COMPARATIVE EVALUATION OF PROGNOSTIC MARKERS IN CANINE AND FELINE MELANOMAS

The present PhD project investigates animal spontaneous models of non-UV induced melanomas, namely canine oral melanoma and feline iris diffuse melanoma (FDIM), which shares unique similarities in biological behavior with human mucosal melanoma and human iris melanoma, respectively. The project investigates selected markers related to the pathogenesis and prognosis of these tumors, i.e. gene and proteins that have been implicated in the progression and metastasis in human, canine and feline melanomas, such as Leukotriene A4 Hydrolase (LTA4H), Fragile X mental retardation-related protein 1 (FXR1) and matrix-metalloproteinases (MMPs). LTA4H is an enzyme of the arachidonic acid cascade, FXR1 is a RNA binding protein, MMPs a family of proteolytic enzymes of the extracellular matrix. The specific aims of the project are: 1) the validation of anti-FXR1 antibodies in the canine species; 2) the investigation of the expression of LTA4H and FXR1 in canine oral melanoma; 3) the study of FXR1-induced modulation of MMPs in canine oral melanoma; 4) the study of MMPs and tumor-matrix interaction in feline diffuse iris melanoma. 1) Two different commercially available polyclonal anti-human FXR1 antibodies were validated for use in dogs. Western blot experiments highlighted the specificity of cross-reaction. Immunohistochemistry described for the first time the specific distribution of FXR1 protein in canine normal tissues, and then the expression of FXR1 in a pool of canine melanocytic tumors. 2) LTA4H and FXR1 genes and proteins expression was investigated in FFPE canine oral melanomas (histology and immunohistochemistry, n=36, from 32 dogs; RT-PCR, subset n=23; clinical follow-up, subset n=13). \u394Ct expression values ranged 0.76-5.11 for LTA4H and 0.22-6.24 range for FXR1 (out of range in 3 cases). The immunohistochemical expression of the proteins was evaluated as IRS-score (percentage of positive cells combined with intensity of the staining). IRS-score of LTA4H and FXR1 proteins did not correlate with the expression of the codifying genes. LTA4H and FXR1 seemed not correlated with the known criteria of malignancy or with the clinical outcome, when available. 3) Since FXR1 belongs to a family of RNA binding protein able to modulate the mRNA coding for the proteolytic enzyme MMP-9, MMP-9 and its inhibitor TIMP-2 were investigated by immunohistochemistry in canine oral melanomas to assess the association of FXR1 with MMP-9 and the association of MMPs activity with the clinical outcome. MMP-9 expression seemed not associated with FXR1 in canine oral melanomas. Anyway, intense levels of MMP-9/TIMP-2 were observed in cases with high expression of FXR1 and with unfavorable clinical outcome in canine oral melanoma. 4) The expression of MMPs in FDIM was investigated. Immunohistochemical expression of MMP-9/TIMP-2 was investigated in 62 FDIM and results were compared with the histological grade and mitotic index. MMP-9 and TIMP-2 were expressed in 77.4% and 71.0% FDIM, respectively. Increasing MMP-9 and TIMP-2 paralleled with high histological grades and high mitotic index

Immunohistochemical Expression of FXR1 in Canine Normal Tissues and Melanomas

Fragile X mental retardation-related protein 1 (FXR1) is a cytoplasmic RNA-binding protein highly conserved among vertebrates. It has been studied for its role in muscle development, inflammation, and tumorigenesis, being related, for example, to metastasizing behavior in human and canine uveal melanoma. Anti-FXR1 antibodies have never been validated in the canine species. To investigate FXR1 expression in canine melanocytic tumors, the present study tested two commercially available polyclonal anti-human FXR1 antibodies, raised in goat and rabbit, respectively. The cross-reactivity of the anti-FXR1 antibodies was assessed by Western blot analysis, and the protein was localized by IHC in a set of normal canine tissues and in canine melanocytic tumors (10 uveal and 10 oral). Western blot results demonstrated that the antibody raised in rabbit specifically recognized the canine FXR1, while the antibody raised in goat did not cross-react with this canine protein. FXR1 protein was immunodetected using rabbit anti-FXR1 antibody, in canine normal tissues with different levels of intensity and distribution. It was also detected in 10/10 uveal and 9/10 oral melanocytic tumors. The present study validated for the first time the use of anti-FXR1 antibody in dogs and highlighted different FXR1 protein expression in canine melanocytic tumors, the significance of which is undergoing further investigations

