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This study addresses the importance of conducting mass movement susceptibility mapping and hazard assessment using quantitative techniques, including machine learning, in the Northern Lima Commonwealth (NLC). A previous exploration of the topographic variables revealed a high correlation and multicollinearity among some of them, which led to dimensionality reduction through a principal component analysis (PCA). Six susceptibility models were generated using weights of evidence, logistic regression, multilayer perceptron, support vector machine, random forest, and naive Bayes methods to produce quantitative susceptibility maps and assess the hazard associated with two scenarios: the first being El Niño phenomenon and the second being an earthquake exceeding 8.8 Mw. The main findings indicate that machine learning models exhibit excellent predictive performance for the presence and absence of mass movement events, as all models surpassed an AUC value of >0.9, with the random forest model standing out. In terms of hazard levels, in the event of an El Niño phenomenon or an earthquake exceeding 8.8 Mw, approximately 40% and 35% respectively, of the NLC area would be exposed to the highest hazard levels. The importance of integrating methodologies in mass movement susceptibility models is also emphasized; these methodologies include the correlation analysis, multicollinearity assessment, dimensionality reduction of variables, and coupling statistical models with machine learning models to improve the predictive accuracy of machine learning models. The findings of this research are expected to serve as a supportive tool for land managers in formulating effective disaster prevention and risk reduction strategies.

Abstract: Background: Recent studies show that most systemic mastocytosis (SM) patients, including indolent SM (ISM) with (ISMs+) and without skin lesions (ISMs−), carry the KIT D816V mutation in PB leukocytes. We investigated the potential association between the degree of involvement of BM hematopoiesis by the KIT D816V mutation and the distribution of different maturation‐associated compartments of bone marrow (BM) and peripheral blood (PB) CD34+ hematopoietic precursors (HPC) in ISM and identified the specific PB cell compartments that carry this mutation. Methods: The distribution of different maturation‐associated subsets of BM and PB CD34+ HPC from 64 newly diagnosed (KIT‐mutated) ISM patients and 14 healthy controls was analyzed by flow cytometry. In 18 patients, distinct FACS‐purified PB cell compartments were also investigated for the KIT mutation. Results: ISM patients showed higher percentages of both BM and PB MC‐committed CD34+ HPC vs controls, particularly among ISM cases with MC‐restricted KIT mutation (ISMMC); this was associated with progressive blockade of maturation of CD34+ HPC to the neutrophil lineage from ISMMC to multilineage KIT‐mutated cases (ISMML). Regarding the frequency of KIT‐mutated cases and cell populations in PB, variable patterns were observed, the percentage of KIT‐mutated PB CD34+ HPC, eosinophils, neutrophils, monocytes and T cells increasing from ISMs−MC and ISMs+MC to ISMML patients. Conclusion: The presence of the KIT D816V mutation in PB of ISM patients is associated with (early) involvement of circulating CD34+ HPC and multiple myeloid cell subpopulations, KIT‐mutated PB CD34+ HPC potentially contributing to early dissemination of the disease. [ABSTRACT FROM AUTHOR]

