Longitudinal Immune Cell Profiling in Patients With Early Systemic Lupus Erythematosus.

OBJECTIVES: To investigate the immune cell profiles of patients with systemic lupus erythematosus (SLE), and to identify longitudinal changes in those profiles over time. METHODS: We employed mass cytometry with 3 different panels of 38-39 markers (an immunophenotyping panel, a T cell/monocyte panel, and a B cell panel) in cryopreserved peripheral blood mononuclear cells (PBMCs) from 9 patients with early SLE, 15 patients with established SLE, and 14 controls without autoimmune disease. We used machine learning-driven clustering, flow self-organizing maps, and dimensional reduction with t-distributed stochastic neighbor embedding to identify unique cell populations in early SLE and established SLE. We used mass cytometry data of PBMCs from 19 patients with early rheumatoid arthritis (RA) and 23 controls to compare levels of specific cell populations in early RA and SLE. For the 9 patients with early SLE, longitudinal mass cytometry analysis was applied to PBMCs at enrollment, 6 months after enrollment, and 1 year after enrollment. Serum samples were also assayed for 65 cytokines using Luminex multiplex assay, and associations between cell types and cytokines/chemokines were assessed. RESULTS: Levels of peripheral helper T cells, follicular helper T (Tfh) cells, and several Ki-67+ proliferating subsets (ICOS+Ki-67+ CD8 T cells, Ki-67+ regulatory T cells, CD19intermediate Ki-67high plasmablasts, and PU.1high Ki-67high monocytes) were increased in patients with early SLE, with more prominent alterations than were seen in patients with early RA. Longitudinal mass cytometry and multiplex serum cytokine assays of samples from patients with early SLE revealed that levels of Tfh cells and CXCL10 had decreased 1 year after enrollment. Levels of CXCL13 were positively correlated with levels of several of the expanded cell populations in early SLE. CONCLUSION: Two major helper T cell subsets and unique Ki-67+ proliferating immune cell subsets were expanded in patients in the early phase of SLE, and the immunologic features characteristic of early SLE evolved over time.

Differential condensation of sister chromatids acts with Cdc6 to ensure asynchronous S-phase entry in Drosophila male germline stem cell lineage.

During Drosophila melanogaster male germline stem cell (GSC) asymmetric division, preexisting old versus newly synthesized histones H3 and H4 are asymmetrically inherited. However, the biological outcomes of this phenomenon have remained unclear. Here, we tracked old and new histones throughout the GSC cell cycle through the use of high spatial and temporal resolution microscopy. We found unique features that differ between old and new histone-enriched sister chromatids, including differences in nucleosome density, chromosomal condensation, and H3 Ser10 phosphorylation. These distinct chromosomal features lead to their differential association with Cdc6, a pre-replication complex component, and subsequent asynchronous DNA replication initiation in the resulting daughter cells. Disruption of asymmetric histone inheritance abolishes differential Cdc6 association and asynchronous S-phase entry, demonstrating that histone asymmetry acts upstream of these critical cell-cycle progression events. Furthermore, disruption of these GSC-specific chromatin features leads to GSC defects, indicating a connection between histone inheritance, cell-cycle progression, and cell fate determination.

Monocyte-derived S1P in the lymph node regulates immune responses.

The lipid chemoattractant sphingosine 1-phosphate (S1P) guides cells out of tissues, where the concentration of S1P is relatively low, into circulatory fluids, where the concentration of S1P is high1. For example, S1P directs the exit of T cells from lymph nodes, where T cells are initially activated, into lymph, from which T cells reach the blood and ultimately inflamed tissues1. T cells follow S1P gradients primarily using S1P receptor 1 (ref. 1). Recent studies have described how S1P gradients are established at steady state, but little is known about the distribution of S1P in disease or about how changing levels of S1P may affect immune responses. Here we show that the concentration of S1P increases in lymph nodes during an immune response. We found that haematopoietic cells, including inflammatory monocytes, were an important source of this S1P, which was an unexpected finding as endothelial cells provide S1P to lymph1. Inflammatory monocytes required the early activation marker CD69 to supply this S1P, in part because the expression of CD69 was associated with reduced levels of S1pr5 (which encodes S1P receptor 5). CD69 acted as a 'stand-your-ground' signal, keeping immune cells at a site of inflammation by regulating both the receptors and the gradients of S1P. Finally, increased levels of S1P prolonged the residence time of T cells in the lymph nodes and exacerbated the severity of experimental autoimmune encephalomyelitis in mice. This finding suggests that residence time in the lymph nodes might regulate the differentiation of T cells, and points to new uses of drugs that target S1P signalling.

Induction of nerve growth factor by phorbol 12-myristate 13-acetate is dependent upon the mitogen activated protein kinase pathway.

Several small molecules have been identified that induce glial cells to synthesize and secrete nerve growth factor (NGF), a critical neurotrophin that supports neuronal growth and survival, and as such show promise in the development of drugs for the chemoprevention of Alzheimer's disease. To map the signal transduction cascade leading to NGF synthesis and secretion, cultured human glial cells were stimulated by phorbol 12-myristate 13-acetate (PMA), an agonist of Protein Kinase C. Changes in intracellular protein phosphorylation states were evaluated by reverse phase protein microarrays (RPPA), selectively screening over 130 protein endpoints. Of these, 55 proteins showed statistically significant changes in phosphorylation state due to cellular exposure to PMA. A critical signal transduction pathway was identified, and subsequent validation by ELISA and qPCR revealed that the signaling proteins Raf, MEK, ERK, and the signal transduction factor CREB are all essential to the upregulation of NGF gene expression by PMA. Additionally, members of the RSK family of kinases appear to be involved in glial secretion (exocytosis) of the NGF protein. Furthermore, through RPPA, the effects of PMA on apoptosis signaling events and cell proliferation were differentiated from the pathway to NGF upregulation. Overall, this study reveals potential protein targets for the rational design of Alzheimer's therapeutics.

Methods for field measurement of antibiotic concentrations: limitations and outlook.

The growing prevalence of antibiotic resistance poses an increasingly serious threat to human health. Although an important driver of antibiotic resistance is the continuous exposure of bacteria to sublethal concentrations of antibiotics in natural environments, antibiotic pollutants are not currently tracked globally or systematically. This limits the international capacity to address the rise of antibiotic resistance at its source. To address this lack of data, the development of methods to measure antibiotic concentrations on-site is essential. These methods, ideally, must be sensitive to sublethal concentrations of antibiotics and require minimal technical expertise. Furthermore, factors such as cost, selectivity, biosafety and the ability to multiplex must be evaluated in the context of field use. Based on these criteria, we provide a critical review of current methods in antibiotic detection and evaluate their adaptability for use on-site. We categorize these methods into microbiological assays, physical and chemical assays, immunoassays, aptasensors and whole-cell biosensors. We recommend continued development of a dipstick or microfluidics approach with a bacterial promoter-based mechanism and colorimetric output. This technique would incorporate the advantageous aspects of existing methods, maximize shelf-life and ease-of-use, and require minimal resources to implement in the field.