Message in a bubble: the translational potential of extracellular vesicles.

Extracellular vesicles (EVs) are small, membrane-enclosed vesicles released by cells into the extracellular milieu. They are found in all body fluids and contain a variety of functional cargo including DNA, RNA, proteins, glycoproteins and lipids, able to provoke phenotypic responses in cells, both locally and at distant sites. They are implicated in a wide array of physiological and pathological processes and hence have attracted considerable attention in recent years as potential therapeutic targets, drug delivery vehicles and biomarkers of disease. In this review we summarise the major functions of EVs in health and disease and discuss their translational potential, highlighting opportunities of - and challenges to - capitalising on our rapidly increasing understanding of EV biology for patient benefit.

Instruments to assess appropriateness of hip and knee arthroplasty: a systematic review.

OBJECTIVE: To assess criteria and psychometric properties of instruments for assessing appropriateness of elective joint arthroplasty (JA) for adults with primary hip and knee osteoarthritis (OA). METHODS: A systematic review guided by Cochrane methods and PRISMA guidelines. Studies were searched in five databases. Eligible articles include all study designs developing, testing, and/or using an instrument to assess JA appropriateness. Two independent reviewers screened and extracted data. Instruments were compared with Hawker et al. JA consensus criteria. Psychometric properties of instruments were described and appraised guided by Fitzpatrick's and COSMIN approaches. RESULTS: Of 55 instruments included, none met all Hawker et al. JA consensus criteria. Criteria the most met were pain (n = 50), function (n = 49), quality of life (n = 33), and radiography (n = 24). Criteria the least met were clinical evidence of OA (n = 18), expectations (n = 15), readiness for surgery (n = 11), conservative treatments (n = 8), and patient/surgeon agree benefits outweigh risks (n = 0). Instrument by Arden et al. met the most criteria (6 of 9). The most tested psychometric properties were appropriateness (n = 55), face/content validity (n = 55), predictive validity (n = 29), construct validity and feasibility (n = 24). The least tested psychometric properties were intra-rater reliability (n = 3), internal consistency (n = 5), and inter-rater reliability (n = 13). Instruments by Gutacker et al. and Osborne et al. met the most psychometric properties (4 of 10). CONCLUSION: Most instruments included traditional criteria for assessing JA appropriateness but did not include a trial of conservative treatments or shared decision-making elements. There was limited evidence on psychometric properties.

Identification of a Czc-like operon of the periodontal pathobiont Porphyromonas gingivalis involved in metal ion efflux.

OBJECTIVE: The study aimed to investigate the role of the PGN2012 gene of the periodontitis contributing pathobiont Porphyromonas gingivalis. PGN2012 is a homolgue of TolC and is a gene our group previously showed was overexpressed in hyperinvasive cells. METHODS: The study used a combination of bioinformatics, knockout mutagenesis, growth experiments, biofilm assays and human cell invation assays to investigate PGN2012 function. RESULTS: Bioinformatics identified that PGN2012 is part of one of four TolC containing gene loci in P. gingivalis that we predicted may encode a metal resistance RND family tripartite pump, similar to those present in other Gram-negative bacteria, but which are not well understood in anaerobic bacteria. A ΔPGN2012 deletion displayed slightly reduced growth in liquid culture but did not effect biofilm formation or human cell invasion. When metal ions were included in the medium the mutant displayed significantly increased sensitivity to the divalent metal ions Zn2+ (500 µM), Co2+ (2 mM), and Cd2+(0.1 mM) but not Cu2+. CONCLUSIONS: We propose to rename the PGN2012-2014 genes czcCBA, which we suggest plays a role in intracellular stress resistance where zinc is often employed by host cells in antibacterial defence with implications for chronic infection in humans.

Association between allergen component sensitisation and clinical allergic disease in children.