Background: A variable percentage of patients with systemic mast cell (MC) activation symptoms meet criteria for systemic mastocytosis (SM). We prospectively evaluated the clinical utility of the REMA score versus serum baseline tryptase (sBt) levels for predicting MC clonality and SM in 158 patients with systemic MC activation symptoms in the absence of mastocytosis in the skin (MIS). Methods: World Health Organization criteria for SM were applied in all cases. MC clonality was defined as the presence of KIT-mutated MC or by a clonal HUMARA test. The REMA score consisted of the assignment of positive or negative points as follows: male (+1), female (-1), sBt <15 μg/l (-1) or >25 μg/l (+2), presence (-2) or absence (+1) of pruritus, hives or angioedema and presence (+3) of presyncope or syncope. Efficiency of the REMA score for predicting MC clonality and SM was assessed by receiver operating characteristic (ROC) curve analyses and compared to those obtained by means of sBt levels alone. Results: Molecular studies revealed the presence of clonal MC in 68/80 SM cases and in 11/78 patients who did not meet the criteria for SM. ROC curve analyses confirmed the greater sensitivity and a similar specificity of the REMA score versus sBt levels (84 vs. 59% and 74 vs. 70% for MC clonality and 87 vs. 62% and 73 vs. 71% for SM, respectively). Conclusions: Our results confirm the clinical utility of the REMA score to predict MC clonality and SM in patients suffering from systemic MC activation symptoms without MIS. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Systemic mastocytosis (SM) is a heterogeneous disease characterized by an expansion of KIT-mutated mast cells (MC). KIT-mutated MC display activated features and release MC mediators that might act on the tumour microenvironment and other immune cells. Here, we investigated the distribution of lymphocyte subsets in blood of patients with distinct subtypes of SM and determined its association with other disease features., Methods: We studied the distribution of TCD4 + and TCD4 - cytotoxic cells and their subsets, as well as total NK- and B cells, in blood of 115 SM patients-38 bone marrow mastocytosis (BMM), 67 indolent SM (ISM), 10 aggressive SM (ASM)- and 83 age-matched healthy donors (HD), using spectral flow cytometry and the EuroFlow Immunomonitoring panel, and correlated it with multilineage KIT D816V , the alpha-tryptasemia genotype (HαT) and the clinical manifestations of the disease., Results: SM patients showed decreased counts (vs. HD) of TCD4 - cytotoxic cells, NK cells and several functional subsets of TCD4 + cells (total Th1, Th2-effector memory, Th22-terminal effector and Th1-like Tregs), together with increased T-follicular-helper and Th1/Th17-like Treg counts, associated with different immune profiles per diagnostic subtype of SM, in multilineal versus MC-restricted KIT D816V and in cases with a HαT + versus HαT - genotype. Unique immune profiles were found among BMM and ISM patients with MC-restricted KIT D816V who displayed HαT, anaphylaxis, hymenoptera venom allergy, bone disease, pruritus, flushing and GI symptoms., Conclusion: Our results reveal altered T- and NK-cell immune profiles in blood of SM, which vary per disease subtype, the pattern of involvement of haematopoiesis by KIT D816V , the HαT genotype and specific clinical manifestations of the disease., (© 2024 The Authors. Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

Oncolytic virus (OV) clinical trials have demonstrated remarkable efficacy in subsets of patients with glioblastoma (GBM). However, the lack of tools to predict this response hinders the advancement of a more personalized application of OV therapy. In this study, we characterize an ex vivo co-culture system designed to examine the immune response to OV infection of patient-derived GBM neurospheres in the presence of autologous peripheral blood mononuclear cells (PBMCs). Co-culture conditions were optimized to retain viability and functionality of both tumor cells and PBMCs, effectively recapitulating the well-recognized immunosuppressive effects of GBM. Following OV infection, we observed elevated secretion of pro-inflammatory cytokines and chemokines, including interferon γ, tumor necrosis factor α, CXCL9, and CXCL10, and marked changes in immune cell activation markers. Importantly, OV treatment induced unique patient-specific immune responses. In summary, our co-culture platform presents an avenue for personalized screening of viro-immunotherapies in GBM, offering promise as a potential tool for future patient stratification in OV therapy., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Many countries continue to experience pertussis epidemics despite widespread vaccination. Waning protection after booster vaccination has highlighted the need for a better understanding of the immunological factors that promote durable protection. Here we apply systems vaccinology to investigate antibody responses in adolescents in the Netherlands (N = 14; NL) and the United Kingdom (N = 12; UK) receiving a tetanus-diphtheria-acellular pertussis-inactivated poliovirus (Tdap-IPV) vaccine. We report that early antiviral and interferon gene expression signatures in blood correlate to persistence of pertussis-specific antibody responses. Single-cell analyses of the innate response identified monocytes and myeloid dendritic cells (MoDC) as principal responders that upregulate antiviral gene expression and type-I interferon cytokine production. With public data, we show that Tdap vaccination stimulates significantly lower antiviral/type-I interferon responses than Tdap-IPV, suggesting that IPV may promote antiviral gene expression. Subsequent in vitro stimulation experiments demonstrate TLR-dependent, IPV-specific activation of the pro-inflammatory p38 MAP kinase pathway in MoDCs. Together, our data provide insights into the molecular host response to pertussis booster vaccination and demonstrate that IPV enhances innate immune activity associated with persistent, pertussis-specific antibody responses., (© 2024. The Author(s).)

