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Introduction Albuminuric and nonalbuminuric pathways contribute to diabetic kidney disease. Proximal tubule and inflammation play important roles in these processes. Urinary biomarker(s) to detect early kidney damage and predict progression are needed. Methods Nine urinary biomarkers were measured at baseline in 400 patients with diabetes. Correlation and multivariate logistic and linear regression analyses were performed to assess the association of biomarkers with chronic kidney disease and progression. Results In the albumin/creatinine ratio (ACR), Graphical abstract

Background:\ud Patients with severe asthma may require maintenance oral corticosteroids (mOCS) for disease control as well as systemic corticosteroid (SCS) bursts for clinically significant exacerbations (CSEs); however, mOCS and SCS use are associated with adverse effects, which increases patient disease burden.\ud \ud Objective:\ud To assess the real-world corticosteroid-sparing effect of mepolizumab in patients with severe asthma.\ud \ud Methods:\ud REALITI-A is a 24-month, international, prospective, observational cohort study involving 84 centers across Europe, Canada, and the USA with a 1-year pre- and post-mepolizumab treatment pre-planned interim analysis. 822 adults with a clinical diagnosis of asthma, and a physician decision to initiate mepolizumab (100 mg subcutaneously) treatment were included. Endpoints included: daily mOCS dose at baseline (penultimate 28 days of pre-treatment) and 1-year post-treatment; percentage reduction from baseline in mOCS dose; patients discontinuing mOCS 1-year post-treatment and rate of CSEs (those requiring OCS for ≥3 days/parenteral administration, and/or emergency room visit and/or hospital admission) pre- and post-treatment.\ud \ud Results:\ud 319 patients received mOCS at baseline (median [interquartile range]: 10.0 [5.0, 15.0] mg/day). At 1 year post-treatment, median mOCS dose reduced by 75% (2.5 [0.0, 5.0] mg/day); 64% of patients had a reduction in mOCS dose ≥50% versus baseline and 43% discontinued mOCS. CSEs decreased between pre- and post-treatment (rate ratio [95% confidence interval] 0.29 [0.26, 0.32] P

Environmental and occupational exposure to heavy metals and metalloids is a major global health risk. The kidney is often a site of early damage. Nephrotoxicity is both a major consequence of heavy metal exposure and potentially an early warning of greater damage. A paradigm shift occurred at the beginning of the 21st century in the field of renal medicine. The medical model of kidney failure and treatment began to give way to a social model of risk factors and prevention with important implications for environmental health. This development threw into focus the need for better biomarkers: markers of exposure to known nephrotoxins; markers of early damage for diagnosis and prevention; markers of disease development for intervention and choice of therapy. Constituents of electronic waste, e-waste or e-pollution, such as cadmium (Cd), lead (Pb), mercury (HG), arsenic (As) and silica (SiO2) are all potential nephrotoxins; they target the renal proximal tubules through distinct pathways. Different nephrotoxic biomarkers offer the possibility of identifying exposure to individual pollutants. In this review, a selection of prominent urinary markers of tubule damage is considered as potential tools for identifying environmental exposure to some key metallic pollutants.

Asthma worsening and symptom control are clinically important health outcomes in patients with severe eosinophilic asthma. This analysis of COMET evaluated whether stopping versus continuing long-term mepolizumab therapy impacted these outcomes. Patients with severe eosinophilic asthma with ≥3 years continuous mepolizumab treatment (via COLUMBA (NCT01691859) or COSMEX (NCT02135692) open-label studies) were eligible to enter COMET (NCT02555371), a randomised, double-blind, placebo-controlled study. Patients were randomised 1:1 to continue mepolizumab 100 mg subcutaneous every 4 weeks or to stop mepolizumab, plus standard of care asthma treatment. Patients could switch to open-label mepolizumab following an exacerbation. Health outcome endpoints included time to first asthma worsening (composite endpoint: rescue use, symptoms, awakening at night and morning peak expiratory flow (PEF)), patient and clinician assessed global rating of asthma severity and overall perception of response to therapy, and unscheduled healthcare resource utilisation. Patients who stopped mepolizumab showed increased risk of and shorter time to first asthma worsening compared with those who continued mepolizumab (hazard ratio (HR) 1.71; 95% CI 1.17–2.52; p=0.006), including reduced asthma control (increased risk of first worsening in rescue use (HR 1.36; 95% CI 1.00–1.84; p=0.047) and morning PEF (HR 1.77; 95% CI 1.21–2.59; p=0.003). There was a higher probability of any unscheduled healthcare resource use (HR 1.81; 95% CI 1.31–2.49; p, The COMET study investigated whether stopping long-term mepolizumab had an impact on health outcomes in patients with severe eosinophilic asthma; data suggest those who continue long-term mepolizumab treatment sustain clinically important improvements https://bit.ly/3A0bvwu

Objectives There are no published reports for anti‐interleukin‐5 therapy in children

