Discovery of phosphorylated lantibiotics with proimmune activity that regulate the oral microbiome.

Lantibiotics are ribosomally synthesized and posttranslationally modified peptides (RiPPs) that are produced by bacteria. Interest in this group of natural products is increasing rapidly as alternatives to conventional antibiotics. Some human microbiome-derived commensals produce lantibiotics to impair pathogens' colonization and promote healthy microbiomes. Streptococcus salivarius is one of the first commensal microbes to colonize the human oral cavity and gastrointestinal tract, and its biosynthesis of RiPPs, called salivaricins, has been shown to inhibit the growth of oral pathogens. Herein, we report on a phosphorylated class of three related RiPPs, collectively referred to as salivaricin 10, that exhibit proimmune activity and targeted antimicrobial properties against known oral pathogens and multispecies biofilms. Strikingly, the immunomodulatory activities observed include upregulation of neutrophil-mediated phagocytosis, promotion of antiinflammatory M2 macrophage polarization, and stimulation of neutrophil chemotaxis-these activities have been attributed to the phosphorylation site identified on the N-terminal region of the peptides. Salivaricin 10 peptides were determined to be produced by S. salivarius strains found in healthy human subjects, and their dual bactericidal/antibiofilm and immunoregulatory activity may provide new means to effectively target infectious pathogens while maintaining important oral microbiota.

A Primed Neutrophil Subset Predicts the Risk of Bloodstream Infections in Allogeneic Hematopoietic Stem-Cell Transplant Patients: A Prospective Study.

BACKGROUND: Bloodstream infections (BSIs) are the most common infectious complication in patients who receive allogeneic hematopoietic stem-cell transplants (allo-HSCTs). Polymorphonuclear neutrophils (PMNs) are quantified to monitor the susceptibility to BSIs; however, their degree of activation is not. We previously identified a population of primed PMNs (pPMNs) with distinct markers of activation representing approximately 10% of PMNs in circulation. In this study, we investigate whether susceptibility to BSIs is related to the proportion of pPMNs rather than strictly PMN counts. METHODS: In this prospective observational study, we used flow cytometry to assess pPMNs in blood and oral rinse samples collected from patients receiving an allo-HSCT over the course of their treatment. We used the proportion of pPMNs in the blood on day 5 post-transplant to categorize patients into a high- or a low-pPMN group (>10% or <10% pPMNs). These groups were then used as a predictor of BSIs. RESULTS: A total of 76 patients were enrolled in the study with 36 in the high-pPMN group and 40 in the low-pPMN group. Patients in the low-pPMN group had lower expression of PMN activation and recruitment markers and displayed a delay in PMN repopulation of the oral cavity after the transplant. These patients were more susceptible to BSIs compared with patients in the high-pPMN group with an odds ratio of 6.5 (95% confidence interval, 2.110-25.07; P = .002). CONCLUSIONS: In patients who receive an allo-HSCT, having <10% pPMNs early in the post-transplant phase can be an independent predictor of BSI in allo-HSCT patients.

GEF-H1 Is Required for Colchicine Inhibition of Neutrophil Rolling and Recruitment in Mouse Models of Gout.

Gout is a painful arthritic inflammatory disease caused by buildup of monosodium urate (MSU) crystals in the joints. Colchicine, a microtubule-depolymerizing agent that is used in prophylaxis and treatment of acute gout flare, alleviates the painful inflammatory response to MSU crystals. Using i.p. and intra-articular mouse models of gout-like inflammation, we found that GEF-H1/GEF-H1/AHRGEF2, a microtubule-associated Rho-GEF, was necessary for the inhibitory effect of colchicine on neutrophil recruitment. GEF-H1 was required for neutrophil polarization in response to colchicine, characterized by uropod formation, accumulation of F-actin and myosin L chain at the leading edge, and accumulation of phosphorylated myosin L chain, flotillin-2, and P-selectin glycoprotein ligand-1 (PSGL-1) in the uropod. Wild-type neutrophils that were pre-exposed to colchicine failed to roll or accumulate on activated endothelial monolayers, whereas GEF-H1 knockout (GEF-H1-/-) neutrophils were unaffected by treatment with colchicine. In vivo, colchicine blocked MSU-induced recruitment of neutrophils to the peritoneum and the synovium in wild-type mice, but not in GEF-H1-/- mice. Inhibition of macrophage IL-1ß production by colchicine was independent of GEF-H1, supporting a neutrophil-intrinsic mode of action. Our results suggest that the anti-inflammatory effects of colchicine in acute gout-like inflammation can be accounted for by inhibition of neutrophil-rolling interactions with the inflamed vasculature and occurs through GEF-H1-dependent neutrophil stimulation by colchicine. These results contribute to our understanding of the therapeutic action of colchicine, and could inform the application of this drug in other conditions.

