RESUMEN
BACKGROUND AND OBJECTIVE: Host responses in periodontitis span a range of local and emigrating cell types and biomolecules. Accumulating evidence regarding the expression of this disease across the population suggests some component of genetic variation that controls onset and severity of disease, in concert with the qualitative and quantitative parameters of the oral microbiome at sites of disease. However, there remains little information regarding the capacity of accruing environmental stressors or modifiers over a lifespan at both the host genetic and microbial ecology levels to understand fully the population variation in disease. This study evaluated the impact of environmental lead exposure on the responses of oral epithelial cells to challenge with a model pathogenic oral biofilm. METHODS AND RESULTS: Using NanoString technology to quantify gene expression profiles of an array of 511 host response-associated genes in the epithelial cells, we identified an interesting primary panel of basal responses of the cells with numerous genes not previously considered as major response markers for epithelial cells, eg, interleukin (IL)-32, CTNNB1, CD59, MIF, CD44 and CD99. Even high levels of environment lead had little effect on these constitutive responses. Challenge of the cells with the biofilms (Streptococcus gordonii/Fusobacterium nucleatum/Porphyromonas gingivalis) resulted in significant increases in an array of host immune-related genes (134 of 511). The greatest magnitude in differential expression was observed with many genes not previously described as major response genes in epithelial cells, including IL-32, CD44, NFKBIA, CTSC, TNFAIP3, IL-1A, IL-1B, IL-8 and CCL20. The effects of environmental lead on responses to the biofilms were mixed, although levels of IL-8, CCL20 and CD70 were significantly decreased at lead concentrations of 1 and/or 5 µmol/L. CONCLUSION: The results provided new information on a portfolio of genes expressed by oral epithelial cells, targeted substantial increases in an array of immune-related genes post-biofilm challenge, and a focused impact of environmental lead on these induced responses.
Asunto(s)
Biopelículas , Exposición a Riesgos Ambientales/efectos adversos , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Expresión Génica/efectos de los fármacos , Plomo/efectos adversos , Mucosa Bucal/microbiología , Antígenos CD59/genética , Antígenos CD59/metabolismo , Línea Celular , Quimiocina CCL20/genética , Quimiocina CCL20/metabolismo , Células Epiteliales/microbiología , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Mucosa Bucal/citología , Mucosa Bucal/inmunología , Inhibidor NF-kappaB alfa/genética , Inhibidor NF-kappaB alfa/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Maintenance of periodontal health or transition to a periodontal lesion reflects the continuous and ongoing battle between the vast microbial ecology in the oral cavity and the array of resident and emigrating inflammatory/immune cells in the periodontium. This war clearly signifies many 'battlefronts' representing the interface of the mucosal-surface cells with the dynamic biofilms composed of commensal and potential pathogenic species, as well as more recent knowledge demonstrating active invasion of cells and tissues of the periodontium leading to skirmishes in connective tissue, the locality of bone and even in the local vasculature. Research in the discipline has uncovered a concerted effort of the microbiome, using an array of survival strategies, to interact with other bacteria and host cells. These strategies aid in colonization by 'ambushing, infiltrating and outflanking' host cells and molecules, responding to local environmental changes (including booby traps for host biomolecules), communicating within and between genera and species that provide MASINT (Measurement and Signature Intelligence) to enhance sustained survival, sabotage the host inflammatory and immune responses and by potentially adopting a 'Fabian strategy' with a war of attrition and resulting disease manifestations. Additionally, much has been learned regarding the ever-increasing complexity of the host-response armamentarium at both cellular and molecular levels that is addressed in this review. Knowledge regarding how these systems fully interact requires both new laboratory and clinical tools, as well as sophisticated modeling of the networks that help maintain homeostasis and are dysregulated in disease. Finally, the triggers resulting in a 'coup de main' by the microbiome (exacerbation of disease) and the characteristics of susceptible hosts that can result in 'pyrrhic victories' with collateral damage to host tissues, the hallmark of periodontitis, remains unclear. While much has been learned, substantial gaps in our understanding of the 'parameters of this war' remain elusive toward fulfilling the Sun Tzu adage: 'If you know the enemy and know yourself, you need not fear the result of a hundred battles.'
