Cardiac Adverse Events and Remdesivir in Hospitalized Patients with Coronavirus Disease 2019 (COVID-19): A Post Hoc Safety Analysis of the Randomized DisCoVeRy Trial.

BACKGROUND: We aimed to evaluate the cardiac adverse events (AEs) in hospitalized patients with Coronavirus Disease 2019 (COVID-19) receiving remdesivir plus standard of care (SoC) compared to SoC alone (control), as an association was noted in some cohort studies and disproportionality analyses of safety databases. METHODS: This post-hoc safety analysis is based on data from the multicenter, randomized, open-label, controlled DisCoVeRy trial in hospitalized patients with COVID-19 (NCT04315948). Any first AE occurring between randomization and day 29 in the modified intention-to-treat (mITT) population randomized to either remdesivir or control group was considered. Analysis was performed using Kaplan-Meier survival curves and Kaplan-Meier estimates were calculated for event rates. RESULTS: Cardiac AEs were reported in 46 (11.2%) of 410 and 48 (11.3%) of 423 patients in the mITT population (n = 833) enrolled in the remdesivir and control groups, respectively. The difference between both groups was not significant (HR 1.0, 95% CI 0.7-1.5, p = 0.98), even when evaluating serious and non-serious cardiac AEs separately. The majority of reports in both groups were of arrhythmic nature (remdesivir, 84.8%; control, 83.3%) and were associated with a favorable outcome. There was no significant difference between remdesivir and control groups in the occurrence of different cardiac AE subclasses, including arrhythmic events (HR 1.1, 95% CI: 0.7-1.7, p = 0.68). CONCLUSIONS: Remdesivir treatment was not associated with an increased risk of cardiac AEs, whether serious or not, and regardless of AE severity, compared to control, in patients hospitalized with moderate or severe COVID-19. This is consistent with the results of other randomized controlled trials and meta-analyses.

Management of pharmacovigilance during the COVID-19 pandemic crisis by the safety department of an academic sponsor: Lessons learnt and challenges from the EU DisCoVeRy clinical trial.

The current COVID-19 pandemic was an exceptional health situation, including for drug use. As there was no known effective drug for COVID-19 at the beginning of the pandemic, different drug candidates were proposed. In this article, we present the challenges for an academic Safety Department to manage the global safety of a European trial during the pandemic. The National Institute for Health and Medical Research (Inserm) conducted a European multicenter, open-label, randomized, controlled trial involving three repurposed and one-in development drugs (lopinavir/ritonavir, IFN-ß1a, hydroxychloroquine, and remdesivir) in adults hospitalized with COVID-19. From 25 March 2020 to 29 May 2020, the Inserm Safety Department had to manage 585 Serious Adverse Events (SAEs) initial notification and 396 follow-up reports. The Inserm Safety Department's staff was mobilized to manage these SAEs and to report Expedited safety reports to the competent authorities within the legal timeframes. More than 500 queries were sent to the investigators due to a lack of or incoherent information on SAE forms. At the same time, the investigators were overwhelmed by the management of patients suffering from COVID-19 infection. These particular conditions of missing data and lack of accurate description of adverse events made evaluation of the SAEs very difficult, particularly the assessment of the causal role of each investigational medicinal product. In parallel, working difficulties were accentuated by the national lockdown, frequent IT tool dysfunctions, delayed implementation of monitoring and the absence of automatic alerts for SAE form modification. Although COVID-19 is a confounding factor per se, the delay in and quality of SAE form completion and the real-time medical analysis by the Inserm Safety Department were major issues in the quick identification of potential safety signals. To conduct a high-quality clinical trial and ensure patient safety, all stakeholders must take their roles and responsibilities.

Ensuring quality control in a COVID-19 clinical trial during the pandemic: The experience of the Inserm C20-15 DisCoVeRy study.

