Real-Time Impedance-Based Monitoring of the Growth and Inhibition of Osteomyelitis Biofilm Pathogen Staphylococcus aureus Treated with Novel Bisphosphonate-Fluoroquinolone Antimicrobial Conjugates.

Osteomyelitis is a limb- and life-threatening orthopedic infection predominantly caused by Staphylococcus aureus biofilms. Bone infections are extremely challenging to treat clinically. Therefore, we have been designing, synthesizing, and testing novel antibiotic conjugates to target bone infections. This class of conjugates comprises bone-binding bisphosphonates as biochemical vectors for the delivery of antibiotic agents to bone minerals (hydroxyapatite). In the present study, we utilized a real-time impedance-based assay to study the growth of Staphylococcus aureus biofilms over time and to test the antimicrobial efficacy of our novel conjugates on the inhibition of biofilm growth in the presence and absence of hydroxyapatite. We tested early and newer generation quinolone antibiotics (ciprofloxacin, moxifloxacin, sitafloxacin, and nemonoxacin) and several bisphosphonate-conjugated versions of these antibiotics (bisphosphonate-carbamate-sitafloxacin (BCS), bisphosphonate-carbamate-nemonoxacin (BCN), etidronate-carbamate-ciprofloxacin (ECC), and etidronate-carbamate-moxifloxacin (ECX)) and found that they were able to inhibit Staphylococcus aureus biofilms in a dose-dependent manner. Among the conjugates, the greatest antimicrobial efficacy was observed for BCN with an MIC of 1.48 µg/mL. The conjugates demonstrated varying antimicrobial activity depending on the specific antibiotic used for conjugation, the type of bisphosphonate moiety, the chemical conjugation scheme, and the presence or absence of hydroxyapatite. The conjugates designed and tested in this study retained the bone-binding properties of the parent bisphosphonate moiety as confirmed using high-performance liquid chromatography. They also retained the antimicrobial activity of the parent antibiotic in the presence or absence of hydroxyapatite, albeit at lower levels due to the nature of their chemical modification. These findings will aid in the optimization and testing of this novel class of drugs for future applications to pharmacotherapy in osteomyelitis.

Efficacy of chlorhexidine for the prevention and treatment of oral mucositis in cancer patients: a systematic review with meta-analyses.

BACKGROUND: Oral mucositis occurs in patients undergoing chemoradiation for cancer treatment. It is believed that colonization of ulcerated mucosa by bacteria, fungi, and virus results in secondary infections. The effect of chlorhexidine on the incidence and severity of oral mucositis in patients with cancer was evaluated in this review. METHODS: Studies were limited to randomized placebo-controlled trials. Three databases were searched: MEDLINE (via PubMed), Web of Science, and the Cochrane Library up to May 25, 2016. RESULTS: Ninety-eight abstracts were evaluated by three independent reviewers. Twelve studies met the criteria for inclusion. Four of these studies were assessed at unclear risk of bias and eight of them at high risk. Of the 12 studies, nine were included in two meta-analyses. Pooled results showed that chlorhexidine did not significantly reduce incidence of mucositis compared to placebo (P = 0.129), nor chlorhexidine did significantly reduce the severity of mucositis (P = 0.127), although subgroup analysis in the chemotherapy group showed a trend toward significance (P = 0.054). Side effects reported in the included studies were teeth staining and altered taste perception. CONCLUSIONS: This systematic review found that chlorhexidine is not significantly effective in reducing the severity of mucositis (moderate quality of evidence) nor in preventing the incidence of mucositis (low quality of evidence). However, more studies are needed in patients receiving chemotherapy only, as a positive trend toward significance was found (P = 0.054).

Modified protocol including topical minocycline in orabase to manage medication-related osteonecrosis of the jaw cases.

