Comparative characterisation of extracellular vesicles from canine and human plasma: a necessary step in biomarker discovery.

Extracellular Vesicles (EV) have become an interesting focus as novel biomarkers of disease and are increasingly reported upon in humans and other species. The Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV2018) guidelines were published to improve rigor and standardisation within the EV field and provide a framework for the reliable isolation and characterisation of EV populations. However, this rigor and standardisation has been challenging in the area of comparative medicine. Herein we present the successful isolation of EVs from human and canine plasma using Size Exclusion Chromatography and characterise these EVs according to best international practice. This study provides evidence for the reliable comparison of human and canine EVs isolated by this approach, and a baseline description of the EVs from healthy dogs to inform future biomarker studies. This work also demonstrates that the MISEV2018 guidelines can be successfully applied to EVs isolated from canine plasma.

What Can Proton Beam Therapy Achieve for Patients with Pectus Excavatum Requiring Left Breast, Axilla and Internal Mammary Nodal Radiotherapy?

AIMS: Exposure of the heart to radiation increases the risk of ischaemic heart disease, proportionate to the mean heart dose (MHD). Radiotherapy techniques including proton beam therapy (PBT) can reduce MHD. The aims of this study were to quantify the MHD reduction achievable by PBT compared with volumetric modulated arc therapy in breath hold (VMAT-BH) in patients with pectus excavatum (PEx), to identify an anatomical metric from a computed tomography scan that might indicate which patients will achieve the greatest MHD reductions from PBT. MATERIALS AND METHODS: Sixteen patients with PEx (Haller Index ≥2.7) were identified from radiotherapy planning computed tomography images. Left breast/chest wall, axilla (I-IV) and internal mammary node (IMN) volumes were delineated. VMAT and PBT plans were prepared, all satisfying target coverage constraints. Signed-rank comparisons of techniques were undertaken for the mean dose to the heart, ipsilateral lung and contralateral breast. Spearman's rho correlations were calculated for anatomical metrics against MHD reduction achieved by PBT. RESULTS: The mean MHD for VMAT-BH plans was 4.1 Gy compared with 0.7 Gy for PBT plans. PBT reduced MHD by an average of 3.4 Gy (range 2.8-4.4 Gy) compared with VMAT-BH (P < 0.001). PBT significantly reduced the mean dose to the ipsilateral lung (4.7 Gy, P < 0.001) and contralateral breast (2.7 Gy, P < 0.001). The distance (mm) at the most inferomedial extent of IMN volume (IMN to heart distance) negatively correlated with MHD reduction achieved by PBT (Spearman's rho -0.88 (95% confidence interval -0.96 to -0.67, P < 0.001)). CONCLUSION: For patients with PEx requiring left-sided breast and IMN radiotherapy, a clinically significant MHD reduction is achievable using PBT, compared with the optimal photon technique (VMAT-BH). This is a patient group in whom PBT could have the greatest benefit.

Reducing the Risk of Death From Pneumocystis jirovecii Pneumonia After Radical Radiation Therapy to the Lung.

AIMS: Lung cancer is the leading cause of cancer death. Radiotherapy given in the curative setting is associated with a 3% risk of death from Pneumocystis jirovecii pneumonia (PJP). Prolonged courses of high-dose steroids also increase the risk of PJP. International guidelines recommend the use of chemoprophylaxis with trimethoprim-sulfamethoxazole for patients at high risk. We assessed the effect of an intervention designed to reduce the impact of PJP. MATERIALS AND METHODS: Prophylaxis guidelines were introduced in 2016. Case records of patients treated with radical radiotherapy were examined for the periods 2014 to 2015 (pre-intervention) and 2017 to 2018 (post-intervention). In total, 247 patients were treated pre-intervention and 334 post-intervention. RESULTS: Freedom from PJP death at 1 year was 96% before intervention and 99% after (hazard ratio 0.3, 95% confidence interval 0.1-0.9, P = 0.029). Although the rate of use of chemoprophylaxis according to the guideline rose from 1% to 13% (P = 0.003), the use of high-dose steroids also fell from 35% to 16% (P < 0.00001). CONCLUSIONS: Reducing radiotherapy-associated infections is an important component of radical treatment in lung cancer. Highlighting chemoprophylaxis guidelines reduced the death rate from PJP, with an associated more judicious use of steroids. Advocating prophylaxis in patients with lymphocyte count <0.6 × 109/l is the next intervention to be studied.

