JAK inhibition in Aicardi-Goutières syndrome: a monocentric multidisciplinary real-world approach study

International audienceThe paradigm type I interferonopathy Aicardi-Goutières syndrome (AGS) is most typically characterized by severe neurological involvement. AGS is considered an immune-mediated disease, poorly responsive to conventional immunosuppression. Premised on a chronic enhancement of type I interferon signaling, JAK1/2 inhibition has been trialed in AGS, with clear improvements in cutaneous and systemic disease manifestations. Contrastingly, treatment efficacy at the level of the neurological system has been less conclusive. Here, we report our real-word approach study of JAK1/2 inhibition in 11 patients with AGS, providing extensive assessments of clinical and radiological status; interferon signaling, including in cerebrospinal fluid (CSF); and drug concentrations in blood and CSF. Over a median follow-up of 17 months, we observed a clear benefit of JAK1/2 inhibition on certain systemic features of AGS, and reproduced results reported using the AGS neurologic severity scale. In contrast, there was no change in other scales assessing neurological status; using the caregiver scale, only patient comfort, but no other domain of everyday-life care, was improved. Serious bacterial infections occurred in 4 out of the 11 patients. Overall, our data lead us to conclude that other approaches to treatment are urgently required for the neurologic features of AGS. We suggest that earlier diagnosis and adequate central nervous system penetration likely remain the major factors determining the efficacy of therapy in preventing irreversible brain damage, implying the importance of early and rapid genetic testing and the consideration of intrathecal drug delivery

Mutations in KEOPS-Complex Genes Cause Nephrotic Syndrome with Primary Microcephaly

Galloway-Mowat syndrome (GAMOS) is an autosomal-recessive disease characterized by the combination of early-onset nephrotic syndrome (SRNS) and microcephaly with brain anomalies. Here we identified recessive mutations in OSGEP, TP53RK, TPRKB, and LAGE3, genes encoding the four subunits of the KEOPS complex, in 37 individuals from 32 families with GAMOS. CRISPR-Cas9 knockout in zebrafish and mice recapitulated the human phenotype of primary microcephaly and resulted in early lethality. Knockdown of OSGEP, TP53RK, or TPRKB inhibited cell proliferation, which human mutations did not rescue. Furthermore, knockdown of these genes impaired protein translation, caused endoplasmic reticulum stress, activated DNA-damage-response signaling, and ultimately induced apoptosis. Knockdown of OSGEP or TP53RK induced defects in the actin cytoskeleton and decreased the migration rate of human podocytes, an established intermediate phenotype of SRNS. We thus identified four new monogenic causes of GAMOS, describe a link between KEOPS function and human disease, and delineate potential pathogenic mechanisms

Apport du modele de peau humaine reconstruite in vitro pour la pharmacologie cutanee des retinoides. Application a la peau humaine normale, au psoriasis et au vieillissement photo-induit

SIGLEAvailable from INIST (FR), Document Supply Service, under shelf-number : T 78433 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Omics approach to characterize milk-derived extracellular vesicles/exosomes isolated from goats expressing or not alphas1-CN

