Visualization of leukocyte transendothelial and interstitial migration using reflected light oblique transillumination in intravital video microscopy

Dynamic visualization of the intravascular events leading to the extravasation of leukocytes into tissues by intravital microscopy has significantly expanded our understanding of the underlying molecular processes. In contrast, the detailed observation of leukocyte transendothelial and interstitial migration in vivo has been hampered by the poor image contrast of cells within turbid media that is obtainable by conventional brightfield microscopy. Here we present a microscopic method, termed reflected light oblique transillumination microscopy, that makes use of the optical interference phenomena generated by oblique transillumination to visualize subtle gradients of refractive indices within tissues for enhanced image contrast. Using the mouse cremaster muscle, we demonstrate that this technique makes possible the reliable quantification of extravasated leukocytes as well as the characterization of morphological phenomena of leukocyte transendothelial and interstitial migration

Intranet - Einsatz in der betrieblichen Interessenvertretung: Schlussfolgerungen aus exemplarischen Fallbeispielen

Betriebsräte wurden durch unternehmensinterne Intranets zu einer Entscheidung darüber gedrängt, ob sie selbst das Intranet als 'modernes Hilfsmittel' nutzen. Hierbei geht es darum, das Gleichgewicht zwischen den Betriebsparteien hinsichtlich der Informationsverbreitung gegenüber den Belegschaften auszutarieren. Gleichzeitig führt die zunehmend medienwirksame Gestaltung von Nachrichten durch den Einsatz hochwertiger multimedialer Kommunikationsmittel, z.B. im Fernsehen, Internet, zu immer größeren Erwartungshaltungen bei Nachrichtenempfängern. Es verwundert insofern nicht, dass Belegschaften von Betriebsräten den Einsatz des Intranets als 'innovatives' Nachrichtenportal erwarten. Die Gestaltung unternehmensinterner Beziehungen gegenüber den Belegschaften mittels moderner Medien steht somit unweigerlich auf der politischen Agenda der betrieblichen Interessenvertretungen - und die Fragen richten sich letztlich nicht nur an Gesamt- bzw. Konzernbetriebsräte großer Unternehmen, sondern auch an Betriebsräte in mittelständischen Betrieben. Die vorliegende Studie stellt fünf Beispiele vor, die über die Nutzung des Intranets durch Betriebsräte informieren. Außerdem wurden zwanzig Telefoninterviews mit Betriebsräten durchgeführt, wobei die Auswahl der Telefoninterviews nicht systematisch erfolgte, sondern an bestehende Kontakte anknüpfte. Anhand dieser Praxisbeispiele werden zentrale Fragegestellungen erörtert und der Versuch unternommen, zukünftige Trends von Intranets betrieblicher Interessenvertretungen zu skizzieren

Targeting the CBM complex causes Treg cells to prime tumours for immune checkpoint therapy.

Solid tumours are infiltrated by effector T cells with the potential to control or reject them, as well as by regulatory T (Treg) cells that restrict the function of effector T cells and thereby promote tumour growth1. The anti-tumour activity of effector T cells can be therapeutically unleashed, and is now being exploited for the treatment of some forms of human cancer. However, weak tumour-associated inflammatory responses and the immune-suppressive function of Treg cells remain major hurdles to broader effectiveness of tumour immunotherapy2. Here we show that, after disruption of the CARMA1-BCL10-MALT1 (CBM) signalosome complex, most tumour-infiltrating Treg cells produce IFNγ, resulting in stunted tumour growth. Notably, genetic deletion of both or even just one allele of CARMA1 (also known as Card11) in only a fraction of Treg cells-which avoided systemic autoimmunity-was sufficient to produce this anti-tumour effect, showing that it is not the mere loss of suppressive function but the gain of effector activity by Treg cells that initiates tumour control. The production of IFNγ by Treg cells was accompanied by activation of macrophages and upregulation of class I molecules of the major histocompatibility complex on tumour cells. However, tumour cells also upregulated the expression of PD-L1, which indicates activation of adaptive immune resistance3. Consequently, blockade of PD-1 together with CARMA1 deletion caused rejection of tumours that otherwise do not respond to anti-PD-1 monotherapy. This effect was reproduced by pharmacological inhibition of the CBM protein MALT1. Our results demonstrate that partial disruption of the CBM complex and induction of IFNγ secretion in the preferentially self-reactive Treg cell pool does not cause systemic autoimmunity but is sufficient to prime the tumour environment for successful immune checkpoint therapy

