Assessing the influence of distinct culture media on human pre-implantation development using single-embryo transcriptomics

The use of assisted reproductive technologies is consistently rising across the world. However, making an informed choice on which embryo culture medium should be preferred to ensure satisfactory pregnancy rates and the health of future children critically lacks scientific background. In particular, embryos within their first days of development are highly sensitive to their micro-environment, and it is unknown how their transcriptome adapts to different embryo culture compositions. Here, we determined the impact of culture media composition on gene expression in human pre-implantation embryos. By employing single-embryo RNA-sequencing after 2 or 5 days of the post-fertilization culture in different commercially available media (Ferticult, Global, and SSM), we revealed medium-specific differences in gene expression changes. Embryos cultured pre-compaction until day 2 in Ferticult or Global media notably displayed 266 differentially expressed genes, which were related to essential developmental pathways. Herein, 19 of them could have a key role in early development, based on their previously described dynamic expression changes across development. When embryos were cultured after day 2 in the same media considered more suitable because of its amino acid enrichment, 18 differentially expressed genes thought to be involved in the transition from early to later embryonic stages were identified. Overall, the differences were reduced at the blastocyst stage, highlighting the ability of embryos conceived in a suboptimal in vitro culture medium to mitigate the transcriptomic profile acquired under different pre-compaction environments

Assessing the influence of distinct culture media on human pre-implantation development using single-embryo transcriptomics

The use of assisted reproductive technologies is consistently rising across the world. However, making an informed choice on which embryo culture medium should be preferred to ensure satisfactory pregnancy rates and the health of future children critically lacks scientific background. In particular, embryos within their first days of development are highly sensitive to their micro-environment, and it is unknown how their transcriptome adapts to different embryo culture compositions. Here, we determined the impact of culture media composition on gene expression in human pre-implantation embryos. By employing single-embryo RNA-sequencing after 2 or 5 days of the post-fertilization culture in different commercially available media (Ferticult, Global, and SSM), we revealed medium-specific differences in gene expression changes. Embryos cultured pre-compaction until day 2 in Ferticult or Global media notably displayed 266 differentially expressed genes, which were related to essential developmental pathways. Herein, 19 of them could have a key role in early development, based on their previously described dynamic expression changes across development. When embryos were cultured after day 2 in the same media considered more suitable because of its amino acid enrichment, 18 differentially expressed genes thought to be involved in the transition from early to later embryonic stages were identified. Overall, the differences were reduced at the blastocyst stage, highlighting the ability of embryos conceived in a suboptimal in vitro culture medium to mitigate the transcriptomic profile acquired under different pre-compaction environments

Cardiac manifestations of MIS-C: cardiac magnetic resonance and speckle-tracking data

BackgroundCardiac involvement is central in MIS-C and represents the main cause of morbidity. In this study, we aimed to assess myocardial damage in patients with MIS-C using cardiac magnetic resonance (CMR) during the acute phase, as well as left ventricular and atrial longitudinal strain on admission, at discharge, and after 3 months.MethodsWe performed a single-center prospective cohort study and case–control study. Between September 2020 and February 2022, we enrolled 39 patients hospitalized for MIS-C at our center. We performed left ventricular and atrial longitudinal 2D strain analysis on admission and during follow-up; echocardiographic data were compared to a matched control population. Patients above 4 years old with increased troponin underwent CMR.ResultsOf 24 patients (mean age: 8.2 ± 4.9 years) who underwent CMR, 14 (58%) presented myocardial edema and 6 (25%) late gadolinium enhancement (LGE). LGE was associated with older age (p < 0.01), increased BMI (p = 0.03), increased ferritin levels (p < 0.001), lower left ventricular (LV) ejection fraction (p < 0.001), LV longitudinal strain (p = 0.004), left atrial (LA) strain (p = 0.05), and prolonged hospital stay (p = 0.02). On admission, LV ejection fraction, LV longitudinal strain, and LA strain were impaired, but each improved gradually over time; LVEF was the fastest to recover, while global LV longitudinal strain was still impaired as compared to controls after 3 months (p = 0.01).ConclusionOur study demonstrates that myocardial injury is present in a quarter of MIS-C patients, and impaired LA and LV myocardial deformation persist for at least several weeks after the acute phase. CMR and LV/LA strain could help us to individualize follow-up of MIS-C patients

Flavonoid profiling and transcriptome analysis reveals new gene–metabolite correlations in tubers of Solanum tuberosum L.

