Using next-generation sequencing for high resolution multiplex analysis of copy number variation from nanogram quantities of DNA from formalin-fixed paraffin-embedded specimens

The use of next-generation sequencing technologies to produce genomic copy number data has recently been described. Most approaches, however, reply on optimal starting DNA, and are therefore unsuitable for the analysis of formalin-fixed paraffin-embedded (FFPE) samples, which largely precludes the analysis of many tumour series. We have sought to challenge the limits of this technique with regards to quality and quantity of starting material and the depth of sequencing required. We confirm that the technique can be used to interrogate DNA from cell lines, fresh frozen material and FFPE samples to assess copy number variation. We show that as little as 5 ng of DNA is needed to generate a copy number karyogram, and follow this up with data from a series of FFPE biopsies and surgical samples. We have used various levels of sample multiplexing to demonstrate the adjustable resolution of the methodology, depending on the number of samples and available resources. We also demonstrate reproducibility by use of replicate samples and comparison with microarray-based comparative genomic hybridization (aCGH) and digital PCR. This technique can be valuable in both the analysis of routine diagnostic samples and in examining large repositories of fixed archival material

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Le macrophage dans l’arthrite réactionnelle à Chlamydia chez la souris SKG

Objective: The mechanisms underlying Chlamydia-induced reactive arthritis are poorly understood. Macrophages have recently come to light as key effectors in the SKG mouse model, without precising the exact mechanisms. TNFα has also been suggested to play a pivotal role in clinical manifestations. We hypothesize that infection of macrophages induces autophagy and/or endoplasmic reticulum stress leading to an increased production of pro-inflammatory cytokines, including IL-23, leading to arthritis, and that TNFα plays a role in its clinical expression. Methods: We depleted macrophages in a group of SKG mice after infection by Chlamydia muridarum, by injection of clodronate. Infection and macrophage depletion were confirmed, compared to a control group. Arthritis was evaluated clinically for 12 weeks, and confirmed by histology upon sacrifice. Arthritis was similarly evaluated after TNFα blockade by treating two groups of infected SKG mice with certolizumab pegol or etanercept. Autophagy, endoplasmic reticulum stress and IL-23 production were analyzed by RT-qPCR in the genital tracts and joints of infected SKG mice. Results: Macrophage depletion prevents C. muridarum-induced reactive arthritis in SKG mice. TNFα blockade both preventively and therapeutically reduces the intensity of the arthritis. Increased autophagy is correlated to a rise in IL-23 production in the genital tracts of infected SKG mice, 1 week post-infection, whereas endoplasmic reticulum stress remains stable. The inverse correlation was found in the joints of infected SKG mice, 5 weeks post-infection. Conclusion: Collectively, our results show the importance of macrophages in the physiopathology of Chlamydia-induced reactive arthritis in SKG mice, probably via an activation of autophagy leading to increased IL-23 production initially. TNFα plays a pivotal role secondarily with its role in tissue damage and inflammation.Objectif : La physiopathologie de l’arthrite réactionnelle à Chlamydia est encore mal comprise. Le rôle du macrophage a récemment été évoqué dans le modèle de la souris SKG. L’importance du TNFα a également été suggérée. Nous émettons l’hypothèse que l’infection des macrophages induit un phénomène d’autophagie et/ou du stress du réticulum endoplasmique entraînant une majoration de la production de cytokines, dont l’IL-23, responsable de l’arthrite, et que le TNFα joue un rôle dans la symptomatologie clinique de l’arthrite. Méthodes : Après infection par Chlamydia muridarum, certaines souris SKG ont subit une déplétion en macrophages, d'autres ont été traités par anti-TNFα. La survenue d’arthrite a été évaluée cliniquement de manière hebdomadaire, puis histologiquement lors de l’euthanasie à 12 semaines post-infection. L’autophagie, le stress du réticulum endoplasmique et la production d’IL-23 ont été évalués par RT-qPCR dans les tractus génitaux et dans les articulations des souris SKG infectées. Résultats : La déplétion en macrophages empêche l’apparition de l’arthrite réactionnelle. Le blocage du TNFα permet une diminution de la sévérité de l’arthrite. Il existe une surproduction d’IL-23 corrélée à une augmentation de l’autophagie dans les tractus génitaux des souris SKG infectées, 1 semaine post-infection, sans modification du stress du reticulum endoplasmique. Conclusion : Nos résultats montrent l’importance du macrophage dans la physiopathologie de l’arthrite réactionnelle à C. muridarum chez la souris SKG, par une activation de l’autophagie à l’origine d’une surproduction d’IL-23. Le TNFα est fondamental pour les manifestations inflammatoires de l'arthrite

