Kenneth Langlands

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.

A Topology-Based Score for Pathway Enrichment

Investigators require intuitive tools to rationalize complex datasets generated by transcriptional profiling experiments. Pathway analysis methods, in which differentially expressed genes are mapped to databases of reference pathways to facilitate assessment of relative enrichment, lead investigators more effectively to biologically testable hypotheses. However, once a set of differentially expressed genes is isolated, pathway analysis approaches tend to ignore rich gene expression information and, moreover, do not exploit relationships between transcripts. In this article, we report the development of a new method in which both pathway topology and the magnitude of gene expression changes inform the scoring system, thereby providing a powerful filter in the enrichment of biologically relevant information. When four sample datasets were evaluated with this method, literature mining confirmed that those pathways germane to the physiological process under investigation were highlighted by our method relative to z-score overrepresentation calculations. Moreover, non-relevant processes were downgraded using the method described herein. The inclusion of expression and topological data in the calculation of a pathway regulation score (PRS) facilitated discrimination of key processes in real biological datasets. Specifically, by combining fold-change data for those transcripts exceeding a significance threshold, and by taking into account the potential for altered gene expression to impact upon downstream transcription, one may readily identify those pathways most relevant to pathophysiological processes.

A MATLAB tool for pathway enrichment using a topology-based pathway regulation score

BackgroundHandling the vast amount of gene expression data generated by genome-wide transcriptional profiling techniques is a challenging task, demanding an informed combination of pre-processing, filtering and analysis methods if meaningful biological conclusions are to be drawn. For example, a range of traditional statistical and computational pathway analysis approaches have been used to identify over-represented processes in microarray data derived from various disease states. However, most of these approaches tend not to exploit the full spectrum of gene expression data, or the various relationships and dependencies. Previously, we described a pathway enrichment analysis tool created in MATLAB that yields a Pathway Regulation Score (PRS) by considering signalling pathway topology, and the overrepresentation and magnitude of differentially-expressed genes (J Comput Biol 19:563-573, 2012). Herein, we extended this approach to include metabolic pathways, and described the use of a graphical user interface (GUI).ResultsUsing input from a variety of microarray platforms and species, users are able to calculate PRS scores, along with a corresponding z-score for comparison. Further pathway significance assessment may be performed to increase confidence in the pathways obtained, and users can view Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway diagrams marked-up to highlight impacted genes.ConclusionsThe PRS tool provides a filter in the isolation of biologically-relevant insights from complex transcriptomic data.

Gene expression profiling of the ageing rat vibrissa follicle.

Background  The application of gene expression profiling to the study of chronological ageing has the potential to illuminate the molecular mechanisms underlying a complex and active process. For example, ageing of the skin and its constituent organs has myriad phenotypic consequences, and a better understanding of the means by which these changes arise has important corollaries for intervention strategies.

Stimulation of glucose uptake in murine soleus muscle and adipocytes by 5-(4-phenoxybutoxy)psoralen (PAP-1) may be mediated by Kv1.5 rather than Kv1.3

Kv1 channels are shaker-related potassium channels that influence insulin sensitivity. Kv1.3−/− mice are protected from diet-induced insulin resistance and some studies suggest that Kv1.3 inhibitors provide similar protection. However, it is unclear whether blockade of Kv1.3 in adipocytes or skeletal muscle increases glucose uptake. There is no evidence that the related channel Kv1.5 has any influence on insulin sensitivity and its expression in adipose tissue has not been reported. PAP-1 is a selective inhibitor of Kv1.3, with 23-fold, 32-fold and 125-fold lower potencies as an inhibitor of Kv1.5, Kv1.1 and Kv1.2 respectively. Soleus muscles from wild-type and genetically obese ob/ob mice were incubated with 2-deoxy[1-14C]-glucose for 45 min and formation of 2-deoxy[1-14C]-glucose-6-phosphate was measured. White adipocytes were incubated with D-[U-14C]-glucose for 1 h. TNFα and Il-6 secretion from white adipose tissue pieces were measured by enzyme-linked-immunoassay. In the absence of insulin, a high concentration (3 µM) of PAP-1 stimulated 2-deoxy[1-14C]-glucose uptake in soleus muscle of wild-type and obese mice by 30% and 40% respectively, and in adipocytes by 20% and 50% respectively. PAP-1 also stimulated glucose uptake by adipocytes at the lower concentration of 1 µM, but at 300 nM, which is still 150-fold higher than its EC50 value for inhibition of the Kv1.3 channel, it had no effect. In the presence of insulin, PAP-1 (3 µM) had a significant effect only in adipocytes from obese mice. PAP-1 (3 µM) reduced the secretion of TNFα by adipose tissue but had no effect on the secretion of IL-6. Expression of Kv1.1, Kv1.2, Kv1.3 and Kv1.5 was determined by RT-PCR. Kv1.3 and Kv1.5 mRNA were detected in liver, gastrocnemius muscle, soleus muscle and white adipose tissue from wild-type and ob/ob mice, except that Kv1.3 could not be detected in gastrocnemius muscle, nor Kv1.5 in liver, of wild-type mice. Expression of both genes was generally higher in liver and muscle of ob/ob mice compared to wild-type mice. Kv1.5 appeared to be expressed more highly than Kv1.3 in soleus muscle, adipose tissue and adipocytes of wild-type mice. Expression of Kv1.2 appeared to be similar to that of Kv1.3 in soleus muscle and adipose tissue, but Kv1.2 was undetectable in adipocytes. Kv1.1 could not be detected in soleus muscle, adipose tissue or adipocytes. We conclude that inhibition of Kv1 channels by PAP-1 stimulates glucose uptake by adipocytes and soleus muscle of wild-type and ob/ob mice, and reduces the secretion of TNFα by adipose tissue. However, these effects are more likely due to inhibition of Kv1.5 than to inhibition of Kv1.3 channels.

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.

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.