Efficacy of Topical Vancomycin- and Gentamicin-Loaded Calcium Sulfate Beads or Systemic Antibiotics in Eradicating Polymicrobial Biofilms Isolated from Diabetic Foot Infections within an In Vitro Wound Model.

Diabetic foot ulcers are notoriously difficult to heal, with ulcers often becoming chronic, in many cases leading to amputation despite weeks or months of antibiotic therapy in addition to debridement and offloading. Alternative wound biofilm management options, such as topical rather than systemic delivery of antimicrobials, have been investigated by clinicians in order to improve treatment outcomes. Here, we collected blood and tissue from six subjects with diabetic foot infections, measured the concentrations of antibiotics in the samples after treatment, and compared the microbiota within the tissue before treatment and after 7 days of antibiotic therapy. We used an in vitro model of polymicrobial biofilm infection inoculated with isolates from the tissue we collected to simulate different methods of antibiotic administration by simulated systemic therapy or topical release from calcium sulfate beads. We saw no difference in biofilm bioburden in the models after simulated systemic therapy (representative of antibiotics used in the clinic), but we did see reductions in bioburden of between 5 and 8 logs in five of the six biofilms that we tested with topical release of antibiotics via calcium sulfate beads. Yeast is insensitive to antibiotics and was a component of the sixth biofilm. These data support further studies of the topical release of antibiotics from calcium sulfate beads in diabetic foot infections to combat the aggregate issues of infectious organisms taking the biofilm mode of growth, compromised immune involvement, and poor systemic delivery of antibiotics via the bloodstream to the site of infection in patients with diabetes.

Antibody-mediated protection against MERS-CoV in the murine model.

Murine antisera with neutralising activity for the coronavirus causative of Middle East respiratory syndrome (MERS) were induced by immunisation of Balb/c mice with the receptor binding domain (RBD) of the viral Spike protein. The murine antisera induced were fully-neutralising in vitro for two separate clinical strains of the MERS coronavirus (MERS-CoV). To test the neutralising capacity of these antisera in vivo, susceptibility to MERS-CoV was induced in naive recipient Balb/c mice by the administration of an adenovirus vector expressing the human DPP4 receptor (Ad5-hDPP4) for MERS-CoV, prior to the passive transfer of the RBD-specific murine antisera to the transduced mice. Subsequent challenge of the recipient transduced mice by the intra-nasal route with a clinical isolate of the MERS-CoV resulted in a significantly reduced viral load in their lungs, compared with transduced mice receiving a negative control antibody. The murine antisera used were derived from mice which had been primed sub-cutaneously with a recombinant fusion of RBD with a human IgG Fc tag (RBD-Fc), adsorbed to calcium phosphate microcrystals and then boosted by the oral route with the same fusion protein in reverse micelles. The data gained indicate that this dual-route vaccination with novel formulations of the RBD-Fc, induced systemic and mucosal anti-viral immunity with demonstrated in vitro and in vivo neutralisation capacity for clinical strains of MERS-CoV.

Dual route vaccination for plague with emergency use applications.

Here, we report a dual-route vaccination approach for plague, able to induce a rapid response involving systemic and mucosal immunity, whilst also providing ease of use in those resource-poor settings most vulnerable to disease outbreaks. This novel vaccine (VypVaxDuo) comprises the recombinant F1 and V proteins in free association. VypVaxDuo has been designed for administration via a sub-cutaneous priming dose followed by a single oral booster dose and has been demonstrated to induce early onset immunity 14 days after the primary immunisation; full protective efficacy against live organism challenge was achieved in Balb/c mice exposed to 2 × 104 median lethal doses of Yersinia pestis Co92, by the sub-cutaneous route at 25 days after the oral booster immunisation. This dual-route vaccination effectively induced serum IgG and serum and faecal IgA, specific for F1 and V, which constitute two key virulence factors in Y. pestis, and is therefore suitable for further development to prevent bubonic plague and for evaluation in models of pneumonic plague. This is an essential requirement for control of disease outbreaks in areas of the world endemic for plague and is supported further by the observed exceptional stability of the primary vaccine formulation in vialled form under thermostressed conditions (40 °C for 29 weeks, and 40 °C with 75% relative humidity for 6 weeks), meaning no cold chain for storage or distribution is needed. In clinical use, the injected priming dose would be administered on simple rehydration of the dry powder by means of a dual barrel syringe, with the subsequent single booster dose being provided in an enteric-coated capsule suitable for oral self-administration.