Introduction: Monitoring of innate myeloid cells (IMC) is broadly applied in basic and translational research, as well as in diagnostic patient care. Due to their immunophenotypic heterogeneity and biological plasticity, analysis of IMC populations typically requires large panels of markers. Currently, two cytometry-based techniques allow for the simultaneous detection of ≥40 markers: spectral flow cytometry (SFC) and mass cytometry (MC). However, little is known about the comparability of SFC and MC in studying IMC populations., Methods: We evaluated the performance of two SFC and MC panels, which contained 21 common markers, for the identification and subsetting of blood IMC populations. Based on unsupervised clustering analysis, we systematically identified 24 leukocyte populations, including 21 IMC subsets, regardless of the cytometry technique., Results: Overall, comparable results were observed between the two technologies regarding the relative distribution of these cell populations and the staining resolution of individual markers (Pearson's ρ=0.99 and 0.55, respectively). However, minor differences were observed between the two techniques regarding intra-measurement variability (median coefficient of variation of 42.5% vs. 68.0% in SFC and MC, respectively; p<0.0001) and reproducibility, which were most likely due to the significantly longer acquisition times (median 16 min vs. 159 min) and lower recovery rates (median 53.1% vs. 26.8%) associated with SFC vs. MC., Discussion: Altogether, our results show a good correlation between SFC and MC for the identification, enumeration and characterization of IMC in blood, based on large panels (>20) of antibody reagents., Competing Interests: JJMvD and AO are chairmen of the EuroFlow scientific foundation, which receives royalties from licensed patents, which are collectively owned by the participants of the EuroFlow Foundation and are exclusively used for continuation of the EuroFlow collaboration and sustainability of the EuroFlow consortium. JJMvD and AO report an Educational Services Agreement from BD Biosciences San José, CA and a Scientific Advisor Agreement with Cytognos/BD Biosciences; all related fees and honoraria are for the involved university departments at Leiden University Medical Center and University of Salamanca. JJMvD, AO, CT and KvdP are listed as coinventors on the patent “Means and methods for multiparameter cytometry-based leukocyte subsetting” PCT/NL2020/050688, filing date 5 November 2019, owned by the EuroFlow scientific consortium, that formed the basis for part of the antibody combination described in this manuscript. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be considered as a potential conflict of interest., (Copyright © 2023 van der Pan, Khatri, de Jager, Louis, Kassem, Naber, de Laat, Hameetman, Comans, Orfao, van Dongen, Díez and Teodosio.)

Background: The rs72824905 single-nucleotide polymorphism in the PLCG2 gene, encoding the p.P522R residue change in Phospholipase C gamma 2 (PLCγ2), associates with protection against several dementia subtypes and with increased likelihood of longevity. Cell lines and animal models indicated that p.P522R is a functional hypermorph. We aimed to confirm this in human circulating peripheral immune cells., Methods: We compared effects of p.P522R on immune system function between carriers and non-carriers (aged 59-103y), using in-depth immunophenotyping, functional B-cell and myeloid cell assays, and in vivo SARS-CoV-2 vaccination., Results: In line with expectations, p.P522R impacts immune cell function only slightly, but it does so across a wide array of immune cell types. Upon B-cell stimulation, we observed increased PLCγ2 phosphorylation and calcium release, suggesting increased B-cell sensitivity upon antigen recognition. Further, p.P522R-carriers had higher numbers of CD20++CD21-CD24+ naive B cells and IgG1+ memory B cells. In myeloid cells, normalized ROS production was higher upon PLCγ2-dependent stimulation. On classical monocytes, CD33 levels were elevated. Furthermore, carriers expressed lower levels of allergy-related FcεRI on several immune cell subsets. Nevertheless, carriers and non-carriers had similar serological responses to SARS-CoV-2 vaccination., Conclusion: The immune system from p.P522R-carriers is slightly more responsive to stimulation than in non-carriers., (© 2023. The Author(s).)

BACKGROUNDTo date, only limited data are available on the mechanisms of protection against colonization with Bordetella pertussis in humans.METHODSIn this study, the cellular responses to B. pertussis challenge were monitored longitudinally using high-dimensional EuroFlow-based flow cytometry, allowing quantitative detection of more than 250 different immune cell subsets in the blood of 15 healthy donors.RESULTSParticipants who were protected against colonization showed different early cellular responses compared with colonized participants. Especially prominent for colonization-protected participants were the early expansion of CD36- nonclassical monocytes on day 1 (D1), natural killer cells (D3), follicular T helper cells (D1-D3), and plasma cells (D3). Plasma cell expansion on D3 correlated negatively with the CFU load on D7 and D9 after challenge. Increased plasma cell maturation on D11-D14 was found in participants with seroconversion.CONCLUSIONThese early cellular immune responses following experimental infection can now be further characterized and potentially linked to an efficient mucosal immune response, preventing colonization. Ultimately, their presence may be used to evaluate whether new B. pertussis vaccine candidates are protective against B. pertussis colonization, e.g., by bacterial challenge after vaccination.TRIAL REGISTRATIONClinicalTrials.gov NCT03751514.FUNDINGInnovative Medicines Initiative 2 Joint Undertaking and the EuroFlow Consortium.

