Toxicologic Pathology Forum: A Roadmap for Building State-of-the-Art Digital Image Data Resources for Toxicologic Pathology in the Pharmaceutical Industry.

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.

Safety, Tissue Distribution, and Metabolism of LNA-Containing Antisense Oligonucleotides in Rats.

Antisense oligonucleotides (ASOs) are chemically modified nucleic acids with therapeutic potential, some of which have been approved for marketing. We performed a study in rats to investigate mechanisms of toxicity after administration of 3 tool locked nucleic acid (LNA)-containing ASOs with differing established safety profiles. Four male rats per group were dosed once, 3, or 6 times subcutaneously, with 7 days between dosing, and sacrificed 3 days after the last dose. These ASOs were either unconjugated (naked) or conjugated with N-acetylgalactosamine for hepatocyte-targeted delivery. The main readouts were in-life monitoring, clinical and anatomic pathology, exposure assessment and metabolite identification in liver and kidney by liquid chromatography coupled to tandem mass spectrometry, ASO detection in liver and kidney by immunohistochemistry, in situ hybridization, immune electron microscopy, and matrix-assisted laser desorption/ionization mass spectrometry imaging. The highly toxic compounds showed the greatest amount of metabolites and a low degree of tissue accumulation. This study reveals different patterns of cell death associated with toxicity in liver (apoptosis and necrosis) and kidney (necrosis only) and provides new ultrastructural insights on the tissue accumulation of ASOs. We observed that the immunostimulatory properties of ASOs can be either primary from sequence-dependent properties or secondary to cell necrosis.

Nidovirus-Associated Proliferative Pneumonia in the Green Tree Python (Morelia viridis).

In 2014 we observed a noticeable increase in the number of sudden deaths among green tree pythons (Morelia viridis). Pathological examination revealed the accumulation of mucoid material within the airways and lungs in association with enlargement of the entire lung. We performed a full necropsy and histological examination on 12 affected green tree pythons from 7 different breeders to characterize the pathogenesis of this mucinous pneumonia. By histology we could show a marked hyperplasia of the airway epithelium and of faveolar type II pneumocytes. Since routine microbiological tests failed to identify a causative agent, we studied lung tissue samples from a few diseased snakes by next-generation sequencing (NGS). From the NGS data we could assemble a piece of RNA genome whose sequence was <85% identical to that of nidoviruses previously identified in ball pythons and Indian pythons. We then employed reverse transcription-PCR to demonstrate the presence of the novel nidovirus in all diseased snakes. To attempt virus isolation, we established primary cultures of Morelia viridis liver and brain cells, which we inoculated with homogenates of lung tissue from infected individuals. Ultrastructural examination of concentrated cell culture supernatants showed the presence of nidovirus particles, and subsequent NGS analysis yielded the full genome of the novel virus Morelia viridis nidovirus (MVNV). We then generated an antibody against MVNV nucleoprotein, which we used alongside RNA in situ hybridization to demonstrate viral antigen and RNA in the affected lungs. This suggests that in natural infection MVNV damages the respiratory tract epithelium, which then results in epithelial hyperplasia, most likely as an exaggerated regenerative attempt in association with increased epithelial turnover.IMPORTANCE Novel nidoviruses associated with severe respiratory disease were fairly recently identified in ball pythons and Indian pythons. Herein we report on the isolation and identification of a further nidovirus from green tree pythons (Morelia viridis) with fatal pneumonia. We thoroughly characterized the pathological changes in the infected individuals and show that nidovirus infection is associated with marked epithelial proliferation in the respiratory tract. We speculate that this and the associated excess mucus production can lead to the animals' death by inhibiting normal gas exchange in the lungs. The virus was predominantly detected in the respiratory tract, which renders transmission via the respiratory route likely. Nidoviruses cause sudden outbreaks with high rates of mortality in breeding collections, and most affected snakes die without prior clinical signs. These findings, together with those of other groups, indicate that nidoviruses are a likely cause of severe pneumonia in pythons.

