Differential Response of Human Dendritic Cells upon Stimulation with Encapsulated or Non-Encapsulated Isogenic Strains of Porphyromonas gingivalis.

During periodontitis, the extracellular capsule of Porphyromonas gingivalis favors alveolar bone loss by inducing Th1 and Th17 patterns of lymphocyte response in the infected periodontium. Dendritic cells recognize bacterial antigens and present them to T lymphocytes, defining their activation and polarization. Thus, dendritic cells could be involved in the Th1 and Th17 response induced against the P. gingivalis capsule. Herein, monocyte-derived dendritic cells were obtained from healthy individuals and then stimulated with different encapsulated strains of P. gingivalis or two non-encapsulated isogenic mutants. Dendritic cell differentiation and maturation were analyzed by flow cytometry. The mRNA expression levels for distinct Th1-, Th17-, or T-regulatory-related cytokines and transcription factors, as well as TLR2 and TLR4, were assessed by qPCR. In addition, the production of IL-1ß, IL-6, IL-23, and TNF-α was analyzed by ELISA. The encapsulated strains and non-encapsulated mutants of P. gingivalis induced dendritic cell maturation to a similar extent; however, the pattern of dendritic cell response was different. In particular, the encapsulated strains of P. gingivalis induced higher expression of IRF4 and NOTCH2 and production of IL-1ß, IL-6, IL-23, and TNF-α compared with the non-encapsulated mutants, and thus, they showed an increased capacity to trigger Th1 and Th17-type responses in human dendritic cells.

Co-Culture of P. gingivalis and F. nucleatum Synergistically Elevates IL-6 Expression via TLR4 Signaling in Oral Keratinocytes.

Periodontitis, characterized by persistent inflammation in the periodontium, is intricately connected to systemic diseases, including oral cancer. Bacteria, such as Porphyromonas gingivalis and Fusobacterium nucleatum, play a pivotal role in periodontitis development because they contribute to dysbiosis and tissue destruction. Thus, comprehending the interplay between these bacteria and their impacts on inflammation holds significant relevance in clinical understanding and treatment advancement. In the present work, we explored, for the first time, their impacts on the expressions of pro-inflammatory mediators after infecting oral keratinocytes (OKs) with a co-culture of pre-incubated P. gingivalis and F. nucleatum. Our results show that the co-culture increases IL-1ß, IL-8, and TNF-α expressions, synergistically augments IL-6, and translocates NF-kB to the cell nucleus. These changes in pro-inflammatory mediators-associated with chronic inflammation and cancer-correlate with an increase in cell migration following infection with the co-cultured bacteria or P. gingivalis alone. This effect depends on TLR4 because TLR4 knockdown notably impacts IL-6 expression and cell migration. Our study unveils, for the first time, crucial insights into the outcomes of their co-culture on virulence, unraveling the role of bacterial interactions in polymicrobial diseases and potential links to oral cancer.

Chronic Inflammation as a Link between Periodontitis and Carcinogenesis.

Periodontitis is characterized by a chronic inflammation produced in response to a disease-associated multispecies bacterial community in the subgingival region. Although the inflammatory processes occur locally in the oral cavity, several studies have determined that inflammatory mediators produced during periodontitis, as well as subgingival species and bacterial components, can disseminate from the oral cavity, contributing therefore, to various extraoral diseases like cancer. Interestingly, carcinogenesis associated with periodontal species has been observed in both the oral cavity and in extra oral sites. In this review, several studies were summarized showing a strong association between orodigestive cancers and poor oral health, presence of periodontitis-associated bacteria, tooth loss, and clinical signs of periodontitis. Proinflammatory pathways were also summarized. Such pathways are activated either by mono- or polymicrobial infections, resulting in an increase in the expression of proinflammatory molecules such as IL-6, IL-8, IL-1ß, and TNF-α. In addition, it has been shown that several periodontitis-associated species induce the expression of genes related to cell proliferation, cell cycle, apoptosis, transport, and immune and inflammatory responses. Intriguingly, many of these pathways are linked to carcinogenesis. Among them, the activation of Toll-like receptors (TLRs) and antiapoptotic pathways (such as the PI3K/Akt, JAK/STAT, and MAPK pathways), the reduction of proapoptotic protein expression, the increase in cell migration and invasion, and the enhancement in metastasis are addressed. Considering that periodontitis is a polymicrobial disease, it is likely that mixed species promote carcinogenesis both in the oral cavity and in extra oral tissues and probably-as observed in periodontitis-synergistic and/or antagonistic interactions occur between microbes in the community. To date, a good amount of studies has allowed us to understand how monospecies infections activate pathways involved in tumorigenesis; however, more studies are needed to determine the combined effect of oral species in carcinogenesis.