SETTING: Health measures taken during the pandemic deeply modified the clinical research practices. At the same time, the demand for the results of the COVID-19 trials was urgent. Thus, the objective of this article is to share Inserm's experience in ensuring quality control in clinical trials in this challenging context. OBJECTIVES: DisCoVeRy is a phase III randomized study that aimed at evaluating the safety and efficacy of 4 therapeutic strategies in hospitalized COVID-19 adult patients. Between March, 22nd 2020 and January, 20th 2021, 1309 patients were included. In order to guarantee the best quality of data, the Sponsor had to adapt to the current sanitary measures and to their impact on clinical research activity, notably by adapting Monitoring Plan objectives, involving the research departments of the participating hospitals and a network of clinical research assistants (CRAs). RESULTS: Overall, 97 CRAs were involved and performed 909 monitoring visits. The monitoring of 100% of critical data for all patients included in the analysis was achieved, and despite of the pandemic context, a conform consent was recovered for more than 99% of patients. Results of the study were published in May and September 2021. DISCUSSION/CONCLUSION: The main monitoring objective was met thanks to the mobilization of considerable personnel resources, within a very tight time frame and external hurdles. There is a need for further reflection to adapt the lessons learned from this experience to the context of routine practice and to improve the response of French academic research during a future epidemic.

Respiratory support in patients with severe COVID-19 in the International Severe Acute Respiratory and Emerging Infection (ISARIC) COVID-19 study: a prospective, multinational, observational study.

BACKGROUND: Up to 30% of hospitalised patients with COVID-19 require advanced respiratory support, including high-flow nasal cannulas (HFNC), non-invasive mechanical ventilation (NIV), or invasive mechanical ventilation (IMV). We aimed to describe the clinical characteristics, outcomes and risk factors for failing non-invasive respiratory support in patients treated with severe COVID-19 during the first two years of the pandemic in high-income countries (HICs) and low middle-income countries (LMICs). METHODS: This is a multinational, multicentre, prospective cohort study embedded in the ISARIC-WHO COVID-19 Clinical Characterisation Protocol. Patients with laboratory-confirmed SARS-CoV-2 infection who required hospital admission were recruited prospectively. Patients treated with HFNC, NIV, or IMV within the first 24 h of hospital admission were included in this study. Descriptive statistics, random forest, and logistic regression analyses were used to describe clinical characteristics and compare clinical outcomes among patients treated with the different types of advanced respiratory support. RESULTS: A total of 66,565 patients were included in this study. Overall, 82.6% of patients were treated in HIC, and 40.6% were admitted to the hospital during the first pandemic wave. During the first 24 h after hospital admission, patients in HICs were more frequently treated with HFNC (48.0%), followed by NIV (38.6%) and IMV (13.4%). In contrast, patients admitted in lower- and middle-income countries (LMICs) were less frequently treated with HFNC (16.1%) and the majority received IMV (59.1%). The failure rate of non-invasive respiratory support (i.e. HFNC or NIV) was 15.5%, of which 71.2% were from HIC and 28.8% from LMIC. The variables most strongly associated with non-invasive ventilation failure, defined as progression to IMV, were high leukocyte counts at hospital admission (OR [95%CI]; 5.86 [4.83-7.10]), treatment in an LMIC (OR [95%CI]; 2.04 [1.97-2.11]), and tachypnoea at hospital admission (OR [95%CI]; 1.16 [1.14-1.18]). Patients who failed HFNC/NIV had a higher 28-day fatality ratio (OR [95%CI]; 1.27 [1.25-1.30]). CONCLUSIONS: In the present international cohort, the most frequently used advanced respiratory support was the HFNC. However, IMV was used more often in LMIC. Higher leucocyte count, tachypnoea, and treatment in LMIC were risk factors for HFNC/NIV failure. HFNC/NIV failure was related to worse clinical outcomes, such as 28-day mortality. Trial registration This is a prospective observational study; therefore, no health care interventions were applied to participants, and trial registration is not applicable.

The Economic, Medical and Psychosocial Consequences of Whole Genome Sequencing for the Genetic Diagnosis of Patients With Intellectual Disability: The DEFIDIAG Study Protocol.

