Journal of Toxicologic Pathology最新文献

Gene therapy (GT) products created using adeno-associated virus (AAV) vectors tend to exhibit toxicity via immune reactions, but other mechanisms of toxicity remain incompletely understood. We examined the cardiotoxicity of an overexpressed transgenic protein. Male C57BL/6J mice were treated with a single intravenous dose of product X, an AAV-based GT product, at 2.6 × 10¹³ vg/kg. Necropsies were performed at 24 h, 7 days, and 14 days after dosing. Pathological examination and gene expression analysis were performed on the heart. Histopathologically, hypertrophy and vacuolar degeneration of cardiomyocytes and fibrosis were observed 14 days after dosing. Immunohistochemistry for endoplasmic reticulum (ER) stress-related proteins revealed increased positive reactions for glucose-regulated protein 78 and C/EBPR homologous protein in cardiomyocytes 7 days after dosing, without histopathological abnormalities. Fourteen days after dosing, some cardiomyocytes showed positivity for PKR-like endoplasmic reticulum kinase and activating transcription factor 4 expression. Ultrastructurally, increases in the ER and cytosol were observed in cardiomyocytes 7 days after dosing, along with an increase in the number of Golgi apparatus compartments 14 days after dosing. The tissue concentration of the transgene product protein increased 7 days after dosing. Gene expression analysis showed upregulation of ER stress-related genes 7 days after dosing, suggesting activation of the PKR-like ER kinase pathway of the unfolded protein reaction (UPR). Thus, the cardiotoxicity induced by product X was considered to involve cell damage caused by the overexpression of the product protein accompanied by UPR. Marked UPR activation may also cause toxicity of AAV-based GT products.

We report a histiocytic sarcoma originating from the epididymis observed in a 110-week-old male CD-1 mouse in a carcinogenicity study. At necropsy, no lesions were observed in the epididymis. Histologically, a neoplastic lesion was observed in the cauda of the epididymis that was well demarcated from the surrounding tissues. The lesion mainly consisted of spindle-shaped tumor cells with oval to elongated nuclei and abundant eosinophilic or foamy cytoplasm. The tumor cells were arranged in a fascicular pattern, interlacing bundles, or a whorl pattern. The nuclei showed mild atypia with irregular shapes and varied sizes, whereas few mitotic figures and no typical multinucleated cells were observed. The epididymal ducts remained within the neoplastic lesion, and the tumor cells invaded between the epithelium and the smooth muscle layer of the epididymal duct. Immunohistochemically, the tumor cells were positive for vimentin and macrophage markers (Iba1, CD204, F4/80, and Mac-2) but negative for cytokeratin and other mesenchymal cell (α-smooth muscle actin, desmin, CD31, and platelet-derived growth factor receptor-β), neural cell (S-100 and nestin), or Leydig cell markers (calretinin). Proliferating cell nuclear antigen-positive tumor cells were sporadically observed in the lesion. Based on these results, the tumor was diagnosed as a histiocytic sarcoma originating from the epididymis. This report provides additional histopathological evidence of spontaneous histiocytic sarcomas originating from the epididymis of aged mice.

The historical control database of a multinational laboratory services provider was queried for all histopathologic findings in New Zealand White rabbits which were used as control animals during a ten-year period (2011–2020). The query included all evaluated tissues, with or without microscopic findings, in studies conducted for safety testing for regulatory approval by the U.S. Food and Drug Agency (FDA) or the U.S. Environmental Protection Agency. A second query included studies conducted in the United Kingdom for control rabbits used in studies compliant with the Healthcare Products Regulatory Agency (MHRA) and/or the European Medicines Agency (EMA), which provide regulatory oversight in the United Kingdom and European Union, respectively. Infiltrates of inflammatory (mixed or mononuclear) cells were commonly noted in various organs including heart, digestive tract, muscle, thyroid, kidney, urinary bladder, eyelid, ocular structures, harderian gland, lacrimal gland, and lung. Mineralization was noted in aorta, kidney, urinary bladder, and ovary. Also noted were degeneration/necrosis in the myocardium, and intramuscular injection sites of the skin, degeneration/regeneration of muscle and diaphragm, ectopic tissue in the pancreas and thyroid, basophilic foci in salivary gland, increased/decreased vacuolation in adrenal gland, increased/decreased lymphocytic cellularity of lymph nodes, intrasinusoidal erythrocytes in lymph nodes, thymic atrophy, increased adipocytes in bone marrow, inflammatory cell foci in the liver and gall bladder, lacrimal gland atrophy, renal tubule basophilia, degeneration/regeneration, and dilatation; oviduct cyst; in the testis, degeneration/atrophy, cellular debris, dilatation, decreased sperm and segmental hypoplasia of seminiferous tubules; and squamous metaplasia of the testis and seminal vesicle.