The Effect of Intensity-Modulated Radiotherapy to the Head and Neck Region on the Oral Innate Immune Response and Oral Microbiome: A Prospective Cohort Study of Head and Neck Tumour Patients.

Neutrophils, also known as polymorphonuclear leukocytes (PMNs), form a significant component of the innate host response, and the consequence of the interaction between the oral microbiota and PMNs is a crucial determinant of oral health status. The impact of radiation therapy (RT) for head and neck tumour (HNT) treatment on the oral innate immune system, neutrophils in particular, and the oral microbiome has not been thoroughly investigated. Therefore, the objective of this study was to characterize RT-mediated changes in oral neutrophils (oPMNs) and the oral microbiome in patients undergoing RT to treat HNTs. Oral rinse samples were collected prior to, during and post-RT from HNT patients receiving RT at Dental Oncology at Princess Margaret Cancer Centre. The oPMNs counts and activation states were analysed using flow cytometry, and the oral microbiome was analysed using 16S rRNA gene sequencing. Statistically significant (p < 0.05) drops in oPMN counts and the activation states of the CD11b, CD16, CD18, CD64 and H3Cit markers from pre-RT to post-RT were observed. Moreover, exposure to RT caused a significant reduction in the relative abundance of commensal Gram-negative bacteria and increased the commensal Gram-positive microbes. Ionizing radiation for the treatment of HNTs simultaneously decreased the recruitment of oPMNs into the oral cavity and suppressed their activation state. The oral microbiome composition post-RT was altered significantly due to RT which may favour the colonization of specific microbial communities unfavourable for the long-term development of a balanced oral microbiome.

The role of CRISPR-Cas in advancing precision periodontics.

The significant advancement of molecular biology has revolutionized medicine and provided important technologies to further clinical research development. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are DNA sequences derived from bacteriophages which have previously infected the bacterial species. The CRISPR-Cas system plays a key role in bacterial defense by detecting and destroying DNA fragments during subsequent bacteriophage invasions. The Cas9 enzyme recognizes and cleaves new invading CRISPR-complementary DNA sequences. Researchers have taken advantage of this biological device to manipulate microbes' genes and develop novel therapeutics to tackle systemic disease. In this review, we discuss the potential of utilizing CRISPR-Cas systems in the periodontal field to develop personalized periodontal care. We summarize promising attempts to bring this technology to the clinical setting. Finally, we provide insights regarding future developments to best utilize the CRISPR-Cas systems to advance precision periodontics. Although further research is imperative to evaluate the safety and potential of using CRISPR-Cas to develop precision periodontics approaches, few studies showed promising data to support the investment into this important technology in the dental sector. CRISPR-Cas9 can be a useful tool to create knockouts in vitro and in vivo as a screening tool to identify cellular pathways involved in the pathogenesis of periodontitis. Alternative CRISPR systems such as CRISPRa, CRISPRi, and Cas13 can be used to modify the transcriptome and gene expression of genes involved in periodontitis progression. CRISPR systems such as Cas3 can be used to target the periodontal biofilm and to develop new strategies to reduce or eliminate periodontal pathogens. Currently, the utility of CRISPR-Cas applications in clinical settings is limited. Through this review, we hope to foster further discussion in the periodontal research and clinical communities with respect to the potential clinical application of novel, CRISPR-Cas based, therapeutics for periodontitis.

Stressed-Out Oral Immunity: A Gateway From Socioeconomic Adversity to Periodontal Disease.

