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Epithelial damage due to gastrointestinal disorders frequently causes severe disease in horses. To study the underlying pathophysiological processes, we aimed to establish equine jejunum and colon enteroids (eqJE, eqCE) mimicking the in vivo epithelium. Therefore, enteroids were cultivated in four different media for differentiation and subsequently characterized histomorphologically, on mRNA and on protein level in comparison to the native epithelium of the same donor horses to identify ideal culture conditions for an in vitro model system. With increasing enterocyte differentiation, the enteroids showed a reduced growth rate as well as a predominantly spherical morphology and less budding compared to enteroids in proliferation medium. Combined or individual withdrawal of stem cell niche pathway components resulted in lower mRNA expression levels of stem cell markers and concomitant differentiation of enterocytes, goblet cells and enteroendocrine cells. For eqCE, withdrawal of Wnt alone was sufficient for the generation of differentiated enterocytes with a close resemblance to the in vivo epithelium. Combined removal of Wnt, R-spondin and Noggin and the addition of DAPT stimulated differentiation of eqJE at a similar level as the in vivo epithelium, particularly with regard to enterocytes. In summary, we successfully defined a medium composition that promotes the formation of eqJE and eqCE consisting of multiple cell types and resembling the in vivo epithelium. Our findings emphasize the importance of adapting culture conditions to the respective species and the intestinal segment. This in vitro model will be used to investigate the pathological mechanisms underlying equine gastrointestinal disorders in future studies.

One Health describes the importance of considering humans, animals, and the environment in health research. One Health and the 3R concept, i.e., the replacement, reduction, and refinement of animal experimentation, shape today’s research more and more. The development of organoids from many different organs and animals led to the development of highly sophisticated model systems trying to replace animal experiments. Organoids may be used for disease modelling in various ways elucidating the manifold host–pathogen interactions. This review provides an overview of disease modelling approaches using organoids of different kinds with a special focus on animal organoids and gastrointestinal diseases. We also provide an outlook on how the research field of organoids might develop in the coming years and what opportunities organoids hold for in-depth disease modelling and therapeutic interventions.

Anti-VEGF agents were found to have clinical implications for the successful treatment of vascular-driven diseases in humans. In this study, a detailed biological characterization of bevacizumab in a variety of in vitro assays was carried out to determine the effect of bevacizumab on equine umbilical vein endothelial cells (EqUVEC). EqUVECs were harvested from umbilical cords of clinically healthy horses and exposed to different concentrations (1, 2, 4, 6, 8 mg/mL) of bevacizumab (Avastin®). Assays concerning the drug’s safety (cell viability and proliferation assay) and efficacy (cell tube formation assay, cell migration assay, and Vascular endothelial growth factor (VEGF) expression) were carried out reflecting multiple cellular processes. Bevacizumab significantly decreased VEGF expression at all concentrations over a 72 h period. No cytotoxic effect of bevacizumab on EqUVECs was observed at concentrations of 4 mg/mL bevacizumab or lower. Incubated endothelial cells showed delayed tube formation and bevacizumab efficiently inhibited cell migration in a dose-dependent manner. Bevacizumab potently inhibits VEGF-induced cellular processes and could be a promising therapeutic approach in vascular-driven diseases in horses.