OBJECTIVE: Management of medication-related osteone-crosis of the jaw (MRONJ) with active infection can be a serious challenge for clinicians. Based on Association of Oral and Maxillofacial Surgeons (AAOMS) recommendations, we have tested a modified treatment protocol using topical minocycline. STUDY DESIGN: Five patients diagnosed with stage II or III MRONJ lesions were willing to consent to our protocol. In addition to conventional treatment as suggested by the AAOMS, such as, surgical debridement, chlorhexidine irrigation, and systemic antibiotics, we applied 10% minocycline to the lesions once a week for sustained local antibiotic delivery. RESULTS: All five patients reported pain relief after the first minocycline application. Complete healing occurred in three patients; case three healed completely after the third application, one case continues to improve toward resolution and one withdraws due to other non-relevant medical problem. CONCLUSIONS: In this study, we are reporting favorable results using a modified protocol with topical minocycline to treat MRONJ lesions.

Microbial biofilms are able to destroy hydroxyapatite in the absence of host immunity in vitro.

PURPOSE: It is widely thought that inflammation and osteoclastogenesis result in hydroxyapatite (HA) resorption and sequestrum formation during osseous infections, and microbial biofilm pathogens induce the inflammatory destruction of HA. We hypothesized that biofilms associated with infectious bone disease can directly resorb HA in the absence of host inflammation or osteoclastogenesis. Therefore we developed an in vitro model to test this hypothesis. MATERIALS AND METHODS: Customized HA discs were manufactured as a substrate for growing clinically relevant biofilm pathogens. Single-species biofilms of Streptococcus mutans, Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans and mixed-species biofilms of C albicans plus S mutans were incubated on HA discs for 72 hours to grow mature biofilms. Three different non-biofilm control groups also were established for testing. HA discs were then evaluated by means of scanning electron microscopy, micro-computed tomography metrotomography, x-ray spectroscopy, and confocal microscopy with planimetric analysis. In addition, quantitative cultures and pH assessment were performed. Analysis of variance was used to test for significance between treatment and control groups. RESULTS: All investigated biofilms were able to cause significant (P < .05) and morphologically characteristic alterations in HA structure as compared with controls. The highest number of alterations observed was caused by mixed biofilms of C albicans plus S mutans. S mutans biofilm incubated in medium with additional sucrose content was the most detrimental to HA surfaces among single-species biofilms. CONCLUSIONS: Our findings suggest that direct microbial resorption of bone is possible in addition to immune-mediated destruction, which has important translational implications for the pathogenesis of chronic bone infections and for targeted antimicrobial therapeutics.

Oral manifestation of Crohn's disease without concomitant gastrointestinal involvement.

Crohn's disease (CD) is a chronic relapsing inflammatory disorder of unknown etiology and uncertain pathogenesis with no known cure. CD can involve any segment of the gastrointestinal tract, and oral lesions consistent with granulomatous ulcers are considered an important extra-intestinal manifestation. Oral lesions in the absence of gastrointestinal involvement are rarely reported. We report a case of a 64-year-old man with a history of CD that was in remission for three decades, presenting with painful cobblestone-like ulcerations of the oral mucosa, but without gastrointestinal signs or symptoms. Surgical biopsy of the oral lesions revealed non-necrotizing chronic granulomatous ulcers on histopathologic examination, similar to results from a biopsy of his small intestine three decades previously which established his diagnosis of CD. The patient was successfully treated with potent topical corticosteroids which resulted in resolution of the oral lesions and associated symptoms.

Patient-derived tumor organoids to model drug response in gastric cancer.

Gastric cancer poses diverse treatment challenges due to its high tumor heterogeneity. Through the use of patient-derived tumor organoid (PDO) models, new research1 has identified genes and molecular signatures that are predictive of chemotherapeutic response, providing valuable insights for clinical management and translational advancements.

Cytomegalovirus-induced salivary gland pathology: AREG, FGF8, TNF-α, and IL-6 signal dysregulation and neoplasia.