Background: Although preclinical data suggest that combining PI3K inhibitors with endocrine therapy may overcome resistance, results from randomized clinical trials have failed to identify a subgroup of patients that derive a substantial benefit. This preoperative window study assessed whether adding the PI3K inhibitor pictilisib can increase the anti-tumor effects of anastrozole in primary breast cancer and aimed to identify the most appropriate patient population for combination therapy. Methods: In this randomized, open-label, phase 2 study, 167 postmenopausal women with newly diagnosed, operable, ER-positive, HER2-negative breast cancers were recruited. Participants were randomly allocated (2:1, favoring the combination) to two-weeks of preoperative treatment with anastrozole 1 mg once daily or the combination of anastrozole 1mg with pictilisib 260 mg once daily. The primary endpoint was inhibition of tumor cell proliferation, as measured by change in Ki-67 protein expression between tumor samples taken before and at the end of treatment. Secondary endpoints include induction of apoptosis (Caspase3) and safety. Comprehensive biomarkers analyses included targeted NGS of a comprehensive cancer panel of >400 genes (Ampliseq Comprehensive Cancer panel), copy number variation analyses, and pre- and post-treatment reverse-phase protein arrays (RPPA) and RNA profiling (NanoString nCounter platform). Results:There was significantly greater geometric mean Ki67 suppression of 82.5% (90% CI, 78.3%-85.8%) for the combination vs 70.7% (61.0%-78.0%) for anastrozole [geometric mean ratio (combination/ anastrozole) 0.60 (0.58-0.85);p=0.01]. Higher baseline Ki67, Luminal B status and/or negative PR status were associated with increased benefit from adding pictilisib. A significant interaction was observed between PIK3CA mutation subtypes [helical domain mutations (HD), kinase domain mutations (KD), wildtype (WT)] and mean Ki67 suppression; the combination/anastrozole geometric mean ratio of Ki67 suppression was 0.48 (0.27-0.84; p=0.02) for patients with HD mutations and 0.63 (0.39–1.0; p=0.05) for patients with PIK3Ca WT, compared to 1.17 (0.57–2.41; p=0.64) for patients with KD mutations. This was largely due to patients with HD mutations showing a particularly poor response to anastrozole alone [mean Ki67 suppression 53.9% (9.5%-76.5%)], that was reversed by the addition of pictilisib [mean Ki-67 suppression 78.1% (71.0%-83.4%)]. On the other hand, patients with KD mutations responded well to anastrozole alone [mean Ki-67 suppression 77.7% (57.0%-88.4%)] and showed no benefit from the addition of pictilisib [mean Ki-67 suppression 73.9% (59.8%-83.0%)]. There was no significant difference in induction of apoptosis between treatment groups. Comprehensive pre- and post-treatment biomarkers analyses will be presented. Conclusions: Adding pictilisib to anastrozole significantly increases the anti-proliferative response to preoperative treatment with anastrozole. A significant interaction was observed between PIK3CA mutation subtypes, with patients with helical domain mutations showing a particularly poor response to anastrozole alone that was reversed by the addition of pictilisib. Citation Format: Schmid P, Pinder S, Wheatley D, Zummit C, Macaskill EJ, Hu J, Price R, Bundred N, Hadad S, Shia A, Sarker S-J, Lim L, Mousa K, O'Brien C, Wilson TR, Lackner MR, Gendreau S, Gazinska P, Korbie D, Trau M, Mainwaring P, Thompson A, Purushotham A. Interaction of PIK3CA mutation subclasses with response to preoperative treatment with the PI3K inhibitor pictilisib in patients with estrogen receptor-positive breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-08-02.

Background The long-term efficacy and safety of mepolizumab for treatment of severe eosinophilic asthma are well established. Here, we examine the clinical impact of stopping mepolizumab after long-term use. Methods COMET (NCT02555371) was a randomised, double-blind, placebo-controlled, parallel-group, multicentre study. Patients who had completed COLUMBA (NCT01691859) or COSMEX (NCT02135692) and received continuous mepolizumab treatment for ≥3 years were randomised 1:1 to stop (switch to placebo) or continue subcutaneous mepolizumab 100 mg every 4 weeks for 52 weeks. Primary end-point: time to first clinically significant exacerbation; secondary end-points: time to first exacerbation requiring hospitalisation/emergency department visit, time to decrease in asthma control (≥0.5-point increase in Asthma Control Questionnaire-5 score from COMET baseline) and blood eosinophil count ratio to COMET baseline. Safety was assessed. Results Patients stopping (n=151) versus continuing (n=144) mepolizumab had significantly shorter times to first clinically significant exacerbation (hazard ratio 1.61, 95% CI 1.17–2.22; p=0.004) and decrease in asthma control (hazard ratio 1.52, 95% CI 1.13–2.02; p=0.005), and higher blood eosinophil counts at week 52 (270 versus 40 cells·µL−1; ratio (stopping versus continuing) 6.19, 95% CI 4.89–7.83; p, This randomised study demonstrates increased exacerbation risk and a decrease in asthma control in patients with severe eosinophilic asthma who stop mepolizumab treatment after long-term use, when compared with those who continue treatment. https://bit.ly/3fsxGV2

Background: Patients with severe eosinophilic asthma (SEA) often have heterogenous phenotypes with periods of asthma worsening, making it difficult to assess mepolizumab treatment response. Aims: To define patient level variables for mepolizumab treatment response. Methods: In this post-hoc analysis we examined mepolizumab response in patients with SEA (≥2 exacerbations in prior year) in the 32-week, randomised, placebo-controlled MENSA study and the following 52-week, open-label COSMOS study. Patients who completed both studies and received mepolizumab throughout were included (n=311). Results: In MENSA, 67% and 21% of patients had 0 or 1 exacerbations, respectively, and were considered responders; 12% (n=37) with ≥2 exacerbations were considered non-responders and assessed against four variables of clinical benefit. Of these non-responders, 89% (n=33) had evidence of response when other variables were assessed (Table). Of the 37 non-responders in MENSA, 38% (n=14) responded in COSMOS, having either 0 (n=3) or 1 (n=11) exacerbations. Conclusions: These data highlight the difficulty in defining individual response to mepolizumab treatment. Responder classification should ideally take into account several clinical measures of improvement beyond exacerbation reduction. The relative importance of each of these measures may differ between patients, therefore doctors should set realistic treatment goals with each patient. Funding: GSK[NCT01691521/NCT01842607]