Angiogenesis plays a crucial role in various physiological and pathological conditions. However, research in equine angiogenesis is relative limited, necessitating the development of suitable in-vitro models. To effectively analyze angiogenesis in-vitro, it is essential to target the specific cells responsible for this process, namely endothelial cells. Human umbilical vein endothelial cells (HUVECs) are one of the most used in vitro models for studying angiogenesis in humans. Serving as an equivalent to HUVECs, we present a comprehensive isolation protocol for equine umbilical vein endothelial cells (EqUVECs) with relatively minimal requirements, thereby enhancing accessibility for researchers. Umbilical cords obtained from five foals were used to isolate endothelial cells, followed by morphological and immunohistochemical identification. Performance of the cells in various assays commonly used in angiogenesis research was studied. Additionally, EqUVEC expression of vascular endothelial growth factor (VEGF) was assessed using ELISA. EqUVECs exhibited endothelial characteristics, forming a homogeneous monolayer with distinctive morphology. Immunohistochemical staining confirmed positive expression of key endothelial markers including von Willebrand factor (vWF), CD31, and vascular endothelial growth factor receptor-2 (VEGFR-2). Furthermore, performance assessments in in-vitro assays demonstrated the viability, proliferation, migration, tube formation and VEGF-expression capabilities of EqUVECs. The findings suggest that EqUVECs are a promising in-vitro model for studying equine angiogenesis, offering a foundation for further investigations into equine-specific vascular processes and therapeutic interventions.

Cell-free circulating tumor DNA in the plasma of cancer patients has become a common point of interest as indicator of therapy options and treatment response in clinical cancer research. Especially patient- and tumor-specific single nucleotide variants that accurately distinguish tumor DNA from wild type DNA are promising targets. The reliable detection and quantification of these single-base DNA variants is technically challenging. Currently, a variety of techniques is applied, with no apparent \"gold standard\". Here we present a novel qPCR protocol that meets the conditions of extreme sensitivity and specificity that are required for detection and quantification of tumor DNA. By consecutive application of two polymerases, one of them designed for extreme base-specificity, the method reaches unprecedented sensitivity and specificity. Three qPCR assays were tested with spike-in experiments, specific for point mutations BRAF V600E, PTEN T167A and NRAS Q61L of melanoma cell lines. It was possible to detect down to one copy of tumor DNA per reaction (Poisson distribution), at a background of up to 200 000 wild type DNAs. To prove its clinical applicability, the method was successfully tested on a small cohort of BRAF V600E positive melanoma patients.

Background Equine malignant melanoma (EMM) is a frequently occurring dermoepidermal tumor in grey horses. Currently available therapies are either challenging or inefficient. Betulinic acid (BA), a naturally occurring triterpenoid, is a promising compound for cancer treatment. To evaluate the potential of BA as a topical therapy for EMM, its anticancer effects on primary equine melanoma cells and dermal fibroblasts and its percutaneous permeation through isolated equine skin were assessed in vitro. Results BA showed antiproliferative and cytotoxic effects on both primary equine melanoma cells and fibroblasts in a time- and dose-dependent manner. The lowest half-maximal inhibitory concentrations were obtained 96 h after the beginning of drug exposure (12.7 μmol/L and 23.6 μmol/L for melanoma cells eRGO1 and MelDuWi, respectively, in cytotoxicity assay). High concentrations of the compound were reached in the required skin layers in vitro. Conclusion BA is a promising substance for topical EMM treatment. Further clinical studies in horses are necessary to assess safety and antitumoral effects in vivo .

Functional intestinal disorders constitute major, potentially lethal health problems in humans. Consequently, research focuses on elucidating the underlying pathobiological mechanisms and establishing therapeutic strategies. In this context, intestinal organoids have emerged as a potent in vitro model as they faithfully recapitulate the structure and function of the intestinal segment they represent. Interestingly, human-like intestinal diseases also affect dogs, making canine intestinal organoids a promising tool for canine and comparative research. Therefore, we generated organoids from canine duodenum, jejunum and colon, and focused on simultaneous long-term expansion and cell differentiation to maximize applicability. Following their establishment, canine intestinal organoids were grown under various culture conditions and then analyzed with respect to cell viability/apoptosis and multi-lineage differentiation by transcription profiling, proliferation assay, cell staining, and transmission electron microscopy. Standard expansion medium supported long-term expansion of organoids irrespective of their origin, but inhibited cell differentiation. Conversely, transfer of organoids to differentiation medium promoted goblet cell and enteroendocrine cell development, but simultaneously induced apoptosis. Unimpeded stem cell renewal and concurrent differentiation was achieved by culturing organoids in the presence of tyrosine kinase ligands. Our findings unambiguously highlight the characteristic cellular diversity of canine duodenum, jejunum and colon as fundamental prerequisite for accurate in vitro modelling.

