Publications by authors named "Vanessa A Johanssen"

9 Publications

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Selective blood-brain barrier permeabilisation of brain metastases by a type-1 receptor selective tumour necrosis factor mutein.

Neuro Oncol 2021 Jul 23. Epub 2021 Jul 23.

Cancer Research UK and Medical Research Council Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford, UK.

Background: Metastasis to the brain is a major challenge with poor prognosis. The blood-brain barrier (BBB) is a significant impediment to effective treatment, being intact during the early stages of tumour development and heterogeneously permeable at later stages. Intravenous injection of tumour necrosis factor (TNF) selectively induces BBB permeabilisation at sites of brain micrometastasis, in a TNF type-1 receptor (TNFR1) dependent manner. Here, to enable clinical translation, we have developed a TNFR1-selective agonist variant of human TNF that induces BBB permeabilisation, whilst minimising potential toxicity.

Methods: A library of human TNF muteins (mutTNF) were generated and assessed for binding specificity to mouse and human TNFR1/2, endothelial permeabilising activity in vitro, potential immunogenicity and circulatory half-life. The permeabilising ability of the most promising variant was assessed in vivo in a model of brain metastasis.

Results: The primary mutTNF variant showed similar affinity for human TNFR1 than wild-type human TNF, similar affinity for mouse TNFR1 as wild-type mouse TNF, undetectable binding to human/mouse TNFR2, low potential immunogenicity and permeabilisation of an endothelial monolayer. Circulatory half-life was similar to mouse/human TNF and BBB permeabilisation was induced selectively at sites of micrometastases in vivo, with a time window of ≥24h and enabling delivery of agents within a therapeutically-relevant range (0.5-150kDa), including the clinically approved therapy, trastuzumab.

Conclusions: We have developed a clinically-translatable mutTNF that selectively opens the BBB at micrometastatic sites, whilst leaving the rest of the cerebrovasculature intact. This approach will open a window for brain metastasis treatment that currently does not exist.
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http://dx.doi.org/10.1093/neuonc/noab177DOI Listing
July 2021

Microbubbles Containing Lysolipid Enhance Ultrasound-Mediated Blood-Brain Barrier Breakdown In Vivo.

Adv Healthc Mater 2021 02 16;10(3):e2001343. Epub 2020 Nov 16.

Institute of Biomedical Engineering, Department of Engineering Science, University of Oxford, Oxford, OX3 7DQ, UK.

Ultrasound and microbubbles (MBs) offer a noninvasive method of temporarily enhancing blood-brain barrier (BBB) permeability to therapeutics. To reduce off-target effects, it is desirable to minimize the ultrasound pressures required. It has been shown that a new formulation of MBs containing lysolipids (Lyso-MBs) can increase the cellular uptake of a model drug in vitro. The aim of this study is to investigate whether Lyso-MBs can also enhance BBB permeability in vivo. Female BALB/c mice are injected with either Lyso-MBs or control MBs and gadolinium-DTPA (Gd-DTPA) and exposed to ultrasound (500 kHz, 1 Hz pulse repetition frequency, 1 ms pulse length, peak-negative pressures 160-480 kPa) for 2 min. BBB permeabilization is measured via magnetic resonance imaging (7.0 T) of Gd-DTPA extravasation and subsequent histological examination of brain tissue to assess serum immunoglobulin G (IgG) extravasation (n = 8 per group). An approximately twofold enhancement in BBB permeability is produced by the Lyso-MBs at the highest ultrasound pressure compared with the control. These findings indicate that modifying the composition of phospholipid-shelled MBs has the potential to improve the efficiency of BBB opening, without increasing the ultrasound pressure amplitude required. This is particularly relevant for delivery of therapeutics deep within the brain.
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http://dx.doi.org/10.1002/adhm.202001343DOI Listing
February 2021

A novel molecular magnetic resonance imaging agent targeting activated leukocyte cell adhesion molecule as demonstrated in mouse brain metastasis models.

J Cereb Blood Flow Metab 2021 07 5;41(7):1592-1607. Epub 2020 Nov 5.

Cancer Research UK and Medical Research Council Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford, UK.