BackgroundThe long-term efficacy and safety of mepolizumab for treatment of severe eosinophilic asthma are well established. Here, we examine the clinical impact of stopping mepolizumab after long-term use.MethodsCOMET (NCT02555371) was a randomised, double-blind, placebo-controlled, parallel-group, multicentre study. Patients who had completed COLUMBA (NCT01691859) or COSMEX (NCT02135692) and received continuous mepolizumab treatment for ≥3 years were randomised 1:1 to stop (switch to placebo) or continue subcutaneous mepolizumab 100 mg every 4 weeks for 52 weeks. Primary end-point: time to first clinically significant exacerbation; secondary end-points: time to first exacerbation requiring hospitalisation/emergency department visit, time to decrease in asthma control (≥0.5-point increase in Asthma Control Questionnaire-5 score from COMET baseline) and blood eosinophil count ratio to COMET baseline. Safety was assessed.ResultsPatients stopping (n=151) versus continuing (n=144) mepolizumab had significantly shorter times to first clinically significant exacerbation (hazard ratio 1.61, 95% CI 1.17–2.22; p=0.004) and decrease in asthma control (hazard ratio 1.52, 95% CI 1.13–2.02; p=0.005), and higher blood eosinophil counts at week 52 (270 versus 40 cells·µL−1; ratio (stopping versus continuing) 6.19, 95% CI 4.89–7.83; pConclusionPatients who stopped mepolizumab had an increase in exacerbations and reduced asthma control versus those who continued.

Purpose Preclinical data support a key role for the PI3K pathway in estrogen receptor–positive breast cancer and suggest that combining PI3K inhibitors with endocrine therapy may overcome resistance. This preoperative window study assessed whether adding the PI3K inhibitor pictilisib (GDC-0941) can increase the antitumor effects of anastrozole in primary breast cancer and aimed to identify the most appropriate patient population for combination therapy. Patients and Methods In this randomized, open-label phase II trial, postmenopausal women with newly diagnosed operable estrogen receptor–positive, human epidermal growth factor receptor 2 (HER2)–negative breast cancers were recruited. Participants were randomly allocated (2:1, favoring the combination) to 2 weeks of preoperative treatment with anastrozole 1 mg once per day (n = 26) or the combination of anastrozole 1 mg with pictilisib 260 mg once per day (n = 49). The primary end point was inhibition of tumor cell proliferation as measured by change in Ki-67 protein expression between tumor samples taken before and at the end of treatment. Results There was significantly greater geometric mean Ki-67 suppression of 83.8% (one-sided 95% CI, ≥ 79.0%) for the combination and 66.0% (95% CI, ≤ 75.4%) for anastrozole (geometric mean ratio [combination:anastrozole], 0.48; 95% CI, ≤ 0.72; P = .004). PIK3CA mutations were not predictive of response to pictilisib, but there was significant interaction between response to treatment and molecular subtype (P = .03); for patients with luminal B tumors, the combination:anastrozole geometric mean ratio of Ki-67 suppression was 0.37 (95% CI, ≤ 0.67; P = .008), whereas no significant Ki-67 response was observed for pictilisib in luminal A tumors (1.01; P = .98). Multivariable analysis confirmed Ki-67 response to the combination treatment of patients with luminal B tumors irrespective of progesterone receptor status or baseline Ki-67 expression. Conclusion Adding pictilisib to anastrozole significantly increases suppression of tumor cell proliferation in luminal B primary breast cancer.

Background: The OPPORTUNE Study randomized postmenopausal patients (pts) to receive 2-week preoperative treatment with anastrozole (ANA) plus pictilisib ("ANA+PIC" arm) or ANA alone. Patients had newly diagnosed, operable, ER+, HER2- invasive breast cancer of ≥1 cm size. The primary outcome at interim analysis (n=70) revealed that the addition of PIC significantly increased the anti-proliferative response to ANA as measured by reduction in Ki67 immunohistochemistry (IHC). Multivariate analyses suggested benefit of PIC for patients with luminal B disease (Schmid et al. SABCS 2014). Methods: RNA expression analysis of ∼800 breast cancer-related genes was performed on patients analyzed at the interim analysis, including 14 (ANA) and 20 (ANA+PIC) patients with matched pre- and post- treatment paired tumour samples using the nCounter platform (NanoString). Differential expression of individual genes by arm was assessed using paired and moderated t-tests and statistical significance assessed through false discovery rate (FDR). Ingenuity Pathway Analysis (IPA) of differentially expressed transcripts identified pathways of relevance. Protein expression was analyzed by reverse protein array ( RPPA) in pre- and post-treatment samples. Results: In an unsupervised analysis, down-regulation of genes associated with ER signaling was observed in patients who received single-agent ANA and ANA+PIC, which included genes that regulate the cell cycle, cell death, survival, growth and proliferation and known ER target genes (e.g., PGR, GREB1). In addition, transcripts related to growth factor signaling pathway appeared to be specifically modulated in the ANA+PIC arm, possibly via the upregulation of the expression of RTK ligands. There were no clear changes in PI3K-related phosphoproteins (e.g., AKT, S6, 4E-BP1) in the post-treatment samples by RPPA. However, known PI3K-regulated genes, IRS2 and PIK3IP1, were upregulated in the post-treatment samples and a composite PI3K gene expression signature score (O'Brien et al. 2010) was reduced in both study arms following treatment. This PI3K signature was associated with pre-treatment luminal B status (n=27) and, consistent with this finding, the baseline PI3K gene signature score in the ANA arm, but not the ANA+PIC arm, was inversely associated with the decrease in post treatment Ki67. The tumor immune microenvironment was analyzed though the use of composite gene sets. In our initial observations, analysis of pre- and post-treatment samples showed that 2-week treatment with ANA resulted in a modest increase in transcripts associated with multiple immune signatures, which was further enhanced by the addition of PIC. Conclusions: Gene expression analysis of pre- and post-treatment samples in the OPPORTUNE study demonstrates on-target inhibition of ER and PI3K signaling networks. The analysis of additional paired samples is in progress to further assess if 2-weeks of treatment with a regimen containing an AI in patients with early breast cancer impacts the tumor immune microenvironment. Citation Format: Schmid P, Pinder SE, Bundred N, Wheatley D, Macaskill J, Zammit C, Hu J, Price R, Shia A, Lim L, Parker P, Molinero L, Yu J, O'Brien C, Wilson T, Savage H, Derynck M, Lackner MR, Amler L, Purushotham A, Thompson A, Gendreau S. Transcript analysis of PI3K and immune-related genes and gene signatures in the pre- and post-treatment samples from the window of opportunity study of anastrozole and anastrozole with pictilisib (GDC-0941) in patients with HR-positive early breast cancer (OPPORTUNE study). [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-13-01.

