseases worldwide. Current therapeutic options for the disease are only partially and temporarily successful due to periodontal re‐emergence of pathogens such as Porphyromonas gingivalis, a keystone bacterium in the oral microbial communities, which elicits a dysbiosis between the microbiota and the host. Previously, we reported a peptide inhibitor of P. gingivalis (SAPP) that specifically targets P. gingivalis and reduces its virulence potential in vitro. Here, we show that SAPP can modulate the ability of P. gingivalis to suppress the host innate immune system. Using a cytokine array analysis, we found that the levels of several cytokines including IL‐6, IL‐8, and MCP‐1 in the culture media of human oral keratinocytes (HOKs) were significantly diminished in the presence of P. gingivalis. Whereas the levels of these cytokines were restored, at least partially, in the culture media of HOKs by SAPP treatment. Furthermore, we also observed in an ex vivo assay that SAPP efficiently inhibited biofilm primed formation by mixed‐species oral bacteria, and significantly dampened the abnormally innate immune responses induced by these bacteria. We also demonstrated, using a mouse model, that SAPP could prevent alveolar bone loss induced by P. gingivalis. Our results suggest that SAPP specifically targets P. gingivalis and its associated bacterial communities and could be envisioned as an emerging therapy for periodontitis.     

TLR4, NOD1 and NOD2 mediate immune recognition of putative newly identified periodontal pathogens

Periodontitis is a polymicrobial inflammatory disease that results from the interaction between the oral microbiota and the host immunity. Although the innate immune response is important for disease initiation and progression, the innate immune receptors that recognize both classical and putative periodontal pathogens that elicit an immune response have not been elucidated. By using the Human Oral Microbe Identification Microarray (HOMIM), we identified multiple predominant oral bacterial species in human plaque biofilm that strongly associate with severe periodontitis. Ten of the identified species were evaluated in greater depth, six being classical pathogens and four putative novel pathogens. Using human peripheral blood monocytes (HPBM) and murine bone‐marrow‐derived macrophages (BMDM) from wild‐type (WT) and Toll‐like receptor (TLR)‐specific and MyD88 knockouts (KOs), we demonstrated that heat‐killed Campylobacter concisus, Campylobacter rectus, Selenomonas infelix, Porphyromonas endodontalis, Porphyromonas gingivalis, and Tannerella forsythia mediate high immunostimulatory activity. Campylobacter concisus, C. rectus, and S. infelix exhibited robust TLR4 stimulatory activity. Studies using mesothelial cells from WT and NOD1‐specific KOs and NOD2‐expressing human embryonic kidney cells demonstrated that Eubacterium saphenum, Eubacterium nodatum and Filifactor alocis exhibit robust NOD1 stimulatory activity, and that Porphyromonas endodontalis and Parvimonas micra have the highest NOD2 stimulatory activity. These studies allowed us to provide important evidence on newly identified putative pathogens in periodontal disease pathogenesis showing that these bacteria exhibit different immunostimulatory activity via TLR4, NOD1, and NOD2 (Clinicaltrials.gov NCT01154855).     

Correlation of cytomegalovirus and human herpesvirus 7 with CD3+ and CD3+ CD4+ cells in chronic periodontitis patients