Molecular magnetic resonance imaging (MRI) allows visualization of biological processes at the molecular level. Upregulation of endothelial ALCAM (activated leukocyte cell adhesion molecule) is a key element for leukocyte recruitment in neurological disease. The aim of this study, therefore, was to develop a novel molecular MRI contrast agent, by conjugating anti-ALCAM antibodies to microparticles of iron oxide (MPIO), for detection of endothelial ALCAM expression . Binding specificity of ALCAM-MPIO was demonstrated under static and flow conditions. Subsequently, in a proof-of-concept study, mouse models of brain metastasis were induced by intracardial injection of brain-tropic human breast carcinoma, lung adenocarcinoma or melanoma cells to upregulate endothelial ALCAM. At selected time-points, mice were injected intravenously with ALCAM-MPIO, and ALCAM-MPIO induced hypointensities were observed on T*-weighted images in all three models. Post-gadolinium MRI confirmed an intact blood-brain barrier, indicating endoluminal binding. Correlation between endothelial ALCAM expression and ALCAM-MPIO binding was confirmed histologically. Statistical analysis indicated high sensitivity (80-90%) and specificity (79-83%) for detection of endothelial ALCAM with ALCAM-MPIO. Given reports of endothelial ALCAM upregulation in numerous neurological diseases, this advance in our ability to image ALCAM may yield substantial improvements for both diagnosis and targeted therapy.
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http://dx.doi.org/10.1177/0271678X20968943DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8217895PMC
July 2021

Improving Delineation of True Tumor Volume With Multimodal MRI in a Rat Model of Brain Metastasis.

Int J Radiat Oncol Biol Phys 2020 04 17;106(5):1028-1038. Epub 2020 Jan 17.

Department of Oncology, Cancer Research UK and Medical Research Council Oxford Institute for Radiation Oncology, University of Oxford. Electronic address:

Purpose: Brain metastases are almost universally lethal with short median survival times. Despite this, they are often potentially curable, with therapy failing only because of local relapse. One key reason relapse occurs is because treatment planning did not delineate metastasis margins sufficiently or accurately, allowing residual tumor to regrow. The aim of this study was to determine the extent to which multimodal magnetic resonance imaging (MRI), with a simple and automated analysis pipeline, could improve upon current clinical practice of single-modality, independent-observer tumor delineation.

Methods And Materials: We used a single rat model of brain metastasis (ENU1564 breast carcinoma cells in BD-IX rats), with and without radiation therapy. Multimodal MRI data were acquired using sequences either in current clinical use or in clinical trial and included postgadolinium T-weighted images and maps of blood flow, blood volume, T and T relaxation times, and apparent diffusion coefficient.

Results: In all cases, independent observers underestimated the true size of metastases from single-modality gadolinium-enhanced MRI (85 ± 36 μL vs 131 ± 40 μL histologic measurement), although multimodal MRI more accurately delineated tumor volume (132 ± 41 μL). Multimodal MRI offered increased sensitivity compared with independent observer for detecting metastasis (0.82 vs 0.61, respectively), with only a slight decrease in specificity (0.86 vs 0.98). Blood flow maps conferred the greatest improvements in margin detection for late-stage metastases after radiation therapy. Gadolinium-enhanced T-weighted images conferred the greatest increase in accuracy of detection for smaller metastases.

Conclusions: These findings suggest that multimodal MRI of brain metastases could significantly improve the visualization of brain metastasis margins, beyond current clinical practice, with the potential to decrease relapse rates and increase patient survival. This finding now needs validation in additional tumor models or clinical cohorts.
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http://dx.doi.org/10.1016/j.ijrobp.2019.12.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7082766PMC
April 2020

Correction to: Amyloid Precursor Protein Mediates Neuronal Protection from Rotenone Toxicity.

Mol Neurobiol 2019 Oct;56(10):7249

Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, Melbourne, Victoria, Australia.

The original version of this article unfortunately contained a mistake in the author name. The family name of Dr. Vanessa A. Johannsen should be written as "Johanssen."
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http://dx.doi.org/10.1007/s12035-019-01726-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6828065PMC
October 2019

Analysis of Prion Protein Conformation Using Circular Dichroism Spectroscopy.

Methods Mol Biol 2017 ;1658:27-34

Department of Medicine, The University of Melbourne, Parkville, Melbourne, VIC, 3010, Australia.

According to the protein-only hypothesis of prion propagation, the pathogenesis of prion disease is due to the misfolding of cellular PrP (PrP) which gives rise to disease-associated PrP. This misfolding results in the predominantly α-helix secondary structure of PrP becoming increasingly β-sheet. Prion protein researchers often employ circular dichroism (CD) spectroscopy to rapidly analyze and identify the degree of α-helix and β-sheet content in their recombinant protein and peptide samples. CD is a nondestructive method of determining protein secondary structure and can be used to monitor the protein structural changes in various environments, e.g., pH and temperature. CD can also be used to investigate kinetic and thermodynamic characteristics of proteins and peptides.
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http://dx.doi.org/10.1007/978-1-4939-7244-9_3DOI Listing
May 2018

Prion protein "gamma-cleavage": characterizing a novel endoproteolytic processing event.

Cell Mol Life Sci 2016 Feb 23;73(3):667-83. Epub 2015 Aug 23.

Department of Medicine, RMH, The University of Melbourne, Parkville, VIC, 3010, Australia.