QUARANTE ADENOMES PRODUCTEURS D'ALDOSTERONE OPERES (CONFRONTATION DE L'ANATOMO-PATHOLOGIE AUX RESULTATS DE L'ENQUETE HORMONALE, TOMODENSITOMETRIQUE ET SCINTIGRAPHIQUE)

LILLE2-BU Santé-Recherche (593502101) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

Rare causes of hypoglycemia in adults

International audienceHypoglycemia is defined by a low blood glucose level associated to clinical symptoms. Hypoglycemia may be related to treatment of diabetes, but also to drugs, alcohol, critical illness, cortisol insufficiency including hypopituitarism, insulinoma, bariatric or gastric surgery, pancreas transplantation or glucagon deficiency, or may be surreptitious. Some hypoglycemic episodes remain unexplained, and genetic, paraneoplastic and immune causes should be considered. Genetic causes may be related to endogenous hyperinsulinism and to inborn errors of metabolism (IEM). Endogenous hyperinsulinism is related to monogenic congenital hyperinsulinism, and especially to mutations of the glucokinase-activating gene or of insulin receptors, both characterised by postprandial hypoglycemia with major hyperinsulinism. In adulthood, IEM-related hypoglycemia can persist in a previously diagnosed childhood disease or may be a presenting sign. It is suggested by systemic involvement (rhabdomyolysis after fasting or exercising, heart disease, hepatomegaly), sometimes associated to a family history of hypoglycemia. The timing of hypoglycemic episodes with respect to the last meal also helps to orientate diagnosis. Fasting hypoglycemia may be related to type 0, I or III glycogen synthesis disorder, fatty acid oxidation or gluconeogenesis disorder. Postprandial hypoglycemia may be related to inherited fructose intolerance. Exercise-induced hyperinsulinism is mainly related to activating mutation of the SLC16A1 gene. Besides exceptional ectopic insulin secretion, paraneoplastic causes involve NICTH (Non-Islet-Cell Tumour Hypoglycemia), caused by Big-IGF2 secretion by a large tumour, with low blood levels of insulin, C-peptide and IGF1. Autoimmune causes involve antibodies against insulin (HIRATA syndrome), especially in case of Graves' disease, or against the insulin receptor. Medical history, timing, and insulin level orientate the diagnosis

Plasma Methionine and Clinical Severity in Nitrous Oxide Consumption

In the last few years, there has been an increase in the recreational use of nitrous oxide (N2O), which can lead to neurological symptoms such as sensory or motor disorders. The literature links these symptoms to a functional inactivation of vitamin B12 by oxidation of its cobalt ion, which prevents the vitamin B12 from acting as a cofactor for methionine synthase. Thus, demyelination related to methionine deficiency could be responsible for the neurological disorders associated with N2O consumption, including the combined sclerosis of the spinal cord. We aimed to study the correlation between the plasma methionine levels and clinical severity observed in N2O users. We retrospectively collected clinical and biological data from 93 patients who chronically consumed N2O. The patients were divided into four groups based of the severity of their clinical symptoms (based on their Peripheral Neuropathy Disability (PND) score). The plasma amino acids measurement, including methionine, were performed systematically by liquid chromatography coupled with mass spectrometry. Plasma methionine is significantly correlated with the clinical severity (Spearman coefficient: −0.42; p-value −5), however, the average methionine level in the four groups is within the physiological values (N: 16–23 µmol/L). There is a significant inverse correlation between plasma methionine and homocysteine (Spearman coefficient: −0.57; p-value −9), which confirms the action of nitrous oxide on the methionine synthase. A decrease in plasma methionine cannot be imputed as the only mechanism involved in the pathophysiology of the neurological disorders in nitrous oxide consumption. In addition, there are few therapeutic indications for the use of methionine. Thus, we should be careful concerning the potential use of methionine in nitrous oxide consumption. As a consequence, other pathophysiological mechanisms probably need to be identified in order to find potential therapeutic targets

Caractérisation des défauts cristallins de type sphérulite dans un verre métallique massif TiCuZrPd et impacts sur ses propriétés mécaniques et électrochimiques

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eCHORD as a tool to characterize internal misorientation of strained samples

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