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Multiplexed siRNA Immunoassay Unveils Spatial and Quantitative Dimensions of siRNA Function, Abundance, and Localization In Vitro and In Vivo. 多重siRNA免疫分析揭示了siRNA在体外和体内功能、丰度和定位的空间和定量维度。
IF 1.4 4区 医学 Q3 PATHOLOGY Pub Date : 2025-01-19 DOI: 10.1177/01926233241311539
Michael Ly, Sandra Diaz-Garcia, Nathaniel Roscoe, Irina Ushach, Zhigang Hong, Monique França, Stephanie Schaffer, Tong-Yuan Yang, Mathieu Marella, Glenn Marsboom, Donna Klein, Tamar R Grossman, Vinicius Carreira, Michael Ollmann

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.

小干扰rna (sirna)已被成功地用作沉默致病基因的治疗药物,当其与配体结合或在脂质纳米颗粒中配制时,可靶向相关细胞类型以获得疗效,同时保证其他细胞的安全性。为了支持siRNA治疗药物递送新方法的发展,我们开发并鉴定了一组针对用于治疗siRNA分子的化学修饰核苷酸产生的抗体,鉴定了一种单克隆抗体,该抗体可以检测代表不同序列和修饰模式的广泛siRNA。通过将这种抗siRNA抗体与其他试剂整合,我们创建了一种多重siRNA免疫分析法,可以同时量化siRNA的摄取、运输和沉默活性。使用免疫组织化学(IHC),我们将我们的方法应用于未偶联的、galnac偶联的或胆固醇偶联的sirna处理的小鼠组织,并定量评估了sirna在各器官中的生物分布和活性。此外,我们使用了高含量成像(HCI),并在组织培养中应用了多重siRNA免疫分析,以同时量化siRNA的摄取、活性和内核体标记的细胞内共定位。这些方法为体外和体内检测核酸递送方法提供了一个强大的平台,可以精确分析和可视化siRNA治疗药物的细胞和亚细胞分辨率的药代动力学和药效学。
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引用次数: 0
Myelin Methods: A Mini-Review. 髓磷脂方法:一个小型回顾。
IF 1.4 4区 医学 Q3 PATHOLOGY Pub Date : 2025-01-19 DOI: 10.1177/01926233241309332
Brad Bolon, Amber Moser, Elizabeth Chlipala

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.

苏木精和伊红(H&E)染色是检测神经组织实质性结构变化的合适方法,但对于识别亚细胞结构和各种化学成分(包括髓磷脂)的细微变化不太敏感。通过光学显微镜更好地评估髓磷脂完整性的神经组织学方法包括嗜酸染料(例如,eriochrome cyanine R,甲苯胺蓝[用于硬塑料切片]);脂蛋白结合染料(如Luxol耐晒蓝[LFB]、Weil’s铁苏木精);金属脂质浸渍(例如,Marchi's,在包埋前使用四氧化二锇进行整体染色);和免疫组化(IHC)方法来突出各种抗原(如髓鞘碱性蛋白[MBP]和外周髓鞘蛋白22 [PMP22])。一些免疫组化方法显示受损髓磷脂中标记物表达增强(如基质金属蛋白酶-9 [MMP9], S100)。在神经病理学检查中,H&E是第一级筛查方法,而髓磷脂染色(通常单独使用LFB或与突出其他结构元素的染料联合使用)是与其他神经组织学检查联合进行的第二级检查,以检查神经轴突损伤和/或神经胶质反应。髓磷脂方法的选择取决于成本、机构偏好、程序(固定和嵌入介质)和研究目标等因素。
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引用次数: 0
Toxicologic Pathology Forum: Opinion on Digital Primary Read and Peer Review-Are We There Yet? 毒理学病理学论坛:对数字化初级阅读和同行评议的看法——我们到了吗?
IF 1.4 4区 医学 Q3 PATHOLOGY Pub Date : 2025-01-18 DOI: 10.1177/01926233241303911
Kenneth A Schafer, Deepa B Rao

Recent trends in toxicological pathology include implementation of digital platforms that have gained rapid momentum in the field. Are we ready to fully implement this new modality? This opinion piece provides some practical perspectives on digital pathology such as its cost limitations, relative time requirements, and a few technical issues, some of which are encountered for specific lesions, that warrant caution. Although the potential for digital pathology assessment with whole slide images has made great strides, we are of the opinion that it is not yet ready for complete replacement of glass slides in toxicologic pathology safety assessments.

