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AIM: To explore the feasibility of screening for periodontitis by measuring biomarkers, namely total proteolytic activity (TPA), matrix metalloproteinase (MMP)-8, chitinase, lysozyme or their combination, in saliva, oral rinse and gingival crevicular fluid (GCF). MATERIAL AND METHODS: Subjects were recruited among healthy/gingivitis individuals and untreated periodontitis patients in Academic Centre for Dentistry Amsterdam (ACTA). All participants donated samples of unstimulated whole saliva, oral rinse and GCF. The protein concentrations and MMP-8 levels were determined by ELISA. Enzymatic activities were measured using appropriate fluorogenic substrates. RESULTS: In oral rinse samples, periodontitis patients (n = 19) exhibited significantly higher concentrations of MMP-8 and TPA than controls (n = 20). MMP-8 in combination with chitinase explained 88% of the variance and assigned a subject to control or periodontitis group, with best accuracy (87.2%) in oral rinse. CONCLUSIONS: The combination of MMP-8 and chitinase in the current oral rinse procedure has the potential to discriminate periodontitis from periodontal health/gingivitis.
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Gingivitis , Periodontitis , Biomarcadores/análisis , Líquido del Surco Gingival/química , Gingivitis/diagnóstico , Humanos , Metaloproteinasa 8 de la Matriz , Periodontitis/diagnóstico , Proyectos Piloto , Saliva/químicaRESUMEN
BACKGROUND: Since periodontitis is bi-directionally associated with several systemic diseases, such as diabetes mellitus and cardiovascular diseases, it is important for medical professionals in a non-dental setting to be able examine their patients for symptoms of periodontitis, and urge them to visit a dentist if necessary. However, they often lack the time, knowledge and resources to do so. We aim to develop and assess "quick and easy" screening tools for periodontitis, based on self-reported oral health (SROH), demographics and/or salivary biomarkers, intended for use by medical professionals in a non-dental setting. METHODS: Consecutive, new patients from our outpatient clinic were recruited. A SROH questionnaire (8 questions) was conducted, followed by a 30 s oral rinse sampling protocol. A complete clinical periodontal examination provided the golden standard periodontitis classification: no/mild, moderate or severe periodontitis. Total periodontitis was defined as having either moderate or severe. Albumin and matrix metalloproteinase-8 concentrations, and chitinase and protease activities were measured in the oral rinses. Binary logistic regression analyses with backward elimination were used to create prediction models for both total and severe periodontitis. Model 1 included SROH, demographics and biomarkers. The biomarkers were omitted in the analysis for model 2, while model 3 only included the SROH questionnaire. The area under the receiver operating characteristic curves (AUROCC) provided the accuracy of each model. The regression equations were used to create scoring algorithms, composed of the remaining predictors, each with its own weight. RESULTS: Of the 156 patients participating in this study, 67% were classified with total periodontitis and 33% had severe periodontitis. The models for total periodontitis achieved an AUROCC of 0.91 for model 1, 0.88 for model 2 and 0.81 for model 3. For severe periodontitis, this was 0.89 for model 1, 0.82 for model 2 and 0.78 for model 3. The algorithm for total periodontitis (model 2), which we consider valid for the Dutch population, was applied to create a freely accessible, web-based screening tool. CONCLUSIONS: The prediction models for total and severe periodontitis proved to be feasible and accurate, resulting in easily applicable screening tools, intended for a non-dental setting.
