Salvador Nares

Periodontal regeneration - intrabony defects: a systematic review from the AAP Regeneration Workshop.

BACKGROUNDnPrevious systematic reviews of periodontal regeneration with bone replacement grafts and guided tissue regeneration (GTR) were defined as state of the art for clinical periodontal regeneration as of 2002.nnnMETHODSnThe purpose of this systematic review is to update those consensus reports by reviewing periodontal regeneration approaches developed for the correction of intrabony defects with the focus on patient-, tooth-, and site-centered factors, surgical approaches, surgical determinants, and biologics. This review adheres to the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines for systematic reviews. A computerized search of the PubMed and Cochrane databases was performed to evaluate the clinically available regenerative approaches for intrabony defects. The search included screening of original reports, review articles, and reference lists of retrieved articles and hand searches of selected journals. All searches were focused on clinically available regenerative approaches with histologic evidence of periodontal regeneration in humans published in English. For topics in which the literature is lacking, non-randomized observational and experimental animal model studies were used. Therapeutic endpoints examined included changes in clinical attachment level, changes in bone level/fill, and probing depth. For purposes of analysis, change in bone fill was used as the primary outcome measure, except in cases in which this information was not available. The SORT (Strength of Recommendation Taxonomy) grading scale was used in evaluating the body of knowledge.nnnRESULTSn1) Fifty-eight studies provided data on patient, tooth, and surgical-site considerations in the treatment of intrabony defects. 2) Forty-five controlled studies provided outcome analysis on the use of biologics for the treatment of intrabony defects.nnnCONCLUSIONSn1) Biologics (enamel matrix derivative and recombinant human platelet-derived growth factor-BB plus β-tricalcium phosphate) are generally comparable with demineralized freeze-dried bone allograft and GTR and superior to open flap debridement procedures in improving clinical parameters in the treatment of intrabony defects. 2) Histologic evidence of regeneration has been demonstrated with laser therapy; however, data are limited on clinical predictability and effectiveness. 3) Clinical outcomes appear most appreciably influenced by patient behaviors and surgical approach rather than by tooth and defect characteristics. 4) Long-term studies indicate that improvements in clinical parameters are maintainable up to 10 years, even in severely compromised teeth, consistent with a favorable/good long-term prognosis.

miR-24, miR-30b, and miR-142-3p Regulate Phagocytosis in Myeloid Inflammatory Cells

Micro-RNAs (miRNAs) are small noncoding RNAs that regulate various biological pathways. As their role in phagocytosis remains poorly understood, we investigated their impact on phagocytosis in myeloid inflammatory cells. Seven miRNAs (miR-24, -30b, -101, 142-3p, -652-3p, -652-5p, and -1275) that were differentially expressed during monocyte to macrophage (Mφ) and monocyte to dendritic cell (DC) differentiation were screened for their potential role in phagocytosis. Among these, overexpression of miR-24, miR-30b, and miR-142-3p in human monocyte-derived Mφ, DC, monocytes, and PBMCs significantly attenuate phagocytosis of Escherichia coli and Staphylococcus aureus, as well as the secretion of inflammatory mediators, including TNF-α, IL-6, and IL-12p40. miRNA-mediated changes in cytokine profiles were observed at transcriptional and/or posttranscriptional levels and importantly exhibit miRNA-specific impact. To examine the underlying mechanism, we monitored the expression of phagocytosis pathway-associated genes and identified several genes that were altered in Mφ and DC transfected with miR-24, miR-30b, and miR-142-3p mimics. Some of these genes with altered expression also harbor putative miRNA binding sites. We show that miR-142-3p directly regulates protein kinase Cα (PKCα), a key gene involved in phagocytosis. Interestingly, miR-142-3p and PKCα exhibit antagonistic expression during Mφ and DC differentiation. Short interfering RNA-mediated knockdown of PKCα in Mφ leads to reduced bacterial uptake, further highlighting the role of the gene in phagocytosis. Overall, these results demonstrate that miR-24, miR-30b, and miR-142-3p regulate phagocytosis and associated cytokine production in myeloid inflammatory cells through modulation of various genes involved in the pathway.

