The Therapeutic Mechanisms of Mesenchymal Stem Cells in MS-A Review Focusing on Neuroprotective Properties.

In multiple sclerosis (MS), there is a great need for treatment with the ability to suppress compartmentalized inflammation within the central nervous system (CNS) and to promote remyelination and regeneration. Mesenchymal stem cells (MSCs) represent a promising therapeutic option, as they have been shown to migrate to the site of CNS injury and exert neuroprotective properties, including immunomodulation, neurotrophic factor secretion, and endogenous neural stem cell stimulation. This review summarizes the current understanding of the underlying neuroprotective mechanisms and discusses the translation of MSC transplantation and their derivatives from pre-clinical demyelinating models to clinical trials with MS patients.

Aberrant cell signalling in PBMCs upon IFN-α stimulation in primary Sjögren's syndrome patients associates with type I interferon signature.

Primary Sjögren's syndrome (pSS) is a complex systemic autoimmune disease with heterogeneous disease manifestations. Genetic predisposition, hormonal and environmental factors are all thought to contribute to disease etiology and pathogenesis. A better understanding of the disease pathogenesis is required in order to establish new targeted therapies. We analysed MAPK/ERK and JAK/STAT signalling networks in peripheral blood mononuclear cells (PBMCs) upon stimulation with interferon alpha 2b (IFN-α2b) by flow cytometry to define potentially dysfunctional intracellular signalling pathways involved in disease pathogenesis. Cells derived from pSS patients displayed small but significant increases in basal phosphorylation levels of numerous signalling proteins compared to cells from healthy donors. The phosphorylation profiles following stimulation with IFNα2b differed significantly between pSS patients and healthy donors, especially regarding STAT1 Y701. PCA further grouped patients according to clinical characteristics. Type I IFN induced gene expression was found to negatively correlate with the IFN-α2b induced phosphorylation of STAT3 S727 in T cells and positively with pSTAT1 Y701 in B cells. Increases in pSTAT1 Y701 were associated with the presence of autoantibodies. Our results indicate involvement of both STAT3 S727 and STAT1 Y701 pathways in pSS patients. Therapies targeting these pathways might therefore be beneficial for certain subgroups of patients.

Titrating Complex Mass Cytometry Panels.

We describe here a simple and efficient antibody titration approach for cell-surface markers and intracellular cell signaling targets for mass cytometry. The iterative approach builds upon a well-characterized backbone panel of antibodies and analysis using bioinformatic tools such as SPADE. Healthy peripheral blood and bone marrow cells are stained with a pre-optimized "backbone" antibody panel in addition to the progressively diluted (titrated) antibodies. Clustering based on the backbone panel enables the titration of each antibody against a rich hematopoietic background and assures that nonspecific binding and signal spillover can be quantified accurately. Using a slightly expanded backbone panel, antibodies quantifying changes in transcription factors and phosphorylated antigens are titrated on ex vivo stimulated cells to optimize sensitivity and evaluate baseline expression. Based on this information, complex panels of antibodies can be thoroughly optimized for use on healthy whole blood and bone marrow and are easily adaptable to the investigation of samples from for example clinical studies. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Optimization of Receptor Occupancy Assays in Mass Cytometry: Standardization Across Channels with QSC Beads.

Receptor occupancy, the ratio between amount of drug bound and amount of total receptor on single cells, is a biomarker for treatment response to therapeutic monoclonal antibodies. Receptor occupancy is traditionally measured by flow cytometry. However, spectral overlap in flow cytometry limits the number of markers that can be measured simultaneously. This restricts receptor occupancy assays to the analysis of major cell types, although rare cell populations are of potential therapeutic relevance. We therefore developed a receptor occupancy assay suitable for mass cytometry. Measuring more markers than currently available in flow cytometry allows simultaneous receptor occupancy assessment and high-parameter immune phenotyping in whole blood, which should yield new insights into disease activity and therapeutic effects. However, varying sensitivity across the mass cytometer detection range may lead to misinterpretation of the receptor occupancy when drug and receptor are detected in different channels. In this report, we describe a method for optimization of mass cytometry receptor occupancy measurements by using antibody-binding quantum simply cellular (QSC) beads for standardization across channels with different sensitivities. We evaluated the method in a mass cytometry-based receptor occupancy assay for natalizumab, a therapeutic antibody used in multiple sclerosis treatment that binds to α4-integrin, which is expressed on leukocyte cell surfaces. Peripheral blood leukocytes from a treated patient were stained with a panel containing metal-conjugated antibodies for detection of natalizumab and α4-integrin. QSC beads with known antibody binding capacity were stained with the same metal-conjugated antibodies and were used to standardize the signal intensity in the leukocyte sample before calculating receptor occupancy. We found that QSC bead standardization across channels corrected for sensitivity differences for detection of drug and receptor and generated more accurate results than observed without standardization. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Single cell immune profiling by mass cytometry of newly diagnosed chronic phase chronic myeloid leukemia treated with nilotinib.