The ability of human melanoma cells to switch from an epithelial to a mesenchymal phenotype contributes to the metastatic potential of disease. Metalloproteinases (MPs) are crucially involved in this process by promoting the detachment of tumor cells from the primary lesion and their migration to the vasculature. In gray horse melanoma, epithelial–mesenchymal transition (EMT) is poorly understood, prompting us to address MP expression in lesions versus intact skin by transcriptome analyses and the immunofluorescence staining (IF) of gray horse tumor tissue and primary melanoma cells. RNAseq revealed the deregulation of several MPs in gray horse melanoma and, notably, a 125-fold upregulation of matrix metalloproteinase 1 (MMP1) that was further confirmed by RT-qPCR from additional tumor material. The IF staining of melanoma tissue versus intact skin for MMP1 and tumor marker S100 revealed MMP1 expression in all lesions. The co-expression of S100 was observed at different extents, with some tumors scoring S100-negative. The IF staining of primary tumor cells explanted from the tumors for MMP1 showed that the metalloproteinase is uniformly expressed in the cytoplasm of 100% of tumor cells. Overall, the presented data point to MP expression being deregulated in gray horse melanoma, and suggest that MMP1 has an active role in gray horse melanoma by driving EMT-mediated tumor cell dissemination via the degradation of the extracellular matrix. Whilst S100 is considered a reliable tumor marker in human MM, gray horse melanomas do not seem to regularly express this protein.

Equine hepacivirus (EqHV) is the closest known genetic homologue of hepatitis C virus. An effective prophylactic vaccine is currently not available for either of these hepaciviruses. The equine as potential surrogate model for hepacivirus vaccine studies was investigated, while equine host responses following vaccination with EqHV E2 recombinant protein and subsequent EqHV inoculation were elucidated. Four ponies received prime and booster vaccinations (recombinant protein, adjuvant) four weeks apart (day −55 and −27). Two control ponies received adjuvant only. Ponies were inoculated with EqHV RNA-positive plasma on day 0. Blood samples and liver biopsies were collected over 26 weeks (day −70 to +112). Serum analyses included detection of EqHV RNA, isotypes of E2-specific immunoglobulin G (IgG), nonstructural protein 3-specific IgG, haematology, serum biochemistry, and metabolomics. Liver tissue analyses included EqHV RNA detection, RNA sequencing, histopathology, immunohistochemistry, and fluorescent in situ hybridization. Al-though vaccination did not result in complete protective immunity against experimental EqHV inoculation, the majority of vaccinated ponies cleared the serum EqHV RNA earlier than the control ponies. The majority of vaccinated ponies appeared to recover from the EqHV-associated liver insult earlier than the control ponies. The equine model shows promise as a surrogate model for future hepacivirus vaccine research.

Apical‐out intestinal organoids are a relatively simple method of gaining access to the apical cell surface and have faced increasing scientific interest over the last few years. Apical‐out organoids can thus be used for disease modelling to compare differing effects on the basolateral versus the apical cell surface. However, these ‘inside‐out’ organoids die relatively quickly and cannot be propagated as long as their basal‐out counterparts. Here, we show that apical‐out organoids have drastically reduced proliferative potential, as evidenced by immunohistochemical staining and the incorporation of the thymidine analogue EdU. At the same time, cell death levels are increased. Nevertheless, these phenomena cannot be explained by an induction of differentiation, as the gene expression of key marker genes for various cell types does not change over time. Basal out organoid polarity can easily be reversed by extracellular matrix removal to generate fully apical out intestinal organoids. However, this polarity reversal is accompanied by decreased proliferation coupled with increased cell death, ultimately leading to the death of apical out organoids, questioning their applicability and usefulness for disease modelling. Despite these differences, we demonstrate that transcript expression of key intestinal marker genes is unaffected by polarity reversal.