Asunto(s)
Interacciones Huésped-Patógeno/inmunología , Boca/microbiología , Periodontitis/inmunología , Periodontitis/microbiología , Biopelículas , Humanos , Microbiota/inmunologíaRESUMEN
The release of freely loaded small molecules from biomaterials often exhibits an initial burst, inhibiting the ability of these materials to match drug release with the biomaterial's degradation period. In terms of antibiotic release systems, the remaining vehicle may become a substrate for colonization by bacterial biofilms once the payload is depleted, which can become life threatening. Secondary surgeries are typically performed to remove these empty depots as a means of preventing this type of infection. To maintain the effectiveness of a locally delivered antibiotic without the drawback of a second surgery, we propose a hydrogel drug delivery system in which the drug release rate of vancomycin and degradation rate of the hydrogel are linked via covalent incorporation of vancomycin in the hydrogel backbone. This was achieved through coupling PEG based monomer with vancomycin to create poly(ß-amino ester) chemistry and verified through drug release and matrix degradation studies. Antibiotic release and material degradation were tunable via hydrophobic/hydrophilic content of the hydrogel matrix and extended up to 3 weeks in PBS sink conditions. Covalent addition of vancomycin to the hydrogel polymer backbone was verified through mass spectroscopy and HPLC peak addition, as well as radiotracing of collected HPLC fractions. Bioactivity of released vancomycin was also confirmed alongside the resulting antimicrobial activity of the reacted vancomycin releasate.
Asunto(s)
Antibacterianos/síntesis química , Portadores de Fármacos/síntesis química , Vancomicina/química , Antibacterianos/farmacología , Materiales Biocompatibles/química , Supervivencia Celular/efectos de los fármacos , Portadores de Fármacos/química , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Hidrogeles/síntesis química , Hidrogeles/química , Interacciones Hidrofóbicas e Hidrofílicas , Pruebas de Sensibilidad Microbiana , Polímeros , Staphylococcus aureus/efectos de los fármacos , Vancomicina/farmacologíaRESUMEN
During the last two to three decades our understanding of the immunobiology of periodontal disease has increased exponentially, both with respect to the microbial agents triggering the disease process and the molecular mechanisms of the host engagement maintaining homeostasis or leading to collateral tissue damage. These foundational scientific findings have laid the groundwork for translating cell phenotype, receptor engagement, intracellular signaling pathways and effector functions into a 'picture' of the periodontium as the host responds to the 'danger signals' of the microbial ecology to maintain homeostasis or succumb to a disease process. These findings implicate the chronicity of the local response in attempting to manage the microbial challenge, creating a 'Double Indemnity' in some patients that does not 'insure' health for the periodontium. As importantly, in reflecting the title of this volume of Periodontology 2000, this review attempts to inform the community of how the science of periodontal immunology gestated, how continual probing of the biology of the disease has led to an evolution in our knowledge base and how more recent studies in the postgenomic era are revolutionizing our understanding of disease initiation, progression and resolution. Thus, there has been substantial progress in our understanding of the molecular mechanisms of host-bacteria interactions that result in the clinical presentation and outcomes of destructive periodontitis. The science has embarked from observations of variations in responses related to disease expression with a focus for utilization of the responses in diagnosis and therapeutic outcomes, to current investigations using cutting-edge fundamental biological processes to attempt to model the initiation and progression of soft- and hard-tissue destruction of the periodontium. As importantly, the next era in the immunobiology of periodontal disease will need to engage more sophisticated experimental designs for clinical studies to enable robust translation of basic biologic processes that are in action early in the transition from health to disease, those which stimulate microenvironmental changes that select for a more pathogenic microbial ecology and those that represent a rebalancing of the complex host responses and a resolution of inflammatory tissue destruction.
Asunto(s)
Interacciones Huésped-Patógeno/inmunología , Enfermedades Periodontales/inmunología , Inmunidad Adaptativa/inmunología , Biopelículas , Citocinas/inmunología , Humanos , Inmunidad Innata/inmunología , Inmunidad Mucosa/inmunología , Interacciones Microbianas/inmunología , Microbiota/inmunología , Enfermedades Periodontales/microbiologíaRESUMEN
Oral bacterial biofilms trigger chronic inflammatory responses in the host that can result in the tissue destructive events of periodontitis. However, the characteristics of the capacity of specific host cell types to respond to these biofilms remain ill-defined. This report describes the use of a novel model of bacterial biofilms to stimulate oral epithelial cells and profile select cytokines and chemokines that contribute to the local inflammatory environment in the periodontium. Monoinfection biofilms were developed with Streptococcus sanguinis, Streptococcus oralis, Streptococcus gordonii, Actinomyces naeslundii, Fusobacterium nucleatum, and Porphyromonas gingivalis on rigid gas-permeable contact lenses. Biofilms, as well as planktonic cultures of these same bacterial species, were incubated under anaerobic conditions with a human oral epithelial cell line, OKF4, for up to 24h. Gro-1α, IL1α, IL-6, IL-8, TGFα, Fractalkine, MIP-1α, and IP-10 were shown to be produced in response to a range of the planktonic or biofilm forms of these species. P. gingivalis biofilms significantly inhibited the production of all of these cytokines and chemokines, except MIP-1α. Generally, the biofilms of all species inhibited Gro-1α, TGFα, and Fractalkine production, while F. nucleatum biofilms stimulated significant increases in IL-1α, IL-6, IL-8, and IP-10. A. naeslundii biofilms induced elevated levels of IL-6, IL-8 and IP-10. The oral streptococcal species in biofilms or planktonic forms were poor stimulants for any of these mediators from the epithelial cells. The results of these studies demonstrate that oral bacteria in biofilms elicit a substantially different profile of responses compared to planktonic bacteria of the same species. Moreover, certain oral species are highly stimulatory when in biofilms and interact with host cell receptors to trigger pathways of responses that appear quite divergent from individual bacteria.
