Asma A. Khan

Differential Expression of MicroRNAs in Normal and Inflamed Human Pulps

INTRODUCTION MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression via posttranscriptional repression. They are critical to normal cellular function, and bioinformatic predictions indicate that at least one third of all messenger RNAs might be regulated by miRNAs. Although both the innate and adaptive immune responses are known to be regulated by miRNAs, their role in regulating endodontic disease has yet to be explored. The purpose of this study was to examine the differential expression of miRNAs in normal and inflamed human dental pulps and to explore their functional gene targets. METHODS After obtaining informed consent, we collected normal and inflamed human pulps (N = 30). Microarray and molecular biology techniques were then used for gene profiling and identifying functional gene targets. RESULTS Of the 335 human miRNAs identified in the pulp tissues, 3 miRNAs, miR-150∗, miR-584, and miR-766, were significantly up-regulated in inflamed pulps as compared with normal pulps (P < .003). Thirty-three miRNAs were down-regulated in the inflamed pulps (P < .003). The false discovery rate for these findings is estimated to be approximately 5%. The potential gene targets for these miRNAs include proinflammatory cytokines as well as other key mediators of the immune and inflammatory response to infection. CONCLUSIONS Our data identify differential expression of miRNAs in healthy and diseased human dental pulps. These findings highlight the intricate and specific roles of miRNA in inflammation and immunity, both of which are key aspects of pulpal pathology.

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.

Effect of calcium hydroxide on proinflammatory cytokines and neuropeptides.

Calcium hydroxide, a widely used intracanal medicament, is known to exert an antimicrobial effect and to degrade bacterial-derived lipopolysaccharides. However, little is known about the effect of Ca(OH)(2) on endogenous inflammatory mediators such as interleukin-1 alpha (IL-1 alpha), tumor necrosis factor-alpha (TNF-alpha), and calcitonin gene-related peptide (CGRP). This is an important gap in knowledge because these inflammatory mediators play an important role in mediating the pathogenesis of periradicular periodontitis. We tested the hypothesis that Ca(OH)(2) denatures IL-1 alpha, TNF-alpha, and CGRP. Human IL-1 alpha (0.125 ng/mL), TNF-alpha (0.2 ng/mL), and CGRP (0.25 ng/mL) were incubated with Ca(OH)(2) (0.035 mg/mL) for 1-7 days. At the end of the incubation period, the pH of the samples was neutralized, and the concentrations of the mediators were measured by immunoassays. Data were analyzed with one-way analysis of variance and Bonferroni multiple comparison tests. The results indicate that Ca(OH)(2) denatures IL-1 alpha, TNF-alpha, and CGRP by 50%-100% during the testing periods (P < .001). We concluded that denaturation of these proinflammatory mediators is a potential mechanism by which Ca(OH)(2) contributes to the resolution of periradicular periodontitis.

Acetaminophen : Old Drug , New Issues

INTRODUCTION The purpose of this review was to discuss new issues related to safety, labeling, dosing, and a better understanding of the analgesic effect of acetaminophen. METHODS The MEDLINE, Embase, Cochrane, and PubMed databases were searched. Additionally, the bibliography of all relevant articles and textbooks were manually searched. Two reviewers independently selected the relevant articles. RESULTS Concerns about acetaminophen overdose and related liver failure have led the US Food and Drug Administration to mandate new labeling on acetaminophen packaging. In addition, large-scale epidemiologic studies increasingly report evidence for second-generation adverse effects of acetaminophen. Prenatal exposure to acetaminophen is associated with neurodevelopmental and behavioral disorders. Recent studies also suggest that acetaminophen is a hormone disrupter (ie, it interferes with sex and thyroid hormone function essential for normal brain development) and thus may not be considered a safe drug during pregnancy. Finally, emerging evidence suggests that although the predominant mechanism by which acetaminophen exerts its therapeutic effect is by inhibition of cyclooxygenase, multiple other mechanisms also contribute to its analgesic effect. CONCLUSIONS Available evidence suggests that indiscriminate usage of this drug is not warranted. and its administration to a pregnant patient should be considered with great caution.

A standardized novel method to measure radiographic root changes after endodontic therapy in immature teeth.

INTRODUCTION Outcome studies of endodontic treatment of necrotic immature permanent teeth rely on radiographic measures as surrogates of whether the treatment achieved regeneration/revascularization/revitalization. An increase in radiographic root length and/or width is thought to result in a better long-term prognosis for the tooth. In this study, a method to measure radiographic outcomes of endodontic therapies on immature teeth was developed and validated. METHODS A standardized protocol was developed for measuring the entire area of the root of immature teeth. The radiographic root area (RRA) measurement accounts for the entire surface area of the root as observed on a periapical radiograph. Reviewers were given instructions on how to measure RRA, and they completed measurements on a set of standardized radiographs. RESULTS The intraclass correlation between the 4 reviewers was 0.9945, suggesting a high concordance among reviewers. There was no effect of the reviewer on the measured RRA values. High concordance was also observed when 1 rater repeated the measurements, with an intraclass correlation value of 0.9995. There was no significant difference in RRA values measured at the 2 sessions by the same rater. Furthermore, significant differences in RRA were detectable between clinical cases that showed obvious continued root development and cases that did not demonstrate discernible root development. CONCLUSIONS These results suggest that RRA is a valid measure to assess radiographic outcomes in endodontically treated immature teeth, and RRA should be useful in future clinical studies of regenerative endodontic outcomes.

