Nandrolone and estradiol biomarkers identification in bovine urine applying a liquid chromatography high-resolution mass spectrometry metabolomics approach.

With the aim of specifically investigating patterns associated with three steroid treatments (17ß-nandrolone, 17ß-estradiol, and 17ß-nandrolone + 17ß-estradiol) in bovine, an reversed phase liquid chromatography (RPLC)-electrospray ionization (ESI)(+/-)-high-resolution mass spectrometry (HRMS) study was conducted to characterize the urinary profiles of involved animals. Although specific fingerprints with strong differences could be highlighted between urinary metabolite profiles within urine samples collected on control and treated animals, it appeared further that significant discriminations could also be observed between steroid treatments, evidencing thus specific patterns and candidate biomarkers associated to each treatment. An MS-2 structural elucidation step enabled level-1 identification of two biomarkers mainly involved in energy pathways, in relation to skeletal muscle functioning. These results make it possible to envisage a global strategy for the detection of anabolic practices involving steroids, while at the same time providing clues as to the compounds used, which would facilitate the confirmation stage to follow.

A new diet supplement formulation containing cranberry extract for the treatment of feline idiopathic cystitis.

This study aimed to investigate whether cranberry extract could reduce lower urinary tract (LUT) and gastro-intestinal (GI) signs in feline idiopathic cystitis (FIC). Twenty-one client-owned cats were randomly allocated to two groups: a treated group (T, n = 10) receiving daily an oral nutritional supplement containing cranberry extract and a control group (C, n = 11). Owners were trained to recognise daily LUT and GI signs. Physical examination, urinalysis and bladder ultrasonography were performed at day 0 (T0), 15 (T15), 30 (T30), 60 (T60). Both groups showed an improvement for dysuria and periuria from T0 to T30 (p < 0.05), but only in cats of the T group, LUT signs disappeared at T60. A significant improvement in the T group was also observed for GI signs and bladder ultrasonography at T60 (p = 0.03). Urinalysis did not show any significant differences. This preliminary study suggests that cranberry could be effective in reducing LUT and GI signs in FIC.

5-Aminolevulinic Acid Triggered by Ultrasound Halts Tumor Proliferation in a Syngeneic Model of Breast Cancer.

Sonodynamic therapy is a bimodal therapeutic approach in which a chemical compound and ultrasound (US) synergistically act to elicit oxidative damage, triggering cancer cell death. Despite encouraging results, mainly for anticancer treatment, sonodynamics is still far from having a clinical application. Therefore, to close the gap between the bench and bedside, more in vivo studies are needed. In this investigation, the combined effect of 5-aminolevulinic acid (Ala), a natural porphyrin precursor, plus exposure to US, was investigated in vivo on a syngeneic breast cancer model. Real-time RT-PCR, Western blotting, and immunohistochemistry assays were performed to evaluate the effect of sonodynamic treatment on the main cancer hallmarks. The sonodynamic-treated group had a significant reduction (p ≤ 0.0001) in tumor size compared to the untreated group, and the Ala- and US-only treated groups, where a strong decrease (p ≤ 0.0001) in Ki67 protein expression was the most relevant feature of sonodynamic-treated cancer tissues. Moreover, oxidative stress was confirmed as the pivotal driver of the anticancer effect through cell cycle arrest, apoptosis, and autophagy; thus, sonodynamics should be explored further for cancer treatment.

Feline morbillivirus in northwestern Italy: first detection of genotype 1-B.

