SummaryRecent studies highlighted the importance of astrocytes in neuroinflammatory diseases, interacting closely with other CNS cells but also with the immune system. However, due to the difficulty in obtaining human astrocytes, their role in these pathologies is still poorly characterized. Here, we develop a serum-free protocol to differentiate human induced pluripotent stem cells (hiPSCs) into astrocytes. Gene expression and functional assays show that our protocol consistently yields a highly enriched population of resting mature astrocytes across the 13 hiPSC lines differentiated. Using this model, we first highlight the importance of serum-free media for astrocyte culture to generate resting astrocytes. Second, we assess the astrocytic response to IL-1β, TNF-α, and IL-6, all cytokines important in neuroinflammation, such as multiple sclerosis. Our study reveals very specific profiles of reactive astrocytes depending on the triggering stimulus. This model provides ideal conditions for in-depth and unbiased characterization of astrocyte reactivity in neuroinflammatory conditions.
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j Anti-Aspergillus IgG antibodies are important biomarkers for the diagnosis of chronic pulmonary aspergillosis (CPA) and allergic bronchopulmonary aspergillosis (ABPA). We compared the performance of a new commercial enzyme immunoassay (EIA) (Bordier Affinity Products) with that of the Bio-Rad and VirionگSerion EIAs. This assay is novel in its association of two recombinant antigens with somatic and metabolic antigens of Aspergillus fumigatus. In a prospective multicenter study, 436 serum samples from 147 patients diagnosed with CPA (136 samples/104 patients) or ABPA (94 samples/43 patients) and from 205 controls (206 samples) were tested. We obtained sensitivities of 97%, 91.7%, and 86.1%, and specificities of 90.3%, 91.3%, and 81.5% for the Bordier, Bio-Rad, and VirionگSerion tests, respectively. The Bordier kit was more sensitive than the Bio-Rad kit (P < 0.01), which was itself more sensitive than the VirionگSerion kit (P ؍ 0.04). The Bordier and Bio-Rad kits had similar specificity (P ؍ 0.8), both higher than that of the VirionگSerion kit (P ؍ 0.02). The area under the receiver operating characteristic (ROC) curves confirmed the superiority of the Bordier kit over the Bio-Rad and the VirionگSerion kits (0.977, 0.951, and 0.897, respectively; P < 0.01 for each comparison). In a subset analysis of 279 serum samples tested with the Bordier and Bio-Rad kits and an in-house immunoprecipitin assay (IPD), the Bordier kit had the highest sensitivity (97.7%), but the IPD tended to be more specific (71.2 and 84.7%, respectively; P ؍ 0.10). The use of recombinant, somatic, and metabolic antigens in a single EIA improved the balance of sensitivity and specificity, resulting in an assay highly suitable for use in the diagnosis of chronic and allergic aspergillosis.
Blood-brain barrier (BBB) breakdown and immune cell infiltration into the central nervous system (CNS) are early hallmarks of multiple sclerosis (MS). The mechanisms leading to BBB dysfunction are incompletely understood and generally thought to be a consequence of neuroinflammation. Here, we have challenged this view and asked if intrinsic alterations in the BBB of MS patients contribute to MS pathogenesis. To this end, we made use of human induced pluripotent stem cells (hiPSCs) derived from healthy controls (HC) and MS patients and differentiated them into brain microvascular endothelial cell (BMEC)-like cells as in vitro model of the BBB. MS-derived BMEC-like cells showed impaired junctional integrity, barrier properties and efflux pump activity when compared to HC. Also, MS-derived BMEC-like cells displayed an inflammatory phenotype with increased adhesion molecule expression and immune cell interactions. Activation of Wnt/β-catenin signaling in MS-derived endothelial progenitor cells enhanced barrier characteristics and reduced the inflammatory phenotype. Our study provides evidence for an intrinsic impairment of BBB function in MS patients that can be modeled in vitro. Human iPSC-derived BMEC-like cells are thus suitable to explore the molecular underpinnings of BBB dysfunction in MS and will assist in the identification of potential novel therapeutic targets for BBB stabilization.