Mucoepidermoid carcinoma (MEC) is the most common malignant tumor originating in major and minor salivary glands (SGs). Although the precise multifactorial etiology of human SG-MEC is largely unknown, we have recently shown that cytomegalovirus (CMV) is an important component of MEC tumorigenesis. Despite the well-documented overexpression of the EGFR → ERK signaling pathway in SG-MEC, there has been limited to no clinical success with inhibition of this pathway. Using our previously characterized mouse model of CMV-induced SG dysplasia/neoplasia, we report that inhibitors of the EGFR → ERK pathway do not ameliorate or rescue well-established pathology, either singly or in combination, but they do inhibit the evolution of progressive pathogenesis ("disease tolerance") in the face of mounting CMV burden. Failure to rescue SG pathology, suggested a possible increase in the ligand levels of alternative pathways that share cell proliferation and survival effectors (e.g. ERK and PI3K). Here we present evidence of a highly significant upregulation of ligands for the EGFR, FGFR, IL-6R, and TNFR signaling pathways, all of which converge upon the Raf/MEK/ERK amplifier module. This explains our finding that even in the presence of the highest nontoxic dose of an ERK phosphorylation inhibitor, pERK is undiminished. Given the considerable pathway crosstalk, a deep understanding of subversion and dysregulation of the SG interactome by CMV is a priori quite daunting. Circumventing this dilemma, we present evidence that concurrent inhibition of ERK phosphorylation (U0126) and CMV replication (acyclovir) obviates progressive pathogenesis and results in complete SG rescue (tumor regression). These findings provide a mechanistic foundation for potential clinical trials that utilize similar concurrent treatment with extant FDA-approved drugs.

Osseointegration of dental implants and osteonecrosis of the jaw in patients treated with bisphosphonate therapy: a systematic review.

Bisphosphonate (BP) drugs are a commonly prescribed group of medications used in the treatment of metabolic and oncologic bone disorders. The aim of this study was to conduct a systematic review in order to evaluate whether patients on BP therapy are appropriate candidates for dental implants as compared to patients not taking BP drugs with respect to successful implant osseointegration and the risk of developing bisphosphonate-related osteonecrosis of the jaw. Based on the current literature, a history of oral or intravenous BP use is not an absolute contraindication for dental implant placement, and dental implants can osseointegrate successfully in this patient population. Importantly, the studies currently available on this topic are of moderate to weak strength of evidence with inherent bias and limitations, and hence results must be interpreted in this context. Well-controlled studies with higher strength of evidence and larger population sizes are required to address this topic more accurately in the future.

Evaluation of serum biomarkers IL-17 and CTX for BRONJ: a pilot clinical case-control study.

A serious complication of bisphosphonate (BP) therapy is BP-related osteonecrosis of the jaw (BRONJ). Currently, no biomarkers exist to identify patients at risk. We evaluated whether interleukin-17 and C-telopeptide correlate with BRONJ development. We conducted a case-control study using patients with a history of BP therapy. Quantitative enzyme-linked immunosorbent assay and Student's t-test were done. Both markers were significantly higher in BRONJ, suggesting altered immune responses and bone remodeling may play roles in BRONJ development.

Human cytomegalovirus and mucoepidermoid carcinoma of salivary glands: cell-specific localization of active viral and oncogenic signaling proteins is confirmatory of a causal relationship.

