Diverse but unique astrocytic phenotypes during embryonic stem cell differentiation, culturing and development

Astrocytes are resident glia cells of the central nervous system (CNS) that play complex and heterogeneous roles in brain development, homeostasis and disease. Since their vast involvement in health and disease is becoming increasingly recognized, suitable and reliable tools for studying these cells in vivo and in vitro are of utmost importance. One of the key challenges hereby is to adequately mimic their context-dependent in vivo phenotypes and functions in vitro. To better understand the spectrum of astrocytic variations in defined settings we performed a side-by-side-comparison of embryonic stem cell (ESC)-derived astrocytes as well as primary neonatal and adult astrocytes, revealing major differences on a functional and transcriptomic level, specifically on proliferation, migration, calcium signaling and cilium activity. Our results highlight the need to carefully consider the choice of astrocyte origin and phenotype with respect to age, isolation and culture protocols based on the respective biological question

International comparative study of low back pain care pathways and analysis of key interventions

Purpose Low back pain (LBP) is a major public health problem worldwide. Significant practice variation exists despite guidelines, including strong interventionist focus by some practitioners. Translation of guidelines into pathways as integrated treatment plans is a next step to improve implementation. The goal of the present study was to analyze international examples of LBP pathways in order to identify key interventions as building elements for care pathway for LBP and radicular pain. Methods International examples of LBP pathways were searched in literature and grey literature. Authors of pathways were invited to fill a questionnaire and to participate in an in-depth telephone interview. Pathways were quantitatively and qualitatively analyzed, to enable the identification of key interventions to serve as pathway building elements. Results Eleven international LBP care pathways were identified. Regional pathways were strongly organized and included significant training efforts for primary care providers and an intermediate level of caregivers in between general practitioners and hospital specialists. Hospital pathways had a focus on multidisciplinary collaboration and stepwise approach trajectories. Key elements common to all pathways included the consecutive screening for red flags, radicular pain and psychosocial risk factors, the emphasis on patient empowerment and self-management, the development of evidence-based consultable protocols, the focus on a multidisciplinary work mode and the monitoring of patient-reported outcome measures. Conclusion Essential building elements for the construction of LBP care pathways were identified from a transversal analysis of key interventions in a study of 11 international examples of LBP pathways

Neuroprotective tissue adaptation induced by IL-12 attenuates CNS inflammation

IL-12 is a well-established driver of type 1 immune responses. Paradoxically, in several autoimmune conditions including neuroinflammation, IL-12 reduces pathology and exhibits regulatory properties. Yet, the mechanism and the involved cellular players behind this immune regulation remain elusive. To identify the IL-12-responsive elements which prevent immunopathology, we generated mouse models lacking a functional IL-12 receptor either in all cells or in specific populations within the immune or central nervous system (CNS) compartments, and induced experimental autoimmune encephalomyelitis (EAE), which models human Multiple Sclerosis (MS). This revealed that the CNS tissue-protective features of IL-12 are mediated by cells of the neuroectoderm, and not immune cells. Importantly, sections of brain from patients with MS show comparable patterns of expression, indicating parallel mechanisms in humans. By combining spectral flow cytometry, bulk and single-nucleus RNA sequencing, we uncovered an IL-12-induced neuroprotective adaption of the neuroectoderm critically involved in maintaining CNS tissue integrity during inflammation

IL-12 sensing in neurons induces neuroprotective CNS tissue adaptation and attenuates neuroinflammation in mice

Interleukin-12 (IL-12) is a potent driver of type 1 immunity. Paradoxically, in autoimmune conditions, including of the CNS, IL-12 reduces inflammation. The underlying mechanism behind these opposing properties and the involved cellular players remain elusive. Here we map IL-12 receptor (IL-12R) expression to NK and T cells as well as neurons and oligodendrocytes. Conditionally ablating the IL-12R across these cell types in adult mice and assessing their susceptibility to experimental autoimmune encephalomyelitis revealed that the neuroprotective role of IL-12 is mediated by neuroectoderm-derived cells, specifically neurons, and not immune cells. In human brain tissue from donors with multiple sclerosis, we observe an IL-12R distribution comparable to mice, suggesting similar mechanisms in mice and humans. Combining flow cytometry, bulk and single-nucleus RNA sequencing, we reveal an IL-12-induced neuroprotective tissue adaption preventing early neurodegeneration and sustaining trophic factor release during neuroinflammation, thereby maintaining CNS integrity in mice

Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition)

The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer‐reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state‐of‐the‐art handbook for basic and clinical researchers.DFG, 389687267, Kompartimentalisierung, Aufrechterhaltung und Reaktivierung humaner Gedächtnis-T-Lymphozyten aus Knochenmark und peripherem BlutDFG, 80750187, SFB 841: Leberentzündungen: Infektion, Immunregulation und KonsequenzenEC/H2020/800924/EU/International Cancer Research Fellowships - 2/iCARE-2DFG, 252623821, Die Rolle von follikulären T-Helferzellen in T-Helferzell-Differenzierung, Funktion und PlastizitätDFG, 390873048, EXC 2151: ImmunoSensation2 - the immune sensory syste