Galaxy growth in a massive halo in the first billion years of cosmic history.

According to the current understanding of cosmic structure formation, the precursors of the most massive structures in the Universe began to form shortly after the Big Bang, in regions corresponding to the largest fluctuations in the cosmic density field. Observing these structures during their period of active growth and assembly-the first few hundred million years of the Universe-is challenging because it requires surveys that are sensitive enough to detect the distant galaxies that act as signposts for these structures and wide enough to capture the rarest objects. As a result, very few such objects have been detected so far. Here we report observations of a far-infrared-luminous object at redshift 6.900 (less than 800 million years after the Big Bang) that was discovered in a wide-field survey. High-resolution imaging shows it to be a pair of extremely massive star-forming galaxies. The larger is forming stars at a rate of 2,900 solar masses per year, contains 270 billion solar masses of gas and 2.5 billion solar masses of dust, and is more massive than any other known object at a redshift of more than 6. Its rapid star formation is probably triggered by its companion galaxy at a projected separation of 8 kiloparsecs. This merging companion hosts 35 billion solar masses of stars and has a star-formation rate of 540 solar masses per year, but has an order of magnitude less gas and dust than its neighbour and physical conditions akin to those observed in lower-metallicity galaxies in the nearby Universe. These objects suggest the presence of a dark-matter halo with a mass of more than 100 billion solar masses, making it among the rarest dark-matter haloes that should exist in the Universe at this epoch.

Feeding the fire: Tracing the mass-loading of 107 K galactic outflows with O vi absorption.

Galactic outflows regulate the amount of gas galaxies convert into stars. However, it is difficult to measure the mass outflows remove because they span a large range of temperatures and phases. Here, we study the rest-frame ultraviolet spectrum of a lensed galaxy at z ~ 2.9 with prominent interstellar absorption lines from O i, tracing neutral gas, up to O vi, tracing transitional phase gas. The O vi profile mimics weak low-ionization profiles at low velocities, and strong saturated profiles at high velocities. These trends indicate that O vi gas is co-spatial with the low-ionization gas. Further, at velocities blueward of -200 km s-1 the column density of the low-ionization outflow rapidly drops while the O vi column density rises, suggesting that O vi is created as the low-ionization gas is destroyed. Photoionization models do not reproduce the observed O vi, but adequately match the low-ionization gas, indicating that the phases have different formation mechanisms. Photoionized outflows are more massive than O vi outflows for most of the observed velocities, although the O vi mass outflow rate exceeds the photoionized outflow at velocities above the galaxy's escape velocity. Therefore, most gas capable of escaping the galaxy is in a hot outflow phase. We suggest that the O vi absorption is a temporary by-product of conduction transferring mass from the photoionized phase to an unobserved hot wind, and discuss how this mass-loading impacts the observed circum-galactic medium.

Adaptation to acetaminophen exposure elicits major changes in expression and distribution of the hepatic proteome.

Acetaminophen overdose is the leading cause of acute liver failure. One dose of 10-15 g causes severe liver damage in humans, whereas repeated exposure to acetaminophen in humans and animal models results in autoprotection. Insight of this process is limited to select proteins implicated in acetaminophen toxicity and cellular defence. Here we investigate hepatic adaptation to acetaminophen toxicity from a whole proteome perspective, using quantitative mass spectrometry. In a rat model, we show the response to acetaminophen involves the expression of 30% of all proteins detected in the liver. Genetic ablation of a master regulator of cellular defence, NFE2L2, has little effect, suggesting redundancy in the regulation of adaptation. We show that adaptation to acetaminophen has a spatial component, involving a shift in regionalisation of CYP2E1, which may prevent toxicity thresholds being reached. These data reveal unexpected complexity and dynamic behaviour in the biological response to drug-induced liver injury.

