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.

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.

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.

GUBS: Graph-Based Unsupervised Brain Segmentation in MRI Images.

Brain segmentation in magnetic resonance imaging (MRI) images is the process of isolating the brain from non-brain tissues to simplify the further analysis, such as detecting pathology or calculating volumes. This paper proposes a Graph-based Unsupervised Brain Segmentation (GUBS) that processes 3D MRI images and segments them into brain, non-brain tissues, and backgrounds. GUBS first constructs an adjacency graph from a preprocessed MRI image, weights it by the difference between voxel intensities, and computes its minimum spanning tree (MST). It then uses domain knowledge about the different regions of MRIs to sample representative points from the brain, non-brain, and background regions of the MRI image. The adjacency graph nodes corresponding to sampled points in each region are identified and used as the terminal nodes for paths connecting the regions in the MST. GUBS then computes a subgraph of the MST by first removing the longest edge of the path connecting the terminal nodes in the brain and other regions, followed by removing the longest edge of the path connecting non-brain and background regions. This process results in three labeled, connected components, whose labels are used to segment the brain, non-brain tissues, and the background. GUBS was tested by segmenting 3D T1 weighted MRI images from three publicly available data sets. GUBS shows comparable results to the state-of-the-art methods in terms of performance. However, many competing methods rely on having labeled data available for training. Labeling is a time-intensive and costly process, and a big advantage of GUBS is that it does not require labels.

Systemic and local innate immune responses to surgical co-transplantation of mesenchymal stromal cells and biphasic calcium phosphate for bone regeneration.

Bone regeneration from mesenchymal stromal cells (MSC) is attributed to comprehensive immune modulation mediated by the MSC. However, the temporal and spatial regulation of these immune responses has not yet been described. The aim of the present study was to assess the local and systemic innate immune responses to implantation of biphasic calcium phosphate biomaterial (BCP) alone, or with bone marrow derived MSC (BCP+MSC), in critical-sized calvarial bone defects of Lewis rats. Four weeks after implantation, flow cytometry analysis of innate immune cells revealed increased numbers of circulating classical monocyte-macrophages (MM) and decreased non-classical MM in the BCP+MSC group. At week 8, this differential systemic MM response was associated with an increased presence of local tissue anti-inflammatory macrophages expressing CD68 and CD163 markers (M2-like). In the BCP group without MSC, NK cells increased at weeks 1 and 4, and neutrophils increased in circulation at weeks 2 and 8. At week 8, the increase in number of neutrophils in circulation was associated with decreased local tissue neutrophils, in the BCP+MSC group. Gene expression analysis of tissue biopsies from defects implanted with BCP+MSC, in comparison to BCP alone, revealed upregulated expression of early osteogenesis genes along with macrophage differentiation-related genes at weeks 1 and 8 and neutrophil chemotaxis-related genes at week 1. This study is the first to demonstrate that surgical implantation of BCP or BCP+MSC grafts differentially regulate both systemic and local tissue innate immune responses which enhance bone formation. The results provide new insights into immune mechanisms underlying MSC-mediated bone regeneration. STATEMENT OF SIGNIFICANCE: The suitability of biphasic calcium phosphate and mesenchymal stromal cell construct (BCP+MSC) transplantation is evident from their progress in clinical trials for treating challenging maxillofacial bone defects. But less is known about the overall immune response generated by this surgical process and how it later impacts the bone formation. To this end, it is crucial to understand for both clinicians and researchers, the systemic immune response to transplanting MSC in patients for ensuring both the safety and efficacy of cell therapies. In this study, we used rat calvarial bone defect model and showed that both systemic and local innate immunes responses (monocyte-macrophages and neutrophils) are favorably directed towards enhanced bone formation in BCP+MSC implanted defects, as compared to BCP alone.

Wearing-off symptoms during standard and extended natalizumab dosing intervals: Experiences from the COVID-19 pandemic.

Natalizumab effectively prevents disease activity in relapsing-remitting multiple sclerosis, but many treated patients report subjective wearing-off symptoms at the end of the 4-week interval between infusions. Extended interval dosing (EID) is a promising strategy to mitigate the risk of natalizumab-associated progressive multifocal leukoencephalopathy, but it is unknown whether EID affects wearing-off symptoms. In this observational study, we evaluated if prevalence or intensity of wearing-off symptoms changed when natalizumab dosing intervals were extended from 4 to 6 weeks in 30 treated patients during the outbreak of COVID-19 in Norway. New or increased wearing-off symptoms during EID were reported by 50%. Symptom increase was more frequent among patients with pre-existing wearing-off symptoms during standard dosing compared to patients without such pre-existing symptoms [p = 0.0005]. Our observations support the need to study the effect of EID on wearing-off symptoms in randomized controlled trials.

Integrated trajectories of the maternal metabolome, proteome, and immunome predict labor onset.

