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Advances in fluorescence imaging coupled with the generation of near infrared probes have significantly improved the capabilities of non-invasive, real-time imaging in whole animals. In this study we were able to overcome a limitation of in vivo fluorescence imaging and have established a dual cell tracking method where two different cell types can be monitored according to the spectral signature of the cell labelling fluorophore. Using a mouse model of acute liver injury, we have characterised the in vivo migration patterns of wild type and transgenic neutrophils with impaired chemotaxis. Here, we were able to demonstrate that IVIS provides a sensitive multiplexing technology to differentiate two different cell populations based on the spectral signature of the cell labelling fluorophores. This spectral unmixing methodology has the potential to uncover multidimensional cellular interactions involved in many diseases such as fibrosis and cancer. In vivo spectral un-mixing provides a useful tool for monitoring multiple biological process in real-time in the same animal.

The progression of fibrosis in chronic liver disease is dependent upon hepatic stellate cells (HSCs) transdifferentiating to a myofibroblast-like phenotype. This pivotal process is controlled by enzymes that regulate histone methylation and chromatin structure, which may be targets for developing anti-fibrotics. There is limited pre-clinical experimental support for the potential to therapeutically manipulate epigenetic regulators in fibrosis. In order to learn if epigenetic treatment can halt the progression of pre-established liver fibrosis, we treated mice with the histone methyltransferase inhibitor 3-deazaneplanocin A (DZNep) in a naked form or by selectively targeting HSC-derived myofibroblasts via an antibody-liposome-DZNep targeting vehicle. We discovered that DZNep treatment inhibited multiple histone methylation modifications, indicative of a broader specificity than previously reported. This broad epigenetic repression was associated with the suppression of fibrosis progression as assessed both histologically and biochemically. The anti-fibrotic effect of DZNep was reproduced when the drug was selectively targeted to HSC-derived myofibroblasts. Therefore, the in vivo modulation of HSC histone methylation is sufficient to halt progression of fibrosis in the context of continuous liver damage. This discovery and our novel HSC-targeting vehicle, which avoids the unwanted effects of epigenetic drugs on parenchymal liver cells, represents an important proof-of-concept for epigenetic treatment of liver fibrosis. Fibrosis results in progressive deposition of scar tissue leading to cirrhosis. The histone methyltransferase EZH2 regulates the activity of scar-forming myofibroblasts. By developing a liposome vehicle decorated with an antibody selective for myofibroblasts, we demonstrate that in vivo pharmacological targeting of myofibroblast EZH2 is sufficient to block fibrosis progression in chronic liver disease.

Synaptophysin is expressed on fibrogenic hepatic myofibroblasts. C1–3 is a single chain human antibody (scAb) that binds specifically to synaptophysin on hepatic myofibroblasts, providing a targeting vector for novel in vivo imaging agents of chronic liver disease. C1–3 and a negative control scAb, CSBD9, were radiolabelled with zirconium-89 via desferrioxamine chelation to enable non-invasive molecular imaging with positron emission tomography (PET). DFO-scAb conjugates were characterised by gel electrophoresis (SDS-PAGE) and MALDI-TOF spectrometry, and 89 Zr-labelled with high radiolabelling efficiency (99%). [ 89 Zr]Zr-DFO-C1–3 exhibited high in vitro stability (> 99%) in mouse and human sera over 3 days at 25 and 37 °C. Activated hepatic myofibroblasts incubated with [ 89 Zr]Zr-DFO-C1–3 displayed significantly higher internalised activity (59.46%, P = 0.001) compared to the [ 89 Zr]Zr-DFO-CSBD9 control, indicating synaptophysin-mediated uptake and high binding specificity of [ 89 Zr]Zr-DFO-C1–3. Mice with CCl 4 -induced acute liver damage exhibited significantly higher liver uptake of [ 89 Zr]Zr-DFO-C1–3, compared to controls, confirmed by both Cerenkov imaging and ex vivo gamma counting (4.41 ± 0.19%ID/g, P < 0.0001). CCl 4 -induced liver damage and the number of hepatic myofibroblasts was confirmed by αSMA staining of liver sections. These findings indicate that [ 89 Zr]Zr-DFO-C1–3 has promising utility as a PET imaging agent for non-invasive detection of hepatic myofibroblasts following acute liver injury.

