Combined effects of glyphosate and chemical hypoxia in zebrafish: A new toxicological point of view.

Glyphosate (Gly), a systemic and non-selective post-emergence herbicide used worldwide, has emerged as a pollutant. However, its toxic effects are debated by regulatory authorities. In addition, in the aquatic environment, often the presence of pollutants is associated with a hypoxia condition that could change their toxicological effects. We used zebrafish embryos to evaluate the toxic effects of Gly and its mechanisms in a hypoxic condition chemically induced by cobalt chloride (CoCl2). We found that Gly induced toxicity in a time and concentration-dependent manner. The toxicity of Gly was determined at 96 h post fertilization as a lethal concentration (LC), and LC10, LC20, and LC50 values were 85.7, 97, and 122.9 mg/L, respectively. When Gly was combined with CoCl2 the toxicological endpoints were lower than values referred to the Gly alone indicating the worse effects of chemical hypoxia on Gly toxicity. Histological observations were performed at 25, 50, 75, and 100 mg/L for Gly both alone and in combination with 10 mM CoCl2. Fisher's exact test showed significant differences in the presence of hepatic and gut inflammation at 75 and 100 mg/L of Gly both alone and in combination with CoCl2. To deeply investigate the effects of hypoxia on Gly toxicity we decided to test the lowest dose of Gly, 50 mg/L, alone or in combination with CoCl2 10 mM on liver glycogen storage and oxidative stress. Again the results obtained indicate the worse effects of chemical hypoxia on Gly toxicity. Thus Gly toxicity could be reconsidered in light of the damage it causes to the liver and intestines and its effect in combination with factors that induce chemical hypoxia.

Antibiotic-induced dysbiosis in the SCIME™ recapitulates microbial community diversity and metabolites modulation of in vivo disease.

Establishing the context: Intestinal dysbiosis is a significant concern among dog owners, and the gut health of pets is an emerging research field. In this context, the Simulator of the Canine Intestinal Microbial Ecosystem (SCIME™) was recently developed and validated with in vivo data. Stating the purpose/introducing the study: The current study presents a further application of this model by using amoxicillin and clavulanic acid to induce dysbiosis, aiming to provoke changes in microbial community and metabolite production, which are well-known markers of the disease in vivo. Describing methodology: Following the induction of dysbiosis, prebiotic supplementation was tested to investigate the potential for microbiota recovery under different dietary conditions. Presenting the results: The results showed that antibiotic stimulation in the SCIME™ model can produce significant changes in microbial communities and metabolic activity, including a decrease in microbial richness, a reduction in propionic acid production, and alterations in microbial composition. Additionally, changes in ammonium and butyric acid levels induced by the tested diets were observed. Discussing the findings: This alteration in microbial community and metabolites production mimicks in vivo canine dysbiosis patterns. A novel dynamic in vitro model simulating canine antibiotic-induced dysbiosis, capable of reproducing microbial and metabolic changes observed in vivo, has been developed and is suitable for testing the effects of nutritional changes.

Unusual tongue metastasis from lung adenocarcinoma in a cat with feline lung-digit syndrome.

This report describes the pathological findings in a 15-year-old spayed female Domestic Shorthaired cat with a pulmonary adenocarcinoma characterized by feline lung-digit syndrome (FLDS) and unusual tongue metastasis. Felis catus papillomavirus type 3 (FcaPV-3) DNA was amplified from the lingual sample but not from samples of the pulmonary mass or digital or splenic metastatic lesions, indicating the presence of FcaPV-3 in the oral cavity but not suggesting a role for FcaPVs in tumour pathogenesis. FLDS is a clinical entity in which primary lung tumours present because of metastatic digital lesions. In humans, tongue metastasis may be a rare initial presentation of lung cancer, whereas, to the best of our knowledge, tongue metastasis of feline tumours has not been reported. Although lingual metastases are rare, the present findings serve to remind clinicians that metastatic manifestations of primary lung tumours in cats may involve multiple extrapulmonary sites, including the tongue.

p63 immunoexpression in hair follicles of normal and alopecia X-affected skin of Pomeranian dogs.