Changes in lipopolysaccharide profile of Porphyromonas gingivalis clinical isolates correlate with changes in colony morphology and polymyxin B resistance.

Virulence factors on the surface of Porphyromonas gingivalis constitute the first line of interaction with host cells and contribute to immune modulation and periodontitis progression. In order to characterize surface virulence factors present on P. gingivalis, we obtained clinical isolates from healthy and periodontitis subjects and compared them with reference strains. Colony morphology, aggregation in liquid medium, surface charge, membrane permeability to bactericidal compounds, novobiocin and polymyxin B resistance, capsule presence and lipopolysaccharide (LPS) profiles were evaluated. By comparing isolates from healthy and periodontitis subjects, differences in colony morphology and aggregation in liquid culture were found; the latter being similar to two reference strains. These differences were not a consequence of variations in bacterial surface charge. Furthermore, isolates also presented differences in polymyxin B and novobiocin resistance; isolates from healthy subjects were susceptible to polymyxin B and resistant to novobiocin and, in contrast, isolates from periodontitis subjects were resistant to polymyxin B and susceptible to novobiocin. These changes in antimicrobial resistance levels correlate with variations in LPS profiles, since -unlike periodontitis isolates-isolates from healthy samples synthesize LPS molecules lacking both O-antigen moieties and anionic polysaccharide. Additionally, this phenotype correlated with the absence of O-antigen ligase activity. Altogether, our results reveal novel variations on surface components of P. gingivalis isolates obtained from healthy and periodontitis subjects that could be associated with differences in bacterial virulence and periodontitis progression.

Antimicrobial Effect of Copper Nanoparticles on Relevant Supragingival Oral Bacteria.

Copper nanoparticles (Cu NPs) show promise in dentistry for combating bacterial dysbiosis and tooth decay. Understanding their effects on commensal versus pathogenic bacteria is vital for maintaining oral health balance. While Cu NPs demonstrate antibacterial properties against various oral bacteria, including common pathogens associated with tooth decay, their impact on commensal bacteria requires careful examination. In our work, we analyzed three types of Cu NPs for their effects on the growth, viability, and biofilm formation of representative caries-associated and commensal oral bacteria. S. sanguinis showed high tolerance to all Cu NPs, while L. rhamnosus was highly sensitive. Oxide-Cu NPs exhibited a stronger inhibitory effect on pathobionts compared with commensal bacteria. Moreover, the biofilm formation of the key cariogenic bacteria S. mutans was reduced, with minimal negative effects on commensal species' biofilm formation. All our results showed that CuO nanoparticles (CuO NPs) exhibit reduced toxicity toward commensal bacteria growth and development but have a strong impact on pathogens. This suggests their potential for targeted treatments against pathogenic bacteria, which could help in maintaining the balance of the oral bacterial community.

Porphyromonas gingivalis-Helicobacter pylori co-incubation enhances Porphyromonas gingivalis virulence and increases migration of infected human oral keratinocytes.

Background: Porphyromonas gingivalis is part of the subgingival biofilm and a keystone species in the development of periodontitis. Interactions between P.gingivalis and other bacteria in biofilms have been shown to affect bacterial virulence. Helicobacter pylori also inhabits the subgingival biofilm, but the consequences of interactions there with P.gingivalis remain unknown. Here, we investigated how the pre-incubation of P.gingivalis with H.pylori affects P.gingivalis virulence. Methods: We assayed P.gingivalis internalization by oral keratinocytes (OKs), hemagglutination and biofilm formation to identify alterations in virulence after pre-incubation with H. pylori. Also, we evaluated viability and migration of OKs infected with P. gingivalis, as well as the role of toll-like receptor 4 (TLR4).   In addition, we quantified the mRNA of genes associated with P.gingivalis virulence. Results: Pre-incubation of P.gingivalis with H.pylori enhanced P.gingivalis biofilm formation, bacterial internalization into OKs and hemagglutination. Infection with pre-incubated P.gingivalis increased OK migration in a manner dependent on the O-antigen and linked to  increased expression of the gingipain RgpB. Also, OK TLR4 participates in these events, because upon TLR4 knock-down, pre-incubated P.gingivalis no longer stimulated OK migration. Discussion: We provide here for the first time insight to the consequences of direct interaction between P.gingivalis and H.pylori. In doing so, we shed light on the mechanism by which H. pylori presence in the oral cavity increases the severity or progression of periodontitis.

