Biology subjects

Andresen, T. L.

Publications and source records attributed to Andresen, T. L..

6 recordsLinked to original sources

bioRxiv2024

Dietary iron deficiency in the adult mouse increases brain endothelial uptake and blood-brain barrier transport of a high-affinity, anti-transferrin receptor antibody

Background and objectivesBrain capillary endothelial cells (BCECs) express transferrin receptor 1 (TfR1) to ensure sufficient iron transport into the brain across the blood-brain barrier (BBB). Our main objective was to examine adult mice subjected to dietary iron deficiency (ID) for possible changes in the content of TfR1 in BCECs and the influence thereof on the uptake and transport of high-affinity anti-transferrin receptor IgG1 antibodies (clone Ri7217). Material and methodsWe subjected adult, female mice to dietary ID for 8 weeks. Iron and copper were measured using inductively coupled plasma mass spectrometry (ICP-MS) in various tissues, including total brain and isolated brain capillaries. Possible effects of ID on cerebral angioarchitecture were estimated using 3D confocal microscopy of optically cleared brain samples with endothelium labelled using intravenous injection of wheat germ agglutinin with subsequent machine learning-based segmentation and vascular tracing. TfR1 was quantified using ELISA. Ri7217 antibodies were conjugated with 1 nm nanogold and brain uptake quantified using ICP-MS. ResultsID significantly reduced the iron content in isolated brain capillaries, liver, spleen, kidney, heart and skeletal muscles. ID increased the copper content in the brain. Analysis of cerebral cortex angioarchitecture revealed no changes following dietary ID except for a minor increase in tortuosity of small-caliber vessels. TfR1 protein was unchanged in the total brain and isolated brain capillaries. In contrast, uptake of nanogold-conjugated Ri7217 increased in the total brain, the supernatant fraction of isolated brain capillaries representing the post-vascular compartment, liver, spleen, and dissected retinae. ConclusionsTargeting TfR1 in ID revealed increased uptake and transport across the BBB of Ri7217 antibodies. Possibly the elevated transport of transferrin receptors through BCECs is due to the increased trafficking of transferrin receptor-containing vesicles in ID, which appeared to have no major effects on the brain angioarchitecture.

neuroscience↗

bioRxiv2024

Remodeling of the brain angioarchitecture in experimental chronic neurodegeneration

BackgroundChronic neurodegenerative diseases are characterized by substantial neuroinflammation with accumulation of macrophages, reactive microglia, and reactive astrocytes. Impairment of the brain vasculature is also commonly seen in chronic neurodegeneration with causal links warranting further investigation. MethodsTo address the effects of chronic neurodegeneration on regional vasculature, we performed a unilateral injection of a glutamate receptor agonist ibotenic acid into striatum of adult rats, which caused excitotoxicity in the substantia nigra pars reticulata (SNpr) due to imbalance between inhibitory inputs from the striatum and excitatory signals from the subthalamic nucleus. Brains were examined at 28 days (short-term neurodegeneration) and 91 days (long-term neurodegeneration). Dissected brain samples were analyzed for protein and gene expression using immunohistochemistry and qPCR. Brains were further analyzed for remodeling of vasculature labeled with wheat germ agglutinin (WGA) Alexa Fluor 647 conjugate using 3D deep confocal microscopy of optically cleared samples combined with machine learning-based image analysis. ResultsThe resulting neurodegeneration was accompanied by neuroinflammation, verified by the expression of inflammatory markers with gradual, regional loss of brain tissue. An in-depth analysis of the angioarchitecture of the degenerating SNpr revealed substantial changes of the vasculature with higher density, increased diameter, and number of tortuous vessels already after 28 days continuing at 91 days. Interestingly, the vascular remodeling changes occurred without changes in the expression of endothelial tight junction proteins, vascular basement membrane proteins, or markers of angiogenesis. ConclusionsThese results demonstrate how neurodegeneration causing prominent tissue loss in SNpr also leads to substantial remodeling of the angioarchitecture, while not altering the structural integrity of the vessel wall judged from the continuous expression of hallmarks of brain endothelial cells and the vascular basement membrane. We propose that this remodeling occurs as a consequence of the loss of brain tissue and with the resulting changes leaving the vasculature prone to additional vascular pathologies like vessel occlusion or formation of aneurysms.

neuroscience↗

bioRxiv2023

Tuning the double lipidation of salmon calcitonin to introduce a pore-like membrane translocation mechanism

