Repurposing proteasome inhibitors for improved treatment of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is associated with poor prognosis and limited treatment options due to the lack of important receptors (estrogen receptor [ER], progesterone receptor [PR], and human epidermal growth factor receptor 2 [HER2]) used for targeted therapy. However, high-throughput in vitro drug screening of cell lines is a powerful tool for identifying effective drugs for a disease. Here, we determine the intrinsic chemosensitivity of TNBC cell lines to proteasome inhibitors (PIs), thereby identifying potentially potent 2-drug combinations for TNBC. Eight TNBC cell lines (BT-549, CAL-148, HCC1806, HCC38, HCC70, MDA-MB-436, MDA-MB-453, and MDA-MB-468) and two controls (MCF-10A and MCF-7) were first exposed to 18 drugs (11 PIs and 7 clinically relevant chemotherapeutic agents) as monotherapy, followed by prediction of potent 2-drug combinations using the IDACombo pipeline. The synergistic effects of the 2-drug combinations were evaluated with SynergyFinder in four TNBC cell lines (CAL-148, HCC1806, HCC38, and MDA-MB-468) and three controls (BT-474, MCF-7, and T47D) in vitro, followed by further evaluation of tumor regression in zebrafish tumor models established using HCC1806 and MCF-7 cells. Monotherapy identified nine effective drugs (bortezomib, carfilzomib, cisplatin, delanzomib, docetaxel, epoxomicin, MLN-2238, MLN-9708, and nedaplatin) across all cell lines. PIs (e.g., bortezomib, delanzomib, and epoxomicin) were highly potent drugs in TNBC cells, of which bortezomib and delanzomib inhibited the chymotrypsin-like activity of the 20 S proteasome by 100% at 10 µM. Moreover, several potent 2-drug combinations (e.g., bortezomib+nedaplatin and epoxomicin+epirubicin) that killed virtually 100% of cells were also identified. Although HCC1806- and MCF-7-derived xenografts treated with bortezomib+nedaplatin and carboplatin+paclitaxel were smaller, HCC1806 cells frequently metastasized to the trunk region. Taken together, we show that PIs used in combination with platinum agents or topoisomerase inhibitors exhibit increased efficiency with almost 100% inhibition in TNBC cell lines, indicating that PIs are therefore promising compounds to use as combination therapy for TNBC.

Cancer stem cells are prevalent in the basal-like 2 and mesenchymal triple-negative breast cancer subtypes in vitro.

Background: Triple-negative breast cancer (TNBC) is an aggressive subtype with the most unfavorable clinical outcomes, in part due to tumor heterogeneity, treatment resistance, and tumor relapse. The TNBC subtypes [basal-like 1 (BL1), basal-like 2 (BL2), mesenchymal (M), and luminal androgen receptor (LAR)] are biologically and clinically distinct entities that respond differently to local and systemic therapies. Therefore, we need to have a better understanding of cancer stemness relating to drug-resistant populations in the TNBC subtypes. Methods: Breast cancer stem cell (BCSC) distribution was investigated using an integrated flow cytometry approach with the ALDEFLUOR™ assay (ALDH) and CD24/CD44 antibodies. In total, 27 commercially available cell lines derived from normal and malignant mammary tissue were characterized into differentiated tumor cells and/or BCSC subpopulations (ALDH-CD44+CD24-/low enriched mesenchymal-like BCSCs, ALDH+non-CD44+CD24-/low enriched epithelial-like BCSCs, and highly purified ALDH+CD44+CD24-/low BCSCs). Results: BCSCs were not only enriched in estrogen receptor (ER) negative (mean, 49.6% versus 6.9% in ER+) and TNBC cell lines (51.3% versus 2.1% in Luminal A), but certain BCSC subpopulations (e.g., enriched mesenchymal-like BCSCs) were also significantly more common in the M (64.0% versus 6.2% in BL1; 64.0% versus 0% in LAR) and BL2 (77.4% versus 6.2% in BL1; 77.4% versus 0% in LAR; 77.4% versus 10.4% in TNBC UNS) TNBC subtypes. In contrast, ALDH status alone was not indicative of ER status or BC subtype. Conclusion: Taken together, these findings demonstrate the enrichment of potentially treatment-resistant BCSC subpopulations in the M and BL2 triple-negative breast cancer subtypes.