Reference ranges of blood-circulating leukocyte populations by, e.g., age and sex, are required for monitoring immune-cell kinetics. Most previous reports in which flow cytometry has been used to define the reference ranges for leukocyte counts included a limited number of donors and/or cell populations and/or did not consider age and sex simultaneously. Moreover, other factors not previously considered in the definition of normal ranges, such as the presence of chronic-lymphocytic-leukemia (CLL)-like low-count monoclonal B-cell lymphocytosis (MBLlo), might also be associated with an altered distribution of leukocytes in blood in association with an immunodeficiency and increased risk of infection and cancer. Here, we established reference cell-count ranges for the major populations of leukocytes in blood of non-MBL and MBLlo adult Caucasians matched by age and sex using the EuroFlow Lymphocyte Screening Tube (LST). A total of 706 Caucasian adult donors—622 non-MBL and 84 MBLlo—were recruited from the general population. Among non-MBL donors, the total leukocyte, neutrophil, basophil dendritic cell and monocyte counts remained stable through adulthood, while the absolute numbers of T- and B-cell populations and plasma cells decreased with age. The number of eosinophils and NK-cell increased over time, with clear differences according to sex for certain age ranges. In MBLlo subjects, few differences in the absolute cell counts by age (vs. non-MBL) were observed, and MBLlo men and women showed similar trends to non-MBL subjects except for the B-cell count drop observed in >70 y-men, which was more pronounced in MBLlo vs. non-MBL controls. Building robust age- and sex-matched reference ranges for the most relevant immune-cell populations in the blood of non-MBL donors is essential to appropriately identify an altered immune status in different clinical settings and highlight the altered immune-cell profiles of MBLlo subjects.

T cell development in the mouse thymus has been studied extensively, but less is known regarding T cell development in the human thymus. We used a combination of single-cell techniques and functional assays to perform deep immune profiling of human T cell development, focusing on the initial stages of prelineage commitment. We identified three thymus-seeding progenitor populations that also have counterparts in the bone marrow. In addition, we found that the human thymus physiologically supports the development of monocytes, dendritic cells, and NK cells, as well as limited development of B cells. These results are an important step toward monitoring and guiding regenerative therapies in patients after hematopoietic stem cell transplantation.

Mass spectrometry (MS)-based proteomics profiling has undoubtedly increased the knowledge about cellular processes and functions. However, its applicability for paucicellular sample analyses is currently limited. Although new approaches have been developed for single-cell studies, most of them have not (yet) been standardized and/or require highly specific (often home-built) devices, thereby limiting their broad implementation, particularly in non-specialized settings. To select an optimal MS-oriented proteomics approach applicable in translational research and clinical settings, we assessed 10 different sample preparation procedures in paucicellular samples of closely-related cell types. Particularly, five cell lysis protocols using different chemistries and mechanical forces were combined with two sample clean-up techniques (C18 filter- and SP3-based), followed by tandem mass tag (TMT)-based protein quantification. The evaluation was structured in three phases: first, cell lines from hematopoietic (THP-1) and non-hematopoietic (HT-29) origins were used to test the approaches showing the combination of a urea-based lysis buffer with the SP3 bead-based clean-up system as the best performer. Parameters such as reproducibility, accessibility, spatial distribution, ease of use, processing time and cost were considered. In the second phase, the performance of the method was tested on maturation-related cell populations: three different monocyte subsets from peripheral blood and, for the first time, macrophages/microglia (MAC) from glioblastoma samples, together with T cells from both tissues. The analysis of 50,000 cells down to only 2,500 cells revealed different protein expression profiles associated with the distinct cell populations. Accordingly, a closer relationship was observed between non-classical monocytes and MAC, with the latter showing the co-expression of M1 and M2 macrophage markers, although pro-tumoral and anti-inflammatory proteins were more represented. In the third phase, the results were validated by high-end spectral flow cytometry on paired monocyte/MAC samples to further determine the sensitivity of the MS approach selected. Finally, the feasibility of the method was proven in 194 additional samples corresponding to 38 different cell types, including cells from different tissue origins, cellular lineages, maturation stages and stimuli. In summary, we selected a reproducible, easy-to-implement sample preparation method for MS-based proteomic characterization of paucicellular samples, also applicable in the setting of functionally closely-related cell populations., Competing Interests: Authors JD and AO are chairmen of the EuroFlow scientific foundation, which receives royalties from licensed patents, which are collectively owned by the participants of the EuroFlow foundation, to be exclusively used for the continuation of the EuroFlow collaboration and sustainability of the EuroFlow consortium, originally supported by the European Commission (EU-STREP Project LSHB-CT-2006-018708). Authors JD and AO report an Educational Services Agreement from BD Biosciences (San José, CA) and a Scientific Advisor Agreement with Cytognos; all related fees and honoraria are for the involved university departments at Leiden University Medical Center and University of Salamanca. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 van der Pan, Kassem, Khatri, de Ru, Janssen, Tjokrodirijo, al Makindji, Stavrakaki, de Jager, Naber, de Laat, Louis, van den Bossche, Vogelezang, Balvers, Lamfers, van Veelen, Orfao, van Dongen, Teodosio and Díez.)