BACKGROUND: Allergen component sensitisation testing is becoming increasingly important in the diagnosis of peanut allergy. The aim of the present study was to evaluate the relationship between sensitisation and symptoms of allergic disease in children by testing a large panel of inhalants, food allergens, and allergen components. METHODS: For 287 children visiting our laboratory for allergy testing, symptoms of allergic disease were recorded by standardised validated questionnaires. Specific IgE to 11 whole allergens was assessed by ImmunoCAP, and to 112 allergen components by ISAC ImmunoCAP assay. We used latent class analysis (LCA) to distinguish clinical phenotypes. RESULTS: Inhalant and food allergen sensitisation was common, irrespective of the children's allergic symptom type. Less than 10% of the variance in symptom scores was explained by variations in the number of allergens (components) that the child was sensitised to. In LCA, 135 children (50.2%) had mild allergy, with few symptoms and sensitisation to no or few allergens, 74 children (27.5%) had more symptoms and sensitisation to inhalant allergens (respiratory allergy) and 60 children (22.3%) showed polysensitisation to a median of six allergens and had more severe symptoms of different organ systems. Adding allergen component test results to LCA failed to result in identifiable classes of allergic disease in children. CONCLUSIONS: In this group of children with allergic symptoms, referred for allergy testing by their physician, broad screening for allergen component sensitisation did not contribute to distinguishing phenotypes of allergic disease.

[Uveitis and tattoos]. / Uvéites et tatouages.

The complications of tattoos are multiple and known for many years. However, their success explains their exponential multiplication in all countries. This confirmed the development of two new complications: a localized or remote skin sarcoidosis reaction, as well as the development of clinical and histological uveitis. This is in the context of a delayed hypersensitivity reaction, or a granulomatous reaction. Their prevention remains rather poorly known. It requires that each tattoo-carrying subject perform a systemic sarcoidosis check-up as well as an ophthalmological investigation in search of possible uveitis. If confirmed, preventive and therapeutic measures will be carried out in emergencies to avoid blindness. These new complications confirm the extreme severity of the use of uncontrolled inks, real toxic and sensitizing mixtures, especially apparently during extensive colorful tattoos. These particularly aggressive colored inks release multiple substances and nanoparticles into the body, not all of which are measured in the medium and long term.

Nociceptin/orphanin FQ receptor ligands and translational challenges: focus on cebranopadol as an innovative analgesic.

Opioids are characterised as classical (mu, delta, and kappa) along with the non-classical nociceptin/orphanin FQ (N/OFQ) receptor or NOP. Targeting NOP has therapeutic indications in control of the cardiovascular and respiratory systems and micturition, and a profile as an antidepressant. For all of these indications, there are translational human data. Opioids such as morphine and fentanyl (activating the mu receptor) are the mainstay of pain treatment in the perioperative period, despite a challenging side-effect profile. Opioids in general have poor efficacy in neuropathic pain. Moreover, longer term use is associated with tolerance. There is good evidence interactions between opioid receptors, and receptor co-activation can reduce side-effects without compromising analgesia; this is particularly true for mu and NOP co-activation. Recent pharmaceutical development has produced a mixed opioid/NOP agonist, cebranopadol. This new chemical entity is effective in animal models of nociceptive and neuropathic pain with greater efficacy in the latter. In animal models, there is little evidence for respiratory depression, and tolerance (compared with morphine) only develops after long treatment periods. There is now early phase clinical development in diabetic neuropathy, cancer pain, and low back pain where cebranopadol displays significant efficacy. In 1996, N/OFQ was formally identified with an innovative analgesic profile. Approximately 20 yr later, cebranopadol as a clinical ligand is advancing through the human trials process.

Detection of iron atoms by emission spectroscopy and laser-induced fluorescence in solid propellant flames.

Planar laser-induced fluorescence on atomic iron is investigated in this paper, and a measurement strategy is proposed to monitor the fluorescence of iron atoms with good sensitivity. A model is proposed to fit the experimental fluorescence spectra, and good agreement is found between simulated and experimental spectra. Emission and laser-induced fluorescence measurements are performed in the flames of ammonium perchlorate composite propellants containing iron-based catalysts. A fluorescence signal from iron atoms after excitation at 248 nm is observed for the first time in propellant flames. Images of the spatial distribution of iron atoms are recorded in the flame in which turbulent structures are generated. Iron fluorescence is detected up to 1.0 MPa, which opens the way to application in propellant combustion.

Cancer-associated fibroblasts promote bone invasion in oral squamous cell carcinoma.

