In vitro studies of factors affecting debridement of dental implants by tricalcium phosphate powder abrasive treatment.

Peri-implantitis is a common complication characterized by inflammation in tissues surrounding dental implants due to plaque accumulation, which can lead to implant failure. While air flow abrasive treatment has been found to be effective for debriding implant surfaces, little is known about the factors that affect its cleaning capacity. This study systematically examined the cleaning capacity of air powder abrasive (APA) treatment with ß-tricalcium phosphate (ß-TCP) powder, using various powder jetting strengths and different particle sizes. Three sizes of ß-TCP powder (S, M, and L) were prepared, and different powder settings (low, medium, and high) were tested. The cleaning capacity was determined by quantifying ink removal, which simulated biofilm removal from the implant surfaces at different time points. The results of the systematic comparisons showed that the most efficient cleaning of implant surfaces was achieved using size M particles with medium setting. Additionally, the amount of powder consumed was found to be critical to cleaning efficiency, and the implant surfaces were altered in all tested groups. These systematically analyzed outcomes may provide insights into the development of potential non-surgical strategies for treating peri-implant diseases.

Persister-mediated emergence of antimicrobial resistance in agriculture due to antibiotic growth promoters.

The creation and continued development of antibiotics have revolutionized human health and disease for the past century. The emergence of antimicrobial resistance represents a major threat to human health, and practices that contribute to the development of this threat need to be addressed. Since the 1950s, antibiotics have been used in low doses to increase growth and decrease the feed requirement of animal-derived food sources. A consequence of this practice is the accelerated emergence of antimicrobial resistance that can influence human health through its distribution via animal food products. In the laboratory setting, sublethal doses of antibiotics promote the expansion of bacterial persister populations, a low energy, low metabolism phenotype characterized broadly by antibiotic tolerance. Furthermore, the induction of persister bacteria has been positively correlated with an increased emergence of antibiotic-resistant strains. This body of evidence suggests that the use of antibiotics in agriculture at subtherapeutic levels is actively catalyzing the emergence of antimicrobial-resistant bacteria through the expansion of bacterial persister populations, which is potentially leading to increased infections in humans and decreased antibiotic potency. There is an urgent need to address this debilitating effect on antibiotics and its influence on human health. In this review, we summarize the recent literature on the topic of emerging antimicrobial resistance and its association with bacterial persister populations.

Adenosine Awakens Metabolism to Enhance Growth-Independent Killing of Tolerant and Persister Bacteria across Multiple Classes of Antibiotics.

Metabolic and growth arrest are primary drivers of antibiotic tolerance and persistence in clinically diverse bacterial pathogens. We recently showed that adenosine (ADO) suppresses bacterial growth under nutrient-limiting conditions. In the current study, we show that despite the growth-suppressive effect of ADO, extracellular ADO enhances antibiotic killing in both Gram-negative and Gram-positive bacteria by up to 5 orders of magnitude. The ADO-potentiated antibiotic activity is dependent on purine salvage and is paralleled with a suppression of guanosine tetraphosphate synthesis and the massive accumulation of ATP and GTP. These changes in nucleoside phosphates coincide with transient increases in rRNA transcription and proton motive force. The potentiation of antibiotic killing by ADO is manifested against bacteria grown under both aerobic and anaerobic conditions, and it is exhibited even in the absence of alternative electron acceptors such as nitrate. ADO potentiates antibiotic killing by generating proton motive force and can occur independently of an ATP synthase. Bacteria treated with an uncoupler of oxidative phosphorylation and NADH dehydrogenase-deficient bacteria are refractory to the ADO-potentiated killing, suggesting that the metabolic awakening induced by this nucleoside is intrinsically dependent on an energized membrane. In conclusion, ADO represents a novel example of metabolite-driven but growth-independent means to reverse antibiotic tolerance. Our investigations identify the purine salvage pathway as a potential target for the development of therapeutics that may improve infection clearance while reducing the emergence of antibiotic resistance. IMPORTANCE Antibiotic tolerance, which is a hallmark of persister bacteria, contributes to treatment-refractory infections and the emergence of heritable antimicrobial resistance. Drugs that reverse tolerance and persistence may become part of the arsenal to combat antimicrobial resistance. Here, we demonstrate that salvage of extracellular ADO reduces antibiotic tolerance in nutritionally stressed Escherichia coli, Salmonella enterica, and Staphylococcus aureus. ADO potentiates bacterial killing under aerobic and anaerobic conditions and takes place in bacteria lacking the ATP synthase. However, the sensitization to antibiotic killing elicited by ADO requires an intact NADH dehydrogenase, suggesting a requirement for an energized electron transport chain. ADO antagonizes antibiotic tolerance by activating ATP and GTP synthesis, promoting proton motive force and cellular respiration while simultaneously suppressing the stringent response. These investigations reveal an unprecedented role for purine salvage stimulation as a countermeasure of antibiotic tolerance and the emergence of antimicrobial resistance.

