No abstract

Our data show stage-specific remodeling of human dystrophin-deficient muscle, with inflammatory pathways predominating in the presymptomatic stages and acute activation of TGFbeta and failure of metabolic pathways later in the disease.

Disuse atrophy is a common clinical phenomenon that significantly impacts muscle function and activities of daily living. The purpose of this study was to implement genome-wide expression profiling to identify transcriptional pathways associated with muscle remodeling in a clinical model of disuse. Skeletal muscle biopsies were acquired from the medial gastrocnemius in patients with an ankle fracture and from healthy volunteers subjected to 4-11 days of cast immobilization. We identified 277 misregulated transcripts in immobilized muscles of patients, of which the majority were downregulated. The most broadly affected pathways were involved in energy metabolism, mitochondrial function, and cell cycle regulation. We also found decreased expression in genes encoding proteolytic proteins, calpain-3 and calpastatin, and members of the myostatin and IGF-I pathway. Only 26 genes showed increased expression in immobilized muscles, including apolipoprotein (APOD) and leptin receptor (LEPR). Upregulation of APOD (5.0-fold, P < 0.001) and LEPR (5.7-fold, P < 0.05) was confirmed by quantitative RT-PCR and immunohistochemistry. In addition, atrogin-1/MAFbx was found to be 2.4-fold upregulated (P < 0.005) by quantitative RT-PCR. Interestingly, 96% of the transcripts differentially regulated in immobilized limbs also showed the same trend of change in the contralateral legs of patients but not the contralateral legs of healthy volunteers. Information obtained in this study complements findings in animal models of disuse and provides important feedback for future clinical studies targeting the restoration of muscle function following limb disuse in humans.

Corticosteroids have been used for decades to modulate inflammation therapeutically, yet there is a paucity of data on their effects in humans. We examined the changes in cellular and molecular immune system parameters, or “immunome”, in healthy humans after systemic corticosteroid administration. We used multiplexed techniques to query the immunome in 20 volunteers at baseline, and after intravenous hydrocortisone (HC) administered at moderate (250 mg) and low (50 mg) doses, to provide insight into how corticosteroids exert their effects. We performed comprehensive phenotyping of 120 lymphocyte subsets by high dimensional flow cytometry, and observed a decline in circulating specific B and T cell subsets, which reached their nadir 4–8 hours after administration of HC. However, B and T cells rebounded above baseline 24 hours after HC infusion, while NK cell numbers remained stable. Whole transcriptome profiling revealed down regulation of NF-κB signaling, apoptosis, and cell death signaling transcripts that preceded lymphocyte population changes, with activation of NK cell and glucocorticoid receptor signaling transcripts. Our study is the first to systematically characterize the effects of corticosteroids on the human immunome, and we demonstrate that HC exerts differential effects on B and T lymphocytes and natural killer cells in humans.

In the above article, we did not include a GEO accession number for the gene expression data from our study. The expression data can be accessed under the accession number GSE47353 at http://www.ncbi.nlm.nih.gov/geo/. In addition, all data used in our analyses can be obtained at http://chi.nhlbi.nih.gov.

Kostek MC, Chen Y-W, Cuthbertson DJ, Shi R, Fedele MJ, Esser KA, Rennie MJ. Gene expression responses over 24 h to lengthening and shortening contractions in human muscle: major changes in CSRP3, MUSTN1, SIX1, and FBXO32.

BackgroundInterest in single-cell transcriptomic analysis is growing rapidly, especially for profiling rare or heterogeneous populations of cells. In almost all reported works investigators have used live cells, which introduces cell stress during preparation and hinders complex study designs. Recent studies have indicated that cells fixed by denaturing fixative can be used in single-cell sequencing, however they did not usually work with most types of primary cells including immune cells.MethodsThe methanol-fixation and new processing method was introduced to preserve human peripheral blood mononuclear cells (PBMCs) for single-cell RNA sequencing (scRNA-Seq) analysis on 10× Chromium platform.ResultsWhen methanol fixation protocol was broken up into three steps: fixation, storage and rehydration, we found that PBMC RNA was degraded during rehydration with PBS, not at cell fixation and up to 3-month storage steps. Resuspension but not rehydration in 3× saline sodium citrate (SSC) buffer instead of PBS preserved PBMC RNA integrity and prevented RNA leakage. Diluted SSC buffer did not interfere with full-length cDNA synthesis. The methanol-fixed PBMCs resuspended in 3× SSC were successfully implemented into 10× Chromium standard scRNA-seq workflows with no elevated low quality cells and cell doublets. The fixation process did not alter the single-cell transcriptional profiles and gene expression levels. Major subpopulations classified by marker genes could be identified in fixed PBMCs at a similar proportion as in live PBMCs. This new fixation processing protocol also worked in several other fixed primary cell types and cell lines as in live ones.ConclusionsWe expect that the methanol-based cell fixation procedure presented here will allow better and more effective batching schemes for a complex single cell experimental design with primary cells or tissues.Electronic supplementary materialThe online version of this article (10.1186/s12967-018-1578-4) contains supplementary material, which is available to authorized users.

Increased risk of premature cardiovascular disease (CVD) is well recognized in systemic lupus erythematosus (SLE). Aberrant type I-Interferon (IFN)-neutrophil interactions contribute to this enhanced CVD risk. In lupus animal models, the Janus kinase (JAK) inhibitor tofacitinib improves clinical features, immune dysregulation and vascular dysfunction. We conducted a randomized, double-blind, placebo-controlled clinical trial of tofacitinib in SLE subjects (ClinicalTrials.gov NCT02535689). In this study, 30 subjects are randomized to tofacitinib (5 mg twice daily) or placebo in 2:1 block. The primary outcome of this study is safety and tolerability of tofacitinib. The secondary outcomes include clinical response and mechanistic studies. The tofacitinib is found to be safe in SLE meeting study’s primary endpoint. We also show that tofacitinib improves cardiometabolic and immunologic parameters associated with the premature atherosclerosis in SLE. Tofacitinib improves high-density lipoprotein cholesterol levels (p = 0.0006, CI 95%: 4.12, 13.32) and particle number (p = 0.0008, CI 95%: 1.58, 5.33); lecithin: cholesterol acyltransferase concentration (p = 0.024, CI 95%: 1.1, −26.5), cholesterol efflux capacity (p = 0.08, CI 95%: −0.01, 0.24), improvements in arterial stiffness and endothelium-dependent vasorelaxation and decrease in type I IFN gene signature, low-density granulocytes and circulating NETs. Some of these improvements are more robust in subjects with STAT4 risk allele.