BACKGROUND: Alopecia X in Pomeranians is caused by a hair cycle deregulation, associated with downregulation of key regulatory genes of the Wnt and Shh pathways, and stem-cell markers. However, the pathogenesis remains unclear. p63 is an important transcription factor correlated with the aforementioned hair cycle modulating genes. HYPOTHESIS/OBJECTIVES: The aim of this study was to highlight possible changes of p63 immunohistochemical expression within the hair follicles in canine alopecia X compared with normal skin. ANIMALS: Skin biopsies from 19 alopecia X-affected and six control Pomeranians were analysed. MATERIALS AND METHODS: Serial histological sections of skin biopsies harbouring anagen, telogen and kenogen hair follicles were immunohistochemically evaluated for differences in p63 expression in the affected and control samples. RESULTS: Dogs with alopecia X had a significantly decreased immunoexpression of p63 in telogen and kenogen hair follicles. CONCLUSIONS AND CLINICAL RELEVANCE: The decrease of p63 immunoexpression observed in canine alopecia X suggests an involvement of p63 in hair cycle.

Strain and Shear-Wave Elastography and Their Relationship to Histopathological Features of Canine Mammary Nodular Lesions.

Mammary gland tumours have a significant impact on the health of dogs, requiring diagnostic tools to support clinicians to develop appropriate therapeutic strategies. Sonoelastography is an emerging technology that is able to define the stiffness of the tissue and has promising applications in the evaluation of mammary gland lesions. In the present study, strain elastography (STE) and shear-wave (SWE) elastography were compared in 38 mammary nodular lesions for their ability to define the histopathological features of canine mammary lesions. Among the techniques, SWE showed better repeatability (intraclass correlation coefficient: 0.876), whereas STE was found to be only acceptable (intraclass correlation coefficient: 0.456). Mammary nodular lesions showed a wide range of tissue stiffening with a similar mean value for STE and SWE in benign (4 ± 0.3 and 115.4 ± 12.6 kPa, respectively) and malignant lesions (3.8 ± 0.1 and 115.5 ± 4.5 kPa, respectively). A significant correlation was found between lesion fibrosis and STE (STE-I: r = 0.513, p < 0.001; STE-R: r = 0.591, p < 0.001) or SWE-S (r = 0.769; p < 0.001). In conclusion, SWE was reliable and correlated with fibrosis and was similar for both benign and malignant lesions, suggesting that other collateral diagnostic techniques should be considered in conjunction with SWE to characterize mammary nodular lesions in dogs.

A Set of Dysregulated Target Genes to Reduce Neuroinflammation at Molecular Level.

Increasing evidence links chronic neurodegenerative diseases with neuroinflammation; it is known that neuroprotective agents are capable of modulating the inflammatory processes, that occur with the onset of neurodegeneration pathologies. Here, with the intention of providing a means for active compounds' screening, a dysregulation of neuronal inflammatory marker genes was induced and subjected to neuroprotective active principles, with the aim of selecting a set of inflammatory marker genes linked to neurodegenerative diseases. Considering the important role of microglia in neurodegeneration, a murine co-culture of hippocampal cells and inflamed microglia cells was set up. The evaluation of differentially expressed genes and subsequent in silico analysis showed the main dysregulated genes in both cells and the principal inflammatory processes involved in the model. Among the identified genes, a well-defined set was chosen, selecting those in which a role in human neurodegenerative progression in vivo was already defined in literature, matched with the rate of prediction derived from the Principal Component Analysis (PCA) of in vitro treatment-affected genes variation. The obtained panel of dysregulated target genes, including Cxcl9 (Chemokine (C-X-C motif) ligand 9), C4b (Complement Component 4B), Stc1 (Stanniocalcin 1), Abcb1a (ATP Binding Cassette Subfamily B Member 1), Hp (Haptoglobin) and Adm (Adrenomedullin), can be considered an in vitro tool to select old and new active compounds directed to neuroinflammation.

