Andrew P. Hibbert

The localization, trafficking and retrograde transport of BDNF bound to p75NTR in sympathetic neurons

BDNF, through p75NTR, promotes apoptosis and inhibits axonal growth of sympathetic neurons, antagonizing the pro-survival and axon growth-promoting actions of NGF through TrkA. While the trafficking of the TrkA:NGF complex is well characterized, little is known about p75NTR:BDNF trafficking in these neurons. Here we show that BDNF binds to and appears inside sympathetic neurons relatively slowly, although the temperature-sensitive internalization step itself is rapid. P75NTR internalization is partially sensitive to disruption of clathrin- or raft-mediated internalization, while that of TrkA is entirely clathrin-mediated. P75NTR, but not Trk, associates with neurotrophins in lipid rafts and coimmunoprecipitates with the truncated beta-caveolin-1 isoform. Finally, we directly visualize the retrograde transport of p75NTR ligands to cell bodies, which is insensitive to inhibitors of Trk retrograde transport, suggesting mechanistic differences. We postulate that beta-caveolin-1-containing lipid rafts and possibly intracellular endosomes might be compartments to which p75NTR:BDNF complexes are trafficked separately from Trk.

Macrophage sub-populations and the lipoxin A4 receptor implicate active inflammation during equine tendon repair.

Macrophages (Mϕ) orchestrate inflammatory and reparatory processes in injured connective tissues but their role during different phases of tendon healing is not known. We investigated the contribution of different Mϕ subsets in an equine model of naturally occurring tendon injury. Post mortem tissues were harvested from normal (uninjured), sub-acute (3–6 weeks post injury) and chronically injured (>3 months post injury) superficial digital flexor tendons. To determine if inflammation was present in injured tendons, Mϕ sub-populations were quantified based on surface antigen expression of CD172a (pan Mϕ), CD14highCD206low (pro-inflammatory M1Mϕ), and CD206high (anti-inflammatory M2Mϕ) to assess potential polarised phenotypes. In addition, the Lipoxin A4 receptor (FPR2/ALX) was used as marker for resolving inflammation. Normal tendons were negative for both Mϕ and FPR2/ALX. In contrast, M1Mϕ predominated in sub-acute injury, whereas a potential phenotype-switch to M2Mϕ polarity was seen in chronic injury. Furthermore, FPR2/ALX expression by tenocytes was significantly upregulated in sub-acute but not chronic injury. Expression of the FPR2/ALX ligand Annexin A1 was also significantly increased in sub-acute and chronic injuries in contrast to low level expression in normal tendons. The combination of reduced FPR2/ALX expression and persistence of the M2Mϕ phenotype in chronic injury suggests a potential mechanism for incomplete resolution of inflammation after tendon injury. To investigate the effect of pro-inflammatory mediators on lipoxin A4 (LXA4) production and FPR2/ALX expression in vitro, normal tendon explants were stimulated with interleukin-1 beta and prostaglandin E2. Stimulation with either mediator induced LXA4 release and maximal upregulation of FPR2/ALX expression after 72 hours. Taken together, our data suggests that although tenocytes are capable of mounting a protective mechanism to counteract inflammatory stimuli, this appears to be of insufficient duration and magnitude in natural tendon injury, which may potentiate chronic inflammation and fibrotic repair, as indicated by the presence of M2Mϕ.

Enterococcus faecalis subverts and invades the host urothelium in patients with chronic urinary tract infection.

