In vivo Microbial Diversity Analysis on Different Surfaces of Dental Restorative Materials via 16S rDNA Sequencing.

BACKGROUND This study aimed to provide precise material selection guidance for proper clinical restoration and treatment of plaque-related oral diseases, such as dental caries and periodontal diseases. MATERIAL AND METHODS Four groups (n=24) of restorative material sheets (n=24) were prepared using 3M Z350 composite resin (ZR), zinc phosphate cement (ZPC), glass-ionomer (GI), and ICON permeable resin (IPR). Six volunteers wore a plaque-collection device equipped with the 4 restorative material sheets for 48 hours. Plaque samples were collected, and Miseq sequencing was applied to obtain template DNA fragments for microbial diversity analysis. The data were analyzed with nonparametric tests. RESULTS The microbial diversity on the ZPC surface was significantly lower than that on GI and IPR surfaces. The abundance of Firmicutes and Streptococcus on the ZPC surface was significantly higher than on the surfaces of GI and IPR. In contrast, the abundance of Porphyromonas on the surface of ZPC was significantly lower than that on GI and IPR surfaces. (P<0.05). CONCLUSIONS The results of the present study might serve as a basis for material selection under different oral microbial conditions to provide more accurate treatments and restorative procedures in the oral cavity.

Improved High-Throughput Sequencing of the Human Oral Microbiome: From Illumina to PacBio.

BACKGROUND: A comprehensive understanding of the commensal microflora and its relation to health is essential for preventing and combating diseases. The aim of this study was to examine the structure of the oral microbiome by using different sequencing technologies. Material and Methods. Five preschool children with no symptoms of oral and systemic diseases were recruited. Samples of saliva were collected. A 468 bp insert size library was constructed on the MiSeq platform and then subjected to 300 bp paired-end sequencing. Libraries with longer insert sizes, including a full-length 16S rDNA gene, were sequenced on the PacBio RS II platform. RESULTS: A total of 122.6 Mb of raw data, including 244,967 high-quality sequences, were generated by the MiSeq platform, while 134.6 Mb of raw data, including 70,030 high-quality reads, were generated by the PacBio RS II platform. Clustering of the unique sequences into OTUs at 3% dissimilarity resulted in an average of 225 OTUs on the MiSeq platform; however, the number of OTUs generated on the PacBio RS II platform was 449, far greater than the number of OTUs generated on the MiSeq platform. A total of 437 species belonging to 10 phyla and 60 genera were detected by the PacBio RS II platform, while 163 species belonging to 12 phyla and 72 genera were detected by the MiSeq platform. CONCLUSIONS: The oral microflora of healthy Chinese children were analyzed. Compared with traditional 16S rRNA sequencing technology, the PacBio system, despite providing a lower amount of clean data, surpassed the resolution of the MiSeq platform by improving the read length and annotating the nucleotide sequences at the species or strain level. This trial is registered with NCT02341352.

The Combinations Chitosan-Pam3CSK4 and Chitosan-Monophosphoryl Lipid A: Promising Immune-Enhancing Adjuvants for Anticaries Vaccine PAc.

We previously demonstrated that recombinant protein PAc could be administered as an anticaries vaccine. However, the relatively weak immunogenicity of PAc limits its application. In the present study, we investigated the effect of two adjuvant combinations of chitosan plus Pam3CSK4 (chitosan-Pam3CSK4) and of chitosan plus monophosphoryl lipid A (chitosan-MPL) in the immune responses to the PAc protein in vivo and in vitro PAc-chitosan-Pam3CSK4 or PAc-chitosan-MPL promoted significantly higher PAc-specific antibody titers in serum and saliva, inhibited Streptococcus mutans colonization onto the tooth surfaces, and endowed better protection effect with significantly less caries activities than PAc alone. Chitosan-Pam3CSK4 and chitosan-MPL showed no statistically significant differences. In conclusion, our study demonstrated that the chitosan-Pam3CSK4 and chitosan-MPL combinations are promising for anticaries vaccine development.

Phenotype and Function of Myeloid-Derived Suppressor Cells Induced by Porphyromonas gingivalis Infection.