Introduction: Like other countries, France has invested in a national medical genomics program. Among the four pilot research studies, the DEFIDIAG project focuses on the use of whole genome sequencing (WGS) for patients with intellectual disability (ID), a neurodevelopmental condition affecting 1-3% of the general population but due to a plethora of genes. However, the access to genomic analyses has many potential individual and societal issues in addition to the technical challenges. In order to help decision-makers optimally introduce genomic testing in France, there is a need to identify the socio-economic obstacles and leverages associated with the implementation of WGS. Methods and Analysis: This humanities and social sciences analysis is part of the DEFIDIAG study. The main goal of DEFIDIAG is to compare the percentage of causal genetic diagnoses obtained by trio WGS (including the patient and both parents) (WGST) to the percentage obtained using the minimal reference strategy currently used in France (Fragile-X testing, chromosomal microarray analysis, and gene panel strategy including 44 ID genes) for patients with ID having their first clinical genetics consultation. Additionally, four complementary studies will be conducted. First, a cost-effectiveness analysis will be undertaken in a subsample of 196 patients consulting for the first time for a genetic evaluation; in a blinded fashion, WGST and solo (index case, only) genomic analysis (WGSS) will be compared to the reference strategy. In addition, quantitative studies will be conducted: the first will estimate the cost of the diagnostic odyssey that could potentially be avoidable with first-line WGST in all patients previously investigated in the DEFIDIAG study; the second will estimate changes in follow-up of the patients in the year after the return of the WGST analysis compared to the period before inclusion. Finally, through semi-directive interviews, we will explore the expectations of 60 parents regarding genomic analyses. Discussion: Humanities and social sciences studies can be used to demonstrate the efficiency of WGS and assess the value that families associate with sequencing. These studies are thus expected to clarify trade-offs and to help optimize the implementation of genomic sequencing in France. Ethics Statement: The protocol was approved by the Ethics Committee Sud Méditerranée I (June 2019)-identification number: 2018-A00680-55 and the French data privacy commission (CNIL, authorization 919361). Clinical Trial Registration: (ClinicalTrials.gov), identifier (NCT04154891).

Molecular profiling of advanced soft-tissue sarcomas: the MULTISARC randomized trial.

BACKGROUND: Soft-tissue sarcomas (STS) represent a heterogeneous group of rare tumors including more than 70 different histological subtypes. High throughput molecular analysis (next generation sequencing exome [NGS]) is a unique opportunity to identify driver mutations that can change the usual one-size-fits-all treatment paradigm to a patient-driven therapeutic strategy. The primary objective of the MULTISARC trial is to assess whether NGS can be conducted for a large proportion of metastatic STS participants within a reasonable time, and, secondarily to determine whether a NGS-guided therapeutic strategy improves participant's outcome. METHODS: This is a randomized, multicentre, phase II/III trial inspired by the design of umbrella and biomarker-driven trials. The setting plans up to 17 investigational centres across France and the recruitment of 960 participants. Participants aged at least 18 years, with unresectable locally advanced and/or metastatic STS confirmed by the French sarcoma pathological reference network, are randomized according to 1:1 allocation ratio between the experimental arm "NGS" and the standard "No NGS". NGS will be considered feasible if (i) NGS results are available and interpretable, and (ii) a report of exome sequencing including a clinical recommendation from a multidisciplinary tumor board is provided to investigators within 7 weeks from reception of the samples on the biopathological platform. A feasibility rate of more than 70% is expected (null hypothesis: 70% versus alternative hypothesis: 80%). In terms of care, participants randomized in "No NGS" arm and who fail treatment will be able to switch to the NGS arm at the request of the investigator. DISCUSSION: The MULTISARC trial is a prospective study designed to provide high-level evidence to support the implementation of NGS in routine clinical practice for advanced STS participants, on a large scale. TRIAL REGISTRATION: clinicaltrial.gov NCT03784014 .

Evaluation of saliva as a source of accurate whole-genome and microbiome sequencing data.

This study sets out to establish the suitability of saliva-based whole-genome sequencing (WGS) through a comparison against blood-based WGS. To fully appraise the observed differences, we developed a novel technique of pseudo-replication. We also investigated the potential of characterizing individual salivary microbiomes from non-human DNA fragments found in saliva. We observed that the majority of discordant genotype calls between blood and saliva fell into known regions of the human genome that are typically sequenced with low confidence; and could be identified by quality control measures. Pseudo-replication demonstrated that the levels of discordance between blood- and saliva-derived WGS data were entirely similar to what one would expect between technical replicates if an individual's blood or saliva had been sequenced twice. Finally, we successfully sequenced salivary microbiomes in parallel to human genomes as demonstrated by a comparison against the Human Microbiome Project.