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Periodontitis/diagnóstico , Humanos , Tamizaje Masivo , Autoinforme , Sensibilidad y Especificidad , Encuestas y CuestionariosRESUMEN
BACKGROUND: Maintaining oral health is a continuous and dynamic process that also involves the immune system. Polymorphonuclear neutrophils (PMNs) migrate from blood circulation and become apparent in the oral fluid. Controversies exist regarding the specific role of the oral PMNs (oPMNs) in the presence of chronic oral inflammation, such as periodontitis. In this study we characterized cell counts, activation status, apoptosis, and reactive oxygen species (ROS) generation by oPMNs and circulatory (cPMNs), and the salivary protease activity, in subjects with and without periodontitis. METHODS: Venous blood and oral rinse samples were obtained from 19 patients with untreated periodontitis and 16 control subjects for PMN isolation. Apoptosis and expression of cell activation markers CD11b, CD63, and CD66b were analyzed using flow cytometry. Constitutive ROS generation was detected using dihydrorhodamine123. Additionally, ROS production in response to stimulation was evaluated in samples incubated with 10 µM phorbol myristate acetate (PMA) or Fusobacterium nucleatum. Total protease activity was measured using substrate PEK-054. RESULTS: Periodontitis patients presented with over 4 times higher oPMN counts compared to controls (p = 0.007), which was a predictor for the total protease activity (r2 = 0.399, P = 0.007). More oPMNs were apoptotic in periodontitis patients compared to the controls (P = 0.004). All three activation markers were more expressed on the oPMNs compared to the cPMNs (p < 0.05), and a higher expression of CD11b on the oPMNs from periodontitis patients was observed compared to the control subjects (P = 0.024). Constitutive ROS production per oPMN was higher compared to the cPMN (P < 0.001). Additional analysis showed that the oPMNs retained their ability to respond to stimulation, with no apparent differences between the periodontitis and control subjects. CONCLUSIONS: Higher numbers of oral PMNs, being more apoptotic and having increased levels of degranulation markers were found in periodontitis compared to periodontal health. However, since the oPMNs in periodontitis were responsive to ex vivo stimulation, we conclude that the oPMNs are active in the oral ecosystem. It is currently unknown whether the oPMN counts, which correlated with the detected protease levels, are detrimental in the long term for the oral mucosa integrity. TRIAL REGISTRATION: This study was retrospectively registered at the ISRCTN registry (trial ID ISRCTN15252886 ). Registration date August 11, 2017.
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Neutrófilos/inmunología , Periodontitis/inmunología , Adulto , Apoptosis , Estudios de Casos y Controles , Femenino , Citometría de Flujo , Humanos , Masculino , Persona de Mediana Edad , Neutrófilos/metabolismo , Péptido Hidrolasas/metabolismo , Periodontitis/metabolismo , Periodontitis/microbiología , Especies Reactivas de Oxígeno/metabolismo , Saliva/químicaRESUMEN
BACKGROUND: Platelets from untreated periodontitis patients are hyper-reactive and form more platelet-leukocyte complexes compared to cells from individuals without periodontitis. It is not known whether the improvement of the periodontal condition achievable by therapy has beneficial effects on the platelet function. We aimed to assess the effects of periodontal therapy on platelet reactivity. METHODS: Patients with periodontitis (n = 25) but unaffected by any other medical condition or medication were included and donated blood before and after periodontal therapy. Reactivity to ADP or oral bacteria was assessed by flow cytometric analysis of membrane markers (binding of PAC-1, P-selectin, CD63) and platelet-leukocyte complex formation. Reactivity values were expressed as ratio between the stimulated and unstimulated sample. Plasma levels of soluble (s) P-selectin were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: Binding of PAC-1, the expression of P-selectin and CD63 in response to the oral bacterium P. gingivalis were lower at recall (1.4 ± 1.1, 1.5 ± 1.2, and 1.0 ± 0.1) than at baseline (2.7 ± 4.1, P = 0.026, 6.0 ± 12.5, P = 0.045, and 2.7 ± 6.7, P = 0.042, respectively). Formation of platelet-leukocyte complexes in response to P. gingivalis was also reduced at recall compared to baseline (1.2 ± 0.7 vs. 11.4 ± 50.5, P = 0.045). sP-selectin levels were significantly increased post-therapy. CONCLUSIONS: In periodontitis patients, the improvement of the periodontal condition is paralleled by a reduction in platelet hyper-reactivity. We suggest that periodontal therapy, as an intervention for improved oral health, can facilitate the management of thrombotic risk, and on the long term can contribute to the prevention of cardiovascular events in patients at risk. TRIAL REGISTRATION: Current Controlled Trials identifier ISRCTN36043780. Retrospectively registered 25 September 2013.