International audienceBackgroundGoats homozygous for a null allele (O/O) at the CSN1S1 locus encoding αs1-casein display a chronic endoplasmic reticulum (ER) stress (Unfolded Protein Response) due to an accumulation of the other caseins in this compartment, thus triggering a general MEC dysfunction with a strong impact on milk composition. Milk-derived extracellular vesicles (MEVs) contain molecular information, which are thus recognized as mediators of intercellular communication. We made the assumption that the absence of CSN1S1 expression may influence MEVs cargos including miRNA, proteins, lipids and metabolites. The objective of this study was to assess the impact of αs1-casein deficit on MEVs cargos and to compare the biological material they convey.Material & MethodsWe have developed an improved method based on a density gradient ultracentrifugation to isolate MEVs. The quality of MEVs was analyzed morphologically by transmission electron microscopy (TEM) with negative staining (uranyl acetate), the specific “exosome” protein markers were detected by Western blot and ELISA and the size distribution and particle concentration were measured by NTA. The MEV’s proteome was acquired by LC-MS/MS and nucleic acid content (mRNA and miRNA) by NGS and qPCR. EV lipid content was extracted with the MTBE method. Data acquisition was performed by an Orbitrap-MS and analyzed using Lipid Data Analyzer. MEV metabolites were extracted using MPLEx protocol, data acquired by LC coupled with HRMS and analyzed using Metaboanalyst online workbenches. Differential statistical and bioinformatic analyses were performed using appropriate softwares.ResultsThe novel purification method gives MEV populations, free of contamination by other EVs and milk components, at sufficient concentrations to perform subsequent analyses. Nearly 280 proteins involved in the biogenesis of exosomes and MVB formation, their adhesion and internalization as well as proteins associated with membrane transport and enzymes involved in cellular metabolism were identified, among which 41 exosomal proteins differed between CSN1S1 O/O (null) and A/A (wildtype) genotypes. Ongoing profiling of RNA from MEVs has already identified over 230 miRNA and confirmed MEC origin due to the presence of mRNA encoding specific major milk proteins. The comparison of exosomal miRNomes of goat homozygous for A and O alleles at the CSN1S1 locus pointed out 15 miRNAs differentially abundant, potentially related to the MEC phenotype. Sphingomyelin and phosphatidylcholin were the major phospholipids observed in MEV populations. We have totally identified ca. 4,000 compounds using pHILIC and RPLC, 79 of which were significantly up or down-regulated in studied genotypes.ConclusionSeveral differences distinguishing goats according to the genotype at the CSN1S1 locus were found at each level of the omic analysis of MEVs. Differentially abundant miRNAs and transcriptome analyses are in agreement with UPR phenotype and confirmed their involvement in post-transcriptional regulatory mechanisms

Expression of calcineurin activity after lung transplantation: a 2-year follow-up.

The objective of this pharmacodynamic study was to longitudinally assess the activity of calcineurin during the first 2 years after lung transplantation. From March 2004 to October 2008, 107 patients were prospectively enrolled and their follow-up was performed until 2009. Calcineurin activity was measured in peripheral blood mononuclear cells. We report that calcineurin activity was linked to both acute and chronic rejection. An optimal activity for calcineurin with two thresholds was defined, and we found that the risk of rejection was higher when the enzyme activity was above the upper threshold of 102 pmol/mg/min or below the lower threshold of 12 pmol/mg/min. In addition, we report that the occurrence of malignancies and viral infections was significantly higher in patients displaying very low levels of calcineurin activity. Taken together, these findings suggest that the measurement of calcineurin activity may provide useful information for the management of the prevention therapy of patients receiving lung transplantation

Fasting upregulates the monocarboxylate transporter MCT1 at the rat blood-brain barrier through PPAR δ activation

International audienceBackground: The blood-brain barrier (BBB) is pivotal for the maintenance of brain homeostasis and it strictly regulates the cerebral transport of a wide range of endogenous compounds and drugs. While fasting is increasingly recognized as a potential therapeutic intervention in neurology and psychiatry, its impact upon the BBB has not been studied. This study was designed to assess the global impact of fasting upon the repertoire of BBB transporters.Methods: We used a combination of in vivo and in vitro experiments to assess the response of the brain endothelium in male rats that were fed ad libitum or fasted for one to three days. Brain endothelial cells were acutely purified and transcriptionaly profiled using RNA-Seq. Isolated brain microvessels were used to assess the protein expression of selected BBB transporters through western blot. The molecular mechanisms involved in the adaptation to fasting were investigated in primary cultured rat brain endothelial cells. MCT1 activity was probed by in situ brain perfusion. Results: Fasting did not change the expression of the main drug efflux ATP-binding cassette transporters or P-glycoprotein activity at the BBB but modulated a restrictive set of solute carrier transporters. These included the ketone bodies transporter MCT1, which is pivotal for the brain adaptation to fasting. Our findings in vivo suggested that PPAR δ, a major lipid sensor, was selectively activated in brain endothelial cells in response to fasting. This was confirmed in vitro where pharmacological agonists and free fatty acids selectively activated PPAR δ, resulting in the upregulation of MCT1 expression. Moreover, dosing rats with a specific PPAR δ antagonist blocked the upregulation of MCT1 expression and activity induced by fasting.Conclusions: Altogether, our study shows that fasting affects a selected set of BBB transporters which does not include the main drug efflux transporters. Moreover, we describe a previously unknown selective adaptive response of the brain vasculature to fasting which involves PPAR δ and is responsible for the up-regulation of MCT1 expression and activity. Our study opens new perspectives for the metabolic manipulation of the BBB in the healthy or diseased brain