Innate myeloid cell (IMC) populations form an essential part of innate immunity. Flow cytometric (FCM) monitoring of IMCs in peripheral blood (PB) has great clinical potential for disease monitoring due to their role in maintenance of tissue homeostasis and ability to sense micro-environmental changes, such as inflammatory processes and tissue damage. However, the lack of standardized and validated approaches has hampered broad clinical implementation. For accurate identification and separation of IMC populations, 62 antibodies against 44 different proteins were evaluated. In multiple rounds of EuroFlow-based design-testing-evaluation-redesign, finally 16 antibodies were selected for their non-redundancy and separation power. Accordingly, two antibody combinations were designed for fast, sensitive, and reproducible FCM monitoring of IMC populations in PB in clinical settings (11-color; 13 antibodies) and translational research (14-color; 16 antibodies). Performance of pre-analytical and analytical variables among different instruments, together with optimized post-analytical data analysis and reference values were assessed. Overall, 265 blood samples were used for design and validation of the antibody combinations and in vitro functional assays, as well as for assessing the impact of sample preparation procedures and conditions. The two (11- and 14-color) antibody combinations allowed for robust and sensitive detection of 19 and 23 IMC populations, respectively. Highly reproducible identification and enumeration of IMC populations was achieved, independently of anticoagulant, type of FCM instrument and center, particularly when database/software-guided automated ( vs. manual "expert-based") gating was used. Whereas no significant changes were observed in identification of IMC populations for up to 24h delayed sample processing, a significant impact was observed in their absolute counts after >12h delay. Therefore, accurate identification and quantitation of IMC populations requires sample processing on the same day. Significantly different counts were observed in PB for multiple IMC populations according to age and sex. Consequently, PB samples from 116 healthy donors (8-69 years) were used for collecting age and sex related reference values for all IMC populations. In summary, the two antibody combinations and FCM approach allow for rapid, standardized, automated and reproducible identification of 19 and 23 IMC populations in PB, suited for monitoring of innate immune responses in clinical and translational research settings., Competing Interests: JD and AO report to be chairmen of the EuroFlow scientific foundation, which receives royalties from licensed patents, which are collectively owned by the participants of the EuroFlow Foundation. These royalties are exclusively used for continuation of the EuroFlow collaboration and sustainability of the EuroFlow consortium. JD and AO report an Educational Services Agreement from BD Biosciences (San José, CA) and a Scientific Advisor Agreement with Cytognos; all related fees and honoraria are for the involved university departments at Leiden University Medical Center and University of Salamanca. AH-D is an employee of Cytognos (Salamanca, Spain). Lastly, JD, CT, AO, JA, WB, KP, MB, AD, DD, and AH-D, are listed as (co)inventors on the patent “Means and methods for multiparameter cytometry-based leukocyte subsetting” (NL2844751, filing date 5 November 2019), owned by the EuroFlow scientific consortium, which describes the flow cytometry panels developed in this study. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 van der Pan, de Bruin-Versteeg, Damasceno, Hernández-Delgado, van der Sluijs-Gelling, van den Bossche, de Laat, Díez, Naber, Diks, Berkowska, de Mooij, Groenland, de Bie, Khatri, Kassem, de Jager, Louis, Almeida, van Gaans-van den Brink, Barkoff, He, Ferwerda, Versteegen, Berbers, Orfao, van Dongen and Teodosio.)

Pertussis is a vaccine-preventable disease caused by the bacterium Bordetella pertussis . Over the past years, the incidence and mortality of pertussis increased significantly. A possible cause is the switch from whole-cell to acellular pertussis vaccines, although other factors may also contribute. Here, we applied high-dimensional flow cytometry to investigate changes in B cells in individuals of different ages and distinct priming backgrounds upon administration of an acellular pertussis booster vaccine. Participants were divided over four age cohorts. We compared longitudinal kinetics within each cohort and between the different cohorts. Changes in the B-cell compartment were correlated to numbers of vaccine-specific B- and plasma cells and serum Ig levels. Expansion and maturation of plasma cells 7 days postvaccination was the most prominent cellular change in all age groups and was most pronounced for more mature IgG1+ plasma cells. Plasma cell responses were stronger in individuals primed with whole-cell vaccine than in individuals primed with acellular vaccine. Moreover, IgG1+ and IgA1+ plasma cell expansion correlated with FHA-, Prn-, or PT- specific serum IgG or IgA levels. Our study indicates plasma cells as a potential early cellular marker of an immune response and contributes to understanding differences in immune responses between age groups and primary vaccination backgrounds.

