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

Galectin-3 promotes lamellipodia formation in epithelial cells by interacting with complex N-glycans on α3β1 integrin

Recent studies have shown that galectin-3 (Gal-3; also known as LGALS3), a β-galactoside-binding lectin, promotes cell migration during re-epithelialization of corneal wounds. The goal of this study was to characterize the molecular mechanism by which Gal-3 stimulates cell migration. We demonstrate here that exogenous Gal-3, but not Gal-1 or Gal-8, promotes cell scattering and formation of lamellipodia in human corneal epithelial cells in a β-lactose-inhibitable manner. α3β1 integrin was identified as the major Gal-3-binding protein in corneal epithelial cells by affinity chromatography of cell lysates on a Gal-3-Sepharose column. Preincubation of cells with anti-α3 integrin function-blocking antibody significantly inhibited the induction of lamellipodia by Gal-3. Furthermore, exogenous Gal-3 activated both focal adhesion kinase, a key regulator of integrin-dependent intracellular signaling, and Rac1 GTPase, a member of the family of Rho GTPases, well known for its role in the reorganization of the actin cytoskeleton and formation of lamellipodial extensions. Experiments involving knockdown of β-1,6-N-acetylglucosaminytransferase V, an enzyme that synthesizes high-affinity glycan ligands for Gal-3, revealed that carbohydrate-mediated interaction between Gal-3 and complex N-glycans on α3β1 integrin plays a key role in Gal-3-induced lamellipodia formation. We propose that Gal-3 promotes epithelial cell migration by cross-linking MGAT5-modified complex N-glycans on α3β1 integrin and subsequently activating α3β1-integrin–Rac1 signaling to promote lamellipodia formation

Research Relevant Conditions and Pathology in Nonhuman Primates

Biomedical research involving animal models continues to provide important insights into disease pathogenesis and treatment of diseases that impact human health. In particular, nonhuman primates (NHPs) have been used extensively in translational research due to their phylogenetic proximity to humans and similarities to disease pathogenesis and treatment responses as assessed in clinical trials. Microscopic changes in tissues remain a significant endpoint in studies involving these models. Spontaneous, expected (ie, incidental or background) histopathologic changes are commonly encountered and influenced by species, genetic variations, age, and geographical origin of animals, including exposure to infectious or parasitic agents. Often, the background findings confound study-related changes, because numbers of NHPs used in research are limited by animal welfare and other considerations. Moreover, background findings in NHPs can be exacerbated by experimental conditions such as treatment with xenobiotics (eg, infectious morphological changes related to immunosuppressive therapy). This review and summary of research-relevant conditions and pathology in rhesus and cynomolgus macaques, baboons, African green monkeys, common marmosets, tamarins, and squirrel and owl monkeys aims to improve the interpretation and validity of NHP studies

Characterization of intra-articular dose administration in the stifle joint of rabbits for toxicity testing

Intra-articular administration is a standard therapeutic option for patients with rheumatoid arthritis, and pharmaceutical companies are increasing the research and development of injectable treatments. Therefore, there is an unmet need for reliable dosing models in toxicity safety testing. The rabbit stifle joint is attractive for toxicology testing of injectable treatments for rheumatoid arthritis because it is the largest synovial joint in the animal's body and is equivalent in structure and function to the human knee. Intra-articular dosing in rabbits is one model currently being used in general toxicology studies to assess the safety of injectable compounds. In this study, intra-articular dose administration was characterized in the stifle joints of New Zealand white rabbits. An anesthesia regimen was developed for the intra-articular injection procedure. Volumes of ≤400 =L were successfully injected into the stifle joint of rabbits. Injection success was confirmed by visual inspection of contrasting agent within the articular space, with minimal leakage and no rupturing of the synovial membrane observed. A post-procedural analgesia regimen was developed to alleviate pain and discomfort. Bilateral and multiple injections were also explored and determined feasible. In addition, successful synovial fluid recovery without flushing was demonstrated. Histopathology of the stifle joint demonstrated that the injection procedure caused minimal inflammation or effects on the stifle joint or articular cartilage. In summary, intra-articular dose administration by injection to the stifle joint(s) of rabbits is a successful procedure for use in preclinical safety evaluation