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Oral health is characterized by functional oral polymorphonuclear neutrophils (oPMNs). Edentulism might be associated with a loss of oPMNs because these cells enter the oral cavity primarily through the gingival crevices. The main aim of this study was to investigate the numbers of oPMNs in rinse samples obtained from edentulous (n = 21) and dentate (n = 20) subjects. A second study aim was to investigate possible differences between oPMNs and peripheral blood polymorphonuclear neutrophils (cPMNs). Apoptosis/necrosis and cell-activation markers (CD11b, CD63 and CD66b) were analyzed using flow cytometry. Reactive oxygen species (ROS) production was determined either without stimulation (constitutive) or in response to 10 µM phorbol myristate acetate or Fusobacterium nucleatum. The edentulous subjects presented with lower oPMN counts and higher percentages of apoptotic/necrotic oPMNs compared with dentate subjects. Furthermore, oPMNs from edentulous donors expressed low levels of all three activation markers and low constitutive ROS. In contrast, oPMNs from dentate subjects expressed high levels of all three activation markers and a higher level of constitutive ROS than cPMNs. When challenged, oPMNs from edentulous subjects showed no upregulation in ROS production, whereas oPMNs from dentate subjects retained their ability to respond to stimulation. The functional characteristics of cPMNs were comparable between edentulous and dentate subjects. This study demonstrates that despite having functional cPMNs, edentulous subjects have low oPMN numbers that are functionally impaired.
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Boca Edéntula/metabolismo , Neutrófilos/metabolismo , Anciano , Antígenos CD/metabolismo , Apoptosis , Antígeno CD11b/metabolismo , Estudios de Casos y Controles , Moléculas de Adhesión Celular/metabolismo , Femenino , Citometría de Flujo , Proteínas Ligadas a GPI/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Necrosis , Especies Reactivas de Oxígeno/metabolismo , Tetraspanina 30/metabolismoRESUMEN
The main focus of this review is polymorphonuclear neutrophilic granulocytes. Polymorphonuclear neutrophils play a pivotal role in normal host resistance to subgingival dental-plaque biofilm. Both hyper- and hypo-responsiveness of the immune system toward the microbial challenge in periodontitis have been described. We review polymorphonuclear neutrophil physiology with emphasis on the role of neutrophil functions and dysfunctions in periodontitis. Text boxes are given at the end of each subsection, which present the current knowledge on neutrophil-modulating agents as a potential therapeutic approach in periodontitis.
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Neutrófilos/inmunología , Neutrófilos/patología , Periodontitis/inmunología , Periodontitis/terapia , Animales , Granulocitos/inmunología , Humanos , Periodontitis/sangre , Periodontitis/microbiología , FagocitosisRESUMEN
AIM: This 3-year prospective randomized controlled trial compared the clinical, microbiological and biochemical outcome of minimally (Turned, Tur) and moderately rough (TiUnite(®) , TiU) implant surfaces in a split-mouth design. MATERIAL AND METHODS: The study population included 14 subjects: nine fully edentulous and five partially edentulous subjects with a history of periodontitis. Implants (n = 78, 39 Tur and 39 TiU) were installed randomly in each patient. Peri-implant clinical parameters and intra-oral radiographs were recorded after 3 years of loading. Subgingival plaque and peri-implant crevicular fluid samples were collected and analysed using culture and quantitative polymerase chain reaction for the biofilm, and enzyme-linked immunosorbent assay for the concentration of osteoprotegerin and receptor activator of nuclear factor kappa-B ligand, respectively. RESULTS: No statistically significant differences in clinical, microbiological and biochemical parameters could be observed when comparing the Tur and TiU implant surfaces. CONCLUSION: After 3 years of loading, in periodontitis susceptible patients, the moderately rough, TiU implants demonstrated a similar clinical outcome compared with the smoother, turned implants. Longer follow-up and studies using different implant types are needed to confirm the statement that minimally and moderately rough implant surfaces perform similar, both from a clinical and from a microbiological point of view.