Sézary syndrome (SS) is an aggressive leukemic form of cutaneous T-cell lymphoma with neoplastic CD4+ T cells present in skin, lymph nodes, and blood. Despite advances in therapy, prognosis remains poor, with a 5-year overall survival of 30%. The immunophenotype of Sézary cells is diverse, which hampers efficient diagnosis, sensitive disease monitoring, and accurate assessment of treatment response. Comprehensive immunophenotypic profiling of Sézary cells with an in-depth analysis of maturation and functional subsets has not been performed thus far. We immunophenotypically profiled 24 patients with SS using standardized and sensitive EuroFlow-based multiparameter flow cytometry. We accurately identified and quantified Sézary cells in blood and performed an in-depth assessment of their phenotypic characteristics in comparison with their normal counterparts in the blood CD4+ T-cell compartment. We observed inter- and intrapatient heterogeneity and phenotypic changes over time. Sézary cells exhibited phenotypes corresponding with classical and nonclassical T helper subsets with different maturation phenotypes. We combined multiparameter flow cytometry analyses with fluorescence-activated cell sorting and performed RNA sequencing studies on purified subsets of malignant Sézary cells and normal CD4+ T cells of the same patients. We confirmed pure monoclonality in Sézary subsets, compared transcriptomes of phenotypically distinct Sézary subsets, and identified novel downregulated genes, most remarkably THEMIS and LAIR1, which discriminate Sézary cells from normal residual CD4+ T cells. Together, these findings further unravel the heterogeneity of Sézary cell subpopulations within and between patients. These new data will support improved blood staging and more accurate disease monitoring., (© 2021 by The American Society of Hematology.)

Systemic mastocytosis (SM) is a KIT-driven hematopoietic neoplasm characterized by the excessive accumulation of neoplastic mast cells (MCs) in various organs and, mainly, the bone marrow (BM). Multiple genetic and epigenetic mechanisms contribute to the onset and severity of SM. However, little is known to date about the metabolic underpinnings underlying SM aggressiveness, which has thus far impeded the development of strategies to leverage metabolic dependencies when existing KIT-targeted treatments fail. Here, we show that plasma metabolomic profiles were able to discriminate indolent from advanced forms of the disease. We identified N-acetyl-d-glucosamine (GlcNAc) as the most predictive metabolite of SM severity. High plasma levels of GlcNAc in patients with advanced SM correlated with the activation of the GlcNAc-fed hexosamine biosynthesis pathway in patients BM aspirates and purified BM MCs. At the functional level, GlcNAc enhanced human neoplastic MCs proliferation and promoted rapid health deterioration in a humanized mouse model of SM. In addition, in the presence of GlcNAc, immunoglobulin E-stimulated MCs triggered enhanced release of proinflammatory cytokines and a stronger acute response in a mouse model of passive cutaneous anaphylaxis. Mechanistically, elevated GlcNAc levels promoted the transcriptional accessibility of chromatin regions that contain genes encoding mediators of receptor tyrosine kinases cascades and inflammatory responses, thus leading to a more aggressive phenotype. Therefore, GlcNAc is an oncometabolite driver of SM aggressiveness. This study suggests the therapeutic potential for targeting metabolic pathways in MC-related diseases to manipulate MCs effector functions., (© 2021 by The American Society of Hematology.)

Nowadays, massive genomics and transcriptomics data can be generated at the single-cell level. However, proteomics in this setting is still a big challenge. Despite the great improvements in sensitivity and performance of mass spectrometry instruments and the better knowledge on sample preparation processing, it is widely acknowledged that multistep proteomics workflows may lead to substantial sample loss, especially when working with paucicellular samples. Still, in clinical fields, frequently limited sample amounts are available for downstream analysis, thereby hampering comprehensive characterization at protein level. To aim at better protein and peptide recoveries, we compare existing and novel approaches in the multistep sample preparation protocols for mass spectrometry studies, from sample collection, cell lysis, protein quantification, and electrophoresis/staining to protein digestion, peptide recovery, and LC-MS/MS instruments. From this critical evaluation, we conclude that the recent innovations and technologies, together with high quality management of samples, make proteomics on paucicellular samples possible, which will have immediate impact for the proteomics community.

