European Society of Toxicologic Pathology (Pathology 2.0 Molecular Pathology Special Interest Group): Review of In Situ Hybridization Techniques for Drug Research and Development

In situ hybridization (ISH) is used for the localization of specific nucleic acid sequences in cells or tissues by complementary binding of a nucleotide probe to a specific target nucleic acid sequence. In the last years, the specificity and sensitivity of ISH assays were improved by innovative techniques like synthetic nucleic acids and tandem oligonucleotide probes combined with signal amplification methods like branched DNA, hybridization chain reaction and tyramide signal amplification. These improvements increased the application spectrum for ISH on formalin-fixed paraffin-embedded tissues. ISH is a powerful tool to investigate DNA, mRNA transcripts, regulatory noncoding RNA, and therapeutic oligonucleotides. ISH can be used to obtain spatial information of a cell type, subcellular localization, or expression levels of targets. Since immunohistochemistry and ISH share similar workflows, their combination can address simultaneous transcriptomics and proteomics questions. The goal of this review paper is to revisit the current state of the scientific approaches in ISH and its application in drug research and development

Orbit Image Analysis: An open-source whole slide image analysis tool.

We describe Orbit Image Analysis, an open-source whole slide image analysis tool. The tool consists of a generic tile-processing engine which allows the execution of various image analysis algorithms provided by either Orbit itself or from other open-source platforms using a tile-based map-reduce execution framework. Orbit Image Analysis is capable of sophisticated whole slide imaging analyses due to several key features. First, Orbit has machine-learning capabilities. This deep learning segmentation can be integrated with complex object detection for analysis of intricate tissues. In addition, Orbit can run locally as standalone or connect to the open-source image server OMERO. Another important characteristic is its scale-out functionality, using the Apache Spark framework for distributed computing. In this paper, we describe the use of Orbit in three different real-world applications: quantification of idiopathic lung fibrosis, nerve fibre density quantification, and glomeruli detection in the kidney

Gastrointestinal round cell tumours in cats and dogs: a retrospective and comparative analysis.

Introduction: Alimentary lymphoma (AL) in cats is the most common type of gastrointestinal (GI) neoplasm and also the most frequent form of lymphoma. On the contrary, in dogs primary GI tumours are mostly epithelial in origin, and AL accounts for approximately only 5-7% of all canine lymphomas. Other primary GI round cell tumours are rare in both species, almost exclusively represented by plasma cell and mast cell tumours (in dogs and cats, respectively). The purpose of this study was to classify round cell tumours of the GI tract in dogs and cats, to determine their immunophenotype and to compare the results between the two species. Materials and Methods: Haematoxylin and eosin stained sections from formalin fixed paraffin embedded samples of GI tumours archived in our Department from 12 year period (1992-2004) have been reviewed, and round cell tumours extrapolated. In the selected 82 cases (68 cats, 18 dogs), CD3 and CD79 immunohistochemistry was performed, and these were classified histologically according to the recent WHO classification system for the hematopoietic tumours. Results: In our series of 109 feline GI neoplasms, round cell tumours accounted for 62.3% (30.4% epithelial, 7.3% mesenchymal tumours), and mainly consisted of AL (65/68, 96%) located in the intestine in 59 out of 65 cases. The only other primary non-lymphoid neoplasms were two malignant histiocytomas and one mast cell tumour. In the 96 canine GI neoplasms, round cell tumours accounted for 19% (59.3% epithelial, 21.9% mesenchymal tumours), all represented by AL (17 out of 18 cases located in the intestine). Most of feline GI lymphomas showed a CD3+ T cell phenotype, while a clear prevalence was not observed in dogs. In both dogs and cats, T cell lymphomas were mainly represented by intestinal T cell lymphoma, although some cases of peripheral T cell lymphoma and angiotropic lymphoma were also present. As regard the B cell lymphomas, B large cell lymphomas (diffuse large cell lymphomas and large cell immunoblastic lymphoma) were the most frequent in dogs and cats, followed by follicular (centre cell and MALT lymphoma). Conclusion: Our results showed in the feline GI neoplasms the uppermost prevalence of round cell tumours, whereas in the dog epithelial tumours were most common. Within round cell tumours, almost all were GI lymphomas in both species. The absence of prevalence of T out of B cell lymphomas in the dog and the marked prevalence of T cell lymphomas in the cat observed in our study are not aligned with the most recent literature data. These preliminary results lay the basis to further studies including a wider number of cases

Effects of feeding free or microencapsulated gluconic acid on growth performance of weanling pigs

