Marilena Leis

Detection of xanthine oxidase activity products by EPR and HPLC in bronchoalveolar lavage fluid from patients with chronic obstructive pulmonary disease

Xanthine oxidase (xanthine: oxygen oxidoreductase, EC 1.1.3.22), a molybdenum-containing hydroxylase that produces superoxide and uric acid from purine substrates and molecular oxygen, is involved in the oxidative stress underlying several human pathologies including lung diseases. An enzymatic activity similar to xanthine oxidase was previously reported in bronchoalveolar lavage fluid of patients with chronic obstructive pulmonary disease (COPD-BAL), by fluorometric analysis of DNA unwinding and cytochrome c reduction kinetics. Here we report the detection of xanthine oxidase activity products by electron paramagnetic resonance (EPR) in presence of the spin-trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and reversed-phase high-performance liquid chromatography (RP-HPLC) in COPD-BAL (n = 14, average age of patients 65 years, range 38-81) and BAL from healthy nonsmoker controls (n = 6, average age 64 years, range 44-73). Superoxide DMPO adducts were detected in COPD-BAL and in an in vitro system containing xanthine and xanthine oxidase (XA/XO), but not in BAL controls and when superoxide dismutase (SOD, 1000 I.U./ml) was added to COPD-BAL. The HPLC analyses after addition of xanthine showed production of uric acid in COPD-BAL and in the XA/XO system but not in BAL controls. These results support the involvement of xanthine oxidase in the mechanisms of superoxide production by BAL supernatant, which increases oxidative stress in chronic obstructive pulmonary disease.

Role of Xanthine Oxidase Activation and Reduced Glutathione Depletion in Rhinovirus Induction of Inflammation in Respiratory Epithelial Cells

Rhinoviruses are the major cause of the common cold and acute exacerbations of asthma and chronic obstructive pulmonary disease. We previously reported rapid rhinovirus induction of intracellular superoxide anion, resulting in NF-κB activation and pro-inflammatory molecule production. The mechanisms of rhinovirus superoxide induction are poorly understood. Here we found that the proteolytic activation of the xanthine dehydrogenase/xanthine oxidase (XD/XO) system was required because pretreatment with serine protease inhibitors abolished rhinovirus-induced superoxide generation in primary bronchial and A549 respiratory epithelial cells. These findings were confirmed by Western blotting analysis and by silencing experiments. Rhinovirus infection induced intracellular depletion of reduced glutathione (GSH) that was abolished by pretreatment with either XO inhibitor oxypurinol or serine protease inhibitors. Increasing intracellular GSH with exogenous H2S or GSH prevented both rhinovirus-mediated intracellular GSH depletion and rhinovirus-induced superoxide production. We propose that rhinovirus infection proteolytically activates XO initiating a pro-inflammatory vicious circle driven by virus-induced depletion of intracellular reducing power. Inhibition of these pathways has therapeutic potential.

Insecticidal activity and fungitoxicity of plant extracts and components of horseradish (Armoracia rusticana) and garlic (Allium sativum).

To avoid environmental pollution and health problems caused by the use of traditional synthetic pesticides, there is a trend to search for naturally occurring toxicants from plants. Among the compounds discussed for anti-fungal and insecticidal activity, the natural extracts from garlic and horseradish have attracted considerable attention. The objective of this study is to determine the insecticidal and anti-fungal activity of Armoracia rusticana and Allium sativum L. extracts against larvae of Aedes albopictus (Skuse) and some pathogenic fungi. For the insecticidal test, horseradish and garlic extracts were prepared from fresh plants (cultivated in Emilia Romagna region) in a solution of ethanol 80 % and the two different solutions were used at different concentrations (for the determination of the lethal dose) against the fourth instar mosquitos larvae. The fungicidal test was carried out by the agar plates technique using garlic and horseradish extracts in a 10 % ethanol solution against the following organisms: Sclerotium rolfsii Sacc., Trichoderma longibrachiatum, Botrytis cinerea Pers., Fusarium oxysporum Schlecht. and Fusarium culmorum (Wm. G. Sm.) Sacc. The first results demonstrated that the horseradish ethanol extracts present only a fungistatic activity against Sclerotium rolfsii Sacc., Fusarium oxysporum Schlecht. and F. culmorum (Wm.G. Sm) Sacc. while garlic extracts at the same concentration provided a good fungicidal activity above all against Botrytis cinerea Pers. and S. rolfsii. A. rusticana and A. sativum preparations showed also an interesting and significant insecticidal activity against larvae of A. albopictus, even if horseradish presented a higher efficacy (LC50 value of 2.34 g/L), approximately two times higher than garlic one (LC50 value of 4.48 g/L).