Immunoglobulin (IG) loci harbor inter-individual allelic variants in many different germline IG variable, diversity and joining genes of the IG heavy (IGH), kappa (IGK) and lambda (IGL) loci, which together form the genetic basis of the highly diverse antigen-specific B-cell receptors. These allelic variants can be shared between or be specific to human populations. The current immunogenetics resources gather the germline alleles, however, lack the population specificity of the alleles which poses limitations for disease-association studies related to immune responses in different human populations. Therefore, we systematically identified germline alleles from 26 different human populations around the world, profiled by "1000 Genomes" data. We identified 409 IGHV, 179 IGKV, and 199 IGLV germline alleles supported by at least seven haplotypes. The diversity of germline alleles is the highest in Africans. Remarkably, the variants in the identified novel alleles show strikingly conserved patterns, the same as found in other IG databases, suggesting over-time evolutionary selection processes. We could relate the genetic variants to population-specific immune responses, e.g. IGHV1-69 for flu in Africans. The population matched IG (pmIG) resource will enhance our understanding of the SHM-related B-cell receptor selection processes in (infectious) diseases and vaccination within and between different human populations., (© 2021. The Author(s).)

Antigen-specific serum immunoglobulin (Ag-specific Ig) levels are broadly used as correlates of protection. However, in several disease and vaccination models these fail to predict immunity. In these models, in-depth knowledge of cellular processes associated with protective versus poor responses may bring added value. We applied high-throughput multicolor flow cytometry to track over-time changes in circulating immune cells in 10 individuals following pertussis booster vaccination (Tdap, Boostrix ® , GlaxoSmithKline). Next, we applied correlation network analysis to extensively investigate how changes in individual cell populations correlate with each other and with Ag-specific Ig levels. We further determined the most informative cell subsets and analysis time points for future studies. Expansion and maturation of total IgG1 plasma cells, which peaked at day 7 post-vaccination, was the most prominent cellular change. Although these cells preceded the increase in Ag-specific serum Ig levels, they did not correlate with the increase of Ig levels. In contrast, strong correlation was observed between Ag-specific IgGs and maximum expansion of total IgG1 and IgA1 memory B cells at days 7 to 28. Changes in circulating T cells were limited, implying the need for a more sensitive approach. Early changes in innate immune cells, i.e. expansion of neutrophils, and expansion and maturation of monocytes up to day 5, most likely reflected their responses to local damage and adjuvant. Here we show that simultaneous monitoring of multiple circulating immune subsets in blood by flow cytometry is feasible. B cells seem to be the best candidates for vaccine monitoring., Competing Interests: AD, CT, JD, AO, MP-A and MB report inventorship of the patent “Means and methods for multiparameter cytometry-based leukocyte subsetting” (NL2844751, filing date 5 November 2019) (21), owned by the EuroFlow Consortium. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Diks, Khatri, Oosten, de Mooij, Groenland, Teodosio, Perez-Andres, Orfao, Berbers, Zwaginga, van Dongen and Berkowska.)

The impact of sleep deprivation on muscular strength and power remains poorly understood. We aimed to determine the acute effects of 24 hr of sleep deprivation on H-reflex and V-wave excitability. Fourteen healthy young adults (eight men, six women) were included. Participants visited the laboratory on two different occasions, without and with 24 hr of sleep deprivation. In each session, participants were tested for maximal voluntary contraction (MVC) of the plantar flexors and dorsiflexors, soleus H- and M-recruitment curves, and evoked V wave, as well as tibialis anterior/soleus electromyographic co-activation. Twenty-four hours of sleep deprivation did not affect either plantarflexion MVC or soleus electromyographic normalized amplitude (p > .05). Moreover, H-reflex and V-wave peak-to-peak normalized amplitude did not change with sleep deprivation (p > .05). Conversely, we obtained a significant increase in antagonist/agonist level of co-activation during MVC post-sleep deprivation (6.2 ± 5.2%, p < .01). In conclusion, we found that H-reflex and V-wave responses are well preserved after 24 hr of sleep deprivation, revealing that descending neural drive and/or modulation in Ia afferent input remains largely unaffected under these circumstances. Yet, sleep deprivation affects motor control by exacerbating the magnitude of antagonist/agonist co-activation during forceful muscle contractions and this is novel., (© 2020 European Sleep Research Society.)