毒理学病理学的最新趋势包括在该领域获得快速发展势头的数字平台的实施。我们准备好全面实施这种新模式了吗?这篇观点文章提供了一些关于数字病理学的实用观点,如其成本限制、相对时间要求和一些技术问题,其中一些是针对特定病变的,需要谨慎处理。尽管利用全载玻片图像进行数字病理评估的潜力已经取得了很大的进步,但我们认为在毒理学病理安全评估中完全取代玻璃载玻片还没有准备好。
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引用次数: 0
Development of a Deep Learning Tool to Support the Assessment of Thyroid Follicular Cell Hypertrophy in the Rat. 一种支持大鼠甲状腺滤泡细胞肥大评估的深度学习工具的开发。
IF 1.4 4区 医学 Q3 PATHOLOGY Pub Date : 2025-01-17 DOI: 10.1177/01926233241309328
Stuart W Naylor, Elizabeth F McInnes, James Alibhai, Scott Burgess, James Baily

Thyroid tissue is sensitive to the effects of endocrine disrupting substances, and this represents a significant health concern. Histopathological analysis of tissue sections of the rat thyroid gland remains the gold standard for the evaluation for agrochemical effects on the thyroid. However, there is a high degree of variability in the appearance of the rat thyroid gland, and toxicologic pathologists often struggle to decide on and consistently apply a threshold for recording low-grade thyroid follicular hypertrophy. This research project developed a deep learning image analysis solution that provides a quantitative score based on the morphological measurements of individual follicles that can be integrated into the standard pathology workflow. To achieve this, a U-Net convolutional deep learning neural network was used that not just identifies the various tissue components but also delineates individual follicles. Further steps to process the raw individual follicle data were developed using empirical models optimized to produce thyroid activity scores that were shown to be superior to the mean epithelial area approach when compared with pathologists' scores. These scores can be used for pathologist decision support using appropriate statistical methods to assess the presence or absence of low-grade thyroid hypertrophy at the group level.

甲状腺组织对内分泌干扰物质的影响很敏感,这是一个重大的健康问题。大鼠甲状腺组织切片的组织病理学分析仍然是评估农药对甲状腺影响的金标准。然而,大鼠甲状腺的外观存在高度的可变性,毒理学病理学家经常难以确定并一致应用记录低级别甲状腺滤泡肥大的阈值。该研究项目开发了一种深度学习图像分析解决方案,该解决方案基于单个卵泡的形态测量提供定量评分,可集成到标准病理工作流程中。为了实现这一目标,使用了U-Net卷积深度学习神经网络,该网络不仅可以识别各种组织成分,还可以描绘单个卵泡。进一步处理原始个体卵泡数据的步骤是使用经验模型进行优化,以产生甲状腺活动评分,与病理学家的评分相比,该评分显示优于平均上皮面积法。这些评分可用于病理学家决策支持,使用适当的统计方法来评估在组水平上是否存在低级别甲状腺肥大。
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引用次数: 0
Immunohistochemistry-Free Enhanced Histopathology of the Rat Spleen Using Deep Learning. 基于深度学习的无免疫组织化学增强大鼠脾脏组织病理学研究。
IF 1.4 4区 医学 Q3 PATHOLOGY Pub Date : 2024-12-26 DOI: 10.1177/01926233241303907
Shima Mehrvar, Kevin Maisonave, Wayne Buck, Magali Guffroy, Bhupinder Bawa, Lauren Himmel

Enhanced histopathology of the immune system uses a precise, compartment-specific, and semi-quantitative evaluation of lymphoid organs in toxicology studies. The assessment of lymphocyte populations in tissues is subject to sampling variability and limited distinctive cytologic features of lymphocyte subpopulations as seen with hematoxylin and eosin (H&E) staining. Although immunohistochemistry is necessary for definitive characterization of T- and B-cell compartments, routine toxicologic assessments are based solely on H&E slides. Here, a deep learning (DL) model was developed using normal rats to quantify relevant compartments of the spleen, including periarteriolar lymphoid sheaths, follicles, germinal centers, and marginal zones from H&E slides. Slides were scanned, destained, dual labeled with CD3 and CD79a chromogenic immunohistochemistry, and rescanned to generate exact co-registered images that served as the ground truth for training and validation. The DL model identified individual splenic compartments with high accuracy (97.8% Dice similarity coefficient) directly from H&E-stained tissue. The DL model was utilized to study the normal range of lymphoid compartment area and cellularity. Future implementation of our DL model and expanding this approach to other lymphoid tissues have the potential to improve accuracy and precision in enhanced histopathology evaluation of the immune system with concurrent gains in time efficiency for the pathologist.

