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.

Spontaneous testicular tubular hypoplasia/atrophy in the Göttingen minipig: a retrospective study.

The aim of this retrospective study was to assess the incidence and severity of tubular atrophy/hypoplasia in the testes of 104 control Göttingen minipigs aged 4.5 to 15 months. The finding was termed "tubular hypoplasia/atrophy" according to published descriptions for the dog. It included different microscopic changes that were considered part of a continuum, namely seminiferous epithelium vacuolation, presence of multinucleated germ cells in the tubular lumen, and decreased numbers (hypospermatogenesis) or absence of germ cells. This retrospective study demonstrates that tubular hypoplasia/atrophy is present in more than 70% of Göttingen minipigs and can occur at a marked severity in control animals. It correlated with a higher level of cell debris and a decrease in sperm content in the epididymides and with lower absolute and relative testes weights. There was no correlation with the weight of other sexual organs, total bodyweight, or age, which demonstrates that this change was not related to sexual immaturity. The distinction between this background finding and a compound-related effect could be challenging for the pathologist.

Multiple atypical mucosal xanthomas in a dog similar to human verruciform xanthoma.

A case of atypical mucosal xanthomas in a 14-year-old Shi-Tzu dog is reported. Grossly, 1-6-mm granular plaques or slightly elevated papillary nodules were found in the oral cavity (dorsal and ventral sides of the tongue, inner upper lip) and in the upper digestive tract (esophagus, stomach). Microscopically, subepithelial aggregates of large foamy cells were found in strong association with papillary epithelial hyperplasia. By immunohistochemistry, the majority of these cells tested positive for lysozyme and ACM1. The cells had cytoplasmic lipid content that stained positively with oil red O. These findings confirmed a monocyte/macrophage lineage. On the basis of macroscopic observations, microscopic changes, and the absence of a clinical metabolic defect, the condition in this dog appears similar to that of humans with oral verruciform xanthoma. The pathogenesis of these xanthomas in the dog remains obscure, although this condition appears to affect people and dogs with an Asian origin.

Tumor Cells Hijack Macrophage-Produced Complement C1q to Promote Tumor Growth.

Clear-cell renal cell carcinoma (ccRCC) possesses an unmet medical need, particularly at the metastatic stage, when surgery is ineffective. Complement is a key factor in tissue inflammation, favoring cancer progression through the production of complement component 5a (C5a). However, the activation pathways that generate C5a in tumors remain obscure. By data mining, we identified ccRCC as a cancer type expressing concomitantly high expression of the components that are part of the classical complement pathway. To understand how the complement cascade is activated in ccRCC and impacts patients' clinical outcome, primary tumors from three patient cohorts (n = 106, 154, and 43), ccRCC cell lines, and tumor models in complement-deficient mice were used. High densities of cells producing classical complement pathway components C1q and C4 and the presence of C4 activation fragment deposits in primary tumors correlated with poor prognosis. The in situ orchestrated production of C1q by tumor-associated macrophages (TAM) and C1r, C1s, C4, and C3 by tumor cells associated with IgG deposits, led to C1 complex assembly, and complement activation. Accordingly, mice deficient in C1q, C4, or C3 displayed decreased tumor growth. However, the ccRCC tumors infiltrated with high densities of C1q-producing TAMs exhibited an immunosuppressed microenvironment, characterized by high expression of immune checkpoints (i.e., PD-1, Lag-3, PD-L1, and PD-L2). Our data have identified the classical complement pathway as a key inflammatory mechanism activated by the cooperation between tumor cells and TAMs, favoring cancer progression, and highlight potential therapeutic targets to restore an efficient immune reaction to cancer.

Presumed primary muscular lymphoma in a dog.

A case of presumed primary muscular lymphoma in an 8-year-old, intact, male Newfoundland dog is reported. The dog was presented for evaluation of an infiltrating ventral cervical mass, respiratory distress, and anorexia of 1-month duration. Fine-needle aspiration of the mass revealed anaplastic large cell lymphoma. Despite chemotherapy, health status declined and the animal was euthanized a few weeks later. At necropsy, the mass infiltrated the cervical muscles and extended ventrally to the left forelimb and cranially to the tongue and laryngeal musculature. Other muscles were infiltrated by the same neoplasm (diaphragm and intercostal, abdominal, and gluteal muscles) indicating a probable multicentric origin. Histological examination confirmed the diagnosis of anaplastic large cell lymphoma, which showed a strong muscular tropism. Immunohistochemical staining revealed neoplastic cell reactivity for cluster of differentiation 3 (CD3) and Ki-67 antigens (70% and 90%, respectively). The neoplastic cells were negative for CD79a. The presumed histological diagnosis in this dog was primary muscular anaplastic large T-cell lymphoma.

Histopathological lesions following intramuscular administration of saline in laboratory rodents and rabbits.

This review was undertaken to assess the nature and incidence of procedure-related changes in mice, rats and rabbits which received saline solution by intramuscular injection. Data were collected on the injection sites from 7 studies representing 152 animals. The original observations by the different study pathologists from both control and treated animals were evaluated in order to create a glossary of preferred terms to be used in toxicology studies. These standardized terms were then applied to changes observed in the saline-treated animals. The review showed that the most severe of the procedure-related lesions were only of a slight level. Two days post-injection, the local reactions were mainly composed of minimal infiltration by mononuclear cells (lymphocytes and macrophages) with occasional degeneration of myofibres. From 10 to 42 days post-injection, lesions showed regeneration of myofibres and some fibrosis. In rats, the number of injections at each site influenced inflammatory infiltrate and degenerative lesions.