Bacterial urinary tract infections (UTI) are a major growing concern worldwide. Uropathogenic Escherichia coli has been shown to invade the urothelium during acute UTI in mice and humans, forming intracellular reservoirs that can evade antibiotics and the immune response, allowing recurrence at a later date. Other bacterial species, such as Staphylococcus saprophyticus, Klebsiella pneumonia and Salmonella enterica have also been shown to be invasive in acute UTI. However, the role of intracellular infection in chronic UTI causing more subtle lower urinary tract symptoms (LUTS), a particular problem in the elderly population, is poorly understood. Moreover, the species of bacteria involved remains largely unknown. A previous study of a large cohort of non-acute LUTS patients found that Enterococcus faecalis was frequently found in urine specimens. E. faecalis accounts for a significant proportion of chronic bladder infections worldwide, although the invasive lifestyle of this uropathogen has yet to be reported. Here, we wanted to explore this question in more detail. We harvested urothelial cells shed in response to inflammation and, using advanced imaging techniques, inspected them for signs of bacterial pathology and invasion. We found strong evidence of intracellular E. faecalis harboured within urothelial cells shed from the bladder of LUTS patients. Furthermore, using a culture model system, these patient-isolated strains of E. faecalis were able to invade a transitional carcinoma cell line. In contrast, we found no evidence of cellular invasion by E. coli in the patient cells or the culture model system. Our data show that E. faecalis is highly competent to invade in this context; therefore, these results have implications for both the diagnosis and treatment of chronic LUTS.

Neurotrophin-4, Alone or Heterodimerized with Brain-derived Neurotrophic Factor, Is Sorted to the Constitutive Secretory Pathway

Nerve growth factor and neurotrophin-3 (NT-3) are processed within the constitutive secretory pathway of neurons and neuroendocrine cells and are released continuously in an activity-independent fashion. In contrast, brain-derived neurotrophic factor (BDNF) is processed in the regulated secretory pathway, stored in vesicles, and released in response to neuronal activity, consistent with its role in modulating synaptic plasticity. In this study, we used vaccinia virus infection and transfection methods to monitor the processing and sorting of neurotrophin-4 (NT-4) in AtT-20 cells, which have been used as a model for the sorting of secretory proteins in neurons. Our data show that NT-4 is processed in the constitutive secretory pathway. The molecule is diffusely distributed within the cells and released, soon after being synthesized, in a manner that is not affected by cell depolarization. We further show that NT-4 and BDNF, when co-expressed, can form heterodimers that are constitutively released. In contrast, heterodimers of NT-3 and BDNF have been shown to be released through the regulated secretory pathway. Thus, NT-4, alone or when co-expressed with BDNF, is processed within and secreted by the constitutive secretory pathway.

A Comprehensive Pathological Survey of Duodenal Biopsies from Dogs with Diet‐Responsive Chronic Enteropathy

BACKGROUND The detailed pathological phenotype of diet-responsive chronic enteropathy (CE) and its modulation with dietary therapy remain poorly characterized. HYPOTHESIS/OBJECTIVES Key mucosal lesions of diet-responsive CE resolve with dietary therapy. METHODS This was a prospective observational study of 20 dogs with diet-responsive CE. Endoscopic duodenal biopsies collected before and 6 weeks after the start of a dietary trial were assessed by means of qualitative and quantitative histopathological, immunohistochemical, and ultrastructural criteria. Control duodenal biopsies were obtained from 10 healthy Beagle dogs on 1 occasion. RESULTS Compared with control dogs, the CE dogs had higher villus stunting scores and higher overall WSAVA scores, a lower villus height-to-width ratio, and higher lamina propria density of eosinophils. The CE dogs also had ultrastructural lesions of the mitochondria and brush border. In common with other studies in which the disease and control populations are not matched for breed, age, sex, and environment, these comparisons should be interpreted with caution. Comparing biopsies collected at presentation and 6 weeks after starting the dietary trial, mean lamina propria mononuclear cell score and lamina propria densities of eosinophils and mononuclear cells decreased. Dietary therapy also improved ultrastructural lesions of the mitochondria and brush border, eliciting a decrease in intermicrovillar space and an increase in microvillus height. CONCLUSIONS AND CLINICAL IMPORTANCE In dogs with diet-responsive CE, the remission of clinical signs with dietary therapy is associated with subtle decreases in lamina propria density of eosinophils and mononuclear cells, and resolution of ultrastructural lesions of the enterocyte.