免疫系统强化组织病理学在毒理学研究中对淋巴器官进行精确的、特定区域的半定量评估。组织中淋巴细胞群的评估受取样变化和淋巴细胞亚群细胞学特征的限制,如苏木精和伊红(H&E)染色。虽然免疫组化是确定 T 细胞和 B 细胞区系特征的必要条件,但常规毒理学评估仅基于 H&E 切片。在此,我们利用正常大鼠开发了一种深度学习(DL)模型,以量化脾脏的相关区段,包括H&E切片中的小动脉周围淋巴鞘、滤泡、生发中心和边缘区。对切片进行扫描、去染色、CD3 和 CD79a 色原免疫组化双重标记并重新扫描,以生成精确的共混图像,作为训练和验证的基本真相。DL 模型能直接从 H&E 染色组织中高精度(97.8% Dice 相似系数)地识别出单个脾脏分区。我们利用 DL 模型研究了淋巴区面积和细胞度的正常范围。未来实施我们的 DL 模型并将这种方法扩展到其他淋巴组织,有可能提高免疫系统组织病理学评估的准确性和精确性,同时提高病理学家的时间效率。
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引用次数: 0
Trends and Challenges of the Modern Pathology Laboratory for Biopharmaceutical Research Excellence. 现代病理实验室追求卓越生物制药研究的趋势与挑战。
IF 1.4 4区 医学 Q3 PATHOLOGY Pub Date : 2024-12-13 DOI: 10.1177/01926233241303898
Sílvia Sisó, Anoop Murthy Kavirayani, Suzana Couto, Birgit Stierstorfer, Sunish Mohanan, Caroline Morel, Mathiew Marella, Dinesh S Bangari, Elizabeth Clark, Annette Schwartz, Vinicius Carreira

Pathology, a fundamental discipline that bridges basic scientific discovery to the clinic, is integral to successful drug development. Intrinsically multimodal and multidimensional, anatomic pathology continues to be empowered by advancements in molecular and digital technologies enabling the spatial tissue detection of biomolecules such as genes, transcripts, and proteins. Over the past two decades, breakthroughs in spatial molecular biology technologies and advancements in automation and digitization of laboratory processes have enabled the implementation of higher throughput assays and the generation of extensive molecular data sets from tissue sections in biopharmaceutical research and development research units. It is our goal to provide readers with some rationale, advice, and ideas to help establish a modern molecular pathology laboratory to meet the emerging needs of biopharmaceutical research. This manuscript provides (1) a high-level overview of the current state and future vision for excellence in research pathology practice and (2) shared perspectives on how to optimally leverage the expertise of discovery, toxicologic, and translational pathologists to provide effective spatial, molecular, and digital pathology data to support modern drug discovery. It captures insights from the experiences, challenges, and solutions from pathology laboratories of various biopharmaceutical organizations, including their approaches to troubleshooting and adopting new technologies.

病理学是连接基础科学发现和临床应用的一门基础学科,是成功药物开发不可或缺的组成部分。解剖病理学本质上是多模态和多维的,分子和数字技术的进步使解剖病理学能够对生物分子(如基因、转录本和蛋白质)进行空间组织检测。在过去的二十年里,空间分子生物学技术的突破以及实验室过程自动化和数字化的进步,使得生物制药研究和开发研究单位能够实施更高通量的分析,并从组织切片中生成广泛的分子数据集。我们的目标是为读者提供一些基本原理,建议和想法,以帮助建立一个现代分子病理学实验室,以满足生物制药研究的新兴需求。本文提供了(1)对病理学研究实践的现状和未来愿景的高层次概述;(2)分享了如何最佳地利用发现、毒理学和转化病理学家的专业知识来提供有效的空间、分子和数字病理学数据来支持现代药物发现的观点。它从各种生物制药组织的病理实验室的经验、挑战和解决方案中获得见解,包括他们的故障排除和采用新技术的方法。
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引用次数: 0
Toxicologic Pathology Forum: Opinion on Digital Primary Read and Peer Review-Diving Head-First Into the Deep Digital Pool! 毒理病理学论坛:关于数字化初读和同行评审的意见--一头扎进数字化深潭!
IF 1.4 4区 医学 Q3 PATHOLOGY Pub Date : 2024-12-12 DOI: 10.1177/01926233241303909
Krista M D La Perle