Health-related quality of life in genotype 1 treatment-naïve chronic hepatitis C patients receiving telaprevir combination treatment in the ADVANCE study.

BACKGROUND: Chronic hepatitis C virus (HCV) infection and its treatment impact patients' health-related quality of life (HRQL). AIM: To report on treatment impact and predictors of HRQL among treatment-naïve patients with genotype 1 chronic HCV infection who received 12-week telaprevir (T) with 24 (T12PR24) or 48 weeks (T12PR48) peginterferon alpha-2a/ribavirin (PR), or 48 weeks of PR in the ADVANCE study. METHODS: The EQ-5D-3L (EQ-5D) questionnaire (index range: 0-1) was completed at baseline and weeks 4, 12, 24, 36, 48 and 72. Patients indicated their health state on five dimensions: mobility, self-care, usual activities, pain/discomfort and anxiety/depression. Descriptive statistics for the EQ-5D index and descriptive system and area under the curve from baseline to week 12 were calculated. Predictors of EQ-5D index were identified using multivariate analyses. RESULTS: Data from 722 patients were included. The mean EQ-5D index decreased during the first 12 weeks and returned to baseline by week 72 (T12PR24 by week 36) across treatments. In multivariate analysis, sustained virological response (SVR) at week 72 was associated (P < 0.0001) with improved EQ-5D index [mean; SVR+ (0.90), SVR- (0.86)], a 4% difference, within the published range of minimal clinically important difference. CONCLUSIONS: Post hoc analyses of data from ADVANCE suggested that HRQL worsened during the first 12 weeks of therapy and returned to baseline by week 72 across treatments. Improvements were observed early following completion of a 24-week treatment (T12PR24). Telaprevir combination therapy was associated with slightly higher reductions in HRQL during the first 12 weeks (vs. PR). SVR was a statistically significant and meaningful predictor of HRQL at week 72.

Dusty starburst galaxies in the early Universe as revealed by gravitational lensing.

In the past decade, our understanding of galaxy evolution has been revolutionized by the discovery that luminous, dusty starburst galaxies were 1,000 times more abundant in the early Universe than at present. It has, however, been difficult to measure the complete redshift distribution of these objects, especially at the highest redshifts (z > 4). Here we report a redshift survey at a wavelength of three millimetres, targeting carbon monoxide line emission from the star-forming molecular gas in the direction of extraordinarily bright millimetre-wave-selected sources. High-resolution imaging demonstrates that these sources are strongly gravitationally lensed by foreground galaxies. We detect spectral lines in 23 out of 26 sources and multiple lines in 12 of those 23 sources, from which we obtain robust, unambiguous redshifts. At least 10 of the sources are found to lie at z > 4, indicating that the fraction of dusty starburst galaxies at high redshifts is greater than previously thought. Models of lens geometries in the sample indicate that the background objects are ultra-luminous infrared galaxies, powered by extreme bursts of star formation.

A conceptual and disease model framework for osteoporotic kyphosis.

UNLABELLED: This paper presents a multi-method research project to develop a conceptual framework for measuring outcomes in studies of osteoporotic kyphosis. The research involved literature research and qualitative interviews among clinicians who treat patients with kyphosis and among patients with the condition. INTRODUCTION: Kyphosis due to at least one vertebral compression fracture is prevalent among osteoporotic patients, resulting in well-documented symptoms and impact on functioning and well-being. A three-part study led to development of a conceptual measurement framework for comprehensive assessment of symptoms, impact, and treatment benefit for kyphosis. METHODS: A literature-based disease model (DM) was developed and tested with physicians (n = 10) and patients (n = 10), and FDA guidelines were used to develop a final disease model and a conceptual framework. RESULTS: The DM included signs, symptoms, causes/triggers, exacerbations, and functional status associated with kyphosis. The DM was largely confirmed, but physicians and patients added several concepts related to impact on functioning, and some concepts were not confirmed and removed from the DM. CONCLUSIONS: This study confirms the need for more comprehensive assessment of health outcomes in kyphosis, as most current studies omit key concepts.

A massive, cooling-flow-induced starburst in the core of a luminous cluster of galaxies.