A thin layer angiogenesis assay: a modified basement matrix assay for assessment of endothelial cell differentiation

BackgroundBasement matrices such as Matrigel™ and Geltrex™ are used in a variety of cell culture assays of anchorage-dependent differentiation including endothelial cell tube formation assays. The volumes of matrix recommended for these assays (approximately 150 μl/cm2) are costly, limit working distances for microscopy, and require cell detachment for subsequent molecular analysis. Here we describe the development and validation of a thin-layer angiogenesis (TLA) assay for assessing the angiogenic potential of endothelial cells that overcomes these limitations.ResultsGeltrex™ basement matrix at 5 μl/cm2 in 24-well (10 μl) or 96-well (2 μl) plates supports endothelial cell differentiation into tube-like structures in a comparable manner to the standard larger volumes of matrix. Since working distances are reduced, high-resolution single cell microscopy, including DIC and confocal imaging, can be used readily. Using MitoTracker dye we now demonstrate, for the first time, live mitochondrial dynamics and visualise the 3-dimensional network of mitochondria present in differentiated endothelial cells. Using a standard commercial total RNA extraction kit (Qiagen) we also show direct RNA extraction and RT-qPCR from differentiated endothelial cells without the need to initially detach cells from their supporting matrix.ConclusionsWe present here a new thin-layer assay (TLA) for measuring the anchorage-dependent differentiation of endothelial cells into tube-like structures which retains all the characteristics of the traditional approach but with the added benefit of a greatly lowered cost and better compatibility with other techniques, including RT-qPCR and high-resolution microscopy.

Prevalence of FoxP3+ Cells in Canine Tumours and Lymph Nodes Correlates Positively with Glucose Transporter 1 Expression

Hypoxia and regulatory T cells (Tregs) in tumours are both known to be negative prognostic factors in cancer, and this study demonstrated a correlation between the two factors in canine neoplasia. Samples of 57 canine tumours and 29 canine lymph nodes categorized as tumour-draining, with or without metastasis, or reactive and not tumour-associated, were examined. Sequential sections were labelled by immunohistochemistry for glucose transporter 1 (Glut1) and FoxP3 as markers of hypoxia and Tregs, respectively. Up to 21 regions of interest (ROI) were selected in each section in a representative pattern and were assigned a semiquantitative score based on Glut1 labelling. The number of FoxP3(+) cells within each ROI was counted. A generalized estimating equation with negative binomial log link function was used to determine an association between Glut1 expression and FoxP3(+) cell count. Higher Glut1 immunoreactivity was correlated with significantly higher numbers of FoxP3(+) cells in the total tumour sample pool and total lymph node sample pool. Analysis of various subcategories of tumours and lymph nodes showed that this correlation was also present within samples characterized as malignant, haemopoietic mesenchymal tumours, non-haemopoietic mesenchymal tumours, epithelial tumours, lymphoma, lymph nodes containing metastases and reactive lymph nodes. These results indicate that hypoxia in canine tumours may result in an increased infiltration by Tregs.

Myosin VI and Associated Proteins Are Expressed in Human Macrophages but Do Not Play a Role in Foam Cell Formation in THP-1 Cells.

Myosin VI (Myo6) functions in endocytosis in conjunction with binding partners including adaptor protein (AP)-2, disabled 2 (Dab2), and GAIP interacting protein C terminus 1 (GIPC1). This study aimed to investigate the expression and function of Myo6 in macrophages and its possible role in the endocytosis of lipoproteins during the induction of foam cell formation. Expression of Myo6, AP-2 (α2 subunit), and Dab2 in THP-1 macrophages and primary human monocyte-derived macrophages was demonstrated at the mRNA and protein level, but GIPC1 was only detected at the mRNA level. Immunofluorescence showed that Myo6 was distributed similarly to F-actin in both macrophage types. AP-2α2 was found to have a similar subcellular distribution to Myo6 and Dab2 in THP-1 cells. Myo6 was located within membrane ruffles and protrusions of the plasma membrane. These results suggest that in macrophages Myo6 is required for several functions including cell adhesion, cell progression, and macropinocytosis. Low-density lipoprotein (LDL) and oxidised LDL (oxLDL) decreased Myo6 and GIPC1 mRNA expression in THP-1 cells, but uptake of the fluorescence-labelled lipoproteins was unaffected by knockdown of the expression of Myo6 or associated proteins with siRNA. Our findings, therefore, do not support the idea that Myo6 plays a major role in foam cell formation.