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Implantes Dentales , Diseño de Prótesis Dental , Prótesis Dental de Soporte Implantado , Arcada Parcialmente Edéntula/cirugía , Arcada Edéntula/cirugía , Anciano , Pérdida de Hueso Alveolar/etiología , Análisis de Varianza , Periodontitis Crónica , Materiales Biocompatibles Revestidos , Prótesis Dental de Soporte Implantado/efectos adversos , Femenino , Estudios de Seguimiento , Líquido del Surco Gingival/química , Recesión Gingival/etiología , Humanos , Masculino , Persona de Mediana Edad , Osteoprotegerina/análisis , Óxidos , Índice Periodontal , Proyectos Piloto , Estudios Prospectivos , Ligando RANK/análisis , Propiedades de SuperficieRESUMEN
OBJECTIVE: To assess the available scientific evidence regarding the placental microbial composition of a healthy pregnancy, the quality of this evidence, and the potential relation between placental and oral microbiome. MATERIALS AND METHODS: Data sources: MEDLINE and EMBASE up to August 1, 2019. STUDY ELIGIBILITY CRITERIA: Human subjects; healthy women; term deliveries; healthy normal birth weight; assessment of microorganisms (bacteria) in placental tissue; full research papers in English. The quality of the included studies was assessed by a modified Joanna Briggs Institute checklist for analytical cross-sectional studies. RESULTS: 57 studies passed the inclusion criteria. Of these, 33 had a high risk of quality bias (e.g., insufficient infection control, lack of negative controls, poor description of the healthy cases). The remaining 24 studies had a low (N = 12) to moderate (N = 12) risk of bias and were selected for in-depth analysis. Of these 24 studies, 22 reported microorganisms in placental tissues, where Lactobacillus (11 studies), Ureaplasma (7), Fusobacterium (7), Staphylococcus (7), Prevotella (6) and Streptococcus (6) were among the most frequently identified genera. Methylobacterium (4), Propionibacterium (3), Pseudomonas (3) and Escherichia (2), among others, although frequently reported in placental samples, were often reported as contaminants in studies that used negative controls. CONCLUSIONS: The results support the existence of a low biomass placental microbiota in healthy pregnancies. Some of the microbial taxa found in the placenta might have an oral origin. The high risk of quality bias for the majority of the included studies indicates that the results of individual papers should be interpreted with caution.
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Fusobacterium/fisiología , Lactobacillus/fisiología , Microbiota/genética , Placenta/microbiología , Embarazo , ARN Ribosómico 16S/genética , Ureaplasma/fisiología , Adulto , Animales , Femenino , Voluntarios Sanos , HumanosRESUMEN
OBJECTIVE: Periodontitis is associated with cardiovascular diseases (CVD). In our previous studies a prothrombotic state has been observed in periodontitis, which contributes to the risk of CVD. The aim of this study was to investigate whether serum IgG levels against Aggregatibacter actinomycetemcomitans (Aa) and Porphyromonas gingivalis (Pg) in periodontitis were associated with a prothrombotic state. MATERIALS AND METHODS: Patients with moderate (n = 38) and severe periodontitis (n = 30) and controls (n = 24) were recruited. We explored correlations between serum anti-Aa and anti-Pg IgG and plasma levels of markers of prothrombotic state (von Willebrand Factor [vWF], prothrombin fragment 1+2 [F1+2], plasminogen activator inhibitor-1 [PAI-1] and D-dimer). Multivariate analyses were performed considering several major potential contributing factors. RESULTS: Periodontitis patients showed higher anti-Aa IgG (p = 0.015) than controls but not for Pg (p = 0.320). In periodontitis patients, body mass index and anti-Aa IgG showed a positive correlation with vWF (ß = 0.297, p = 0.010 and ß = 0.248, p = 0.033 respectively). CONCLUSIONS: In periodontitis, infection with Aa together with other well accepted risk factors for CVD, may play a role in increasing the risk for prothrombotic state.
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Oral health and dentistry are essential components of systems medicine, which has received lesser attention in comparison to other medical fields, such as cancer biology. In this context, oral polymorphonuclear neutrophils (oPMNs) play an important role in the maintenance of oral health. To the best of our knowledge, this is the first study to report original observations on the transcriptional responses of oPMNs during experimentally induced gingivitis, by temporarily refraining from regular oral care. Oral rinses were prospectively collected at four different time points for oPMNs isolation from healthy volunteers: day 1 (start of the experimental gingivitis challenge), day 9 (during challenge), day 14 (end of the challenge), and day 21 (postchallenge). Transcriptome of oPMNs was determined by RNA sequencing. Differentially expressed genes (DEGs) were selected at p < 0.01 level, and evaluated for pathway regulation using Ingenuity Pathway Analysis suite. We found four major clusters of DEGs, consisting of 256 initial response DEGs (day 9 only), 221 late response DEGs (day 14 only), 53 persistent responsive DEGs (consistent at day 9 and 14), and 524 DEGs showing responses only in the postchallenge phase (day 21 only). Pathway analysis of the initial and late response DEGs showed involvement in many immune regulatory pathways and PMN function, whereas DEGs at day 21 were associated with epithelial adherence signaling and other miscellaneous related signaling pathways. The results from this pilot study showed that oPMNs mediate oral inflammatory processes, suggesting their immunomodulatory role in oral equilibrium.