BACKGROUND: The molecular mechanisms involved in the invasion of bone by oral squamous cell carcinomas (OSCC) are poorly understood, and little is known about the role of cancer-associated fibroblasts (CAF), the presence of which confers a poor prognosis. METHODS: Clinicopathological data from 277 OSCC cases involving bone resections were reviewed, and 32 cases thoroughly analysed histologically. Immunohistochemistry was used to examine αSMA, RANKL and OPG. Western blotting and qPCR were used to assess myofibroblast (CAF-like) differentiation, RANKL and OPG expression in vitro, and RANKL secretion was analysed by ELISA. Osteoclastogenesis was examined using TRAP staining, multinucleation and pit forming assays. RESULTS: Fibrous stroma intervened between tumour and bone in the majority of cases, with no direct contact between cancer cells and bone. RANKL and OPG, two proteins key to regulating bone resorption, were expressed in tumour cells as well as fibrous stroma adjacent to bone and αSMA-positive myofibroblastic CAF were consistently seen infiltrating into bone ahead of tumour cells. Human primary osteoblasts cultured with conditioned media from human OSCC-derived cells and human primary CAF showed a significant increase in RANKL and a decline in OPG mRNA expression. RANKL secretion was significantly increased in primary oral fibroblasts induced to differentiate into a CAF-like phenotype by transforming growth factor-ß1 (TGF-ß1) treatment and in primary CAF. Indirect co-culture of murine macrophages with conditioned media from CAF (experimentally derived and isolated from OSCCs) resulted in a marked increase in osteoclastogenesis (in excess of that provoked by cancer cells) determined by tartrate-resistant acid phosphatase activity, multinucleation and resorption pit formation. CONCLUSIONS: This study is the first to describe a functional role for CAFs in bone invasion and turnover, identifying a novel potential therapeutic target and diagnostic indicator in this difficult to treat bone invasive malignancy.

Fibroblast activation and senescence in oral cancer.

There is now compelling evidence that the tumour stroma plays an important role in the pathogenesis of cancers of epithelial origin. The pre-eminent cell type of the stroma is carcinoma-associated fibroblasts. These cells demonstrate remarkable heterogeneity with activation and senescence being common stress responses. In this review, we summarise the part that these cells play in cancer, particularly oral cancer, and present evidence to show that activation and senescence reflect a unified programme of fibroblast differentiation. We report advances concerning the senescent fibroblast metabolome, mechanisms of gene regulation in these cells and ways in which epithelial cell adhesion is dysregulated by the fibroblast secretome. We suggest that the identification of fibroblast stress responses may be a valuable diagnostic tool in the determination of tumour behaviour and patient outcome. Further, the fact that stromal fibroblasts are a genetically stable diploid cell population suggests that they may be ideal therapeutic targets and early work in this context is encouraging.

Angiotensin 1-7 inhibits angiotensin II-stimulated head and neck cancer progression.

Angiotensin II (Ang II) is the product of the proteolytic action of angiotensin-converting enzyme (ACE) on the precursor peptide, angiotensin I (Ang I). In addition to its vasoactive properties, Ang II is able to stimulate angiogenesis and act as a mitogen, promoting cellular proliferation. Recently, evidence has emerged that Ang II is also able to promote tumour invasion, a key step in the metastatic cascade, although the mechanisms by which it does so remain largely obscure. Here we show that Ang II is able to promote the invasion and migration of head and neck squamous cell carcinoma (HNSCC) cells both in an autocrine manner and by triggering stromal tumour-paracrine interactions. The effects of Ang II on autocrine and paracrine signalling pathways are mediated by angiotensin receptor 1 (AT1 R) and inhibited by angiotensin 1-7 (Ang 1-7), a peptide produced from Ang II by the action of angiotensin-converting enzyme 2 (ACE2). These data are the first to demonstrate a role for the renin-angiotensin system in oral carcinogenesis and raise the possibility of utilizing AT1 R receptor antagonists and/or Ang 1-7 as novel therapeutic agents for HNSCC.

Evidence for nociceptin/orphanin FQ (NOP) but not µ (MOP), δ (DOP) or κ (KOP) opioid receptor mRNA in whole human blood.