The effect of the native kinematics of the knee on the outcome following total knee arthroplasty.

AIMS: The aim of this study was to investigate differences in pain, range of movement function and satisfaction at three months and one year after total knee arthroplasty (TKA) in patients with an oblique pattern of kinematic graph of the knee and those with a varus pattern. PATIENTS AND METHODS: A total of 91 patients who underwent TKA were included in this retrospective study. Patients (59 women and 32 men with mean age of 68.7 years; 38.6 to 88.4) were grouped according to kinematic graphs which were generated during navigated TKA and the outcomes between the groups were compared. RESULTS: The graphs were varus in 50 patients (55%), oblique in 19 (21%), neutral in 17 (18.5%) and valgus in five (5.5%). After adjustment for pre-operative scores and gender, compared with patients with varus knee kinematics, patients with an oblique kinematic graph had a poorer outcome with lower Knee Society scores at three months (9.2 points, p = 0.038). CONCLUSION: We found four distinct kinematic graphs in knees and that patients with an oblique graph have a poorer outcome in the short-term after TKA. Cite this article: Bone Joint J 2016;98-B:1471-8.

Effects of acute and chronic sunitinib treatment on cardiac function and calcium/calmodulin-dependent protein kinase II.

BACKGROUND AND PURPOSE: Calcium/calmodulin-dependent protein kinase IIδ (CaMKIIδ) is an important regulator of cardiac contractile function and dysfunction and may be an unwanted secondary target for anti-cancer drugs such as sunitinib and imatinib that have been reported to alter cardiac performance. This study aimed to determine whether anti-cancer kinase inhibitors may affect CaMKII activity and expression when administered in vivo. EXPERIMENTAL APPROACH: Cardiovascular haemodynamics in response to acute and chronic sunitinib treatment, and chronic imatinib treatment, were assessed in guinea pigs and the effects compared with those of the known positive and negative inotropes, isoprenaline and verapamil. Parallel studies from the same animals assessed CaMKIIδ expression and CaMKII activity following drug treatments. KEY RESULTS: Acute administration of sunitinib decreased left ventricular (LV) dP/dtmax. Acute administration of isoprenaline increased LVdP/dtmax dose-dependently, while LVdP/dtmax was decreased by verapamil. CaMKII activity was decreased by acute administration of sunitinib and was increased by acute administration of isoprenaline, and decreased by acute administration of verapamil. CaMKIIδ expression following all acute treatments remained unchanged. Chronic imatinib and sunitinib treatments did not alter fractional shortening; however, both CaMKIIδ expression and CaMKII activity were significantly increased. Chronic administration of isoprenaline and verapamil decreased LV fractional shortening with parallel increases in CaMKIIδ expression and CaMKII activity. CONCLUSIONS AND IMPLICATIONS: Chronic sunitinib and imatinib treatment increased CaMKIIδ expression and CaMKII activity. As these compounds are associated with cardiac dysfunction, increased CaMKII expression could be an early indication of cellular cardiotoxicity marking potential progression of cardiac contractile dysfunction.

First case of Chlorella wound infection in a human in Australia.

A 30-year-old man developed an infected knee wound 2 days after jumping his bicycle into a freshwater dam. He required repeated debridement and tissue grew bright green colonies typical of the alga Chlorella plus Aeromonas hydrophila. This, and one previously reported case, responded to surgical debridement and careful wound management.

Substandard and falsified anti-tuberculosis drugs: a preliminary field analysis.

SETTING: Pharmacies in 19 cities in Angola, Brazil, China, Democratic Republic of Congo, Egypt, Ethiopia, Ghana, India (n = 3), Kenya, Nigeria, Russia, Rwanda, Thailand, Turkey, Uganda, United Republic of Tanzania and Zambia. OBJECTIVE: To assess the quality of the two main first-line anti-tuberculosis medicines, isoniazid and rifampicin, procured from private-sector pharmacies, to determine if substandard and falsified medicines are available and if they potentially contribute to drug resistance in cities in low- and middle-income countries. DESIGN: Local nationals procured 713 treatment packs from a selection of pharmacies in 19 cities. These samples were tested for quality using 1) thin-layer chromatography to analyze levels of active pharmaceutical ingredient (API), and 2) disintegration testing. RESULTS: Of 713 samples tested, 9.1% failed basic quality testing for requisite levels of API or disintegration. The failure rate was 16.6% in Africa, 10.1% in India, and 3.9% in other middle-income countries. CONCLUSIONS: Substandard and falsified drugs are readily available in the private marketplace and probably contribute to anti-tuberculosis drug resistance in low- and middle-income countries. This issue warrants further investigation through large-scale studies of drug quality in all markets.