In the cores of some clusters of galaxies the hot intracluster plasma is dense enough that it should cool radiatively in the cluster's lifetime, leading to continuous 'cooling flows' of gas sinking towards the cluster centre, yet no such cooling flow has been observed. The low observed star-formation rates and cool gas masses for these 'cool-core' clusters suggest that much of the cooling must be offset by feedback to prevent the formation of a runaway cooling flow. Here we report X-ray, optical and infrared observations of the galaxy cluster SPT-CLJ2344-4243 (ref. 11) at redshift z = 0.596. These observations reveal an exceptionally luminous (8.2 × 10(45) erg s(-1)) galaxy cluster that hosts an extremely strong cooling flow (around 3,820 solar masses a year). Further, the central galaxy in this cluster appears to be experiencing a massive starburst (formation of around 740 solar masses a year), which suggests that the feedback source responsible for preventing runaway cooling in nearby cool-core clusters may not yet be fully established in SPT-CLJ2344-4243. This large star-formation rate implies that a significant fraction of the stars in the central galaxy of this cluster may form through accretion of the intracluster medium, rather than (as is currently thought) assembling entirely via mergers.

Paradoxical embolism: a rare complication of thrombolysis.

Paradoxical embolus is a rare complication of thrombolysis. With a high prevalence of clinically silent septal defects and widespread use of thrombolysis, recognition of the risks, clinical signs and symptoms is important. A case is reported of paradoxical embolus following thrombolysis in a woman with a previously undiagnosed patent foramen ovale.

Paradoxical embolism: a rare complication of thrombolysis.

Paradoxical embolus is a rare complication of thrombolysis. With a high prevalence of clinically silent septal defects and widespread use of thrombolysis, recognition of the risks, clinical signs and symptoms is important. A case is reported of paradoxical embolus following thrombolysis in a woman with a previously undiagnosed patent foramen ovale.

The metabolism of the 5HT3 antagonists ondansetron, alosetron and GR87442 I: a comparison of in vitro and in vivo metabolism and in vitro enzyme kinetics in rat, dog and human hepatocytes, microsomes and recombinant human enzymes.

The metabolism of the structurally related 5HT3 antagonists ondansetron, alosetron and GR87442 in the rat, dog and human was determined in hepatocytes, liver microsomes and human recombinant microsomes. The profiles of phase I metabolites were similar in human hepatocytes and microsomes. The metabolites of all three compounds produced in rat, dog and human microsomes and hepatocytes were similar to those seen in vivo, with the major routes of metabolism being N-dealkylation and/or hydroxylation. There was more extensive metabolic processing in hepatocytes than in microsomes; however, sequential metabolism was less extensive in vitro compared with in vivo. The pharmacokinetics of the three 5HT3 antagonists investigated were dominated by CYP3A4 (and/or 2C9) compared with CYP1A2 in man, possibly determined by enzyme capacity rather than relative enzyme affinity. These data support the use of rat, dog and human hepatocytes for the prediction of in vivo metabolites of ondansetron, alosetron and GR87442.

The metabolism of the 5HT3 antagonists, ondansetron, alosetron and GR87442 II: investigation into the in vitro methods used to predict the in vivo hepatic clearance of ondansetron, alosetron and GR87442 in the rat, dog and human.

The in vitro clearances of the 5HT3 antagonists, ondansetron, alosetron and GR87442 were investigated. Intrinsic clearances using either metabolite formation or substrate depletion methods were equivalent (R2 = 0.95). Hepatocytes from preclinical species were superior to microsomes for the prediction of hepatic clearance (CL(H)), whereas the predictions from human microsomes and hepatocytes were similar. Using a non-restrictive model, seven of the nine CL(H) predictions using hepatocytes were within 2-fold of the in vivo CL(H) values. If the unbound fraction was included, the clearance of the compounds was generally under-predicted by both in vitro models. However, for the most metabolically stable compound, GR87442, the non-restrictive model over-predicted CLp. This and the possibility of extrahepatic metabolism indicate that the restrictive model is more appropriate for prediction of CL(H). The rank order of metabolic stability correlated with that in vivo. All three compounds were more metabolically stable in human than in the preclinical animal species examined.