Background: Monocyte/macrophages have been shown to be altered in monoclonal gammopathy of undetermined significance (MGUS), smoldering (SMM) and active multiple myeloma (MM), with an impact on the disruption of the homeostasis of the normal bone marrow (BM) microenvironment., Methods: We investigated the distribution of different subsets of monocytes (Mo) in blood and BM of newly-diagnosed untreated MGUS ( n = 23), SMM ( n = 14) and MM ( n = 99) patients vs. healthy donors (HD; n = 107), in parallel to a large panel of cytokines and bone-associated serum biomarkers., Results: Our results showed normal production of monocyte precursors and classical Mo (cMo) in MGUS, while decreased in SMM and MM ( p ≤ 0.02), in association with lower blood counts of recently-produced CD62L + cMo in SMM ( p = 0.004) and of all subsets of (CD62L + , CD62L - and FcεRI + ) cMo in MM ( p ≤ 0.02). In contrast, intermediate and end-stage non-classical Mo were increased in BM of MGUS ( p ≤ 0.03), SMM ( p ≤ 0.03) and MM ( p ≤ 0.002), while normal (MGUS and SMM) or decreased (MM; p = 0.01) in blood. In parallel, increased serum levels of interleukin (IL)1β were observed in MGUS ( p = 0.007) and SMM ( p = 0.01), higher concentrations of serum IL8 were found in SMM ( p = 0.01) and MM ( p = 0.002), and higher serum IL6 ( p = 0.002), RANKL ( p = 0.01) and bone alkaline phosphatase (BALP) levels ( p = 0.01) with decreased counts of FcεRI + cMo, were restricted to MM presenting with osteolytic lesions. This translated into three distinct immune/bone profiles: (1) normal (typical of HD and most MGUS cases); (2) senescent-like (increased IL1β and/or IL8, found in a minority of MGUS, most SMM and few MM cases with no bone lesions); and (3) pro-inflammatory-high serum IL6, RANKL and BALP with significantly ( p = 0.01) decreased blood counts of immunomodulatory FcεRI + cMo-, typical of MM presenting with bone lesions., Conclusions: These results provide new insight into the pathogenesis of plasma cell neoplasms and the potential role of FcεRI + cMo in normal bone homeostasis.

Diagnosis and monitoring of primary brain tumours, brain metastasis and acute ischaemic stroke all require invasive, burdensome and costly diagnostics, frequently lacking adequate sensitivity, particularly during disease monitoring. Monocytes are known to migrate to damaged tissues, where they act as tissue macrophages, continuously scavenging, phagocytizing and digesting apoptotic cells and other tissue debris. We hypothesize that upon completion of their tissue-cleaning task, these tissue macrophages might migrate via the lymph system to the bloodstream, where they can be detected and evaluated for their phagolysosomal contents. We discovered a blood monocyte subpopulation carrying the brain-specific glial fibrillary acidic protein in glioma patients and in patients with brain metastasis and evaluated the diagnostic potential of this finding. Blood samples were collected in a cross-sectional study before or during surgery from adult patients with brain lesions suspected of glioma. Together with blood samples from healthy controls, these samples were flowing cytometrically evaluated for intracellular glial fibrillary acidic protein in monocyte subsets. Acute ischaemic stroke patients were tested at multiple time points after onset to evaluate the presence of glial fibrillary acidic protein-carrying monocytes in other forms of brain tissue damage. Clinical data were collected retrospectively. High-grade gliomas ( N  = 145), brain metastasis ( N  = 21) and large stroke patients (>100 cm 3 ) ( N  = 3 versus 6; multiple time points) had significantly increased frequencies of glial fibrillary acidic protein+CD16+ monocytes compared to healthy controls. Based on both a training and validation set, a cut-off value of 0.6% glial fibrillary acidic protein+CD16+ monocytes was established, with 81% sensitivity (95% CI 75-87%) and 85% specificity (95% CI 80-90%) for brain lesion detection. Acute ischaemic strokes of >100 cm 3 reached >0.6% of glial fibrillary acidic protein+CD16+ monocytes within the first 2-8 h after hospitalization and subsided within 48 h. Glioblastoma patients with >20% glial fibrillary acidic protein+CD16+ non-classical monocytes had a significantly shorter median overall survival (8.1 versus 12.1 months). Our results and the available literature, support the hypothesis of a tissue-origin of these glial fibrillary acidic protein-carrying monocytes. Blood monocytes carrying glial fibrillary acidic protein have a high sensitivity and specificity for the detection of brain lesions and for glioblastoma patients with a decreased overall survival. Furthermore, their very rapid response to acute tissue damage identifies large areas of ischaemic tissue damage within 8 h after an ischaemic event. These studies are the first to report the clinical applicability for brain tissue damage detection through a minimally invasive diagnostic method, based on blood monocytes and not serum markers, with direct consequences for disease monitoring in future (therapeutic) studies and clinical decision making in glioma and acute ischaemic stroke patients., (© The Author(s) (2020). Published by Oxford University Press on behalf of the Guarantors of Brain.)