Organic acids belong to the supplements that can be used as an alternative to antibiotics fed to farm animals as growth promoters. Gluconic acid (GA) has been shown to reach the large intestine in rats where it can be fermented by the microflora (1). In piglets, when GA was fed at 0.3 and 0.6%, average daily gain of the animals was improved (2). Aim of this study was the evaluation of the effect of feeding GA in its free form or microencapsulated on piglet growth and intestinal microflora. Methods: Immediately after weaning, 48 piglets were divided into 3 groups (16 animals per group, housed in individual cages) for a 32 d trial. Treatments were a commercial diet with a) no addition (control diet) or with b) 0.3% of microencapsulated GA (MGA), and c) 0.3% of free GA (FGA). Feed and water were provided ad libitum. Animals were weighed every week and feed consumption was recorded. At the end of the trial, 8 animals per group were killed. Samples of jejunum and caecum content were cultured for viable bacteria (coliforms, clostridia, enterococci, and lactobacilli) and the content and the mucosa from the middle section of the jejunum and from ileum and cecum were sampled for pH, ammonia and SCFA determination, and for intestinal mucosa morphology analysis. Results: Feeding GA did not influence live weight, average daily gain (ADG), and daily feed intake of piglets. Feed to gain ratio (FG) was improved by GA between Day 14 and 21 (1.68, 1.44, and 1.34 for control, MGA, and FGA, respectively; P < 0.05) but differences were not significant in the period 0-32 d (Table 1). Intestinal counts of viable bacteria were not significantly influenced by treatment. Nevertheless, caecal clostridia showed a tendency towards a reduction when GA was fed (6.9, 6.4, and 6.2 log10 CFU/g for control, MGA, and FGA, respectively). Ammonia concentration in the caecum was higher when MGA was fed (P < 0.01). Compared with control, feeding MGA reduced the concentration of iso-butyric acid in jejunum and ileum (P < 0.10) but no other differences in SCFA intestinal concentrations were observed. Animals receiving the FGA diet had longer ileal villi (+ 18%; P < 0.10) and shorter caecal crypts ( 17%; P < 0.05) than control animals. Conclusion: Despite the fact that during a previous study GA had improved piglets growth performances (2), both the free and the microencapsulated form of GA failed to enhance animal growth. Further studies will be needed to achieve a deeper understanding of the effect that GA has on growth and intestinal ecology and morphology of piglets. 1) ASANO, T, YUASA, K, YOSHIMURA, Y, TAKENAWA, S and FUKUBA, H (1997): J. Jpn. Soc. Nutr. Food Sci. 50, 287-294. 2) BIAGI, G., PIVA, A., MOSCHINI, M., VEZZALI, E., and ROTH, F. X. (2005): J. Anim. Sci. Accepted for publication

Ectopic expression of PLC-β2 in non-invasive breast tumor cells plays a protective role against malignant progression and is correlated with the deregulation of miR-146a

Cells in non-invasive breast lesions are widely believed to possess molecular alterations that render them either susceptible or refractory to the acquisition of invasive capability. One such alteration could be the ectopic expression of the β2 isoform of phosphoinositide-dependent phospholipase C (PLC-β2), known to counteract the effects of hypoxia in low-invasive breast tumor-derived cells. Here, we studied the correlation between PLC-β2 levels and the propensity of non-invasive breast tumor cells to acquire malignant features. Using archival FFPE samples and DCIS-derived cells, we demonstrate that PLC-β2 is up-regulated in DCIS and that its forced down-modulation induces an epithelial-to-mesenchymal shift, expression of the cancer stem cell marker CD133, and the acquisition of invasive properties. The ectopic expression of PLC-β2 in non-transformed and DCIS-derived cells is, to some extent, dependent on the de-regulation of miR-146a, a tumor suppressor miRNA in invasive breast cancer. Interestingly, an inverse relationship between the two molecules, indicative of a role of miR-146a in targeting PLC-β2, was not detected in primary DCIS from patients who developed a second invasive breast neoplasia. This suggests that alterations of the PLC-β2/miR-146a relationship in DCIS may constitute a molecular risk factor for the appearance of new breast lesions. Since neither traditional classification systems nor molecular characterizations are able to predict the malignant potential of DCIS, as is possible for invasive ductal carcinoma (IDC), we propose that the assessment of the PLC-β2/miR-146a levels at diagnosis could be beneficial for identifying whether DCIS patients may have either a low or high propensity for invasive recurrence

A fully automated image analysis method to quantify lung fibrosis in the bleomycin-induced rat model