An open-label randomized controlled trial of the effect of lopinavir/ritonavir, lopinavir/ritonavir plus IFN-ß-1a and hydroxychloroquine in hospitalized patients with COVID-19.

OBJECTIVES: We evaluated the clinical, virological and safety outcomes of lopinavir/ritonavir, lopinavir/ritonavir-interferon (IFN)-ß-1a, hydroxychloroquine or remdesivir in comparison to standard of care (control) in coronavirus 2019 disease (COVID-19) inpatients requiring oxygen and/or ventilatory support. METHODS: We conducted a phase III multicentre, open-label, randomized 1:1:1:1:1, adaptive, controlled trial (DisCoVeRy), an add-on to the Solidarity trial (NCT04315948, EudraCT2020-000936-23). The primary outcome was the clinical status at day 15, measured by the WHO seven-point ordinal scale. Secondary outcomes included quantification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in respiratory specimens and pharmacokinetic and safety analyses. We report the results for the lopinavir/ritonavir-containing arms and for the hydroxychloroquine arm, trials of which were stopped prematurely. RESULTS: The intention-to-treat population included 583 participants-lopinavir/ritonavir (n = 145), lopinavir/ritonavir-IFN-ß-1a (n = 145), hydroxychloroquine (n = 145), control (n = 148)-among whom 418 (71.7%) were male, the median age was 63 years (IQR 54-71), and 211 (36.2%) had a severe disease. The day-15 clinical status was not improved with the investigational treatments: lopinavir/ritonavir versus control, adjusted odds ratio (aOR) 0.83, (95% confidence interval (CI) 0.55-1.26, p 0.39), lopinavir/ritonavir-IFN-ß-1a versus control, aOR 0.69 (95%CI 0.45-1.04, p 0.08), and hydroxychloroquine versus control, aOR 0.93 (95%CI 0.62-1.41, p 0.75). No significant effect of investigational treatment was observed on SARS-CoV-2 clearance. Trough plasma concentrations of lopinavir and ritonavir were higher than those expected, while those of hydroxychloroquine were those expected with the dosing regimen. The occurrence of serious adverse events was significantly higher in participants allocated to the lopinavir/ritonavir-containing arms. CONCLUSION: In adults hospitalized for COVID-19, lopinavir/ritonavir, lopinavir/ritonavir-IFN-ß-1a and hydroxychloroquine improved neither the clinical status at day 15 nor SARS-CoV-2 clearance in respiratory tract specimens.

Genome Sequencing for Genetics Diagnosis of Patients With Intellectual Disability: The DEFIDIAG Study.

Introduction: Intellectual Disability (ID) is the most common cause of referral to pediatric genetic centers, as it affects around 1-3% of the general population and is characterized by a wide genetic heterogeneity. The Genome Sequencing (GS) approach is expected to achieve a higher diagnostic yield than exome sequencing given its wider and more homogenous coverage, and, since theoretically, it can more accurately detect variations in regions traditionally not well captured and identify structural variants, or intergenic/deep intronic putatively pathological events. The decreasing cost of sequencing, the progress in data-management and bioinformatics, prompted us to assess GS efficiency as the first line procedure to identify the molecular diagnosis in patients without obvious ID etiology. This work is being carried out in the framework of the national French initiative for genomic medicine (Plan France Médecine Génomique 2025). Methods and Analysis: This multidisciplinary, prospective diagnostic study will compare the diagnostic yield of GS trio analysis (index case, father, mother) with the French core minimal reference strategy (Fragile-X testing, chromosomal microarray analysis and Gene Panel Strategy of 44 selected ID genes). Both strategies are applied in a blinded fashion, in parallel, in the same population of 1275 ID index cases with no obvious diagnosis (50% not previously investigated). Among them, a subgroup of 196 patients are randomized to undergo GS proband analysis in addition to GS trio analysis plus the French core minimal reference strategy, in order to compare their efficiency. The study also aims to identify the most appropriate strategy according to the clinical presentation of the patients, to evaluate the impact of deployment of GS on the families' diagnostic odyssey and the modification of their care, and to identify the advantages/difficulties for the patients and their families. Ethics Statement: The protocol was approved by the Ethics Committee Sud Méditerranée I and the French data privacy commission (CNIL, authorization 919361). Trial Registration: ClinicalTrials.gov identifier NCT04154891 (07/11/2019).