During chronic kidney disease (CKD) there is a dysregulation of extracellular matrix (ECM) homeostasis leading to renal fibrosis. Lysosomal proteases such as cathepsins (Cts) regulate this process in other organs, however, their role in CKD is still unknown. Here we describe a novel role for cathepsins in CKD. CtsD and B were located in distal and proximal tubular cells respectively in human disease. Administration of CtsD (Pepstatin A) but not B inhibitor (Ca074-Me), in two mouse CKD models, UUO and chronic ischemia reperfusion injury, led to a reduction in fibrosis. No changes in collagen transcription or myofibroblasts numbers were observed. Pepstatin A administration resulted in increased extracellular urokinase and collagen degradation. In vitro and in vivo administration of chloroquine, an endo/lysosomal inhibitor, mimicked Pepstatin A effect on renal fibrosis. Therefore, we propose a mechanism by which CtsD inhibition leads to increased collagenolytic activity due to an impairment in lysosomal recycling. This results in increased extracellular activity of enzymes such as urokinase, triggering a proteolytic cascade, which culminates in more ECM degradation. Taken together these results suggest that inhibition of lysosomal proteases, such as CtsD, could be a new therapeutic approach to reduce renal fibrosis and slow progression of CKD.

Vegetation alters soil fabric by providing biological reinforcement and enhancing the overall mechanical behaviour of slopes, thereby controlling shallow mass movement. To predict the behaviour of vegetated slopes, parameters representing the root system structure, such as root distribution, length, orientation and diameter, should be considered in slope stability models. This study quantifies the relationship between soil physical characteristics and root growth, giving special emphasis on (1) how roots influence the physical architecture of the surrounding soil structure and (2) how soil structure influences the root growth. A systematic experimental study is carried out using high-resolution X-ray micro-computed tomography (µCT) to observe the root behaviour in layered soil. In total, 2 samples are scanned over 15 days, enabling the acquisition of 10 sets of images. A machine learning algorithm for image segmentation is trained to act at 3 different training percentages, resulting in the processing of 30 sets of images, with the outcomes prompting a discussion on the size of the training data set. An automated in-house image processing algorithm is employed to quantify the void ratio and root volume ratio. This script enables post processing and image analysis of all 30 cases within few hours. This work investigates the effect of stratigraphy on root growth, along with the effect of image-segmentation parameters on soil constitutive properties.

Background Preclinical models of spinal cord stimulation (SCS) are lacking objective measurements to inform translationally applicable SCS parameters. The evoked compound action potential (ECAP) represents a measure of dorsal column fiber activation. This measure approximates the onset of SCS-induced sensations in humans and provides effective analgesia when used with ECAP-controlled closed-loop (CL)-SCS systems. Therefore, ECAPs may provide an objective surrogate for SCS dose in preclinical models that may support better understanding of SCS mechanisms and further translations to the clinics. This study assessed, for the first time, the feasibility of recording ECAPs and applying ECAP-controlled CL-SCS in freely behaving rats subjected to an experimental model of neuropathic pain. Methods Adult male Sprague–Dawley rats (200–300 g) were subjected to spared nerve injury (SNI). A custom-made six-contact lead was implanted epidurally covering T11-L3, as confirmed by computed tomography or X-ray. A specially designed multi-channel system was used to record ECAPs and to apply ECAP-controlled CL-SCS for 30 min at 50 Hz 200 µs. The responses of dorsal column fibers to SCS were characterized and sensitivity towards mechanical and cold stimuli were assessed to determine analgesic effects from ECAP-controlled CL-SCS. Comparisons between SNI rats and their controls as well as between stimulation parameters were made using omnibus analysis of variance (ANOVA) tests and t -tests . Results The recorded ECAPs showed the characteristic triphasic morphology and the ECAP amplitude (mV) increased as higher currents (mA) were applied in both SNI animals and controls (SNI SCS-ON and sham SCS-ON). Importantly, the use of ECAP-based SCS dose, implemented in ECAP-controlled CL-SCS, significantly reduced mechanical and cold hypersensitivity in SNI SCS-ON animals through the constant and controlled activation of dorsal column fibers. An analysis of conduction velocities of the evoked signals confirmed the involvement of large, myelinated fibers. Conclusions The use of ECAP-based SCS dose implemented in ECAP-controlled CL-SCS produced analgesia in animals subjected to an experimental model of neuropathic pain. This approach may offer a better method for translating SCS parameters between species that will improve understanding of the mechanisms of SCS action to further advance future clinical applications.

Root growth alters soil fabric and consequently its mechanical and physical properties. Recent studies show that roots induce compaction of soil in their immediate vicinity, a region that is central for plant health. However, high quality quantification of root influence on the soil fabric, able to inform computational models is lacking from the literature. This study quantifies the relationship between soil physical characteristics and root growth, giving special emphasis on how roots in early stage formation influence the physical architecture of the surrounding soil structure. High-resolution X-ray micro-Computed Tomography (µCT) is used to acquire three dimensional images of two homogeneously-packed samples. It is observed that the void ratio profile extending from the soil-root interface into the bulk soil is altered by root growth. The roots considerably modify the immediate soil physical characteristics by creating micro cracks at the soil-root interface and by increasing void ratio. This paper presents the mechanisms that led to the observed structure as well as some of the implications that it has in such a dynamic zone.