Asunto(s)
Biopelículas , Quimiocinas/biosíntesis , Citocinas/biosíntesis , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Actinomyces/fisiología , Anaerobiosis , Biopelículas/crecimiento & desarrollo , Línea Celular , Células Epiteliales/efectos de los fármacos , Fusobacterium nucleatum/fisiología , Humanos , Boca/microbiología , Plancton/fisiología , Porphyromonas gingivalis/fisiología , Streptococcus/fisiología , Streptococcus gordonii/fisiología , Streptococcus oralis/fisiologíaRESUMEN
Oral biofilms comprise complex multispecies consortia aided by specific inter- and intraspecies interactions occurring among commensals and pathogenic bacterial species. Oral biofilms are primary initiating factors of periodontal disease, although complex multifactorial biological influences, including host cell responses, contribute to the individual outcome of the disease. To provide a system to study initial stages of interaction between oral biofilms and the host cells that contribute to the disease process, we developed a novel in vitro model system to grow biofilms on rigid gas-permeable contact lenses (RGPLs), which enable oxygen to permeate through the lens material. Bacterial species belonging to early- and late-colonizing groups were successfully established as single- or three-species biofilms, with each group comprising Streptococcus gordonii, Streptococcus oralis, and Streptococcus sanguinis; S. gordonii, Actinomyces naeslundii, and Fusobacterium nucleatum; or S. gordonii, F. nucleatum, and Porphyromonas gingivalis. Quantification of biofilm numbers by quantitative PCR (qPCR) revealed substantial differences in the magnitude of bacterial numbers in single-species and multispecies biofilms. We evaluated cell-permeable conventional nucleic acid stains acridine orange, hexidium iodide, and Hoechst 33258 and novel SYTO red, blue, and green fluorochromes for their effect on bacterial viability and fluorescence yield to allow visualization of the aggregates of individual bacterial species by confocal laser scanning microscopy (CLSM). Substantial differences in the quantity and distribution of the species in the multispecies biofilms were identified. The specific features of these biofilms may help us better understand the role of various bacteria in local challenge of oral tissues.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Lentes de Contacto/microbiología , Técnicas Microbiológicas/métodos , Oxígeno/metabolismo , Bacterias/crecimiento & desarrollo , Carga Bacteriana , Viabilidad Microbiana , Microscopía Confocal , Coloración y Etiquetado/métodosRESUMEN
This study examined the oral epithelial immunotranscriptome response patterns modulated by oral bacterial planktonic or biofilm challenge. We assessed gene expression patterns when epithelial cells were challenged with a multispecies biofilm composed of Streptococcus gordonii, Fusobacterium nucleatum, and Porphyromonas gingivalis representing a type of periodontopathic biofilm compared to challenge with the same species of planktonic bacteria. Of the 579 human immunology genes, a substantial signal of the epithelial cells was observed to 181 genes. Biofilm challenged stimulated significant elevations compared to planktonic bacteria for IL32, IL8, CD44, B2M, TGFBI, NFKBIA, IL1B, CD59, IL1A, CCL20 representing the top 10 signals comprising 55% of the overall signal for the epithelial cell responses. Levels of PLAU, CD9, IFITM1, PLAUR, CD24, TNFSF10, and IL1RN were all elevated by each of the planktonic bacterial challenge vs the biofilm responses. While the biofilms up-regulated 123/579 genes (>2-fold), fewer genes were increased by the planktonic species (36 [S gordonii], 30 [F nucleatum], 44 [P gingivalis]). A wide array of immune genes were regulated by oral bacterial challenge of epithelial cells that would be linked to the local activity of innate and adaptive immune response components in the gingival tissues. Incorporating bacterial species into a structured biofilm dramatically altered the number and level of genes expressed. Additionally, a specific set of genes were significantly decreased with the multispecies biofilms suggesting that some epithelial cell biologic pathways are down-regulated when in contact with this type of pathogenic biofilm.