Letter to the Editor Regarding “Pathology Informatics Education Committee of the American College of Veterinary Pathologists (ACVP)”

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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

HistoNet: A Deep Learning-Based Model of Normal Histology

We introduce HistoNet, a deep neural network trained on normal tissue. On 1690 slides with rat tissue samples from 6 preclinical toxicology studies, tissue regions were outlined and annotated by pathologists into 46 different tissue classes. From these annotated regions, we sampled small 224 × 224 pixels images (patches) at 6 different levels of magnification. Using 4 studies as training set and 2 studies as test set, we trained VGG-16, ResNet-50, and Inception-v3 networks separately at each magnification level. Among these model architectures, Inception-v3 and ResNet-50 outperformed VGG-16. Inception-v3 identified the tissue from query images, with an accuracy up to 83.4%. Most misclassifications occurred between histologically similar tissues. Investigation of the features learned by the model (embedding layer) using Uniform Manifold Approximation and Projection revealed not only coherent clusters associated with the individual tissues but also subclusters corresponding to histologically meaningful structures that had not been annotated or trained for. This suggests that the histological representation learned by HistoNet could be useful as the basis of other machine learning algorithms and data mining. Finally, we found that models trained on rat tissues can be used on non-human primate and minipig tissues with minimal retraining

Society of Toxicologic Pathology Digital Pathology and Image Analysis Special Interest Group Article*: Opinion on the Application of Artificial Intelligence and Machine Learning to Digital Toxicologic Pathology

Toxicologic pathology is transitioning from analog to digital methods. This transition seems inevitable due to a host of ongoing social and medical technological forces. Of these, artificial intelligence (AI) and in particular machine learning (ML) are globally disruptive, rapidly growing sectors of technology whose impact on the long-established field of histopathology is quickly being realized. The development of increasing numbers of algorithms, peering ever deeper into the histopathological space, has demonstrated to the scientific community that AI pathology platforms are now poised to truly impact the future of precision and personalized medicine. However, as with all great technological advances, there are implementation and adoption challenges. This review aims to define common and relevant AI and ML terminology, describe data generation and interpretation, outline current and potential future business cases, discuss validation and regulatory hurdles, and most importantly, propose how overcoming the challenges of this burgeoning technology may shape toxicologic pathology for years to come, enabling pathologists to contribute even more effectively to answering scientific questions and solving global health issues.*This article is a product of a Special Interest Group of the Society of Toxicologic Pathology (STP). The views expressed in this article are those of the authors and do not necessarily represent the policies, positions, or opinions of the STP

sj-docx-1-spp-10.1177_01926233231178282 – Supplemental material for European Society of Toxicologic Pathology (Pathology 2.0 Molecular Pathology Special Interest Group): Review of In Situ Hybridization Techniques for Drug Research and Development

Supplemental material, sj-docx-1-spp-10.1177_01926233231178282 for European Society of Toxicologic Pathology (Pathology 2.0 Molecular Pathology Special Interest Group): Review of In Situ Hybridization Techniques for Drug Research and Development by Josep M. Monné Rodríguez, Anna-Lena Frisk, Robert Kreutzer, Thomas Lemarchand, Stephane Lezmi, Chandrassegar Saravanan, Birgit Stierstorfer, Céline Thuilliez, Enrico Vezzali, Grazyna Wieczorek, Seong-Wook Yun and Dirk Schaudien in Toxicologic Pathology</p

sj-tif-5-tpx-10.1177_01926233231182115 – Supplemental material for Results of the European Society of Toxicologic Pathology Survey on the Use of Artificial Intelligence in Toxicologic Pathology

Supplemental material, sj-tif-5-tpx-10.1177_01926233231182115 for Results of the European Society of Toxicologic Pathology Survey on the Use of Artificial Intelligence in Toxicologic Pathology by Xavier Palazzi, Erio Barale-Thomas, Bhupinder Bawa, Jonathan Carter, Kyathanahalli Janardhan, Heike Marxfeld, Abraham Nyska, Chandrassegar Saravanan, Dirk Schaudien, Vanessa L. Schumacher, Robert H. Spaet, Simone Tangermann, Oliver C Turner and Enrico Vezzali in Toxicologic Pathology</p