Microbiota-Sourced Purines Support Wound Healing and Mucous Barrier Function.

The intestinal mucosa requires high levels of nucleotides for energy procurement, proliferation, and innate immunity. This need for nucleotide substrates substantially increases during injury, infection, and wound healing. In the present studies, we profile potential sources of purine nucleotides in murine mucosal tissue. This work reveals the gut microbiota as a prominent source of exogenous purines and that such microbiota-sourced purines (MSPs) are available to the intestinal mucosa. The MSPs are utilized for nucleotide genesis and promote energy balance. Further analyses reveal that colitic tissues lacking MSPs are proliferatively stunted, with notable energetic and endoplasmic reticulum stress to the detriment of mucous barrier integrity. Purine reconstitution either directly or through colonization of germ-free/antibiotic-treated mice with MSP-sufficient E. coli alleviates such deficits, establishing MSP as a critical source of substrate for tissue metabolism, wound healing, and mucous barrier sterile integrity.

Markers of Hypoxia Correlate with Histologic and Endoscopic Severity of Colitis in Inflammatory Bowel Disease.

BACKGROUND: Inflammation results in significant shifts in tissue metabolism. Recent studies indicate that inflammation and hypoxia occur concomitantly. We examined whether circulating and tissue markers of hypoxia could serve as surrogate indicators of disease severity in adult and pediatric patients with inflammatory bowel disease (IBD). METHODS: Serum and colonic biopsies were obtained from pediatric subjects with active IBD colitis and adult subjects with active and inactive ulcerative colitis, along with healthy non-colitis controls of all ages. Disease activity was evaluated by endoscopy and histopathology. Levels of serum hypoxia markers (macrophage inflammatory protein-3α [MIP-3α], vascular endothelial growth factor [VEGF], and erythropoietin [EPO]) were measured. RESULTS: Children with active IBD colitis had higher levels of serum MIP-3α and VEGF compared to non-colitis controls (p<0.01 and p<0.05, respectively). In adult subjects with endoscopically active ulcerative colitis, serum MIP-3α and EPO were significantly elevated compared to non-colitis controls (both p<0.01). In parallel, analysis of colon tissue MIP-3α mRNA and protein in pediatric subjects revealed increased expression in those with IBD colitis compared to controls (p<0.05 and p<0.01 for mRNA and protein, respectively). Serum MIP-3α and VEGF significantly increased with histology grade. CONCLUSION: Peripheral blood hypoxia markers may be useful indicators of disease activity for pediatric and adult IBD patients.

Neutrophils as sources of dinucleotide polyphosphates and metabolism by epithelial ENPP1 to influence barrier function via adenosine signaling.

Extracellular adenosine signaling is established as a protective component in mucosal inflammatory responses. The sources of extracellular adenosine include enzymatic processing from nucleotides, such as ATP and AMP, that can be liberated from a variety of cell types, including infiltrating leukocytes. Here we demonstrate that activated human neutrophils are a source of diadenosine triphosphate (Ap3A), providing an additional source of nucleotides during inflammation. Profiling murine enteroids and intestinal epithelial cell lines revealed that intestinal epithelia prominently express apical and lateral ectonucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1), a member of the ENPP family of enzymes that metabolize diadenosine phosphates, especially Ap3A. Extensions of these studies demonstrated that intestinal epithelia metabolize Ap3A to ADP and AMP, which are further metabolized to adenosine and made available to activate surface adenosine receptors. Using loss and gain of ENPP1 approaches, we revealed that ENPP1 coordinates epithelial barrier formation and promotes epithelial wound healing responses. These studies demonstrate the cooperative metabolism between Ap3A and ENPP1 function to provide a significant source of adenosine, subserving its role in inflammatory resolution.

