Nicolás Dutzan

The subgingival microbiome in health and periodontitis and its relationship with community biomass and inflammation

The goals of this study were to better understand the ecology of oral subgingival communities in health and periodontitis and elucidate the relationship between inflammation and the subgingival microbiome. Accordingly, we used 454-pyrosequencing of 16S rRNA gene libraries and quantitative PCR to characterize the subgingival microbiome of 22 subjects with chronic periodontitis. Each subject was sampled at two sites with similar periodontal destruction but differing in the presence of bleeding, a clinical indicator of increased inflammation. Communities in periodontitis were also compared with those from 10 healthy individuals. In periodontitis, presence of bleeding was not associated with different α-diversity or with a distinct microbiome, however, bleeding sites showed higher total bacterial load. In contrast, communities in health and periodontitis largely differed, with higher diversity and biomass in periodontitis. Shifts in community structure from health to periodontitis resembled ecological succession, with emergence of newly dominant taxa in periodontitis without replacement of primary health-associated species. That is, periodontitis communities had higher proportions of Spirochetes, Synergistetes, Firmicutes and Chloroflexi, among other taxa, while the proportions of Actinobacteria, particularly Actinomyces, were higher in health. Total Actinomyces load, however, remained constant from health to periodontitis. Moreover, an association existed between biomass and community structure in periodontitis, with the proportion of specific taxa correlating with bacterial load. Our study provides a global-scale framework for the ecological events in subgingival communities that underline the development of periodontitis. The association, in periodontitis, between inflammation, community biomass and community structure and their role in disease progression warrant further investigation.

Characterization of progressive periodontal lesions in chronic periodontitis patients: levels of chemokines, cytokines, matrix metalloproteinase‐13, periodontal pathogens and inflammatory cells

BACKGROUND AND AIMS Periodontitis is an infection with an episodic nature of tissue support destruction. The aim of this work was to determine the levels of chemokines, cytokines, matrix metalloproteinase-13, periodontal pathogens and inflammatory cells in periodontal sites characterized by active periodontal connective tissue destruction. MATERIAL AND METHOD Fifty-six patients with moderate or advanced severity of chronic periodontitis were selected. Periodontitis was characterized by at least six sites with probing depth > or =5 mm, clinical attachment level > or =3 mm and radiographic bone loss. Periodontitis progression was determined by the tolerance method. Receptor activator for nuclear factor kappa B-ligand (RANK-L), monocyte chemoattractant protein-1 (MCP-1), tumour necrosis factor-alpha (TNF-alpha), IL-1beta, MMP-13, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsithia and inflammatory cells levels were determined. Statistical analysis was performed using the Stata 7.0 software. Data were expressed as mean+/-SD and paired samples t-test and chi(2) tests were used. RESULTS Higher RANK-L, IL-1beta and MMP-13 activity levels were observed in active sites (p<0.05). The proportion of P. gingivalis, A. actinomycetemcomitans, T. forsythia and the number of CD4(+) T were higher in active than in inactive sites (p>0.05). CONCLUSION The detection of periodontopathic bacteria, host matrix metalloproteinases and cytokines in periodontitis patients with lesions undergoing episodic attachment loss could partially explain the mechanisms associated with the destruction of the supporting tissues of the tooth.

Over-expression of forkhead box P3 and its association with receptor activator of nuclear factor-kappa B ligand, interleukin (IL) -17, IL-10 and transforming growth factor-beta during the progression of chronic periodontitis.

AIM T regulatory (Treg) cells have been detected in periodontitis lesions, and forkhead box P3 (Foxp3) expression has been negatively correlated to receptor activator of nuclear factor-kappa B ligand (RANKL). The aim of this study was to correlate T-helper type 1 (Th1), Th2, Th17 and Treg transcription factor expressions, in gingival tissues from patients undergoing active periodontal tissue destruction, with bone loss-associated cytokines. MATERIALS AND METHODS In 10 chronic periodontitis patients undergoing disease progression, the mRNA expressions of T-bet, GATA-3, Foxp3, RORC2, interleukin (IL)-1beta, IL-10, IL-17, RANKL, interferon (IFN)-gamma and transforming growth factor (TGF)-beta1 were quantified using real-time reverse transcription-polymerase chain reaction. The levels of these markers were compared between active and inactive periodontal lesions. RESULTS In active periodontal lesions, Foxp3, T-bet, RANKL, IL-17, IL-1beta and IFN-gamma were significantly over-expressed compared with inactive lesions. The expression of IFN-gamma was the highest within the active periodontal lesions, similar to that of TGF-beta1 within the inactive ones. There was a positive correlation between RANKL and IL-17. Additionally, RANKL and IL-17 were positively correlated with RORC2, but no correlation was detected with Foxp3. CONCLUSIONS These results lead us to speculate that Foxp3(+) cells that do not have a regulatory function might have a role in the pathogenesis of active periodontal lesions by down-regulating TGF-beta1 and IL-10 synthesis that lead to the over-expression of Th17-associated cytokines RANKL and IL-17.