Thomasini RL, Bonon SH, Durante P, Costa SCB. Correlation of cytomegalovirus and human herpesvirus 7 with CD3 ⁺ and CD3 ⁺ CD4 ⁺ cells in chronic periodontitis patients. J Periodont Res 2012; 47: 114–120. © 2011 John Wiley & Sons A/S Background and Objective: Human chronic periodontitis is an inflammatory process characterized by dense accumulation of immune cells in the periodontal tissue. The periodontitis can lead to loss of teeth in the patient and the pathogenesis of this disease is not completely known. This study tested the hypothesis that chronic periodontitis‐affected sites can harbor betaherpesviruses and that viruses are linked to a profile of the inflammatory infiltrate. Material and Methods: Biopsies of periodontal tissue were taken from periodontitis‐affected patients and from healthy subjects. Immunohistochemistry was performed to count CD19⁺ B cells, CD3⁺ total T cells, T‐CD4⁺ and T‐CD8⁺ cell subsets, and PCR was performed to detect cytomegalovirus and human herpesvirus 6 and 7 in the samples. One slide of each sample was stained with Giemsa for histopathological examination and to evaluate the quality of the cellular infiltrate. Results: As expected, tissues collected from healthy subjects presented no significant level of inflammatory infiltration and were therefore excluded from immunostaining procedures. Results showed that CD19⁺ B cells were in higher number than CD3⁺ T cells in the periodontitis‐affected tissue, but this was not statistically significant. The T‐CD4⁺ lymphocyte subset was significantly higher than the T‐CD8⁺ lymphocyte subset (p = 0.004) in the samples. Cytomegalovirus and human herpesvirus 7 were found at periodontitis‐affected sites, but not in tissue collected from healthy subjects (p = 0.04 and p = 0.04, respectively). Human herpesvirus 6 was rarely detected. We found a correlation between cytomegalovirus and lower CD19⁺/CD3⁺ ratios (ratio < 0.9, p = 0.003) and between human herpesvirus 7 and lower CD19⁺/CD3⁺ ratios (ratio < 0.9, p = 0.003) and higher CD4⁺/CD8⁺ ratios (ratio > 1.1, p = 0.002). Conclusion: This study shows that cytomegalovirus and human herpesvirus 7 can be present at periodontitis‐affected sites but are uncommon at healthy periodontal sites. Moreover, our data suggest that cytomegalovirus can be related to an inflammatory infiltrate with predominance of CD3⁺ T cells, whereas human herpesvirus 7 can be associated with an infiltrate with predominance of T‐CD4⁺ cells. However, further studies are necessary to support this hypothesis. Herpesviruses could play a role in human chronic periodontitis by modulation of the T cell response.     

Application of specialized pro‐resolving mediators in periodontitis and peri‐implantitis: a review

Scaling and root planning is a key element in the mechanical therapy used for the eradication of biofilm, which is the major etiological factor for periodontitis and peri‐implantitis. However, periodontitis is also a host mediated disease, therefore, removal of the biofilm without adjunctive therapy may not achieve the desired clinical outcome due to persistent activation of the innate and adaptive immune cells. Most recently, even the resident cells of the periodontium, including periodontal ligament fibroblasts, have been shown to produce several inflammatory factors in response to bacterial challenge. With increased understanding of the pathophysiology of periodontitis, more research is focusing on opposing excessive inflammation with specialized pro‐resolving mediators (SPMs). This review article covers the major limitations of current standards of care for periodontitis and peri‐implantitis, and it highlights recent advances and prospects of SPMs in the context of tissue reconstruction and regeneration. Here, we focus primarily on the role of SPMs in restoring tissue homeostasis after periodontal infection.     

Polymorphisms in the 5' flanking region of IL12RB2 are associated with susceptibility to periodontal diseases in the Japanese population

Objectives: The expression of interleukin (IL)‐12Rβ2 molecule is a crucial regulatory factor in the T‐helper type (Th) 1 differentiation of T cells. To elucidate the role of the cell‐mediated immune (CMI) response in the pathogenesis of periodontitis, Japanese periodontal patients were subjected to single nucleotide polymorphism (SNP) analyses of the 5′ flanking region of IL12RB2, whose variants are frequently detected in lepromatous leprosy patients, in which the very weak cellular immune response is caused by low expression of IL‐12Rβ2. Material and Methods: The gene polymorphisms of the 5′ flanking region of IL12RB2 were examined in subjects with several types of periodontal disease and in healthy controls. Serum immunoglobulin (Ig) G antibody titres against periodontopathic bacteria were measured and compared in periodontal patients with and without variant alleles of IL12RB2. Results: The frequencies of variant alleles of IL12RB2 were significantly higher in aggressive periodontitis patients as compared with healthy controls or chronic periodontitis patients. Serum IgG titres against all periodontal bacteria examined in subjects carrying variant alleles were higher than those in subjects without variant alleles. Conclusion: IL‐12Rβ2 SNPs could be useful as genetic markers to access the susceptibility of the general population to periodontal disease. Low CMI responses or high humoral responses are associated with the pathogenesis of inflammatory periodontal diseases.     

