Experimental Pathology Laboratories

The 2025 annual Division of Translational Toxicology (DTT) Satellite Symposium, entitled ''Pathology Potpourri'' was held in Chicago, IL, at the Society of Toxicologic Pathology's (STP) 44th annual meeting. This year marked the 25th anniversary of the DTT Satellite Symposium. The goal of this symposium was to present and discuss challenging or interesting diagnostic pathology and/or nomenclature issues. This article presents summaries of the speakers' talks along with select images and data that were used by the audience for voting and discussion. Various lesions and topics covered during the symposium included inflammatory lung lesions in rats due to inhalation of silica dust; excipient-related renal tubular vacuolation in rats; spheroids in the cerebellum of dogs associated with administration of an O-GlcNAc inhibitor; hyperplasia and papillomas in the bladder of mice treated with a fragrance component; hematology analyzer error flags related to increased numbers of large unstained cells (LUCs), as well as analyzer dysfunction in the measurement of the white blood cell (WBC) count and differential, paradoxical effect of cyclosporine in allergic lung inflammation in mice, and vertebral deformation and gas gland adenomas in killifish.

Historically, safety and efficacy assessment of medical devices began and has continued as standards under the International Organization for Standardization (ISO) rather than under regulatory agency guidelines applied to developing other biomedical product classes. These parallel and unequal pathways have led to multiple and substantive differences in methods and endpoints to determine adverse biological responses among therapeutic classes. Toxicologic pathologists with medical device experience consider standardized nomenclature and diagnostic criteria for medical devices and device-containing combination products as a critical unmet need for nonclinical pathology evaluations. The International Harmonization of Nomenclature and Diagnostic Criteria for Lesions (INHAND) initiative has established globally accepted terminology for proliferative and nonproliferative lesions in various laboratory animal species. Experienced pathologists have identified that some existing INHAND terms for rodents and particularly nonrodents are already used or can be modified for use in medical device studies, but new terms for diagnostic features unique to medical device studies are needed to close gaps in existing INHAND nomenclature. The best approach to establishing appropriate INHAND terms for medical devices (and by extension, device-containing combination products) will be to develop and implement suitable terminology (modified and new, as warranted) to address unmet needs for this distinctive therapeutic class.

Despite effective countermeasures, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) persists worldwide because of its ability to diversify and evade human immunity. This evasion stems from amino acid substitutions, particularly in the receptor binding domain (RBD) of the spike protein that confers resistance to vaccine-induced antibodies and antibody therapeutics. To constrain viral escape through resistance mutations, we combined antibody variable regions that recognize different RBD sites into multispecific antibodies. Here, we describe multispecific antibodies, including a trivalent trispecific antibody that potently neutralized diverse SARS-CoV-2 variants and prevented virus escape more effectively than single antibodies or mixtures of the parental antibodies. Despite being generated before the appearance of Omicron, this trispecific antibody neutralized all major Omicron variants through BA.4/BA.5 at nanomolar concentrations. Negative stain electron microscopy suggested that synergistic neutralization was achieved by engaging different epitopes in specific orientations that facilitated binding across more than one spike protein. Moreover, a tetravalent trispecific antibody containing the same variable regions as the trivalent trispecific antibody also protected Syrian hamsters against Omicron variants BA.1, BA.2, and BA.5 challenge, each of which uses different amino acid substitutions to mediate escape from therapeutic antibodies. These results demonstrated that multispecific antibodies have the potential to provide broad SARS-CoV-2 coverage, decrease the likelihood of escape, simplify treatment, and provide a strategy for antibody therapies that could help eliminate pandemic spread for this and other pathogens.

Chikungunya virus (CHIKV), which induces chikungunya fever and chronic arthralgia, is an emerging public health concern. Safe and efficient vaccination strategies are needed to prevent or mitigate virus-associated acute and chronic morbidities for preparation of future outbreaks. Eilat (EILV)/CHIKV, a chimeric alphavirus which contains the structural proteins of CHIKV and the non-structural proteins of EILV, does not replicate in vertebrate cells. The chimeric virus was previously reported to induce protective adaptive immunity in mice. Here, we assessed the capacity of the virus to induce quick and durable protection in cynomolgus macaques. EILV/CHIKV protected macaques from wild-type (WT) CHIKV infection one year after a single dose vaccination. Transcriptome and in vitro functional analyses reveal that the chimeric virus triggered toll-like receptor signaling and T cell, memory B cell and antibody responses in a dose-dependent manner. Notably, EILV/CHIKV preferentially induced more durable, robust, and broader repertoire of CHIKV-specific T cell responses, compared to a live attenuated CHIKV 181/25 vaccine strain. The insect-based chimeric virus did not cause skin hypersensitivity reactions in guinea pigs sensitized to mosquito bites. Furthermore, EILV/CHIKV induced strong neutralization antibodies and protected cynomolgus macaques from WT CHIKV infection within six days post vaccination. Transcriptome analysis also suggest that the chimeric virus induction of multiple innate immune pathways, including Toll-like receptor signaling, type I IFN and IL-12 signaling, antigen presenting cell activation, and NK receptor signaling. Our findings suggest that EILV/CHIKV is a safe, highly efficacious vaccine, and provides both rapid and long-lasting protection in cynomolgus macaques.