Optimization of Cell Viability Assays for Drug Sensitivity Screens.

During the preclinical stages of the drug discovery process, cell viability assays are fundamental tools for studying the phenotypic properties and overall health of cells following in vitro drug sensitivity screens. Therefore, it is important to optimize your viability assay of choice to obtain reproducible and replicable results, as well as use relevant drug response metrics (e.g., IC50, AUC, GR50, and GRmax) to identify candidate drugs for further evaluation in vivo. Herein, we used the resazurin reduction assay which is a quick, cost-effective, simple-to-use, and sensitive method for examining the phenotypic properties of cells. Using the MCF7 breast cancer cell line, we provide a detailed step-by-step protocol for optimizing drug sensitivity screens using the resazurin assay.

APR-246 Enhances Colorectal Cancer Sensitivity to Radiotherapy.

p53 mutation is common and highly related to radiotherapy resistance in rectal cancer. APR-246, as a small molecule, can restore the tumor-suppressor function to mutant p53. As there is currently no existing study on combining APR-246 with radiation in rectal cancer, our objective was to investigate whether APR-246 could enhance the sensitivity of colorectal cancer cells, regardless of their p53 status, to radiation treatment. The combination treatment had synergistic effects on HCT116p53-R248W/- (p53Mut) cells, followed by HCT116p53+/+ [wild-type p53 (p53WT)] cells, and exhibited an additive effect on HCT116p53-/- (p53Null) cells through inhibiting proliferation, enhancing reactive oxygen species, and apoptosis. The results were confirmed in zebrafish xenografts. Mechanistically, p53Mut and p53WT cells shared more activated pathways and differentially expressed genes following the combination treatment, compared with p53Null cells, although the combination treatment regulated individual pathways in the different cell lines. APR-246 mediated radiosensitization effects through p53-dependent and -independent ways. The results may provide evidence for a clinical trial of the combination in patients with rectal cancer.

Intranasal dexmedetomidine sedation for paediatric MRI by radiology personnel: A retrospective observational study.

BACKGROUND: MRI often requires sedation or anaesthesia to ensure good image quality in paediatric patients. Access to paediatric anaesthesia services is, however, a limiting factor for effective paediatric MRI service, and alternative sedation methods are, therefore, warranted. OBJECTIVE: To investigate the efficacy and safety of an intranasal dexmedetomidine sedation program for paediatric MRI, without immediate presence of anaesthesia personnel. DESIGN: Single institution retrospective observational study. SETTING: Tertiary care paediatric hospital. PATIENTS: Children 0 to 12 years, ASA risk class 1 or 2 with heart rate within age-appropriate limit. INTERVENTION: Radiology personnel administered an initial dose of intranasal dexmedetomidine of 4 µg kg -1 followed by a second dose of 2 µg kg -1 to the patients if needed. Recordings of image quality, critical events, heart rate, pulse oximetry saturation and noninvasive blood pressure before and after dexmedetomidine administration were made. MAIN OUTCOME MEASURES: Changes in haemodynamic and respiratory data before vs. after intranasal dexmedetomidine were analysed for changes, and the incidence of critical events was evaluated as well as rate of successful MRI scans. RESULTS: One thousand and ninety-one MRIs under intranasal dexmedetomidine sedation were included (mean age 34 months, 95% confidence interval (CI), 33 to 36, 599 male individuals). A success rate of 93% (95% CI, 91 to 94%) was found. No major critical events were recorded, total incidence of minor issues was 0.2% (95% CI, 0 to 0.7%). Five children had a heart rate under a preset minimal limit after dexmedetomidine (0.4%; 95% CI, 0.1 to 0.9%). Significant decreases in heart rate and mean arterial pressure, within acceptable limits not requiring intervention, was seen after dexmedetomidine administration. CONCLUSION: Intranasal dexmedetomidine sedation without immediate presence of anaesthesia personnel appears to be well tolerated and associated with minimal interference on MRI image quality. TRIAL REGISTRATION: clinicaltrials.org NCT05163704, retrospectively registered.