Estimating the time of delivery is of high clinical importance because pre- and postterm deviations are associated with complications for the mother and her offspring. However, current estimations are inaccurate. As pregnancy progresses toward labor, major transitions occur in fetomaternal immune, metabolic, and endocrine systems that culminate in birth. The comprehensive characterization of maternal biology that precedes labor is key to understanding these physiological transitions and identifying predictive biomarkers of delivery. Here, a longitudinal study was conducted in 63 women who went into labor spontaneously. More than 7000 plasma analytes and peripheral immune cell responses were analyzed using untargeted mass spectrometry, aptamer-based proteomic technology, and single-cell mass cytometry in serial blood samples collected during the last 100 days of pregnancy. The high-dimensional dataset was integrated into a multiomic model that predicted the time to spontaneous labor [R = 0.85, 95% confidence interval (CI) [0.79 to 0.89], P = 1.2 × 10-40, N = 53, training set; R = 0.81, 95% CI [0.61 to 0.91], P = 3.9 × 10-7, N = 10, independent test set]. Coordinated alterations in maternal metabolome, proteome, and immunome marked a molecular shift from pregnancy maintenance to prelabor biology 2 to 4 weeks before delivery. A surge in steroid hormone metabolites and interleukin-1 receptor type 4 that preceded labor coincided with a switch from immune activation to regulation of inflammatory responses. Our study lays the groundwork for developing blood-based methods for predicting the day of labor, anchored in mechanisms shared in preterm and term pregnancies.

Cerebrospinal fluid proteome shows disrupted neuronal development in multiple sclerosis.

Despite intensive research, the aetiology of multiple sclerosis (MS) remains unknown. Cerebrospinal fluid proteomics has the potential to reveal mechanisms of MS pathogenesis, but analyses must account for disease heterogeneity. We previously reported explorative multivariate analysis by hierarchical clustering of proteomics data of MS patients and controls, which resulted in two groups of individuals. Grouping reflected increased levels of intrathecal inflammatory response proteins and decreased levels of proteins involved in neural development in one group relative to the other group. MS patients and controls were present in both groups. Here we reanalysed these data and we also reanalysed data from an independent cohort of patients diagnosed with clinically isolated syndrome (CIS), who have symptoms of MS without evidence of dissemination in space and/or time. Some, but not all, CIS patients had intrathecal inflammation. The analyses reported here identified a common protein signature of MS/CIS that was not linked to elevated intrathecal inflammation. The signature included low levels of complement proteins, semaphorin-7A, reelin, neural cell adhesion molecules, inter-alpha-trypsin inhibitor heavy chain H2, transforming growth factor beta 1, follistatin-related protein 1, malate dehydrogenase 1 cytoplasmic, plasma retinol-binding protein, biotinidase, and transferrin, all known to play roles in neural development. Low levels of these proteins suggest that MS/CIS patients suffer from abnormally low oxidative capacity that results in disrupted neural development from an early stage of the disease.

Wearing-off at the end of natalizumab dosing interval and risk of MS disease activity: A prospective 1-year follow-up study.

Natalizumab effectively prevents disease activity in relapsing-remitting multiple sclerosis by binding α4 integrin and inhibiting leukocyte migration to the central nervous system. We recently reported an association between low natalizumab receptor occupancy and subjective wearing-off symptoms at the end of the 4-week dosing interval. Here, we aimed to evaluate the short-term risk of disease activity in a 1-year prospective follow-up of the same patient cohort (n = 40). We found that all patients available for follow-up after one year (n = 35) fulfilled the criteria for no evidence of disease activity (NEDA). Thus, wearing-off symptoms were not associated with increased short-term risk of disease activity. Longer follow-up in a larger patient cohort is required to establish whether therapeutic efficacy is maintained in patients with wearing-off symptoms.

Wearing-off at the end of natalizumab dosing intervals is associated with low receptor occupancy.

OBJECTIVE: We aimed to investigate whether wearing-off symptoms at the end of the natalizumab dosing interval were associated with clinical and demographic patient characteristics or natalizumab receptor occupancy (RO) on leukocytes. METHODS: In this cross-sectional study of 40 patients with relapsing-remitting MS (RRMS) receiving natalizumab at the Department of Neurology, Haukeland University Hospital, we recorded clinical and demographic data including age, body mass index (BMI), working status, smoking habits, disease characteristics, treatment duration, vitamin D levels, and wearing-off symptoms. We quantified neurofilament light chain in serum and measured natalizumab RO in leukocyte subtypes by high-parameter mass cytometry. Associations with wearing-off symptoms were analyzed. RESULTS: Eight (20.0%) patients who reported regular occurrence of wearing-off symptoms, 9 (22.5%) who sometimes had wearing-off symptoms, and 23 (57.5%) who did not have wearing-off symptoms were evaluated. Patients who regularly had wearing-off symptoms had lower natalizumab RO than patients who reported having such symptoms sometimes or never. The former group also had higher BMI and higher frequency of sick leave. High BMI was associated with low RO. No other demographic or disease characteristics were associated with the phenomenon. CONCLUSIONS: Low RO may explain the wearing-off phenomenon observed in some patients with RRMS treated with natalizumab, and high BMI may be the underlying cause.