Periodontal microbiology in Latin America

This review article describes the microbiota associated with periodontal disease in Latin America. This vast territory includes 22 nations, which show great ethnic diversity, with large groups of White people, Black people, Mestizo people and Native people. Widespread poverty and limited access to education and health‐care services, including periodontal care, are prominent predisposing factors for destructive periodontal disease in Latin America. Black people and Mestizo people seem to have particularly severe periodontal disease and are frequently colonized by the major periodontal pathogens Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. The ‘red complex’ bacterial pathogens and A. actinomycetemcomitans predominate in chronic and aggressive periodontitis, but gram‐negative enteric rods and herpesviruses can also play important periodontopathic roles in Latin America. The key to minimizing the risk of periodontal disease is control of the pathogens, and new low‐cost periodontal treatments deserve serious consideration in Latin America.     

Induction of calprotectin release by Porphyromonasgingivalis lipopolysaccharide in human neutrophils

Calprotectin, a major cytosolic protein of leukocytes, is detected in neutrophils, monocytes/macrophages, and epithelial cells. This protein is known to be a marker for several inflammatory diseases and is detected in inflammatory gingival tissue with periodontal disease. Recently, we found that the calprotectin level in gingival crevicular fluid from periodontitis patients was significantly higher than that of healthy subjects. However, the regulation of calprotectin in periodontal disease is unclear. In the present study, we investigated the effect of lipopolysaccharides of periodontopathic bacteria on calprotectin release from human neutrophils. Neutrophils from healthy donors were treated with lipopolysaccharides from Porphyromonas gingivalis (P‐LPS), Actinobacillus actinomycetemcomitans, Prevotella intermedia, Fusobacterium nucleatum, and Escherichia coli. Calprotectin of neutrophil was identified by immunoblotting and calprotectin amount was determined by ELISA. Two subunits (10 and 14 kDa) of calprotectin were observed in the cell and medium fractions from neutrophils. P‐LPS increased calprotectin release from seven to 16 times the control level after 30 min and its effect appeared in a dose‐dependent manner (10–1000 ng/ml). Lipopolysaccharides from A. actinomycetemcomitans, P. intermedia, F. nucleatum, and E. coli also induced calprotectin release from neutrophils. These results suggest that lipopolysaccharides from periodontopathic bacteria induce calprotectin release from human neutrophils.     

Microbiological features of Papillon-Lefèvre syndrome periodontitis

Abstract. Papillon-Lefèvre syndrome patients exhibit hyperkeratosis palmoplantaris and severe periodontitis. The syndrome is an autosomal recessive trait, but the mechanism of periodontal destruction is not known. This report presents the clinical and microbiological features of an 11-year old girl with Papillon-Lefěvre syndrome. Clinical examination included conventional periodontal measurements and radiographic analysis. In samples from 3 deep periodontal lesions, the occurrence of major suspected periodontopathic bacteria was determined by selective and non-selective culture and polymerase chain reaction (PCR) identification, and the presence of cytomegalovirus and Epstein-Barr type 1 virus by a nested-PCR detection method. 10 of 22 available teeth demonstrated severe periodontal breakdown. Major cultivable bacteria included Actinobacillus actinomycetemcomitans (3.4% of total isolates), Prevotella nigrescens (16.4%), Fusobacterium nucleatum (14.3%) and Peptostreptococcus micros (10.6%). A. actinomycetemcomitans, P. nigrescens, Porphyromonas gingivalis and Eikenella corrodens were identified by PCR analysis. The patient's non-affected parents and older brother revealed several periodontal pathogens but not A. actinomycetemcomitans. The viral examination demonstrated cytomegalovirus and Epstein-Barr type 1 virus in the subgingival sample of the Papillon-Lefèvre syndrome patient. The father and brother yielded subgingival cytomegalovirus but not Epstein-Barr type 1 virus. We hypothesize that human herpesviruses in concert with A. actinomycetemcomitans play important rǒles in the development of Papillon-Lefèvre syndrome periodontitis.     

Interleukin‐1 β, interleukin‐12 and interleukin‐18 levels in gingival fluid and serum of patients with gingivitis and periodontitis