OBJECTIVES: A novel morbillivirus was recently described in stray and domestic cats in Asia, the USA and Europe. Most cats infected with feline morbillivirus (FeMV) showed lower urinary tract or kidney disease. Although the association of FeMV infection and kidney diseases has been suggested, the virus pathogenicity remains unclear. The present study aimed to investigate the distribution of FeMV infection, as well as the relationship between FeMV infection and kidney diseases in cats from northwestern Italy. METHODS: A total of 153 urine samples (150 individuals and three pools) and 50 kidney samples were collected and included in the study; total RNA was extracted and a reverse transcription quantitative PCR (RT-qPCR) was performed in order to identify FeMV. Kidneys were also submitted to anatomopathological examination. Phylogenetic analysis and isolation attempts were carried out on positive samples. In FeMV-positive cats, urinalysis and blood analysis were performed. RESULTS: FeMV RNA was detected in 7.3% of urine samples and in 8% of kidney samples, both in healthy cats and in cats with clinical signs/post-mortem lesions compatible with kidney disease. At histopathological examination, tubulointerstitial nephritis (TIN) was shown in 3/4 positive kidney samples, but a clear relationship between FeMV and TIN was not observed. Isolation attempts were unsuccessful, although the urine sample of one castrated male cat hosted in a cattery showed a positive signal in RT-qPCR until the fourth cell passage. Phylogenetic analysis revealed that this FeMV strain belonged to genotype 1-B. In the same cattery, a second genotype 1-B variant was detected from a urine pool. Urinalysis showed proteinuria in three cats, while at blood analysis three cats presented altered creatinine levels. CONCLUSIONS AND RELEVANCE: Data reported suggest the presence of a FeMV sub-cluster distinct from the strain previously isolated in Italy, whose role in renal disorders remains uncertain.

Cistus salviifolius Toxicity in Cattle.

Toxicity related to consumption of Cistus sp. pl. has been described in ruminants in some countries. This report describes the clinical and pathological findings of Cistus salviifolius toxicosis in 3 beef cattle herds located in 2 different areas of Sicily, Italy. Outbreaks were observed after grazing in poor winter pasture where C. salviifolius was abundant. Mean morbidity and mortality were 29% and 21%, respectively. Most of the affected animals (6 to 36 months old) showed anorexia, weight loss, and pollakiuria culminating in recumbency and death. Occasionally, abortion and neurological signs were observed. In animals with acute signs, there was a moderate decrease of sodium and chloride concentrations in serum. Animals with chronic signs showed an increase of serum urea, creatine phosphokinase (CPK), aspartate transaminase (AST), lactate dehydrogenase (LDH), and phosphorus and a decrease in total serum protein, calcium, chloride, and magnesium concentrations. Moderate anemia and slight neutropenia, lymphocytosis, and eosinophilia were detected in all groups. At necropsy, the main lesion was severe distention of the urinary bladder with turbid hemorrhagic urine and crystalluria. Histologically, chronic cystitis, interstitial nephritis, eosinophilic enteritis, and nonsuppurative necrotizing hepatitis were observed. To our knowledge, this is the first report of C. salviifolius toxicosis in cattle in Italy.

Solid Lipid Nanoparticles Carrying Temozolomide for Melanoma Treatment. Preliminary In Vitro and In Vivo Studies.

AIM: To develop an innovative delivery system for temozolomide (TMZ) in solid lipid nanoparticles (SLN), which has been preliminarily investigated for the treatment of melanoma. MATERIALS AND METHODS: SLN-TMZ was obtained through fatty acid coacervation. Its pharmacological effects were assessed and compared with free TMZ in in vitro and in vivo models of melanoma and glioblastoma. RESULTS: Compared to the standard free TMZ, SLN-TMZ exerted larger effects, when cell proliferation of melanoma cells, and neoangiogeneis were evaluated. SLN-TMZ also inhibited growth and vascularization of B16-F10 melanoma in C57/BL6 mice, without apparent toxic effects. CONCLUSION: SLN could be a promising strategy for the delivery of TMZ, allowing an increased stability of the drug and thereby its employment in the treatment of aggressive malignacies.

Comparison of Allogeneic and Syngeneic Rat Glioma Models by Using MRI and Histopathologic Evaluation.