<div><p>Intratracheal administration of bleomycin induces fibrosis in the lung, which is mainly assessed by histopathological grading that is subjective. Current literature highlights the need of reproducible and quantitative pulmonary fibrosis analysis. If some quantitative studies looked at fibrosis parameters separately, none of them quantitatively assessed both aspects: lung tissue remodeling and collagenization. To ensure reliable quantification, support vector machine learning was used on digitalized images to design a fully automated method that analyzes two important aspects of lung fibrosis: (i) areas having substantial tissue remodeling with appearance of dense fibrotic masses and (ii) collagen deposition. Fibrotic masses were identified on low magnification images and collagen detection was performed at high magnification. To insure a fully automated application the tissue classifier was trained on several independent studies that were performed over a period of four years. The detection method generates two different values that can be used to quantify lung fibrosis development: (i) percent area of fibrotic masses and (ii) percent of alveolar collagen. These two parameters were validated using independent studies from bleomycin- and saline-treated animals. A significant change of these lung fibrosis quantification parameters- increased amount of fibrotic masses and increased collagen deposition- were observed upon intratracheal administration of bleomycin and subsequent significant beneficial treatments effects were observed with BIBF-1120 and pirfenidone.</p></div

Fibrosis features used for morphometric detection.

<p>Digitalized slides of Masson’s tri-chrome stained lung paraffin sections. Collagen is stained in blue and cell nuclei in red. <b>A</b>: alveolar thickening by collagen deposition. <b>B</b>: fibrotic masses composed of collagen, fibroblasts, and other components.</p

Quantitative analysis of anti-fibrotic compound effects.

<p>Lungs from Wistar rats treated with bleomycin in combination with pirfenidone (0,50%) and BIBF-1120 (50mg/kg) were collected at Day 14 and a Masson’s trichrome staining was performed for fibrosis parameter assessment using automated image analysis. <b>A</b>: percentage of fibrotic masses, <b>B</b>: percentage of alveolar collagen. Mean with +/- SEM, Mann-Whitney statistical test. Each dot represents an individual animal and color represents independent studies. Study 2 in red, and study 6 in blue.</p

Fibrosis feature detection.

<p>Masson’s trichrome stained lung sections from study 3, Bleomycin (A, C, E, F) and Saline (B, D, F, H) animals, <b>A and B</b> at low magnification, scale bar 2.5mm <b>E and F</b> alveolar structure at high magnification, scale bar 50μm. Upper right square: zoom in one alveola. <b>C and D</b>: low scale detection model; false colour image indicates areas of dense fibrosis (green), alveolar tissue (blue), bronchi (red), background (yellow). <b>G and H:</b> high magnification detection; false colour image indicates collagen (blue), lung tissue (red), background (yellow). Upper right square: zoom in one alveola collagen detection.</p

Cenerimod, a selective S1P receptor modulator, improves organ-specific disease outcomes in animal models of Sjögren's syndrome.

BACKGROUND Sjögren's syndrome is a systemic autoimmune disease characterized by immune cells predominantly infiltrating the exocrine glands and frequently forming ectopic lymphoid structures. These structures drive a local functional immune response culminating in autoantibody production and tissue damage, associated with severe dryness of mucosal surfaces and salivary gland hypofunction. Cenerimod, a potent, selective and orally active sphingosine-1-phosphate receptor 1 modulator, inhibits the egress of lymphocytes into the circulation. Based on the mechanism of action of cenerimod, its efficacy was evaluated in two mouse models of Sjögren's syndrome. METHODS Cenerimod was administered in two established models of Sjögren's syndrome; firstly, in an inducible acute viral sialadenitis model in C57BL/6 mice, and, secondly, in the spontaneous chronic sialadenitis MRL/lpr mouse model. The effects of cenerimod treatment were then evaluated by flow cytometry, immunohistochemistry, histopathology and immunoassays. Comparisons between groups were made using a Mann-Whitney test. RESULTS In the viral sialadenitis model, cenerimod treatment reduced salivary gland immune infiltrates, leading to the disaggregation of ectopic lymphoid structures, reduced salivary gland inflammation and preserved organ function. In the MRL/lpr mouse model, cenerimod treatment decreased salivary gland inflammation and reduced T cells and proliferating plasma cells within salivary gland ectopic lymphoid structures, resulting in diminished disease-relevant autoantibodies within the salivary glands. CONCLUSIONS Taken together, these results suggest that cenerimod can reduce the overall autoimmune response and improve clinical parameters in the salivary glands in models of Sjögren's syndrome and consequently may reduce histological and clinical parameters associated with the disease in patients