The cellular prion protein (PrP(C)) is a ubiquitously expressed protein of currently unresolved but potentially diverse function. Of putative relevance to normal biological activity, PrP(C) is recognized to undergo both α- and β-endoproteolysis, producing the cleavage fragment pairs N1/C1 and N2/C2, respectively. Experimental evidence suggests the likelihood that these processing events serve differing cellular needs. Through the engineering of a C-terminal c-myc tag onto murine PrP(C), as well as the selective use of a far-C-terminal anti-PrP antibody, we have identified a new PrP(C) fragment, nominally 'C3', and elaborating existing nomenclature, 'γ-cleavage' as the responsible proteolysis. Our studies indicate that this novel γ-cleavage event can occur during transit through the secretory pathway after exiting the endoplasmic reticulum, and after PrP(C) has reached the cell surface, by a matrix metalloprotease. We found that C3 is GPI-anchored like other C-terminal and full length PrP(C) species, though it does not localize primarily at the cell surface, and is preferentially cleaved from an unglycosylated substrate. Importantly, we observed that C3 exists in diverse cell types as well as mouse and human brain tissue, and of possible pathogenic significance, γ-cleavage may increase in human prion diseases. Given the likely relevance of PrP(C) processing to both its normal function, and susceptibility to prion disease, the potential importance of this previously underappreciated and overlooked cleavage event warrants further consideration.
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http://dx.doi.org/10.1007/s00018-015-2022-zDOI Listing
February 2016

Glycosaminoglycan sulfation determines the biochemical properties of prion protein aggregates.

Glycobiology 2015 Jul 20;25(7):745-55. Epub 2015 Feb 20.

Department of Pathology

Prion diseases are transmissible neurodegenerative disorders associated with the conversion of the cellular prion protein, PrP(C), to a misfolded isoform called PrP(Sc). Although PrP(Sc) is a necessary component of the infectious prion, additional factors, or cofactors, have been shown to contribute to the efficient formation of transmissible PrP(Sc). Glycosaminoglycans (GAGs) are attractive cofactor candidates as they can be found associated with PrP(Sc) deposits, have been shown to enhance PrP misfolding in vitro, are found in the same cellular compartments as PrP(C) and have been shown to be disease modifying in vivo. Here we investigated the effects of the sulfated GAGs, heparin and heparan sulfate (HS), on disease associated misfolding of full-length recombinant PrP. More specifically, the degree of sulfation of these molecules was investigated for its role in modulating the disease-associated characteristics of PrP. Both heparin and HS induced a β-sheet conformation in recombinant PrP that was associated with the formation of aggregated species; however, the biochemical properties of the aggregates formed in the presence of heparin or HS varied in solubility and protease resistance. Furthermore, these properties could be modified by changes in GAG sulfation, indicating that subtle changes in the properties of prion disease cofactors could initiate disease associated misfolding.
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http://dx.doi.org/10.1093/glycob/cwv014DOI Listing
July 2015

C-terminal peptides modelling constitutive PrPC processing demonstrate ameliorated toxicity predisposition consequent to α-cleavage.

Biochem J 2014 Apr;459(1):103-15

§Mental Health Research Institute, University of Melbourne, Parkville, VIC 3010, Australia.

Misfolding of PrPC (cellular prion protein) to β-strand-rich conformations constitutes a key event in prion disease pathogenesis. PrPC can undergo either of two constitutive endoproteolytic events known as α- and β-cleavage, yielding C-terminal fragments known as C1 and C2 respectively. It is unclear whether C-terminal fragments generated through α- and β-cleavage, especially C2, influence pathogenesis directly. Consequently, we compared the biophysical properties and neurotoxicity of recombinant human PrP fragments recapitulating α- and β-cleavage, namely huPrP-(112-231) (equating to C1) and huPrP-(90-231) (equating to C2). Under conditions we employed, huPrP-(112-231) could not be induced to fold into a β-stranded isoform and neurotoxicity was not a feature for monomeric or multimeric assemblies. In contrast, huPrP-(90-231) easily adopted a β-strand conformation, demonstrated considerable thermostability and was toxic to neurons. Synthetic PrP peptides modelled on α- and β-cleavage of the unique Y145STOP (Tyr145→stop) mutant prion protein corroborated the differential toxicity observed for recombinant huPrP-(112-231) and huPrP-(90-231) and suggested that the persistence of soluble oligomeric β-strand-rich conformers was required for significant neurotoxicity. Our results additionally indicate that α- and β-cleavage of PrPC generate biophysically and biologically non-equivalent C-terminal fragments and that C1 generated through α-cleavage appears to be pathogenesis-averse.
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http://dx.doi.org/10.1042/BJ20131378DOI Listing
April 2014
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