Epstein-Barr Virus-Oral Bacterial Link in the Development of Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) is the most common type of oral cancer. Its development has been associated with diverse factors such as tobacco smoking and alcohol consumption. In addition, it has been suggested that microorganisms are risk factors for oral carcinogenesis. Epstein-Barr virus (EBV), which establishes lifelong persistent infections and is intermittently shed in the saliva, has been associated with several lymphomas and carcinomas that arise in the oral cavity. In particular, it has been detected in a subset of OSCCs. Moreover, its presence in patients with periodontitis has also been described. Porphyromonas gingivalis (P. gingivalis) is an oral bacterium in the development of periodontal diseases. As a keystone pathogen of periodontitis, P. gingivalis is known not only to damage local periodontal tissues but also to evade the host immune system and eventually affect systemic health. Persistent exposure to P. gingivalis promotes tumorigenic properties of oral epithelial cells, suggesting that chronic P. gingivalis infection is a potential risk factor for OSCC. Given that the oral cavity serves as the main site where EBV and P. gingivalis are harbored, and because of their oncogenic potential, we review here the current information about the participation of these microorganisms in oral carcinogenesis, describe the mechanisms by which EBV and P. gingivalis independently or synergistically can collaborate, and propose a model of interaction between both microorganisms.

Variability in Genomic and Virulent Properties of Porphyromonas gingivalis Strains Isolated From Healthy and Severe Chronic Periodontitis Individuals.

Porphyromonas gingivalis has been extensively associated with both the onset and progression of periodontitis. We previously isolated and characterized two P. gingivalis strains, one from a patient exhibiting severe chronic periodontitis (CP3) and another from a periodontally healthy individual (H3). We previously showed that CP3 and H3 exhibit differences in virulence since H3 showed a lower resistance to cationic peptides compared with CP3, and a lower ability to induce proliferation in gingival epithelial cells. Here, we aimed to determine whether differences in virulence between these two strains are associated with the presence or absence of specific genes encoding virulence factors. We sequenced the whole genomes of both P. gingivalis CP3 and H3 and conducted a comparative analysis regarding P. gingivalis virulence genetic determinants. To do so, we performed a homology search of predicted protein sequences in CP3 and H3 genomes against the most characterized virulence genes for P. gingivalis available in the literature. In addition, we performed a genomic comparison of CP3 and H3 with all the 62 genomes of P. gingivalis found in NCBI's RefSeq database. This approach allowed us to determine the evolutionary relationships of CP3 and H3 with other virulent and avirulent strains; and additionally, to detect variability in presence/absence of virulence genes among P. gingivalis genomes. Our results show genetic variability in the hemagglutinin genes. While CP3 possesses one copy of hagA and two of hagC, H3 has no hagA and only one copy of hagC. Experimentally, this finding is related to lower in vitro hemmaglutination ability of H3 compared to CP3. Moreover, while CP3 encodes a gene for a major fimbrium subunit FimA type 4 (CP3_00160), H3 possess a FimA type 1 (H3_01400). Such genetic differences are in agreement with both lower biofilm formation ability and less intracellular invasion to oral epithelial cells exhibited by H3, compared with the virulent strain CP3. Therefore, here we provide new results on the genome sequences, comparative genomics analyses, and phenotypic analyses of two P. gingivalis strains. The genomics comparison of these two strains with the other 62 genomes included in the analysis provided relevant results regarding genetic determinants and their association with P. gingivalis virulence.

Capsular-defective Porphyromonas gingivalis mutant strains induce less alveolar bone resorption than W50 wild-type strain due to a decreased Th1/Th17 immune response and less osteoclast activity.