A Near-Infrared Cell Tracker Reagent for Multiscopic In Vivo Imaging and Quantification of Leukocyte Immune Responses

The complexity of the tumor microenvironment necessitates that cell behavior is studied in a broad, multi-scale context. Although tomographic and microscopy-based far and near infrared fluorescence (NIRF, >650 nm) imaging methods offer high resolution, sensitivity, and depth penetration, there has been a lack of optimized NIRF agents to label and track cells in their native environments at different scales. In this study we labeled mammalian leukocytes with VivoTag 680 (VT680), an amine reactive N-hydroxysuccinimide (NHS) ester of a (benz) indolium-derived far red fluorescent probe. We show that VT680 diffuses into leukocytes within minutes, covalently binds to cellular components, remains internalized for days in vitro and in vivo, and does not transfer fluorescence to adjacent cells. It is biocompatible, keeps cells fully functional, and fluoresces at high intensities. In a tumor model of cytotoxic T lymphocyte (CTL) immunotherapy, we track and quantify VT680-labeled cells longitudinally at the whole-body level with fluorescence-mediated molecular tomography (FMT), within tissues at single cell resolutions by multiphoton and confocal intravital microscopy, and ex vivo by flow cytometry. Thus, this approach is suitable to monitor cells at multiple resolutions in real time in their native environments by NIR-based fluorescence imaging.Version of Recor

Sphingosine-1-phosphate promotes the persistence of activated CD4 T cells in inflamed sites

Inflammation can be protective or pathogenic depending on context and timeframe. Acute inflammation, including the accumulation of CD4 T cells, accompanies protective immune responses to pathogens, but the presence of activated CD4 T cells at sites of inflammation is associated with chronic inflammatory disease. While significant progress has been made in understanding the migration of CD4 T cells into inflamed sites, the signals that lead to their persistence are poorly characterized. Using a murine ear model of acute inflammation and intravital two-photon imaging, we have dissected the signals that mediate CD4 T cell persistence. We report the unexpected finding that the bioactive lipid, sphingosine-1-phosphate (S1P), is both necessary and sufficient for the persistence of activated CD4 T cells at peripheral tissues in acute inflammation. S1P mediated the enhanced motility of CD4 T cells at inflamed tissues but did not affect their migration to the downstream draining lymph node. We found that sphingosine kinase-1, which regulates S1P production is increased at inflamed sites in mice and in patients with the chronic inflammatory disease, rheumatoid arthritis. Together, these data suggest that S1P, or its regulators, may be key targets to promote or disrupt accumulation of CD4 T cells at inflamed tissues

Probing the Effects of the Well-mixed Assumption on Viral Infection Dynamics

Viral kinetics have been extensively studied in the past through the use of spatially well-mixed ordinary differential equations describing the time evolution of the diseased state. However, emerging spatial structures such as localized populations of dead cells might adversely affect the spread of infection, similar to the manner in which a counter-fire can stop a forest fire from spreading. In a previous publication (Beauchemin et al., 2005), a simple 2-D cellular automaton model was introduced and shown to be accurate enough to model an uncomplicated infection with influenza A. Here, this model is used to investigate the effects of relaxing the well-mixed assumption. Particularly, the effects of the initial distribution of infected cells, the regeneration rule for dead epithelial cells, and the proliferation rule for immune cells are explored and shown to have an important impact on the development and outcome of the viral infection in our model.Comment: LaTeX, 12 pages, 22 EPS figures, uses document class REVTeX 4, and packages float, graphics, amsmath, and SIunit

Imaging interactions between the immune and cardiovascular systems in vivo by multiphoton microscopy