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Odontología/métodos , Genómica , Gingivitis/etiología , Boca/microbiología , Neutrófilos/inmunología , Neutrófilos/metabolismo , Higiene Bucal , Comunicación Celular , Odontología/normas , Susceptibilidad a Enfermedades , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Genómica/métodos , Gingivitis/metabolismo , Gingivitis/patología , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Neutrófilos/patología , Transducción de SeñalRESUMEN
In periodontitis, polymorphonuclear leucocytes (PMNs) are activated. They entrap and eliminate pathogens by releasing neutrophil extracellular traps (NETs). Abnormal NET degradation is part of a pro-inflammatory status, affecting co-morbidities such as cardiovascular disease. We aimed to investigate the ex vivo NET degradation capacity of plasma from periodontitis patients compared to controls (part 1) and to quantify NET degradation before and after periodontal therapy (part 2). Fresh NETs were obtained by stimulating blood-derived PMNs with phorbol 12-myristate 13-acetate. Plasma samples from untreated periodontitis patients and controls were incubated for 3 h onto freshly generated NETs (part 1). Similarly, for part 2, NET degradation was studied for 91 patients before and 3, 6 and 12 mo after non-surgical periodontal therapy with and without adjunctive systemic antibiotics. Finally, NET degradation was fluorospectrometrically quantified. NET degradation levels did not differ between periodontitis patients and controls, irrespective of subject-related background characteristics. NET degradation significantly increased from 65.6 ± 1.7% before periodontal treatment to 75.7 ± 1.2% at 3 mo post periodontal therapy, and this improvement was maintained at 6 and 12 mo, irrespective of systemic usage of antibiotics. Improved NET degradation after periodontitis treatment is another systemic biomarker reflecting a decreased pro-inflammatory status, which also contributes to an improved cardiovascular condition.
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Trampas Extracelulares/inmunología , Neutrófilos/inmunología , Periodontitis/inmunología , Adulto , Amoxicilina/uso terapéutico , Animales , Antibacterianos/uso terapéutico , Células Cultivadas , Enfermedad Crónica , Femenino , Humanos , Inmunidad Innata , Masculino , Metronidazol/uso terapéutico , Persona de Mediana Edad , Periodontitis/terapiaRESUMEN
Maintenance of oral health is in part managed by the immune-surveillance and antimicrobial functions of polymorphonuclear leukocytes (PMNs), which migrate from the circulatory system through the oral mucosal tissues as oral PMNs (oPMNs). In any microorganism-rich ecosystem, such as the oral cavity, PMNs migrate toward various exogenous chemoattractants, phagocytose bacteria, and produce neutrophil extracellular traps (NETs) to immobilize and eliminate pathogens. PMNs obtained from the circulation through venipuncture (hereafter called cPMNs) have been widely studied using various functional assays. We aimed to study the potential of oPMNs in maintaining oral health and therefore compared their chemotactic and antimicrobial functions with cPMNs. To establish chemotactic, phagocytic, and NET forming capacities, oPMNs and cPMNs were isolated from healthy subjects without obvious oral inflammation. Directional chemotaxis toward the chemoattractant fMLP was analyzed using an Insall chamber and video microscopy. fMLP expression was assessed by flow cytometry. Phagocytosis was analyzed by flow cytometry, following PMN incubation with heat-inactivated FITC-labeled micro-organisms. Furthermore, agar plate-based killing assays were performed with Escherichia coli (Ec). NET formation by oPMNs and cPMNs was quantified fluorimetrically using SYTOX™ Green, following stimulation with either PMA or RPMI medium (unstimulated control). In contrast to cPMNs, the chemotactic responses of oPMNs to fMLP did not differ from controls (mean velocity ± SEM of cPMNs: 0.79 ± 0.24; of oPMNs; 0.10 ± 0.07 micrometer/min). The impaired directional movement toward fMLP by oPMNs was explained by significantly lower fMLP receptor expression. Increased adhesion and internalization of various micro-organisms by oPMNs was observed. oPMNs formed 13 times more NETs than stimulated cPMNs, in both unstimulated and stimulated conditions. Compared to cPMNs, oPMNs showed a limited ability for intracellular killing of Ec. In conclusion, oPMNs showed exhausted capacity for efficient chemotaxis toward fMLP which may be the result of migration through the oral tissues into the oral cavity, being a highly "hostile" ecosystem. Overall, oPMNs' behavior is consistent with hyperactivity and frustrated killing. Nevertheless, oPMNs most likely contribute to maintaining a balanced oral ecosystem, as their ability to internalize microbes in conjunction with their abundant NET production remains after entering the oral cavity.