Introduction: Cytokines are of major importance in periodontal disease progression. Interleukin‐12 (IL‐12) stimulates interferon‐γ production by T helper type 1 (Th1) cells while IL‐18 induces Th1 responses when present with IL‐12 but Th2 responses in the absence of IL‐12. IL‐1β has been correlated with periodontal disease destruction. This study determined the local concentrations of these cytokines in sites of gingivitis and periodontitis. Methods: Gingival crevicular fluid was collected from two sites in each of 10 gingivitis patients and from two gingivitis sites and two periodontitis sites from each of 10 periodontitis patients. Serum samples were also collected. IL‐1β, biologically active IL‐12 p70, the IL‐12 p40 subunit and IL‐18 concentrations were determined by enzyme‐linked immunoabsorbent assay. Results: IL‐1β and IL‐18 concentrations were higher in the gingival crevicular fluid from periodontitis patients than in that from gingivitis patients; IL‐18 concentrations were higher than those of IL‐1β. Very little IL‐12, either p40 or p70, was detected in the gingival crevicular fluid samples. In the serum, very low levels of cytokines were found. The level of serum IL‐12 p40, however, was higher than in the fluid from periodontitis sites of periodontitis patients. Conclusion: The local production of IL‐1β and IL‐18 in the gingival crevicular fluid increased with increasing inflammation and IL‐18 was the predominant cytokine at both gingivitis and periodontitis sites. Very little IL‐12 was detected with levels decreasing with increasing inflammation. These results suggest that there is an association between severity of periodontal disease and levels of IL‐1, IL‐12 and IL‐18.     

Herpesviruses: a unifying causative factor in periodontitis?

Human cytomegalovirus and Epstein‐Barr virus type 1 are discussed in this review as they relate to destructive periodontal disease in humans. Genomes of the two herpesviruses occur frequently in severe adult periodontitis, localized and generalized juvenile periodontitis, Papillon‐Lefèvre syndrome periodontitis, Down’s syndrome periodontitis, HIV‐associated periodontitis and acute necrotizing ulcerative gingivitis. Herpesvirus infections generally involve a mild or asymptomatic primary phase followed by an asymptomatic latent phase interrupted sporadically by periods of activation, where viral replication and possibly clinical disease become manifest. Herpesvirus reactivation is triggered by a number of immunosuppressing factors, some of which have also been shown to be risk indicators of periodontal disease. Available evidence argues for the involvement of active cytomegalovirus infection in the initiation and progression of localized juvenile periodontitis and possibly other types of periodontal disease. In periodontal disease, herpesviruses may cause release of tissue‐destructive cytokines, overgrowth of pathogenic periodontal bacteria, and initiation of cytotoxic or immunopathogenic events. Understanding the significance of herpesviruses in the causation and pathogenesis of destructive periodontal diseases may have important implications in future prevention and treatment of the diseases.     

Salivary infectious agents and periodontal disease status

Saygun I, Nizam N, Keskiner I, Bal V, Kubar A, Aç?kel C, Serdar M, Slots J. Salivary infectious agents and periodontal disease status. J Periodont Res 2011; 46: 235–239. © 2011 John Wiley & Sons A/S Background and Objectives: The potential of salivary microorganisms to diagnose periodontal disease and to guide periodontal treatment is a research topic of current interest. This study aimed to determine whether the salivary counts of periodontopathic microbes correlated with the periodontal pocket counts of the same infectious agents, and whether the salivary counts of the test infectious agents could distinguish among individuals with periodontal health and various types of periodontal disease. Material and Methods: The study included 150 systemically healthy adults, of whom 37 were periodontally healthy, 31 had gingivitis, 46 had chronic periodontitis and 36 had aggressive periodontitis. Each study subject contributed microbial samples from the two deepest periodontal pockets of the dentition and from whole saliva. Aggregatibacter actinomycetemcomitans, Campylobacter rectus, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia and Epstein–Barr virus were identified using the TaqMan real‐time PCR methodology. Statistical analysis was performed using the Mann–Whitney U‐test and the receiver operating characteristic statistics. Results: C. rectus, F. nucleatum, P. gingivalis, P. intermedia and T. forsythia occurred with significantly higher copy‐counts in salivary samples from patients with gingivitis, chronic periodontitis and aggressive periodontitis than from periodontally healthy individuals. A. actinomycetemcomitans only showed higher salivary copy‐counts in subjects with aggressive periodontitis compared with subjects with healthy periodontium, and the salivary copy‐counts of Epstein–Barr virus did not reveal any significant difference among the four subject groups studied. The diagnostic sensitivity for periodontitis was 89.19 for P. gingivalis and for T. forsythia and 86.49 for P. intermedia, with specificities ranging from 83.78 to 94.59. The optimal copy‐counts per mL saliva for identifying periodontitis were 40,000 for P. gingivalis, 700,000 for T. forsythia and 910,000 for P. intermedia. Conclusion: Salivary copy‐counts of P. gingivalis, T. forsythia and P. intermedia appear to have the potential to identify the presence of periodontitis, whereas the salivary level of the other test infectious agents may possess little or no diagnostic utility. Longitudinal studies are warranted to determine the ability of salivary copy‐counts of major periodontopathic bacteria to predict future periodontal breakdown.     