Pan-cancer analysis of genomic and transcriptomic data reveals the prognostic relevance of human proteasome genes in different cancer types.

BACKGROUND: The human proteasome gene family (PSM) consists of 49 genes that play a crucial role in cancer proteostasis. However, little is known about the effect of PSM gene expression and genetic alterations on clinical outcome in different cancer forms. METHODS: Here, we performed a comprehensive pan-cancer analysis of genetic alterations in PSM genes and the subsequent prognostic value of PSM expression using data from The Cancer Genome Atlas (TCGA) containing over 10,000 samples representing up to 33 different cancer types. External validation was performed using a breast cancer cohort and KM plotter with four cancer types. RESULTS: The PSM genetic alteration frequency was high in certain cancer types (e.g. 67%; esophageal adenocarcinoma), with DNA amplification being most common. Compared with normal tissue, most PSM genes were predominantly overexpressed in cancer. Survival analysis also established a relationship with PSM gene expression and adverse clinical outcome, where PSMA1 and PSMD11 expression were linked to more unfavorable prognosis in ≥ 30% of cancer types for both overall survival (OS) and relapse-free interval (PFI). Interestingly, PSMB5 gene expression was associated with OS (36%) and PFI (27%), and OS for PSMD2 (42%), especially when overexpressed. CONCLUSION: These findings indicate that several PSM genes may potentially be prognostic biomarkers and novel therapeutic targets for different cancer forms.

Incidence of venous thromboembolic events not related to vascular catheters in a prospective cohort of critically ill children.

The risk for venous thromboembolism (VTE) is considered to be low in the general paediatric intensive care unit (PICU) population, and pharmacological thromboprophylaxis is not routinely used. PICU patients considered at high-risk of VTE could possibly benefit from pharmacological thromboprophylaxis, but the incidence of VTE in this group of patients is unclear. This was an observational, prospective study at a tertiary multi-disciplinary paediatric hospital. We used comprehensive ultrasonography screening for VTE in critically ill children with multiple risk factors for VTE. Patients admitted to PICU ≥ 72 h and with ≥ two risk factors for VTE were included. Patients receiving pharmacological thromboprophylaxis during their entire PICU stay were excluded. The primary outcome of the study was VTEs not related to the use of a CVC. Ultrasonography screening of the great veins was performed at PICU discharge. Seventy patients with median (interquartile range) 3 (2-4) risk factors for VTE were evaluated. Median age was 0.3 years (0.03-4.3) and median PICU length of stay 9 days (5-17). Regarding the primary outcome, no symptomatic VTEs occurred and no asymptomatic VTEs were found on ultrasonography screening, resulting in an incidence of VTEs not related to a vascular catheter of 0% (95% CI: 0-5.1%). CONCLUSION: Our results indicate that VTEs not related to a vascular catheter are a rare event even in a selected group of severely ill small children considered to be at high risk of VTE. WHAT IS KNOWN: • Children in the PICU often have several risk factors for venous thromboembolism (VTE). • The incidence of VTE in PICU patients is highly uncertain, and there are no evidence-based guidelines regarding VTE prophylaxis. WHAT IS NEW: • This study found an incidence of VTEs not related to a vascular catheter of 0% (95% CI: 0-5.1%). • This indicates that such VTE events are rare even in PICU patients with multiple risk factors for VTE.

Pan-cancer analysis identifies BIRC5 as a prognostic biomarker.