Widespread extrahepatic expression of acute-phase proteins in healthy chicken (Gallus gallus) tissues

Acute phase proteins (APP) are plasma proteins that can modify their expression in response to inflammation caused by tissue injury, infections, immunological disorders or stress. Although APP are produced mainly in liver, extrahepatic production has also been described. As a prerequisite to get insight the expression of APP in chicken during diseases, this study investigated the presence of five APP, including alpha1-acid glycoprotein (AGP), Serum Amyloid A (SAA), PIT54, C-Reactive protein (CRP) and Ovotransferrin (OVT) in twenty tissues collected from healthy chicken (Gallus gallus) by quantitative Real Time PCR and immunohistochemistry. As expected, APP gene abundance was higher in liver compared with other tissues. The mRNA coding for CRP, OVT and SAA was detected in all analyzed tissues with a higher expression in gastrointestinal tract, respiratory and lymphatic samples. SAA expression was particularly high in cecal tonsil, lung, spleen and Meckel's diverticulum, whereas OVT in lung, bursa of Fabricius and pancreas. AGP and PIT54 mRNA expression were detected in all tissues but at negligible levels. Immunohistochemical expression of AGP and OVT was variably detected in different organs, being identified in endothelium of every tissue. Positive cells were present in the epithelium of the mucosal layer of gastrointestinal tract and kidney. Lung and central nervous system stained for both proteins. No positive staining was detected in lymphoid tissues and muscle. These results suggest that most tissues can express different amount of APP even in healthy conditions and are therefore capable to mount a local acute phase reaction

Frequent Storm Surges Affect the Groundwater of Coastal Ecosystems

Recent studies have focused on the effect of large tropical cyclones (hurricanes) on the shore, neglecting the role of less intense but more frequent events. Here we analyze the effect of the offshore tropical storm Melissa on groundwater data collected along the North America Atlantic coast. Our meta-analysis indicates that both groundwater level and specific conductivity significantly increased during Melissa, respectively reaching maximum values of 1.09 m and 25.2 mS/cm above pre-storm levels. Time to recover to pre-storm levels was 10 times greater for groundwater specific conductivity, with a median value of 20 days, while groundwater level had a median recovery time of 2 days. A frequency-magnitude analysis indicates that the percent of time with salinization is higher for Melissa than for energetic hurricanes. Given the high frequency of these events (return period of 1–2 years), and the long time needed for groundwater conditions to return to normal levels, we conclude that increasingly frequent moderate storms will have a significant impact on the ecology of vegetated shorelines

Fibrosarcoma of the eyelid in two sibling Czech wolfdogs

Most canine tumors of the eyelid are tumors generally encountered in the skin. They are most commonly of epithelial origin and benign. In this report, we describe the cases of two sibling Czech wolfdogs presented, one year apart, with a subcutaneous mass involving the left eyelid. Both lesions were histologically consistent with a diagnosis of subcutaneous fibrosarcoma. Immunohistochemical analyses of the tumors revealed a mild positivity for vimentin and negativity for GFAP, desmin, \u3b1SMA, myoglobin, S100, PNL2 and calponin, excluding all differential diagnosis (i.e. peripheral nerve sheath tumor, melanoma, perivascular sarcoma, myofibroblastic sarcoma, rhabdomyosarcoma). To the best of authors\u2019 knowledge, this is the first report of canine eyelid fibrosarcoma. Since this rare tumor has been observed in two full siblings, we could speculate the existence of some genetic predisposition to sarcoma, however the present data did not allow any definite conclusion on the etiopathogenesis or genetic basis of these tumors

MMP-9 immunohistochemical expression is correlated with histologic grade in feline diffuse iris melanoma