Regulation of miR-24, miR-30b, and miR-142-3p during macrophage and dendritic cell differentiation potentiates innate immunity

miRNAs are ubiquitous regulators of human biology. Parallel profiling of in vitro monocyte‐to‐Mφ and monocyte‐to‐DC differentiation revealed static, convergent, and divergent expression of miRNA. Bioinformatic and network analysis of differentially expressed miRNAs implicated miR‐24, miR‐30b, and miR‐142‐3p as negative regulators of intracellular signaling pathways, triggered not only by differentiation factors (M‐CSF/GM‐CSF/IL‐4) but also from PRRs. Manipulation of miR‐24, miR‐30b, and miR‐142‐3p expression during the differentiation of mD‐Mφ and mD‐DC differentiation had minimal impact on the acquisition of phenotype but significantly abrogated the ability of these cells to mount inflammatory responses to pathogen‐associated stimuli. Forced expression of these miRNAs, which are down‐regulated during differentiation, inhibited release of inflammatory cytokines [TNF‐α, IL‐12(p40), IL‐6] upon stimulation with LPS. Functional analysis revealed overlapping mechanisms of inhibition, including surface expression of TLR4/CD14/MD‐1 and intracellular PKCα/NF‐κB activation. Potential intermediary targets of the TLR4‐NF‐κB axis included members of the PI3K and MAPK families and PKC isoforms. These results demonstrate the requirement of miR‐24, miR‐30b, and miR‐142‐3p down‐regulation for the generation of fully functional Mφs and DCs.

Periodontal Regeneration – Intrabony Defects: A Consensus Report From the AAP Regeneration Workshop

BACKGROUNDnTreatment of intrabony defects is an important therapeutic goal of periodontal therapy. The goal of this consensus report was to critically appraise the evidence for the available approaches for promoting periodontal regeneration in intrabony defects. In addition to evaluating the effectiveness of new regenerative approaches for intrabony defects, recommendations for future research were defined for this area.nnnMETHODSnA systematic review was conducted using computerized searches of PubMed and Cochrane databases, supplemented with screening of references in original reports, review articles, and a hand search in selected journals. All searches were focused on regenerative approaches with histologic evidence of periodontal regeneration (proof of principle), clinical trials, and case reports. For purposes of analysis, change in intrabony defect fill was considered the primary outcome variable, with change in clinical attachment as a secondary outcome. The SORT (Strength of Recommendation Taxonomy) grade was used to evaluate the quality and strength of the evidence. During the consensus meeting, the group agreed on the outcomes of the systematic review, pertinent sources of evidence, clinical recommendations, and areas requiring future research.nnnRESULTSnThe systematic review, which was conducted for the consensus conference, evaluated the effectiveness of the use of biologics for the treatment of intrabony defects. Enamel matrix derivative (EMD) and recombinant human platelet-derived growth factor-BB (rhPDGF-BB) with β-tricalcium phosphate were shown to be efficacious in regenerating intrabony defects. The level of evidence is supported by multiple studies documenting effectiveness. The clinical application of biologics supports improvements in clinical parameters comparable with selected bone replacement grafts and guided tissue regeneration (GTR). Factors negatively affecting regeneration included smoking and excessive tooth mobility.nnnCONCLUSIONSnPeriodontal regeneration in intrabony defects is possible on previously diseased root surfaces, as evidenced by a gain in clinical attachment, decreased pocket probing depth, gain in radiographic bone height, and overall improvement in periodontal health. These clinical findings are consistent with available histologic evidence. Clinical improvements can be maintained over long periods (>10 years). Although bone replacement grafts have been the most commonly investigated modality, GTR, biologics, and combination therapies have also been shown to be effective. Future research should emphasize patient-reported outcomes, individual response differences, and emerging technologies to enhance treatment results.nnnCLINICAL RECOMMENDATIONSnEarly management of intrabony defects with regenerative therapies offers the greatest potential for successful periodontal regeneration. The clinical selection and application of a regenerative therapy or combination of therapies for periodontal regeneration should be based on the clinicians experiences and understanding of the regenerative biology and technology. This decision-making process should take into consideration the potential adverse influence of factors, such as smoking, poor oral hygiene, tooth mobility, and defect morphology, on regeneration. Management should be coupled with an effective maintenance program for long-term success.

Leukocyte Production of Inflammatory Mediators Is Inhibited by the Antioxidants Phloretin, Silymarin, Hesperetin, and Resveratrol

Antioxidants possess significant therapeutic potential for the treatment of inflammatory disorders. One such disorder is periodontitis characterised by an antimicrobial immune response, inflammation, and irreversible changes to the supporting structures of the teeth. Recognition of conserved pathogen-associated molecular patterns is a crucial component of innate immunity to Gram-negative bacteria such as Escherichia coli, as well as the periodontal pathogen Aggregatibacter actinomycetemcomitans. In this study, we investigated the antioxidants Phloretin, Silymarin, Hesperetin, and Resveratrol to ascertain whether they altered the production of inflammatory mediators by innately-activated leukocytes. Peripheral blood mononuclear cells were stimulated with lipopolysaccharide purified from Aggregatibacter actinomycetemcomitans, and the production of cytokines, chemokines, and differentiation factors was assayed by enzyme-linked immunosorbent assay, cytometric bead array, and RT-PCR. Significant inhibition of these factors was achieved upon treatment with Phloretin, Silymarin, Hesperetin, and Resveratrol. These data further characterise the potent anti-inflammatory properties of antioxidants. Their ability to inhibit the production of inflammatory cytokines, chemokines, and differentiation factors by a heterogeneous population of leukocytes has clear implications for their therapeutic potential in vivo.