Feasibility of high-throughput sequencing in clinical routine cancer care: lessons from the cancer pilot project of the France Genomic Medicine 2025 plan.

BACKGROUND: Whole exome sequencing and RNA sequencing (WES/RNASeq) should now be implemented in the clinical practice in order to increase access to optimal care for cancer patients. Providing results to Tumour Boards in a relevant time frame-that is, compatible with the clinical pathway-is crucial. Assessing the feasibility of this implementation in the French care system is the primary objective of the Multipli study, as one of the four pilot projects of the national France Genomic Medicine 2025 (FGM 2025) plan. The Multipli study encompasses two innovative trials which will be driven in around 2400 patients suffering from a soft-tissue sarcoma (Multisarc) or a metastatic colorectal carcinoma (Acompli). METHODS: Prior to launching the FGM 2025 cancer pilot study itself, the performance of the Multipli genomic workflow has been evaluated through each step, from the samples collection to the Molecular Tumour Board (MTB) report. Two Multipli-assigned INCa-labelled molecular genetics centres, the CEA-CNRGH sequencing platform and the Institut Bergonié's Bioinformatics Platform were involved in a multicentric study. The duration of each step of the genomic workflow was monitored and bottlenecks were identified. RESULTS: Thirty barriers which could affect the quality of the samples, sequencing results and the duration of each step of the genomic pathway were identified and mastered. The global turnaround time from the sample reception to the MTB report was of 44 calendar days. CONCLUSION: Our results demonstrate the feasibility of tumour genomic analysis by WES/RNASeq within a time frame compatible with the current cancer patient care. Lessons learnt from the Multipli WES/RNASeq Platforms Workflow Study will constitute guidelines for the forthcoming Multipli study and more broadly for the future clinical routine practice in the first two France Genomic Medicine 2025 platforms.

Development and psychometric testing of a scale assessing the sharing of medical information and interprofessional communication: the CSI scale.

BACKGROUND: Interprofessional collaboration is essential in creating a safer patient environment. It includes the need to develop communication and coordination between professionals, implying a better sharing of medical information. Several questionnaires exist in the literature, but none of them have been developed in the French context. The objective was to develop and test the psychometric properties of the communication and sharing information (CSI) scale which assesses specifically interprofessional communication, especially the sharing of medical information and the effectiveness of communication between members of the team. METHODS: The questionnaire construction process used a literature review and involved a panel of voluntary professionals. A list of 32 items explored the quality of shared information delivered to patients and the effectiveness of interprofessional communication. The study was conducted in 16 voluntary units in a University Hospital (France), which included medical, surgical, obstetrics, intensive care, pediatrics, oncology and rehabilitation care. The scale-development process comprised an exploratory principal component analysis, Cronbach's α-coefficients and structural equation modeling (SEM). RESULTS: From these 16 units, a total of 503 health professionals took part in the study. Among them, 23.9% were physicians (n = 120), 43.9% nurses (n = 221) and 32.2% nurse assistants (n = 162).The validated questionnaire comprised 13 items and 3 dimensions relative to "the sharing of medical information" (5 items), "communication between physicians" (4 items) and "communication between nurses and nurse assistants" (4 items). The 3 dimensions accounted for 63.7% of the variance of the final questionnaire. Their respective Cronbach's alpha coefficients were 0.80, 0.87 and 0.81. SEM confirmed the existence of the 3 latent dimensions but the best characteristics were obtained with a hierarchical model including the three latent factors and a global "communication between healthcare professionals" latent factor, bringing the 8 items linked to communication together. All the structural coefficients were highly significant (P < 0.001). CONCLUSIONS: This self-perception CSI scale assessing several facets of interprofessional communication is the first one developed in the French context. The development study exhibited excellent psychometric properties. Further psychometric analysis is needed to establish test-retest reliability, sensibility to change and concurrent validity.

Prognostic factors in patients with non resectable metastatic colorectal cancer in the era of targeted biotherapies: relevance of Köhne's risk classification.