Feline diffuse iris melanoma (FDIM) is the most common primary intraocular neoplasm in cats (Dubielzig, 2017). It is usually a malignant tumor, even if slowly progressive, thus representing an unique spontaneous model of the aggressive, although rare, human iris melanoma (Demirci et al., 2002). In cats, the extent of the tumor within the eye, expressed as histological grade, is considered a good predictor of survival (Kalishman et al., 1998). In the context of the neoplastic cells-tumor microenvironment interaction, Matrix Metalloproteinase-9 (MMP-9) is an endopeptidase able to digest the extracellular matrix with involvement in tumor invasion (Nagase et al., 2006). MMP-9 expression has been positively correlated with metastasizing behavior in human posterior uveal melanoma (El-Shabrawi et al., 2001). The present study investigates the expression of MMP-9 in a caseload of formalin-fixed paraffin-embedded FDIMs in relation to the histological grade (Kalishman et al., 1998) and mitotic index (MI) (threshold=7/10 hpf) (Wiggans et al., 2016). Sixty-one samples of FDIM evaluated on light microscopy (Figure 1) were selected (grade I n=22, grade II n=20, grade III n=19). Immunohistochemical staining with standard ABC method was performed using a mouse anti-MMP-9 antibody (Porcellato et al., 2014). Results were semi-quantitatively scored and compared by Mann-Whitney U test. MMP-9 was expressed in 59,1% grade I FDIM, 90,0% grade II and 80,0% grade III. Tumors with MMP-9 expression in more than 50% of neoplastic cells were 13,6% in grade I cases, 40,0% in grade II and 36,8% in grade III. MMP-9 was expressed in 71,4% of FDIM with MI 647 and 92,3% of FDIM with MI>7. MMP-9 expression differed significantly between grade I and the other two grades, and between groups with low and high MI. In conclusion, intense expression of MMP-9 seems to correlate with the histological aggressiveness of FDIM

Lacrimal Gland Tumours in Dogs: A Histological and Immunohistochemical Study

Previous article in issueNext article in issue Introduction: Lacrimal gland tumours (LGTs) are well-known neoplasms affecting both the nictitating membrane and the main lacrimal glands in dogs; however, extensive histological and immunohistochemical studies are currently lacking. In recent years an increasing number of LGTs with a complex (i.e. epithelial and myoepithelial) phenotype have been recorded in our routine histopathology service. The aim of the present study was to review histological and immunohistochemical features of LGTs in dogs. Material and Methods: Nictitating membrane (NM) and main lacrimal gland (LG) biopsy samples from the departmental archive (2010\u20132016) were reviewed. HE sections were re-evaluated by light microscopy. Since a specific classification for LGTs is currently lacking, the classification proposed by Goldschmidt (2011) for mammary gland tumours was applied. Immunohistochemistry for pancytokeratin (CK AE1/AE3), CK14, \u3b1-smooth muscle actin, calponin and p63 was performed. Results: One-hundred and sixty-one NM and LG specimens were reviewed: 106 neoplastic lesions were diagnosed, 28 were epithelial gland tumours (25 NM; three LG). Twenty-four of 28 were carcinomas: seven complex, five solid, two simple tubular, two tubulopapillary, two comedocarcinoma, one ductal, four carcinoma and malignant myoepithelioma, one malignant myoepithelioma. Four were benign tumours: two complex adenomas, two multilobular adenomas. CKAE1/AE3 labelled tubular cells, while CK14, \u3b1-SMA, calponin and p63 labelled myoepithelial cells, when present. Conclusions: In the present cohort of cases, NM and LG complex carcinomas and adenomas were highly represented among LGTs. NM and LG carcinomas outnumbered adenomas and were characterized by marked intratumoural variability. A specific classification for lacrimal tumours is needed

Water Adsorption Effect on Carbon Molecular Sieve Membranes in H2-CH4 Mixture at High Pressure