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Quimiotaxis/inmunología , Trampas Extracelulares/inmunología , Mucosa Bucal/inmunología , Neutrófilos/inmunología , Fagocitosis , Humanos , Inflamación/inmunologíaRESUMEN
The ligand of the receptor activator of NF-κB (RANKL) is a key molecule in the formation of osteoclasts, the key cells that cause the disease-associated alveolar bone resorption in periodontitis. We hypothesized that polymorphonuclear leukocytes (PMNs), found as the most prominent cells of inflamed periodontal tissues, could play an important role in providing signals to trigger osteoclastogenesis and thus activating pathological bone resorption in periodontitis. RANKL expression was investigated on circulatory PMNs (cPMNs) and oral PMNs (oPMNs) taken from both controls and periodontitis patients. On average, 2.3% and 2.4% RANKL expression was detected on the cPMNs and oPMNs from periodontitis patients, which did not differ significantly from healthy controls. Since cPMNs may acquire a more osteoclastogenesis-facilitating phenotype while migrating into the inflamed periodontium, we next investigated whether stimulated (with LPS, TNF-α, or IL-6) cPMNs have the capacity to contribute to osteoclastogenesis. Enduring surface expression of RANKL for short-lived cells as cPMNs was achieved by fixating stimulated cPMNs. RANKL expression on stimulated cPMNs, as assessed by flow cytometry and immunohistochemistry, was limited (6.48 ± 0.72%, mean expression ± SEM) after 24 and 48 hours of stimulation with LPS. Likewise, stimulation with TNF-α and IL-6 resulted in limited RANKL expression levels. These limited levels of expression did not induce osteoclastogenesis when cocultured with preosteoclasts for 10 days. We report that, under the aforementioned experimental conditions, neither cPMNs nor oPMNs directly induced osteoclastogenesis. Further elucidation of the key cellular players and immune mediators that stimulate alveolar bone resorption in periodontitis will help to unravel its pathogenesis.
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Neutrófilos/metabolismo , Osteogénesis/inmunología , Ligando RANK/metabolismo , Adulto , Anciano , Pérdida de Hueso Alveolar/etiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Periodontitis/inmunología , Periodontitis/fisiopatologíaRESUMEN
PURPOSE OF REVIEW: Oral health is maintained in a dynamic equilibrium between the host immunity and the oral microbiome. Oral polymorphonuclear neutrophils (oPMNs) are important innate immune cells in the oral cavity. RECENT FINDINGS: The oPMNs play a co-controlling part in the maintenance of oral equilibrium. In human saliva, the oPMNs integrity is preserved, and their function remains unaffected. In general, oPMNs are in a higher state of baseline activation compared to peripheral PMNs. However, in periodontitis, the oPMNs' activation state can result in excessive release of damaging molecules in the extracellular environment. SUMMARY: The presence of oPMNs may unwittingly negatively impact the integrity of the oral tissues. While most of the oPMN functions occur intracellularly, release of their potent active mediators into the extracellular environment may jeopardize oral homeostasis and its integrity. The dual nature of oPMNs, both beneficial and detrimental, remains a challenging and understudied topic.
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Periodontitis, a chronic inflammatory disease of the periodontium, is characterized by osteoclast-mediated alveolar bone destruction. Gingival fibroblasts (GFs) present in the bone-lining mucosa have the capacity to activate the formation of osteoclasts, but little is known about which local immune cells (co-)mediate this process. The aim of this study was to investigate the cellular interactions of GFs with immune cells, including the contribution of GFs to osteoclast formation and their possible role in the proliferation of these immune cells. In addition, we investigated the expression of adhesion molecules and the inflammatory cytokines that are evoked by this interaction. GFs were cocultured with peripheral blood mononuclear cells (PBMCs), CD14+ monocytes or peripheral blood lymphocytes (PBLs) for 7, 14, and 21 days. After 21 days, comparable numbers of multinucleated cells (osteoclasts) were found in gingival fibroblast (GF)-PBMC and GF-monocyte cocultures. No osteoclasts were formed in GF-PBL cocultures, indicating that the PBLs present in GF-PBMC cocultures do not contribute to osteoclastogenesis. Persisting mononuclear cells were interacting with osteoclasts in GF-PBMC cocultures. Remarkably, a predominance of CD3+ T cells was immunohistochemically detected in GF cocultures with PBLs and PBMCs for 21 days that frequently interacted with osteoclasts. Significantly more T, B (CD19+), and NK (CD56+CD3-) cells were identified with multicolor flow cytometry in both GF-PBMC and GF-PBL cocultures compared to monocultures without GFs at all time points. GFs retained PBLs independently of the presence of monocytes or osteoclasts over time, showing a stable population of T, B, and NK cells between 7 and 21 days. T helper and cytotoxic T cell subsets remained stable over time in GF cocultures, while the number of Th17 cells fluctuated. Lymphocyte retention is likely mediated by lymphocyte-function-associated antigen-1 (LFA-1) expression, which was significantly higher in GF-PBL cultures compared to GF-monocyte cultures. When assessing inflammatory cytokine expression, high tumor necrosis alpha expression was only observed in the GF-PBMC cultures, indicating that this tripartite presence of GFs, monocytes, and lymphocytes is required for such an induction. Carboxyfluorescein succinimidyl ester-labeling showed that only the CD3+ cells proliferated in presence of GFs. This study demonstrates a novel role for GFs in the survival, retention, and selective proliferation of lymphocytes.