A widespread strategy to increase the transport of therapeutic peptides across cellular membranes has been to attach lipid moieties to the peptide backbone (lipidation) to enhance their intrinsic membrane interaction. Efforts in vitro and in vivo investigating the correlation between lipidation characteristics and peptide membrane translocation efficiency have traditionally relied on end-point read-out assays and trial-and-error-based optimization strategies. Consequently, the molecular details of how therapeutic peptide lipidation affects its membrane permeation and translocation mechanisms remain unresolved. Here we employed salmon calcitonin as a model therapeutic peptide and synthesized nine double lipidated analogs with varying lipid chain lengths. We used single giant unilamellar vesicle (GUV) calcein influx time-lapse fluorescence microscopy to determine how tuning the lipidation length can lead to an All-or-None GUV filling mechanism, indicative of a peptide mediated pore formation. Finally, we used a GUVs-containing-inner-GUVs assay to demonstrate that only peptide analogs capable of inducing pore formation show efficient membrane translocation. Our data provided the first mechanistic details on how therapeutic peptide lipidation affects their membrane perturbation mechanism and demonstrated that fine-tuning lipidation parameters could induce an intrinsic pore-forming capability. These insights and the microscopy based workflow introduced for investigating structure-function relations could be pivotal for optimizing future peptide design strategies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=197 SRC="FIGDIR/small/548826v1_ufig1.gif" ALT="Figure 1"> View larger version (87K): org.highwire.dtl.DTLVardef@23430org.highwire.dtl.DTLVardef@b06145org.highwire.dtl.DTLVardef@1c22579org.highwire.dtl.DTLVardef@12a56eb_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights- Lipidating the therapeutic peptide salmon calcitonin alters its biophysical characteristics, including oligomer size, hydrophobicity and membrane activity. - Fluorescent microscopy of single GUVs enables the determination of peptide mediated reporter dye influx behavior as either graded or All-or-None, which is coupled to either smaller membrane perturbations or peptide pore formation. - Modulating the number of hydrocarbons constituting the lipidation moieties determines the membrane permeation mechanism. - By increasing the lipid chain length lipidated of salmon calcitonin goes from displaying smaller membrane perturbations to a peptide pore formation mechanism. - Effective membrane translocation of lipidated salmon calcitonin requires a peptide mediated pore forming mechanism.

biophysics↗

bioRxiv2023

Cancer Immunotherapy through Tissue Adhering Polymers

TLR 7/8 agonists are highly potent immunostimulators, though their clinical translation has been met with mixed success, due to their high toxicity as a result of an unregulated systemic immune activation. There is enormous potential to augment cancer immunotherapies with synthetic TLR 7/8 agonists, though a thorough control of pharmacokinetics and localization is needed for the general use of TLR 7/8 agonists in cancer immunotherapy. Herein, we control localization of TLR 7/8 agonists, by exploiting the extensive tissue retention of poly(acrylic acid-co-styrene). In a murine CT26 model, we find that covalently attaching TLR 7/8 agonists to the copolymer allows for retaining the drug in the tumor microenvironment for at least 15 weeks, after intratumoral injection, and results in a curative monotherapy. The copolymer itself is a new avenue for attaining prolonged tissue rentention for covalently attached drugs.

cancer biology↗

bioRxiv2021

T-cell Receptor Diversity Estimates for Repertoires (TCRDivER) uses sequence similarity to find signatures of immune response

We propose TCRDivER, a global approach to T-cell repertoire comparison using diversity profiles sensitive to both clone size and sequence similarity. As immunotherapies improve, the long standing biological interest in connecting outcome with T cell receptor (TCR) repertoire status has become more urgent. Here we show that new insights can be extracted from high throughput repertoire sequencing data. Most current efforts focus on identification of immunisation-specific sequence motifs or on monitoring changes in frequency of individual clones. Applying TCRDivER to murine spleen samples shows it characterises an additional dimension of repertoire variation, beyond conventional diversity estimates, allowing distinction between immunised and non-immunised samples. We further apply TCRDivER to repertoires from human blood. In both cases we show characteristic relationships between repertoire features. These reveal biologically interpretable relationships between sequence similarity and clonal expansions. We thereby demonstrate a new tool for investigation in clinical and research applications.

immunology↗

bioRxiv2020

Post-capillary venules is the locus for transcytosis of therapeutic nanoparticles to the brain.

Treatments of neurodegenerative diseases require biologic drugs to be actively transported across the blood-brain barrier (BBB). To answer outstanding questions regarding transport mechanisms, we determined how and where transcytosis occurs at the BBB. Using two-photon microscopy, we characterized the transport of therapeutic nanoparticles at all steps of delivery to the brain and at the nanoscale resolution in vivo. Transferrin receptor-targeted nanoparticles were taken up by endothelium at capillaries and venules, but not at arterioles. The nanoparticles moved unobstructed within endothelial cells, but transcytosis across the BBB occurred only at post-capillary venules, where endothelial and glial basement membranes form a perivascular space that can accommodate biologics. In comparison, transcytosis was absent in capillaries with closely apposed basement membranes. Thus, post-capillary venules, not capillaries, provide an entry point for transport of large molecules across the BBB, and targeting therapeutic agents to this locus may be an effective way for treating brain disorders. HIGHLIGHTSO_LIIntegration of drug carrier nanotechnology with two-photon microscopy in vivo C_LIO_LIReal-time nanoscale-resolution imaging of nanoparticle transcytosis to the brain C_LIO_LIDistinct trafficking pattern in the endothelium of cerebral venules and capillaries C_LIO_LIVenules, not capillaries, is the locus for brain uptake of therapeutic nanoparticles C_LI

neuroscience↗