MiRNA-181a regulates Toll-like receptor agonist-induced inflammatory response in human fibroblasts.

MicroRNAs (miRNAs) regulate the synthesis of cytokines in response to Toll-like receptor (TLR) activation. Our recent microarray study comparing normal and inflamed human dental pulps showed that miRNA-181 (miR-181) family is differentially expressed in the presence of inflammation. Prior studies have reported that the dental pulp, which is composed primarily of TLR4/2+ fibroblasts, expresses elevated levels of cytokines including interleukin-8 (IL-8) when inflamed. In this study, we employed an in-vitro model to determine the role of the miRNA-181 family in the TLR agonist-induced response in human fibroblasts. TLR4/2+ primary human dental pulp fibroblasts were stimulated with lipopolysaccharide from Porphyromonas gingivalis (Pg LPS), a known oral pathogen, and IL-8 and miR-181 expression measured. An inversely proportional relationship between IL-8 and miR-181a was observed. In-silico analysis identified a miR-181a-binding site on the 3′ untranslated region (UTR) of IL-8, which was confirmed by dual-luciferase assays. MiR-181a directly binds to the 3′UTR of IL-8, an important inflammatory component of the immune response, and modulates its levels. This is the very first report demonstrating miR-181a regulation of IL-8.

MicroRNA: Dynamic Regulators of Macrophage Polarization and Plasticity

The ability of a healthy immune system to clear the plethora of antigens it encounters incessantly relies on the enormous plasticity displayed by the comprising cell types. Macrophages (MΦs) are crucial member of the mononuclear phagocyte system (MPS) that constantly patrol the peripheral tissues and are actively recruited to the sites of injury and infection. In tissues, infiltrating monocytes replenish MΦ. Under the guidance of the local micro-milieu, MΦ can be activated to acquire specialized functional phenotypes. Similar to T cells, functional polarization of macrophage phenotype viz., inflammatory (M1) and reparative (M2) is proposed. Equipped with diverse toll-like receptors (TLRs), these cells of the innate arm of immunity recognize and phagocytize antigens and secrete cytokines that activate the adaptive arm of the immune system and perform key roles in wound repair. Dysregulation of MΦ plasticity has been associated with various diseases and infection. MicroRNAs (miRNAs) have emerged as critical regulators of transcriptome output. Their importance in maintaining health, and their contribution toward disease, encompasses virtually all aspects of human biology. Our understanding of miRNA-mediated regulation of MΦ plasticity and polarization can be utilized to modulate functional phenotypes to counter their role in the pathogenesis of numerous disease, including cancer, autoimmunity, periodontitis, etc. Here, we provide an overview of current knowledge regarding the role of miRNA in shaping MΦ polarization and plasticity through targeting of various pathways and genes. Identification of miRNA biomarkers of diagnostic/prognostic value and their therapeutic potential by delivery of miRNA mimics or inhibitors to dynamically alter gene expression profiles in vivo is highlighted.

