Biology subjects

Persson, H.

Publications and source records attributed to Persson, H..

5 recordsLinked to original sources

bioRxiv2023

Sensitivity and validation of porous membrane electrical cell substrate impedance spectroscopy (PM-ECIS) for measuring endothelial barrier properties

Conventional trans-endothelial electrical resistance (TEER) setups are invasive and cannot directly measure monolayer integrity in co-culture. These limitations are addressed by porous membrane electrical cell-substrate impedance sensing (PM-ECIS), which measures barrier integrity in cell monolayers grown directly on permeable membranes patterned with electrodes. Here we advance the design and utility of PM-ECIS by investigating its sensitivity to working electrode size and correlation to TEER. Gold electrodes were fabricated on porous membrane inserts using hot embossing and UV lithography, with working electrode diameters of 250, 500, and 750 {micro}m within the same insert. Frequency scans of confluent primary endothelial monolayers showed normalized resistance peaking at 4 kHz, consistent with traditional solid substrate ECIS. Sensitivity to resistance changes (4 kHz) during endothelial barrier formation was inversely proportional to electrode size, with the smallest electrodes being the most sensitive (p<0.001). Similarly, smaller electrodes were most sensitive to changes in impedance (40 kHz) corresponding to cell spreading and proliferation (p<0.001). Barrier disruption with EGTA was detectable by 250 and 750 {micro}m (p<0.01), and 500 {micro}m electrodes (p=0.058). Resistances measured by PM-ECIS vs. TEER for sodium chloride solutions were positively and significantly correlated for all electrode sizes (r>0.9; p<0.0001), but only with 750 {micro}m electrodes for endothelial monolayers (r=0.71; p=0.058). These data inform design and selection of PM-ECIS electrodes for specific applications, and support PM-ECIS as a promising alternative to conventional TEER due to its capacity for direct, non-invasive, and real-time assessment of cells cultured on porous membranes.

bioengineering↗

bioRxiv2022

Autoantigenic properties of the aminoacyl tRNA synthetase family in idiopathic inflammatory myopathies

ObjectivesAutoantibodies are thought to play a key role in the pathogenesis of idiopathic inflammatory myopathies (IIM). However, up to 40% of IIM patients, even those with clinical manifestations of anti-synthetase syndrome (ASSD), test seronegative to all known myositis-specific autoantibodies (MSAs). We hypothesized the existence of new potential autoantigens among human cytoplasmic aminoacyl tRNA synthetases (aaRS) in patients with IIM. MethodsPlasma samples and clinical data from 217 patients with, 50 patients with ASSD, 165 without, and two with unknown ASSD status were included retrospectively, as well as serum from 156 age/sex-matched population controls. Samples were screened using a multiplex bead array assay for presence of autoantibodies against a panel of 118 recombinant protein variants, representing 33 myositis-related proteins, including all 19 cytoplasmic aaRS. ResultsWe identified reactivity towards 16 aaRS in 72 of the 217 patients. Twelve patients displayed reactivity against nine novel aaRS. The novel autoantibody specificities were detected in four patients previously seronegative for MSAs and in eight with previously detected MSAs. We also confirmed reactivity to four of the most common aaRS (Jo1, PL12, PL7, and EJ (n=45)) and identified patients positive for anti-Zo, -KS, and -HA (n=10) that were not previously tested. A low frequency of anti-aaRS autoantibodies was detected in controls. ConclusionOur results suggest that most, if not all, cytoplasmic aaRS may become autoantigenic. Autoantibodies against new aaRS may be found in plasma of patients previously classified as seronegative with potential high clinical relevance.

immunology↗

bioRxiv2021

Vascular tropism models of blood-borne microbial dissemination

Similar to circulating tumour and immune cells, many blood-borne microbes preferentially "home" to specific vascular sites and tissues during hematogenous dissemination 1-5. For many pathogens, the "postal codes" and mechanisms responsible for tissue-specific vascular tropism are unknown and have been challenging to unravel. Members of the Lyme disease Borreliella burgdorferi species complex infect a broad range of mammalian tissues and exhibit complex strain-, species- and host-specific tissue tropism patterns. Intravenous perfusion experiments and intravital microscopy studies suggest that heterogeneous tissue tropism properties may depend on tissue-specific differences in host and microbial molecules supporting vascular interaction and extravasation. However, interpreting these studies can be complicated because of the immune-protective moonlighting (multitasking) properties of many B. burgdorferi adhesins. Here, we investigated whether B. burgdorferi vascular interaction properties measured by live cell imaging and particle tracking in aorta, bladder, brain, joint and skin microvascular flow chamber models predict strain- and tissue-specific dissemination patterns in vivo These studies identified strain- and endothelial cell type-specific interaction properties that accurately predicted in vivo dissemination of B. burgdorferi to bladder, brain, joint and skin but not aorta, and indicated that dissemination mechanisms in all of these tissues are distinct. Thus, the ability to interact with vascular surfaces under physiological shear stress is a key determinant of tissue-specific tropism for Lyme disease bacteria. The methods and model systems reported here will be invaluable for identifying and characterizing the diverse, largely undefined molecules and mechanisms supporting dissemination of Lyme disease bacteria. These methods and models may be useful for studying tissue tropism and vascular dissemination mechanisms of other blood-borne microbes.

microbiology↗

bioRxiv2021

Selection and structural characterisation of anti-TREM2 scFvs that reduce levels of shed ectodomain

Single point mutations in TREM2, a receptor expressed by microglia in the brain, are associated with an increased risk of neurodegeneration including Alzheimers disease. Numerous studies support a role for TREM2 in sensing damaging stimuli and triggering signalling cascades necessary for neuroprotection. Despite its significant role, ligands and regulators of TREM2 activation, and the mechanisms governing TREM2-dependent responses and its cleavage from the membrane, remain poorly characterised. Here, we present phage display generated scFv antibody binders to human TREM2 ectodomain. Cocrystal structures revealed the binding of two scFvs to an epitope on the globular TREM2 domain distal to the putative ligand-binding site. Enhanced functional activity was observed for oligomeric scFv species which inhibited the production of soluble TREM2 in a HEK293 cell model. We hope that detailed characterisation of their epitopes and properties will facilitate the use of these renewable binders as structural and functional biology tools for TREM2 research.

molecular biology↗

bioRxiv2019

Strategies to improve scFvs as crystallization chaperones suggested by analysis of a complex with the human PHD-bromodomain SP140

Antibody fragments have great potential as crystallization chaperones for structural biology due to their ability to either stabilise targets, trap certain conformations and/or promote crystal packing. Here we present an example of using a single-chain variable fragment (scFv) to determine the previously unsolved structure of the multidomain protein SP140. This nuclear leukocyte-specific protein contains domains related to chromatin-mediated gene expression and has been implicated in various disease states. The structure of two of the domains (PHD-bromodomain) was solved by crystallizing them as a complex with a scFv generated by phage display technology. SP140 maintains a similar overall fold to previous PHD-bromodomains and the scFv CDR loops predominately interact with the PHD, while the framework regions of the scFv makes numerous interactions with the bromodomain. Analysis of our and other complex structures suggest various protein engineering strategies that might be employed to improve the usefulness of scFvs as crystallization chaperones.

biochemistry↗