Joanne L. Selway

A novel automated image analysis method for accurate adipocyte quantification.

Increased adipocyte size and number are associated with many of the adverse effects observed in metabolic disease states. While methods to quantify such changes in the adipocyte are of scientific and clinical interest, manual methods to determine adipocyte size are both laborious and intractable to large scale investigations. Moreover, existing computational methods are not fully automated. We, therefore, developed a novel automatic method to provide accurate measurements of the cross-sectional area of adipocytes in histological sections, allowing rapid high-throughput quantification of fat cell size and number. Photomicrographs of H&E-stained paraffin sections of murine gonadal adipose were transformed using standard image processing/analysis algorithms to reduce background and enhance edge-detection. This allowed the isolation of individual adipocytes from which their area could be calculated. Performance was compared with manual measurements made from the same images, in which adipocyte area was calculated from estimates of the major and minor axes of individual adipocytes. Both methods identified an increase in mean adipocyte size in a murine model of obesity, with good concordance, although the calculation used to identify cell area from manual measurements was found to consistently over-estimate cell size. Here we report an accurate method to determine adipocyte area in histological sections that provides a considerable time saving over manual methods.

A novel method to assess collagen architecture in skin

BackgroundTexture within biological specimens may reveal critical insights, while being very difficult to quantify. This is a particular problem in histological analysis. For example, cross-polar images of picrosirius stained skin reveal exquisite structure, allowing changes in the basketweave conformation of healthy collagen to be assessed. Existing techniques measure gross pathological changes, such as fibrosis, but are not sufficiently sensitive to detect more subtle and progressive pathological changes in the dermis, such as those seen in ageing. Moreover, screening methods for cutaneous therapeutics require accurate, unsupervised and high-throughput image analysis techniques.ResultsBy analyzing spectra of images post Gabor filtering and Fast Fourier Transform, we were able to measure subtle changes in collagen fibre orientation intractable to existing techniques. We detected the progressive loss of collagen basketweave structure in a series of chronologically aged skin samples, as well as in skin derived from a model of type 2 diabetes mellitus.ConclusionsWe describe a novel bioimaging approach with implications for the evaluation of pathology in a broader range of biological situations.

Evidence That Ca2+ within the Microdomain of the L-Type Voltage Gated Ca2+ Channel Activates ERK in MIN6 Cells in Response to Glucagon-Like Peptide-1

Glucagon like peptide-1 (GLP-1) is released from intestinal L-cells in response to nutrient ingestion and acts upon pancreatic β-cells potentiating glucose-stimulated insulin secretion and stimulating β-cell proliferation, differentiation, survival and gene transcription. These effects are mediated through the activation of multiple signal transduction pathways including the extracellular regulated kinase (ERK) pathway. We have previously reported that GLP-1 activates ERK through a mechanism dependent upon the influx of extracellular Ca2+ through L-type voltage gated Ca2+ channels (VGCC). However, the mechanism by which L-type VGCCs couple to the ERK signalling pathway in pancreatic β-cells is poorly understood. In this report, we characterise the relationship between L-type VGCC mediated changes in intracellular Ca2+ concentration ([Ca2+]i) and the activation of ERK, and demonstrate that the sustained activation of ERK (up to 30 min) in response to GLP-1 requires the continual activation of the L-type VGCC yet does not require a sustained increase in global [Ca2+]i or Ca2+ efflux from the endoplasmic reticulum. Moreover, sustained elevation of [Ca2+]i induced by ionomycin is insufficient to stimulate the prolonged activation of ERK. Using the cell permeant Ca2+ chelators, EGTA-AM and BAPTA-AM, to determine the spatial dynamics of L-type VGCC-dependent Ca2+ signalling to ERK, we provide evidence that a sustained increase in Ca2+ within the microdomain of the L-type VGCC is sufficient for signalling to ERK and that this plays an important role in GLP-1- stimulated ERK activation.

Molecular mechanisms of muscarinic acetylcholine receptor-stimulated increase in cytosolic free Ca(2+) concentration and ERK1/2 activation in the MIN6 pancreatic β-cell line.

Muscarinic acetylcholine receptor (mAChR) activation of pancreatic β-cells elevates intracellular Ca2+ and potentiates glucose-stimulated insulin secretion. In addition, it activates a number of signaling molecules, including ERK1/2, whose activation has been shown to play an important role in regulating pancreatic β-cell function and mass. The aim of this work was to determine how mAChR activation elevates intracellular Ca2+ concentration ([Ca2+]i) and activates ERK1/2 in the pancreatic β-cell line MIN6. We demonstrate that agonist-stimulated ERK1/2 activation is dependent on the activation of phospholipase C and an elevation in [Ca2+]i, but is independent of the activation of diacylglycerol-dependent protein kinase C isoenzymes. Using a pharmacological approach, we provide evidence that agonist-induced increases in [Ca2+]i and ERK activity require (1) IP3 receptor-mediated mobilization of Ca2+ from the endoplasmic reticulum, (2) influx of extracellular Ca2+ through store-operated channels, (3) closure of KATP channels, and (4) Ca2+ entry via L-type voltage-operated Ca2+ channels. Moreover, this Ca2+-dependent activation of ERK is mediated via both Ras-dependent and Ras-independent mechanisms. In summary, this study provides important insights into the multifactorial signaling mechanisms linking mAChR activation to increases in [Ca2+]i and ERK activity.