Combined pedunculopontine-subthalamic stimulation in Parkinson disease.

OBJECTIVE: To assess the effect of deep brain stimulation (DBS) in the pedunculopontine nucleus (PPN) and caudal zona incerta (cZi)-both separately and in combination-on motor symptoms and regional cerebral blood flow (rCBF) in patients with Parkinson disease (PD). METHODS: Four patients with bilateral cZi and PPN DBS electrodes were rated with the Unified Parkinson's Disease Rating Scale motor subscale (UPDRS-III) when taking and withdrawn from medication. A block of 16 [(15)O]-H(2)O PET resting measurements of rCBF were performed in 4 different states with patients withdrawn from medication: 1) no stimulation, 2) cZi stimulation alone, 3) PPN stimulation alone, 4) combined PPN/cZi stimulation. RESULTS: When patients were medicated, combined PPN/cZi stimulation produced a statistically significant improvement in UPDRS-III score compared to cZi stimulation alone. In the "off" medication state, the clinical effect of combined stimulation was not significantly different from that induced by cZi stimulation alone. Concomitant PPN/cZi stimulation had a cumulative effect on levels of rCBF, effectively combining subcortical and cortical changes induced by stimulation of either target in isolation. CONCLUSIONS: These findings suggest that concomitant low frequency stimulation of PPN and cZi regions induces additive brain activation changes and provides improved control of PD symptoms when medicated. CLASSIFICATION OF EVIDENCE: This study provides Class IV evidence that concomitant low frequency stimulation of PPN and cZI improves motor symptoms in patients with PD on dopamine replacement. It provides Class III evidence that concomitant low frequency stimulation of PPN and cZi induces additive rCBF changes in motor areas of brain.

Parental attributions of controllability as a moderator of the relationship between developmental disability and behaviour problems.

BACKGROUND: Children with developmental disabilities present behaviour problems to a greater extent than do typically developing children. Psychosocial models of child development suggest that parental attributions of child and adult controllability could moderate this relationship between child disability status and behaviour. METHODS: The influence of parental attributions of adult and child controllability on the relationship between problem behaviours and disability was explored in mothers of children with developmental disabilities (DD) (N = 20) with a mean age of 9 years 3 months (SD 24.6 months), and in mothers of typically developing (TD) children (N = 26) with a mean age of 9 years 4 months (standard deviation 23.7 months). The DD group comprised 11 children with autistic spectrum disorders or other communication impairments, three children with Down Syndrome, one with cerebral palsy, one with attentional problems, and four with specific or complex developmental problems. Child behaviour was measured by the Child Behaviour Checklist. Parental attributions were measured using a modified version of the Parent Attribution Test and mothers were divided into higher and lower controllability groups on the basis of their responses on this test. RESULTS: Multivariate analysis of variance found significant group × adult controllability interaction effects for 'aggressive behaviour', 'rule-breaking behaviour', as well as borderline significant effects for 'social problems' and 'other problems'. Simple effects analysis suggested that when mothers had lower attributions of adult controllability, there were indeed significantly more problem behaviours in the DD group, but when mothers had attributions of higher adult controllability there was no longer any significant difference in problematic behaviour between the two groups. CONCLUSIONS: Parental attributions of controllability may moderate the well-established effect of disability on problem behaviour. Implications for parent intervention programmes are discussed.

The comparative efficacy of four anthelmintics against a natural acquired Fasciola hepatica infection in hill sheep flock in the west of Ireland.