BACKGROUND: The BIRC5 gene encodes for the Survivin protein, which is a member of the inhibitor of apoptosis family. Survivin is found in humans during fetal development, but generally not in adult cells thereafter. Previous studies have shown that Survivin is abundant in most cancer cells, thereby making it a promising target for anti-cancer drugs and a potential prognostic tool. METHODS: To assess genetic alterations and mutations in the BIRC5 gene as well as BIRC5 co-expression with other genes, genomic and transcriptomic data were downloaded via cBioPortal for approximately 9000 samples from The Cancer Genome Atlas (TCGA) representing 33 different cancer types and 11 pan-cancer organ systems, and validated using the ICGC Data Portal and COSMIC. TCGA BIRC5 RNA sequencing data from 33 different cancer types and matching normal tissue samples for 16 cancer types were downloaded from Broad GDAC Firehose and validated using breast cancer microarray data from our previous work and data sets from the GENT2 web-based tool. Survival data were analyzed with multivariable Cox proportional hazards regression analysis and validated using KM plotter for breast-, ovarian-, lung- and gastric cancer. RESULTS: Although genetic alterations in BIRC5 were not common in cancer, BIRC5 expression was significantly higher in cancer tissue compared to normal tissue in the 16 different cancer types. For 14/33 cancer types, higher BIRC5 expression was linked to worse overall survival (OS, 4/14 after adjusting for both age and tumor grade and 10/14 after adjusting only for age). Interestingly, higher BIRC5 expression was associated with better OS in lung squamous cell carcinoma and ovarian serous cystadenocarcinoma. Higher BIRC5 expression was also linked to shorter progressive-free interval (PFI) for 14/33 cancer types (4/14 after adjusting for both age and tumor grade and 10/14 after adjusting only for age). External validation showed that high BIRC5 expression was significantly associated with worse OS for breast-, lung-, and gastric cancer. CONCLUSIONS: Our findings suggest that BIRC5 overexpression is associated with the initiation and progression of several cancer types, and thereby a promising prognostic biomarker.

Complications of Pediatric Midline Catheters: A Prospective Observational Pilot Study.

BACKGROUND: Midline catheters are peripheral intravenous (IV) catheters in which the tip of the catheter does not reach the central circulation. In children, the use of midline catheters could lead to decreased complications from central venous catheters. To validate the safety of midline catheter use in children, we aimed to describe the complications and dwell time of pediatric midline catheters. The primary outcome was the incidence of catheter-related venous thromboembolism (VTE). METHODS: We conducted an observational, prospective study including consecutive patients at a tertiary multidisciplinary pediatric hospital. One hundred pediatric midline catheters were followed for thrombotic, infectious, and mechanical complications. After catheter removal, Doppler ultrasonography was performed to detect asymptomatic VTE. RESULTS: The mean age was 6.0 years (standard deviation [SD], 4.7), and median catheter dwell time was 6 (4-8) days. Most midline catheters were inserted in arm veins, most commonly in the basilic vein (56%). Catheter-related VTE was diagnosed in 30 (30%; 95% confidence interval [CI], 21%-40%) cases, corresponding to an incidence rate of 39 (95% CI, 26-55) cases per 1000 catheter days. Eight of 14 saphenous vein catheters were complicated by VTE compared to 22 of 86 arm vein catheters, suggesting an imbalance in favor of arm vein insertion site. Two patients needed anticoagulation therapy due to catheter-related VTE. Thirty (30%) catheters were removed unintentionally or due to complications, 22 of these needed additional IV access to complete the intended therapy. No catheter-related bloodstream infection (95% CI, 0%-4%) occurred. Mechanical complications occurred in 33 (33%; 95% CI, 24%-43%) midline catheters. CONCLUSIONS: In children, thrombotic and mechanical complications of midline catheters are common, but only few VTEs are severe enough to warrant anticoagulation therapy. Systemic infectious complications are rare. Seventy-eight percent of patients did not need additional venous access to complete short-term IV therapy. Considering the rate of clinically relevant complications and the catheter dwell time, pediatric midline catheters could be an alternative to central venous access for short-term (5-10 days) IV therapy.

Levels of mannose-binding lectin (MBL) associates with sepsis-related in-hospital mortality in women.