Porphyromonas gingivalis, a major etiologic agent of periodontitis, has been reported to induce the expansion of myeloid-derived suppressor cells (MDSC); however, little is known regarding the subpopulations of MDSC expanded by P. gingivalis infection. Flow cytometry was used to evaluate bone marrow and spleen cells from mice infected with P. gingivalis and controls for surface expression of CD11b, Ly6G, and Ly6C. To characterize the phenotype of MDSC subpopulations induced by infection, cells were sorted based on the differential expression of Ly6G and Ly6C. Moreover, since MDSC are suppressors of T cell immune activity, we determined the effect of the induced subpopulations of MDSC on the proliferative response of OVA-specific CD4+ T cells. Lastly, the plasticity of MDSC to differentiate into osteoclasts was assessed by staining for tartrate-resistant acid phosphatase activity. P. gingivalis infection induced the expansion of three subpopulations of MDSC (Ly6G++ Ly6C+, Ly6G+ Ly6C++, and Ly6G+ Ly6C+); however, only CD11b+ Ly6G+ Ly6C++-expressing cells exerted a significant suppressive effect on T cell proliferation. Inhibition of proliferative responses required T cell-MDSC contact and was mediated by inducible nitric oxide synthase and cationic amino acid transporter 2 via gamma interferon. Furthermore, only the CD11b+ Ly6G+ Ly6C++ subpopulation of MDSC induced by P. gingivalis infection was able to differentiate into osteoclasts. Thus, the inflammatory response induced by P. gingivalis infection promotes the expansion of immune-suppressive cells and consequently the development of regulatory inhibitors that curtail the host response. Moreover, monocytic MDSC have the plasticity to differentiate into OC, thus perhaps contributing to the OC pool in states of periodontal disease.

IL-1R/TLR2 through MyD88 Divergently Modulates Osteoclastogenesis through Regulation of Nuclear Factor of Activated T Cells c1 (NFATc1) and B Lymphocyte-induced Maturation Protein-1 (Blimp1).

Toll-like receptors (TLR) and the receptor for interleukin-1 (IL-1R) signaling play an important role in bacteria-mediated bone loss diseases including periodontitis, rheumatoid arthritis, and osteomyelitis. Recent studies have shown that TLR ligands inhibit the receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation from un-committed osteoclast precursors, whereas IL-1 potentiates RANKL-induced osteoclast formation. However, IL-1R and TLR belong to the same IL-1R/TLR superfamily, and activate similar intracellular signaling pathways. Here, we investigate the molecular mechanisms underlying the distinct effects of IL-1 and Porphyromonas gingivalis lipopolysaccharide (LPS-PG) on RANKL-induced osteoclast formation. Our results show that LPS-PG and IL-1 differentially regulate RANKL-induced activation of osteoclast genes encoding Car2, Ctsk, MMP9, and TRAP, as well as expression of NFATc1, a master transcription factor of osteoclastogenesis. Regulation of osteoclast genes and NFATc1 by LPS-PG and IL-1 is dependent on MyD88, an important signaling adaptor for both TLR and IL-1R family members. Furthermore, LPS-PG and IL-1 differentially regulate RANKL-costimulatory receptor OSCAR (osteoclast-associated receptor) expression and Ca(2+) oscillations induced by RANKL. Moreover, LPS-PG completely abrogates RANKL-induced gene expression of B lymphocyte-induced maturation protein-1 (Blimp1), a global transcriptional repressor of anti-osteoclastogenic genes encoding Bcl6, IRF8, and MafB. However, IL-1 enhances RANKL-induced blimp1 gene expression but suppresses the gene expression of bcl6, irf8, and mafb. Our study reveals the involvement of multiple signaling molecules in the differential regulation of RANKL-induced osteoclastogenesis by TLR2 and IL-1 signaling. Understanding the signaling cross-talk among TLR, IL-1R, and RANK is critical for identifying therapeutic strategies to control bacteria-mediated bone loss.

Intranasal co-delivery of IL-6 gene enhances the immunogenicity of anti-caries DNA vaccine.