The aim of this study was to evaluate the efficacy of four different anthelmintics against F. hepatica in a naturally infected hill sheep flock in the west of Ireland. In a randomised trial, 138 ewes were divided into four groups. On day 0 each group was dosed with one of four anthelmintics: triclabendazole, closantel, oxyclozanide or nitroxynil. Faecal samples were obtained from each animal per rectum on the day of treatment and again at 7, 14, 21 and 56 days post-treatment. The number of F. hepatica eggs per gram of faeces was determined using the sedimentation technique and the efficacy of each anthelmintic was calculated in terms of the percentage reduction in egg count at each time point. The results for closantel, oxyclozanide and nitroxynil indicate that these drugs are effective with faecal egg count being reduced by 100% by day 14 post-treatment. However, the results for triclabendazole group yielded lower efficacy levels, with faecal egg count reductions of between 49% and 66% based on arithmetic means, over the period 7-56 days post-treatment. These results are highly indicative of triclabendazole resistant F. hepatica in sheep on this farm.

Young people's views of mental health education in secondary schools: a Scottish study.

BACKGROUND: This exploratory study used mixed methods to investigate young people's preferences in the delivery of mental health education and to investigate possible age and gender differences. METHOD: Information was gathered about the delivery of mental health education in three secondary schools. Nine pupil focus groups were carried out to identify key themes which were then further developed and administered through questionnaires to a larger sample of 773 pupils. RESULTS: Gender and age differences were found in young people's preferences about who should deliver mental health education, and what, when, where and how this should be delivered. CONCLUSION: Mental health education should reflect the needs of young people. Age and gender preferences should be considered when designing these programmes.

A case-control study of community-associated Clostridium difficile infection.

OBJECTIVES: The aim of this study was to determine the incidence of and risk factors for community-associated Clostridium difficile infection (CDI). METHODS: Prospective surveillance of community-derived faecal samples for C. difficile cytotoxin, followed by a questionnaire-based case-control study in two distinct patient cohorts (one semi-rural and the other urban). RESULTS: The proportion of randomly selected faecal samples positive for C. difficile cytotoxin was 2.1% in both patient cohorts (median ages 73 and 45 years for the urban and semi-rural cohorts, respectively). Exposure to antibiotics in the previous 4 weeks, particularly multiple agents (P < 0.001), aminopenicillins (P < 0.05) and oral cephalosporins (P < 0.05), was significantly more frequent among cases than controls. Hospitalization in the preceding 6 months was significantly associated with CDI (45% versus 23%; P = 0.022). However, almost half the cases had not received antibiotic therapy in the month before C. difficile detection, and approximately one-third neither had exposure to antibiotics nor recent hospitalization. Contact with infants aged < or =2 years was significantly associated with CDI (14% versus 2%; P = 0.02). Prior exposure to gastrointestinal-acting drugs (proton pump inhibitor, H2 antagonist or non-steroidal anti-inflammatory) was not significantly more common in CDI cases. C. difficile PCR ribotype 001 caused 60% and 13% of urban and semi-rural community-associated CDI cases, respectively. CONCLUSIONS: Reliance on antibiotic history and age (> or =65 years) will contribute to missed diagnoses of community-associated CDI. Potential risk factors for community-associated CDI should be explored further to explain the large proportion of cases not linked to recent antibiotic therapy or hospitalization.

Determination of pattern centre in EBSD using the moving-screen technique.

The 'moving-screen' or 'pattern magnification' method of calibration for electron backscatter diffraction (EBSD) was reformulated to develop a high-precision technique requiring no crystallographic knowledge of the specimen and no initial estimates of the calibration parameters. The technique depends upon the accurate displacement of the screen and camera assembly. Corresponding points are selected, interactively, from EBSD patterns. It is suggested that, as an alternative, the selection of points from the Hough transform could lead to a completely automated routine.

The association between benzo[a]pyrene-DNA adducts and body mass index, calorie intake and physical activity.

Prior work suggests that body size and fat content may influence carcinogen-DNA adduct levels measured in white blood cells. Here we consider energy balance more broadly by assessing the impact of body mass index (BMI), physical activity and calorie intake on the presence of benzo[a]pyrene-DNA (BP-DNA) adducts in white blood cell DNA. Our cross-sectional study employed subjects from a separately conducted intervention trial. Physical activity and food intake data were collected at 12 and 15 months of follow-up, respectively. BP-DNA adducts were measured by high-performance liquid chromatography (HPLC) in white blood cell samples collected at 12 months of follow-up. Complete data on all variables were available from 143 subjects. Logistic regression showed that BMI was inversely associated with the presence of detectable adducts (OR = 0.90, p = 0.02), and that hours of moderate-intensity physical activity were positively associated with the presence of detectable adducts (OR = 1.04, p = 0.04). These results provide further evidence that body fat content influences carcinogen-DNA adduct levels, probably by altering the distribution of the lipophilic parent compound.

