Christopher John Von Ruhland

Defects in cell polarity underlie TSC and ADPKD-associated cystogenesis.

Clinical trials are underway for the treatment of tuberous sclerosis (TSC)-associated tumours using mTOR inhibitors. Here, we show that many of the earliest renal lesions from Tsc1+/- and Tsc2+/- mice do not exhibit mTOR activation, suggesting that pharmacological targeting of an alternative pathway may be necessary to prevent tumour formation. Patients with TSC often develop renal cysts and those with inherited co-deletions of the autosomal dominant polycystic kidney disease (ADPKD) 1 gene (PKD1) develop severe, early onset, polycystic kidneys. Using mouse models, we showed a genetic interaction between Tsc1 and Tsc2 with Pkd1 and confirmed an mTOR-independent pathway of renal cystogenesis. We observed that the Tsc and Pkd1 gene products helped regulate primary cilia length and, consistent with the function of this organelle in modulating cell polarity, found that many dividing pre-cystic renal tubule and hepatic bile duct cells from Tsc1, Tsc2 and Pkd1 heterozygous mice were highly misoriented. We therefore propose that defects in cell polarity underlie TSC and ADPKD-associated cystic disease and targeting of this pathway may be of key therapeutic benefit.

Opa1 is essential for retinal ganglion cell synaptic architecture and connectivity

Retinal ganglion cell dendritic pruning has been reported in association with a 50% reduction in Opa1 transcript and protein in retinal and neural tissue, which manifests as visual dysfunction in the heterozygous mutant mouse, B6;C3-Opa1(Q285STOP). Here we report a marked reduction in retinal ganglion cell synaptic connectivity in the absence of soma loss and explore the mechanism and relationship between mitochondrial integrity and synaptic connectivity. We observed decreased levels of postsynaptic density protein 95 in Opa1(+/-) mutant mice consistent with synaptic loss in the inner plexiform layer. Glutamatergic but not γ-aminobutyric acid-ergic synaptic sites were reduced in Opa1(+/-) mice. We observed increased synaptic vesicle number in bipolar cell terminal arbours assessed by immunohistochemistry, electron microscopy and western blot analysis. These changes occur without significant loss of mitochondrial membrane potential in retina and optic nerve. Analysis of biolistically transfected retinal ganglion cells shows the retraction of mitochondria towards the soma, and mitochondrial fragmentation, preceding dendritic loss. These processes cast light on the intimate relationship between normal mitochondrial fusion and fission balances, as influenced by the OPA1 protein, in neural cell connectivity in the mammalian retina.

Characterisation of adipocyte-derived extracellular vesicles released pre- and post-adipogenesis

Extracellular vesicles (EVs) are submicron vesicles released from many cell types, including adipocytes. EVs are implicated in the pathogenesis of obesity-driven cardiovascular disease, although the characteristics of adipocyte-derived EVs are not well described. We sought to define the characteristics of adipocyte-derived EVs before and after adipogenesis, hypothesising that adipogenesis would affect EV structure, molecular composition and function. Using 3T3-L1 cells, EVs were harvested at day 0 and day 15 of differentiation. EV and cell preparations were visualised by electron microscopy and EVs quantified by nanoparticle tracking analysis (NTA). EVs were then assessed for annexin V positivity using flow cytometry; lipid and phospholipid composition using 2D thin layer chromatography and gas chromatography; and vesicular protein content by an immuno-phenotyping assay. Pre-adipogenic cells are connected via a network of protrusions and EVs at both time points display classic EV morphology. EV concentration is elevated prior to adipogenesis, particularly in exosomes and small microvesicles. Parent cells contain higher proportions of phosphatidylserine (PS) and show higher annexin V binding. Both cells and EVs contain an increased proportion of arachidonic acid at day 0. PREF-1 was increased at day 0 whilst adiponectin was higher at day 15 indicating EV protein content reflects the stage of adipogenesis of the cell. Our data suggest that EV production is higher in cells before adipogenesis, particularly in vesicles <300 nm. Cells at this time point possess a greater proportion of PS (required for EV generation) whilst corresponding EVs are enriched in signalling fatty acids, such as arachidonic acid, and markers of adipogenesis, such as PREF-1 and PPARγ.