AIM: To investigate the effects of co-delivering IL-6 expressing plasmid pCI-IL-6 on the immunogenicity of the anti-caries DNA vaccine pCIA-P, which encodes the surface protein antigen PAc of Streptococcus mutans. METHODS: Plasmid pCI-IL-6 was constructed by inserting the murine IL-6 gene into the pCI vector. Expression of IL-6 in vitro was assessed using Western blot analysis. BALB/c mice were intranasally co-immunized with pCIA-P plus pCI-IL-6 on d 0 and 14. Anti-PAc IgG and secretory IgA (sIgA) were assessed by ELISA. Splenocytes from the mice were re-stimulated with the PAc protein, and IFN-γ and IL-4 production was measured using ELISA. Splenocyte proliferation was analyzed with flow cytometry. Rats were similarly immunized, and dental caries scores were determined using the Keyes method. RESULTS: Marked expression of IL-6 was found in COS-7 cells transfected with pCI-IL-6. In the pCI-IL-6 co-immunized mice, the specific IgG antibodies in serum and sIgA antibodies in saliva were significantly higher than those in the control mice at weeks 4 and 8. Moreover, the secretion of IFN-γ from splenocytes in response to re-stimulation with PAc protein was significantly higher in the pCI-IL-6 co-immunized mice than that in the control mice, whereas the secretion of IL-4 had no significant difference. The proliferation of splenocytes from the pCI-IL-6 co-immunized mice was significantly higher than that from the mice immunized with pCIA-P and pCI vector. In the rat caries model, the pCI-IL-6 co-immunization rats displayed lower caries scores than the control rats. CONCLUSION: Intranasal co-delivery of IL-6 gene significantly enhances the immunogenicity of the anti-caries DNA vaccine.

Co-delivery of ccl19 gene enhances anti-caries DNA vaccine pCIA-P immunogenicity in mice by increasing dendritic cell migration to secondary lymphoid tissues.

AIM: To investigate how co-delivery of the gene encoding C-C chemokine ligand-19 (CCL-19) affected the systemic immune responses to an anti-caries DNA vaccine pCIA-P in mice. METHODS: Plasmid encoding CCL19-GFP fusion protein (pCCL19/GFP) was constructed by inserting murine ccl19 gene into GFP-expressing vector pAcGFP1-N1. Chemotactic effect of the fusion protein on murine dendritic cells (DCs) was assessed in vitro and in vivo using transwell and flow cytometric analysis, respectively. BALB/c mice were administered anti-caries DNA vaccine pCIA-P plus pCCL19/GFP (each 100 µg, im) or pCIA-P alone. Serum level of anti-PAc IgG was assessed with ELISA. Splenocytes from the mice were stimulated with PAc protein for 48 h, and IFN-γ and IL-4 production was measured with ELISA. The presence of pCCL19/GFP in spleen and draining lymph nodes was assessed using PCR. The expression of pCCL19/GFP protein in these tissues was analyzed under microscope and with flow cytometry. RESULTS: The expression level of CCL19-GFP fusion protein was considerably increased 48 h after transfection of COS-7 cells with pCCL19/GFP plasmids. The fusion protein showed potent chemotactic activity on DCs in vitro. The level of serum PAc-specific IgG was significantly increased from 4 to 14 weeks in the mice vaccinated with pCIA-P plus pCCL19/GFP. Compared to mice vaccinated with pCIA-P alone, the splenocytes from mice vaccinated with pCIA-P plus pCCL19/GFP produced significantly higher level of IFN-γ, but IL-4 production had no significant change. Following intromuscular co-delivery, pCCL19/GFP plasmid and fusion protein were detected in the spleen and draining lymph nodes. Administration of CCL19 gene in mice markedly increased the number of mature DCs in secondary lymphoid tissues. CONCLUSION: CCL19 serves as an effective adjuvant for anti-caries DNA vaccine by inducing chemotactic migration of DCs to secondary lymphoid tissues.

Porphyromonas gingivalis­derived lipopolysaccharide inhibits brown adipocyte differentiation via lncRNA­BATE10.

Epidemiological studies have suggested an association between obesity and periodontal disease. Brown adipose tissue (BAT) has an anti-obesity effect. However, the effects of periodontitis on obesity and BAT remain unclear. Therefore, the present study aimed to determine the effects of lipopolysaccharide derived from Porphyromonas gingivalis (P. gingivalis LPS) on brown adipocytes. For this purpose, the present study examined the effects of the intravenous administration of Porphyromonas gingivalis (P. gingivalis) in mice, the treatment of brown adipocytes with P. gingivalis LPS during differentiation, and the administration of small interfering RNA targeting interferon on brown preadipocytes by assessing the expression of genes involved in differentiation, using a long non-coding (lnc)RNA, and pro-inflammatory factors using reverse transcription-quantitative PCR. In addition, the accumulation of lipid droplets was examined using Oil Red O staining. P. gingivalis LPS reduced the expression of uncoupling protein 1 (UCP1) and lncRNA-BATE10 in brown adipocytes during differentiation. Consistent with this finding, P. gingivalis reduced UCP1 and lncRNA-BATE10 expression in the BAT of mice. lncRNA-BATE10 may thus be involved in the regulation of UCP1 expression that occurs during the differentiation of brown adipocytes treated with P. gingivalis LPS. Thus, P. gingivalis LPS may inhibit BAT differentiation by reducing lncRNA-BATE10 expression.