A comparison of the expression and metabolizing activities of phase I and II enzymes in freshly isolated human lung parenchymal cells and cryopreserved human hepatocytes.

The pulmonary and hepatic expression and catalytic activities of phase I and II drug-metabolizing enzymes were compared using human lung and liver tissue, and lung parenchymal cells (LPCs) and cryopreserved hepatocytes. Cytochrome P450 gene expression was generally lower in lung than in liver and CYP3A4 expression in lung was negligible. Esterase gene expression was similar in lung and liver. Expression of all sulfotransferase isoforms in lung was similar to or higher than that in liver. Lung tissue expressed low levels of UGT. However, the expression of UGT2A1 in lung was higher than that in liver. There was a range of catalytic activities in LPCs, including cytochrome P450, esterase, and sulfation pathways. Phase I activities were generally less than 10% of those determined in hepatocytes. Rates of ester hydrolysis and sulfation in LPCs were similar to those in hepatocytes. When measurable, glucuronidation in LPCs was present at very low levels, reflecting the gene expression data. The metabolism of salbutamol, formoterol, and budesonide was also investigated. Production of salbutamol-4-O-sulfate and budesonide oleate was observed in LPCs from at least two of three donor preparations studied. Formoterol sulfate and low levels of formoterol glucuronide were detected in one of three donors. In general, drug-metabolizing capability of LPCs is low compared with liver, although some evidence for substantial sulfation and deesterification capacity was observed. Therefore, these data support the use of this cell-based system for the investigation of key routes of xenobiotic metabolism in human lung parenchyma.

A photometric redshift of z = 6.39 +/- 0.12 for GRB 050904.

Gamma-ray bursts (GRBs) and their afterglows are the most brilliant transient events in the Universe. Both the bursts themselves and their afterglows have been predicted to be visible out to redshifts of z approximately 20, and therefore to be powerful probes of the early Universe. The burst GRB 000131, at z = 4.50, was hitherto the most distant such event identified. Here we report the discovery of the bright near-infrared afterglow of GRB 050904 (ref. 4). From our measurements of the near-infrared afterglow, and our failure to detect the optical afterglow, we determine the photometric redshift of the burst to be z = 6.39 - 0.12 + 0.11 (refs 5-7). Subsequently, it was measured spectroscopically to be z = 6.29 +/- 0.01, in agreement with our photometric estimate. These results demonstrate that GRBs can be used to trace the star formation, metallicity, and reionization histories of the early Universe.

TNFalpha-stimulated gene product (TSG-6) and its binding protein, IalphaI, in the human intervertebral disc: new molecules for the disc.

Inflammation and irritation of the nerve roots has been indicated as an important factor in the pain associated with symptomatic disc herniations. Tumour necrosis factor alpha (TNFalpha) is now believed to be involved in this pathway. TNFalpha causes connective tissue cells in culture to synthesise a glycoprotein, TNFalpha-stimulated gene-6 (TSG-6). TSG-6 is found in inflammatory diseases of related connective tissues, such as articular cartilage in rheumatoid arthritis, but is not present in unaffected individuals. In order to determine if TSG-6 occurred in intervertebral disc (and cartilage endplate), we have investigated the presence of TSG-6 and its binding protein, inter-alpha-inhibitor (IalphaI), in 58 herniated and 15 non-herniated discs. Immunostaining for the cytokines, IL-1alpha, IL-1beta and TNFalpha, has also been carried out. We have demonstrated that both TSG-6 and IalphaI occur commonly in human intervertebral disc matrix with at least some TSG-6 in 98% of discs studied and IalphaI in all of them. Staining for TSG-6 was greatest in herniated discs, particularly close to blood vessels. IalphaI immunostaining was frequently widespread throughout the disc but there was little in the cartilage endplate. It has been proposed that these molecules have widespread effects, including extracellular matrix stabilisation, down-regulation of the protease network and reduction of inflammation. Hence, the occurrence of TSG-6 and IalphaI in disc tissue could have implications in the aetiopathogenesis and future therapeutics of intervertebral disc disease.

Effect of matrix depleting agents on the expression of chondrocyte metabolism by equine chondrocytes.