OBJECTIVE: It has been suggested that adverse socioeconomic conditions "get under the skin" by eliciting a stress response that can trigger periodontal inflammation. We aimed to a) estimate the extent to which socioeconomic position (SEP) is associated with periodontal disease (PD) and proinflammatory oral immunity, and b) determine the contribution of psychosocial stress and stress hormones to these relationships. METHODS: In this cross-sectional study (n = 102), participants (20-59 years old) completed financial and perceived stress questionnaires and underwent full-mouth periodontal examinations. SEP was characterized by annual household income and educational attainment. Cortisol, a biological correlate of chronic stress, was assessed in hair samples. Oral immunity was characterized by assessing oral inflammatory load and proinflammatory oral neutrophil function. Blockwise Poisson and logistic regression models were applied. RESULTS: Compared with lower SEP, individuals in the middle- and higher-income categories had a significantly lower probability of PD (incidence rate ratio [IRR] = 0.5 [confidence interval {CI} = 0.3-0.7] and IRR = 0.4 [95% CI = 0.2-0.7]) and oral inflammatory load (IRR = 0.6 [95% CI = 0.3-0.8] and IRR = 0.5 [95% CI = 0.3-0.7]) and were less likely to have a proinflammatory oral immune function (odds ratio [OR] = 0.1 [95% CI = 0.0-0.7] and OR = 0.1 [95% CI = 0.0-0.9]). PD and oral immune parameters were significantly associated with financial stress and cortisol. Adjusting for financial stress and cortisol partially attenuated the socioeconomic differences in PD to IRR = 0.7 (95% CI = 0.5-0.8) and IRR = 0.6 (95% CI = 0.5-0.7) for the middle- and higher-income categories, respectively. Similar results were observed for proinflammatory immunity (OR = 0.2 [95% CI = 0.0-1.8] and OR = 0.3 [95% CI = 0.0-2.3]). CONCLUSION: These findings suggest that psychosocial stress may contribute to a proinflammatory immunity that is implicated in PD pathobiology and provide insight into social-to-biological processes in oral health.

Periodontitis is an inflammatory disease of oxidative stress: We should treat it that way.

Periodontitis is a highly prevalent disease. As it progresses, it causes serious morbidity in the form of periodontal abscesses and tooth loss and, in the latter stages, pain. It is also now known that periodontitis is strongly associated with several nonoral diseases. Thus, patients with periodontitis are at greater risk for the development and/or exacerbation of diabetes, chronic obstructive pulmonary disease, and cardiovascular diseases, among other conditions. Although it is without question that specific groups of oral bacteria which populate dental plaque play a causative role in the development of periodontitis, it is now thought that once this disease has been triggered, other factors play an equal, and possibly more important, role in its progression, particularly in severe cases or in cases that prove difficult to treat. In this regard, we allude to the host response, specifically the notion that the host, once infected with oral periodontal pathogenic bacteria, will mount a defense response mediated largely through the innate immune system. The most abundant cell type of the innate immune system - polymorphonuclear neutrophils - can, when protecting the host from microbial invasion, mount a response that includes upregulation of proinflammatory cytokines, matrix metalloproteinases, and reactive oxygen species, all of which then contribute to the tissue damage and loss of teeth commonly associated with periodontitis. Of the mechanisms referred to here, we suggest that upregulation of reactive oxygen species might play one of the most important roles in the establishment and progression of periodontitis (as well as in other diseases of inflammation) through the development of oxidative stress. In this overview, we discuss both innate and epigenetic factors (eg, diabetes, smoking) that lead to the development of oxidative stress. This oxidative stress then provides an environment conducive to the destructive processes observed in periodontitis. Therefore, we shall describe some of the fundamental characteristics of oxidative stress and its effects on the periodontium, discuss the diseases and other factors that cause oxidative stress, and, finally, review potentially novel therapeutic approaches for the management (and possibly even the reversal) of periodontitis, which rely on the use of therapies, such as resveratrol and other antioxidants, that provide increased antioxidant activity in the host.

Mechanistic insight into the microtubule and actin cytoskeleton coupling through dynein-dependent RhoGEF inhibition.