Research in neurooncology traditionally requires appropriate in vivo animal models, on which therapeutic strategies are tested before human trials are designed and proceed. Several reproducible animal experimental models, in which human physiologic conditions can be mimicked, are available for studying glioblastoma multiforme. In an ideal rat model, the tumor is of glial origin, grows in predictable and reproducible patterns, closely resembles human gliomas histopathologically, and is weakly or nonimmunogenic. In the current study, we used MRI and histopathologic evaluation to compare the most widely used allogeneic rat glioma model, C6-Wistar, with the F98-Fischer syngeneic rat glioma model in terms of percentage tumor growth or regression and growth rate. In vivo MRI demonstrated considerable variation in tumor volume and frequency between the 2 rat models despite the same stereotactic implantation technique. Faster and more reproducible glioma growth occurred in the immunoresponsive environment of the F98-Fischer model, because the immune response is minimized toward syngeneic cells. The marked inability of the C6-Wistar allogeneic system to generate a reproducible model and the episodes of spontaneous tumor regression with this system may have been due to the increased humoral and cellular immune responses after tumor implantation.

Solid lipid nanoparticles by coacervation loaded with a methotrexate prodrug: preliminary study for glioma treatment.

AIM: Methotrexate-loaded biocompatible nanoparticles were tested for preliminary efficacy in glioma treatment. MATERIALS & METHODS: Behenic acid nanoparticles, prepared by the coacervation method, were loaded with the ester prodrug didodecylmethotrexate, which was previously tested in vitro against glioblastoma human primary cultures. Nanoparticle conjugation with an ApoE mimicking chimera peptide was performed to obtain active targeting to the brain. RESULTS & CONCLUSION: Biodistribution studies in healthy rats assessed the superiority of ApoE-conjugated formulation, which was tested on an F98/Fischer glioma model. Differences were observed in tumor growth rate (measured by MRI) between control and treated rats. In vitro tests on F98 cultured cells assessed their susceptibility to treatment, with consequent apoptosis, and allowed us to explain the apoptosis observed in glioma models.

An outbreak of bovine tuberculosis in a fallow deer herd (Dama dama) in Sicily.

Wild ruminants have an important role in the epidemiology of bovine tuberculosis (bTB). This study describes an outbreak of bovine tuberculosis occurring in a fallow deer herd in Sicily. In 2012 a Sicilian herd of 47 animals was referred for cachexia. Pathological examination of 2 dead animals revealed disseminated granulomas predominantly involving the skin and subcutaneous tissues. Tissue samples were submitted for histological analysis, bacteriological culture, and biomolecular assay. PCR analysis identified Mycobacterium strains. Genotyping by spoligotyping and MIRU-VNTR profiles identified Mycobacterium bovis spoligotype SB0120 in both animals. In 2014, bTB skin testing of 28 fallow deer from the same group was positive in 4 and inconclusive in another 4. All 8 positive/inconclusive reactors were euthanized. Disseminated granulomatous lesions were noted in 6 of these animals, 3 of which (2 positive and 1 negative to skin tests) also presented cutaneous lesions. M. bovis spoligotype SB0120 was identified from all animals in which tuberculous-like lesions were observed, including 2 negative reactors. Many of the animals involved in this outbreak presented diffuse skin lesions, a potential route of transmission of M. bovis infection. Given the epidemiological role wildlife play in the maintenance of bTB infection and its potential risk for humans, a comprehensive monitoring plan for this zoonosis in wildlife species in Sicily is needed.

Severe meningeal fibrinoid vasculitis associated with Theileria taurotragi infection in two short-horned Zebu cattle.

The Authors describe a severe vasculitis with fibrinoid necrosis of the meningeal arteries observed in two brains of indigenous short-horn zebu (Bos indicus) cattle, with bovine cerebral theileriosis (BCT) caused by a tick-transmitted hemoprotozoan, Theileria taurotragi, from Northern Tanzania. In the Author's opinion, the role of T. taurotragi infection in the angiocentric and angiodestructive detected features remains to be evaluated. A possible immunopathologic cancerous mechanism, secondary to the lymphoid deregulation, could be involved. This report suggests further studies to better characterize the lymphoid cell involvement in the pathogenesis of the meningeal vascular lesions by T. taurotragi.