Tissue cross-reactivity (TCR) studies for the development of therapeutic antibodies are conducted to estimate any possible binding sites within the human body that can be affected by the antibody when assessing safety in humans. Any possible binding sites include specific binding sites of the antibody to its target antigen and nonspecific or off-target binding sites. In TCR studies the therapeutic antibodies and immunohistochemistry (IHC) of frozen tissues must be applied in assays. However, there are technical issues with applying a therapeutic antibody or test article to IHC, such as human-on-human staining, difficulty in applying the test article to IHC, and retention of the target antigen in frozen sections. In the current review, we introduce three case studies in which these technical issues were addressed, and propose a practical scheme for points to consider when conducting a TCR study. Information on the target antigen distribution obtained through robust assays and case-by-case strategies were found to be useful for understanding and assessing the relevance of toxic effects between animals and humans. Thus, we anticipate that by considering the points discussed in the current review and combining the data with information on the biological features of the target antigens and therapeutic antibodies, it will be possible to predict safety risks in humans with higher accuracy.

Duplicate testes lined in series were observed in the right scrotum of a 6-week-old Sprague-Dawley rat in a single-dose toxicity study. Of the two right testicles, one was spherical and less than half the size of a normal testis. The other was oval-shaped, slightly smaller than a normal testis, and possessed clear, tortuous blood vessels similar to those of a normal testis. Each right testis was grossly separated but faced the intertesticular adipose tissue and was sparsely joined by thin cord-like structures. Only one epididymis covered or encompassed the two right testes. The caput epididymis was attached to the smaller spherical testis, whereas the cauda epididymis was attached to the oval testis. Histopathological examination revealed that the smaller spherical testis on the right side and the testis on the left side were normal. The oval-shaped testis on the right exhibited markedly dilated degenerative seminiferous tubules with one to two layers of Sertoli or germ cells, and almost no spermatogenesis was observed. Multinucleated germ cells were observed in the lumen of the degenerated seminiferous tubules. The right epididymis was morphologically normal and contained few sperm in the epididymal duct of the tail. The cord-like structures between duplicate testes comprised fibrous and adipose tissues. Single efferent ductules, ectopic cartilage, and skeletal muscle tissues were buried in the adipose tissue. To our knowledge, this is the first report of spontaneous polyorchidism in a rodent.

This study aimed to establish an exposure method that can induce homogeneous lesions with minimal inter-individual variability. The distribution of lesions induced by bleomycin (BLM) administration was also analyzed. C57BL mice were intrabronchially administered 20 µL of BLM (3 mg/mL) using a bronchoscope in the left or right bronchus. The mice were sacrificed 14 days after administration, and their lungs were evaluated histopathologically. BLM-induced inflammatory lesions were widely observed in the lungs. In the left bronchus-treated group, lesions were uniformly observed throughout the lobe, and no individual differences were noted. Meanwhile, in the right bronchus-treated group, individual differences in the distribution of the pulmonary lesions were observed. The distribution of lesions differed among the four lobes of the right lung owing to their anatomical features. Administration into the left bronchus is recommended for highly homogeneous lung exposure and for establishing models that contribute to highly accurate toxicity and efficacy evaluations.

In neurosurgical interventions, effective closure of the dura mater is essential to prevent cerebrospinal fluid leakage and minimize post-operative complications. Biodegradable synthetic materials have the potential to be used as dura mater grafts owing to their regenerative properties and low immunogenicity. This study evaluated the safety of ArtiFascia, a synthetic dura mater graft composed of poly(l-lactic-co-caprolactone acid) and poly(d-lactic-co-caprolactone acid), in a rabbit durotomy model. Previously, ArtiFascia demonstrated positive local tolerance and biodegradability in a 12-month preclinical trial. Here, specialized stains were used to evaluate potential brain damage associated with ArtiFascia use. Histochemical and immunohistochemical assessments included Luxol Fast Blue, cresyl Violet, Masson’s Trichrome, neuronal nuclei,, Glial Fibrillary Acidic Protein, and ionized calcium-binding adaptor molecule 1 stains. The stained slides were graded based on the brain-specific reactions. The results showed no damage to the underlying brain tissue for either the ArtiFascia or control implants. Neither inflammation nor neuronal loss was evident, corroborating the safety of the ArtiFascia. This approach, combined with previous histopathological analyses, strengthens the safety profile of ArtiFascia and sets a benchmark for biodegradable material assessment in dura graft applications. This study aligns with the Food and Drug Administration guidelines and offers a comprehensive evaluation of the potential neural tissue effects of synthetic dura mater grafts.