Monitoring of single cell signal transduction in leukemic cellular subsets has been proposed to provide deeper understanding of disease biology and prognosis, but has so far not been tested in a clinical trial of targeted therapy. We developed a complete mass cytometry analysis pipeline for characterization of intracellular signal transduction patterns in the major leukocyte subsets of chronic phase chronic myeloid leukemia. Changes in phosphorylated Bcr-Abl1 and the signaling pathways involved were readily identifiable in peripheral blood single cells already within three hours of the patient receiving oral nilotinib. The signal transduction profiles of healthy donors were clearly distinct from those of the patients at diagnosis. Furthermore, using principal component analysis, we could show that phosphorylated transcription factors STAT3 (Y705) and CREB (S133) within seven days reflected BCR-ABL1IS at three and six months. Analyses of peripheral blood cells longitudinally collected from patients in the ENEST1st clinical trial showed that single cell mass cytometry appears to be highly suitable for future investigations addressing tyrosine kinase inhibitor dosing and effect. (clinicaltrials.gov identifier: 01061177).

Signaling effects of sodium hydrosulfide in healthy donor peripheral blood mononuclear cells.

Hydrogen sulfide (H2S) is an endogenous gasotransmitter in human physiology and inflammatory disease, however, with limited knowledge of how signal transduction pathways are involved in immune cells. To examine the effects of sulfide on relevant intracellular signaling in human peripheral blood mononuclear cells (PBMCs), we stimulated healthy donor PBMCs with sodium hydrosulfide (NaHS, 1-1000µM) to mimic H2S stimulation, and analyzed phosphorylation of p38 mitogen activated protein kinase (MAPK) (pT180/pY182), NF-κB p65 (pS529), Akt (pS473) and CREB/ATF1 (pS133/pS63) with flow and mass cytometry. In contrast to transient effects in subsets of lymphocytes, classical monocytes demonstrated sustained phosphorylation of p38, Akt and CREB/ATF1. NaHS induced calcium dependent phosphorylation of p38, Akt and CREB, but not NF-κB, and the phosphorylation of Akt was partly dependent on p38, indicative of p38-Akt crosstalk. Attenuation of these effects by molecules targeting p38 and Hsp90 indicated Hsp90 as a possible target for H2S-induced activation of p38. These results provide a description of a NaHS-induced signal transduction pathway in human primary immune cells that may have relevance for the role of sulfides in inflammation.

Neural regeneration in the human central nervous system-from understanding the underlying mechanisms to developing treatments. Where do we stand today?

Mature neurons in the human central nervous system (CNS) fail to regenerate after injuries. This is a common denominator across different aetiologies, including multiple sclerosis, spinal cord injury and ischemic stroke. The lack of regeneration leads to permanent functional deficits with a substantial impact on patient quality of life, representing a significant socioeconomic burden worldwide. Great efforts have been made to decipher the responsible mechanisms and we now know that potent intra- and extracellular barriers prevent axonal repair. This knowledge has resulted in numerous clinical trials, aiming to promote neuroregeneration through different approaches. Here, we summarize the current understanding of the causes to the poor regeneration within the human CNS. We also review the results of the treatment attempts that have been translated into clinical trials so far.

Automated cell type annotation and exploration of single-cell signaling dynamics using mass cytometry.

Mass cytometry by time-of-flight (CyTOF) is an emerging technology allowing for in-depth characterization of cellular heterogeneity in cancer and other diseases. Unfortunately, high-dimensional analyses of CyTOF data remain quite demanding. Here, we deploy a bioinformatics framework that tackles two fundamental problems in CyTOF analyses namely (1) automated annotation of cell populations guided by a reference dataset and (2) systematic utilization of single-cell data for effective patient stratification. By applying this framework on several publicly available datasets, we demonstrate that the Scaffold approach achieves good trade-off between sensitivity and specificity for automated cell type annotation. Additionally, a case study focusing on a cohort of 43 leukemia patients reported salient interactions between signaling proteins that are sufficient to predict short-term survival at time of diagnosis using the XGBoost algorithm. Our work introduces an automated and versatile analysis framework for CyTOF data with many applications in future precision medicine projects.

Early response evaluation by single cell signaling profiling in acute myeloid leukemia.

Aberrant pro-survival signaling is a hallmark of cancer cells, but the response to chemotherapy is poorly understood. In this study, we investigate the initial signaling response to standard induction chemotherapy in a cohort of 32 acute myeloid leukemia (AML) patients, using 36-dimensional mass cytometry. Through supervised and unsupervised machine learning approaches, we find that reduction of extracellular-signal-regulated kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (MAPK) phosphorylation in the myeloid cell compartment 24 h post-chemotherapy is a significant predictor of patient 5-year overall survival in this cohort. Validation by RNA sequencing shows induction of MAPK target gene expression in patients with high phospho-ERK1/2 24 h post-chemotherapy, while proteomics confirm an increase of the p38 prime target MAPK activated protein kinase 2 (MAPKAPK2). In this study, we demonstrate that mass cytometry can be a valuable tool for early response evaluation in AML and elucidate the potential of functional signaling analyses in precision oncology diagnostics.