Asunto(s)
Biopelículas , Células Epiteliales/inmunología , Expresión Génica/efectos de los fármacos , Expresión Génica/inmunología , Transcriptoma/genética , Transcriptoma/inmunología , Inmunidad Adaptativa , Antígenos CD59/genética , Antígenos CD59/metabolismo , Quimiocina CCL20/genética , Quimiocina CCL20/metabolismo , Regulación hacia Abajo , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Fusobacterium nucleatum/metabolismo , Expresión Génica/genética , Encía/inmunología , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/metabolismo , Inmunidad Innata , Interleucina-1alfa/genética , Interleucina-1alfa/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Microbiota , Boca/microbiología , Inhibidor NF-kappaB alfa/genética , Inhibidor NF-kappaB alfa/metabolismo , Porphyromonas gingivalis/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Streptococcus gordonii/metabolismo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Regulación hacia ArribaRESUMEN
Large infected bone defects, often resulting from high energy traumas, are difficult to treat due to their variability in complexity and location. Standard treatment for infected bone defects begins with a protocol that includes a series of debridements in conjunction with an extended course of systemic antibiotics. Only after the infection has been eliminated will repair of the defect commence, typically with implantation of autologous bone. To address some of the shortcomings of the standard treatment methods, such as serial procedures, limited grafting material, and the need for a second surgical site for autologous bone, a sequential, dual drug-releasing, moldable, calcium sulfate-based bone graft substitute was developed previously. In the present studies, the effectiveness of the material for treating both the infection with vancomycin and bone defect with simvastatin was evaluated in vivo using a critically sized, infected segmental defect model in rat femurs. Although the infection was not fully eliminated, the local release of vancomycin increased survivorship of infected animals by 464% compared to nontreated controls. Infected animals receiving antimicrobial treatment showed comparable amounts of new bone formation within the defect site when compared to noninfected controls. Incorporating agents capable of disrupting established biofilms into bone graft substitutes may enhance effectiveness in treating a biofilm infection within a bone defect. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1878-1886, 2018.
Asunto(s)
Sustitutos de Huesos , Sulfato de Calcio , Fémur , Vancomicina , Animales , Sustitutos de Huesos/química , Sustitutos de Huesos/farmacología , Sulfato de Calcio/química , Sulfato de Calcio/farmacología , Fémur/lesiones , Fémur/metabolismo , Fémur/microbiología , Fémur/patología , Control de Infecciones , Infecciones/microbiología , Masculino , Ratas , Ratas Sprague-Dawley , Vancomicina/química , Vancomicina/farmacologíaRESUMEN
BACKGROUND: Neuropeptides (NPs) are innate pivotal regulators of the immunoinflammatory response. Nevertheless, their role in the pathogenesis of periodontal disease remains unknown. Changes in gene expression of 10 NPs and 16 NP receptors (NPRs) coincident with the initiation, progression, and resolution of periodontitis were determined. METHODS: The ligature-induced periodontitis model was used in rhesus monkeys (n = 18). Gingival tissue samples were taken at baseline (preligatures), at 2 weeks and at 1 month (initiation), and at 3 months (progression) postligation. Ligatures were removed and samples taken 2 months later (resolution). Total RNA was isolated from tissues and NP/NPR gene expression microarray analysis was performed. Gene expression changes were validated by quantitative polymerase chain reaction and immunohistochemistry. RESULTS: Unexpectedly, the expression of pro-inflammatory NPs/NPRs did not change during periodontitis or with resolution. However, increased expression of the anti-inflammatory NPs adrenomedullin (ADM) and galanin (GAL), and the NPRs calcitonin receptor-like (CALCRL) and receptor activity-modifying protein-2 and -3 (RAMP2 and RAMP3) were observed during initiation and progression of disease. The expression of the same NPs/NPRs exhibited a significant positive correlation with both molecular (interleukin-1ß, matrix mettaloproteinase-9, and receptor activator of nuclear factor-kappa B ligand) and clinical measures of gingival inflammation and tissue destruction. CONCLUSION: Initiation and progression of periodontitis involve significant overexpression of ADM, GAL, CALCRL, RAMP2, and RAMP3. These anti-inflammatory NPs/NPRs could play a role in the unresolved infection and inflammation that normally drives tissue destruction in periodontitis. Both ADM and GAL potentially are new candidates to consider as biomolecules associated with periodontal disease activity.