Introduction L’efficacite et la tolerance du mepolizumab dans ASE etant bien etablies, COMET a examine les resultats apres la poursuite ou l’arret du mepolizumab a long terme. Methodes COMET etait une etude multicentrique, randomisee, en double aveugle, controle par placebo. Les patients eligibles avaient complete COLUMBA/COSMEX, recu un traitement continu par mepolizumab pendant au moins 3 ans. Les patients ont ete randomises 1 :1 pour continuer mepolizumab 100 mg par voie SC toutes les 4 semaines pendant 52 semaines ou pour arreter mepolizumab. Les patients pouvaient revenir au traitement par mepolizumab en ouvert a la suite d’une exacerbation. Le critere primaire etait la duree jusqu’a la premiere exacerbation cliniquement significative. Les criteres secondaires etaient : la duree jusqu’a la diminution du controle de l’asthme ; la reduction de taux d’eosinophiles sanguins. Les criteres ont ete analyses en utilisant le modele des risques proportionnels de Cox et des modeles repetes de modele mixte ajustes pour les covariables. Resultats Cent quarante-quatre patients ont recu mepolizumab et 151 placebo. Quarante-six pour cent (66/144) patients ayant continue mepolizumab versus 59 % (89/151) patients ayant arrete mepolizumab ont eu une exacerbation. Les patients ayant continue versus ceux ayant arrete mepolizumab ont eu une duree plus longue jusqu’a la premiere exacerbation (HR : 0,62 [(IC)95 % : 0,45 ; 0,86] ; p = 0,004) et une duree significativement plus longue jusqu’a la diminution du controle de l’asthme (HR : 0,66 [0,49 ; 0,88] ; p =0,005), sans difference entre les traitements pour la duree jusqu’a la premiere exacerbation necessitant une venue aux urgences ou une consultation (HR : 1,33 [0,50 ; 3,51]). Les patients continuant mepolizumab ont maintenu leur taux d’eosinophiles entre 40 et 60 cellules/μL, les taux d’eosinophiles ont augmente jusqu’a 270 cellules/μL a la semaine 12 pour les patients ayant arrete mepolizumab (ratio : 0,19 [0,15 ; 0,24] ; p Conclusion Les patients ayant arrete mepolizumab ont presente une augmentation du taux d’eosinophiles sanguins, une augmentation des exacerbations, une duree plus courte jusqu’a la premiere exacerbation, et une reduction du controle de l’asthme compare a ceux ayant continue mepolizumab. Ces resultats montrent que le traitement continu par mepolizumab a des benefices cliniques durables chez la plupart des patients avec ASE.

Background Mepolizumab has demonstrated favorable safety and efficacy profiles in placebo-controlled trials of 12 months' duration or less; however, long-term data are lacking. Objective We sought to evaluate the long-term safety and efficacy of mepolizumab in patients with severe eosinophilic asthma (SEA). Methods COLUMBA (Open-label Long Term Extension Safety Study of Mepolizumab in Asthmatic Subjects, NCT01691859 ) was an open-label extension study in patients with SEA previously enrolled in DREAM (Dose Ranging Efficacy And Safety With Mepolizumab in Severe Asthma, NCT01000506 ). Patients received 100 mg of subcutaneous mepolizumab every 4 weeks plus standard of care until a protocol-defined stopping criterion was met. Safety end points included frequency of adverse events (AEs), serious AEs, and AEs of special interest. Efficacy end points included annualized exacerbation rates, changes from baseline in Asthma Control Questionnaire 5 scores, and blood eosinophil counts. Immunogenicity was also assessed. Results Overall, 347 patients were enrolled for an average of 3.5 years (maximum, 4.5 years; total exposure, 1201 patient-years). On-treatment AEs were reported in 94% of patients (exposure-adjusted rate, 3688 events/1000 patient-years). The most frequently reported on-treatment AEs were respiratory tract infection, headache, bronchitis, and asthma worsening. Seventy-nine (23%) patients experienced 1 or more on-treatment serious AEs; there were 6 deaths, none of which were assessed as related to mepolizumab. For patients with 156 weeks or greater enrollment, the exacerbation rate was 0.74 events/y (weeks 0–156), a 56% reduction from the off-treatment period between DREAM and COLUMBA. For all patients, at the first postbaseline assessment, the mean Asthma Control Questionnaire 5 score was reduced by 0.47 points, and blood eosinophil counts were reduced by 78%, with similar improvements maintained throughout the study. The immunogenicity profile (8% anti-drug antibodies) was consistent with previous studies. Conclusion These data support the long-term safety and efficacy of mepolizumab in patients with SEA.