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Comunicación Celular , Fibroblastos/metabolismo , Encía/citología , Linfocitos/inmunología , Linfocitos/metabolismo , Biomarcadores , Supervivencia Celular , Técnicas de Cocultivo , Citocinas/biosíntesis , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Inmunofenotipificación , Molécula 1 de Adhesión Intercelular/metabolismo , Activación de Linfocitos/inmunología , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Osteoclastos/citología , Osteoclastos/metabolismo , Osteogénesis/genética , Osteogénesis/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
OBJECTIVE: Polymorphonuclear neutrophils (PMNs) are the most abundant innate immune cells and are also important effectors in the maintenance of oral health. However, little is known about the effects of saliva on the PMN. We therefore aimed to investigate the effect of saliva on the PMNs' morphology and functioning. DESIGN: Effect of saliva on the membrane integrity of PMNs isolated from blood was evaluated with FACS using Annexin V (apoptosis marker) and propidum iodide (membrane integrity marker). The effect on cell morphology was examined using transmission electron imaging. Binding and phagocytosis of the oral bacterium Fusobacterium nucleatum by PMNs was analysed by FACS. Reactive oxygen species (ROS) production was measured with chemiluminescence. RESULTS: Incubation with saliva for 60â¯min had no detectable effects on the membrane integrity or the morphology of PMNs. In contrast, preincubation of F. nucleatum with saliva inhibited its subsequent interaction with PMNs, resulting in a diminished production of ROS. CONCLUSIONS: Saliva does not impair the function of PMNs. However, interaction of salivary components with F. nucleatum may affect their recognition by PMNs resulting in a diminished functional response.
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Neutrófilos/fisiología , Saliva/metabolismo , Adulto , Apoptosis , Adhesión Bacteriana , Femenino , Fusobacterium nucleatum/metabolismo , Voluntarios Sanos , Humanos , Masculino , Microscopía Electrónica de Transmisión , Persona de Mediana Edad , Fagocitosis , Fenotipo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Inflammatory response is a protective biological process intended to eliminate the harmful effect of the insulting influx. Resolution of inflammation constitutes an active sequence of overlapping events mediated by specialized proresolving mediators, such as lipoxins, resolvins, protectins, and maresins, which originate from the enzymatic conversion of polyunsaturated fatty acids (PUFAs). An unresolved acute inflammatory response results in chronic inflammation, which is a leading cause of several common pathological conditions. Periodontitis is a biofilm-induced chronic inflammatory disease, which results in loss of periodontal connective tissue and alveolar bone support around the teeth, leading to tooth exfoliation. An inadequate proresolving host response may constitute a mechanism explaining the pathogenesis of periodontal disease. An emerging body of clinical and experimental evidence has focused on the underlying molecular mechanisms of resolvins and particularly Resolvin E1 (RvE1) in periodontitis. Recently, RvE1 has been directly correlated with the resolution of inflammation in periodontal disease. Herein, we provide a comprehensive overview of the literature regarding the role and possible mechanisms of action of RvE1 on different cell populations recruited in periodontal inflammation as well as its potential therapeutic implications. Along with recent data on the benefits of PUFAs supplementation in periodontal clinical parameters, we touch upon suggested future directions for research.