Pathogenic mechanisms implicated in the intravascular coagulation in the lungs of BVDV-infected calves challenged with BHV-1.

Resistance to respiratory disease in cattle requires host defense mechanisms that protect against pathogens which have evolved sophisticated strategies to evade them, including an altered function of pulmonary macrophages (MΦs) or the induction of inflammatory responses that cause lung injury and sepsis. The aim of this study was to clarify the mechanisms responsible for vascular changes occurring in the lungs of calves infected with bovine viral diarrhea virus (BVDV) and challenged later with bovine herpesvirus type 1 (BHV-1), evaluating the role of MΦs in the development of pathological lesions in this organ. For this purpose, pulmonary lesions were compared between co-infected calves and healthy animals inoculated only with BHV-1 through immunohistochemical (MAC387, TNFα, IL-1α, iNOS, COX-2 and Factor-VIII) and ultrastructural studies. Both groups of calves presented important vascular alterations produced by fibrin microthrombi and platelet aggregations within the blood vessels. These findings were earlier and more severe in the co-infected group, indicating that the concomitance of BVDV and BHV-1 in the lungs disrupts the pulmonary homeostasis by facilitating the establishment of an inflammatory and procoagulant environment modulated by inflammatory mediators released by pulmonary MΦs. In this regard, the co-infected calves, in spite of presenting a greater number of IMΦs than single-infected group, show a significant decrease in iNOS expression coinciding with the presence of more coagulation lesions. Moreover, animals pre-inoculated with BVDV displayed an alteration in the response of pro-inflammatory cytokines (TNFα and IL-1), which play a key role in activating the immune response, as well as in the local cell-mediated response.

MMO-Net (Multi-Magnification Organ Network): A use case for Organ Identification using Multiple Magnifications in Preclinical Pathology Studies.

Identifying organs within histology images is a fundamental and non-trivial step in toxicological digital pathology workflows as multiple organs often appear on the same whole slide image (WSI). Previous works in automated tissue classification have investigated the use of single magnifications, and demonstrated limitations when attempting to identify small and contiguous organs at low magnifications. In order to overcome these shortcomings, we present a multi-magnification convolutional neural network (CNN), called MMO-Net, which employs context and cellular detail from different magnifications to facilitate the recognition of complex organs. Across N=320 WSI from 3 contract research organization (CRO) laboratories, we demonstrate state-of-the-art organ detection and segmentation performance of 7 rat organs with and without lesions: liver, kidney, thyroid gland, parathyroid gland, urinary bladder, salivary gland, and mandibular lymph node (AUROC=0.99-1.0 for all organs, Dice≥0.9 except parathyroid (0.73)). Evaluation takes place at both inter- and intra CRO levels, suggesting strong generalizability performance. Results are qualitatively reviewed using visualization masks to ensure separation of organs in close proximity (e.g., thyroid vs parathyroid glands). MMO-Net thus offers organ localization that serves as a potential quality control tool to validate WSI metadata and as a preprocessing step for subsequent organ-specific artificial intelligence (AI) use cases. To facilitate research in this area, all associated WSI and metadata used for this study are being made freely available, forming a first of its kind dataset for public use.

BVDV permissiveness and lack of expression of co-stimulatory molecules on PBMCs from calves pre-infected with BVDV.

Bovine viral diarrhea virus (BVDV) has been detected in peripheral blood mononuclear cells (PBMCs) of immunocompetent animals, not being clear whether the development of a specific humoral immune response can prevent BVDV infection. The aim of this study was to evaluate the ability of non-cytopathic BVDV to replicate and produce infectious virus in PBMCs from calves pre-infected with BVDV and to elucidate the immunomodulatory effect of BVDV on these cells in an in vitro model. Quantification of virus was by quantitative PCR, while its replicative capacity and shedding into the extracellular environment was evaluated by viral titration. Apoptosis was assessed by flow cytometry analysis of annexin V and propidium iodide, and by expression of caspase-3/7. Flow cytometry was used to analyze the expression of CD14/CD11b/CD80, CD4/CD8/CD25, MHC-I/MHC-II and B-B2 markers. Our results showed that PBMCs from cattle naturally infected with BVDV were more susceptible to in vitro BVDV infection and showed a more severe apoptosis response than those from naïve animals. Non-cytopathic BVDV in vitro infection also resulted in a lack of effect in the expression of antigen presentation surface markers. All these findings could be related to the immunosuppressive capacity of BVDV and the susceptibility of cattle to this infection.

