Toxicologic Pathology最新文献

Mass Spectrometry Imaging (MSI) is a powerful tool to understand molecular pathophysiology and therapeutic and toxicity mechanisms, as well as for patient stratification and precision medicine. MSI, a label-free technique offering detailed spatial information on a large number of molecules in different tissues, encompasses various techniques including Matrix-Assisted Laser Desorption Ionization (MALDI), Desorption Electrospray Ionization (DESI), and Secondary Ion Mass Spectrometry (SIMS) that can be applied in diagnostic and toxicologic pathology. Given the utmost importance of high-quality samples, pathologists play a pivotal role in providing comprehensive pathobiology and histopathology knowledge, as well as information on tissue sampling, orientation, morphology, endogenous biomarkers, and pathogenesis, which are crucial for the correct interpretation of targeted experiments. This article introduces MSI and its fundamentals, and reports on case examples, determining the best suited technology to address research questions. High-level principles and characteristics of the most used modalities for spatial metabolomics, lipidomics and proteomics, sensitivity and specific requirements for sample procurement and preparation are discussed. MSI applications for projects focused on drug metabolism, nonclinical safety assessment, and pharmacokinetics/pharmacodynamics and various diagnostic pathology cases from nonclinical and clinical settings are showcased.

The safety of a 2'-O-methoxyethyl antisense oligonucleotide (ASO) was investigated in Mauritius cynomolgus monkeys in a 41-week Good Laboratory Practice (GLP) toxicity study after multiple intrathecal (IT) administrations. Histopathological examination revealed ectopic formation of lymphoid follicles in the spinal cord (SC) at the injection site at all doses and the presence of granular material in neurons of the SC in high-dose animals. The granular material was seen in all the segments of the SC, but mainly in the lumbar segment and persisted at the end of the 26-week recovery period, while the lymphoid follicles showed a reversibility trend. Findings associated with repeated IT administration of ASOs have been described in nonhuman primate (NHP) toxicity studies, specifically in the brain, but findings in the SC are rarely reported. In the present study, we report a high incidence of findings in the SC compared to brain, especially in the lumbar segment in proximity to IT injection sites. An extensive panel of immunohistochemistry markers showed that the ectopic lymphoid follicle formation (LFF) had a cellular composition and organization consistent with tertiary lymphoid structure (TLS) without associated axonal damage in the adjacent nervous tissue. In situ hybridization with an miRNA probe complementary to the ASO revealed that the granular material represented a dose-dependent ASO accumulation in the cytoplasm of neurons without inducing cell death or apoptosis. Glial and ependymal cells in the SC also showed dose-dependent accumulation of the ASO preceding detection of granular material by hematoxylin and eosin (H&E). Based on these molecular localization data, the presence of LFF in SC suggests a chronic local immune activation. Considering the absence of neuronal dysfunction or injury and transient clinical signs previously reported with other 2'-MOE ASOs, the presence of TLS and ASO was considered non-adverse.

Nonhuman primates (NHPs) have been and remain a highly valuable animal model with an essential role in translational research and pharmaceutical drug development. Based on current regulatory guidelines, the nonclinical safety of novel therapeutics should be evaluated in relevant nonclinical species, which commonly includes NHPs for biotherapeutics. Given the practical and ethical limitations on availability and/or use of NHPs and in line with the widely accepted guiding "3Rs" (replace, reduce, and refine) principles, many approaches have been considered to optimize toxicity study designs to meaningfully reduce the number of NHPs used. Standard general toxicity studies usually include four groups of equal size, including one group of vehicle control animals. Here, we describe an approach to achieve an overall significant reduction in control animal use, while also resolving many of the issues that may limit application of fully virtual control animals. We propose in Good Laboratory Practice (GLP)-compliant toxicity studies to maintain concurrent control group animals for the in-life phase of the studies, but to limit euthanasia to a subset of control animals. The nonterminated control animals can then be returned to the facility colony for reuse in subsequent studies. The proposed study design could lead to a 15% to 20% reduction in NHP usage. The scientific, logistical, and animal welfare considerations associated with such an approach and suggested solutions are discussed in detail.

Off-target evaluation is essential in preclinical safety assessments of novel biotherapeutics, supporting lead molecule selection, endpoint selection in toxicology studies, and regulatory requirements for first-in-human trials. Off-target interaction of a therapeutic antibody and antibody derivatives has been historically assessed via the Tissue Cross-Reactivity (TCR) study, in which the candidate molecule is used as a reagent in immunohistochemistry (IHC) to assess binding of the candidate molecule to a panel of human tissue sections. The TCR approach is limited by the performance of the therapeutic as an IHC reagent, which is often suboptimal to outright infeasible. Furthermore, binding of the therapeutic in IHC conditions typically has poor in vitro to in vivo translation and lacks qualitative data of the identity of putative off-targets limiting the decisional value of the data. More recently, cell-based protein arrays (CBPA) that allow for screening against a large portion of the human membrane proteome and secretome have emerged as a complement, and likely a higher value alternative, to IHC-based off-target assessment. These arrays identify specific protein interactions and may be useful for testing nontraditional antibody-based therapeutic formats that are unsuitable for TCR studies. This article presents an overview of CBPA technologies in the context of TCR and off-target assessment studies. Selected case examples and strategic considerations covering a range of different modalities are presented.

