Evaluation of quantity and purity of miRNAs extracted from different matrices collected from dogs with Mast Cell Tumours.

MicroRNAs (miRNAs) are a class of short non-coding RNA, which interact with the 3’ UTR region of complementary mRNA to decrease or inhibit the translation of proteins (Lai, 2002). MiRNAs regulate pathways in various pathophysiological status, and are regarded as biomarkers for early diagnosis of several diseases, including cancer (Di Leva et al., 2014).The study aims to evaluate the quality and purity of miRNAs extracted from a) 11 archival Formalin Fixed and Paraffin Embedded (FFPE) samples of Mast Cell Tumour (MCT) at stage I, II, III and IV, and 8 intra-patient healthy controls; b) samples collected during surgery, including 6 samples of saliva, primary tumour biopsy and serum/plasma. The quality of miRNA largely influence the downstream experiments, and must be carefully evaluated before performing for examples, the sequencing reaction. MiRNA extraction was carried out using commercial kits (Qiagen) and quantify using Small RNA Kit (Agilent) on Agilent 2100 Bioanalyzer. The results showed that the concentration of miRNAs from FFPE, saliva,  primary tumor biopsy and serum was acceptable with a Median (Me)= 56,91 ng/ml, Me=10,30 ng/ml, Me=3,44 ng/ml and  Me=0,71 ng/ml, and a miRNA/Small RNA ratio of 48%, 61%, 17% and 76%, respectively. The concentration of miRNAs from plasma was not detectable. Studies reveal that plasma ranks as the first choice source for diagnostic purpose, much more than serum (Aung et al., 2014), but the debate remains open and subsequent analyses are needed.The concentration of miRNAs from FFPE and saliva samples is higher than that from other matrices. Possible explanations include a) different quantity and quality of starting materials; b) nucleic acids fragmentation, due to the formalin fixation and paraffin embedded procedure; c) presence of nucleases in saliva, which produce small fragments recognized as miRNAs or smallRNAs.In conclusion, the quantity and the purity of miRNAs, obtained using Qiagen commercial kits, are reliable for further NGS analysis

Characterization of skin surface and dermal microbiota in dogs with mast cell tumor

The skin microbiota interacts with the host immune response to maintain the homeostasis. Changes in the skin microbiota are linked to the onset and the progression of several diseases, including tumors. We characterized the skin surface and dermal microbiota of 11 dogs affected by spontaneous mast cell tumor (MCT), using skin contralateral sites as intra-animal healthy controls. The microbial profile differed between healthy and tumor skin surfaces and dermis, demonstrating that the change in microbiota composition is related to the presence of MCT. The number of observed taxa between MCT and healthy skin surfaces was detected, showing a decrease in number and heterogeneity of taxa over the skin surface of MCT, at both inter- and intra-individual level. Preliminary data on bacterial population of MCT dermis, obtained only on three dogs, demonstrated an intra-individual reduction of taxa number when compared to the skin surface. Taxonomy reveals an increase of Firmicutes phylum and Corynebacteriaceae family in MCT skin surface when compared to the healthy contralateral. In conclusion, we demonstrate that microbial population of skin surface and dermis is related to mast cell tumor. Our study provides the basis for future investigations aiming to better define the interaction between mast cell tumors, microbiota and host immune response

Identification of Altered miRNAs in Cerumen of Dogs Affected by Otitis Externa

Otitis externa is one of the most common diseases in dogs. It is associated with bacteria and yeast, which are regarded as secondary causes. Cerumen is a biological substance playing an important role in the protection of ear skin. The involvement of cerumen in immune defense is poorly understood. MicroRNAs can modulate the host immune response and can provide promising biomarkers for several inflammatory and infectious disorder diagnosis. The aims of this study were to profile the cerumen miRNA signature associated with otitis externa in dogs, integrate miRNAs to their target genes related to immune functions, and investigate their potential use as biomarkers. Cerumen was collected from healthy and otitis affected dogs and the expression of miRNAs was profiled by Next Generation Sequencing; the validation of the altered miRNAs was performed using RT-qPCR. The potential ability of miRNAs to modulate immune-related genes was investigated using bioinformatics tools. The results pointed out that 32 miRNAs, of which 14 were up- and 18 down-regulated, were differentially expressed in healthy vs. otitis-affected dogs. These results were verified by RT-qPCR. To assess the diagnostic value of miRNAs, ROC analysis was carried out, highlighting that 4 miRNAs are potential biomarkers to discriminate otitis-affected dogs. Bioinformatics showed that cerumen miRNAs may be involved in the modulation of host immune response. In conclusion, we have demonstrated for the first time that miRNAs can be efficiently extracted and quantified from cerumen, that their profile changes between healthy and otitis affected dogs, and that they may serve as potential biomarkers. Further studies are necessary to confirm their diagnostic value and to investigate their interaction with immune-related genes

Circulating MiR-30b-5p is upregulated in Cavalier King Charles Spaniels affected by early myxomatous mitral valve disease.