BACKGROUND: While it is well known that opioids depress the immune system, the site(s) of action for this depression is highly controversial. Immune modulation could occur directly at the immune cell or centrally via the hypothalamic-pituitary-adrenal axis. In a number of studies using individual enriched immune cell populations we have failed to detect classical µ (MOP), δ (DOP) and κ (KOP) receptors. The non-classical nociceptin/orphanin FQ (N/OFQ) receptor (NOP) is expressed on all cells examined thus far. Our hypothesis was that immune cells do not express classical opioid receptors and that using whole blood would definitively answer this question. METHODS: Whole blood (containing all immune cell types) was incubated with opioids (morphine and fentanyl) commonly encountered in anaesthesia and with agents mimicking sepsis [lipopolysaccharide (LPS) and peptidoglycan G (PepG)]. Opioid receptor mRNA expression was assessed by endpoint polymerase chain reaction (PCR) with gel visualisation and quantitative PCR. RESULTS: Classical MOP, DOP, and KOP receptors were not detected in any of the samples tested either at rest or when challenged with opioids, LPS or PepG. Commercial primers for DOP did not perform well in quantitative PCR, so the absence of expression was confirmed using a traditional gel-based approach. NOP receptors were detected in all samples; expression was unaffected by opioids and reduced by LPS/PepG combinations. CONCLUSIONS: Classical opioid receptors are not expressed on circulating immune cells.

End-tidal carbon monoxide as an indicator of the hemolytic rate.

In the first days of life, low grade jaundice is essentially universal. The source of the elevated bilirubin level giving rise to "physiological jaundice of the newborn" is only partly known. We hypothesized that it is, at least in part, the result of active and specific hemolysis involving a physiological mechanism to lower the high fetal hematocrit, appropriate for the relatively low oxygen environment in utero, to a lower level appropriate for the state of oxygen abundance after birth. We tested this by quantifying end tidal carbon monoxide (ETCO) as a marker of the rate of heme metabolism to bilirubin. We found that ETCO values of 20 neonates and children with known hemolytic disorders were higher than 20 age-matched healthy controls (p<0.0001), indicating that this instrumentation recognizes hemolysis in neonates and children. We also found that ETCO reference intervals were indeed higher in healthy neonates during the first three days after birth (5th to 95th percentile reference range, 1.4 to 1.7ppm) than after 1month of age (all ≤1.0ppm, p<0.0001). These results suggest to us that hemolysis is physiological during the first days after birth. The cellular and molecular mechanisms responsible for transient hemolysis after birth are topics of current investigation.

Clostridium botulinum Group II Isolate Phylogenomic Profiling Using Whole-Genome Sequence Data.

Clostridium botulinum group II isolates (n = 163) from different geographic regions, outbreaks, and neurotoxin types and subtypes were characterized in silico using whole-genome sequence data. Two clusters representing a variety of botulinum neurotoxin (BoNT) types and subtypes were identified by multilocus sequence typing (MLST) and core single nucleotide polymorphism (SNP) analysis. While one cluster included BoNT/B4/F6/E9 and nontoxigenic members, the other comprised a wide variety of different BoNT/E subtype isolates and a nontoxigenic strain. In silico MLST and core SNP methods were consistent in terms of clade-level isolate classification; however, core SNP analysis showed higher resolution capability. Furthermore, core SNP analysis correctly distinguished isolates by outbreak and location. This study illustrated the utility of next-generation sequence-based typing approaches for isolate characterization and source attribution and identified discrete SNP loci and MLST alleles for isolate comparison.

Development and characterisation of novel fentanyl-delta opioid receptor antagonist based bivalent ligands.