Histological Evaluation of Long-Term Collagen Type I Culture.

Histological approach to long-term culture on collagen type I permits the evaluation of vasculogenic mimicry morphological features and the identification of endothelial-like cell-specific antigens. Here, we show the preparation of collagen type I solution, the embedding and the sections cutting of D17 osteosarcoma cells long-term culture, and then the hematoxylin and eosin (H&E) staining to identify endothelial-like structure. Moreover, we provide the protocols for periodic acid-Schiff (PAS) staining to evidence glycoproteins and CD31 immunohistochemistry to exclude the presence of this endothelial marker, as per definition by vasculogenic mimicry concept. This method allows to consider long-term culture as tissue, promoting the deeper study of vascular-like structures in vitro.

Health and Mortality Monitoring in Threatened Mammals: A First Post Mortem Study of Otters (Lutra lutra L.) in Italy.

Dead specimens provide valuable data for the conservation of threatened species, allowing investigations of mortality, health conditions, and demographic parameters. The Eurasian otter (Lutra lutra) is a semiaquatic carnivore listed as endangered in Italy. In 2009, we started the first post mortem (PM) study of otters in Italy, through collaborative research between mammal ecologists and veterinary pathologists, using standardized protocols. Twenty-eight otters, mostly collected between 2009 and 2017, were examined. Most otters were males (67%), between 1 and 3 years old (64%), and predominantly in good nutritional condition. Adult males were significantly larger than adult females (p < 0.02), as expected for the species, although both sexes appeared to be smaller than otters examined in Central−northern Europe. The youngest sexually mature female was 3 years old. Road traffic collisions were the major cause of death, especially in young individuals, and mainly occurred in autumn−winter, particularly for females. Investigations of the scene of death contributed to revealing factors forcing otters to travel out of the water and move over the road, suggesting appropriate measures to reduce vehicle collision risk. Other causes of death included blunt chest trauma of uncertain origin, dog and conspecific attacks, or diseases of infectious or non-infectious origin, such as ulcerative gastritis, pleuropneumonia and peritonitis. Other diagnosed diseases included lymphoma. Ecto- and endoparasites were rarely detected, although we report the first documentation of heartworm and Ixodes hexagonus infestation in Italian otters. It is important to continue comprehensive, standardized PM investigations of otters in Italy to define baseline health, biometric and demographic parameters, collect biological samples for comparative analyses, and to reduce road-kill mortality. The present study suggests that the timely collection of carcasses and collaborative and coordinated research efforts are essential for obtaining useful data for the conservation of otters.

An Update on Molecular Pathways Regulating Vasculogenic Mimicry in Human Osteosarcoma and Their Role in Canine Oncology.

Canine tumors are valuable comparative models for human counterparts, especially to explore novel biomarkers and to understand pathways and processes involved in metastasis. Vasculogenic mimicry (VM) is a unique property of malignant cancer cells which promote metastasis. Thus, it represents an opportunity to investigate both the molecular mechanisms and the therapeutic targets of a crucial phenotypic malignant switch. Although this biological process has been largely investigated in different human cancer types, including osteosarcoma, it is still largely unknown in veterinary pathology, where it has been mainly explored in canine mammary tumors. The presence of VM in human osteosarcoma is associated with poor clinical outcome, reduced patient survival, and increased risk of metastasis and it shares the main pathways involved in other type of human tumors. This review illustrates the main findings concerning the VM process in human osteosarcoma, search for the related current knowledge in canine pathology and oncology, and potential involvement of multiple pathways in VM formation, in order to provide a basis for future investigations on VM in canine tumors.