Human cytomegalovirus (hCMV) infection is common. Although still controversial, there is growing evidence that active hCMV infection is associated with a variety of malignancies, including brain, breast, lung, colon, and prostate. Given that hCMV is frequently resident in salivary gland (SG) ductal epithelium, we hypothesized that hCMV would be important to the pathogenesis of SG mucoepidermoid carcinoma (MEC). This was initially supported by our finding that purified CMV induces malignant transformation in SG cells in an in vitro mouse model, and utilizes a pathogenic pathway previously reported for human MEC. Here we present the histologic and molecular characterizations of 39 human SG MECs selected randomly from a repository of cases spanning 2004-2011. Serial sections were obtained from formalin-fixed, paraffin embedded, tissue blocks from previous incisional or excisional biopsies. Immunohistochemical assays were performed for active hCMV proteins (IE1 and pp65) and the activated COX/AREG/EGFR/ERK signaling pathway. All four prospective causal criteria for viruses and cancer are fully satisfied: (1) protein markers for active hCMV are present in 97% of MECs; (2) markers of active hCMV are absent in non-neoplastic SG tissues; (3) hCMV-specific proteins (IE1, pp65) are in specific cell types and expression is positively correlated with severity; (4) hCMV correlates and colocalizes with an upregulation and activation of an established oncogenic signaling pathway (COX/AREG/EGFR/ERK). Thus, the evidential support reported here and previously in a mouse model is strongly confirmatory of a causal relationship between hCMV and SG mucoepidermoid carcinoma. To our knowledge, this is the first demonstration of hCMV's role in human oncogenesis that fully responds to all of Koch's Postulates as revised for viruses and cancer. In the absence of any contrary evidence, hCMV can reasonably be designated an "oncovirus."

Bisphosphonates: The role of chemistry in understanding their biological actions and structure-activity relationships, and new directions for their therapeutic use.

The bisphosphonates ((HO)2P(O)CR1R2P(O)(OH)2, BPs) were first shown to inhibit bone resorption in the 1960s, but it was not until 30 years later that a detailed molecular understanding of the relationship between their varied chemical structures and biological activity was elucidated. In the 1990s and 2000s, several potent bisphosphonates containing nitrogen in their R2 side chains (N-BPs) were approved for clinical use including alendronate, risedronate, ibandronate, and zoledronate. These are now mostly generic drugs and remain the leading therapies for several major bone-related diseases, including osteoporosis and skeletal-related events associated with bone metastases. The early development of chemistry in this area was largely empirical and only a few common structural features related to strong binding to calcium phosphate were clear. Attempts to further develop structure-activity relationships to explain more dramatic pharmacological differences in vivo at first appeared inconclusive, and evidence for mechanisms underlying cellular effects on osteoclasts and macrophages only emerged after many years of research. The breakthrough came when the intracellular actions on the osteoclast were first shown for the simpler bisphosphonates, via the in vivo formation of P-C-P derivatives of ATP. The synthesis and biological evaluation of a large number of nitrogen-containing bisphosphonates in the 1980s and 1990s led to the key discovery that the antiresorptive effects of these more complex analogs on osteoclasts result mostly from their potency as inhibitors of the enzyme farnesyl diphosphate synthase (FDPS/FPPS). This key branch-point enzyme in the mevalonate pathway of cholesterol biosynthesis is important for the generation of isoprenoid lipids that are utilized for the post-translational modification of small GTP-binding proteins essential for osteoclast function. Since then, it has become even more clear that the overall pharmacological effects of individual bisphosphonates on bone depend upon two key properties: the affinity for bone mineral and inhibitory effects on biochemical targets within bone cells, in particular FDPS. Detailed enzyme-ligand crystal structure analysis began in the early 2000s and advances in our understanding of the structure-activity relationships, based on interactions with this target within the mevalonate pathway and related enzymes in osteoclasts and other cells have continued to be the focus of research efforts to this day. In addition, while many members of the bisphosphonate drug class share common properties, now it is more clear that chemical modifications to create variations in these properties may allow customization of BPs for different uses. Thus, as the appreciation for new potential opportunities with this drug class grows, new chemistry to allow ready access to an ever-widening variety of bisphosphonates continues to be developed. Potential new uses of the calcium phosphate binding mechanism of bisphosphonates for the targeting of other drugs to the skeleton, and effects discovered on other cellular targets, even at non-skeletal sites, continue to intrigue scientists in this research field.

Bisphosphonates in dentistry: Historical perspectives, adverse effects, and novel applications.