Candidate Markers for Stratification and Classification in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune, inflammatory disease, characterized by synovitis in small- and medium-sized joints and, if not treated early and efficiently, joint damage, and destruction. RA is a heterogeneous disease with a plethora of treatment options. The pro-inflammatory cytokine tumor necrosis factor (TNF) plays a central role in the pathogenesis of RA, and TNF inhibitors effectively repress inflammatory activity in RA. Currently, treatment decisions are primarily based on empirics and economic considerations. However, the considerable interpatient variability in response to treatment is a challenge. Markers for a more exact patient classification and stratification are lacking. The objective of this study was to identify markers in immune cell populations that distinguish RA patients from healthy donors with an emphasis on TNF signaling. We employed mass cytometry (CyTOF) with a panel of 13 phenotyping and 10 functional markers to explore signaling in unstimulated and TNF-stimulated peripheral blood mononuclear cells from 20 newly diagnosed, untreated RA patients and 20 healthy donors. The resulting high-dimensional data were analyzed in three independent analysis pipelines, characterized by differences in both data clean-up, identification of cell subsets/clustering and statistical approaches. All three analysis pipelines identified p-p38, IkBa, p-cJun, p-NFkB, and CD86 in cells of both the innate arm (myeloid dendritic cells and classical monocytes) and the adaptive arm (memory CD4+ T cells) of the immune system as markers for differentiation between RA patients and healthy donors. Inclusion of the markers p-Akt and CD120b resulted in the correct classification of 18 of 20 RA patients and 17 of 20 healthy donors in regression modeling based on a combined model of basal and TNF-induced signal. Expression patterns in a set of functional markers and specific immune cell subsets were distinct in RA patients compared to healthy individuals. These signatures may support studies of disease pathogenesis, provide candidate markers for response, and non-response to TNF inhibitor treatment, and aid the identification of future therapeutic targets.

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.

Single Cell Based Phosphorylation Profiling Identifies Alterations in Toll-Like Receptor 7 and 9 Signaling in Patients With Primary Sjögren's Syndrome.

Primary Sjögren's syndrome (pSS) is associated with polymorphisms and mRNA expression profiles that are indicative of an exaggerated innate and type I IFN immune response. Excessive activation potential of signaling pathways may play a role in this profile, but the intracellular signaling profile of the disease is not well characterized. To gain insights into potentially dysfunctional intracellular signaling profiles of pSS patients we conducted an exploratory analysis of MAPK/ERK and JAK/STAT signaling networks in peripheral blood mononuclear cells (PBMC) from 25 female pSS patients and 25 female age-matched healthy donors using phospho-specific flow cytometry. We analyzed unstimulated samples, as well as samples during a 4 h time period following activation of Toll-like receptor (TLR) 7 and 9. Expression levels of MxA, IFI44, OAS1, GBP1, and GBP2 in PBMC were analyzed by real-time PCR. Cytokine levels in plasma were determined using a 25-plex Luminex-assay. Principal component analysis (PCA) showed that basal phosphorylation profiles could be used to differentiate pSS patients from healthy donor samples by stronger intracellular signaling pathway activation in NK and T cells relative to B cells. Stimulation of PBMC with TLR7 and -9 ligands showed significant differences in the phosphorylation profiles between samples from pSS patients and healthy donors. Including clinical parameters such as extraglandular manifestations (EGM), we observed stronger responses of NF-κB and STAT3 S727 in B cells from EGM-negative patients compared to EGM-positive patients and healthy controls. Plasma cytokine levels were correlated to the basal phosphorylation levels in these patients. In addition, 70% of the patients had a positive IFN score. These patients differed from the IFN score negative patients regarding their phosphorylation profiles and their plasma cytokine levels. In conclusion, we here report increased signaling potentials in peripheral B cells of pSS patients in response to TLR7 and -9 stimulation through STAT3 S727 and NF-κB that correlate with a type I IFN signature. Induction of these pathways could contribute to the generation of a type I IFN signature in pSS. Patients displaying elevated potentiation of STAT3 S727 and NF-κB signaling could therefore benefit from therapies targeting these pathways.

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.

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.

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.

Single-cell proteomics: potential implications for cancer diagnostics.

Single-cell proteomics in cancer is evolving and promises to provide more accurate diagnoses based on detailed molecular features of cells within tumors. This review focuses on technologies that allow for collection of complex data from single cells, but also highlights methods that are adaptable to routine cancer diagnostics. Current diagnostics rely on histopathological analysis, complemented by mutational detection and clinical imaging. Though crucial, the information gained is often not directly transferable to defined therapeutic strategies, and predicting therapy response in a patient is difficult. In cancer, cellular states revealed through perturbed intracellular signaling pathways can identify functional mutations recurrent in cancer subsets. Single-cell proteomics remains to be validated in clinical trials where serial samples before and during treatment can reveal excessive clonal evolution and therapy failure; its use in clinical trials is anticipated to ignite a diagnostic revolution that will better align diagnostics with the current biological understanding of cancer.