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.

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.

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.

Detection of human papillomavirus in cases of head and neck squamous cell carcinoma by RNA-seq and VirTect.

Next-generation sequencing provides an opportunity to detect viral species from RNA-seq data of human tissues, but existing computational approaches do not perform optimally on clinical samples. We developed a bioinformatic method called VirTect for detecting viruses in neoplastic human tissues using RNA-seq data. Here, we used VirTect to analyze RNA-seq data from 363 head and neck squamous cell carcinoma (HNSCC) patients and identified 22 human papillomavirus (HPV)-induced HNSCCs. These predictions were validated by manual review of pathology reports on histopathologic specimens. VirTect showed better performance in recall and accuracy compared to the two existing prediction methods, VirusFinder and VirusSeq, in identifying viral sequences from RNA-seq data. The majority of HPV carcinogenesis studies thus far have been performed on cervical cancer and generalized to HNSCC. Our results suggest that carcinogenesis of HPV-induced HNSCC and other cases of HNSCC involve different genes, so understanding the underlying molecular mechanisms will have a significant impact on therapeutic approaches and outcomes. In summary, RNA-seq together with VirTect can be an effective solution for the detection of viruses from tumor samples and can facilitate the clinicopathologic characterization of various types of cancers with broad applications for oncology.

Risk-reductive dental strategies for medication related osteonecrosis of the jaw among cancer patients: A systematic review with meta-analyses.

The purpose of this systematic review with meta-analysis was to assess the effectiveness of dental interventions in preventing or reducing the incidence of medication-related osteonecrosis of the jaw (MRONJ) in cancer patients receiving antiresorptive therapy, compared to similar control groups receiving no intervention. Randomized controlled trials (RCT), case-controls and cohorts on cancer patients with primary outcome being the prevalence of MRONJ were included. Four electronic databases were searched (Cochrane Library, PubMed, EMBASE and Web of Science) up to February 12, 2018. A total of 409 abstracts were assessed and one case-control, one RCT and four cohort studies with 2332 cancer patients met our inclusion criteria. Risk of bias analysis followed Cochrane's handbook. Risk of bias was unclear for the case-control study and high risk for the RCT and all cohort studies. Five studies utilized preventive measures consisting of an initial examination and performing all necessary dental treatment before patients initiated antiresorptive therapy; one study used specialized post-extraction protocols utilizing plasma-rich in growth factors (PRGF) on cancer patients receiving antiresorptive therapy. Though dental preventive measures decreased MRONJ incidence by 77.3% in six studies with 2332 cancer patients (95% CI = 47.4-90.2%; p = .001) compared to control groups, quality of the evidence was low due to high or unclear risk of bias and the observational nature of five of the included studies. In conclusion, high-quality long-term prospective large sample size studies are needed to confirm these results due to high risk of bias and heterogeneous interventions. No funding.

Single-cell genomic analysis of head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) incidence or rates have increased dramatically recently with little improvement in patient outcomes. There is an unmet need in HNSCC to develop reliable molecular markers capable of evaluating patient risks and advising treatments. This review focuses on recent developments in single-cell molecular analysis of cancer, and its applications for HNSCC diagnosis and treatments. For proof of concept, we examined gene expression levels of 62 patients with HNSCC, and correlate the gene expression profiles to single-cell gene expression profiles obtained from a pilot single-cell study of CCR5-positive breast carcinoma cells. The single-cell molecular analyses complemented the lysate data and reveals heterogeneity of oncogenesis pathways with the cancer cell population. Our single-cell molecular analysis indicated that molecular heterogeneity exists in HNSCC and should be addressed in treatment strategy of HNSCC. Single-cell molecular technology can have significant impact on diagnosis, therapeutic decision making, and prognosis of HNSCC.

Opportunistic Oral Infections.

An opportunistic infection (OI) is a disease of microbial cause or pathogenesis generally thought to occur in hosts with weakened immunity. Oral OIs are associated with many risk factors and pathogens. Causative organisms for oral OIs have unique modes of transmission. The clinical presentation of oral OIs is heterogeneous and diagnosis can be challenging. Therefore, laboratory identification of causative pathogens is useful for definitive diagnosis and targeted therapeutics, and can be achieved by biological, serologic, histologic, and/or molecular methods. Clinical risk assessment and history with review of systems, and accurate diagnosis, treatment, and follow-up, are essential.