Positive-charged solid lipid nanoparticles as paclitaxel drug delivery system in glioblastoma treatment.

Paclitaxel loaded solid lipid nanoparticles (SLN) of behenic acid were prepared with the coacervation technique. Generally, spherical shaped SLN with mean diameters in the range 300­600 nm were obtained. The introduction of charged molecules, such as stearylamine and glycol chitosan into the formulation allowed to obtain positive SLN with Zeta potential in the 8-20 mV range and encapsulation efficiency in the 25­90% range.Blood­brain barrier (BBB) permeability, tested in vitro through hCMEC/D3 cells monolayer, showed a significantly increase in the permeation of Coumarin-6, used as model drug, when vehicled in SLN. Positive-charged SLN do not seem to enhance permeation although stearylamine-positive SLN resulted the best permeable formulation after 24 h.Cytotoxicity studies on NO3 glioblastoma cell line demonstrated the maintenance of cytotoxic activity of all paclitaxel-loaded SLN that was always unmodified or greater compared with free drug. No difference in cytotoxicity was noted between neutral and charged SLN.Co-culture experiments with hCMEC/D3 and different glioblastoma cells evidenced that, when delivered in SLN, paclitaxel increased its cytotoxicity towards glioblastoma cells.

Solid lipid nanoparticles for potential doxorubicin delivery in glioblastoma treatment: preliminary in vitro studies.

The major obstacle to glioblastoma pharmacological therapy is the overcoming of the blood-brain barrier (BBB). In literature, several strategies have been proposed to overcome the BBB: in this experimental work, solid lipid nanoparticles (SLN), prepared according to fatty acid coacervation technique, are proposed as the vehicle for doxorubicin (Dox), to enhance its permeation through an artificial model of BBB. The in vitro cytotoxicity of Dox-loaded SLN has been measured on three different commercial and patient-derived glioma cell lines. Dox was entrapped within SLN thanks to hydrophobic ion pairing with negatively charged surfactants, used as counterions. Results indicate that Dox entrapped in SLN maintains its cytotoxic activity toward glioma cell lines; moreover, its permeation through hCMEC/D3 cell monolayer, assumed as a model of the BBB, was increased when the drug was entrapped in SLN. In conclusion, SLN proved to be a promising vehicle for the delivery of Dox to the brain in glioblastoma treatment.

Epidemiological significance of the domestic black pig (Sus scrofa) in maintenance of bovine tuberculosis in Sicily.

Bovine tuberculosis (bTB) is an emerging disease among wild animals in many parts of the world. Wildlife reservoir hosts may thus represent a potential source of infection for livestock and humans. We investigated the role played by the Sicilian black pig, an autochthonous free- or semi-free-ranging domestic pig breed, as a potential source of bTB infection in an area where bTB prevalence in cattle is high. We initially performed a preliminary field study to assess the occurrence of bTB in such animals. We sampled 119 pigs at abattoir and found 6.7% and 3.4% of them to be affected by gross tuberculous-like lesions (TBL) and Mycobacterium bovis culture positive, respectively. We then proceeded to investigate the dissemination and characteristics of lesions in a second field study performed on 100 animals sampled from infected herds. Here, tissues collected at the abattoir were examined macroscopically, microscopically, and by culture tests. Most pigs with TBL showed generalized lesions in both gross and histological examinations (53% and 65.5%, respectively). Head lymph nodes were the most frequently affected in both localized and generalized TB cases observed macroscopically and microscopically. M. bovis was the most frequently isolated etiologic agent. The molecular characterization of isolates from both field studies by spoligotyping and analysis of 12 mycobacterial interspersed repetitive-unit-variable number tandem repeat (MIRU-VNTR) loci, followed by their comparison to isolates of cattle origin, suggested a potential transmission of mycobacteria from domestic animals to black pigs and vice versa. Our findings, along with ethological, ecological, and management considerations, suggest that the black pig might act as a bTB reservoir in the ecosystem under study. However, additional studies will be necessary to establish the true epidemiological significance of the Sicilian black pig.