BACKGROUND: Mannose-binding lectin (MBL) mediates the innate immune response either through direct opsonisation of microorganisms or through activation of the complement system. There are conflicting data whether MBL deficiency leads to increased susceptibility to infections or not. The aim of this study was to determine if low levels of mannose-binding lectin (MBL) predict sepsis development, sepsis severity and outcome from severe sepsis or septic shock. METHOD: Patients aged 18 years or more with documented sepsis within 24 h after admission to the intensive care unit were included if they had participated in a health survey and donated blood samples prior to the sepsis event. A subset of these patients had stored plasma also from the acute phase. Two matched referents free of known sepsis were selected for each case. Plasma levels MBL were determined in stored samples from health surveys (baseline) and from ICU admission (acute phase). The association between MBL and sepsis, sepsis severity and in-hospital mortality were determined with 1300 ng/mL as cut-off for low levels. RESULTS: We identified 148 patients (61.5% women) with a first-time sepsis event 6.5 years (median with IQR 7.7) after participation in a health survey, of which 122 also had samples from the acute septic phase. Both high MBL levels in the acute phase (odds ratio [95% confidence interval]) (2.84 [1.20-6.26]), and an increase in MBL levels from baseline to the acute phase (3.76 [1.21-11.72]) were associated with increased risk for in-hospital death in women, but not in men (0.47 [0.11-2.06]). Baseline MBL levels did not predict future sepsis, sepsis severity or in-hospital mortality. CONCLUSIONS: An increase from baseline to the acute phase as well as high levels in the acute phase associated with an unfavourable outcome in women.

Integrative genomics approach identifies molecular features associated with early-stage ovarian carcinoma histotypes.

Ovarian cancer comprises multiple subtypes (clear-cell (CCC), endometrioid (EC), high-grade serous (HGSC), low-grade serous (LGSC), and mucinous carcinomas (MC)) with differing molecular and clinical behavior. However, robust histotype-specific biomarkers for clinical use have yet to be identified. Here, we utilized a multi-omics approach to identify novel histotype-specific genetic markers associated with ovarian carcinoma histotypes (CCC, EC, HGSC, and MC) using DNA methylation, DNA copy number alteration and RNA sequencing data for 96 primary invasive early-stage (stage I and II) ovarian carcinomas. More specifically, the DNA methylation analysis revealed hypermethylation for CCC in comparison with the other histotypes. Moreover, copy number imbalances and novel chromothripsis-like rearrangements (n = 64) were identified in ovarian carcinoma, with the highest number of chromothripsis-like patterns in HGSC. For the 1000 most variable transcripts, underexpression was most prominent for all histotypes in comparison with normal ovarian samples. Overall, the integrative approach identified 46 putative oncogenes (overexpressed, hypomethylated and DNA gain) and three putative tumor suppressor genes (underexpressed, hypermethylated and DNA loss) when comparing the different histotypes. In conclusion, the current study provides novel insights into molecular features associated with early-stage ovarian carcinoma that may improve patient stratification and subclassification of the histotypes.

Optimization of cell viability assays to improve replicability and reproducibility of cancer drug sensitivity screens.

Cancer drug development has been riddled with high attrition rates, in part, due to poor reproducibility of preclinical models for drug discovery. Poor experimental design and lack of scientific transparency may cause experimental biases that in turn affect data quality, robustness and reproducibility. Here, we pinpoint sources of experimental variability in conventional 2D cell-based cancer drug screens to determine the effect of confounders on cell viability for MCF7 and HCC38 breast cancer cell lines treated with platinum agents (cisplatin and carboplatin) and a proteasome inhibitor (bortezomib). Variance component analysis demonstrated that variations in cell viability were primarily associated with the choice of pharmaceutical drug and cell line, and less likely to be due to the type of growth medium or assay incubation time. Furthermore, careful consideration should be given to different methods of storing diluted pharmaceutical drugs and use of DMSO controls due to the potential risk of evaporation and the subsequent effect on dose-response curves. Optimization of experimental parameters not only improved data quality substantially but also resulted in reproducible results for bortezomib- and cisplatin-treated HCC38, MCF7, MCF-10A, and MDA-MB-436 cells. Taken together, these findings indicate that replicability (the same analyst re-performs the same experiment multiple times) and reproducibility (different analysts perform the same experiment using different experimental conditions) for cell-based drug screens can be improved by identifying potential confounders and subsequent optimization of experimental parameters for each cell line.