Actin-based stress fiber formation is coupled to microtubule depolymerization through the local activation of RhoA. While the RhoGEF Lfc has been implicated in this cytoskeleton coupling process, it has remained elusive how Lfc is recruited to microtubules and how microtubule recruitment moderates Lfc activity. Here, we demonstrate that the dynein light chain protein Tctex-1 is required for localization of Lfc to microtubules. Lfc residues 139-161 interact with Tctex-1 at a site distinct from the cleft that binds dynein intermediate chain. An NMR-based GEF assay revealed that interaction with Tctex-1 represses Lfc nucleotide exchange activity in an indirect manner that requires both polymerized microtubules and phosphorylation of S885 by PKA. We show that inhibition of Lfc by Tctex-1 is dynein dependent. These studies demonstrate a pivotal role of Tctex-1 as a negative regulator of actin filament organization through its control of Lfc in the crosstalk between microtubule and actin cytoskeletons.

Inhibition of BRD4 Reduces Neutrophil Activation and Adhesion to the Vascular Endothelium Following Ischemia Reperfusion Injury.

Renal ischemia reperfusion injury (IRI) is associated with inflammation, including neutrophil infiltration that exacerbates the initial ischemic insult. The molecular pathways involved are poorly characterized and there is currently no treatment. We performed an in silico analysis demonstrating changes in NFκB-mediated gene expression in early renal IRI. We then evaluated NFκB-blockade with a BRD4 inhibitor on neutrophil adhesion to endothelial cells in vitro, and tested BRD4 inhibition in an in vivo IRI model. BRD4 inhibition attenuated neutrophil adhesion to activated endothelial cells. In vivo, IRI led to increased expression of cytokines and adhesion molecules at 6 h post-IRI with sustained up-regulated expression to 48 h post-IRI. These effects were attenuated, in part, with BRD4 inhibition. Absolute neutrophil counts increased significantly in the bone marrow, blood, and kidney 24 h post-IRI. Activated neutrophils increased in the blood and kidney at 6 h post-IRI and remained elevated in the kidney until 48 h post-IRI. BRD4 inhibition reduced both total and activated neutrophil counts in the kidney. IRI-induced tubular injury correlated with neutrophil accumulation and was reduced by BRD4 inhibition. In summary, BRD4 inhibition has important systemic and renal effects on neutrophils, and these effects are associated with reduced renal injury.

Novel Assay To Characterize Neutrophil Responses to Oral Biofilms.

Neutrophils, the most numerous leukocytes, play an important role in maintaining oral health through interactions with oral microbial biofilms. Both neutrophil hyperactivity and the bacterial subversion of neutrophil responses can cause inflammation-mediated tissue damage like that seen in periodontal disease. We describe here an assay that assesses neutrophil activation responses to monospecies biofilm bacteria in vitro based on the surface expression of cluster of differentiation (CD) markers associated with various neutrophil functions. Most of what we know about neutrophil responses to bacteria is based on in vitro assays that use planktonic bacteria and isolated/preactivated neutrophils, which makes interpretation of the neutrophil responses to bacteria a challenge. An understanding of how neutrophils differentially interact with and respond to commensal and pathogenic oral bacteria is necessary in order to further understand the neutrophil's role in maintaining oral health and the pathogenesis of periodontal disease. In this study, a flow cytometry-based in vitro assay was developed to characterize neutrophil activation states based on CD marker expressions in response to oral monospecies bacterial biofilms. Using this approach, changes in CD marker expressions in response to specific prominent oral commensal and pathogenic bacteria were assayed. Several functional assays, including assays for phagocytosis, production of reactive oxygen species, activation of the transcription factor Nrf2, neutrophil extracellular trap formation, and myeloperoxidase release, were also performed to correlate neutrophil function with CD marker expression. Our results demonstrate that neutrophils display bacterial species-specific responses. This assay can be used to characterize how specific biofilms alter specific neutrophil pathways associated with their activation.

Morphological characterization of para- and proinflammatory neutrophil phenotypes using transmission electron microscopy.