Characterization and regulation of osteoclast precursors following chronic Porphyromonas gingivalis infection.

Bone destruction in inflammatory osteolytic diseases including periodontitis is related to excessive activity of osteoclasts (OC), which originate from precursor cells of the myeloid lineage, termed osteoclast precursors (OCP). In contrast to ample knowledge that we currently have on mature OC, little is known about OCP and their regulation during bacterial infection. Therefore, this study aimed to identify and characterize OCP following chronic infection with a periodontal bacteria Porphyromonas gingivalis (Pg). We used a micro-osmotic pump to continually release Pg subcutaneously in a murine model. Two weeks after Pg infection, the frequency of CD11b+c-fms+Ly6Chi population is significantly elevated within the bone marrow, spleen and peripheral blood. In vitro and in vivo studies identified these cells as the OCP-containing population and Pg infection significantly enhanced the osteoclastogenic activity of these cells. Furthermore, mRNA sequencing analysis indicated a unique gene and pathway profile in CD11b+c-fms+Ly6Chi population following Pg infection, with changes in genes and pathways related to OC differentiation, cell proliferation and apoptosis, inflammatory response, phagocytosis and immunity, as well as antigen processing and presentation. Moreover, using IL-6 knockout mice, we found that IL-6 is important for Pg-induced accumulation of CD11b+c-fms+Ly6Chi population from the bone marrow and periphery. Our results provide new insights into the characterization and regulation of OCP following a chronic bacterial infection. This knowledge is relevant to the understanding of the pathogenesis of bacteria-induced bone loss, and to the identification of potential therapeutic targets of bone loss diseases.

Frontline Science: Characterization and regulation of osteoclast precursors following chronic Porphyromonas gingivalis infection.

Bone destruction in inflammatory osteolytic diseases including periodontitis is related to excessive activity of osteoclasts (OC), which originate from precursor cells of the myeloid lineage, termed osteoclast precursors (OCP). In contrast to ample knowledge that we currently have on mature OC, little is known about OCP and their regulation during bacterial infection. Therefore, this study aimed to identify and characterize OCP following chronic infection with a periodontal bacteria Porphyromonas gingivalis (Pg). We used a microosmotic pump to continually release Pg subcutaneously in a murine model. Two weeks after Pg infection, the frequency of CD11b+c-fms+Ly6Chi population is significantly elevated within the bone marrow, spleen, and peripheral blood. In vitro and in vivo studies identified these cells as the OCP-containing population and Pg infection significantly enhanced the osteoclastogenic activity of these cells. Furthermore, mRNA sequencing analysis indicated a unique gene and pathway profile in CD11b+c-fms+Ly6Chi population following Pg infection, with changes in genes and pathways related to OC differentiation, cell proliferation and apoptosis, inflammatory response, phagocytosis, and immunity, as well as antigen processing and presentation. Moreover, using IL-6 knockout mice, we found that IL-6 is important for Pg-induced accumulation of CD11b+c-fms+Ly6Chi population from the bone marrow and periphery. Our results provide new insight into the characterization and regulation of OCP following a chronic bacterial infection. This knowledge is relevant to the understanding of the pathogenesis of bacteria-induced bone loss, and to the identification of potential therapeutic targets of bone loss diseases.

Exosomes derived from preadipocytes improve osteogenic differentiation, potentially via reduced miR­223 expression.