Anthocyanin content of potato tubers is a trait that is attracting increasing attention as the potential nutritional benefits of this class of compound become apparent. However, our understanding of potato tuber anthocyanin accumulation is not complete. The aim of this study was to use a potato microarray to investigate gene expression patterns associated with the accumulation of purple tuber anthocyanins. The advanced potato selections, CO97216-3P/PW and CO97227-2P/PW, developed by conventional breeding procedures, produced tubers with incomplete expression of tuber flesh pigmentation. This feature permits sampling pigmented and non-pigmented tissues from the same tubers, in essence, isolating the factors responsible for pigmentation from confounding genetic, environmental, and developmental effects. An examination of the transcriptome, coupled with metabolite data from purple pigmented sectors and from non-pigmented sectors of the same tuber, was undertaken to identify these genes whose expression correlated with elevated or altered polyphenol composition. Combined with a similar study using eight other conventional cultivars and advanced selections with different pigmentation, it was possible to produce a refined list of only 27 genes that were consistently differentially expressed in purple tuber tissues compared with white. Within this list are several new candidate genes that are likely to impact on tuber anthocyanin accumulation, including a gene encoding a novel single domain MYB transcription factor

Integrative epigenomics in Sjögren´s syndrome reveals novel pathways and a strong interaction between the HLA, autoantibodies and the interferon signature

Primary Sjögren's syndrome (SS) is a systemic autoimmune disease characterized by lymphocytic infiltration and damage of exocrine salivary and lacrimal glands. The etiology of SS is complex with environmental triggers and genetic factors involved. By conducting an integrated multi-omics study, we confirmed a vast coordinated hypomethylation and overexpression effects in IFN-related genes, what is known as the IFN signature. Stratified and conditional analyses suggest a strong interaction between SS-associated HLA genetic variation and the presence of Anti-Ro/SSA autoantibodies in driving the IFN epigenetic signature and determining SS. We report a novel epigenetic signature characterized by increased DNA methylation levels in a large number of genes enriched in pathways such as collagen metabolism and extracellular matrix organization. We identified potential new genetic variants associated with SS that might mediate their risk by altering DNA methylation or gene expression patterns, as well as disease-interacting genetic variants that exhibit regulatory function only in the SS population. Our study sheds new light on the interaction between genetics, autoantibody profiles, DNA methylation and gene expression in SS, and contributes to elucidate the genetic architecture of gene regulation in an autoimmune population

Complement component C4 structural variation and quantitative traits contribute to sex-biased vulnerability in systemic sclerosis

Altres ajuts: Fondo Europeo de Desarrollo Regional (FEDER), "A way of making Europe".Copy number (CN) polymorphisms of complement C4 play distinct roles in many conditions, including immune-mediated diseases. We investigated the association of C4 CN with systemic sclerosis (SSc) risk. Imputed total C4, C4A, C4B, and HERV-K CN were analyzed in 26,633 individuals and validated in an independent cohort. Our results showed that higher C4 CN confers protection to SSc, and deviations from CN parity of C4A and C4B augmented risk. The protection contributed per copy of C4A and C4B differed by sex. Stronger protection was afforded by C4A in men and by C4B in women. C4 CN correlated well with its gene expression and serum protein levels, and less C4 was detected for both in SSc patients. Conditioned analysis suggests that C4 genetics strongly contributes to the SSc association within the major histocompatibility complex locus and highlights classical alleles and amino acid variants of HLA-DRB1 and HLA-DPB1 as C4-independent signals

Serum profiling identifies CCL8, CXCL13, and IL-1RA as markers of active disease in patients with systemic lupus erythematosus

IntroductionSystemic lupus erythematosus (SLE) is a clinically heterogeneous disease that presents a challenge for clinicians. To identify potential biomarkers for diagnosis and disease activity in SLE, we investigated a selected yet broad panel of cytokines and autoantibodies in patients with SLE, healthy controls (HC), and patients with other autoimmune diseases (AIDs).MethodsSerum samples from 422 SLE patients, 546 HC, and 1223 other AIDs were analysed within the frame of the European PRECISESADS project (NTC02890121). Cytokine levels were determined using Luminex panels, and autoantibodies using different immunoassays.ResultsOf the 83 cytokines analysed, 29 differed significantly between patients with SLE and HC. Specifically, CCL8, CXCL13, and IL-1RA levels were elevated in patients with active, but not inactive, SLE versus HC, as well as in patients with SLE versus other AIDs. The levels of these cytokines also correlated with SLE Disease Activity Index 2000 (SLEDAI-2K) scores, among five other cytokines. Overall, the occurrence of autoantibodies was similar across SLEDAI-2K organ domains, and the correlations between autoantibodies and activity in different organ domains were weak.DiscussionOur findings suggest that, upon validation, CCL8, CXCL13, and IL-1RA could serve as promising serum biomarkers of activity in SLE

O31 Integrative analysis reveals a molecular stratification of systemic autoimmune diseases

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Assessment of a strategy to inhibit clearance of proteins by alveolar macrophages