BACKGROUND AND OBJECTIVE: Bacterial challenge is constant in the oral cavity. To contain the commensal biofilm, partly activated neutrophils are continuously recruited as part of a normal physiologic process, without exposing the host to the harmful effect of a fully active neutrophil response. This intermediate immune state has been termed para-inflammation, as opposed to the fully activated proinflammatory state in oral disease. Directly visualizing these cells and their components via transmission electron microscopy (TEM) enhances our understanding of neutrophil activation state differences in oral health and disease, as obtained from molecular studies. The aim of this study was to describe the morphology of the para-inflammatory phenotype displayed by oral neutrophils in health, and compare it to the morphology of the naïve blood neutrophil, and the proinflammatory oral neutrophils in chronic periodontitis. This morphology was characterized by differences in granule content, phagosome content and cytoplasm and nuclear changes. We also examined the morphological changes induced in naïve neutrophils, which were stimulated in vitro by bacteria, and in oral neutrophils in full tissue samples in vivo. MATERIAL AND METHODS: Neutrophils were isolated from blood and saliva samples of patients with chronic periodontitis and healthy individuals. The cells were viewed under TEM and analyzed in imaging software examining granularity, cytoplasm density, euchromatin amount in the nucleus and phagosome content. A separate cohort of blood neutrophils was incubated with Streptococcus oralis and analyzed under TEM in the same manner. Gingival tissue samples were obtained from patients with chronic periodontitis and viewed under TEM, with the neutrophils present analyzed in the same manner. RESULTS: The proinflammatory cells showed less granulation, lighter cytoplasm and higher amount of nuclear euchromatin. These changes were accentuated in the proinflammatory oral chronic periodontitis neutrophils compared to the para-inflammatory oral health neutrophils. The oral chronic periodontitis neutrophils also contained more phagosomes and had more phagosomes containing undigested bacteria. These changes were partially reproduced in the naïve blood cells after exposing them to S. oralis. The neutrophils in the gingival tissues displayed naïve morphology when viewed in the blood vessels and gradually showed proinflammatory morphological changes as they traveled through the connective tissue into the epithelium. CONCLUSION: Oral neutrophils display morphological changes consistent with partial or full activation, corresponding to their para- or proinflammatory states. These changes can also be induced in naïve cells by incubating them with commensal bacteria. Neutrophils change their morphology towards an activated state as they travel through the gingival tissue.

Lack of p47(phox) in Akita Diabetic Mice Is Associated with Interstitial Pneumonia, Fibrosis, and Oral Inflammation.

Excess reactive oxygen species production is central to the development of diabetic complications. The contribution of leukocyte reactive oxygen species produced by the NADPH oxidase to altered inflammatory responses associated with uncontrolled hyperglycemia is poorly understood. To get insight into the role of phagocytic superoxide in the onset of diabetic complications, we used a model of periodontitis in mice with chronic hyperglycemia and lack of leukocyte p47(phox) (Akita/Ncf1) bred from C57BL/6-Ins2(Akita)/J (Akita) and neutrophil cytosolic factor 1 knockout (Ncf1) mice. Akita/Nfc1 mice showed progressive cachexia starting at early age and increased mortality by six months. Their lungs developed infiltrative interstitial lesions that obliterated air spaces as early as 12 weeks when fungal colonization of lungs also was observed. Neutrophils of Akita/Ncf1 mice had normal degranulation and phagocytic efficiency when compared with wild-type mice. Although Akita/Ncf1 mice had increased prevalence of oral infections and more severe periodontitis compared with wild-type mice, bone loss was only marginally higher compared with Akita and Ncf1 null mice. Altogether these results indicate that lack of leukocyte superoxide production in mice with chronic hyperglycemia results in interstitial pneumonia and increased susceptibility to infections.

Identification of neutrophil surface marker changes in health and inflammation using high-throughput screening flow cytometry.

Neutrophils are the most abundant white blood cell and are an essential component of the innate immune system. A complete cataloguing of cell surface markers has not been undertaken for neutrophils isolated from circulation as well as healthy and inflamed tissues. To identify cell-surface markers specific to human neutrophils, we used high-throughput flow cytometry to screen neutrophil populations isolated from blood and oral rinses from healthy and chronic periodontitis patients against a panel of 374 known cluster of differentiation (CD) antibodies. This screen identified CD11b, CD16, and CD66b as markers that are consistently expressed on neutrophils independent of the cell location, level of activation and disease state. Cell sorting against CD11b, CD16 and CD66b allowed for the enrichment of mature neutrophils, yielding neutrophil populations with up to 99% purity. These findings suggest an ideal surface marker set for isolating mature neutrophils from humans. The screen also demonstrated that tissue neutrophils from chronically inflamed tissue display a unique surface marker set compared to tissue neutrophils present in healthy, non-inflamed tissues.

