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Unsuccessful clinical translation of orally delivered biological drugs remains a challenge in pharmaceutical development and has been linked to insufficient mechanistic understanding of intestinal drug transport. Live cell imaging could provide such mechanistic insights by directly tracking drug transport across intestinal barriers at subcellular resolution, however traditional intestinal in vitro models are not compatible with the necessary live cell imaging modalities. Here, we employed a novel microfluidic platform to develop an in vitro intestinal epithelial barrier compatible with advanced widefield- and confocal microscopy. We established a quantitative, multiplexed and high-temporal resolution imaging assay for investigating the cellular uptake and cross-barrier transport of biologics while simultaneously monitoring barrier integrity. As a proof-of-principle, we use the generic model to monitor the transport of co-administrated cell penetrating peptide (TAT) and insulin. We show that while TAT displayed a concentration dependent difference in its transport mechanism and efficiency, insulin displayed cellular internalization, but was restricted from transport across the barrier. This illustrates how such a sophisticated imaging based barrier model can facilitate mechanistic studies of drug transport across intestinal barriers and aid in vivo and clinical translation in drug development.

The Wadden Sea World Heritage site is one of the world’s most important wetlands for migratory waterbirds. It is the single most important staging and moulting area and an important wintering area for waterbirds on the East Atlantic Flyway from the Arctic to South Africa. The Expert Group Migratory Birds (EG MB) program is carried out in the framework of the Trilateral Monitoring and Assessment Program (TMAP) and constitutes an internationally coordinated long-term monitoring program. It covers a large connected ecoregion stretching from Den Helder in the Netherlands to Esbjerg in Denmark; regular ground counts for most species and areas plus aerial and boat counts for sea ducks involves hundreds of observers and several institutes and agencies.

Several types of pathogenic bacteria produce genotoxins that induce DNA damage in host cells. Accumulating evidence suggests that a central function of these genotoxins is to dysregulate the host's immune response, but the underlying mechanisms remain unclear. To address this issue, we investigated the effects of the most widely expressed bacterial genotoxin, the cytolethal distending toxin (CDT), on T cells—the key mediators of adaptive immunity. We show that CDT induces premature senescence in activated CD4 T cells in vitro and provide evidence suggesting that infection with genotoxin-producing bacteria promotes T cell senescence in vivo. Moreover, we demonstrate that genotoxin-induced senescent CD4 T cells assume a senescence-associated secretory phenotype (SASP) which, at least partly, is orchestrated by the ATM-p38 signaling axis. These findings provide insight into the immunomodulatory properties of bacterial genotoxins and uncover a putative link between bacterial infections and T cell senescence.

Membrane-active peptides (MAPs) have several potential therapeutic uses, including as antimicrobial drugs. Many traditional methods used to evaluate the membrane interactions of MAPs have limited applicability. Low-throughput methods, such as microscopy, provides detailed information but often relies on fluorophore-labeled MAPs, and high-throughput assays, such as the calcein release assay, cannot assess the mechanism behind the disruption of vesicular-based lipid membranes. Here we present a flow cytometric assay that provides detailed information about the peptide-lipid membrane interactions on single artificial lipid vesicles while being high-throughput (1000–2000 vesicles/s) and based on label-free MAPs. We synthesized and investigated six MAPs with different modes of action to evaluate the versatility of the assay. The assay is based on the flow cytometric readouts from artificial lipid vesicles, including the fluorescence from membrane-anchored and core-encapsulated fluorophores, and the vesicle concentration. From these parameters, we were able to distinguish between MAPs that induce vesicle solubilization, permeation (pores/membrane distortion), and aggregation or fusion. Our flow cytometry findings have been verified by traditional methods, including the calcein release assay, dynamic light scattering, and fluorescence microscopy on giant unilamellar vesicles. We envision that the presented flow cytometric assay can be used for various types of peptide-lipid membrane studies, e.g. to identify new antibiotics. Moreover, the assay can easily be expanded to derive additional valuable information.