Insufficient bone volume remains a key issue when using dental implants. Adipose tissue­derived stem cells (ADSCs) can accelerate bone healing when combined with dental implants. To improve the application of ADSCs for dental uses, the present study aimed to identify optimal implantation conditions. Mesenchymal stem cell­derived exosomes can induce naïve stem cells to differentiate through the osteogenic lineage. In the present study, exosomes derived from 3T3L1 preadipocytes (3T3L1­exo) were purified and characterized. The effects and potential mechanisms of 3T3L1­exo on 3T3L1 cell ossification were examined by reverse transcription­quantitative polymerase chain reaction, western blotting, electron microscopy, RNA sequencing and histological analysis. The current study confirmed that 3T3L1­exo enhanced 3T3L1 preadipocyte osteogenic differentiation, as revealed by upregulation of osteogenic differentiation­associated genes and increased Alizarin Red staining. Furthermore, the microRNA (miR) expression profiles of 3T3L1­exo and 3T3L1 preadipocytes were sequenced and compared. The results of a further analysis demonstrated that miR­223 expression was reduced in 3T3L1 preadipocytes stimulated by 3T3L1­exo compared with in unstimulated cells. This finding suggested that 3T3L1­exo promoted 3T3L1 bone formation by decreasing miR­223 through a competitive mechanism, another miRNA, or another factor. The mechanism by which miR­223 is decreased warrants further investigation. In conclusion, the application of 3T3L1­exo may be useful for investigating preadipocyte­induced bone regeneration.

Oral Microbiome Alterations Associated with Early Childhood Caries Highlight the Importance of Carbohydrate Metabolic Activities.

Globally, dental caries is the most prevalent chronic oral disease and affects roughly half of all children. The aim of this report was to use metagenomic analyses to investigate the relationship between the oral microbiome and caries in preschool children. A total of 25 preschoolers, aged 3 to 5 years old with severe early childhood caries (ECC), and 19 age-matched, caries-free children as controls were recruited. Saliva samples were collected from the participants and were subjected to metagenomic analyses, whereby the oral microbial communities were investigated. The metagenomic analyses revealed substantial microbiota differences between the two groups, indicating apparent shifts of the oral microbiome present in the ECC group. At the species level, the ECC-enriched microbes included Prevotella amnii, Shuttleworthia satelles, Olsenella uli, and Anaeroglobus geminatus Interestingly, Actinomyces odontolyticus and Actinomyces graevenitzii exhibited apparent differences at the strain level but not the species level between the ECC and control groups. Functional examination showed that the ECC group displayed extensive alterations in metabolic genes/pathways/modules, including enriched functions in sugar metabolism. Finally, an SVM (support vector machine) classifier comprising seven species was developed and generated a moderately good performance in predicting caries onset (area under the receiver operating characteristic curve [AUC] = 78.33%). Together, these findings indicate that caries is associated with considerable changes in the oral microbiome, some of which can potentially be exploited as therapeutic targets or diagnostic markers. (This study has been registered at ClinicalTrials.gov under registration no. NCT02341352.)IMPORTANCE Dental caries is a highly prevalent oral disease that can lead to severe dental damage and may greatly compromise the quality of life of the affected individuals. Previous studies, including those based on 16S rRNA gene, have revealed that the oral microbiota plays a prominent role in development of the disease. But the approach of those studies was limited in analyzing several key microbiome traits, including species- or strain-level composition and functional profile. Here, we performed metagenomic analyses for a cohort of preschool children with or without caries. Our results showed that caries was associated with extensive microbiota differences at various taxonomic and functional levels. Some caries-associated species had not been previously reported, some of which may have significant clinical implications. A microbiome gene catalogue from children with caries was constructed for the first time. The results demonstrated that caries is associated with alterations of the oral microbiome, including changes in microbial composition and metabolic functional profile.

Association between cadmium exposure and diabetes mellitus risk: a prisma-compliant systematic review and meta-analysis.

Cadmium (Cd) is a pollutant with multiple adverse health effects: cancer, renal dysfunction, osteoporosis and fracture, and cardiovascular disease. Several population-based studies found an association between Cd and diabetes mellitus (DM), but this association is inconsistent with other research. We conducted meta-analysis to examine relationship between urinary/blood Cd exposure and DM risk. Pertinent studies were identified by searching PubMed and Embase databases, and combined odds ratio (OR) and corresponding 95% confidence interval (CI) were applied to evaluate said association. Meta-analysis showed that high U-Cd exposure is not correlated with DM risk (OR = 1.19; 95% CI = 0.83-1.71), and high B-Cd exposure is also not associated with increased risk of DM (OR = 1.16; 95% CI = 0.84-1.62) in the general population. Subgroup and sensitivity analysis proved similar results, with little evidence of publication bias. This meta-analysis suggests that high U-Cd/B-Cd exposure may not be risk factor for DM in general populations. However, large prospective studies are needed to confirm this finding.