BACKGROUND: Opioid tolerance is a limiting factor in chronic pain. Delta opioid peptide (DOP)(δ) receptor antagonism has been shown to reduce tolerance. Here, the common clinical mu opioid peptide (MOP)(µ) receptor agonist fentanyl has been linked to the DOP antagonist Dmt-Tic (2',6'-dimethyl-L-tyrosyl-1,2,3,4-tetrahydrisoquinoline-3-carboxylic acid) to create new bivalent compounds. METHODS: Binding affinities of bivalents(#9, #10, #11, #12 and #13) were measured in Chinese hamster ovary (CHO) cells expressing recombinant human MOP, DOP, Kappa opioid peptide (KOP)(κ) and nociceptin/orphanin FQ opioid peptide (NOP) receptors. Functional studies, measuring GTPγ[(35)S] or ß-arrestin recruitment, were performed in membranes or whole cells respectively expressing MOP and DOP. RESULTS: The new bivalents bound to MOP (pKi : #9:7.31; #10:7.58; #11:7.91; #12:7.94; #13:8.03) and DOP (#9:8.03; #10:8.16; #11:8.17; #12:9.67; #13:9.71). In GTPγ[(35)S] functional assays, compounds #9(pEC50:6.74; intrinsic activity:0.05) #10(7.13;0.34) and #11(7.52;0.27) showed weak partial agonist activity at MOP. Compounds #12 and #13, with longer linkers, showed no functional activity at MOP. In antagonist assays at MOP, compounds #9 (pKb:6.87), #10(7.55) #11(7.81) #12(6.91) and #13(7.05) all reversed the effects of fentanyl. At DOP, all compounds showed antagonist affinity (#9:6.85; #10:8.06; #11:8.11; #12:9.42; #13:9.00), reversing the effects of DPDPE ([D-Pen(2,5)]enkephalin). In ß-arrestin assays, compared with fentanyl (with response at maximum concentration (RMC):13.62), all compounds showed reduced ability to activate ß-arrestin (#9 RMC:1.58; #10:2.72; #11:2.40; #12:1.29; #13:1.58). Compared with fentanyl, the intrinsic activity was: #9:0.12; #10:0.20; #11:0.18; #12:0.09 and #13:0.12. CONCLUSIONS: The addition of a linker between fentanyl and Dmt-Tic did not alter the ability to bind to MOP and DOP, however a substantial loss in MOP functional activity was apparent. This highlights the difficulty in multifunctional opioid development.

The roles of HOXD10 in the development and progression of head and neck squamous cell carcinoma (HNSCC).

BACKGROUND: HOX gene expression is altered in many cancers; previous microarray revealed changes in HOX gene expression in head and neck squamous cell carcinoma (HNSCC), particularly HOXD10. METHODS: HOXD10 expression was assessed by qPCR and immunoblotting in vitro and by immunohistochemistry (IHC) in tissues. Low-expressing cells were stably transfected with HOXD10 and the phenotype assessed with MTS, migration and adhesion assays and compared with the effects of siRNA knockdown in high-HOXD10-expressing cells. Novel HOXD10 targets were identified using expression microarrays, confirmed by reporter assay, and validated in tissues using IHC. RESULTS: HOXD10 expression was low in NOKs, high in most primary tumour cells, and low in lymph node metastasis cells, a pattern confirmed using IHC in tissues. Overexpression of HOXD10 decreased cell invasion but increased proliferation, adhesion and migration, with knockdown causing reciprocal effects. There was no consistent effect on apoptosis. Microarray analysis identified several putative HOXD10-responsive genes, including angiomotin (AMOT-p80) and miR-146a. These were confirmed as HOXD10 targets by reporter assay. Manipulation of AMOT-p80 expression resulted in phenotypic changes similar to those on manipulation of HOXD10 expression. CONCLUSIONS: HOXD10 expression varies by stage of disease and produces differential effects: high expression giving cancer cells a proliferative and migratory advantage, and low expression may support invasion/metastasis, in part, by modulating AMOT-p80 levels.

Two novel toxin variants revealed by whole-genome sequencing of 175 Clostridium botulinum type E strains.

We sequenced 175 Clostridium botulinum type E strains isolated from food, clinical, and environmental sources from northern Canada and analyzed their botulinum neurotoxin (bont) coding sequences (CDSs). In addition to bont/E1 and bont/E3 variant types, neurotoxin sequence analysis identified two novel BoNT type E variants termed E10 and E11. Strains producing type E10 were found along the eastern coastlines of Hudson Bay and the shores of Ungava Bay, while strains producing type E11 were only found in the Koksoak River region of Nunavik. Strains producing BoNT/E3 were widespread throughout northern Canada, with the exception of the coast of eastern Hudson Bay.