A retrospective study was performed to determine the incidences of spontaneous findings in control laboratory New Zealand White (NZW) and Dutch Belted (DB) rabbits. Terminal body and organ weights data were also collected. A total of 2170 NZW (526 males/1644 females), 100 DB rabbits (50 animals per sex), aged 4- to 7-month-old were obtained from 158 non-clinical studies evaluated between 2013 and 2022. The NZW rabbits had greater mean terminal body weights than DB strain. Mixed cell infiltration in the lung was the most recorded finding in both strains, followed by pulmonary inflammation/mononuclear cell infiltration. Differentiation between pulmonary "infiltration"/"inflammation" remained challenging as interpretation of guidelines for diagnostic terminology may vary amongst pathologists. Other common findings included mineralization and basophilia of the renal tubules; hepatic/renal mononuclear cell infiltration, all more common in females. Cysts were commonly recorded, with high prevalence in the oviduct, thyroid gland, ovary in NZW strain, while uterine, pituitary gland, and thyroid gland cysts were the most identified in DB rabbits. Neoplasms and infectious etiologies were absent. Most of the animals were sexually mature. To our knowledge, this is the most recent comprehensive study of spontaneous lesions and organ weights in both rabbit strains and should facilitate the differentiation of spontaneous and induced lesions in safety studies.

Characterizing the expression of novel targets in normal and diseased tissues is a fundamental component of a target validation data package. Often these targets are presented to the pathology team for assessment with bulk or single-cell RNAseq data and limited to no spatial tissue expression data. In situ hybridization to detect mRNA (RNAscope) is a valuable tool to (1) identify cells that may express the target protein and to corroborate protein expression during immunohistochemical (IHC) assay development or (2) to use as surrogate for single-cell expression IHC when antibodies are not available. Chromogenic RNAscope in situ hybridization (CISH) can be performed on frozen or formalin-fixed, paraffin-embedded (FFPE) tissues. This CISH workflow starts with RNA qualification of the tissue (to assess RNA integrity) by measuring the expression of housekeeping genes. RNA-qualified tissues then undergo CISH for the target in question, and positive CISH signals are quantified in VisioPharm by a combination of color deconvolution, size gating, and dot density thresholding. This RNA workflow can complement IHC or standalone in target validation for spatial characterization of novel targets.

Small interfering RNAs (siRNAs) have been successfully used as therapeutics to silence disease-causing genes when conjugated to ligands or formulated in lipid nanoparticles to target relevant cell types for efficacy while sparing other cells for safety. To support the development of new methods for delivery of siRNA therapeutics, we developed and characterized a panel of antibodies generated against chemically modified nucleotides used in therapeutic siRNA molecules, identifying a monoclonal antibody that detects a broad range of siRNA representing distinct sequences and modification patterns. By integrating this anti-siRNA antibody with additional reagents, we created a multiplex siRNA immunoassay that simultaneously quantifies siRNA uptake, trafficking, and silencing activity. Using immunohistochemistry (IHC), we applied our method on tissues from mice treated with unconjugated, GalNAc-conjugated, or cholesterol-conjugated siRNAs and quantitatively assessed the biodistribution and activity of siRNAs in various organs. In addition, we used high-content imaging (HCI) and applied our multiplex siRNA immunoassay in tissue culture to enable simultaneous quantification of siRNA uptake, activity, and intracellular colocalization with endosome markers. These methods provide a robust platform for testing nucleic acid delivery methods in vitro and in vivo, allowing precise analysis and visualization of the pharmacokinetics and pharmacodynamics of siRNA therapeutics with cellular and subcellular resolution.

Hematoxylin and eosin (H&E) staining is a suitable approach for detecting substantial structural changes in neural tissues but is less sensitive for identifying subtle alterations to subcellular structures and various chemical constituents, including myelin. Neurohistological methods to better evaluate myelin integrity by light microscopy include acidophilic dyes (eg, eriochrome cyanine R, toluidine blue [used with hard plastic sections]); lipoprotein-binding dyes (eg, Luxol fast blue [LFB], Weil's iron hematoxylin); lipid impregnation with metals (eg, Marchi's, which uses osmium tetroxide for en bloc staining before embedding); and immunohistochemical (IHC) methods to highlight various antigens (eg, myelin basic protein [MBP] and peripheral myelin protein 22 [PMP22]). Some IHC methods reveal enhanced marker expression in damaged myelin (eg, matrix metalloproteinase-9 [MMP9], S100). In neuropathology investigations, H&E is the first-tier screening method, whereas myelin stains (often LFB alone or in combination with dyes that highlight other structural elements) are second-tier procedures performed in combination with other neurohistological procedures to examine neuroaxonal injury and/or glial responses. The choice of myelin method depends on such considerations as cost, institutional preference, the procedure (fixation and embedding medium), and the study objective.