Carbon molecular sieve membranes (CMSMs) are emerging as promising solution to overcome the drawbacks of Pd-based membranes for H2 separation since (i) they are relatively easy to manufacture; (ii) they have low production and raw material costs; (iii) and they can work at conditions where polymeric and palladium membranes are not stable. In this work CMSMs have been investigated in pure gas and gas mixture tests for a proper understanding of the permeation mechanism, selectivity and purity towards hydrogen. No mass transfer limitations have been observed with these membranes, which represents an important advantage compared to Pd-Ag membranes, which suffer from concentration polarization especially at high pressure and low hydrogen concentrations. H2, CH4, CO2 and N2 permeation at high pressures and different temperatures in presence of dry and humidified stream (from ambient and water vapour) have been carried out to investigate the effect of the presence of water in the feed stream. Diffusion is the main mechanism observed for hydrogen, while methane, nitrogen and especially carbon dioxide permeate through adsorption-diffusion at low temperatures and high pressures. Finally, H2 permeation from H2-CH4 mixtures in presence of water has been compared at different temperatures and pressure, which demonstrates that water adsorption is an essential parameter to improve the performance of carbon molecular sieve membranes, especially when working at high temperature. Indeed, a hydrogen purity of 98.95% from 10% H2—90% CH4 was achieved. The main aim of this work is to understand the permeation mechanisms of CMSMs in different operating conditions and find the best conditions to optimize the separation of hydrogen.This project has received funding from the Fuel Cells and Hydrogen 2 Joint Undertaking under grant Agreement no. 700355. This Joint Undertaking receves support from the European Union´s Horizon 2020 research

From Monoamine Oxidase Inhibition to Antiproliferative Activity: New Biological Perspectives for Polyamine Analogs

Monoamine oxidases (MAOs) are well-known pharmacological targets in neurological and neurodegenerative diseases. However, recent studies have revealed a new role for MAOs in certain types of cancer such as glioblastoma and prostate cancer, in which they have been found overexpressed. This finding is opening new frontiers for MAO inhibitors as potential antiproliferative agents. In light of our previous studies demonstrating how a polyamine scaffold can act as MAO inhibitor, our aim was to search for novel analogs with greater inhibitory potency for human MAOs and possibly with antiproliferative activity. A small in-house library of polyamine analogs (2-7) was selected to investigate the effect of constrained linkers between the inner amine functions of a polyamine backbone on the inhibitory potency. Compounds 4 and 5, characterized by a dianiline (4) or dianilide (5) moiety, emerged as the most potent, reversible, and mainly competitive MAO inhibitors (Ki < 1 μM). Additionally, they exhibited a high antiproliferative activity in the LN-229 human glioblastoma cell line (GI50 < 1 μM). The scaffold of compound 5 could represent a potential starting point for future development of anticancer agents endowed with MAO inhibitory activity

Immune Response After Cochlear Implantation

A cochlear implant (CI) is an electronic device that enables hearing recovery in patients with severe to profound hearing loss. Although CIs are a successful treatment for profound hearing impairment, their effectivity may be improved by reducing damages associated with insertion of electrodes in the cochlea, thus preserving residual hearing ability. Inner ear trauma leads to inflammatory reactions altering cochlear homeostasis and reducing post-operative audiological performances and electroacoustic stimulation. Strategies to preserve residual hearing ability led to the development of medicated devices to minimize CI-induced cochlear injury. Dexamethasone-eluting electrodes recently showed positive outcomes. In previous studies by our research group, intratympanic release of dexamethasone for 14 days was able to preserve residual hearing from CI insertion trauma in a Guinea pig model. Long-term effects of dexamethasone-eluting electrodes were therefore evaluated in the same animal model. Seven Guinea pigs were bilaterally implanted with medicated rods and four were implanted with non-eluting ones. Hearing threshold audiograms were acquired prior to implantation and up to 60 days by recording compound action potentials. For each sample, we examined the amount of bone and fibrous connective tissue grown within the scala tympani in the basal turn of the cochlea, the cochleostomy healing, the neuronal density, and the correlation between electrophysiological parameters and histological results. Detection of tumor necrosis factor alpha, interleukin-6, and foreign body giant cells showed that long-term electrode implantation was not associated with an ongoing inflammation. Growth of bone and fibrous connective tissue around rods induced by CI was reduced in the scala tympani by dexamethasone release. For cochleostomy sealing, dexamethasone-treated animals showed less bone tissue growth than negative. Dexamethasone did not affect cell density in the spiral ganglion. Overall, these results support the use of dexamethasone as anti-inflammatory additive for eluting electrodes able to protect the cochlea from CI insertion trauma