The 3BP2 adapter protein is required for chemoattractant-mediated neutrophil activation.

3BP2 is a pleckstrin homology and Src homology 2 domain-containing adapter protein mutated in cherubism, a rare autosomal-dominant human bone disorder. Previously, we have demonstrated a functional role for 3BP2 in peripheral B cell development and in peritoneal B1 and splenic marginal zone B cell-mediated Ab responses. In this study, we show that 3BP2 is required for G protein-coupled receptor-mediated neutrophil functions. Neutrophils derived from 3BP2-deficient (Sh3bp2-/-) mice failed to polarize their actin cytoskeleton or migrate in response to a gradient of chemotactic peptide, fMLF. Sh3bp2-/- neutrophils failed to adhere, crawl, and emigrate out of the vasculature in response to fMLF superfusion. 3BP2 is required for optimal activation of Src family kinases, small GTPase Rac2, neutrophil superoxide anion production, and for Listeria monocytogenes bacterial clearance in vivo. The functional defects observed in Sh3bp2-/- neutrophils may partially be explained by the failure to fully activate Vav1 guanine nucleotide exchange factor and properly localize P-Rex1 guanine nucleotide exchange factor at the leading edge of migrating cells. Our results reveal an obligate requirement for the adapter protein 3BP2 in G protein-coupled receptor-mediated neutrophil function.

Analysis of Human and Mouse Neutrophil Phagocytosis by Flow Cytometry.

Neutrophils are primary phagocytes that recognize their targets through surface chemistry, either through pattern recognition receptor (PPR) interaction with pathogen-associated molecular patterns (PAMPs) or through immunoglobulin (Ig) or complement mediated recognition. Opsonization can be important for target recognition and phagocytosis by neutrophils. Therefore, phagocytosis assays performed using neutrophils in whole blood, compared to isolated cells, will differ due to the presence of opsonizing blood serum components, as well as other blood components like platelets. Powerful and sensitive flow cytometry-based methods are presented to measure phagocytosis by human blood neutrophils and mouse peritoneal neutrophils.

Intervertebral disc degeneration and osteoarthritis: a common molecular disease spectrum.

Intervertebral disc degeneration (IDD) and osteoarthritis (OA) affecting the facet joint of the spine are biomechanically interdependent, typically occur in tandem, and have considerable epidemiological and pathophysiological overlap. Historically, the distinctions between these degenerative diseases have been emphasized. Therefore, research in the two fields often occurs independently without adequate consideration of the co-dependence of the two sites, which reside within the same functional spinal unit. Emerging evidence from animal models of spine degeneration highlight the interdependence of IDD and facet joint OA, warranting a review of the parallels between these two degenerative phenomena for the benefit of both clinicians and research scientists. This Review discusses the pathophysiological aspects of IDD and OA, with an emphasis on tissue, cellular and molecular pathways of degeneration. Although the intervertebral disc and synovial facet joint are biologically distinct structures that are amenable to reductive scientific consideration, substantial overlap exists between the molecular pathways and processes of degeneration (including cartilage destruction, extracellular matrix degeneration and osteophyte formation) that occur at these sites. Thus, researchers, clinicians, advocates and policy-makers should consider viewing the burden and management of spinal degeneration holistically as part of the OA disease continuum.

Effects of a stabilized stannous fluoride dentifrice on clinical, immunomodulatory, and microbial outcomes in a human experimental gingivitis model.

BACKGROUND: Stannous fluoride dentifrice is well established for its beneficial clinical effects. In this study, we evaluated the effects of stannous fluoride on inflammation and oral microbiome. METHODS: In this randomized, parallel-arm, double-blind, controlled clinical trial, we compared clinical resolution of experimental gingivitis by evaluating bleeding on probing, gingival index, and plaque index between stannous fluoride stabilized with zinc phosphate (test) and sodium fluoride (control) dentifrices. Further, these groups were compared for oral neutrophil counts, systemic priming of neutrophils, gingival crevicular fluid (GCF) expression of inflammatory markers, and the oral microbiome. RESULTS: We found significant reduction in bleeding on probing in the test group compared to the control group in experimental gingivitis when participants used the test dentifrice prior to induction of experimental gingivitis. The test group also showed significant reductions in GCF levels of inflammatory markers (matrix metalloproteinase 8 [MMP8], receptor activator of nuclear factor kappa-Β ligand [RANKL]), oral polymorphonuclear neutrophil (PMN) counts, and systemic neutrophil priming (CD11b expression) during experimental gingivitis. Further, significant reductions in the gram-negative genera Porphyromonas, Tannerella, and Treponema were noted in the test group. CONCLUSION: The stannous fluoride stabilized with zinc phosphate dentifrice formulation demonstrated clinical reduction in gingival inflammation and a beneficial effect on microbiome and immune markers. This intervention should be explored as a preventive aid in the progression of plaque-induced gingivitis to periodontitis.