Polyphenols and Cannabidiol Modulate Transcriptional Regulation of Th1/Th2 Inflammatory Genes Related to Canine Atopic Dermatitis.

Canine atopic dermatitis (AD) is a multifactorial allergic disease associated with immune and abnormal skin barrier dysfunction and it is one of the primary causes of pruritus. Using a novel in vitro model of AD, here we tried to revert the alteration of transcriptional regulation of AD canine key genes testing a nutraceutical mixture containing flavonoids, stilbene, and cannabinoids, which are already well-known for their applications within dermatology diseases. The nutraceutical mixture induced in inflamed cells a significant downregulation (p < 0.05) of the gene expression of ccl2, ccl17, and tslp in keratinocytes and of ccl2, ccl17, and il31ra in monocytes. Consistent with the observed alterations of tslp, ccl2, ccl17, and il31ra messenger RNA (mRNA) levels, a significant increase (p < 0.05) of DNA methylation at specific CpG sites on the gene regulatory regions was found. These results lay the foundation for the use of these natural bioactives in veterinary medicine and provide a model for deeper understanding of their mechanisms of action, with potential translation to human research.

Impact of Nutritional Supplementation on Canine Dermatological Disorders.

Nutritional supplements, also known as complementary feeds, are products administered with the aim of furnishing health benefits, regardless of nutritional needs. They have been used since ancient times in veterinary dermatology, and a number of studies have focused on investigating the health benefits of some ingredients found in commercially available complementary feed for dogs. The aim of this paper is to review the literature available on the use of nutritional supplementation for the management of canine skin diseases, critically appraising the clinical efficacy of such interventions and summarizing the current state of knowledge. This review highlights how these feeds can be considered useful in the management of dermatological disorders and outlines their beneficial effects in the prevention of dietary deficiencies and treatment of diseases, alone, or in addition to conventional pharmacological therapy. In recent years, nutritional supplements have found increasing potential application in veterinary medicine, and the scientific proofs of their beneficial effects are described in this review.

Inflammatory myofibroblastic tumor of the pancreas in a dog.

A large, ill-defined, firm, multinodular mass involving the pancreas was confirmed on postmortem examination of a 5-y-old, male Rottweiler that died following acute respiratory distress syndrome, after a period of anorexia and lethargy. Histologically, the mass consisted of plump spindle cells admixed with a variable number of macrophages, lymphocytes, plasma cells, and neutrophils. Foci of coagulative necrosis and hemorrhage were also observed. Spindle cells strongly reacted to antibodies against vimentin, α-smooth muscle actin, and calponin, whereas desmin was expressed only mildly and focally. Pan-cytokeratin, KIT, glial fibrillary acidic protein, and S100 protein were nonreactive. Variable numbers of MAC 387-positive cells, CD3+ lymphocytes, and numerous blood vessels were also detected throughout the mass. Histologic and IHC findings were consistent with a diagnosis of inflammatory myofibroblastic tumor of the pancreas.

Left atrial appendage rupture due to blunt chest trauma in an Eurasian otter (Lutra lutra).

An adult male Eurasian otter, found dead on the roadside, was submitted for post-mortem examination in April 2014 at the Veterinary Pathology Unit of the Faculty of Veterinary Medicine of Teramo, as part of the RECAL [RECovery and post mortem Analysis of Eurasian otters (Lutra lutra) in the National Park of Cilento, Vallo di Diano and Alburni (Salerno, Italy), and surrounding areas] project. Necropsy revealed an abundant hemothorax associated with multifocal, bilateral pulmonary contusions and lacerations, and a severe hemopericardium characterised by the presence of a wide blood clot in the intact pericardial sac. Two small laceration wounds of the left auricle were found at the base, along the atrioventricular groove, and on the outer free wall. Since myocardial and endocardial tissues showed no other gross and histopathological abnormalities, a left atrial appendage rupture resulting from a blunt chest trauma was diagnosed. Blunt traumatic cardiac rupture is a rarely reported, life-threatening condition in humans. To the best of our knowledge, this is the first report on a left atrial appendage rupture due to blunt chest trauma in veterinary literature. The possible occurrence of a cardiac rupture following a blunt thoracic injury should be taken into consideration in veterinary emergency care.