There is a growing interest in developing new molecular markers of heart disease in young dogs affected by myxomatous mitral valve disease. The study aimed to measure 3 circulating microRNAs and their application as potential biomarkers in the plasma of Cavalier King Charles Spaniels with early asymptomatic myxomatous mitral valve disease. The hypothesis is that healthy Cavalier King Charles Spaniels have different microRNA expression profiles than affected dogs in American College of Veterinary Internal Medicine (ACVIM) stage B1. The profiles can differ within the same class among subjects of different ages. This is a prospective cross-sectional study. Thirty-three Cavalier King Charles Spaniels in ACVIM stage B1 were divided into three groups (11 younger than 3 years, 11 older than 3 years and younger than 7 years, and 11 older than 7 years), and 11 healthy (ACVIM stage A) dogs of the same breed were included as the control group. Three circulating microRNAs (miR-1-3p, miR30b-5p, and miR-128-3p) were measured by quantitative real-time PCR using TaqMan® probes. Diagnostic performance was evaluated by calculating the area under the receiver operating curve (AUC). MiR-30b-5p was significantly higher in ACVIM B1 dogs than in ACVIM A subjects, and the area under the receiver operating curve was 0.79. According to the age of dogs, the amount of miR-30b-5p was statistically significantly higher in group B17y (2.7 folds, P = 0.018) than in group A. The area under the receiver operating curves were fair in discriminating between group B17y and A (AUC 0.822). Identifying dogs with early asymptomatic myxomatous mitral valve disease through the evaluation of miR-30b-5p represents an intriguing possibility that certainly merits further research. Studies enrolling a larger number of dogs with preclinical stages of myxomatous mitral valve disease are needed to expand further and validate conclusively the preliminary findings from this report

MicroRNA Expression in Formalin-Fixed, Paraffin-Embedded Samples of Canine Cutaneous and Oral Melanoma by RT-qPCR

MicroRNAs (miRNAs) are a class of small, noncoding RNA that post-transcriptionally regulate protein expression. miRNAs are emerging as clinical biomarkers of many diseases, including tumors. The aim of this study was to investigate whether miRNA expression could vary in melanoma samples derived from formalin-fixed, paraffin-embedded (FFPE) tissues. The study included 4 groups: (1) 9 samples of oral canine malignant melanoma, (2) 10 samples of cutaneous malignant melanoma, (3) 5 samples of healthy oral mucosa, and (4) 7 samples of healthy skin. The expression levels of 6 miRNAs-miR-145, miR-146a, miR-425-5p, miR-223, miR-365, and miR-134-were detected and assessed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) using TaqMan probes. Cutaneous canine malignant melanoma showed a decrease of the expression level of miR-145 and miR-365 and an increase of miR-146a and miR-425-5p compared to control samples. MiR-145 was also downregulated in oral canine malignant melanoma. The miRNAs with decreased expression may regulate genes involved in RAS, Rap1, and transforming growth factor β (TGF-β) signaling pathways, as well as upregulated genes associated with phosphatidylinositol signaling system, adherens junction, and RAS signaling pathways. In conclusion, miR-145, miR-365, miR-146a, and miR-425-5p were differentially expressed in canine malignant melanoma and healthy FFPE samples, suggesting that they may play a role in canine malignant melanoma pathogenesis

