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BackgroundSkin injuries in horses frequently lead to chronic wounds that lack a keratinocyte cover essential for healing. The limited proliferation of equine keratinocytes using current protocols has limited their use for regenerative medicine. Previously, equine induced pluripotent stem cells (eiPSCs) have been produced, and eiPSCs could be differentiated into equine keratinocytes suitable for stem cell-based skin constructs. However, the procedure is technically challenging and time-consuming. The present study was designed to evaluate whether conditional reprogramming (CR) could expand primary equine keratinocytes rapidly in an undifferentiated state but retain their ability to differentiate normally and form stratified epithelium.MethodsConditional reprogramming was used to isolate and propagate two equine keratinocyte cultures. PCR and FISH were employed to evaluate the equine origin of the cells and karyotyping to perform a chromosomal count. FACS analysis and immunofluorescence were used to determine the purity of equine keratinocytes and their proliferative state. Three-dimensional air-liquid interphase method was used to test the ability of cells to differentiate and form stratified squamous epithelium.ResultsConditional reprogramming was an efficient method to isolate and propagate two equine keratinocyte cultures. Cells were propagated at the rate of 2.39 days/doubling for more than 40 population doublings. A feeder-free culture method was also developed for long-term expansion. Rock-inhibitor is critical for both feeder and feeder-free conditions and for maintaining the proliferating cells in a stem-like state. PCR and FISH validated equine-specific markers in the cultures. Karyotyping showed normal equine 64, XY chromosomes. FACS using pan-cytokeratin antibodies showed a pure population of keratinocytes. When ROCK inhibitor was withdrawn and the cells were transferred to a three-dimensional air-liquid culture, they formed a well-differentiated stratified squamous epithelium, which was positive for terminal differentiation markers.ConclusionsOur results prove that conditional reprogramming is the first method that allows for the rapid and continued in vitro propagation of primary equine keratinocytes. These unlimited supplies of autologous cells could be used to generate transplants without the risk of immune rejection. This offers the opportunity for treating recalcitrant horse wounds using autologous transplantation.

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. [ABSTRACT FROM AUTHOR]

Aims: Inflammation and angiogenesis play an important role in the development of early diabetic kidney disease. We investigated the association of soluble Tumour Necrosis Factor Receptor 1 (sTNF‐R1), sTNF‐R2 and endostatin with new onset microalbuminuria in normoalbuminuric patients with diabetes mellitus type 2. Methods: We conducted a case control study to assess serum levels of sTNF‐R1, sTNF‐R2 and endostatin in 169 patients with new onset microalbuminuria and in 188 matched normoalbuminuric, diabetic controls. Baseline serum samples from participants of the ROADMAP (Randomized Olmesartan and Diabetes Microalbuminuria Prevention) and observational follow‐up (ROADMAP‐OFU) studies were used. Results: Endostatin and sTNF‐R1 but not sTNF‐R2 were increased at baseline in patients with future microalbuminuria. In the multivariate analysis, each log2 increment in endostatin levels was associated with an increase of only 6% in the risk of development of microalbuminuria (adjusted HR (95% CI) 1.006 (1.001–1011). sTNF‐R1 and sTNF‐R2 levels were conversely associated with microalbuminuria, but the results did not reach statistical significance. The respective adjusted HRs (95% CI) were 1.305 (0.928–1.774) and 0.874 (0.711–1.074). Conclusions: sTNF‐R1 and sTNF‐R2 failed to predict the occurrence of microalbuminuria in normoalbuminuric patients with type 2 diabetes. Likewise, the utility of endostatin in predicting new onset proteinuria is limited. [ABSTRACT FROM AUTHOR]

Equine sarcoids are caused by infection with bovine papillomavirus (BPV) types 1, 2, and possibly 13. However, a number of sarcoids lack BPV DNA, and new potential etiological agents for sarcoid diseases need to be considered. High-performance digital droplet polymerase chain reaction (ddPCR) was used for the quantitative detection of ovine papillomavirus (OaPV) types 1–4 DNA from 63 sarcoid DNA samples collected in Austria. All samples were comparatively evaluated for OaPV DNA loads by qPCR. Conventional PCR and amplicon sequencing were used to validate the data. Of the 63 sarcoid DNA isolates, ddPCR was able to detect 22 samples harboring OaPV DNA (34.92%), whereas only five of the OaPV-positive samples were revealed by qPCR (22.72%). The differences in detection by ddPCR and qPCR were statistically significant (p < 0.05). The detected OaPV types were OaPV1, 3, and 4. Both methods failed to detect OaPV2 DNA, which could be due to the limited number of examined samples. Importantly, ddPCR detected multiple types of OaPV DNA in seven cases, whereas the qPCR failed to detect multiple infections. This study is the first to provide evidence of the presence of OaPV types 1, 3, and 4 DNA in a subset of equine sarcoids. The comparative detection approach underscores the superior sensitivity of ddPCR compared to that of qPCR. [ABSTRACT FROM AUTHOR]