E. Biasibetti

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.

Antimicrobial Activity of Lactoferrin-Related Peptides and Applications in Human and Veterinary Medicine

Antimicrobial peptides (AMPs) represent a vast array of molecules produced by virtually all living organisms as natural barriers against infection. Among AMP sources, an interesting class regards the food-derived bioactive agents. The whey protein lactoferrin (Lf) is an iron-binding glycoprotein that plays a significant role in the innate immune system, and is considered as an important host defense molecule. In search for novel antimicrobial agents, Lf offers a new source with potential pharmaceutical applications. The Lf-derived peptides Lf(1–11), lactoferricin (Lfcin) and lactoferrampin exhibit interesting and more potent antimicrobial actions than intact protein. Particularly, Lfcin has demonstrated strong antibacterial, anti-fungal and antiparasitic activity with promising applications both in human and veterinary diseases (from ocular infections to osteo-articular, gastrointestinal and dermatological diseases).

Feeding a diet contaminated with ochratoxin A for broiler chickens at the maximum level recommended by the EU for poultry feeds (0.1 mg/kg). 2. Effects on meat quality, oxidative stress, residues and histological traits.

The European Commission Recommendation 2006/576/EC indicates that the maximum tolerable level of ochratoxin A (OTA) in poultry feeds is 0.1 mg OTA/kg. Thirty-six 1-day-old male broiler chicks were divided into two groups, a control (basal diet) and an OTA (basal diet + 0.1 mg OTA/kg) group. The OTA concentration was quantified in serum, liver, kidney, breast and thigh samples. The thiobarbituric acid reactive substances (TBARS) content were evaluated in the liver, kidney, breast and thigh samples. The glutathione (GSH) content, and catalase (CAT) and superoxide dismutase (SOD) activity were measured in the liver and kidney samples. Histopathological traits were evaluated for the spleen, bursa of Fabricius and liver samples. Moreover, the chemical composition of the meat was analysed in breast and thigh samples. In the OTA diet-fed animals, a serum OTA concentration of 1.15 ± 0.35 ng/ml was found, and OTA was also detected in kidney and liver at 3.58 ± 0.85 ng OTA/g f.w. and 1.92 ± 0.21 ng OTA/g f.w., respectively. The TBARS content was higher in the kidney of the ochratoxin A group (1.53 ± 0.18 nmol/mg protein vs. 0.91 ± 0.25 nmol/mg protein). Feeding OTA at 0.1 mg OTA/kg also resulted in degenerative lesions in the spleen, bursa of Fabricius and liver. The maximum tolerable level of 0.1 mg OTA/kg, established for poultry feeds by the EU, represents a safe limit for the final consumer, because no OTA residues were found in breast and thigh meat. Even though no clinical signs were noticed in the birds fed the OTA-contaminated diet, moderate histological lesions were observed in the liver, spleen and bursa of Fabricius.

Diprosopia/dicephalia in calves in northern Italy: clinical and aetio-pathological features.

With 1 table and 4 figures

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.

Effects of verbascoside supplemented diets on growth performance, blood traits, meat quality, lipid oxidation and histological features in broiler chickens

A trial has been performed to evaluate the effects of dietary verbascoside (VB) on the growth performance, blood traits, meat quality and histological features of broiler chickens. Seventy-two male broiler chickens (Ross 708) were divided into three experimental groups (8 replicates): a control group (C; basal diet), a low VB group (LVB; basal diet+2.5 mg VB/kg feed) and a high VB group (HVB; basal diet+5.0 mg VB/kg feed). The animals were kept in cages, three birds per cage. The trial lasted 35 days. Growth performance and carcass yield were recorded. Blood samples were collected at day 35 in order to evaluate the blood, serum and plasma parameters. As a result of this study, growth and slaughter performances were not affected by the dietary treatments. The LVB and HVB groups showed significantly lower concentrations of total protein (P=0.000), albumin (P=0.000), α-globulin (P=0.001) and β-globulin (P=0.023) than C group. The heterophil to lymphocyte ratio was higher in HVB group than in LVB and C groups (P=0.005). The thiobarbituric acid reactive substances results indicated lower lipid oxidation in the LVB group than in C group. The most relevant histological feature was the lymphoid depletion observed in the thymus and bursa of Fabricius in LVB and HVB. Other studies are still needed to identify the optimal dosage and exposure time of VB dietary supplementation and to further investigate the effects of VB on lymphoid tissue as well as to establish the VB antioxidant effect on broiler chickens.

Lipid nanoparticles for intranasal administration: application to nose-to-brain delivery

ABSTRACT Introduction: The blood brain barrier is a functional barrier allowing the entry into the brain of only essential nutrients, excluding other molecules. Its structure, although essential to keep the harmful entities out, is also a major roadblock for pharmacological treatment of brain diseases. Several alternative invasive drug delivery approaches, such as transcranial drug delivery and disruption of blood brain barrier have been explored, with limited success and several challenges. Intranasal delivery is a non-invasive methodology, which bypasses the systemic circulation, and, through the intra- and extra- neuronal pathways, provides direct brain drug delivery. Colloidal drug delivery systems, particularly lipidic nanoparticles offer several unique advantages for this goal . Areas covered: This review focuses on key brain diseases such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and amyotrophic lateral sclerosis, and provide a detailed overview of the current lipid nanoparticle based treatment options explored thus far. The review also delves into basic preparation, challenges and evaluation methods of lipid drug delivery systems. Expert opinion: Brain diseases present complex pathophysiology, in addition to the practically inaccessible brain tissues, hence according to the authors, a two-pronged approach utilizing new target discovery coupled with new drug delivery systems such as lipid carriers must be adopted.

