Opioid-induced proinflammatory glial activation modulates wide-ranging aspects of opioid pharmacology including: opposition of acute and chronic opioid analgesia, opioid analgesic tolerance, opioid-induced hyperalgesia, development of opioid dependence, opioid reward, and opioid respiratory depression. However, the mechanism(s) contributing to opioid-induced proinflammatory actions remains unresolved. The potential involvement of toll like receptor 4 (TLR4) was examined using in vitro, in vivo, and in silico techniques. Morphine non-stereoselectively induced TLR4 signaling in vitro, blocked by a classical TLR4 antagonist and non-stereoselectively by naloxone. Pharmacological blockade of TLR4 signaling in vivo potentiated acute intrathecal morphine analgesia, attenuated development of analgesic tolerance, hyperalgesia, and opioid withdrawal behaviors. TLR4 opposition to opioid actions was supported by morphine treatment of TLR4 knockout mice, which revealed a significant threefold leftward shift in the analgesia dose response function, versus wildtype mice. A range of structurally diverse clinically employed opioid analgesics was found to be capable of activating TLR4 signaling in vitro. Selectivity in the response was identified since morphine-3-glucuronide, a morphine metabolite with no opioid receptor activity, displayed significant TLR4 activity, whilst the opioid receptor active metabolite, morphine-6-glucuronide, was devoid of such properties. In silico docking simulations revealed ligands bound Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptBrain Behav Immun. Author manuscript; available in PMC 2011 January 1. preferentially to the LPS binding pocket of MD-2 rather than TLR4. An in silico to in vitro prediction model was built and tested with substantial accuracy. These data provide evidence that select opioids may non-stereoselectively influence TLR4 signaling and have behavioral consequences resulting, in part, via TLR4 signaling.

Burkholderia is a diverse and dynamic genus, containing pathogenic species as well as species that form complex interactions with plants. Pathogenic strains, such as B. pseudomallei and B. mallei, can cause serious disease in mammals, while other Burkholderia strains are opportunistic pathogens, infecting humans or animals with a compromised immune system. Although some of the opportunistic Burkholderia pathogens are known to promote plant growth and even fix nitrogen, the risk of infection to infants, the elderly, and people who are immunocompromised has not only resulted in a restriction on their use, but has also limited the application of non-pathogenic, symbiotic species, several of which nodulate legume roots or have positive effects on plant growth. However, recent phylogenetic analyses have demonstrated that Burkholderia species separate into distinct lineages, suggesting the possibility for safe use of certain symbiotic species in agricultural contexts. A number of environmental strains that promote plant growth or degrade xenobiotics are also included in the symbiotic lineage. Many of these species have the potential to enhance agriculture in areas where fertilizers are not readily available and may serve in the future as inocula for crops growing in soils impacted by climate change. Here we address the pathogenic potential of several of the symbiotic Burkholderia strains using bioinformatics and functional tests. A series of infection experiments using Caenorhabditis elegans and HeLa cells, as well as genomic characterization of pathogenic loci, show that the risk of opportunistic infection by symbiotic strains such as B. tuberum is extremely low.

Introduction: Pseudomonas aeruginosa infections are prevalent amongst chronic rhinosinusitis (CRS) sufferers. Many P. aeruginosa strains form biofilms, leading to treatment failure. Lytic bacteriophages (phages) are viruses that infect, replicate within, and lyse bacteria, causing bacterial death.Aim: To assess the activity of a phage cocktail in eradicating biofilms of ex vivo P.aeruginosa isolates from CRS patients.Methods: P. aeruginosa isolates from CRS patients with and without cystic fibrosis (CF) across three continents were multi-locus sequence typed and tested for antibiotic resistance. Biofilms grown in vitro were treated with a cocktail of four phages (CT-PA). Biofilm biomass was measured after 24 and 48 h, using a crystal violet assay. Phage titrations were performed to confirm replication of the phages. A linear mixed effects model was applied to assess the effects of treatment, time, CF status, and multidrug resistance on the biomass of the biofilm.Results: The isolates included 44 strain types. CT-PA treatment significantly reduced biofilm biomass at both 24 and 48 h post-treatment (p < 0.0001), regardless of CF status or antibiotic resistance. Biomass was decreased by a median of 76% at 48 h. Decrease in biofilm was accompanied by a rise in phage titres for all except one strain.Conclusion: A single dose of phages is able to significantly reduce biofilms formed in vitro by a range of P.aeruginosa isolates from CRS patients. This represents an exciting potential and novel targeted treatment for P. aeruginosa biofilm infections and multidrug resistant bacteria.

IMPORTANCE Staphylococcus aureus infections are associated with recalcitrant chronic rhinosinusitis (CRS). The emerging threat of multidrug-resistant S aureus infections has revived interest in bacteriophage (phage) therapy. OBJECTIVE To investigate the safety, tolerability, and preliminary efficacy of ascending multiple intranasal doses of investigational phage cocktail AB-SA01 in patients with recalcitrant CRS due to S aureus. DESIGN, SETTING, AND PARTICIPANTS This phase 1, first-in-humans, open-label clinical trial of multiple ascending doses was conducted at a single tertiary referral center from December 1, 2015, through September 30, 2016, with follow-up completed on December 31, 2016. Patients with recalcitrant CRS (aged 18-70 years) in whom surgical and medical treatment had failed and who had positive S aureus cultures sensitive to AB-SA01 were recruited. Findings were analyzed from February 2 through August 31, 2017. INTERVENTIONS Three patient cohorts (3 patients/cohort) received serial doses of twice-daily intranasal irrigations with AB-SA01 at a concentration of 3 × 10 8 plaque-forming units (PFU) for 7 days (cohort 1), 3 × 10 8 PFU for 14 days (cohort 2), and 3 × 10 9 PFU for 14 days (cohort 3). MAIN OUTCOMES AND MEASURES The primary study outcome was the safety and tolerability of intranasal AB-SA01. Safety observations included vital signs, physical examinations, clinical laboratory test results, and adverse events. The secondary outcome was preliminary efficacy assessed by comparing pretreatment and posttreatment microbiology results, disease-relevant endoscopic Lund-Kennedy Scores, and symptom scores using a visual analog scale and Sino-Nasal Outcome Test-22. RESULTS All 9 participants (4 men and 5 women; median age, 45 years [interquartile range, 41.0-71.5 years]) completed the trial. Intranasal phage treatment was well tolerated, with no serious adverse events or deaths reported in any of the 3 cohorts. No change in vital signs occurred before and 0.5 and 2.0 hours after administration of AB-SA01 and at the exit visit. No changes in biochemistry were found except for 1 participant in cohort 3 who showed a decrease in blood bicarbonate levels on exit visit, with normal results of physical examination and vital signs. All biochemistry values were normalized 8 days later. No changes in temperature were recorded before, during, or after treatment. Six adverse effects were reported in 6 participants; all were classified as mild treatment-emergent adverse effects and resolved by the end of the study. Preliminary efficacy results indicated favorable outcomes across all cohorts, with 2 of 9 patients showing clinical and microbiological evidence of eradication of infection. CONCLUSIONS AND RELEVANCE Intranasal irrigation with AB-SA01 of doses to 3 × 10 9 PFU for 14 days was safe and well tolerated, with promising preliminary efficacy observations. Phage therapy could be an alternative to antibiotics for patients with CRS.