Microbiota-Derived Indole Metabolites Promote Human and Murine Intestinal Homeostasis through Regulation of Interleukin-10 Receptor.

Interactions between the gut microbiota and the host are important for health, where dysbiosis has emerged as a likely component of mucosal disease. The specific constituents of the microbiota that contribute to mucosal disease are not well defined. The authors sought to define microbial components that regulate homeostasis within the intestinal mucosa. Using an unbiased, metabolomic profiling approach, a selective depletion of indole and indole-derived metabolites was identified in murine and human colitis. Indole-3-propionic acid (IPA) was selectively diminished in circulating serum from human subjects with active colitis, and IPA served as a biomarker of disease remission. Administration of indole metabolites showed prominent induction of IL-10R1 on cultured intestinal epithelia that was explained by activation of the aryl hydrocarbon receptor. Colonization of germ-free mice with wild-type Escherichia coli, but not E. coli mutants unable to generate indole, induced colonic epithelial IL-10R1. Moreover, oral administration of IPA significantly ameliorated disease in a chemically induced murine colitis model. This work defines a novel role of indole metabolites in anti-inflammatory pathways mediated by epithelial IL-10 signaling and identifies possible avenues for utilizing indoles as novel therapeutics in mucosal disease.

Cannabinoid Receptor-2 Ameliorates Inflammation in Murine Model of Crohn's Disease.

BACKGROUND AND AIMS: Cannabinoid receptor stimulation may have positive symptomatic effects on inflammatory bowel disease [IBD] patients through analgesic and anti-inflammatory effects. The cannabinoid 2 receptor [CB2R] is expressed primarily on immune cells, including CD4+ T cells, and is induced by active inflammation in both humans and mice. We therefore investigated the effect of targeting CB2R in a preclinical IBD model. METHODS: Employing a chronic ileitis model [TNFΔARE/+ mice], we assessed expression of the CB2R receptor in ileal tissue and on CD4+ T cells and evaluated the effect of stimulation with CB2R-selective ligand GP-1a both in vitro and in vivo. Additionally, we compared cannabinoid receptor expression in the ilea and colons of healthy human controls with that of Crohn's disease patients. RESULTS: Ileal expression of CB2R and the endocannabinoid anandamide [AEA] was increased in actively inflamed TNF∆ARE/+ mice compared with controls. CB2R mRNA was preferentially induced on regulatory T cells [Tregs] compared with T effector cells, approximately 2.4-fold in wild-type [WT] and 11-fold in TNF∆ARE/+ mice. Furthermore, GP-1a enhanced Treg suppressive function with a concomitant increase in IL-10 secretion. GP-1a attenuated murine ileitis, as demonstrated by improved histological scoring and decreased inflammatory cytokine expression. Lastly, CB2R is downregulated in both chronically inflamed TNF∆ARE/+ mice and in IBD patients. CONCLUSIONS: In summary, the endocannabinoid system is induced in murine ileitis but is downregulated in chronic murine and human intestinal inflammation, and CB2R activation attenuates murine ileitis, establishing an anti-inflammatory role of the endocannabinoid system.

Intestinal Epithelial Ecto-5'-Nucleotidase (CD73) Regulates Intestinal Colonization and Infection by Nontyphoidal Salmonella.