ObjectiveCoronavirus disease (COVID-19) has been associated with a large variety of neurologic disorders. However, the mechanisms underlying these neurologic complications remain elusive. In this study, we aimed at determining whether neurologic symptoms were caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) direct infection or by either systemic or local proinflammatory mediators.MethodsIn this cross-sectional study, we checked for SARS-CoV-2 RNA by quantitative reverse transcription PCR, SARS-CoV-2–specific antibodies, and 49 cytokines/chemokines/growth factors (by Luminex) in the CSF +/− sera of a cohort of 22 COVID-19 patients with neurologic presentation and 55 neurologic control patients (inflammatory neurologic disorder [IND], noninflammatory neurologic disorder, and MS).ResultsWe detected anti–SARS-CoV-2 immunoglobulin G in patients with severe COVID-19 with signs of intrathecal synthesis for some of them. Of the 4 categories of tested patients, the CSF of IND exhibited the highest level of cytokines, chemokines, and growth factors. By contrast, patients with COVID-19 did not present overall upregulation of inflammatory mediators in the CSF. However, patients with severe COVID-19 (intensive care unit patients) exhibited higher concentrations of CCL2, CXCL8, and vascular endothelium growth factor A (VEGF-A) in the CSF than patients with a milder form of COVID-19. In addition, we could show that intrathecal CXCL8 synthesis was linked to an elevated albumin ratio and correlated with the increase of peripheral inflammation (serum hepatocyte growth factor [HGF] and CXCL10).ConclusionsOur results do not indicate active replication of SARS-CoV-2 in the CSF or signs of massive inflammation in the CSF compartment but highlight a specific impairment of the neurovascular unit linked to intrathecal production of CXCL8.
Endometriosis is a multifactorial disease with possible genetic predisposition and involvement of environmental factors in its pathogenesis. The genetic polymorphism of glutathione S-transferase M1 (GSTM1) gene, which codes for glutathione S-transferase 1, class mu foreign compound conjugating enzyme of phase II detoxification system, was studied by polymerase chain reaction from the blood spots in patients with different stages of endometriosis (n = 50) and in controls (n = 72) of French origin. A total of 86.0% of patients appeared to lack GSTM1 enzyme activity due to the presence of an extended deletion (GSTM1 0/0 genotype), compared with 45.8% in a control group (P < 0.0001), which was consistent with the frequency of GSTM1 deletion in French population. Moreover, the distribution of GSTM1-active genotypes was significantly different in patients and controls (P < 0.0001), as no patient with GSTM1A/B genotype, which is correlated with the highest activity of GSTM1 enzyme, has been found so far (18.1% in a control group). The unusually high frequency of homozygotes for the GSTM1 gene deletion among patients with endometriosis suggests a possible contribution of environmental toxins in the pathogenesis of this disease due to the absence or low activity of GSTM1 enzyme.
Objective:To evaluate the long-term effects of treatments used in MS on the T-cell trafficking profile.Methods:We enrolled 83 patients with MS under fingolimod (FTY), natalizumab (NTZ), dimethyl fumarate (DMF), or other disease-modifying treatments (DMTs). Blood was drawn before treatment onset and up to 36–48 months. The ex vivo expression of CNS-related integrins (α4β1 and αL subunit of LFA-1) and the gut-related integrin (α4β7) was assessed using flow cytometry on CD4+ and CD8+ T cells. The adhesion profiles of CD3+ T cells to specific integrin ligands (vascular cell adhesion molecule-1 [VCAM-1], intercellular adhesion molecule-1 [ICAM-1], and mucosal vascular addressin cell adhesion molecule-1 [MAdCAM-1]) were measured in vitro before and after 12 and 36–48 months.Results:NTZ decreased the frequency of α4β1+ and α4β7+ integrin expressing T cells and the binding of these cells to VCAM-1 and MAdCAM-1, respectively. After 12 months, DMF induced a decreased frequency of αLhighCD4+ T cells combined with reduced binding to ICAM-1. By contrast, with FTY, there was a doubling of the frequency of α4β1+ and αLhigh, but a decreased frequency of α4β7+ T cells. Strikingly, the binding of α4β1+, α4β7+, and to a lesser extent of αLhigh T cells to VCAM-1, MAdCAM-1, and ICAM-1, respectively, was decreased at month 12 under FTY treatment. The presence of manganese partially restored the binding of these T cells to VCAM-1 in vitro, suggesting that FTY interferes with integrin activation.Conclusions:In addition to NTZ, DMF and FTY but not other tested DMTs may also decrease T-cell–mediated immune surveillance of the CNS. Whether this mechanism may contribute to the onset of CNS opportunistic infections remains to be shown.
Summary This protocol describes how to obtain human astrocytes from human-induced pluripotent stem cells (hiPSCs) in chemically defined media, without the use of fetal bovine serum (FBS). FBS eases the differentiation of astrocytes but also deeply alters their phenotype, as compared with their in vivo counterparts. Our protocol generates hiPSC-derived astrocytes displaying a phenotype and functions similar to human primary astrocytes, including adequate response to inflammation, neurotransmitter uptake, and trophic support to neurons. For complete details on the use and execution of this protocol, please refer to Perriot et al. (2018) .
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