Neutrophils are the most abundant inflammatory cells at the earliest stages of wound healing and play important roles in wound repair and fibrosis. Formyl peptide receptor 1 (FPR-1) is abundantly expressed on neutrophils and has been shown to regulate their function, yet the importance of FPR-1 in fibrosis remains ill defined. FPR-1-deficient (fpr1-/-) mice were protected from bleomycin-induced pulmonary fibrosis but developed renal and hepatic fibrosis normally. Mechanistically, we observed a failure to effectively recruit neutrophils to the lungs of fpr1-/- mice, whereas neutrophil recruitment was unaffected in the liver and kidney. Using an adoptive transfer model we demonstrated that the defect in neutrophil recruitment to the lung was intrinsic to the fpr1-/- neutrophils, as C57BL/6 neutrophils were recruited normally to the damaged lung in fpr1-/- mice. Finally, C57BL/6 mice in which neutrophils had been depleted were protected from pulmonary fibrosis. In conclusion, FPR-1 and FPR-1 ligands are required for effective neutrophil recruitment to the damaged lung. Failure to recruit neutrophils or depletion of neutrophils protects from pulmonary fibrosis.

C3 glomerulopathy (C3G) is associated with dysregulation of the alternative pathway (AP) of complement and treatment options remain inadequate. Factor H (FH) is a potent regulator of the AP. An in-depth analysis of FH-related protein dimerised minimal (mini)-FH constructs has recently been published. This analysis showed that addition of a dimerisation module to mini-FH not only increased serum half-life but also improved complement regulatory function, thus providing a potential treatment option for C3G. Herein, we describe the production of a murine version of homodimeric mini-FH [mHDM-FH (mFH 1–5^18–20^R1–2 )], developed to reduce the risk of anti-drug antibody formation during long-term experiments in murine models of C3G and other complement-driven pathologies. Our analysis of mHDM-FH indicates that it binds with higher affinity and avidity to WT mC3b when compared to mouse (m)FH (mHDM-FH K D =505 nM; mFH K D =1370 nM) analogous to what we observed with the respective human proteins. The improved binding avidity resulted in enhanced complement regulatory function in haemolytic assays. Extended interval dosing studies in CFH -/- mice (5mg/kg every 72hrs) were partially effective and bio-distribution analysis in CFH -/- mice, through in vivo imaging technologies, demonstrates that mHDM-FH is preferentially deposited and remains fixed in the kidneys (and liver) for up to 4 days. Extended dosing using an AAV- human HDM-FH (hHDM-FH) construct achieved complete normalisation of C3 levels in CFH -/- mice for 3 months and was associated with a significant reduction in glomerular C3 staining. Our data demonstrate the ability of gene therapy delivery of mini-FH constructs to enhance complement regulation in vivo and support the application of this approach as a novel treatment strategy in diseases such as C3G.

BackgroundMolecular characterisation of hepatocellular carcinoma (HCC) is central to the development of novel therapeutic strategies for the disease. We have previously demonstrated mutagenic consequences of Long-Interspersed Nuclear Element-1 (LINE1s/L1) retrotransposition. However, the role of L1 in HCC, besides somatic mutagenesis, is not well understood.MethodsWe analysed L1 expression in the TCGA-HCC RNAseq dataset (n = 372) and explored potential relationships between L1 expression and clinical features. The findings were confirmed by immunohistochemical (IHC) analysis of an independent human HCC cohort (n = 48) and functional mechanisms explored using in vitro and in vivo model systems.ResultsWe observed positive associations between L1 and activated TGFβ-signalling, TP53 mutation, alpha-fetoprotein and tumour invasion. IHC confirmed a positive association between pSMAD3, a surrogate for TGFβ-signalling status, and L1 ORF1p (P < 0.0001, n = 32). Experimental modulation of L1 ORF1p levels revealed an influence of L1 ORF1p on key hepatocarcinogenesis-related pathways. Reduction in cell migration and invasive capacity was observed upon L1 ORF1 knockdown, both in vitro and in vivo. In particular, L1 ORF1p increased PIN1 cytoplasmic localisation. Blocking PIN1 activity abrogated L1 ORF1p-induced NF-κB-mediated inflammatory response genes while further activated TGFβ-signalling confirming differential alteration of PIN1 activity in cellular compartments by L1 ORF1p.DiscussionOur data demonstrate a causal link between L1 ORF1p and key oncogenic pathways mediated by PIN1, presenting a novel therapeutic avenue.