Ecto-5'-nucleotidase (CD73) is expressed abundantly on the apical surface of intestinal epithelial cells (IECs) and functions as the terminal enzyme in the generation of extracellular adenosine. Previous work demonstrated that adenosine signaling in IECs results in a number of tissue-protective effects during inflammation; however, a rationale for its apical expression has been lacking. We hypothesized that the highly polarized expression of CD73 is indicative of an important role for extracellular adenosine as a mediator of host-microbe interactions. We show that adenosine harbors bacteriostatic activity against Salmonella enterica serovar Typhimurium that is not shared by the related purine metabolite 5'-AMP, inosine, or hypoxanthine. Analysis of Salmonella colonization in IEC-specific CD73 knockout mice (CD73f/fVillinCre ) revealed a nearly 10-fold increase in colonization compared to that in controls. Despite the increased luminal colonization by Salmonella, CD73f/fVillinCre mice were protected against Salmonella colitis and showed reduced Salmonella burdens in viscera, suggesting that adenosine promotes dissemination. The knockdown of CD73 expression in cultured IECs resulted in dramatic defects in intraepithelial localization and replication as well as defective transepithelial translocation by Salmonella In conclusion, we define a novel antimicrobial activity of adenosine in the gastrointestinal tract and unveil an important role for adenosine as a regulator of host-microbe interactions. These findings have broad implications for the development of new therapeutic agents for infectious disease.

Tissue metabolism and the inflammatory bowel diseases.

The intestinal mucosa provides a selective barrier between the anaerobic lumen and a highly metabolic lamina propria. A number of recent studies indicate that acute inflammation of the mucosa can result in tissue hypoxia and associated shifts in tissue metabolism. The activation of hypoxia-inducible factor (HIF) under these conditions has been demonstrated to function as an endogenous molecular cue to promote resolution of inflammation, particularly through the orchestration of barrier repair toward homeostasis. Given the central role of oxygen in tissue metabolism, ongoing studies have defined metabolic endpoints of HIF stabilization as important biomarkers of disease activity. Such findings make HIF and HIF-associated metabolic pathways particularly attractive therapeutic targets in inflammatory bowel disease (IBD). Here, we review the recent literature related to tissue metabolism in IBD.

Neutrophils and the inflammatory tissue microenvironment in the mucosa.

The interaction of neutrophils (PMNs) and epithelial cells are requisite lines of communication during mucosal inflammatory responses. Consequences of such interactions often determine endpoint organ function, and for this reason, much interest has developed around defining the constituents of the tissue microenvironment of inflammatory lesions. Physiologic in vitro and in vivo models have aided in the discovery of components that define the basic inflammatory machinery that mold the inflammatory tissue microenvironment. Here, we will review the recent literature related to the contribution of PMNs to molding of the tissue microenvironment, with an emphasis on the gastrointestinal (GI) tract. We focus on endogenous pathways for promoting tissue homeostasis and the molecular determinants of neutrophil-epithelial cell interactions during ongoing inflammation. These recent studies highlight the dynamic nature of these pathways and lend insight into the complexity of treating mucosal inflammation.

HPLC-Based Metabolomic Analysis of Normal and Inflamed Gut.

The idiopathic inflammatory bowel diseases, which include Crohn's disease and ulcerative colitis, are multifactorial chronic conditions that result in numerous perturbations of metabolism in the gastrointestinal mucosa. Thus, methodologies for the qualitative and quantitative analysis of small molecule metabolites in mucosal tissues are important for further elucidation of mechanisms driving inflammation and the metabolic consequences of inflammation. High-performance liquid chromatography (HPLC) is a ubiquitous analytical technique that can be adapted for both targeted and non-targeted metabolomic analysis. Here, protocols for reversed-phase (RP) HPLC-based methods using two different detection modalities are presented. Ultraviolet detection is used for the analysis of adenine nucleotide metabolites, whereas electrochemical detection is used for the analysis of multiple amino acid metabolites. These methodologies provide platforms for further characterization of the metabolic changes that occur during gastrointestinal inflammation.

NMR-Based Metabolomic Analysis of Normal and Inflamed Gut.

Crohn's disease and ulcerative colitis, the two major forms of idiopathic inflammatory bowel disease (IBD), are thought to occur through a loss of intestinal barrier leading to an inappropriate immune response toward intestinal microbiota. While genome-wide association studies (GWAS) have provided much information about susceptibility loci associated with these diseases, the etiology of IBD is still unknown. Metabolomic analysis allows for the comprehensive measurement of multiple small molecule metabolites in biological samples. During the past decade, metabolomic techniques have yielded novel and potentially important findings, revealing insight into metabolic perturbations associated with these diseases. This chapter provides metabolomic methodologies describing a nuclear magnetic resonance (NMR)-based non-targeted approach that has been utilized to make important contributions toward a better understanding of IBD.