The presence of naturally occurring cytokine-specific autoantibodies (c-aAb) in humans is well established, as well as associations to selected pathologies. However, the overall influence of c-aAb on immunocompetence remains largely unknown. In this paper, we performed a large-scale investigation of c-aAb association with infection risk. A cohort of healthy Danish blood donors was screened for c-aAb against IL-1α, IL-6, IL-10, IFNα, and GM-CSF using a Luminex-based multiplex assay, and results were linked to data from the Danish National Prescription Registry. The filing of an antimicrobial prescription following c-aAb measurement was used as a proxy for impaired immunocompetence. We found that c-aAb against pro-inflammatory cytokines IFNα and GM-CSF tended to associate with increased risk of prescription filings in women, whereas antibodies against anti-inflammatory IL-10 were associated with a lower predicted risk of antimicrobial prescriptions, as well as higher self-perceived health scores. We also observed an association of cumulative c-aAb presence with prescription risk. Our data show that cytokine autoantibodies in healthy individuals associate with various proxies for immunomodulation, with the exact association dependent on the pattern of pro- or anti-inflammatory cytokines targeted. This suggests that c-aAb may express cytokine-modulatory properties in healthy individuals and may be critical to further investigate as biomarkers of immunodeficiency.

Introduction: The aim of this study was to give quantitative insight into the number of cytokine molecules needed to activate a target cell and relate this to the physiological consequences of the amounts of cytokines typically detectable in humans. As a model system blood interleukin-6 (IL-6) was chosen since this cytokine is one of the most studied and clinically monitored cytokines, and because of the tools for the present investigations such as fully bioactive iodinated recombinant IL-6, cellular cytokine binding assays, and bioassays have been thoroughly validated. Methods: The key intermediates of the basic equilibrium principles that govern cytokine binding and exchange were deduced and applied on concrete estimations of cellular and extracellular IL-6 binding in the bloodstream based on experimental binding data and data from the literature. In parallel, in vitro cellular IL-6 binding data was substantiated by paired measurements of IL-6 bioactivity on IL-6 sensitive B9 hybridoma cells. Results: Blood leucocytes and B9 cells expressed 50 to 300, 10 to 20 picomolar affinity, IL-6 binding sites per cell and at physiological concentrations of IL-6 less than 10 IL-6 molecules seemed to be bound to blood cells. Nonetheless, binding off as few as four IL-6 molecules per cell seemed to result in statistically significant bioactivity, whereas binding of 16 IL-6 molecules triggered extensive cellular responses. Conclusion: Together, the estimations and the measurements support the notion that target cells with more than 100 bioactive cytokine receptors per cell, such as T cells and hepatocytes, are likely to be under steady and substantial cytokine-induced endocrine activation.

IL-6-specific autoantibodies (aAb-IL-6) have been reported in diseased and healthy individuals. We recently established a model for aAb-IL-6 in different mouse strains, based on vaccination with immunogenic IL-6 analogues, in which titers of aAb-IL-6 above 1,000 resulted in an in vivo IL-6 deficiency. Here, we examined aAb-IL-6 in 4,230 blood donors. Stable low titers of aAb-IL-6 were found in 9% of the donors, while 1% had titers ranging from 64 to greater than 10,000. Such aAb-IL-6-positive donors appeared normal with no overt signs of pathology. Natural and recombinant forms of IL-6 bound avidly to their IgG, and their plasma strongly neutralized IL-6 in vitro. Slightly elevated concentrations of IL-6 exclusively in the form of IL-6-IgG complexes were present in their circulation. The complexes did not contain soluble IL-6 receptors. Titers of 0.1% of the blood donors were as positive as the vaccination-induced IL-6-deficient mice. Such donors might be IL-6 deficient, and if so, IL-6 seems be dispensable for several months in otherwise healthy individuals. Such highly positive donors also explain why normal human IgG for pharmaceutical use may contain high anti-IL-6 activity. Finally, transfusion of plasma with a high titer of aAb-IL-6 might, temporally, render a recipient IL-6 deficient.

Udgivelsesdato: 2004-Jan, Inappropriate expression of IL-6 plays a role in various inflammatory conditions, degenerative diseases, and cancers. Several model systems have been developed that can specifically block IL-6-receptor interactions. Here we present a simple and highly effective approach based on vaccination with a pool of specifically mutated IL-6 analogues to induce a neutralizing IL-6 antibody response in mice. Judged by the ability of the analogues to bind to heterologous anti-IL-6 antibodies and cellular IL-6 receptors the IL-6 analogues seemed to have a three-dimensional structure comparable to that of wild-type IL-6. Injection of them broke self-tolerance and induced an immune response to IL-6, presumably because of the amino acid differences between the analogues and wild-type IL-6. This resulted in a long-lasting anti-IL-6 antibody-mediated IL-6 deficiency that blocked experimentally induced IL-6-mediated pathology.

Anatomi