Candidate gene analysis of 21q22: support for S100B as a susceptibility gene for bipolar affective disorder with psychosis.

A genome-wide scan in 60 bipolar affective disorder (BPAD) affected sib-pairs (ASPs) identified linkage on chromosome 21 at 21q22 (D21S1446, NPL = 1.42, P = 0.08), a BPAD susceptibility locus supported by multiple studies. Although this linkage only approaches significance, the peak marker is located 12 Kb upstream of S100B, a neurotrophic factor implicated in the pathology of psychiatric disorders, including BPAD and schizophrenia. We hypothesized that the linkage signal at 21q22 may result from pathogenic disease variants within S100B and performed an association analysis of this gene in a collection of 125 BPAD type I trios. S100B single nucleotide polymorphisms (SNPs) rs2839350 (P = 0.022) and rs3788266 (P = 0.031) were significantly associated with BPAD. Since variants within S100B have also been associated with schizophrenia susceptibility, we reanalyzed the data in trios with a history of psychosis, a phenotype in common between the two disorders. SNPs rs2339350 (P = 0.016) and rs3788266 (P = 0.009) were more significantly associated in the psychotic subset. Increased significance was also obtained at the haplotype level. Interestingly, SNP rs3788266 is located within a consensus-binding site for Six-family transcription factors suggesting that this variant may directly affect S100B gene expression. Fine-mapping analyses of 21q22 have previously identified transient receptor potential gene melastatin 2 (TRPM2), which is 2 Mb upstream of S100B, as a possible BPAD susceptibility gene at 21q22. We also performed a family-based association analysis of TRPM2 which did not reveal any evidence for association of this gene with BPAD. Overall, our findings suggest that variants within the S100B gene predispose to a psychotic subtype of BPAD, possibly via alteration of gene expression.

Preliminary studies on the effect of moderate physical activity on blood levels of glutathione.

Molecular epidemiological approaches are being used to study how physical activity may protect against cancer. Prior epidemiological data suggest that physical activity protects against lung cancer; however, interpretation of these data is complicated by potential confounding by smoking. Glutathione (GSH) detoxifies cigarette smoke carcinogens and the paper tests whether physical activity levels are associated with blood GSH levels. Study subjects were enrolled in a chemoprevention trial testing whether antioxidant micronutrient supplementation reduces genetic damage from cigarette smoking. Physical activity data were collected by questionnaire from 178 subjects at 12 months of follow-up in the trial. Total GSH (tGSH), which is the sum of free and protein-bound GSH and glutathione disulfide levels, was measured using the 5,5'-dithiobis-(2-nitrobenzenoic acid) colormetric assay with red blood cell samples collected at the 12-month time point. In multivariate linear regression analyses that controlled for gender and cigarettes smoked per day, tGSH was positively associated with hours per week of moderate intensity activity (beta=0.005, p=0.02). Hours per week of vigorous intensity activity were unassociated with tGSH and the effect of moderate activity remained after control for vigorous activity. The results are consistent with prior research showing differential effects of moderate and vigorous activity and suggest a mechanism through which physical activity may influence lung cancer risk.

Stenotrophomonas maltophilia contamination of nebulizers used to deliver aerosolized therapy to inpatients with cystic fibrosis.

There is circumstantial evidence that nebulizer equipment may be a source of Stenotrophomonas maltophilia for patients with cystic fibrosis. Eighty-nine inpatient nebulizers were examined for evidence of S. maltophilia contamination of which nine (10%) yielded 14 strains of the bacterium. Environmental samples were obtained from 73 different sites on the ward, of which 17 (23%) yielded a further 21 strains. Positive sites included taps, sink drains, and potable water. Genotyping using ERIC-PCR and pulsed-field gel electrophoresis revealed that two pairs of patients' nebulizers were contaminated with closely related strains. None of the S. maltophilia isolates obtained from the ward environment shared genotypes with those obtained from the nebulizers. The frequency of isolation of S. maltophilia from potable water sources on the ward suggests that contamination may result from using it to clean reusable nebulizer equipment, particularly if this is followed by inadequate drying. Although the actual source of S. maltophilia contamination of hospital-use nebulizer equipment in this study remained elusive, these results have important infection control implications.