Background: Mepolizumab is approved for severe eosinophilic asthma in adults and, in some regions, adolescents (12-17 years old). Efficacy results in children (6-11 years old) have yet to be compared to data from adults and adolescents. Methods: Asthma exacerbation incidence and asthma control questionnaire (ACQ-5) responder rates were taken from adult/adolescents in double-blind, placebo controlled studies (NCT01000506, NCT01691521, NCT02281318, NCT01691508) of mepolizumab 75 mg to 750 mg while children (40 mg SC if Results: Subjects had similar mean exacerbation rates in the year prior to study: 4.0 in children and 2.8-3.6 in adolescents/adults. All subjects reported at least 2 exacerbations in the prior year. The incidence of exacerbations after 12 weeks of mepolizumab treatment was consistent between children and adolescents/adults (Table). The ACQ-5 responder rate (minimal clinically important reduction ≥0.5 points) at 12 weeks was 55% in children, 33-63% in adolescents, and 52-56% in adults. Conclusions: Mepolizumab in children with severe eosinophilic asthma results in similar efficacy (exacerbations and ACQ-5) when compared to adolescents/adults after 12 weeks. Funding: GSK

Rationale: Immune responses against biologics can result in adverse events or a lack of efficacy. We summarize here the immunogenicity profile of mepolizumab during the phase III clinical development program in severe asthma. Methods: We evaluated immunogenicity data from five mepolizumab studies ranging from 24 to 228 weeks. There were three randomized studies: DREAM (75, 250, or 750mg IV); MENSA (75mg IV or 100mg SC); SIRIUS (100mg SC) versus placebo, and two open-label studies of 100mg SC: COSMOS (52-week extension from MENSA and SIRIUS) and COLUMBA (228-week extension from DREAM). Anti-drug antibodies (ADA) and neutralizing antibodies (NAb) were measured pre-study and at multiple time points throughout the trials. Results: Of the 1,327 patients from the three randomized studies 915 received mepolizumab and 412 received placebo. Overall, the incidence of ADA ranged from 1% to 9%, was not affected by route of administration (IV vs. SC) and was similar across the 10-fold dose range. In COSMOS (N=651), and COLUMBA (N=347) 5% and 8% of patients tested ADA positive post-baseline, respectively. One subject on 100mg SC tested positive for NAb (SIRIUS). There was no correlation between ADA titres or change in blood eosinophil counts. Adverse events evaluated as potential systemic allergic reactions were uncommon (£2%) and unrelated to study drug in antibody-positive patients. Conclusion: Following extensive testing in phase III mepolizumab has been shown to be well-tolerated with minimal potential to elicit anti-drug antibodies. Sponsored by Glaxo Smith Kline: NCT01000506 (DREAM), NCT01691521 (MENSA), NCT01691508 (SIRIUS), NCT01842607 (COSMOS), NCT01691859 (COLUMBA)

Resume Introduction L’anticorps monoclonal anti-IL5 mepolizumab a ete approuve par l’UE, les Etats-Unis et d’autres marches internationaux comme traitement additionnel au traitement de fond chez les patients adultes avec un asthme severe eosinophilique (ASE). Dans plusieurs pays, la population approuvee inclut les adolescents (12– Methodes Trente-six sujets âges 6–11 ans avec ASE ont ete inclus et ont recu le mepolizumab. La PK (nombre de prelevement sanguin reduit), PD, la tolerance et la tolerabilite ont ete etudiees apres l’administration du mepolizumab par voie sous-cutanee (SC). Le schema posologique SC de 40 mg (sujets Resultats Parmi les 36 sujets qui ont recu le mepolizumab, 26 ont recu 40 mg et 10 ont recu 100 mg. Les expositions derivees (surface sous la courbe des concentrations du temps 0 a l’infini) normalisees au poids moyen dans chaque groupe de doses SC etaient de 454 μg*jour/m (40 mg [sujets Conclusion Le schema posologique etudie dans l’ASE pediatrique est juge acceptable etant donne l’index therapeutique du mepolizumab. La reduction du taux d’eosinophiles sanguins etait similaire a celle observee dans les etudes chez l’adulte avec 100 mg SC. La PKPD est predite par celle observee chez les adultes apres ajustement de la biodisponibilite. Le mepolizumab a ete bien tolere sans probleme particulier de toxicite compare aux adultes avec ASE.