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Since the etiology of periodontitis in humans is not fully understood, genetic mouse models may pinpoint indispensable genes for optimal immunological protection of the periodontium against tissue destruction. This review describes the current knowledge of genes that are involved for a proper maintenance of a healthy periodontium in mice. Null mutations of genes required for leukocyte cell-cell recognition and extravasation (e.g., Icam-1, P-selectin, Beta2-integrin/Cd18), for pathogen recognition and killing (e.g., Tlr2, Tlr4, Lamp-2), immune modulatory molecules (e.g., Cxcr2, Ccr4, IL-10, Opg, IL1RA, Tnf-α receptor, IL-17 receptor, Socs3, Foxo1), and proteolytic enzymes (e.g., Mmp8, Plasmin) cause periodontitis, most likely due to an inefficient clearance of bacteria and bacterial products. Several mechanisms resulting in periodontitis can be recognized: (1) inefficient bacterial control by the polymorphonuclear neutrophils (defective migration, killing), (2) inadequate antigen presentation by dendritic cells, or (3) exaggerated production of pro-inflammatory cytokines. In all these cases, the local immune reaction is skewed toward a Th1/Th17 (and insufficient activation of the Th2/Treg) with subsequent osteoclast activation. Finally, genotypes are described that protect the mice from periodontitis: the SCID mouse, and mice lacking Tlr2/Tlr4, the Ccr1/Ccr5, the Tnf-α receptor p55, and Cathepsin K by attenuating the inflammatory reaction and the osteoclastogenic response.
RESUMEN
A dysbiotic state is believed to be a key factor in the onset of oral disease. Although oral diseases have been studied for decades, our understanding of oral health, the boundaries of a healthy oral ecosystem and ecological shift toward dysbiosis is still limited. Here, we present the ecobiological heterogeneity of the salivary ecosystem and relations between the salivary microbiome, salivary metabolome and host-related biochemical salivary parameters in 268 healthy adults after overnight fasting. Gender-specific differences in the microbiome and metabolome were observed and were associated with salivary pH and dietary protein intake. Our analysis grouped the individuals into five microbiome and four metabolome-based clusters that significantly related to biochemical parameters of saliva. Low salivary pH and high lysozyme activity were associated with high proportions of streptococcal phylotypes and increased membrane-lipid degradation products. Samples with high salivary pH displayed increased chitinase activity, higher abundance of Veillonella and Prevotella species and higher levels of amino acid fermentation products, suggesting proteolytic adaptation. An over-specialization toward either a proteolytic or a saccharolytic ecotype may indicate a shift toward a dysbiotic state. Their prognostic value and the degree to which these ecotypes are related to increased disease risk remains to be determined.
Asunto(s)
Metaboloma , Microbiota , Saliva/metabolismo , Saliva/microbiología , Adolescente , Adulto , Bacterias/clasificación , Bacterias/aislamiento & purificación , Estudios Transversales , Disbiosis , Ecosistema , Femenino , Humanos , Masculino , Adulto JovenRESUMEN
Polymorphonuclear neutrophils (PMNs) have a major role in the innate immune system. However, little is known about PMN contribution in relation to oral health. The objective of this study was to investigate the numbers and functional characteristics of oral PMNs (oPMNs) compared with circulatory PMNs (cPMNs). Oral rinse and venous blood samples were obtained from 268 systemically and orally healthy volunteers in a cross-sectional observational study. PMN counts, cell cycle analysis and cellular activation state were investigated. Also, reactive oxygen species (ROS) production was analyzed, with and without bacterial stimulation (Fusobacterium nucleatum). In males, 1.2 × 10(6)±1.0 × 10(6) oPMNs were collected, and showed a tendency to correlate with the levels of gingival bleeding (r=0.215, P=0.008). Comparable oPMNs counts were found among females (1.0 × 10(6)±0.7 × 10(6)). More late-stage apoptotic/necrotic cells were found among the oPMNs (53.1%) compared with the cPMNs (8.5%; P<0.001). Without additional stimulation, oPMNs were more activated than cPMNs, as indicated by higher expression of CD11b, CD63 and CD66b, and higher constitutive ROS levels (P<0.001). Notably, in response to bacterial stimulation, oPMNs released comparable ROS levels as cPMNs (P=0.042). In conclusion, this study provides data on viable oPMNs showing high levels of activation in orally and systemically healthy individuals, free of apparent caries lesions and periodontal disease. These data suggests that although the oPMNs are in a more mature stage of their life cycle compared with the cPMNs, oPMNs are still responsive to stimulation, which indicates their functional potential and possible contribution to a healthy oral ecosystem.