Asunto(s)
Mucosa Bucal , Neuropéptidos , Animales , Primates , Proteína 3 Modificadora de la Actividad de Receptores , Receptores de CalcitoninaRESUMEN
Periodontal disease is highly prevalent, with 90% of the world population affected by either periodontitis or its preceding condition, gingivitis. These conditions are caused by bacterial biofilms on teeth, which stimulate a chronic inflammatory response that leads to loss of alveolar bone and, ultimately, the tooth. Current treatment methods for periodontitis address specific parts of the disease, with no individual treatment serving as a complete therapy. The present research sought to demonstrate development of a multiple drug delivery system for stepwise treatment of different stages of periodontal disease. More specifically, multilayered films were fabricated from an association polymer comprising cellulose acetate phthalate and Pluronic F-127 to achieve sequential release of drugs. The four types of drugs used were metronidazole, ketoprofen, doxycycline, and simvastatin to eliminate infection, inhibit inflammation, prevent tissue destruction, and aid bone regeneration, respectively. Different erosion times and adjustable sequential release profiles were achieved by modifying the number of layers or by inclusion of a slower-eroding polymer layer. Analysis of antibiotic and anti-inflammatory bioactivity showed that drugs released from the devices retained 100% bioactivity. The multilayered CAPP delivery system offers a versatile approach for releasing different drugs based on the pathogenesis of periodontitis and other conditions.
Asunto(s)
Preparaciones de Acción Retardada/farmacología , Periodontitis/tratamiento farmacológico , Antibacterianos/farmacología , Antiinflamatorios/farmacología , Celulosa/análogos & derivados , Celulosa/química , Preparaciones de Acción Retardada/química , Doxiciclina/farmacología , Diseño de Fármacos , Humanos , Cetoprofeno/farmacología , Metronidazol/farmacología , Modelos Teóricos , Poloxámero/química , Simvastatina/farmacologíaRESUMEN
The efficacy of a novel synthetic antimicrobial peptide (WLBU2) was evaluated against three oral microorganisms (grown planktonically): Streptococcus gordonii, Fusobacterium nucleatum, and Porphyromonas gingivalis. WLBU2 killed all three species, with F. nucleatum being the most susceptible. WLBU2 also reduced the bacterial burden of S. gordonii and F. nucleatum biofilms.
Asunto(s)
Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Fusobacterium nucleatum/efectos de los fármacos , Péptidos/farmacología , Porphyromonas gingivalis/efectos de los fármacos , Streptococcus/efectos de los fármacos , Biopelículas/efectos de los fármacos , Humanos , Pruebas de Sensibilidad MicrobianaRESUMEN
SUMMARY Gray leaf spot of perennial ryegrass (Lolium perenne) is a severe foliar disease caused by the ascomycete fungus Magnaporthe oryzae (formerly known as Magnaporthe grisea). Control of gray leaf spot is completely dependent on the use of fungicides because currently available perennial ryegrass cultivars lack genetic resistance to this disease. M. oryzae isolates from perennial ryegrass (prg) were unable to cause disease on rice cultivars CO39 and 51583, and instead triggered a hypersensitive response. Southern hybridization analysis of DNA from over 50 gray leaf spot isolates revealed that all of them contain sequences corresponding to AVR1-CO39, a host specificity gene that confers avirulence to rice cultivar CO39, which carries the corresponding resistance gene Pi-CO39(t). There was also an almost complete lack of restriction site polymorphism at the avirulence locus. Cloning and sequencing of the AVR1-CO39 gene (AVR1-CO39(Lp)) from 16 different gray leaf spot isolates revealed just two point mutations, both of which were located upstream of the predicted open reading frame. When an AVR1-CO39(Lp) gene copy was transferred into ML33, a rice pathogenic isolate that is highly virulent to rice cultivar CO39, the transformants were unable to cause disease on CO39 but retained their virulence to 51583, a rice cultivar that lacks Pi-CO39(t). These data demonstrate that the AVR1-CO39 gene in the gray leaf spot pathogens is functional, and suggest that interaction of AVR1-CO39(Lp) and Pi-CO39(t) is responsible, at least in part, for the host specificity expressed on CO39. This indicates that it may be possible to use the Pi-CO39(t) resistance gene as part of a transgenic strategy to complement the current deficiency of gray leaf spot resistance in prg. Furthermore, our data indicate that, if Pi-CO39(t) can function in prg, the resistance provided should be broadly effective against a large proportion of the gray leaf spot pathogen population.