This study was carried out to investigate the effect of two enzymes (collagenase and chondroitinase) and two cytokines/metabolites (interleukin-1beta and retinoic acid) of known catabolic activity on the expression of cartilage metabolism/phenotype in equine articular cartilage. Articular cartilage explants from 11 horses (5-13 years old) were treated for 48 h and assayed for total sulphated glycosaminoglycan (GAG), the incorporation of 35S-sulphate, collagen degradation and mRNA expression of the proteoglycans collagen II, collagen IIA, collagen III, collagen IX, collagen X, collagen XI and glyceraldehyde-3-phosphate (GAPDH). Purified collagenase and retinoic acid were responsible for increased GAG loss from the tissues. Chondroitinase, responsible for catalysing the elimination of glucuronate residues from chondroitin A, B and C (Chondroitinase ABC) and retinoic acid treatment induced an inhibition of proteoglycan synthesis, whereas collagenase treatment did not. Collagenase activity was correlated with increased appearance of the CB11B epitope and type II collagen denaturation. By RT-PCR there was evidence of expression of altered collagen type IIA in purified collagenase treated tissues.

The use of chondrogide membrane in autologous chondrocyte implantation.

UNLABELLED: Autologous chondrocyte implantation is a new technique for the treatment of chondral defects in the knee. The exact procedure involved is continuously being developed with the ultimate aim of achieving hyaline cartilage regeneration. We present the outcome of our series of 31 patients, focussing on the use of the chondrogide membrane in the implantation process. Assessment is presented both in the form of arthroscopic appearance at approximately 1 year, and in the form of clinical outcome measures at 1 year and at 2 years after the second stage of the procedure. CONCLUSION: the use of chondrogide membrane in the fixation of cells during the implantation process is associated with satisfactory clinical outcome and does not appear to show evidence of hypertrophy at one-year arthroscopy, as compared to periosteum.

A study of the feasibility of Internet administration of a computerized health survey: the headache impact test (HIT).

BACKGROUND: Headache impact test (HIT) is a precise, practical tool that quantifies the impact of headache on respondents' lives. It is the first widely-available dynamic health assessment (DynHA). Applications of this brief, precise survey include population based screening for disabling headaches, tracking of individual patient scores over time, disease management programs and others. We use data from Internet HIT assessments during the fall of 2000 to (1) evaluate characteristics of respondents and assessments, (2) assess the utility of joint administration of HIT and the SF-8 Health Survey (SF-8) to screen for migraine and depression, and (3) explore associations between HIT scores and subsequent healthcare-related attitudes and behaviors. METHODS: We analyzed Internet HIT surveys completed between 9/1 and 11/30/2000 (n = 19,195). Subsamples include respondents who also completed (1) a 12-item Internet survey assessing severity, frequency, cause and management of headaches; (2) an e-mail survey measuring healthcare-related behaviors; (3) the SF-8; or (4) the website registration process, providing age and gender data. We used analysis of variance (ANOVA) to evaluate HIT score differences associated with age, gender, headache severity or frequency, and healthcare-related behaviors and attitudes and chi2 tests to assess the prevalence and comorbidity of migraine and depression. RESULTS: Three-quarters of respondents achieved a precise HIT score in < or = 5 items. Most had moderate/severe headaches; 65% had headaches at least monthly. HIT scores were directly related to headache severity and frequency. Most respondents were females, with significantly higher HIT scores than males. Most HIT respondents were between ages 25 and 54 (HIT scores were higher for younger respondents). Sixty four percent screened positive for migraine; 20% for depression. Both conditions were more prevalent among females than males. Comorbid migraine and depression was 50% more prevalent among females and increased with age until age 50. Patients with worse headache impact were more likely to seek care, discuss headaches with their providers and find HIT useful. CONCLUSIONS: It is feasible to use Internet-based dynamic assessments to measure health status. These data complement previous results showing that HIT differentiates respondents according to headache characteristics (severity and frequency). HIT plus SF-8 yields a practical screen for migraine and depression in headache patients and may lead to more effective treatment for patients with these conditions. Preliminary findings suggest that the experience of taking HIT on the Internet may motivate headache patients to seek care and discuss headaches with their providers.