Salivary miR-21 is a potential biomarker for canine mast cell tumors

MicroRNAs (miRNAs) are a class of noncoding RNA molecules playing a crucial role in tumor modulation targeting mRNA. This study aimed to validate the diagnostic potential of a panel of 3 miRNAs previously identified in canine mast cell tumors (MCTs), miR-21, miR-379, and miR-885, as markers of lymph node involvement in terms of histological absence (nonmetastatic: HN0; premetastatic: HN1) and presence (early-metastatic: HN2; overt-metastatic: HN3) of metastasis, in the saliva of mast cell tumor (MCT)-affected dogs by quantitative polymerase chain reaction (PCR). Forty-seven saliva samples were analyzed: 36 from MCT-affected dogs (12 subcutaneous [3 HN0-1 and 9 HN2-3] and 24 cutaneous [9 HN0-1 and 15 HN2-3—MCT]) and 11 from healthy dogs. MCT-group effects were investigated using analysis of variance (ANOVA). The origin of the tumor affected the expression of salivary miR-21 ( P = .011) with an increase in cases with subcutaneous MCTs compared with the healthy group ( P = .0005) and those with cutaneous MCTs ( P = .004). Salivary miR-21 was higher in the HN2-3 class compared with the healthy group ( P = .004). Salivary miR-885 was not affected by the presence of MCT, while miR-379 was not detected in saliva. The diagnostic potential of salivary miR-21 in discriminating MCT-affected dogs from the healthy group (AUC = 0.8917), cutaneous from subcutaneous (AUC = 0.8111), and subcutaneous HN0-1 (AUC = 0.7250) and HN2-3 (AUC = 0.9750) classes from healthy samples was demonstrated by receiver operating characteristic curve analysis. Overall, salivary miR-21 was identified as a promising tool, representing a novel approach to detecting MCT-associated epigenetic alterations in a minimally invasive manner

Treating iPSC-Derived β Cells with an Anti-CD30 Antibody–Drug Conjugate Eliminates the Risk of Teratoma Development upon Transplantation

Insulin-producing cells derived from induced pluripotent stem cells (iPSCs) are promising candidates for β cell replacement in type 1 diabetes. However, the risk of teratoma formation due to residual undifferentiated iPSCs contaminating the differentiated cells is still a critical concern for clinical application. Here, we hypothesized that pretreatment of iPSC-derived insulin-producing cells with an anti-CD30 antibody–drug conjugate could prevent in vivo teratoma formation by selectively killing residual undifferentiated cells. CD30 is expressed in all human iPSCs clones tested by flow cytometry (n = 7) but not in iPSC-derived β cells (iβs). Concordantly, anti-CD30 treatment in vitro for 24 h induced a dose-dependent cell death (up to 90%) in human iPSCs while it did not kill iβs nor had an impact on iβ identity and function, including capacity to secrete insulin in response to stimuli. In a model of teratoma assay associated with iβ transplantation, the pretreatment of cells with anti-CD30 for 24 h before the implantation into NOD-SCID mice completely eliminated teratoma development (0/10 vs. 8/8, p < 0.01). These findings suggest that short-term in vitro treatment with clinical-grade anti-CD30, targeting residual undifferentiated cells, eliminates the tumorigenicity of iPSC-derived β cells, potentially providing enhanced safety for iPSC-based β cell replacement therapy in clinical scenarios

Effect of inactivated cultures of Lactobacillus rhamnosus on subclinical mastitis quarter milk microbiota

Water buffaloes mastitis represents a major issue in terms of animal health, cost of therapy, premature culling and decreased milk yeld. The emergence of antibiotic resistance has led to investigate strategies in order to avoid or minimize the antibiotic use, especially during subclinical mastitis disease (SM) (1). Lactobacillus rhamnosus is part of the normal gut microflora, having meanwhile an immunostimulatory activity (2). The aim of this study was to investigate the change of milk microbiota after the theraputic treatment with inactivated cultures of Lactobacillus rhamnosus of mammary gland quarters affected by subclinical mastitis. A number of 43 quarters were included in the study, and were treated with antibiotics, Lactobacillus rhamnosus  and PBS as negative control. Samples were collected at two time points, T0 and T5 (days) and V4 region of 16S rRNA gene was amplified by PCR and sequenced using Ion Torrent Personal Genome Machine. The microbiota structure of SM quarters showed no major changes after PBS treatment, while differed after antibiotic treatment where Staphylococcus decreased its relative abundance from 41% at T0 to 3% at T5. Lactobacillus rhamnosus induced a less dramatic change in milk microbiota, although the relative abundance of some genera were found to be modifidied, among which an increase of Pseudomonas from 1.5% at T0 up to 4% at T5. No differences were present between the microbiota structure of quarters treated with Lactobacillus rhamnosus and PBS. This study allowed to characterize the changes of microbiota in milk from animals with Lactobacillus rhamnosus and antibiotics. While changes in milk microbiota after antibiotic treatment were evident, changes after Lactobacillus rhamnosus were more limited. Further investigations are needed to evaluate alternative strategies to mastitis treatment.