Introduction Le mepolizumab est approuve pour le traitement de l’asthme severe a eosinophiles (ASE) chez les adultes et, dans certaines regions, chez les adolescents (12–17 ans). En aout 2018, l’Agence europeenne des medicaments a approuve le mepolizumab a 40 mgSC pour les enfants de 6 a 11 ans avec un ASE. L’efficacite et la tolerance du mepolizumab a long terme chez les enfants de 6 a 11 ans n’ont pas ete evaluees. Methodes Il s’agissait d’une etude ouverte, non controlee avec 2 parties A et B, chez les enfants atteint d’ASE. Les patients ayant termine la partie A (semaine 0–20) de l’etude sur la pharmacocinetique et pharmacodynamique etaient eligibles a la partie B de 52 semaines (semaine 20–72) de l’etude pour evaluer la tolerance et la pharmacodynamie a long terme du mepolizumab. Dans la partie B, les patients ont recu 40 mgSC de mepolizumab si 40 kg en plus de leur traitement de fond de depart. La dose de mepolizumab etait augmentee a 100 mgSC si le patient atteignait 40 kg pendant la phase B. Les criteres d’evaluation comprenaient le taux d’exacerbations, le controle de l’asthme et la tolerance. Resultats Trente patients sont entres dans la partie B et 29 ont termine les 52 semaines de traitement. Une amelioration du controle de l’asthme a ete observee via les questionnaires ACQ-7 et ACQ-5, avec respectivement 55 % et 59 % de repondeurs (amelioration ≥ 0,5 points par rapport a l’inclusion) a la visite de fin d’etude (semaine 72). Une augmentation du score ACT pour les enfants a ete egalement observee, qui montre une amelioration du controle de l’asthme et ce independamment du groupe de traitement (sur le poids des patients). Les taux annuels d’exacerbations pendant le traitement etaient inferieurs aux valeurs initiales pour chaque groupe. Dans l’ensemble, 80 % des sujets ont presente une reduction ≥ 50 % du taux d’exacerbations pendant la partie B par rapport a l’annee precedant l’inclusion. Le mepolizumab a ete bien tolere dans la partie B, aucun nouveau probleme de tolerance n’a ete rapporte chez les enfants par rapport au profil de tolerance connu des adultes/adolescents. La bronchite (30 %) etait l’effet indesirable le plus souvent rapporte. Aucun patient n’a eu d’anticorps anti-mepolizumab pendant la partie B ( Tableau 1 ). Conclusion Chez les enfants de 6 a 11 ans, le traitement par mepolizumab a entraine pendant 52 semaines une amelioration du controle de l’asthme et une reduction du taux annuel d’exacerbations par rapport a l’annee precedant l’inclusion. Le mepolizumab a ete bien tolere chez les enfants de 6 a 11 ans.

Although atrazine has been banned in the European Union since 2007 it still persists in soil from where it can enter the food chain. Milk-producing animals accumulate atrazine from contaminated feed and water and since large quantities of milk and milk products are consumed its quality should be constantly monitored. The objective of this investigation was to develop a simple tube ELISA procedure suitable for use in non-specialised laboratories and in the field. A polyclonal antibody raised in sheep and the hapten-gelatine conjugate was immobilised onto polystyrene tubes. This enables the colour produced to be read on a basic spectrophotometer. Milk samples were collected from three farms in different regions of Poland and diluted before immunoassay was performed. Samples were extracted with hexane-acetone for HPLC analysis. The amount of fat in the milk samples interferes with the dose response so it essential that the standards are prepared in the same samples matrix. A good correlation between 1% and 2% was found between the two methods in the analysis of real samples. However the ELISA procedure was more sensitive that the HPLC method since atrazine was detected in some samples by the ELISA but was not confirmed by the HPLC method. The study demonstrated that the simple antigen-coated tube assay provides a cost effective and valuable screening test that can be easily modified for direct use as a screening tool in the field.

Contamination of public water ways with sewage represents a serious environmental and health risk. We monitored pollution of the river Thames by enumerating the indicator organism Escherichia coli. Samples were taken from a site in central London near Waterloo Bridge in different seasons. E. coli were quantified using a membrane filtration method, and correlated with the tidal variations of the river and meteorological data on rainfall and temperature. More frequent and severe incidents of pollution occurred in the autumn. Heavy rainfall resulted in sharp peaks of E. coli contamination that implies a potential increase of numbers of pathogenic micro-organisms. Sixty percent of all samples were found to be in excess of the accepted upper limit of pollution set by European Union (EU) legislation for bathing water. This study demonstrated that frequent sewage pollution of the Thames results in high numbers of E. coli and incidents of detectable levels of pathogenic bacteria demonstrating the need for regular monitoring of bacterial pollution.

Chronic non-healing wounds are a major health problem with resident bacteria strongly implicated in their impaired healing. A rapid-screen to provide detailed knowledge of wound bacterial populations would therefore be of value and help prevent unnecessary and indiscriminate use of antibiotics-a process associated with promoting antibiotic resistance. We analysed chronic wound fluid samples, which had been assessed for microbial content, using 20 different fluorescent labelled peptide substrates to determine whether protease activity correlated with the bacterial load. Eight of the peptide substrates showed significant release of fluorescence after reaction with some of the wound samples. Comparison of wound fluid protease activities with the microbiological data indicated that there was no correlation between bacterial counts and enzyme activity for most of the substrates tested. However, two of the peptide substrates produced a signal corresponding with the microbial data revealing a strong positive correlation with Pseudomonas aeruginosa numbers. This demonstrated that short fluorescent labelled peptides can be used to detect protease activity in chronic wound fluid samples. The finding that two peptides were specific indicators for the presence of P. aeruginosa may be the basis for a diagnostic test to determine wound colonisation by this organism.