Host-Pathogen Interactions in Progressive Chronic Periodontitis

Periodontitis is an infection characterized by the occurrence of supporting tissue destruction with an episodic nature. Disease progression is often determined by the loss of attachment level or alveolar bone, and sequential probing of periodontal attachment remains the most commonly utilized method to diagnose progressive destruction of the periodontium. The tolerance method has been the most extensive clinical method used in recent years to determine site-specific attachment level changes. There is abundant evidence that major tissue destruction in periodontal lesions results from the recruitment of immune cells. Considerable effort has been made to study the host cell and mediator profiles involved in the pathogenesis of chronic periodontitis, but the definition of active sites, where current periodontal breakdown occurs, and consecutive characterization of the mediators involved are still among the main concerns. In the present review, we summarize periodontopathic bacteria and host factors, including infiltrating cell populations, cytokines, and host matrix metalloproteinases, associated with under-going episodic attachment loss that could partly explain the mechanisms involved in destruction of the supporting tissues of the tooth.

Host response mechanisms in periodontal diseases

Periodontal diseases usually refer to common inflammatory disorders known as gingivitis and periodontitis, which are caused by a pathogenic microbiota in the subgingival biofilm, including Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythia and Treponema denticola that trigger innate, inflammatory, and adaptive immune responses. These processes result in the destruction of the tissues surrounding and supporting the teeth, and eventually in tissue, bone and finally, tooth loss. The innate immune response constitutes a homeostatic system, which is the first line of defense, and is able to recognize invading microorganisms as non-self, triggering immune responses to eliminate them. In addition to the innate immunity, adaptive immunity cells and characteristic cytokines have been described as important players in the periodontal disease pathogenesis scenario, with a special attention to CD4+ T-cells (T-helper cells). Interestingly, the T cell-mediated adaptive immunity development is highly dependent on innate immunity-associated antigen presenting cells, which after antigen capture undergo into a maturation process and migrate towards the lymph nodes, where they produce distinct patterns of cytokines that will contribute to the subsequent polarization and activation of specific T CD4+ lymphocytes. Skeletal homeostasis depends on a dynamic balance between the activities of the bone-forming osteoblasts (OBLs) and bone-resorbing osteoclasts (OCLs). This balance is tightly controlled by various regulatory systems, such as the endocrine system, and is influenced by the immune system, an osteoimmunological regulation depending on lymphocyte- and macrophage-derived cytokines. All these cytokines and inflammatory mediators are capable of acting alone or in concert, to stimulate periodontal breakdown and collagen destruction via tissue-derived matrix metalloproteinases, a characterization of the progression of periodontitis as a stage that presents a significantly host immune and inflammatory response to the microbial challenge that determine of susceptibility to develop the destructive/progressive periodontitis under the influence of multiple behavioral, environmental and genetic factors.

Associations Between Matrix Metalloproteinase-8 and -14 and Myeloperoxidase in Gingival Crevicular Fluid From Subjects With Progressive Chronic Periodontitis: A Longitudinal Study

BACKGROUND Matrix metalloproteinase (MMP)-8 is a central mediator in chronic periodontitis. MMP-8 can be activated by the cooperative action of other MMPs such as MMP-14, reactive oxygen species, and microbial proteases. The aim of this study is to associate the levels, molecular forms, isoenzyme distribution, and degree of activation of MMP-8 and -14, myeloperoxidase (MPO), and tissue inhibitor of MMP (TIMP)-1 in gingival crevicular fluid (GCF) from patients with progressive periodontitis at baseline and after periodontal therapy. METHODS In this longitudinal study, GCF samples from active (n = 25) and inactive (n = 25) sites of subjects with periodontitis were screened at baseline for GCF levels of MMP-8 by immunofluorometric assay, of MMP-14 by specific activity assay, and of MPO and TIMP-1 by enzyme-linked immunosorbent assay. MMP-8 and MPO were also measured after periodontal treatment. Molecular forms were determined by immuno-Western blot analyses and subjected to densitometric scanning and statistical analyses. RESULTS High MMP-8 and MPO levels and a strong MPO/MMP-8 positive correlation were found in active and inactive sites at baseline. After treatment, decreases in MPO and MMP-8 were seen, except for active sites in which MMP-8 differences were not significant (P <0.05). CONCLUSIONS We present initial data that show that GCF levels and associations between MPO and MMP-8 are related to progression episodes and treatment responses in patients with chronic periodontitis. Our results suggest an interaction between the MPO oxidative pathway and MMP-8 activation, and this cascade might be useful as a potential biomarker for treatment outcomes.