Zielzellen und Mechanismen des IL-12/IL-23 Signalweges in der Alzheimer Erkrankung

Alzheimer’s disease (AD) is a severe neurodegenerative disorder characterised by the accumulation of aggregated amyloid-β (Aβ) and tau proteins as well as the activation of inflammatory processes in the brain, leading to neuronal cell death. Modulation of inflammatory factors presents an attractive therapeutic target since a direct interplay between inflammation and AD pathogenesis has been identified. In this study, we confirm previously published data showing that inhibition of the interleukin (IL)-12 and IL-23 signalling pathway leads to an amelioration of Aβ pathology. Genetic deletion of the shared IL-12/IL-23 IL12p40 subunit in APP23 AD-like mice caused a gender-specific reduction in Aβ burden, highlighting the significance of the IL-12/IL-23 pathway as well as gender considerations in AD. Furthermore, we identified neurons and oligodendrocytes as the IL-12/IL-23 receptor-bearing cells, uncovering a yet unknown intercellular inflammatory signalling pathway. Based on these observations, we generated AD-like mouse lines deleting the IL-23-specific receptor (APPPS1;NestinCre;IL23Rfl/fl and APPPS1;Aldh1l1CreERT;IL23Rfl/fl) as well as the IL-12-specific receptor (APPPS1;NestinCre;IL12Rβ2fl/fl and APPPS1;Aldh1l1CreERT;IL12Rβ2fl/fl) only in neural cells. Since initial results indicate that IL-23 signalling is not the key player in IL-12/IL-23-driven pathogenic aspects of AD, future research is required to prove the importance of IL-12 signalling in AD. Obtaining a detailed understanding of the precise downstream signalling mechanisms of the IL-12/IL-23 pathway is essential since it can provide a basis for target-directed therapy of AD, a disease that is not yet curable.Die Alzheimer Erkrankung (AE) ist eine schwerwiegende neurodegenerative Erkrankung, die charakterisiert wird durch die Akkumulation von aggregierten Amyloid-β (Aβ) und Tau Proteinen sowie der Aktivierung inflammatorischer Prozesse im Gehirn, welche schließlich zu neuronalem Zelltod führen. Modulierung von inflammatorischen Faktoren gilt als attraktiver therapeutischer Angriffspunkt, da ein direkter Zusammenhang zwischen Inflammation und AE Pathogenese besteht. In dieser Arbeit konnten wir bereits publizierte Daten validieren, welche zeigten, dass die Inhibition des Interleukin (IL) 12/IL 23 Signalwegs zu einer Verbesserung der Aβ Pathologie führte. Genetische Deletion der von IL-12/IL-23 geteilten IL12p40 Untereinheit im APP23 AE-Mausmodell erwirkte eine geschlechter-spezifische Reduktion der Aβ Belastung, welches die Signifikanz des IL 12/IL-23 Signalwegs sowie von Geschlechterunterschieden in der AE hervorhebt. Zusätzlich identifizierten wir, dass Neurone und Oligodendrozyten die IL 12/IL-23 Rezeptoren exprimierten, und entdeckten somit einen neuen interzellulären inflammatorischen Signalweg. Basierend auf diesen Ergebnissen generierten wir AE-ähnliche Mauslinien, in denen der IL-23-spezifische Rezeptor (APPPS1;NestinCre;IL23Rfl/fl und APPPS1;Aldh1l1CreERT;IL23Rfl/fl) sowie der IL 12 spezifische Rezeptor (APPPS1;NestinCre;IL12Rβ2fl/fl und APPPS1;Aldh1l1CreERT;IL12Rβ2fl/fl) ausschließlich auf neuralen Zellen deletiert ist. Erste Ergebnisse zeigten, dass IL-23 wohl nicht das entscheidende Zytokin im pathogenetisch relevanten IL-12/IL-23 Signalweg darstellt, weshalb sich unsere weiteren Forschungsvorhaben auf die Rolle von IL-12 in der AE fokussieren werden. Ein exaktes Verständnis der nachgeschalteten Mechanismen des IL-12/IL-23 Signalwegs ist essentiell um eine valide und präzise Grundlage für künftige Therapieansätze der bislang unheilbaren AE zu entwickeln

Diverse but unique astrocytic phenotypes during embryonic stem cell differentiation, culturing and development

Functional and transcriptomic comparison of primary neonatal, adult and ESC-derived astrocytes reveals major variations, highlighting the importance for an informed choice of astrocyte origin for research questions

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition)

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion

Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition)

Cossarizza A, Chang H‐D, Radbruch A, et al. Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition). European Journal of Immunology. 2021;51(12):2708-3145.The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer-reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state-of-the-art handbook for basic and clinical researchers

Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition)

The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer-reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state-of-the-art handbook for basic and clinical researchers.ISSN:0014-2980ISSN:1521-414