Aims: To determine the effect of pH, temperature, desiccation, ethylenediaminetetraacetic acid (EDTA) and Desferrioxamine B (DFO) on Panton-Valentine Leukocidin positive Community Acquired Methicillin Susceptible Staphylococcus aureus (PVL +ve CA-MSSA) biofilm formation. Methods and Results: Biofilms from PVL +ve CA-MSSA (clinical isolate) were subjected to pH, temperature, desiccation, EDTA and DFO. PVL +ve CA-MSSA were more resistant to pH and heat than their planktonic equivalents. Desiccation studies demonstrated that PVL +ve CA-MSSA biofilms were more refractory to the treatment than planktonic cells. Significant inhibition of PVL +ve CA-MSSA biofilm formation was observed in the presence of 1 mM EDTA. Low concentrations (2.5 μM) of DFO enhanced the growth of PVL +ve CA-MSSA biofilms. At higher concentrations (1 mM) DFO did inhibit the growth but not as much as EDTA. A combination of EDTA and DFO inhibited PVL +ve CA-MSSA biofilm formation at lower concentrations than either alone. Conclusions: This study demonstrates that PVL +ve CA-MSSA biofilms are resistant to environmental stress but their growth can inhibited effectively by a mixture of EDTA and DFO. Significance and Impact of Study: The inhibition of biofilm formation by PVL +ve CA-MSSA using chelating agents has not been previously reported and provides a practical approach to achieve the disruption of these potentially important biofilms formed by an emerging pathogen.

Bacterial proteases, Type XXIV from Bacillus licheniformens and Type XIV from Streptomyces griseus, were used to investigate the utility and optimisation of a solid phase assay for proteases, using immunoglobulin proteins as substrates. Immunoglobulins IgA and IgG were adsorbed on to surfaces of ELISA plates and exposed to various levels of the bacterial proteases which led to digestion and desorption of proportional amounts of the immunoglobulins. The assay signal was developed by measuring the remaining proteins on the polystyrene surface with appropriate enzyme-labelled anti-immunoglobulin reagents. The assay was fully optimised in terms of substrate levels employing ELISA techniques to titrate levels of adsorbed substrates and protease analytes. The critical factor which influences assay sensitivity was found to be the substrate concentration, the levels of adsorbed immunoglobulins. The estimated detection limits for protease XXIV and XIV were 10micro units/test and 9micro units/test using IgA as a substrate. EC(50) values were calculated as 213 and 48micro units/test for each protease respectively. Using IgG as a substrate, the estimated detection limits were 104micro units/test for protease XXIV and 9micro units/test for protease XIV. EC(50) values were calculated at 529micro units/test and 28micro units/test for protease XXIV and XIV respectively. The solid phase protease assay required no modification of the substrates and the adsorption step is merely simple addition of immunoglobulins to ELISA plates. Adsorption of the immunoglobulins to polystyrene enabled straightforward separation of reaction mixtures prior to development of assay signal. The assay exploits the advantages of the technical facilities of ELISA technology and commercially available reagents enabling the detection and measurement of a wide range of proteases. However, the key issue was found to be that in order to achieve the potential performance of the simple assay, optimisation of the method was essential.

Aims: To evaluate a new range of chromogenic substrates for the detection of β-galactosidase activity in coliforms and to compare their performance in agar media and broths. Methods and Results: Sixteen novel galactoside substrates were prepared and incorporated into agar and broth. Their performance was compared using Escherichia coli (five strains), Salmonella (two strains), Enterobacter (two strains), Klebsiella, Pseudomonas, Listeria, Serratia, Shigella, Citrobacter, Proteus and Staphylococcus as well as pathological urine samples. The six substrates out of the initial 16 that showed the greatest sensitivity were VQE-gal, VQM-gal, VLPr-gal, VLE-gal, VLM-gal and VBzTM-gal, whose released chromophores were red, brown or purple. VQE-gal and VLPr-gal were studied in greater detail and were incorporated into agar medium. Coliform colonies appeared red and brown respectively, following incubation at 37°C for 24 h; however, positive results were obtained within a working day. The VQE-gal medium was compared with some commercially available media. Conclusions: The range of substrates described can be used in broths as well as in agars. The VQE agar allows the detection of coliforms within a working day. VQE-gal medium proved to be more sensitive when compared to other available chromogenic media and allows the unambiguous detection of coliforms.

Eight novel chromogenic substrates were evaluated for their efficacy in detecting lipase activity in clinical isolates of Staphylococcus aureus from the United Kingdom and Malta. All isolates metabolized the chromogenic lipase substrates 5-(4-hydroxy-3,5-dimethoxyphenylmethylene)-2-thioxothia-zolidin-4-one-3-ethanoic acid (SRA)-propionate, SRA-butyrate, SRA-octanoate and 2-[2-(4-hydroxy-3,5-dimethoxyphenyl)-vinyl]-3-methy-benzothiazolium salt (SB(Z)TM)-acetate. Over 90% of the isolates metabolized the lipase substrates SRA-decanoate and SRA-laurate. However, only 0.6% of UK isolates and 2% of Maltese isolates metabolized the lipase substrate SRA-myristate; none of the isolates tested metabolized SB(Z)TM-butyrate. Traditional Tween 80 assays showed that over 73% of the UK methicillin-resistant Staphylococcus aureus (MRSA) isolates and 83% of the UK methicillin-sensitive Staphylococcus aureus (MSSA) isolates demonstrated lipolytic activity. In contrast, Maltese isolates showed lipase activity in 94% and 88% of the MRSA and MSSA strains, respectively. Lipases in MRSA and MSSA demonstrated substrate specificity whose activity appeared dependent upon hydrocarbon chain length of the chromogen. These novel chromogens can be used for lipase enzyme detection and have application for full characterization of numerous S. aureus lipases.