Before the COVID-19 pandemic, digital pathology was increasingly used in veterinary education, diagnostics, and research. The pandemic accelerated this adoption as institutions needed to maintain operations amidst lockdowns. It also enabled pharmaceutical companies to conduct peer reviews digitally, circumventing travel restrictions. At the 2023 Society of Toxicologic Pathology Annual Symposium, a Town Hall Meeting highlighted the current use of digital pathology. A majority of the respondents viewed whole slide images (WSI) favorably. Many institutions use digital pathology primarily for non-GLP and GLP conforming primary reads and peer reviews. Takeda has long utilized digital pathology, incorporating scanners and an image management repository, and recently adopted a cloud-based platform tailored for toxicologic pathology, enhancing efficiency and collaboration. Digital pathology not only saves time but also reduces travel needs and environmental impact. Technological advancements and wider adoption are expected to further enhance the field, promising significant benefits for the overall digital pathology infrastructure.

在2019冠状病毒病大流行之前,数字病理学越来越多地用于兽医教育、诊断和研究。大流行加速了这种做法的采用,因为各机构需要在封锁期间维持运营。它还使制药公司能够以数字方式进行同行评审,从而绕过旅行限制。在2023年毒物病理学学会年度研讨会上,一个市政厅会议强调了数字病理学的当前使用。大多数受访者认为整个幻灯片图像(WSI)是有利的。许多机构主要将数字病理学用于非GLP和符合GLP的初级阅读和同行评审。武田长期以来一直使用数字病理学,包括扫描仪和图像管理存储库,最近采用了专为毒理学病理学定制的基于云的平台,从而提高了效率和协作。数字病理学不仅节省了时间,还减少了旅行需求和对环境的影响。技术进步和更广泛的应用有望进一步加强该领域,为整个数字病理学基础设施带来重大好处。
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引用次数: 0
Mass Spectrometry Imaging Distinguishes Biliary Toxicants on the Basis of Cellular Distribution. 质谱成像在细胞分布的基础上区分胆道毒物。
IF 1.4 4区 医学 Q3 PATHOLOGY Pub Date : 2024-12-12 DOI: 10.1177/01926233241303890
Junhai Yang, Andrew P Bowman, Wayne R Buck, Rebecca Kohnken, Christopher J Good, David S Wagner

Mass spectrometry imaging (MSI) was used to investigate and provide insights into observed biliary pathology found in dogs and rats after administration of two different compounds. Both compounds were associated with peribiliary inflammatory infiltrates and proliferation of the bile duct epithelium. However, MSI revealed very different spatial distribution profiles for the two compounds: Compound A showed significant accumulation within the bile duct epithelium with a much higher concentration than in the parenchymal hepatocytes, while Compound T exhibited only a slight increase in the bile duct epithelium compared to parenchymal hepatocytes. These findings implicate cholangiocyte uptake and accumulation as a key step in the mechanism of biliary toxicity. In both cases, compounds are shown at the site of toxicity in support of a direct mechanism of toxicity on the biliary epithelium. MSI is a powerful tool for localizing small molecules within tissue sections and improvements in sensitivity have enabled localization down to the cellular level in some cases. MSI was also able to identify biomarker candidates of toxicity by differential analysis of ion profiles comparing treated and control cholangiocytes from tissue sections.

质谱成像(MSI)用于研究和提供观察到的胆道病理发现在狗和大鼠给药两种不同的化合物。这两种化合物都与胆管周围炎症浸润和胆管上皮增生有关。然而,MSI显示两种化合物的空间分布特征非常不同:化合物A在胆管上皮内明显积聚,浓度远高于肝实质细胞,而化合物T在胆管上皮内的浓度仅比肝实质细胞轻微增加。这些发现暗示胆管细胞的摄取和积聚是胆道毒性机制的关键步骤。在这两种情况下,化合物都显示在毒性部位,支持胆上皮毒性的直接机制。MSI是一种强大的工具,用于定位组织切片内的小分子,在某些情况下,灵敏度的提高使定位下降到细胞水平。MSI还能够通过比较处理和对照胆管细胞组织切片的离子谱的差异分析来识别毒性的生物标志物候选物。
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引用次数: 0
IHI VICT3R: Developing and Implementing Virtual Control Groups to Reduce Animal Use in Toxicology Research. 开发和实施虚拟控制组以减少毒理学研究中动物的使用。
IF 1.4 4区 医学 Q3 PATHOLOGY Pub Date : 2024-12-12 DOI: 10.1177/01926233241303906
Thomas Steger-Hartmann, Ferran Sanz, Frank Bringezu, Inari Soininen

The virtual control group (VCG) concept was originally developed in the IMI2 project eTRANSAFE, using data of control animals which pharmaceutical companies have accrued over decades from animal toxicity studies. This control data could be repurposed to create virtual control animals to reduce or replace concurrent controls in animal studies. Initial work demonstrated the general feasibility of the VCG concept, but implementation requires significant further collaborative efforts. The new Innovative Health Initiative (IHI) project VICT3R aims to address these challenges and to obtain regulatory acceptance for the VCG concept. To achieve these goals, VICT3R will build a database comprising high-quality, standardized, and duly annotated control animal data from past and forthcoming toxicity studies. The VICT3R project will create workflows and computational tools to generate adequate VCGs based on statistical and artificial intelligence (AI) approaches. The validity, reproducibility, and robustness of the resulting VCGs will be assessed by comparing the performance of their use with that of real control groups.