BACKGROUND: Köhne's prognostic classification has been previously proposed, based on performance status, alkaline phosphatase level, number of metastatic sites and white blood cells count. AIMS: To identify prognostic factors for survival and to assess the validity of Köhne's classification, in the era of targeted biotherapies, in patients treated with chemotherapy for non resectable metastatic colorectal cancer. METHODS: A total of 290 consecutive patients were retrospectively identified in all gastroenterology units of one French county, between 2004 and 2008. Univariate and multivariate analysis for overall survival were performed using pre-treatment patient characteristics. RESULTS: All data were available for prognostic categorization in 133 patients. Median survival was 22.1 months. The distribution and median survival for Köhne's prognostic groups were as following: good (n=73; 24.8 months), intermediate (n=35; 24.2 months), and poor (n=25; 7.0 months). The survival difference was significant between good and poor prognostic groups (p<0.01) and between intermediate and poor prognostic groups (p<0.01), but not between good and intermediate prognostic groups (p=0.5). The two independent prognostic factors of survival in multivariate analysis were performance status 0/1 (p<0.01) and white blood cells count<10×10(9)/L (p<0.01). CONCLUSIONS: The relevance of Köhne's classification is questioned. A simplified score could be validated by largest studies, based on white blood cells count and performance status.

Relapse factors for ileal neuroendocrine tumours after curative surgery: a retrospective French multicentre study.

AIM: To evaluate the characteristics of postoperative relapse, predictive factors and time to relapse after curative surgery for well-differentiated neuroendocrine tumours of the ileum, without hepatic or other distant metastases. METHODS: Clinical data of patients entered into the Groupe d'étude des Tumeurs Endocrines database were collected and analysed retrospectively to identify factors predictive of relapse. RESULTS: Among 100 patients followed for a median of 56.5 (range 1-290) months, 42 relapsed after a median follow-up of 57.5 (range 6-176) months, with liver lesions in 27 (64.3%). Median disease-free survival (Kaplan-Meier) was 88 months (95% confidence interval 72-115). Disease-free survival was shorter for emergency surgery patients (p<0.01), patients with distant mesenteric lymph-node metastases (p<0.01), with fortuitous diagnosis (p=0.02), with tumour diameter >20mm (p=0.02), and those with multiple tumours (p=0.07). Multivariate analysis retained emergency surgery (odds-ratio 4.04 [95% confidence interval 2.01-8.11]), distant mesenteric lymph-node metastases (odds-ratio 2.53 [95% confidence interval 1.22-5.25]), and multiple tumours (odds-ratio 2.14 [95% confidence interval 1.01-4.50]), as being significantly associated with relapse. CONCLUSION: Patients who underwent emergency surgery, with distant mesenteric lymph-node metastases or with multiple ileal tumours relapsed earlier. Closer monitoring for the patients with these risk factors may be required.

Association between television in bedroom and adiposity throughout adolescence.

OBJECTIVE: The objective was to determine if having a television (TV) in the bedroom is associated with physical activity (PA), TV/video viewing, and adiposity throughout adolescence. RESEARCH METHODS AND PROCEDURES: Longitudinal data (September 2002 through June 2005) were analyzed of 379 initially 12-year-old French adolescents participating as controls in the Intervention Centered on Adolescents' Physical activity and Sedentary behavior (ICAPS). Presence of a TV set in the bedroom (TV(bedroom)) and leisure activities were obtained by questionnaire. There was annual assessment of BMI, waist circumference, and body fat by bioimpedance. RESULTS: In boys but not girls, baseline TV(bedroom) was associated with higher TV/video viewing over time [odds ratio (OR) of high TV/video = 1.87; 95% confidence interval, 1.2 to 2.8] and less no-sport club participation (OR = 0.59; 95% confidence interval, 0.35 to 1.0). Both boys and girls with baseline TV(bedroom) had lower reading time (p < 0.0001 in boys; p = 0.04 in girls), while PA did not differ according to TV(bedroom) for boys or for girls. For boys only, baseline TV(bedroom) was associated with higher BMI (mean BMI over time 20.5 +/- 0.5 vs. 19.0 +/- 0.5 kg/m(2); p = 0.001), waist circumference (70.9 +/- 0.9 vs. 67.2 +/- 0.8 cm; p < 0.001), and body fat (15.9 +/- 0.9% vs. 13.5 +/- 0.9%; p < 0.001), without interaction with time. These relationships remained significant after adjustment for socioeconomic status. TV/video viewing explained 26%, 42%, and 36% of the relationships of TV(bedroom) with BMI, waist circumference, and body fat, respectively, while addition of other leisure activities in the models only marginally reduced the effects. DISCUSSION: These results suggest the importance of keeping TV out of an adolescent's bedroom from an obesity prevention perspective but show gender differences.