HIF-dependent regulation of claudin-1 is central to intestinal epithelial tight junction integrity.

Intestinal epithelial cells (IECs) are exposed to profound fluctuations in oxygen tension and have evolved adaptive transcriptional responses to a low-oxygen environment. These adaptations are mediated primarily through the hypoxia-inducible factor (HIF) complex. Given the central role of the IEC in barrier function, we sought to determine whether HIF influenced epithelial tight junction (TJ) structure and function. Initial studies revealed that short hairpin RNA-mediated depletion of the HIF1ß in T84 cells resulted in profound defects in barrier and nonuniform, undulating TJ morphology. Global HIF1α chromatin immunoprecipitation (ChIP) analysis identified claudin-1 (CLDN1) as a prominent HIF target gene. Analysis of HIF1ß-deficient IEC revealed significantly reduced levels of CLDN1. Overexpression of CLDN1 in HIF1ß-deficient cells resulted in resolution of morphological abnormalities and restoration of barrier function. ChIP and site-directed mutagenesis revealed prominent hypoxia response elements in the CLDN1 promoter region. Subsequent in vivo analysis revealed the importance of HIF-mediated CLDN1 expression during experimental colitis. These results identify a critical link between HIF and specific tight junction function, providing important insight into mechanisms of HIF-regulated epithelial homeostasis.

Comparison of ridge expansion and ridge splitting techniques for narrow alveolar ridge in a Swine cadaver model.

Ridge splitting and ridge expansion have been used to expand narrow alveolar ridges. Piezosurgical ridge splitting involves separating the atrophic crests with piezosurgical inserts. Ridge expansion with motor-driven expanders was proposed to achieve the cortical dilation. The purpose of this study was to evaluate the efficacy of ridge gain by ridge expansion or ridge splitting. Eighteen (36 ramus) swine cadaver jaws were first divided into two groups- ridge expansion with a motor-driven expander or ridge splitting with the piezosurgical system. Then, either an active-tapping implant or nonactivetapping cylinder-type implant was inserted. The crestal ridge diameter change was measured with a Boley gauge. The area of bony perforation, which includes fenestrations and dehiscences, was measured with a prefabricated reference grid. The results showed that there was no statistically significant difference in crestal width gain between groups. However, the combination of the motor-driven ridge expansion technique and the active-tapping implant could be beneficial in significantly decreasing the bony perforation area.

Focused physician-performed echocardiography in sports medicine: a potential screening tool for detecting aortic root dilatation in athletes.

OBJECTIVES: The purpose of this study was to investigate whether sports medicine physicians can obtain accurate measurements of the aortic root in young athletes. METHODS: Twenty male collegiate athletes, aged 18 to 21 years, were prospectively enrolled. Focused echocardiography was performed by a board-certified sports medicine physician and a medical student, followed by comprehensive echocardiography within 2 weeks by a cardiac sonographer. A left parasternal long-axis view was acquired to measure the aortic root diameter at the sinuses of Valsalva. Intraclass correlation coefficients (ICCs) were used to assess inter-rater reliability compared to a reference standard and intra-rater reliability of repeated measurements obtained by the sports medicine physician and medical student. RESULTS: The ICCs between the sports medicine physician and cardiac sonographer and between the medical student and cardiac sonographer were strong: 0.80 and 0.76, respectively. Across all 3 readers, the ICC was 0.89, indicating strong inter-rater reliability and concordance. The ICC for the 2 measurements taken by the sports medicine physician for each athlete was 0.75, indicating strong intra-rater reliability. The medical student had moderate intra-rater reliability, with an ICC of 0.59. CONCLUSIONS: Sports medicine physicians are able to obtain measurements of the aortic root by focused echocardiography that are consistent with those obtained by a cardiac sonographer. Focused physician-performed echocardiography may serve as a promising technique for detecting aortic root dilatation and may contribute in this manner to preparticipation cardiovascular screening for athletes.