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).

Design, Synthesis, and Antimicrobial Evaluation of a Novel Bone-Targeting Bisphosphonate-Ciprofloxacin Conjugate for the Treatment of Osteomyelitis Biofilms.

Osteomyelitis is a major problem worldwide and is devastating due to the potential for limb-threatening sequelae and mortality. Osteomyelitis pathogens are bone-attached biofilms, making antibiotic delivery challenging. Here we describe a novel osteoadsorptive bisphosphonate-ciprofloxacin conjugate (BV600022), utilizing a "target and release" chemical strategy, which demonstrated a significantly enhanced therapeutic index versus ciprofloxacin for the treatment of osteomyelitis in vivo. In vitro antimicrobial susceptibility testing of the conjugate against common osteomyelitis pathogens revealed an effective bactericidal profile and sustained release of the parent antibiotic over time. Efficacy and safety were demonstrated in an animal model of periprosthetic osteomyelitis, where a single dose of 10 mg/kg (15.6 µmol/kg) conjugate reduced the bacterial load by 99% and demonstrated nearly an order of magnitude greater activity than the parent antibiotic ciprofloxacin (30 mg/kg, 90.6 µmol/kg) given in multiple doses. Conjugates incorporating a bisphosphonate and an antibiotic for bone-targeted delivery to treat osteomyelitis biofilm pathogens constitute a promising approach to providing high bone-antimicrobial potency while minimizing systemic exposure.

Lack of Utility of Cytokeratins in Differentiating Pseudocarcinomatous Hyperplasia of Granular Cell Tumors from Oral Squamous Cell Carcinoma.

Granular cell tumor (GCT) of the oral cavity is a benign lesion. Half of oral GCTs demonstrate pseudocarcinomatous hyperplasia (PCH) of the mucosa which can mimic invasive islands of oral squamous cell carcinoma (SCC). Such similarity can be confusing when diagnosing or evaluating the two conditions, potentially leading to misdiagnosis or misclassification. Indeed, several misdiagnosed cases of oral GCT have been reported in the literature as OSCC or malignant oral GCT that resulted in unnecessary aggressive treatment for the affected patients. The aim of this study was to investigate if the cytokeratin pattern of the PCH can help in differentiating GCT from oral SCC. To distinguish between these two entities, we examined 12 patient specimens of oral GCT-PCH and oral SCC histologically and via immunohistochemistry (IHC) for CK13, CK17 and P75. The results suggest that the cytokeratin profile of PCH is similar to that of oral SCC. Therefore, consideration of IHC findings for epithelial markers alone may lead to erroneous diagnosis; thus, the presence of the granular tumor underneath the PCH and its immunopositivity for P75 or other neural definition markers can be essential to identify the underlying tumor and exclude oral SCC. Finally we recommend more studies on the molecular biology of PCH to understand how it can mimic oral SCC histologically without harboring its malignant phenotype clinically, which could have significant translational potential for understanding invasive oral SCC.

Assessing the utility of serum C-telopeptide cross-link of type 1 collagen as a predictor of bisphosphonate-related osteonecrosis of the jaw: A systematic review and meta-analysis.

BACKGROUND: The authors of this systematic review and meta-analysis assessed the utility of serum C-telopeptide cross-link of type 1 collagen (sCTX), a biomarker of bone resorption, as a predictor of the development of bisphosphonate-related osteonecrosis of the jaw (BRONJ). TYPES OF STUDIES REVIEWED: The authors searched for studies involving adult participants, written in English, and published through January 20, 2016, using the following electronic databases: the Cochrane Library, MEDLINE via PubMed, and Web of Science. They also searched Google Scholar and the reference lists of all eligible trials and reviews. They identified 16 articles that met their inclusion criteria (9 controlled studies and 7 case series). They applied the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines for systematic reviews and meta-analyses. They independently extracted data in duplicate, including the characteristics of study participants, risk factors, control groups, and outcomes. They assessed risk of bias, and they resolved any disagreements between review authors through discussion. RESULTS: A meta-analysis with 9 controlled studies revealed no significant difference in mean sCTX values between patients with BRONJ and control participants (difference in means, -31.417; 95% confidence interval [CI], -91.560 to 28.726; P = .306). A second meta-analysis with 4 studies showed no significant difference in risk of having an sCTX value below 150 picograms per milliliter for patients with BRONJ compared with control participants (risk ratio, 1.892; 95% CI, 0.636-5.626; P = .251). CONCLUSIONS AND PRACTICAL IMPLICATIONS: A systematic review of the literature with meta-analysis does not support the use of sCTX levels as a predictor of the development of BRONJ. Further prospective large sample studies are needed to understand the role of sCTX as a predictor for BRONJ.