Levels of interleukin-21 in patients with untreated chronic periodontitis

Background: A growing body of evidence suggested that interleukin (IL)‐21 enhances the effector phase during T‐cell responses. The aim of our study is to determine the levels of IL‐21 in periodontal sites from patients with chronic periodontitis and controls. Methods: The population studied consisted of 34 patients (15 with chronic periodontitis and 19 healthy patients). Twenty samples (10 gingival crevicular fluid [GCF] and 10 gingival biopsies) were collected from each group before the patients with periodontitis received periodontal treatment. Total protein concentrations were measured in all samples; the presence of IL‐21 was confirmed by immunohistochemistry and Western blot, and IL‐21 levels were quantified through an enzyme‐linked immunosorbent assay. Statistical analyses were performed using statistical software. Data were expressed as patient means ± SDs or medians (interquartile ranges) by using the χ², Student t, and Mann‐Whitney U tests. Results: GCF IL‐21 was mainly detected in patients with chronic periodontitis (P <0.05). Levels of IL‐21 in gingival tissues were significantly higher in patients with chronic periodontitis compared to healthy individuals (P <0.05). The Western blot and immunohistochemical staining confirmed the presence of IL‐21 in periodontal tissues and GCF. Conclusion: IL‐21 was highly expressed in patients with chronic periodontitis, especially in gingival biopsies; therefore, IL‐21 might play a role in the T‐cell response.     

Update on human cytomegalovirus in destructive periodontal disease

AIM: Human cytomegalovirus (HCMV), a herpesvirus, is discussed in this review as it relates to destructive periodontal disease in humans. RESULTS: HCMV genomic sequences, detected by polymerase chain reaction identification, occur with elevated frequency in severe adult periodontitis, localized and generalized aggressive (juvenile) periodontitis, Papillon-Lefèvre syndrome periodontitis, acute necrotizing ulcerative gingivitis, and periodontal abscesses. DISCUSSION: Herpesviruses establish lifelong persistent infections. HCMV infection involves an asymptomatic latent phase interrupted by periods of recrudescence where viral replication and possibly clinical disease become manifest. HCMV reactivation is triggered by a number of immunosuppressive factors, some of which have been shown also to be risk factors/indicators of periodontitis. HCMV periodontal infection may cause release of tissue-destructive cytokines, overgrowth of pathogenic periodontal bacteria, and initiation of cytotoxic or immunopathologic events. CONCLUSIONS: A growing body of data supports the concept that HCMV contributes to severe types of periodontal disease. HCMV infection of the periodontium may alter the immune control of resident microorganisms and be important in a multistage pathogenesis of periodontitis involving viral activation, periodontopathic bacteria, and host immune responses. Understanding the significance of HCMV and other herpesviruses in the development of periodontal disease may have important therapeutic implications. Vaccines against HCMV, which are in various stages of development, need to be evaluated for their ability to decrease the incidence of destructive periodontal disease.     

Periodontal ligament and gingival fibroblasts from periodontitis patients are more active in interaction with Porphyromonas gingivalis