Studies of the potential role of bisphosphonates in dentistry date back to physical chemical research in the 1960s, and the genesis of the discovery of bisphosphonate pharmacology in part can be linked to some of this work. Since that time, parallel research on the effects of bisphosphonates on bone metabolism continued, while efforts in the dental field included studies of bisphosphonate effects on dental calculus, caries, and alveolar bone loss. While some utility of this drug class in the dental field was identified, leading to their experimental use in various dentrifice formulations and in some dental applications clinically, adverse effects of bisphosphonates in the jaws have also received attention. Most recently, certain bisphosphonates, particularly those with strong bone targeting properties, but limited biochemical effects (low potency bisphosphonates), are being studied as a local remedy for the concerns of adverse effects associated with other more potent members of this drug class. Additionally, low potency bisphosphonate analogs are under study as vectors to target active drugs to the mineral surfaces of the jawbones. These latter efforts have been devised for the prevention and treatment of oral problems, such as infections associated with oral surgery and implants. Advances in the utility and mechanistic understanding of the bisphosphonate class may enable additional oral therapeutic options for the management of multiple aspects of dental health.

Bisphosphonates for delivering drugs to bone.

Advances in the design of potential bone-selective drugs for the treatment of various bone-related diseases are creating exciting new directions for multiple unmet medical needs. For bone-related cancers, off-target/non-bone toxicities with current drugs represent a significant barrier to the quality of life of affected patients. For bone infections and osteomyelitis, bacterial biofilms on infected bones limit the efficacy of antibiotics because it is hard to access the bacteria with current approaches. Promising new experimental approaches to therapy, based on bone-targeting of drugs, have been used in animal models of these conditions and demonstrate improved efficacy and safety. The success of these drug-design strategies bodes well for the development of therapies with improved efficacy for the treatment of diseases affecting the skeleton. LINKED ARTICLES: This article is part of a themed issue on The molecular pharmacology of bone and cancer-related bone diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.9/issuetoc.

Cytomegalovirus induces stage-dependent enamel defects and misexpression of amelogenin, enamelin and dentin sialophosphoprotein in developing mouse molars.

Of the approximately 8,400 children born each year in the US with cytomegalovirus (CMV)-induced birth defects, more than one third exhibit hypoplasia and hypocalcification of tooth enamel. Our prior studies indicated that CMV severely delayed, but did not completely interrupt, early mouse mandibular first molar morphogenesis in vitro. The aim of the present study was to examine the effects of CMV infection on progressive tooth differentiation and amelogenesis. Since initial CMV infection in human fetuses can occur at different developmental times, we varied the stage of initial viral infection (that is, Cap stage, Early Bell stage and Bell stage), as well as the duration of infection. CMV infection of embryonic mouse mandibular first molars in vitro induces tooth dysmorphogenesis and enamel defects in a developmental stage- and duration-dependent manner. Cap stage- and Early Bell stage-infected molars exhibit enamel agenesis and Bell stage-infected molars exhibit enamel hypoplasia. This viral-induced pathology is coincident with stage-dependent changes in Amelx, Enam and Dspp gene expression, distribution of amelogenin, enamelin and DSP proteins, cell proliferation localization and dedifferentiation of secretory ameloblasts. Importantly, our data indicate that specific levels of Amelx and Dspp gene expression define whether mouse CMV induces enamel agenesis or hypoplasia.

The role of microbial biofilms in osteonecrosis of the jaw associated with bisphosphonate therapy.

Microbial biofilms have been observed and described in bone specimens of patients with bisphosphonate (BP)-associated osteonecrosis of the jaw (BONJ) and investigators are more recently suggesting that this condition essentially represents an osteomyelitis of the jaw clinically, with greater susceptibility in some patients on BP therapy. This article explains the role of microbial biofilms in BONJ and also discusses associated factors in the disease pathogenesis, which include BP effects on bone remodeling, anti-angiogenesis, matrix necrosis, microcracks, soft tissue toxicity, and inflammation and wound healing. Recent findings suggest a key role for microbial biofilms in the pathogenesis of BONJ; this has important therapeutic implications because biofilm organisms represent a clinical target for prevention and treatment efforts aimed at reducing the significant morbidity and costs associated with this condition.