Chemotaxis and the cell surface-area problem

can approximately double their apparent surface area. In their review, Kay and colleagues suggest that ‘folds’ in the cell surface as possible reservoirs of additional membrane are unlikely, and focus on endocytic cycling as the potential mechanism. However, scanning electron microscopy of neutrophils show that this cell type has a wrinkled surface

Nano-analyses of Wear Particles from Metal-on-Metal and Non-Metal-on-Metal Dual Modular Neck Hip Arthroplasty

Increased failure rates due to metallic wear particle-associated adverse local tissue reactions (ALTR) is a significant clinical problem in resurfacing and total hip arthroplasty. Retrieved periprosthetic tissue of 53 cases with corrosion/conventional metallic wear particles from 285 revision operations for ALTR was selected for nano-analyses. Three major classes of hip implants associated with ALTR, metal-on-metal hip resurfacing arthroplasty (MoM HRA) and large head total hip replacement (MoM LHTHA) and non-metal-on-metal dual modular neck total hip replacement (Non-MoM DMNTHA) were included. The size, shape, distribution, element composition, and crystal structure of the metal particles were analyzed by conventional histological examination and electron microscopy with analytic tools of 2D X-ray energy dispersive spectrometry and X-ray diffraction. Distinct differences in size, shape, and element composition of the metallic particles were detected in each implant class which correlate with the histological features of severity of ALTR and variability in implant performance.

Can the choice of intermediate solvent or resin affect glomerular basement membrane thickness

BACKGROUND Establishing glomerular basement membrane (GBM) thickness is important in the diagnosis of some renal diseases. It is widely believed that GBM thickness varies according to the processing method, yet there appear to be no published data to support this. In this study we aimed to determine whether the choice of intermediate solvent and embedding resin influenced the thickness of the GBM and to assess the magnitude of any such effect. METHODS Subcapsular renal cortical rat tissue was processed for electron microscopy using four different intermediate solvents [propylene oxide (PO), xylene (Xyl), acetone (Ac) or ethanol (Et)], and three epoxy resins [Araldite (Aral), TAAB embedding resin (TER) or TAAB low viscosity resin (TLV)]. GBM thickness was estimated by the orthogonal intercept method. RESULTS Compared with PO-Aral embedding, GBM was significantly thinner in Xyl-Aral, Ac-Aral and Xyl-TER, and significantly thicker in PO-TER-, Ac-TER-, Et-TER- and PO-TLV-embedded tissue (P < 0.05 in all cases). No significant difference was seen with Xyl-TLV- and Ac-TLV-embedded tissue. Et-Aral and Et-TLV embedding resulted in poor quality blocks. CONCLUSIONS The results suggest that the choice of both intermediate solvent and resin influences shrinkage of the GBM during TEM processing.

Cerebrospinal fluid extracellular vesicle enrichment for protein biomarker discovery in neurological disease; multiple sclerosis

ABSTRACT The discovery of disease biomarkers, along with the use of “liquid biopsies” as a minimally invasive source of biomarkers, continues to be of great interest. In inflammatory diseases of the central nervous system (CNS), cerebrospinal fluid (CSF) is the most obvious biofluid source. Extracellular vesicles (EVs) are also present in CSF and are thought to be potential “biomarker treasure chests”. However, isolating these CSF-derived EVs remains challenging. This small-scale pilot study developed and tested a protocol to enrich for CSF-EVs, both in relapsing remitting multiple sclerosis (RRMS) CSF and controls. These were subsequently compared, using an aptamer based proteomics array, SOMAscan™. EVs were enriched from RRMS patient (n = 4) and non-demyelinating control (idiopathic intracranial hypertension (IIH) (n = 3)) CSF using precipitation and mini size-exclusion chromatography (SEC). EV-enriched fractions were selected using pre-defined EV characteristics, including increased levels of tetraspanins. EVs and paired CSF were analysed by SOMAscan™, providing relative abundance data for 1128 proteins. CSF-EVs were characterised, revealing exosome-like features: rich in tetraspanins CD9 and CD81, size ~100 nm, and exosome-like morphology by TEM. Sufficient quantities of, SOMAscan™ compatible, EV material was obtained from 5 ml CSF for proteomics analysis. Overall, 348 and 580 proteins were identified in CSF-EVs and CSF, respectively, of which 50 were found to be significantly (t-test) and exclusively enriched in RRMS CSF-EVs. Selected proteins, Plasma kallikrein and Apolipoprotein-E4, were further validated by western blot and appeared increased in CSF-EVs compared to CSF. Functional enrichment analysis of the 50 enriched proteins revealed strong associations with biological processes relating to MS pathology and also extracellular regions, consistent with EV enrichment. This pilot study demonstrates practicality for EV enrichment in CSF derived from patients with MS and controls, allowing detailed analysis of protein profiles that may offer opportunities to identify novel biomarkers and therapeutic approaches in CNS inflammatory diseases.