T-cell activation is accompanied by an ubiquitination process occurring at the immunological synapse.

The immunological synapse (IS) is a specialized signaling area formed at the contact site between T-cells and antigen-presenting cells (APC), where sustained engagement and signaling of TCR and accessory molecules occur. A key feature of T-cell antigen recognition is that the process of TCR/peptide-MHC interaction is self-limited by the internalization and degradation of triggered TCR and recruited signaling components. The mechanism of signaling component degradation involves their ubiquitination and targeting for degradation. Yet, the relationship between the ubiquitination process and TCR signaling as well as the cellular localization of TCR-induced ubiquitination are still elusive. In the present work, we visualize for the first time ubiquitination at the TCR signaling area. We show an enrichment of ubiquitin staining in TCR/CD3 caps in T-lymphocytes stimulated by anti-CD3 antibodies. Remarkably, we also show the recruitment of the ubiquitin ligase Cbl-b and a significant ubiquitination at the immunological synapse in antigen-stimulated T-cells. Our results identify the immunological synapse as the cellular area where TCR-induced protein ubiquitination occurs. They imply that the synapse is a specialized site where the activation process is not only triggered, but also controlled via ubiquitination of signaling actors.

MAP kinase-dependent degradation of p27Kip1 by calpains in choroidal melanoma cells. Requirement of p27Kip1 nuclear export.

We investigated the status and the regulation of the cyclin-dependent kinases (CDK) inhibitor p27(Kip1) in a choroidal melanoma tumor-derived cell line (OCM-1). By contrast to normal choroidal melanocytes, the expression level of p27(Kip1) was low in these cells and the mitogen-activated protein (MAP) kinase pathway was constitutively activated. Genetic or chemical inhibition of this pathway induced p27(Kip1) accumulation, whereas MAP kinase reactivation triggered a down-regulation of p27(Kip1) that could be partially reversed by calpain inhibitors. In good accordance, ectopic expression of the cellular calpain inhibitor calpastatin led to an increase of endogenous p27(Kip1) expression. In vitro, p27(Kip1) was degraded by calpains, and OCM-1 cell extracts contained a calcium-dependent p27(Kip1) degradation activity. MAP kinase inhibition partially inhibited both calpain activity and calcium-dependent p27(Kip1) degradation by cellular extracts. Immunofluorescence labeling and subcellular fractionation revealed that p27(Kip1) was in part localized in the cytoplasmic compartment of OCM-1 cells but not of melanocytes, and accumulated into the nucleus upon MAP kinase inhibition. MAP kinase activation triggered a cytoplasmic translocation of the protein, as well as a change in its phosphorylation status. This CRM-1-dependent cytoplasmic translocation was necessary for MAP kinase- and calpain-dependent degradation. Taken together, these data suggest that in tumor-derived cells, p27(Kip1) could be degraded by calpains through a MAP kinase-dependent process, and that abnormal cytoplasmic localization of the protein, probably linked to modifications of its phosphorylation state, could be involved in this alternative mechanism of degradation.

Cell adhesion protects c-Raf-1 against ubiquitin-dependent degradation by the proteasome.

MAP kinase activation by growth factors depends on cell adhesion to the extracellular matrix. Disrupting the cell adhesion process in NIH 3T3 fibroblasts induced an almost complete inhibition of MAP kinase, which was impaired by proteasome inhibitors. In the absence of cell anchorage, c-Raf-1 expression was dramatically decreased after 24 h. This down-regulation was suppressed by proteasome inhibitors, suggesting that a proteasome-dependent degradation of Raf occurred in the absence of cell adhesion. Proteasome inhibitors did not affect Raf-1 levels in adherent cells, indicating that this degradation only occurred in the absence of cell adhesion. Finally, ectopic coexpression of Raf-1 and ubiquitin in HEK-293 and NIH 3T3 cells generated ubiquitylated forms of Raf-1, both in adherent and suspended cells, suggesting a possible ubiquitin-dependent degradation of the protein.