miR-24 Regulates Macrophage Polarization and Plasticity

Objective MicroRNAs (miRNA) are ubiquitous regulators of human biology and immunity. Previously, we have demonstrated an inhibitory role for miR-24 in the phagocytosis of Escherichia coli and Staphylococcus aureus bioparticles and the induction of cytokine secretion in response to lipopolysaccharide (LPS) of the same origin; also, we have identified divergent and convergent miRNA responses to LPS from the periodontopathic pathogens Aggregatibacter actinomycetemcomitams (Aa) and Porphyromonas gingivalis (Pg), and revealed cigarette smoke extract as an environmental modifier of Pg LPS structure (Pg CSE) impacting macrophage miRNA responses. This study was designed to investigate the role of miR-24 on macrophage polarization and plasticity. Methods Primary human macrophages were differentiated from CD14+ monocytes isolated from peripheral blood mononuclear cells (PBMCs) by MACS positive selection and transfected with miR-24 miRNA mimics, inhibitors, or negative control mimic; followed by stimulation with cytokines and/or LPS under various conditions representing key stages of macrophage activation. Macrophage activation and polarization was assessed using assays for cytokine production (ELISA) and protein expression (flow cytometry, immunoblot). MiR-24 expression was assessed by RT-PCR. Results Stimulation of macrophages with LPSs of Aa, Pg, and Pg CSE origin resulted in dissimilar levels of cytokine expression and differential expression of miR-24. Overexpression of miR-24 inhibited cytokine secretion in response to LPS. Priming of macrophages with interferon gamma (IFN-γ) did not overcome this inhibitory effect, but classical activation of macrophages with IFN-γ plus TNF-α, TNF-β, or IL-17, modulated the pattern of miR-24 mediated suppression in a cytokine-specific fashion. Overexpression of miR-24 enhanced CD206 upregulation during alternative macrophage activation and inhibited its downregulation in macrophage transitioning from alternative to classical activation states. Overexpression of miR-24 resulted in reduced expression of the Class 1A PI 3-kinase subunit p110 delta (p110δ). Conclusion Pathogen- and environment-specific modifications in LPS alter the expression of cytokines and miR-24 in human macrophages. MiR-24 is a negative regulator of macrophage classical activation by LPS and promotes alternative activation under conditions of polarization and plasticity. MiR-24 mediated inhibition of LPS-induced cytokine secretion is dependent upon macrophage activation state at the point of stimulation, and this may be due to the degree to which p110δ is involved in the intracellular signaling pathway/s that transduce receptor ligation into cytokine induction. While important differences were observed in the effect of miR-24 on macrophages, these data indicate that overexpression of miR-24 would be predominantly anti-inflammatory

MiR-24, miR-30b and miR-142-3p interfere with antigen processing and presentation by primary macrophages and dendritic cells

Antigen uptake, processing and presentation by antigen presenting cells (APCs) are tightly coupled processes which consequently lead to the activation of innate and adaptive immune responses. However, the regulatory role of microRNA (miRNAs) in these critical pathways is poorly understood. In this study, we show that overexpression of miR-24, miR-30b and miR-142-3p attenuates uptake and processing of soluble antigen ovalbumin (Ova) in primary human macrophages and dendritic cells. MiRNA mimic transfected APCs exhibit defects in antigen presentation (Ova and CMV antigen) to CD4+ T-cells leading to reduced cell proliferation. Using transgenic OT-II mice we demonstrated that this impairment in T-cell proliferation is specific to antigen provided i.e., Ova. Further, human T-cells co-cultured with miRNA transfected dendritic cells secrete low levels of T helper (Th)-1 polarization associated cytokines. Analysis of molecules regulating APC and T-cell receptor interaction shows miRNA-mediated induced expression of Programmed Death-Ligand 1 (PD-L1) which inhibits T-cell proliferation. Blocking PD-L1 with antibodies rescues miRNA-mediated inhibition of T cell priming by DCs. These results uncover regulatory functions of miR-24, miR-30b and miR-142-3p in pairing innate and adaptive components of immunity.

MicroRNA target Fc receptors to regulate Ab-dependent Ag uptake in primary macrophages and dendritic cells

Phagocytosis commences with particle internalization and culminates with the activation of innate and adaptive immune responses. However, the role of miRNAs in phagocytosis remains largely unknown. In this study, we examined the role of miR-24, miR-30b and miR-142-3p in Ab Fc receptor (FcR)-mediated phagocytosis by macrophages (MΦ) and dendritic cells (DC). The expression of these miRNAs was reduced following phagocytosis of both IgG-opsonized beads and Escherichia coli, indicating their regulatory role in the process. Further, overexpression of these miRNAs impaired the uptake of IgG-coated latex beads, which corroborated the reduced secretion of the pro-inflammatory cytokines TNF-α and IL-8 and down-regulation of PKC-α, as well as superoxide-generating enzyme NADPH oxidase 2 expression level. Mechanistically, MΦ and DC transfected with miRNA mimics show marked reduction in expression of FcRs including FCGR2A, FcɛR1G and FCER2. We show that FcɛR1G expression is not affected at the transcription level, rather it is post-transcriptionally regulated by miR-30b. Finally, we demonstrate that siRNA-mediated knockdown of FcɛR1G leads to reduced uptake of IgG-opsonized beads, indicating its involvement on Ab-mediated phagocytosis. These results uncover miR-24, miR-30b and miR-142-3p as an essential component of FcR-mediated phagocytosis and associated innate immune responses.