A Laboratory Method for Determining Bacterially Formed Odorants and Reducing Odor in Absorbent Incontinence Products.

PURPOSE: The purpose of this study was to design a laboratory test method to mimic the formation of bacterially formed odorants during the use of absorbent urinary incontinence products. Three odor inhibitors with different modes of action were tested and evaluated. METHODS: Bacterially formed odorants in incontinence products were evaluated by adding a synthetic urine inoculated with a mixture of 4 bacterial strains to product samples cut from the incontinence products. The product samples were incubated in sealed flasks. The odorants that formed in the head space were sampled onto adsorbent tubes and analyzed by gas chromatography. The inhibitory effects of low pH, ethylenediaminetetraacetic acid (EDTA), and activated carbon were then measured. RESULTS: This technique enabled production of known odorants 3-methylbutanal, guaiacol, diacetyl, and dimethyl disulfide (DMDS) in concentrations of 50 to 600 ng/L in incontinence products. The method was further evaluated by testing 3 types of odor inhibitors; EDTA significantly reduced formation of all 4 odorants (P < .001). Lowering the pH from 6.0 to 4.9 decreased levels of 3-methylbutanal, DMDS, and guaiacol (P < .001); however, diacetyl levels increased (P < .001). Activated carbon significantly reduced the formation of diacetyl, DMDS, guaiacol, and 3-methylbutanal (P < .001). CONCLUSIONS: The technique we developed can be used to evaluate inhibitors with different modes of action to determine odor control in incontinence products. The odorants formed are produced by bacteria and have been identified as key contributors to the odor of used incontinence products. This work can be a step toward establishing a standard in the field of incontinence and odor control; creation of a standard will help the health care sector compare products to be purchased and benefit patients through the development of better products.

Incidence of and risk factors for venous thrombosis in children with percutaneous non-tunnelled central venous catheters.

BACKGROUND: Venous thrombosis (VT) in children is often associated with a central venous catheter (CVC). We aimed to determine the incidence of VT associated with percutaneous non-tunnelled CVCs in a general paediatric population, and to identify risk factors for VT in this cohort. METHODS: Observational, prospective study enrolling consecutive patients at a tertiary multi-disciplinary paediatric hospital. A total of 211 percutaneous, non-tunnelled CVCs were analysed. Data regarding potential risk factors for CVC-related VT were collected. Compression ultrasonography with colour Doppler was used to diagnose VT. RESULTS: Overall, 30.3% of children developed CVC-related VT, with an incidence rate of 29.6 (confidence interval, 22.5-36.9) cases/1000 CVC days. Upper body CVC location, multiple lumen CVCs, and male gender were independent risk factors for VT in multivariate analysis. All upper body VTs were in the internal jugular vein (IJV). The occurrence of CVC-related VT did not affect length of paediatric ICU or hospital stay. In patients with VT, femoral CVCs, young age, paediatric ICU admission, and a ratio of CVC/vein diameter >0.33 were associated with VT being symptomatic, occlusive, or both. IJV VT was often asymptomatic and non-occlusive. CONCLUSIONS: Paediatric non-tunnelled CVCs are frequently complicated by VT. Avoiding IJV CVCs and multiple lumen catheters could potentially reduce the overall risk of VT. However, IJV VT was more likely to be smaller and asymptomatic compared with femoral vein VT. More data are needed on the risk of complications from smaller, asymptomatic VT compared with the group of VT with symptoms or vein occlusion. Femoral vein CVCs and CVC/vein diameter >0.33 could be modifiable risk factors for VT with larger thrombotic mass. CLINICAL TRIAL REGISTRATION: ACTRN12615000442505.

Evaluation of ninety-six periprosthetic hip joint infections seen within five consecutive years.