Preclinical and Clinical Evidence for a Distinct Regulation of Mu Opioid and Type 1 Cannabinoid Receptor Genes Expression in Obesity.

Among endogenous signaling networks involved in both rewarding and homeostatic mechanisms of obesity, a relevant role is played by the endocannabinoid (ECS) and the opioid (EOS) systems. We here studied the transcriptional regulation of ECS and EOS genes in the hypothalamus of Diet-induced obesity rats, a preclinical model of obesity, as well as in humans with obesity and healthy controls. A significant and selective increase in type 1 cannabinoid receptor gene (Cnr1) expression was observed at the beginning of obesity development (5 weeks on high fat diet) as well as after 21 weeks of high diet exposure. After 5 weeks on high fat diet, selective up-regulation of mu opioid receptor gene (Oprm1) expression was also observed. Consistently, epigenetic studies showed a selective and significant decrease in DNA methylation at specific CpG sites at both gene promoters in overweight rats, but only after 5 weeks on high fat diet. Moreover, significantly lower levels of DNA methylation were observed at selected CpG sites of both receptor gene promoters, analyzed in peripheral blood mononuclear cells from younger (<30 years old) humans with obesity, as well as in those with shorter time length from disease onset. Taken together, we here provide evidence of selective, synergistic and time-dependent transcriptional regulation of CNR1 and OPRM1 genes in overweight rats, as well as in human subjects. These alterations in genes regulation could contribute to the development of the obese phenotype, and we thus suggest CNR1 and OPRM1 epigenetic modulation as possible biomarkers of obesity development. Due to the reversible nature of the epigenetic hallmark, our data might also open new avenue to early environmental strategies of intervention.

On the Role of Adenosine A2A Receptor Gene Transcriptional Regulation in Parkinson's Disease.

Adenosine A2A receptors (A2ARs) have attracted considerable attention as an important molecular target for the design of Parkinson's disease (PD) therapeutic compounds. Here, we studied the transcriptional regulation of the A2AR gene in human peripheral blood mononuclear cells (PBMCs) obtained from PD patients and in the striatum of the well-validated, 6-hydroxydopamine (6-OHDA)-induced PD mouse model. We report an increase in A2AR mRNA expression and protein levels in both human cells and mice striata, and in the latter we could also observe a consistent reduction in DNA methylation at gene promoter and an increase in histone H3 acetylation at lysine 9. Of particular relevance in clinical samples, we also observed higher levels in the receptor gene expression in younger subjects, as well as in those with less years from disease onset, and less severe disease according to clinical scores. In conclusion, the present findings provide further evidence of the relevant role of A2AR in PD and, based on the clinical data, highlight its potential role as disease biomarker for PD especially at the initial stages of disease development. Furthermore, our preclinical results also suggest selective epigenetic mechanisms targeting gene promoter as tool for the development of new treatments.

Establishment of three-dimensional canine osteosarcoma cell lines showing vasculogenic mimicry and evaluation of biological properties after treatment with 17-AAG.