Japan has various death investigation systems; however, external examinations, postmortem computed tomography, macroscopic examinations, and microscopic examinations are performed regardless of the system used. These examinations can reveal morphological abnormalities, whereas the cause of death in cases with non-morphological abnormalities can be detected through additional examinations. Molecular autopsy and postmortem genetic analyses are important additional examinations. They are capable of detecting inherited arrhythmias or inherited metabolic diseases, which are representative non-morphological disorders that cause sudden death, especially in infants and young people. In this review, we introduce molecular autopsy reports from Japan and describe our experience with representative cases. The relationships between drug-related deaths and genetic variants are also reviewed. Based on the presented information, molecular autopsy is expected to be used as routine examinations in death investigations because they can provide information to save new lives.

In drug development, assessment of non-clinical peripheral neurotoxicity is important to ensure human safety. Clarifying the pathological features and mechanisms of toxicity enables the management of safety risks in humans by estimating the degree of risk and proposing monitoring strategies. Published guidelines for peripheral neurotoxicity assessment do not provide detailed information on which endpoints should be monitored preferentially and how the results should be integrated and discussed. To identify an optimal assessment method for the characterization of peripheral neurotoxicity, we conducted pathological, biochemical (biomaterials contributing to mechanistic considerations and biomarkers), and behavioral evaluations of isoniazid-treated rats. We found a discrepancy between the days on which marked pathological changes were noted and those on which biochemical and behavioral changes were noted, suggesting the importance of combining these evaluations. Although pathological evaluation is essential for pathological characterization, the results of biochemical and behavioral assessments at the same time points as the pathological evaluation are also important for discussion. In this study, since the measurement of serum neurofilament light chain could detect changes earlier than pathological examination, it could be useful as a biomarker for peripheral neurotoxicity. Moreover, examination of semi-thin specimens and choline acetyltransferase immunostaining were useful for characterizing morphological neurotoxicity, and image analysis of semi-thin specimens enabled us to objectively show the pathological features.

Renal tubular epithelial cell (TEC) injury is the most common cause of drug-induced kidney injury (DIKI). Although TEC regeneration facilitates renal function and structural recovery following DIKI, maladaptive repair of TECs leads to irreversible fibrosis, resulting in chronic kidney disease (CKD). CD44 is specifically expressed in TECs during maladaptive repair in several types of rat CKD models. In this study, we investigated CD44 expression and its role in renal fibrogenesis in a cyclosporine (CyA) rat model of CKD. Seven-week-old male Sprague–Dawley rats fed a low-salt diet were subcutaneously administered CyA (0, 15, or 30 mg/kg) for 28 days. CD44 was expressed in atrophic, dilated, and hypertrophic TECs in the fibrotic lesions of the CyA groups. These TECs were collected by laser microdissection and evaluated by microarray analysis. Gene ontology analysis suggested that these TECs have a mesenchymal phenotype, and pathway analysis identified CD44 as an upstream regulator of fibrosis-related genes, including fibronectin 1 (Fn1). Immunohistochemistry revealed that epithelial and mesenchymal markers of TECs of fibrotic lesions were downregulated and upregulated, respectively, and that these TECs were surrounded by a thickened basement membrane. In situ hybridization revealed an increase in Fn1 mRNA in the cytoplasm of TECs of fibrotic lesions, whereas fibronectin protein was localized in the stroma surrounding these tubules. Enzyme-linked immunosorbent assay revealed increased serum CD44 levels in CyA-treated rats. Collectively, these findings suggest that CD44 contributes to renal fibrosis by inducing fibronectin secretion in TECs exhibiting partial epithelial-mesenchymal transition and highlight the potential of CD44 as a biomarker of renal fibrosis.