The aim of the research was to determine optimal conditions for atrazine determination in trophic chain samples by means of an antigen-coated tube enzyme-linked immunosorbent assay (ELISA). The ELISA method was used for analysis of a selection of samples and the results and method requirement compared with HPLC. The 2 h competitive ELISA showed a minimum detection limit of 0.05 ng mL(-1) and a dynamic range 0.1-2 ng mL(-1). Investigation of atrazine concentration in a selection of trophic chain samples indicated that the content of atrazine (microg kg(-1)) in soil samples was 3.2-85.4, vegetable roots 32.9-148.9, green parts of plants 67.7-136.4, cereals 42.4-91.5 and samples of animal origin 1.3-8.4. The correlation between results obtained by HPLC and ELISA methods was 0.97. In addition, simazine content was determined by the HPLC method in which the detection limits were 0.2 microg g(-1) for atrazine and 0.3 microg g(-1) for simazine. The content (microg kg(-1)) of simazine in soil samples was 13.5-15.5, in vegetables roots 29.5-93.7, in green parts of plants 34.6-72.6 and in cereals 158-189. The study demonstrates the utility and convenience of the simple, practical and cost-effective ELISA method in a non-immunoassay laboratory for the analysis of food and environmental samples. The method is ideal for the rapid screening of large numbers of samples in laboratories where access to HPLC facilities is limited or lacking. In addition the investigation demonstrates the presence of significant levels of atrazine and simazine in trophic chain samples collected from different areas of the region. As expected, the highest concentration of both herbicides was found in plants.

Evidence has accumulated that herbicides in the environment present a significant health hazard to the population. Therefore, the levels of heavily used substances such as atrazine and simazine and their metabolites need to be regularly assessed. The objective was to develop a rapid and simple tube ELISA procedure suitable for use in field studies and non-specialized laboratories. The antisera used were polyclonal antibodies raised in sheep against atrazine or simazine amido caproic acid conjugated to bovine serum albumin. The antibodies were first used to construct a two-step competitive ELISA procedure in 96-well microtitre plates. The 96-well format was then adapted to a coated-tube enzyme immunoassay, by immobilization of hapten-gelatine conjugates on polystyrene tubes. This enabled the colour to be read using a basic spectrophotometer. Soil samples were collected from agricultural and non-agricultural sites in Poland. Atrazine and simazine were extracted by liquid extraction from soil and assayed by tube ELISA. In addition, the samples were extracted by solid-phase extraction before analysis by HPLC. The immunoassays and chemical analysis were carried out by different individuals who were unaware of each other's results, which were then compared at the end of the study. Correlation of the two methods was excellent, with R=98.7 and 81.3 for atrazine and simazine, respectively. The immunoassay yielded the same order of results without having to perform solid-phase extraction before analysis. The study has demonstrated that the simple antigen-coated tube assay provides a cost-effective and valuable screening test. Comparison with the more elaborate, heavily labour-intensive HPLC analysis demonstrated that the results obtained by the simpler enzyme-immunoassay tests were within the same order.

The use of dextran-coated activated charcoal (DCC) powder to absorb solubilising detergents from cell lysates is described. Normal embryonic epithelial cells were lysed in the presence of sodium dodecyl sulphate (SDS). The detergent was then absorbed with DCC to facilitate analysis of polycystin-1 with antibody-based methods. Polycystin-1 is a membrane protein that is involved in the pathogenesis of autosomal dominant polycystic kidney disease (ADPKD). The adverse effect of SDS on antibody-polycystin-1 binding was studied and the improvement resulting from its removal demonstrated using enzyme-linked immunosorbent assays (ELISAs). The results indicate that DCC can be used in a simple manner to remove highly reactive membrane-solubilising reagents from protein mixtures prior to immunological analysis. This procedure may be relevant to a variety of other techniques that are normally affected by detergents.

Polycystin-1 is a putative 460 kDa membrane protein with a unique structure and is possibly representative of a new family of proteins. Its structure suggests an involvement in cell signalling and cell–matrix interactions. The amino acid sequence of polycystin-1 has to date been predicted from its gene sequence. This, to our knowledge, is the first report of the isolation and analysis of polycystin-1 at the protein level using mass spectrometry to confirm its predicted structure. The availability of purified polycystin-1 will allow a new approach to unravelling the complexity of the cell–cell and cell–matrix interactions of this large molecule in normal cells and its perturbation in disease.

A large number of compounds, which are in common usage in industry and medicine, are potentially nephrotoxic. Renal damage and disease resulting from toxic exposure is progressive and will, if unarrested, culminate in irreversible renal disease. There is, therefore, a need to develop a battery of tests with which to monitor and characterise the nephrotoxic cascade. A European-wide study compared biomarker profiles of adult male workers who were exposed to heavy metals or solvents. It became apparent that the urinary profiles varied with the nature of the toxin, reflecting the functional region of the kidney affected and also the severity of the damage. Children are a particularly vulnerable group and the investigation of range of biomarkers indicated that they were indeed susceptible to nephrotoxic pollutants in their environment. It is proposed that a small cohort of tests should be used to monitor the early (pre-clinical stages) of renal damage or dysfunction; these can be supplemented if necessary by additional specific tests. In the future better information on at-risk populations and genetic information will enable the determination of individual susceptibility to be assessed more precisely.

The current investigation is the largest to date concerned with the assessment of the value of different urinary biomarkers to detect nephrotoxic effects in children exposed to cadmium and lead. A battery of tests which had proved valuable in previous studies on men and women where used, together with a number of more recently developed biomarkers. No significant effect of sex and age were found but the location of the children (site) was important. The results indicated that there might have been variability in either the assay procedures or sample handling between the different sites. A small group of tests were found to be elevated following toxic exposure and should be used in future studies. However, there was considerable variation in the degree of exposure amongst the control groups from different countries and in the test groups. This made pooling of the data difficult but the study does highlight the way forward and demonstrates that children can be at risk from environmental exposure to toxins at a lower level than is acceptable for adults.