Several recent studies in immunology have used multiphoton laser-scanning microscopy to visualise the induction of an immune response in real time in vivo. These experiments are illuminating the cellular and molecular interactions involved in the induction, maintenance and regulation of immune responses. Similar approaches are being applied in cardiovascular research where there is an increasing body of evidence to support a significant role for the adaptive immune system in vascular disease. As such, we have begun to dissect the role of T lymphocytes in atherosclerosis in real time in vivo. Here, we provide step-by-step guides to the various stages involved in visualising the migration of T cells within a lymph node and their infiltration into inflamed tissues such as atherosclerotic arteries. These methods provide an insight into the mechanisms involved in the activation and function of immune cells in vivo

PEG-Like Nanoprobes: Multimodal, Pharmacokinetically and Optically Tunable Nanomaterials

“PEG-like Nanoprobes” (PN’s) are pharmacokinetically and optically tunable nanomaterials whose disposition in biological systems can be determined by fluorescence or radioactivity. PN’s feature a unique design where a single PEG polymer surrounds a short fluorochrome and radiometal bearing peptide, and endows the resulting nanoprobe with pharmacokinetic control (based on molecular weight of the PEG selected) and optical tunability (based on the fluorochrome selected), while the chelate provides a radiolabeling option. PN’s were used to image brain capillary angiography (intravital 2-photon microscopy), tumor capillary permeability (intravital fluorescent microscopy), and the tumor enhanced permeability and retention (EPR) effect (111In-PN and SPECT). Clinical applications of PN’s include use as long blood half-life fluorochromes for intraoperative angiography, for measurements of capillary permeability in breast cancer lesions, and to image EPR by SPECT, for stratifying patient candidates for long-circulating nanomedicines that may utilize the EPR mechanism

Targeting IgE in Severe Atopic Dermatitis with a Combination of Immunoadsorption and Omalizumab

Patients with atopic dermatitis (AD) tend to have greatly elevated levels of serum immunoglobulin E (IgE). However, the role of IgE in the pathogenesis of AD is debated. This investigator-initiated open-label pilot study evaluates an anti-IgE-treatment approach by combining extracorporeal immunoadsorption and anti-IgE antibody omalizumab in 10 patients with severe, therapy-refractory AD. IgE levels decreased after immunoadsorption and decreased continuously in all patients during anti-IgE therapy. The reverse trend was observed during 6 months follow-up without treatment. In parallel with these observations, an improvement in AD was observed during the treatment period, with aggravation during follow-up. Further research is needed, based on the principle of reducing IgE levels in order to improve clinical symptoms, using a combination anti-IgE treatment approach, adjusted according to IgE levels.</p

Framing the frontier - Tracing issues related to soybean expansion in transnational public spheres

Unidad de excelencia María de Maeztu CEX2019-000940-MAltres ajuts: Acord transformatiu CRUE-CSICRapid soybean expansion in South America has been linked to numerous socio-environmental problems, including deforestation in sensitive biomes. As a major importing region of soybeans, wider public awareness has also put pressure on the European Union. Different governance initiatives involving various groups of stakeholders have sought to address these issues. However, what is identified as a relevant problem, as a region of interest or which actors are mentioned in this context are all matters of claims-making processes between different groups and mediated through various channels of communication. This study uses a text-mining approach to trace the construction of socio-ecological problems related to soybean expansion and the actors and regions linked with these issues in public discourse. The focus lies on print media from the European Union, but several additional sources are included to investigate the similarities and differences between various communication channels and regions. These include newspaper articles from producing countries and international news agencies, scientific abstracts, corporate statements, and reports from advocacy groups gathered from the mid-1990s to 2020. The results show that European mass media have shifted their focus from consumer labeling, health, and concerns over genetically modified organisms towards more distant or abstract phenomena, such as deforestation and climate change. This has been accompanied with a broader view on different stakeholders, but also with a strong regional focus on the Amazon biome. There has also been much less attention on direct concerns for communities in producing regions, such as land conflicts or disputes over intellectual property rights. We conclude that while European public spheres appear to become more receptive to issues related to impacts in sourcing regions, there remains a narrow focus on specific problems and regions, which reflects a fundamental asymmetry in different stakeholders' ability to shape transnational deliberations and resulting governance processes