Vasculogenic mimicry (VM) is an alternative type of blood perfusion characterized by formation of non-endothelial cell-lined microcirculatory channels and is responsible for aggressive tumour biology and increased tumour-related mortality. VM-correlated genes are associated with vascular endothelial grown factor receptor 1 (VEGFR1), and hypoxia-related (hypoxia inducible factor 1 α-HIF1α) signalling pathways, whose molecules are client proteins of Hsp90 (heat shock protein 90) and are potential therapeutic targets. This pilot study was aimed to investigate vasculogenic mimicry in a three-dimensional (3D) cell culture system of two aggressive canine osteosarcoma (OSA) cell lines (D22 and D17), and to evaluate the response of these cells to 17-AAG (17-N-allylamino-17-demethoxygeldanamycin) treatment in relation to tubular-like structure formation in vitro. Only D17 cell line formed hollow matrix channels in long-term 3D cultures and assumed endothelial morphology, with cells expressing both Hsp90 and VEGFR1, but lacking expression of endothelial marker CD31. 17-AAG treatment inhibited migration of D17 OSA cells, also decreasing VM markers in vitro and inducing a reduction of HIF1α transcript and protein in this cell line. Taken together, these preliminary data indicate that the biological effects of 17-AAG on D17 3D culture and on HIF1α regulation can provide interesting information to translate these findings from the basic research to clinical approach for the treatment of canine OSA as a model in comparative oncology.

Nectin-4 and p63 immunohistochemical expression in canine prostate tumourigenesis.

Nectin-4 is an E-cadherin-based adherens junction protein of normal epithelial cells, as well as a potent mediator of anchorage-independent cancer colony formation. It is considered a tumour-associated histological and serological marker in various human cancers. The transcription factor p63 is a basal cell marker in the normal prostate, involved in cell adhesion, as well as in the formation and survival of circulating tumour cell clusters. The aim of this study was to evaluate Nectin-4 and p63 immunohistochemical expression in 42 canine prostate tissues including 2 normal prostates, 10 benign prostatic hyperplasias (BPHs), 30 prostatic carcinomas (PCs), 1 pulmonary and 1 lymph node metastasis. From normal to neoplastic tissues, Nectin-4 showed a progressive switching from membranous (m-Nectin-4) to cytoplasmic (c-Nectin-4), regardless of the histological subtypes, except for lack of expression in solid PCs. Metastatic cells exhibited both strong membranous and cytoplasmic positivity. c-Nectin-4 expression was significantly (P < 0.0001) increased in PCs/metastasis compared to BPHs cases and a decrease (P < 0.05) of nuclear p63 immunostaining was also detected in the two groups. Furthermore, data showed a significant association (P < 0.05) between p63 and m-Nectin-4 distribution, although their colocalization was detected only in scattered cells by double immunofluorescence. Our results suggest the involvement of m-Nectin-4 in canine prostate tumourigenesis and metastatic potential, while the exact role of c-Nectin-4 expression detectable in primary PCs requires further investigations.

Survivin and Sox9: Potential Stem Cell Markers in Canine Normal, Hyperplastic, and Neoplastic Canine Prostate.

Canine prostatic carcinoma is a relevant model for human prostatic carcinoma. Survivin is proposed as a biomarker of malignancy in human prostatic cancer. Sox9 is a stem cell marker required for prostate development and expressed in several adult tissues. The aims of the present study were to evaluate the patterns and expression levels of 2 putative stem cell markers, survivin and Sox9, in canine benign prostatic hyperplasia (BPH) and prostatic carcinoma to investigate their potential as stem cell markers. Immunohistochemistry with specific antibodies was performed on 3 samples of normal prostate gland, 18 samples of canine BPH, and 16 samples of prostatic carcinoma. The basal cell layer of normal and hyperplastic prostatic lobules had nuclear Sox9 immunolabeling and nuclear and rarely cytoplasmic survivin immunostaining, identifying them as potential stem cell markers. Significantly more frequent survivin and Sox9 expression (≥10% of nuclei) was observed in prostatic carcinoma as compared with BPH. The potential coexpression of survivin with Sox9, androgen receptor, and p63 was also investigated in selected BPH and prostatic carcinoma cases with immunofluorescence, and a partial colocalization was observed. Results indicate that Sox9 and survivin could be considered markers of stemness in canine prostate cells. Given its role in proliferation, cells in the basal cell layer with nuclear survivin expression are likely to be transit-amplifying cells that maintain some stem cell proprieties.