Inhalation aerosols are proving to be a promising alternative to injection for the systemic administration of peptides and proteins. The lungs provide higher bioavailabilities of macromolecules than any other non-inasive routes of delivery. However, bioavailabilities of inhaled therapeutics remain limited and do not exceed 10 % in general, suggesting that uncontrolled biological losses in the respiratory tissue significantly diminish molecular transfer to the bloodstream. Recently, alveolar macrophages (AM) have been shown to be a primary barrier to pulmonary absorption of macromolecules. The uptake and the degradation of inhaled macromolecules by AM competes with their systemic absorption, and thereby, lowers bioavailability to the degree that the rate of degradation is near to or greater than the rate of systemic absorption. The aim of this thesis was to assess an AM-inhibiting strategy in order to decrease AM uptake and clearance of proteins and thereby, increase their pulmonary absorption to the systemic circulation. We aimed to create a sterical hindrance around AM to repel the approach, the binding and the endocytosis of therapeutic proteins. The sterical hindrance was created by grafting poly(ethylene glycol) (PEG) chains on the cell surface of AM using PEGylated monoclonal antibodies (mAbs) directed to sialoadhesin (Sn), a macrophage-restricted adhesion molecule. Anti-mouse Sn mAbs, SER-4 and 3D6, were conjugated to PEG 5 kDa and PEG 20 kDa, resulting in the incorporation of up to 3 molecules of PEG per mAb molecule. PEGylation of antibodies had little effect on their antigen binding activity. Yet, PEGylation strongly increased the potency of antibodies to inhibit red blood cells adhesion mediated by Sn. We concluded that the increase of the potential of SER-4 and 3D6 antibodies by PEGylation was likely due to the steric hindrance provided by the PEG molecules. Sn expression on mouse AM, the population of macrophages targeted by the strategy, was then evaluated. Although Sn expression is restricted to macrophages, it is limited to specific subsets of tissue macrophages and no data clearly demonstrate Sn expression on mouse AM. We showed using flow cytometry that Sn was expressed on murine AM and that this receptor was regulated by cis-interactions. Finally, in order to assess the macrophage-inhibiting strategy in vitro, we used thioglycollate-elicited peritoneal macrophages (TPM) induced to express Sn. These cells can be obtained in large quantities and they show a rapid induction of Sn expression. We first characterised endocytosis modes of albumin and transferrin in these cells and we showed that these molecules were taken up by non-specific adsorptive endocytosis and specific receptor-mediated endocytosis, respectively. Latex beads were also confirmed to be internalised by phagocytosis in TPM induced to express Sn. We showed that PEGylation of macrophages surface using PEGylated SER-4 and 3D6 mAbs did not impair membrane integrity nor cell metabolism and that this local Sn-mediated PEGylation and the associated steric effects of PEG inhibited neither pinocytosis of macromolecules nor phagocytosis of particles. In conclusion, this thesis work showed that PEGylation of Sn on macrophage surface using PEGylated anti-Sn mAbs was not effective to repel the approach, the binding and the uptake of macromolecules and particles. PEGylation of the cell surface needs to be improved in order to make possible our approach of AM inhibition. Yet, our results have however shown for the first time, that PEGylation of antibodies greatly increases their efficacy to inhibit cell adhesion activity. Flexibility and motility of PEG molecules on SER-4 and 3D6 mAbs most likely created a steric hindrance around antibodies that increased the inhibition of red blood cells specific binding to Sn. Thus, PEGylation of antibodies directed to cell surface receptors could be potentially exploited in a therapeutic setting, such as autoimmune or inflammatory diseases, to increase the inhibitory potency of antibodies without impairing vital functions of cells.Doctorat en sciences pharmaceutiques (FARM 3)--UCL, 200

Crucial Biopharmaceutical Issues Facing Macromolecular Candidates for Inhalation: The Role of Macrophages in Pulmonary Protein Clearance

Our laboratory recently demonstrated that alveolar macrophages (AM) comprised a major barrier to the transport of two large proteins, IgG and human chorionic gonadotropin, from the airways into the bloodstream, while they had no impact on the peptide insulin. This study was aimed at assessing the role of AM in the alveolar clearance of human growth hormone (hGH), a smaller therapeutic protein being investigated as an inhalation aerosol in clinical trials. Using intratracheal instillation of liposome-encapsulated clodronate to deplete AM of rat lungs, we studied the pulmonary absorption of hGH in AM-depleted versus normal animals. The systemic absorption of hGH, following pulmonary administration, did not show significant alterations following AM depletion; absolute bioavailabilities reached 11.4%, 10.7% and 9.0% in clodronate liposome-, PBS liposome- and PBS-treated rats. Absorption from the lungs was rapid in all tested conditions, indicating that hGH crossed the alveolar epithelium quickly, presumably preventing major uptake and degradation by AM. AM uptake of proteins appears to be a significant local elimination mechanism for proteins above a certain size which implies a residence time within the alveolar spaces long enough for significant AM endocytosis