Scheres N, Laine ML, Sipos PM, Bosch‐Tijhof CJ, Crielaard W, de Vries TJ, Everts V. Periodontal ligament and gingival fibroblasts from periodontitis patients are more active in interaction withPorphyromonas gingivalis. J Periodont Res 2011; 46: 407–416. © 2011 John Wiley & Sons A/S Background and Objective: Inflammatory responses of host cells to oral pathogenic bacteria, such as Porphyromonas gingivalis, are crucial in the development of periodontitis. Host cells, such as periodontal ligament and gingival fibroblasts, from periodontitis patients may respond to P. gingivalis in a different manner compared with cells from healthy persons. The aim of this study was to investigate inflammatory responses to viable P. gingivalis by periodontal ligament and gingival fibroblasts from periodontitis patients and healthy control subjects. Material and Methods: Primary periodontal ligament and gingival fibroblasts from periodontitis patients (n = 14) and healthy control subjects (n = 8) were challenged in vitro with viable P. gingivalis. Gene expression of Toll‐like receptors (TLRs) 1, 2, 4, 6, 7 and 9, CD14, nuclear factor‐κB1 and its putative inhibitor NF‐κB inhibitor‐like protein 1, and of interleukin‐1β, interleukin‐6, interleukin‐8, tumour necrosis factor‐α, monocyte chemotactic protein‐1 and regulated upon activation, normal T‐cel expressed, and secreted, were assessed by real‐time PCR. Results: Periodontal ligament fibroblasts from periodontitis patients had a higher mRNA expression of TLR1, TLR4, TLR7 and CD14, and a lower expression of NFKBIL1, both before and after P. gingivalis challenge. In contrast, gingival fibroblasts from periodontitis patients had stronger induction of TLR1, TLR2 and TLR7 by P. gingivalis. Cytokine responses were not different between patients and control subjects. Interestingly, periodontal ligament, but not gingival, fibroblasts from P. gingivalis culture‐positive persons responded more strongly to P. gingivalis than periodontal ligament fibroblasts from P. gingivalis‐negative persons. Conclusion: Periodontal ligament and gingival fibroblasts respond to P. gingivalis in a different manner and may play different roles in periodontitis. Both subsets of fibroblasts from patients appear more active in interaction with P. gingivalis. Moreover, periodontal ligament fibroblasts from P. gingivalis‐positive donors are more responsive to an in vitro P. gingivalis challenge.     

Identification and characterization of B‐cell epitopes of a 53‐kDa outer membrane protein from Porphyromonas gingivalis

We have previously reported that Porphyromonas gingivalis FDC 381 possesses a 53‐kDa protein antigen (Ag53) on its outer membrane that evokes a strong humoral immune response in many patients with periodontal disease and that the humoral immune responses to Ag53 differ greatly among patients. To understand how the individual humoral immune system against Ag53 was determined, the regions of Ag53 recognized by specific antibody (B‐cell epitopes) and dominant subclasses of serum immunoglobulin G (IgG) against major B‐cell epitopes were examined by enzyme‐linked immunosorbent assay. This study used sera from six patients with periodontitis, which all reacted strongly with sonic extracts of P. gingivalis 381 and with purified Ag53, and sera from six periodontally healthy children, which did not react with either sonic extracts of P. gingivalis 381 or Ag53. The epitopes were identified using synthetic 5‐residue overlapping decapeptides covering the entire Ag53. Thirteen of 89 synthetic decapeptides showed a strong reaction with sera from the periodontal patients, but no reaction with those from the healthy children. Four peptides of 13 exerted different immune responses among patients. Furthermore, restriction analyses of the highly antigenic regions revealed that three sequences, RAAIRAS, YYLQ and MSPARR, were identified as major B‐cell epitopes. Additionally, these epitopes were recognized mainly by the IgG2 isotype. These data suggest that the difference of B‐cell epitopes might influence individual differences in antibody titer against Ag53 and also that the epitopes recognized commonly by multiple antibodies are quite valuable for peptide vaccine development against P. gingivalis infection.     

The Association of CSF‐1 Gene Polymorphism With Chronic Periodontitis in the Han Chinese Population

Background: Chronic periodontitis (CP) is a multifactorial complex periodontal disease involving immune response, inflammation, alveolar bone resorption, and attachment loss. Colony stimulating factor‐1 (CSF‐1) controls the production, differentiation, and function of macrophages and plays a vital role in the innate immune response to the external microbial infections, suggesting the potential role of CSF‐1 in the pathogenesis of periodontitis. The objective of this study is to determine the association of single nucleotide polymorphisms (SNPs) rs333967, rs2297706, and rs1058885 with CP in the Han Chinese population. Methods: Genomic DNA was isolated from buccal epithelial cells obtained from unrelated Chinese participants (440 patients with CP and 324 controls). The SNPs were genotyped by a matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry method. Results: Three previously identified SNPs were genotyped in Han Chinese with Shanghai origin, but none of them was statistically significantly associated with CP. However, a T‐C‐G haplotype in male participants showed an observed P value of 4.52^E‐08, with an odds ratio of 0.092. Conclusion: None of the individual SNPs among rs333967, rs2297706, and rs1058885 in CSF‐1 was found statistically significantly associated with CP in the Han Chinese population with Shanghai origin, whereas a haplotype T‐C‐G showed an observed statistically significant association with decreased risk of CP susceptibility in males.