Is p16-positive oropharyngeal squamous cell carcinoma associated with favorable prognosis? A systematic review and meta-analysis.

The purpose of this systematic review and meta-analysis was to compare the prognosis of patients with p16 expressing oropharyngeal squamous cell cancers to patients with p16 non-expressing cancers. Clinical outcomes that were evaluated included overall survival, local recurrence, disease-free survival, disease-specific survival, and event-free survival. The following electronic databases were searched: Cochrane Library, MEDLINE (via Pubmed), and Web of Science. Publications were restricted to English language. Studies were limited to controlled clinical trials on the survival rates of patients with oropharyngeal tumors that were p16 expressing, compared to patients with p16 non-expressing tumors, and at least one clinical endpoint reported by trial authors (hazard ratios). Specific ascertainment criteria were applied for inclusion and exclusion of eligible studies. Data was independently extracted in duplicate. This systematic review adhered to the Preferred Reporting Items for Systematic Reviews and Meta-analysis PRISMA checklist. Risk of bias was assessed for all included studies, and disagreements between review authors were discussed until an agreement was reached. Eighteen studies were included for final review and meta-analysis. The subgroup meta-analyses, which included survival and recurrence data, showed significantly favorable outcomes for patients with p16 expressing tumors. There is strong evidence to support that patients with p16 expressing oropharyngeal squamous cell cancers have favorable clinical outcomes and prognosis.

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.

Intracranial bacterial infections of oral origin.

Brain abscesses are rare but potentially deadly complications of odontogenic infections. This phenomenon has been described mainly in the form of case reports, as large-scale studies are difficult to perform. We compiled a total of 60 previously published cases of such a complication to investigate the predisposing factors, microbiology, and clinical outcomes of intracranial abscesses of odontogenic origin. A systematic review of the literature using the PubMed database was performed. Men accounted for 82.1% of cases, and the mean age was 42.1 years. Caries with periapical involvement and periodontitis were the two most common intra-oral sources, and wisdom tooth extraction was the most common preceding dental procedure. In 56.4% of cases, there were obvious signs of dental disease prior to development of intracranial infection. Commonly implicated microorganisms included Streptococcus viridans (especially the anginosus group), Actinomyces, Peptostreptococcus, Prevotella, Fusobacterium, Aggregatibacter actinomycetemcomitans and Eikenella corrodens. There was an 8.3% mortality rate. Intracranial abscesses can form anywhere within the brain, and appear unrelated to the side of dental involvement. This suggests that hematogenous spread is the most likely route of dissemination.

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.

Gaucher disease with jawbone involvement: a case report.

INTRODUCTION: Gaucher disease is an autosomal recessive systemic condition, and the most common of the lysosomal storage disorders. It is characterized by lipid accumulation in certain cells and organs, particularly macrophages, which appear on light microscopy as 'Gaucher cells' or vacuolated lipid-laden reticuloendothelial cells. Long bone involvement is common in Gaucher disease, whereas craniofacial bone involvement is extremely rare. Reports confirming the diagnoses of Gaucher disease involving craniofacial bones by histopathologic evidence are even rarer. CASE PRESENTATION: A 46-year-old Caucasian Ashkenazi Jewish woman with Gaucher disease presented with jawbone pain and lytic radiographic lesions of her mandible. Surgical biopsy of a mandibular lesion revealed Gaucher cells infiltrating the mandible, which correlated with radiographic and clinical findings, supporting a diagnosis of Gaucher disease with jawbone involvement. CONCLUSIONS: Lysosomal storage diseases can have head and neck manifestations, and bone involvement in Gaucher disease is common. Therefore, careful consideration of signs and symptoms and medical history, with a thorough review of systems, is important when evaluating patients with lysosomal storage disorders to rule out head and neck involvement of disease. Biopsy may be warranted in some cases for more definitive diagnosis of painful jawbone lesions and to rule out other odontogenic and non-odontogenic conditions in the differential diagnosis.