Proteolytic roles of matrix metalloproteinase (MMP)‐13 during progression of chronic periodontitis: initial evidence for MMP‐13/MMP‐9 activation cascade

AIM Matrix metalloproteinases (MMP)-13 can initiate bone resorption and activate proMMP-9 in vitro, and both these MMPs have been widely implicated in tissue destruction associated with chronic periodontitis. We studied whether MMP-13 activity and TIMP-1 levels in gingival crevicular fluid (GCF) associated with progression of chronic periodontitis assessed clinically and by measuring carboxy-terminal telopeptide of collagen I (ICTP) levels. We additionally addressed whether MMP-13 could potentiate gelatinase activation in diseased gingival tissue. MATERIALS AND METHODS In this prospective study, GCF samples from subjects undergoing clinical progression of chronic periodontitis and healthy controls were screened for ICTP levels, MMP-13 activity and TIMP-1. Diseased gingival explants were cultured, treated or not with MMP-13 with or without adding CL-82198, a synthetic MMP-13 selective inhibitor, and assayed by gelatin zymography and densitometric analysis. RESULTS Active sites demonstrated increased ICTP levels and MMP-13 activity (p<0.05) in progression subjects. The MMP-9 activation rate was elevated in MMP-13-treated explants (p<0.05) and MMP-13 inhibitor prevented MMP-9 activation. CONCLUSIONS MMP-13 could be implicated in the degradation of soft and hard supporting tissues and proMMP-9 activation during progression of chronic periodontitis. MMP-13 and -9 can potentially form an activation cascade overcoming the protective TIMP-1 shield, which may become useful for diagnostic aims and a target for drug development.

Interleukin-21 Expression and Its Association With Proinflammatory Cytokines in Untreated Chronic Periodontitis Patients

BACKGROUND Interleukin-21 (IL-21) controls the differentiation of T-helper Th17 cells and induces the production of IL-17 in this T-cell subtype. The aim of this study is to determine the relative expression of IL-21 in gingival tissues of chronic periodontitis patients and correlate/associate this expression with proinflammatory cytokines and clinical parameters of disease. METHODS Samples of gingival biopsies were collected from chronic periodontitis patients (n = 10) and controls (n = 8). The mRNA expressions of IL-21, IL-1β, IL-6, IL-17, IL-23, IL-10, and transforming growth factor-β1 (TGF-β1) were quantified using real-time reverse transcription-polymerase chain reaction. IL-21 levels were compared between chronic periodontitis and healthy gingival tissues and correlated with cytokine and clinical parameters of tissue destruction. RESULTS A significant overexpression of IL-21, IL-1β, IL-6, IL-17, and IL-23p19 was detected in periodontal disease-affected tissues compared to healthy gingival tissues. IL-10 and TGF-β1 were, however, downregulated in periodontal lesions. IL-21 yielded significant positive correlations with probing depth, clinical attachment level, IL-1β, and IL-6. In addition, IL-21 was negatively correlated with IL-10 and TGF-β1. CONCLUSIONS IL-21 was overexpressed in chronic periodontitis gingival tissues and correlated with clinical parameters of periodontal destruction and with proinflammatory cytokines. Therefore, IL-21 might play a role in the tissue destruction that characterizes chronic periodontal disease.

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 χ(2), 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.

Melanoma cell lysate induces CCR7 expression and in vivo migration to draining lymph nodes of therapeutic human dendritic cells

We have previously reported a novel method for the production of tumour‐antigen‐presenting cells (referred to as TAPCells) that are currently being used in cancer therapy, using an allogeneic melanoma‐derived cell lysate (referred to as TRIMEL) as an antigen provider and activation factor. It was recently demonstrated that TAPCell‐based immunotherapy induces T‐cell‐mediated immune responses resulting in improved long‐term survival of stage IV melanoma patients. Clinically, dendritic cell (DC) migration from injected sites to lymph nodes is an important requirement for an effective anti‐tumour immunization. This mobilization of DCs is mainly driven by the C‐C chemokine receptor type 7 (CCR7), which is up‐regulated on mature DCs. Using flow cytometry and immunohistochemistry, we investigated if TRIMEL was capable of inducing the expression of the CCR7 on TAPCells and enhancing their migration in vitro, as well as their in vivo relocation to lymph nodes in an ectopic xenograft animal model. Our results confirmed that TRIMEL induces a phenotypic maturation and increases the expression of surface CCR7 on melanoma patient‐derived DCs, and also on the monocytic/macrophage cell line THP‐1. Moreover, in vitro assays showed that TRIMEL‐stimulated DCs and THP‐1 cells were capable of migrating specifically in the presence of the CCR7 ligand CCL19. Finally, we demonstrated that TAPCells could migrate in vivo from the injection site into the draining lymph nodes. This work contributes to an increased understanding of the biology of DCs produced ex vivo allowing the design of new strategies for effective DC‐based vaccines for treating aggressive melanomas.