Herpesvirus of turkey (HVT) increases activation of T cells in 1-day-old chickens when administered in ovo. This study evaluated whether adding cytosine-guanosine oligodeoxynucleotides (CpG ODNs) to the HVT vaccine could enhance the adjuvant effect of HVT. We used a CpG ODN dose of 10 μg per egg. The experimental groups were (1) diluent-only control (sham), (2) HVT, (3) HVT+CpG ODN, (4) HVT+non-CpG ODN, (5) CpG ODN, and (6) non-CpG ODN control. Cellular response evaluation included measuring the frequencies of macrophages (KUL01+MHC-II+), gamma delta T cells (γδTCR+MHC-II+), CD4+, and CD8+ T cell subsets, including double-positive (DP) cells. In addition, CD4+ and CD8+ T cell activation was evaluated by measuring the cellular expression of major histocompatibility complex class II (MHC-II), CD44 or CD28 costimulatory molecules. An adjuvant effect was considered when HVT+CpG ODN, but not HVT+non CpG ODN, or CpG ODN, or non-CpG ODN, induced significantly increased effects on any of the immune parameters examined when compared with HVT. The findings showed that (1) HVT vaccination induced significantly higher frequencies of γδ+MHC-II+ and CD4+CD28+ T cells when compared with sham chickens. Frequencies of DP and CD4+CD28+ T cells in HVT-administered birds were significantly higher than those observed in the non-CpG ODN group. (2) Groups receiving HVT+CpG ODN or CpG ODN alone were found to have significantly increased frequencies of activated CD4+ and CD8+ T cells, when compared with HVT. Our results show that CpG ODN administration in ovo with or without HVT significantly increased frequencies of activated CD4+ and CD8+ T cells.

PredictONCO 1.0 is a unique web server that analyzes effects of mutations on proteins frequently altered in various cancer types. The server can assess the impact of mutations on the protein sequential and structural properties and apply a virtual screening to identify potential inhibitors that could be used as a highly individualized therapeutic approach, possibly based on the drug repurposing. PredictONCO integrates predictive algorithms and state-of-the-art computational tools combined with information from established databases. The user interface was carefully designed for the target specialists in precision oncology, molecular pathology, clinical genetics and clinical sciences. The tool summarizes the effect of the mutation on protein stability and function and currently covers 44 common oncological targets. The binding affinities of Food and Drug Administration/ European Medicines Agency -approved drugs with the wild-type and mutant proteins are calculated to facilitate treatment decisions. The reliability of predictions was confirmed against 108 clinically validated mutations. The server provides a fast and compact output, ideal for the often time-sensitive decision-making process in oncology. Three use cases of missense mutations, (i) K22A in cyclin-dependent kinase 4 identified in melanoma, (ii) E1197K mutation in anaplastic lymphoma kinase 4 identified in lung carcinoma and (iii) V765A mutation in epidermal growth factor receptor in a patient with congenital mismatch repair deficiency highlight how the tool can increase levels of confidence regarding the pathogenicity of the variants and identify the most effective inhibitors. The server is available at https://loschmidt.chemi.muni.cz/predictonco.

The setting of concentrations for testing substances in ecotoxicological studies is often based on fractions of the concentrations that cause 50% mortality (LC50 or LD50) rather than environmentally relevant levels. This practice can result in exposures to animals at test concentrations that are magnitudes of order greater than those experienced in the environment. Often, such unrealistically high concentrations may cause non-specific biochemical or morphologic changes that primarily reflect the near-lethal health condition of the animal subjects, as opposed to effects characteristic of the particular test compound. Meanwhile, it is recognized that for many chemicals, the toxicologic mode of action (MOA) responsible for lethality may differ entirely from the MOAs that cause various sublethal effects. One argument for employing excessively high exposure concentrations in sublethal studies is to ensure the generation of positive toxicological effects, which can then be used to establish safety thresholds; however, it is possible that the pressure to produce exposure-related effects may also contribute to false positive outcomes. The purpose of this paper is to explore issues involving some current usages of acute LC50 data in ecotoxicology testing, and to propose an alternative strategy for performing this type of research moving forward. Toward those ends, a brief literature survey was conducted to gain an appreciation of methods that are currently being used to set test concentrations for sublethal definitive studies.

The amphibian metamorphosis assay (AMA) is a key in vivo endocrine screen to investigate chemicals with potential thyroid activity. The test guidelines and associated guidance consider that treatment-related effects on thyroid gland histomorphology automatically result in the assay being considered positive for thyroid activity, independent of the direction of change or conflicting results in the other biological endpoints. An AMA study was conducted with five different feeding rations equivalent to 50%, 30%, 20%, 10%, and 5% of the recommended feeding rate. Biological endpoints relating to growth and development, including thyroid gland histopathology, were evaluated, and the specificity of these endpoints for the determination of thyroid activity was assessed. There was no effect on survival or clinical signs of toxicity. Effects related to feed reduction generally occurred in a feeding ration-response manner and included reduced development stage; reduced body weight and body length metrics; decreased prevalence of thyroid follicular cell hyperplasia and hypertrophy, and the occurrence of thyroid atrophy; reduced liver vacuolation; and the occurrence of liver atrophy. The results indicate that treatment-related histopathological changes in the AMA can be induced by Non-chemical factors; therefore histopathological results are not necessarily diagnostically specific for chemically induced thyroid endocrine activity. Consequently, the interpretation of data from AMA studies should be adjusted accordingly. We recommend that the decision logic presented in the test guidelines and associated guidance be changed to reflect a requirement for directional agreement between the thyroid histopathology findings and the growth and developmental endpoints before it is concluded that a test substance has thyroid endocrine activity. Environ Toxicol Chem 2023;42:1061-1074. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.