Yellow mealworm larvae (Tenebrio molitor) inclusion in diets for male broiler chickens: effects on growth performance, gut morphology, and histological findings

Abstract This study evaluated the effects of Tenebrio molitor (TM) larvae meal inclusion in diets for broilers. A total of 160 male broiler chicks (Ross 708) at one‐day of age were randomly allotted to four dietary treatments: a control (C) group and three TM groups, in which TM meal was included at 50 (TM5), 100 (TM10), and 150 (TM15) g/kg, respectively. The experimental diets were isonitrogenous and isoenergetic. Each group consisted of five pens as replicates (8 chicks/pen). After the evaluation of growth performance and haematochemical parameters, the animals were slaughtered at 53 days and carcass traits were recorded. Morphometric investigations were performed on duodenum, jejunum, and ileum and histopathological alterations were assessed for liver, spleen, thymus, bursa of Fabricius, kidney, and heart. The live weight (LW) showed a linear (12 and 25 days, P < 0.001 and P < 0.05, maximum with TM15 and TM10) and quadratic (53 days, P < 0.05, maximum with TM5) response to dietary TM meal inclusion. A linear (1 to 12 and 12 to 25 days, P < 0.001, maximum with TM15) and quadratic (12 to 25 days, P = 0.001, maximum with TM15) effect was also observed for the daily feed intake (DFI). The feed conversion ratio (FCR) showed a linear response (25 to 53 and 1 to 53 days, P = 0.001 and P < 0.05, maximum with TM15). Haematological and serum biochemical traits, carcass traits and histopathological findings were not affected by dietary TM meal inclusion (P > 0.05). TM15 birds showed lower villus height (P < 0.05), higher crypt depth (P < 0.05), and lower villus height to crypt depth ratio (P = 0.001) compared with C and TM5. In conclusion, increasing levels of dietary TM meal inclusion in male broiler chickens may improve body weight and feed intake, but negatively affect feed efficiency and intestinal morphology, thus suggesting that low levels may be more suitable. However, no effect on haematochemical parameters, carcass traits, and histological findings were observed in relation to TM meal utilization.

Pathology and genetic findings in a rare case of Mycobacterium caprae infection in a sow

Bovine tuberculosis, a reemerging zoonosis in diverse ecological scenarios, has been reported in the autochthonous Nebrodi black pig breed population used for meat production in Italy. During a routine abattoir inspection in 2013, 24 of 299 carcasses (8%) of Nebrodi black pigs presented tuberculosis-like lesions at pathologic examination. Mycobacterium bovis was isolated from 23 animals and M. caprae from a 3-year-old sow. The sow showed severe diffuse lesions involving the visceral organs, right coxofemoral joint, and mammary glands. Isolation of M. caprae from mammary glands is uncommon, with only one other case involving a sow reported so far; however, Mycobacteria infection of the mammary glands may be transmitted from lactating sows to piglets, contributing to the spread and maintenance of bovine tuberculosis in swine. Genotyping analysis showed M. caprae spoligotype SB0866 and profile 4,1,5,4,4,11,4,2,4,3,8,7 MIRU-VNTR (mycobacterial interspersed repetitive units-variable number of tandem repeats). The worldwide prevalence of this spoligotype is very low. The finding of severe, diffuse tuberculous lesions strongly suggests that Nebrodi black pigs are susceptible for Mycobacterium spp. and that they might act as a distributor for these microorganisms. Since natural ecosystems with multiple contacts among different livestock species and wild animals are very common in Mediterranean regions, current surveillance and eradication plans for bovine tuberculosis will need to be extended to other potential reservoir species in regions where extensive and traditional breeding systems are operated.

A mouse mastitis model to study the effects of the intramammary infusion of a food-grade Lactococcus lactis strain

Lactococcus lactis is one of the most important microorganisms in the dairy industry and has “generally recognized as safe” (GRAS) status. L. lactis belongs to the group of lactic acid bacteria (LAB) and is encountered in a wide range of environments. Recently, the use of the intramammary infusion of a live culture of LAB has been investigated as a new antibiotic alternative for treating mastitis in dairy ruminants. Controversial results are described in literature regarding its efficacy and safety. In this study we conducted in-depth investigation of the mammary gland immune response induced by intramammary inoculum of a live culture of L. lactis LMG 7930 using the mouse mastitis model. Overnight cultures either of L. lactis (≈ 107 CFU) or of the mastitis pathogens Staphylococcus chromogenes (≈ 105 CFU) or S. aureus (≈ 102 CFU/ml) were injected into the mouse inguinal glands. A double injection, consisting of S. chromogenes first and then L. lactis, was also investigated. Bacterial recovery from the gland and inflammatory cell infiltration were assessed. L. lactis-treated and control glands were analysed for proinflammatory cytokine production. Microbiological results showed that L. lactis was able to survive in the mammary gland 24 h post infection, as were the mastitis pathogens S. chromogenes and S. aureus. L. lactis reduced S. chromogenes survival in the glands and increased its own survival ability by coexisting with the pathogen. Histology showed that L. lactis-treated glands presented variable histological features, ranging from undamaged tissue with no inflammatory cell infiltrate to severe PMN infiltrate with focal areas of tissue damage. S. aureus-treated glands showed the most severe histological grade of inflammation despite the fact that the inoculum size was the smallest. In contrast, most S. chromogenes-treated glands showed normal structures with no infiltration or lesions. Significant increases in IL-1β and TNF-α levels were also found in L. lactis-inoculated glands. The above findings seem to suggest that food-grade L. lactis at a high-inoculum dose such as an overnight culture may elicit a suppurative inflammatory response in the mammary gland, thus becoming a potential mastitis-causing pathogen. Because of the unpredictable potential of L. lactis in acting as a potential mastitis pathogen, this organism cannot be considered a safe treatment for bovine mastitis.