MicroRNAs responsive to Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis LPS modulate expression of genes regulating innate immunity in human macrophages

MicroRNAs (miRNAs) are a class of small, noncoding RNAs that regulate post-transcriptional expression of their respective target genes and are responsive to various stimuli, including LPS. Here we examined the early (4 h) miRNA responses of THP1-differentiated macrophages challenged with LPS derived from the periodontal pathogens, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis or environmentally-modified LPS obtained from P. gingivalis grown in cigarette smoke extract. Predicted miRNA-gene target interactions for LPS-responsive miR-29b and let-7f were confirmed using dual-luciferase assays and by transfection experiments using miRNA mimics and inhibitors. Convergent and divergent miRNA profiles were observed in treated samples where differences in miRNA levels related to the type, concentration and incubation times of LPS challenge. Dual-luciferase experiments revealed miR-29b targeting of interleukin-6 receptorα (IL-6Rα) and IFN-γ inducible protein 30 and let-7f targeting of suppressor of cytokine signaling 4 and thrombospondin-1. Transfection experiments confirmed miR-29b and let-7f modulation of IL-6Rα and SOCS4 protein expression levels, respectively. Thus, we have demonstrated convergent/divergent miRNA responses to wild type LPS and its environmentally-modified LPS, and demonstrate miRNA targeting of key genes linked to inflammation and immunity. Our data indicate that these LPS-responsive miRNAs may play a key role in fine-tuning the host response to periodontal pathogens.

Animal Models of Orofacial Pain

Pain is one of the most common reasons for which patients seek dental and medical care. Orofacial pain conditions consist of a wide range of disorders including odontalgia (toothache), temporomandibular disorders, trigeminal neuralgia and others. Most of these conditions are either inflammatory or neuropathic in nature. This chapter provides an overview of the commonly used models to study inflammatory and neuropathic orofacial pain.

Effects of Antibiotics on Bone and Soft-Tissue Healing Following Immediate Single-Tooth Implant Placement Into Sites With Apical Pathology

Overprescription of antibiotics can cause bacterial resistance problems, leading to life-threatening illnesses and public health crises. Clinicians often believe antibiotics can prevent dental implant failure and postoperative complications. In conjunction with implant surgery, antibiotics are therefore routinely prescribed for all cases. In this double-blind, randomized controlled trial, the effects of antibiotics on the clinical outcomes of immediate implant placement upon replacing a tooth with an apical pathology were examined to compare antibiotics (n = 10) and placebo (n = 10). In each subject, a tooth with a chronic apical lesion was extracted, thoroughly curetted, irrigated, and replaced with single implant with a screw-retained custom provisional abutment/crown. Postoperative pain/discomfort was measured at 1- and 4-week postsurgical follow-up visits using visual analog scales. Facial alveolar bone and soft-tissue changes were measured using pre- and postoperative cone-beam computerized tomography and impressions. We found survival rates of 100% (antibiotics) and 78% (control). However, there was no statistical difference in means for any clinical outcome (t tests with Bonferroni adjustment for multiple testing), except for midfacial soft-tissue changes: 0.43 mm (SD, 0.76) in the antibiotics group and 1.70 mm (SD, 1.06) in the placebo group (t15 = -2.89, P = .011). The average change of the midfacial alveolar plate was 0.62 mm (SD, 0.46) and 1.34 mm (SD, 0.91) for the antibiotic and placebo groups, respectively, which did not significantly differ statistically. No significant correlation (Spearman correlation) existed between the changes in facial alveolar bone and the facial gingival margin. Antibiotics appear to have little effect on immediate implant treatment outcomes.

Expression Profiling of LPS Responsive miRNA in Primary Human Macrophages

microRNAs (miRNAs) have emerged as important regulators of the innate and adaptive immune response. The purpose of the present study was to interrogate miRNA profiles of primary human macrophages challenged with bacterial lipopolysaccharide (LPS) with focus on expression kinetics. We employed Nanostring platform to precisely characterize the changes in miRNA expression following different doses and durations of LPS exposure. Differentially expressed miRNAs were identified in response to LPS challenge with convergent and divergent expression profiles. Pathway analysis of LPS-responsive miRNAs revealed regulation of biological processes linked to key cell signaling (including PIK3-Akt, MAP kinase, ErbB) and pathogen response pathways. Our data provide a comprehensive miRNA profiling of human primary macrophages treated with LPS. These results show that bacterial Toll like receptor (TLR) ligands can temporally modulate macrophage miRNA expression.

Temporomandibular Joint Disorders

Temporomandibular joint disorder (TMJD) is a musculoskeletal pain condition affecting the TMJ, masticatory muscles, and/or associated structures. It is characterized by spontaneous pain and jaw function-induced pain in the TMJ and muscles of mastication. Conditions comorbid with TMJD include fibromyalgia, irritable bowel syndrome, chronic headache, interstitial cystitis, and chronic tinnitus. The risk factors consistently associated with TMJD are female gender, depression, and preexisting pain conditions. While TMJD pain is estimated to be 1.5–2 times more common in women than in men, the influence of gender on the diagnosis and treatment of TMJD is yet to be evaluated. Future studies identifying the environmental, genetic, biological, and psychosocial risk factors which seem to ‘protect’ the male sex from the development of TMJD will better the prevention and management of this multifaceted disorder.