The chemorepellent, SLIT2, bolsters innate immunity against Staphylococcus aureus.

Neutrophils are essential for host defense against Staphylococcus aureus (S. aureus). The neuro-repellent, SLIT2, potently inhibits neutrophil chemotaxis, and might, therefore, be expected to impair antibacterial responses. We report here that, unexpectedly, neutrophils exposed to the N-terminal SLIT2 (N-SLIT2) fragment kill extracellular S. aureus more efficiently. N-SLIT2 amplifies reactive oxygen species production in response to the bacteria by activating p38 mitogen-activated protein kinase that in turn phosphorylates NCF1, an essential subunit of the NADPH oxidase complex. N-SLIT2 also enhances the exocytosis of neutrophil secondary granules. In a murine model of S. aureus skin and soft tissue infection (SSTI), local SLIT2 levels fall initially but increase subsequently, peaking at 3 days after infection. Of note, the neutralization of endogenous SLIT2 worsens SSTI. Temporal fluctuations in local SLIT2 levels may promote neutrophil recruitment and retention at the infection site and hasten bacterial clearance by augmenting neutrophil oxidative burst and degranulation. Collectively, these actions of SLIT2 coordinate innate immune responses to limit susceptibility to S. aureus.

The antihelmintic flubendazole inhibits microtubule function through a mechanism distinct from Vinca alkaloids and displays preclinical activity in leukemia and myeloma.

On-patent and off-patent drugs with previously unrecognized anticancer activity could be rapidly repurposed for this new indication given their prior toxicity testing. To identify such compounds, we conducted chemical screens and identified the antihelmintic flubendazole. Flubendazole induced cell death in leukemia and myeloma cell lines and primary patient samples at nanomolar concentrations. Moreover, it delayed tumor growth in leukemia and myeloma xenografts without evidence of toxicity. Mechanistically, flubendazole inhibited tubulin polymerization by binding tubulin at a site distinct from vinblastine. In addition, cells resistant to vinblastine because of overexpression of P-glycoprotein remained fully sensitive to flubendazole, indicating that flubendazole can overcome some forms of vinblastine resistance. Given the different mechanisms of action, we evaluated the combination of flubendazole and vinblastine in vitro and in vivo. Flubendazole synergized with vinblastine to reduce the viability of OCI-AML2 cells. In addition, combinations of flubendazole with vinblastine or vincristine in a leukemia xenograft model delayed tumor growth more than either drug alone. Therefore, flubendazole is a novel microtubule inhibitor that displays preclinical activity in leukemia and myeloma.

Metabolites of the oral microbiome: important mediators of multikingdom interactions.

The oral cavity hosts over 700 different microbial species that produce a rich reservoir of bioactive metabolites critical to oral health maintenance. Over the last two decades, new insights into the oral microbiome and its importance in health and disease have emerged mainly due to the discovery of new oral microbial species using next-generation sequencing. This advancement has revolutionized the documentation of unique microbial profiles associated with different niches and health/disease states within the oral cavity and the relation of the oral bacteria to systemic diseases. However, less work has been done to identify and characterize the unique oral microbial metabolites that play critical roles in maintaining equilibrium between the various oral microbial species and their human hosts. This article discusses the most significant microbial metabolites produced by these diverse communities of oral bacteria that can either foster health or contribute to disease. Finally, we shed light on how advances in genomics and genome mining can provide a high-throughput platform for discovering novel bioactive metabolites derived from the human oral microbiome to tackle emerging infectious and systemic diseases.