The equilibrative nucleoside transporter ENT1 is critical for nucleotide homeostasis and optimal erythropoiesis

This is a related article to: Nucleoside ENTry modulates erythropoiesis (cf ci-dessous)International audienceAbstract The tight regulation of intracellular nucleotides is critical for the self-renewal and lineage specification of hematopoietic stem cells (HSCs). Nucleosides are major metabolite precursors for nucleotide biosynthesis and their availability in HSCs is dependent on their transport through specific membrane transporters. However, the role of nucleoside transporters in the differentiation of HSCs to the erythroid lineage and in red cell biology remains to be fully defined. Here, we show that the absence of the equilibrative nucleoside transporter (ENT1) in human red blood cells with a rare Augustine-null blood type is associated with macrocytosis, anisopoikilocytosis, an abnormal nucleotide metabolome, and deregulated protein phosphorylation. A specific role for ENT1 in human erythropoiesis was demonstrated by a defective erythropoiesis of human CD34+ progenitors following short hairpin RNA-mediated knockdown of ENT1. Furthermore, genetic deletion of ENT1 in mice was associated with reduced erythroid progenitors in the bone marrow, anemia, and macrocytosis. Mechanistically, we found that ENT1-mediated adenosine transport is critical for cyclic adenosine monophosphate homeostasis and the regulation of erythroid transcription factors. Notably, genetic investigation of 2 ENT1null individuals demonstrated a compensation by a loss-of-function variant in the ABCC4 cyclic nucleotide exporter. Indeed, pharmacological inhibition of ABCC4 in Ent1−/− mice rescued erythropoiesis. Overall, our results highlight the importance of ENT1-mediated nucleotide metabolism in erythropoiesis

Impaired lymphocyte function and differentiation in CTPS1-deficient patients result from a hypomorphic homozygous mutation

International audienceCytidine triphosphate (CTP) synthetase 1 (CTPS1) deficiency is caused by a unique homozygous frameshift splice mutation (c.1692-1G>C, p.T566Dfs26X). CTPS1-deficient patients display severe bacterial and viral infections. CTPS1 is responsible for CTP nucleotide de novo production involved in DNA/RNA synthesis. Herein, we characterized in depth lymphocyte defects associated with CTPS1 deficiency. Immune phenotyping performed in 7 patients showed absence or low numbers of mucosal-associated T cells, invariant NKT cells, memory B cells, and NK cells, whereas other subsets were normal. Proliferation and IL-2 secretion by T cells in response to TCR activation were markedly decreased in all patients, while other T cell effector functions were preserved. The CTPS1 T566Dfs26X mutant protein was found to be hypomorphic, resulting in 80%-90% reduction of protein expression and CTPS activity in cells of patients. Inactivation of CTPS1 in a T cell leukemia fully abolished cell proliferation. Expression of CTPS1 T566Dfs26X failed to restore proliferation of CTPS1-deficient leukemia cells to normal, except when forcing its expression to a level comparable to that of WT CTPS1. This indicates that CTPS1 T566Dfs26X retained normal CTPS activity, and thus the loss of function of CTPS1 T566Dfs26X is completely attributable to protein instability. This study supports that CTPS1 represents an attractive therapeutic target to selectively inhibit pathological T cell proliferation, including lymphoma