BACKGROUND: Encapsulation of Porphyromonas gingivalis has been demonstrated as responsible of several host immunological changes, which have been associated with the pathogenesis of periodontitis. Using a murine model of periodontitis and two isogenic non-capsulated mutants of P. gingivalis, this study aimed to analyze whether P. gingivalis encapsulation induces more severe alveolar bone resorption, and whether this bone loss is associated with a T-helper (Th)1 and Th17-pattern of immune response. METHODS: Experimental periodontal infections were generated by oral inoculation with the encapsulated W50 wild-type strain or isogenic non-encapsulated ΔPG0116-PG0120 (GPA) and ΔPG0109-PG0118 (GPC) mutants of P. gingivalis. Periodontal infections induced with the encapsulated HG184 or non-encapsulated ATCC 33277 strains of P. gingivalis were used as controls. Alveolar bone resorption was analyzed using microcomputed tomography and scanning electron microscopy. The expression levels of Th1, Th2, Th17, or T regulatory-associated cytokines and RANKL, as well as the periodontal bacterial load, were quantified by quantitative polymerase chain reaction. The detection of Th1 and Th17 lymphocytes was analyzed by flow cytometry. RESULTS: In the periodontal lesions, both capsular-defective knockout mutant strains of P. gingivalis induced less alveolar bone resorption than the encapsulated W50 wild-type strain. This decreased bone loss was associated with a dismissed RANKL expression, decreased Th1- and Th17-type of cytokine expression, reduced Th1 and Th17 lymphocyte detection, and low osteoclast finding. CONCLUSION: These data demonstrate that encapsulation of P. gingivalis plays a key role in the alveolar bone resorption induced during periodontitis, and this bone loss is associated with a Th1- and Th17-pattern of immune response triggered in the periodontal lesions.

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.

Designing antimicrobial bioactive glass materials with embedded metal ions synthesized by the sol-gel method.

Bioactive glasses (SiO2-P2O5-CaO) having tailored concentrations of different biocide metal ions (copper or silver) were produced by the sol-gel method. All the particles release phosphorous ions when immersed in water and simulated body fluid (SBF). Moreover, a surface layer of polycrystalline hydroxy-carbonate apatite was formed on the particle surfaces after 10 day immersion in SBF as confirmed by X-ray diffraction and scanning electron microscopy (SEM) showing the bioactive materials. Samples with embedded either copper or silver ions were able to further release the biocide ions with a release rate that depends on the metal embedded and the dissolution medium: water or SBF. This biocide ion release from the samples explains the antimicrobial effect of our active particles against Escherichia coli DH5α ampicillin-resistant (Gram-negative) and Streptococcus mutans (Gram-positive) as determined by the Minimum Bactericidal Concentration (MBC) method. The antimicrobial behavior of the particles depends on the bacteria and the biocide ion used. Noteworthy, although samples with copper are able to release more metal ion than samples with silver, they present higher MBC showing the high effect of silver against these bacteria.

Biomimetic, mild chemical synthesis of CdTe-GSH quantum dots with improved biocompatibility.

Multiple applications of nanotechnology, especially those involving highly fluorescent nanoparticles (NPs) or quantum dots (QDs) have stimulated the research to develop simple, rapid and environmentally friendly protocols for synthesizing NPs exhibiting novel properties and increased biocompatibility. In this study, a simple protocol for the chemical synthesis of glutathione (GSH)-capped CdTe QDs (CdTe-GSH) resembling conditions found in biological systems is described. Using only CdCl(2), K(2)TeO(3) and GSH, highly fluorescent QDs were obtained under pH, temperature, buffer and oxygen conditions that allow microorganisms growth. These CdTe-GSH NPs displayed similar size, chemical composition, absorbance and fluorescence spectra and quantum yields as QDs synthesized using more complicated and expensive methods.CdTe QDs were not freely incorporated into eukaryotic cells thus favoring their biocompatibility and potential applications in biomedicine. In addition, NPs entry was facilitated by lipofectamine, resulting in intracellular fluorescence and a slight increase in cell death by necrosis. Toxicity of the as prepared CdTe QDs was lower than that observed with QDs produced by other chemical methods, probably as consequence of decreased levels of Cd(+2) and higher amounts of GSH. We present here the simplest, fast and economical method for CdTe QDs synthesis described to date. Also, this biomimetic protocol favors NPs biocompatibility and helps to establish the basis for the development of new, "greener" methods to synthesize cadmium-containing QDs.

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.