Demonstration of bacterial biofilms in culture-negative silicone stent and jones tube.

PURPOSE: To demonstrate the presence of bacterial biofilms on a dacryocystorhinostomy silicone stent and a Jones tube. METHODS: One dacryocystorhinostomy silicone stent and one Jones tube were removed from 2 patients who presented with an infection of their respective nasolacrimal system. Cultures were obtained, and the implants were processed for scanning electron microscopy and confocal laser scanning microscopy, advanced microscopic methods that are applicable for detection of uncultivable biofilm organisms. RESULTS: Routine bacterial cultures revealed no growth, but bacterial biofilms on outer and inner surfaces of both implants were confirmed by advanced microscopic techniques. CONCLUSIONS: To the authors' knowledge, this is the first article that documents the presence of biofilms on a Crawford stent or a Jones tube on patients who presented with infections involving the nasolacrimal system. Although initial cultures revealed absence of any bacterial growth, confocal laser scanning microscopy and scanning electron microscopy documented bacterial colonization. Clinicians should consider the role of biofilms and the limitation of our standard culturing techniques while treating patients with device- or implant-related infections.

Genomic Analysis of Oral Lichen Planus and Related Oral Microbiome Pathogens.

Oral lichen planus (OLP) is a common chronic inflammatory disease affecting the oral mucosa. The pathogenesis of OLP is incompletely understood but is thought to be related to the immune system. As the oral cavity is a major reservoir and transmission gateway for bacteria, viruses, and fungi, the microbial composition of the oral cavity could play a role in the pathogenesis of OLP. However, limited by analytic technology and knowledge of the microbial community in the oral cavity, it is not yet clear which pathogens are associated with OLP. Next generation sequencing (NGS) is a powerful tool to identify pathogens for many infectious diseases. In this study, we compared the host cell gene expression profiles and the microbial profiles between OLP patients and matched healthy individuals. We identified the activation of the hepatocyte nuclear factor alpha (HNF4A) network in OLP patients and potential pathogens, including Corynebacterium matruchotii, Fusobacterium periodonticum, Streptococcus intermedius, Streptococcus oralis, and Prevotella denticola. Prevotella denticola is capable of activating the HNF4A gene network. Our findings shed light on the previously elusive association of OLP with various diseases like hepatitis, and indicate that OLP is a T-helper type 17 (Th17) mediated mucosal inflammatory process. The identified molecular pathways and microbes could be used to inform future investigations into OLP pathogenesis and to develop novel therapeutics for OLP treatment.

Oral bisphosphonate use and the prevalence of osteonecrosis of the jaw: an institutional inquiry.

BACKGROUND: Initial reports of osteonecrosis of the jaw (ONJ) secondary to bisphosphonate (BP) therapy indicated that patients receiving BPs orally were at a negligible risk of developing ONJ compared with patients receiving BPs intravenously. The authors conducted a study to address a preliminary finding that ONJ secondary to oral BP therapy with alendronate sodium in a patient population at the University of Southern California was more common than previously suggested. METHODS: The authors queried an electronic medical record system to determine the number of patients with a history of alendronate use and all patients receiving alendronate who also were receiving treatment for ONJ. RESULTS: The authors identified 208 patients with a history of alendronate use. They found that nine had active ONJ and were being treated in the school's clinics. These patients represented one in 23 of the patients receiving alendronate, or approximately 4 percent of the population. CONCLUSIONS: This is the first large institutional study in the United States with respect to the epidemiology of ONJ and oral bisphosphonate use. Further studies along this line will help delineate more clearly the relationship between oral BP use and ONJ. CLINICAL IMPLICATIONS: The findings from this study indicated that even short-term oral use of alendronate led to ONJ in a subset of patients after certain dental procedures were performed. These findings have important therapeutic and preventive implications.