虚拟对照组(VCG)概念最初是在IMI2项目eTRANSAFE中开发的,使用了制药公司几十年来从动物毒性研究中积累的对照动物数据。这些对照数据可以重新用于创建虚拟对照动物,以减少或取代动物研究中的并发对照。最初的工作证明了VCG概念的总体可行性,但实施需要进一步的大量合作努力。新的创新健康倡议(IHI)项目VICT3R旨在应对这些挑战,并获得VCG概念的监管认可。为了实现这些目标,VICT3R将建立一个数据库,包括来自过去和即将进行的毒性研究的高质量、标准化和适当注释的对照动物数据。VICT3R项目将创建工作流程和计算工具,以基于统计和人工智能(AI)方法生成足够的vcg。所得到的vcg的有效性、可重复性和稳健性将通过将其与真实对照组的使用性能进行比较来评估。
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引用次数: 0
Characterization of Pulmonary Pathology in the Golden Syrian Hamster Model of COVID-19 Using Micro-Computed Tomography. 用显微计算机断层扫描表征金叙利亚仓鼠新冠肺炎模型的肺部病理。
IF 1.4 4区 医学 Q3 PATHOLOGY Pub Date : 2024-12-05 DOI: 10.1177/01926233241300451
Julita A Ramirez, Micah D Dunlap, Reyna Prosnitz, Anderson Watson, Mary K Montgomery, Matthew Gutman, Timothy M Coskran, Samantha L Levinson, Katharine Yang, Isis Kanevsky, Shambhunath Choudhary

The Golden Syrian hamster is a well-characterized rodent model for severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2)-associated pneumonia. We sought to characterize the pulmonary disease course during SARS-CoV-2 infection (strain USA-WA1/2020) in the hamster model using micro-computed tomography (micro-CT) and compare radiologic observations with histopathologic findings. We observed a range of radiologic abnormalities, including ground glass opacities (GGOs), consolidations, air bronchograms, and pneumomediastinum. The appearance, distribution, and progression of these abnormalities in hamsters were similar to those observed in the lungs of coronavirus disease 2019 (COVID-19) patients by clinical CT and chest X-rays, and correlated with clinical signs and weight loss during the course of disease. Histopathological analysis of infected hamsters revealed lung pathology characteristic of COVID-19 pneumonia, and we observed a strong association between CT and histopathologic scorings. We also analyzed accumulation of air in the thoracic cavity by both manual and automated threshold-based segmentation and found that automated analysis significantly decreases the time needed for data analysis. Data presented here demonstrate that micro-CT imaging can be a major tool in preclinical investigative studies using animal models by providing early and detailed assessment of disease severity and outcomes.

金色叙利亚仓鼠是严重急性呼吸综合征-冠状病毒-2 (SARS-CoV-2)相关肺炎的典型啮齿类动物模型。我们试图利用微型计算机断层扫描(micro-CT)在仓鼠模型中描述SARS-CoV-2感染(菌株USA-WA1/2020)期间的肺部疾病病程,并将放射学观察结果与组织病理学结果进行比较。我们观察到一系列影像学异常,包括磨玻璃混浊(GGOs)、实变、支气管充气征和纵隔气肿。这些异常在仓鼠中的表现、分布和进展与临床CT和胸部x射线在2019冠状病毒病(COVID-19)患者肺部观察到的相似,并与疾病期间的临床症状和体重减轻相关。感染仓鼠的组织病理学分析显示COVID-19肺炎的肺部病理特征,我们观察到CT与组织病理学评分之间有很强的相关性。我们还通过手动和自动阈值分割分析了胸腔内的空气积聚,发现自动分析显著减少了数据分析所需的时间。本文提供的数据表明,通过提供疾病严重程度和结果的早期和详细评估,微型ct成像可以成为使用动物模型进行临床前调查研究的主要工具。
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引用次数: 0
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Toxicologic Pathology
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