Massive Accumulation of Myofibroblasts in the Critical Isthmus Is Associated With Ventricular Tachycardia Inducibility in Post-Infarct Swine Heart

Objectives In this study the authors determined the extent of cellular infiltration and dispersion, and regional vascularization in electrophysiologically (EP) defined zones in post–myocardial infarction (MI) swine ventricle. Background The critical isthmus (CI) in post-MI re-entrant ventricular tachycardia (VT) is a target for catheter ablation. In vitro evidence suggests that myofibroblasts (MFB) within the scar border zone (BZ) may increase the susceptibility to slow conduction and VT, but whether this occurs in vivo remains unproven. Methods Six weeks after mid–left anterior descending coronary artery occlusion, EP catheter-based mapping was used to assess susceptibility to VT induction. EP data were correlated with detailed cellular profiling of ventricular zones using immunohistochemistry and spatial distribution analysis of cardiomyocytes, fibroblasts, MFB, and vascularization. Results In pigs with induced sustained monomorphic VT (mean cycle length: 353 ± 89 ms; n = 6) the area of scar that consisted of the BZ (i.e., between the normal and the low-voltage area identified by substrate mapping) was greater in VT-inducible hearts (iVT) than in noninducible hearts (non-VT) (p < 0.05). Scar in iVT hearts was characterized by MFB accumulation in the CI (>100 times that in normal myocardium and >5 times higher than that in the BZ in non-VT hearts) and by a 1.7-fold increase in blood vessel density within the dense scar region extending towards the CI. Sites of local abnormal ventricular activity potentials exhibited cellularity and vascularization that were intermediate to the CI in iVT and BZ in non-VT hearts. Conclusions The authors reported the first cellular analysis of the VT CI following an EP-based zonal analysis of iVT and non-VT hearts in pigs post-MI. The data suggested that VT susceptibility was defined by a remarkable number of MFB in the VT CI, which appeared to bridge the few remaining dispersed clusters of cardiomyocytes. These findings define the cellular substrate for the proarrhythmic slow conduction pathway.

The use of metal-catalyzed oxidation to suppress background staining caused by marker amplification reagents and the effects of this oxidation on the stability of antibody-antigen complexes in immunohistochemistry

ABSTRACT Marker amplification is a powerful technique for visualizing immunohistochemically deposited markers that otherwise would be invisible. Amplification usually is achieved with physical developers, which are solutions that contain a source of silver(I) plus a reducing agent. When the marker is present in extremely small quantities, prolonged incubation in the developer is required and unwanted background staining in the form of type III argyrophilia becomes problematic. Suppression of type III argyrophilia can be achieved by metal-catalyzed oxidation using the copper/H2O2 system, which normally is applied immediately prior to amplification. Because there is no reason, in principle, why metal-catalyzed oxidation should not be employed at earlier stages in the immunohistochemical staining procedure, we investigated whether earlier oxidation might confer any advantages over the traditional methodology. Immunocolloidal gold combined with two light insensitive physical developers was chosen as the model system, because visualization by light microscopy requires extended periods in the developers. Moreover, the system does not suffer from problems concerning endogenous enzyme- or non-enzyme-catalyzed marker deposition. Applying metal-catalyzed oxidation at each stage of the immunohistochemical procedure revealed that the technique could be employed successfully prior to staining, but not following the primary or secondary antibodies. In the latter cases, specific immunolocalization was lost entirely and only generalized nonspecific staining was seen. A limited investigation into the mechanism of metal-catalyzed oxidation of aldehyde fixed tissue sections suggested that it involved the formation of aldehyde groups. We suggest that the application of metal-catalyzed oxidation prior to immunohistochemical staining would have the advantages of both suppressing type III argyrophilia and inhibiting unwanted endogenous peroxidase activity. We also suggest that metal-catalyzed oxidation might reduce the affinity of tissue for other transition metals, such as copper, whose potential for improving marker amplification techniques has been demonstrated previously in dot-blot model systems.