Periprosthetic hip joint infections (PHJI) are severe complications. In 2003 Zimmerli published a well-noted treatment algorithm for PHJI. The aim of this study is to evaluate outcome, analyze the applied treatment regimen and compare it to the proposed algorithm. We evaluated the outcome of 96 PHJI treated at our institution between 2008 and 2012 and analysed adherence to the algorithm and outcome in coherence with the algorithm. The operations performed were irrigation and debridement with exchange of mobile parts (45%), two-stage exchange (36%), one-stage exchange (12%) and permanent explantation (7%). 47% were acute infections, 53% were chronic. Staphylococcus aureus was the most common pathogen. The overall success rate was 88%. In 12% of the cases the chosen operation didn't follow the algorithm. Of these only 10% was successfully treated with the primary operation. We find that the algorithm proposed by Zimmerli is a useful tool and easy to translate into clinical practice. When followed it yields a high success rate.

Leptin independently predicts development of sepsis and its outcome.

BACKGROUND: Sepsis is a life-threatening condition and obesity is related to the clinical outcome. The underlying reasons are incompletely understood, but the adipocyte derived hormones leptin and adiponectin may be involved. METHODS: Patients aged 18 years or more with documented first time sepsis events were included in a nested case-referent study if they had participated in previous health surveys. Two matched referents free of known sepsis were identified. Circulating levels of leptin and adiponectin were determined in stored plasma, and their impact on a future sepsis event and its outcome was evaluated. RESULTS: We identified 152 patients (62% women) with a sepsis event and a previous participation in a health survey. Eighty-three % had also blood samples from the acute event. Hyperleptinemia at health survey associated with a future sepsis event (OR 1.77, 95% CI 1.04-3.00) and with hospital death. After adjustment for BMI leptin remained associated with sepsis in men, but not in women. High levels in the acute phase associated with increased risk for in hospital death in women (OR 4.18, 95% CI 1.17-15.00), while being protective in men (OR 0.05, 95% CI 0.01-0.48). Furthermore, leptin increased more from baseline to the acute phase in men than in women. Adiponectin did not predict sepsis and did not relate to outcome. CONCLUSIONS: Hyperleptinemia independently predicted the development of sepsis and an unfavourable outcome in men, and inertia in the acute response related to worse outcome.

The CLOSED trial; CLOnidine compared with midazolam for SEDation of paediatric patients in the intensive care unit: study protocol for a multicentre randomised controlled trial.

INTRODUCTION: Sedation is an essential part of paediatric critical care. Midazolam, often in combination with opioids, is the current gold standard drug. However, as it is a far-from-ideal agent, clonidine is increasingly being used in children. This drug is prescribed off-label for this indication, as many drugs in paediatrics are. Therefore, the CLOSED trial aims to provide data on the pharmacokinetics, safety and efficacy of clonidine for the sedation of mechanically ventilated patients in order to obtain a paediatric-use marketing authorisation. METHODS AND ANALYSIS: The CLOSED study is a multicentre, double-blind, randomised, active-controlled non-inferiority trial with a 1:1 randomisation between clonidine and midazolam. Both treatment groups are stratified according to age in three groups with the same size: <28 days (n=100), 28 days to <2 years (n=100) and 2-18 years (n=100). The primary end point is defined as the occurrence of sedation failure within the study period. Secondary end points include a pharmacokinetic/pharmacodynamic relationship, pharmacogenetics, occurrence of delirium and withdrawal syndrome, opioid consumption and neurodevelopment in the neonatal age group. Logistic regression will be used for the primary end point, appropriate statistics will be used for the secondary end points. ETHICS: Written informed consent will be obtained from the parents/caregivers. Verbal or deferred consent will be used in the sites where national legislation allows. The study has institutional review board approval at recruiting sites. The results will be published in a peer-reviewed journal and shared with the worldwide medical community. TRIAL REGISTRATION: EudraCT: 2014-003582-24; Clinicaltrials.gov: NCT02509273; pre-results.