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Digitization of histologic slides brings with it the promise of enhanced toxicologic pathology practice through the increased application of computational methods. However, the development of these advanced methods requires access to substrate image data, that is, whole slide images (WSIs). Deep learning methods, in particular, rely on extensive training data to develop robust algorithms. As a result, pharmaceutical companies interested in leveraging computational methods in their digital pathology workflows must first invest in data infrastructure to enable data access for both data scientists and pathologists. The process of building robust image data resources is challenging and includes considerations of generation, curation, and storage of WSI files, and WSI access including via linked metadata. This opinion piece describes the collective experience of building resources for WSI data in the Roche group. We elaborate on the challenges encountered and solutions developed with the goal of providing examples of how to build a data resource for digital pathology analytics in the pharmaceutical industry.

To ensure reliable quantification of rat lung fibrosis, 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.

Aims Eosinophilic oesophagitis (EoE) is a chronic inflammatory disease characterised clinically by symptoms of oesophageal dysfunction and histopathologically by a prominent eosinophilic inflammation. Despite eosinophils having a histologically predominant position, their role in the immunopathogenesis of the disease is still questionable. Several other inflammatory cells are involved and may also play a critical role. The purpose of this study was to characterise the mast cell infiltration, and to correlate it with the clinical state of EoE. Methods and results Using immunohistochemistry and quantitative morphometry, we investigated eosinophils and mast cells extensively in oesophageal biopsies from patients with active EoE and from patients with EoE in remission, and compared the findings with healthy individuals. In EoE, epithelium and lamina propria were similarly infiltrated with eosinophils. In contrast, mast cells infiltration was limited to the epithelium, displaying a localised immune response. Interestingly, whereas epithelial mast cells and eosinophils were high in active EoE, some patients in remission, e.g. normalised epithelial eosinophils, showed remaining high numbers of mast cells. Patient clustering supported two groups of patients in clinical remission, differentiating based on presence or absence of epithelial mast cells. Conclusions Active EoE is characterised in addition to the well-known tissue eosinophilia by a marked epithelium-restricted mast cell infiltration. Of interest, in a subgroup of patients, mast cell infiltration persisted despite clinical remission. To elucidate the clinical consequence of persistent epithelial mast cells infiltration further studies are required following patients in clinical remission longitudinally.

Automated fibrosis analysis using Orbit module, is based on this modified masson trichrome staining protocol

To ensure reliable quantification of rat lung fibrosis, 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.

One hallmark of the rapid expansion of the polar surface of fungal hyphae is the spatial separation of regions of exocytosis and endocytosis at hyphal tips, as recently shown for Ashbya gossypii and Aspergillus nidulans. To determine where cortex-associated eisosomes form with respect to these two regions, we monitored fluorescently marked eisosomes in A. gossypii. Each minute, 1.6±0.5 eisosomes form within the first 30 μm of each hypha and are exclusively subapical of the endocytosis region. This spatial separation of the processes of eisosome formation and endocytosis, and the much lower frequency of eisosome formation compared with that of endocytic vesicle production do not support a recently proposed role for eisosomes in endocytosis. Levels of mRNA encoding eisosome components are tenfold higher in spores than in hyphae, explaining the observed higher eisosome density at the cortex of germ bubbles. As in Saccharomyces cerevisiae, eisosomes in A. gossypii are very stable. In contrast to S. cerevisiae, however, the A. gossypii homologue of Pil1, one of the main eisosome subunits, is very important for polar growth, whereas the homologue of Nce102, which colocalizes with eisosomes, is not needed for eisosome stability. By testing partial deletions of the A. gossypii homologue of Ymr086w, another component of the eisosome, we identified a novel protein domain essential for eisosome stability. We also compare our results with recent findings about eisosomes in A. nidulans.

Aims Evaluation of intraepidermal nerve fibres (IENFs) in skin biopsies is used in the diagnosis of small-fibre neuropathies. The number of IENFs is assessed manually under a microscope, with an inter-rater variability of ~25%. Unless the images are digitized, there is no documentation. Our aim was to develop a method for standardized semi-automated quantification (SAQ) and documentation of IENF density. Methods and results We analysed samples from four different university centres that were immunostained according to local protocols. Images were acquired through the Z-plane with a whole slide scanner. orbit image analysis software was used to create an analysable image and develop a reliable algorithm for IENF detection. Rebuilt images revealed well-contrasted nerves, allowing detection of IENFs (automated). The software presented the detected nerves for confirmation by the operator (manual). As compared with the conventional microscopy count, the SAQ achieved correlation coefficients of 0.99 and 0.96 and interfacility variabilities of 19% and 23%, respectively. We found better reproducibility with fluorescence-stained specimens than with bright-field images. Conclusions The new semi-automated method has high experimenter-independent reproducibility when based on nerve detection by fluorescence and is easy to perform, even by untrained users. The IENF counting is electronically well documented.

During filamentous fungus development, multinucleated hyphae employ a system for long-range nuclear migration to maintain an equal nuclear density. A decade ago the microtubule motor dynein was shown to play a central role in this process. Previous studies with Ashbya gossypii revealed extensive bidirectional movements and bypassings of nuclei, an autonomous cytoplasmic microtubule (cMT) cytoskeleton emanating from each nucleus, and pulling of nuclei by sliding of cMTs along the cortex. Here, we show that dynein is the sole motor for bidirectional movements and bypassing because these movements are concomitantly decreased in mutants carrying truncations of the dynein heavy-chain DYN1 promoter. The dynactin component Jnm1, the accessory proteins Dyn2 and Ndl1, and the potential dynein cortical anchor Num1 are also involved in the dynamic distribution of nuclei. In their absence, nuclei aggregate to different degrees, whereby the mutants with dense nuclear clusters grow extremely long cMTs. As in budding yeast, we found that dynein is delivered to cMT plus ends, and its activity or processivity is probably controlled by dynactin and Num1. Together with its role in powering nuclear movements, we propose that dynein also plays (directly or indirectly) a role in the control of cMT length. Those combined dynein actions prevent nuclear clustering in A. gossypii and thus reveal a novel cellular role for dynein.

The accumulation of aggregated mutant huntingtin (mHtt) inclusion bodies is involved in Huntigton's disease (HD) progression. Medium sized-spiny neurons (MSNs) in the corpus striatum are highly vulnerable to mHtt aggregate accumulation and degeneration, but the mechanisms and pathways involved remain elusive. Here we have developed a new model to study MSNs degeneration in the context of HD. We produced organotypic cortico-striatal slice cultures (CStS) from HD transgenic mice mimicking specific features of HD progression. We then show that induction of autophagy using catalytic inhibitors of mTOR prevents MSNs degeneration in HD CStS. Furthermore, disrupting autophagic flux by overexpressing Atg4b in neurons and slice cultures, accelerated mHtt aggregation and neuronal death, suggesting that Atg4b-dependent autophagic flux influences HD progression. Under these circumstances induction of autophagy using catalytic inhibitors of mTOR was inefficient and did not affect mHtt aggregate accumulation and toxicity, indicating that mTOR inhibition alleviates HD progression by inducing Atg4b-dependent autophagic flux. These results establish modulators of Atg4b-dependent autophagic flux as new potential targets in the treatment of HD.