Persistence of hepatitis E virus in the liver of non-viremic naturally infected wild boar.

Hepatitis E virus (HEV) is an emerging zoonotic pathogen with pigs and wild boar serving as reservoirs for human infection through direct contact with infected animals or the consumption of raw or undercooked pork products. The liver is considered the main target site of HEV replication in swine and an important organ in the pathogenesis of the disease. The aim of this study was to characterize the target liver cells for HEV entry in naturally infected wild boar and to evaluate the type and severity of the pathological changes in order to reach a better understanding of the hepatic pathogenic mechanisms involved in hepatitis E. In total, 58 livers from hunted wild boar were histopathologically evaluated. The presence of specific HEV antibodies in serum was determined by indirect ELISA. Immunohistochemistry was used for the detection of HEV antigen and Real time RT-PCR to detect HEV RNA in liver and serum. HEV seroprevalence in these animals was of 5.197% (CI95%: 1.77-14.14). By Real time RT-PCR, HEV was detected in the liver tissue of four wild boar (6.8%; CI95%: 2.7-16.4) and only one animal was also positive in serum (1.7%; CI95%: 0.3-9.1). The non-viremic animals naturally infected with HEV presented evidence of liver infection, mainly in Kupffer cells and liver sinusoidal endothelial cells, without apparent associated hepatitis lesions. This study supports the hypothesis that low viral titers may persist in the liver of non-viremic individuals, giving thus the possibility of consumption of contaminated liver of animals diagnosed as HEV-negative in serum. Further immunopathogenic studies are necessary to elucidate the mechanisms responsible for this process and to evaluate the protocols of HEV diagnosis in animals destined for human consumption.

Comparison of pathological changes and viral antigen distribution in tissues of calves with and without preexisting bovine viral diarrhea virus infection following challenge with bovine herpesvirus-1.

OBJECTIVE: To compare pathological changes and viral antigen distribution in tissues of calves with and without preexisting subclinical bovine viral diarrhea virus (BVDV) infection following challenge with bovine herpesvirus-1 (BHV-1). ANIMALS: 24 Friesian calves. PROCEDURES: 12 calves were inoculated intranasally with noncytopathic BVDV-1a; 12 days later, 10 of these calves were challenged intranasally with BHV-1 subtype 1. Two calves were euthanized before and 1, 2, 4, 7, or 14 days after BHV-1 inoculation. Another 10 calves were inoculated intranasally with BHV-1 only and euthanized 1, 2, 4, 7, or 14 days later. Two calves were inoculated intranasally with virus-free tissue culture fluid and euthanized as negative controls. Pathological changes and viral antigen distribution in various tissue samples from calves with and without BVDV infection (all of which had been experimentally inoculated with BHV-1) were compared. RESULTS: Following BHV-1 challenge, calves with preexisting subclinical BVDV infection had earlier development of more severe inflammatory processes and, consequently, more severe tissue lesions (limited to lymphoid tissues and respiratory and digestive tracts) and greater dissemination of BHV-1, compared with calves without preexisting BVDV infection. Moreover, coinfected calves had an intense lymphoid depletion in the Peyer patches of the ileum as well as the persistence of BVDV in target organs and the reappearance of digestive tract changes during disease progression. CONCLUSIONS AND CLINICAL RELEVANCE: In calves, preexisting infection with BVDV facilitated the establishment of BHV-1 infection, just as the presence of BHV-1 favors BVDV persistence, thereby synergistically potentiating effects of both viruses and increasing the severity of the resultant clinical signs.