Automatic layer segmentation of H&E microscopic images of mice skin

Mammalian skin is a complex organ composed of a variety of cells and tissue types. The automatic detection and quantification of changes in skin structures has a wide range of applications for biological research. To accurately segment and quantify nuclei, sebaceous gland, hair follicles, and other skin structures, there is a need for a reliable segmentation of different skin layers. This paper presents an efficient segmentation algorithm to segment the three main layers of mice skin, namely epidermis, dermis, and subcutaneous layers. It also segments the epidermis layer into two sub layers, basal and cornified layers. The proposed algorithm uses adaptive colour deconvolution technique on H&E stain images to separate different tissue structures, inter-modes and Otsu thresholding techniques were effectively combined to segment the layers. It then uses a set of morphological and logical operations on each layer to removing unwanted objects. A dataset of 7000 H&E microscopic images of mutant and wild type mice were used to evaluate the effectiveness of the algorithm. Experimental results examined by domain experts have confirmed the viability of the proposed algorithms.

Collagen remodeling and peripheral immune cell recruitment characterizes the cutaneous Langerhans cell histiocytosis microenvironment.

Langerhans cell histiocytosis (LCH) is a rare and potentially fatal disorder of unknown etiology arising from the accumulation of epidermal Langerhans‐like cells in bone, skin, or other tissues. Tissue damage and morbidity results from lesional cytokine release, and we sought to investigate the LCH microenvironment using a combination of histological stains and immunohistochemistry.

A Novel Mechanism for Activation of GLI1 by Nuclear SMO That Escapes Anti-SMO Inhibitors

Small-molecule inhibitors of the Hedgehog (HH) pathway receptor Smoothened (SMO) have been effective in treating some patients with basal cell carcinoma (BCC), where the HH pathway is often activated, but many patients respond poorly. In this study, we report the results of investigations on PTCH1 signaling in the HH pathway that suggest why most patients with BCC respond poorly to SMO inhibitors. In immortalized human keratinocytes, PTCH1 silencing led to the generation of a compact, holoclone-like morphology with increased expression of SMO and the downstream HH pathway transcription factor GLI1. Notably, although siRNA silencing of SMO in PTCH1-silenced cells was sufficient to suppress GLI1 activity, this effect was not phenocopied by pharmacologic inhibition of SMO, suggesting the presence of a second undefined pathway through which SMO can induce GLI1. Consistent with this possibility, we observed increased nuclear localization of SMO in PTCH1-silenced cells as mediated by a putative SMO nuclear/nucleolar localization signal [N(o)LS]. Mutational inactivation of the N(o)LS ablated this increase and suppressed GLI1 induction. Immunohistologic analysis of human and mouse BCC confirmed evidence of nuclear SMO, although the pattern was heterogeneous between tumors. In PTCH1-silenced cells, >80% of the genes found to be differentially expressed were unaffected by SMO inhibitors, including the putative BCC driver gene CXCL11. Our results demonstrate how PTCH1 loss results in aberrant regulation of SMO-independent mechanisms important for BCC biology and highlights a novel nuclear mechanism of SMO-GLI1 signaling that is unresponsive to SMO inhibitors.Significance: This study describes novel noncanonical Hedgehog signaling, where SMO enters the nucleus to activate GLI1, a mode that is unaffected by SMO inhibitors, thus prompting re-evaluation of current BCC treatment as well as new potential therapies targeting nuclear SMO. Cancer Res; 78(10); 2577-88. ©2018 AACR.

Genetic homogeneity of adult Langerhans cell histiocytosis lesions: Insights from BRAFV600E mutations in adult populations

Langerhans cell histiocytosis (LCH) is a heterologous disease with a recognized disparity in incidence, affected sites and prognosis between adults and children. The recent identification of BRAFV600E mutations in LCH prompted the investigation of the frequency of these mutations in adult and childhood disease with the involvement of single or multiple sites in the present study. The study analysed the BRAFV600E status in a cohort of adult LCH patients by DNA sequencing, and performed a broader meta-analysis of BRAFV600E mutations in LCH in order to investigate any association with disease site and severity. A review of the literature revealed that ~47% of lesions from cases of adult disease (patient age, >18 years) were V600E-positive compared with 53% in those under 18 years. When single and multiple site disease was compared, there was a slight increase in the former (61 vs. 51%, respectively). A greater difference was observed when high- and low-risk organs were compared; for example, 75% of liver biopsies (a high-risk organ) were reported to bear the mutation compared with 47% of lung biopsies. In the adult LCH population, DNA sequencing identified mutations in 38% of 29 individuals, which is slightly lower than the figure identified from the meta-analysis (in which a total of 132 individuals were sampled), although we this value could not be broken down by clinical status. Thus, V600E status at presentation in itself is not predictive of tumour course, but a considerable proportion of LCH patients may respond to targeted V600E therapies.

Histochemical and immunohistochemical evaluation of mouse skin histology: comparison of fixation with neutral buffered formalin and alcoholic formalin

Abstract Histological analysis is a cornerstone of skin disease diagnosis, and the stratified nature of the skin presents many technical problems in the preparation of sections of acceptable quality. Furthermore, histological artifacts may be exacerbated in pathological states that compromise cutaneous integrity. Therefore, great care must be taken when selecting the conditions, particularly fixative type, to ensure that meaningful conclusions may be drawn. The goal of this work was to compare the morphology of mouse skin as revealed using a range of histological stains, and the antigenicity of key cutaneous proteins using immunohistochemistry (IHC) following fixation in either 10% neutral buffered formalin (NBF) or alcoholic formalin (AF). Although 10% NBF fixation is routinely used for histology, providing good retention of morphology and structure for special staining, AF fixation more effectively maintained antigenicity for the cutaneous markers investigated in this study. A comprehensive assessment of the two fixatives and histological techniques to optimize morphology, structure, and antigenicity in the analysis of mouse skin is presented.