Preliminary Resistance Screening on Abamectin in Pear Psylla (Hemiptera: Psyllidae) in Northern Italy

Abstract In northern Italy (Emilia-Romagna region), integrated pest management has been used for several years against pear psylla, Cacopsylla pyri L. (Hemiptera: Psyllidae), a relevant pest of pear (Pyrus spp.) trees. After the outlawing of amitraz in 2005, the most common active ingredient involved is abamectin, a mixture of avermectin B1a and avermectin B1b. After the development of C. pyri resistance to azinphos methyl in southern France, we evaluated, by topical application, the different sensitivities to abamectin on C. pyri populations collected in orchards from Emilia-Romagna, where different field strategies were used, with alternative success in terms of pest management. The LC50 values ranged between 1.61 and 28.37 mg/liter, and they revealed variations more related to collection time than to field strategies. The failure of abamectin treatments against C. pyri in some Emilia-Romagna locations is probably unrelated to resistance development, but rather it is related to incorrect pest defense management, which could interfere with pest parasitoids and predators.

Geochemical characterization of elements in Vitis vinifera cv. Negroamaro grape berries grown under different soil managements

The present geochemical study concerns the impact of viticultural practices in the chemical composition of the grape cultivar “Negroamaro” in Apulia, a southern Italian region renowned for its quality wine. Three types of soil management (SM), two cover cropping with different mixtures, and a soil tillage were considered. For each SM, the vines were irrigated according to two irrigation levels. Chemical composition of soil and of berries of Vitis vinifera cultivar “Negroamaro” were analyzed by X-ray fluorescence, inductively coupled plasma-mass spectrometry and multivariate statistics (linear discrimination analysis). In detail, we investigated major and trace elements behavior in the soil according to irrigation levels, the related index of bioaccumulation (BA) and the relationship between trace element concentration and soil management in “Negroamaro” grapes. The results indicate that soil management affects the mobility of major and trace elements. A specific assimilation of these elements in grapes from vines grown under different soil management was confirmed by BA. Multivariate statistics allowed to associate the vines to the type of soil management. This geochemical characterization of elements could be useful to develop fingerprints of vines of the cultivar “Negroamaro” according to soil management and geographical origin.

Traumatic Myiasis Caused by an Association of Sarcophaga tibialis (Diptera: Sarcophagidae) and Lucilia sericata (Diptera: Calliphoridae) in a Domestic Cat in Italy

We describe here a rare case of traumatic myiasis occurred in August 2014, caused by an association of 2 Diptera species, Sarcophaga tibialis Macquart (Diptera: Sarcophagidae) and Lucilia sericata (Meigen) (Diptera: Calliphoridae), in a domestic cat in northern Italy. Species identification was based on adult male morphology. The present case is the first report of S. tibialis as an agent of myiasis in Italy, and also the first ever report of myiasis caused by an association of S. tibialis and L. sericata. The cat developed an extensive traumatic myiasis in a large wound on the rump, which was treated pharmacologically and surgically. The biology, ecology, and distribution of S. tibialis and L. sericata are also discussed. A literature review is provided on cases of myiasis caused by S. tibialis, and cases of myiasis by L. sericata involving cats worldwide and humans and animals in Italy.

A comparative toxicity study between an autochthonous Artemia and a non native invasive species

Acute heavy metal toxicity was compared in nauplii of an autochthonous Artemia parthenogenetica (Branchiopoda, Anostraca) from Saline di Cervia (Ravenna, Italy) and of a non native commercially available A. franciscana. No significant difference in sensitivity was detected between two species.

Survey of Susceptibility to Abamectin of Pear Psylla (Hemiptera: Psyllidae) in Northern Italy

ABSTRACT The pear psylla, Cacopsylla pyri L. (Hemiptera: Psyllidae), is a relevant pest of pear, Pyrus communis L., trees in Emilia-Romagna region (northern Italy). The susceptibility to the insecticide abamectin was evaluated at different times of the year on C. pyri populations undergoing different control strategies within conventional, integrated, and organic farms. The tests performed were the egg spray and the topic and dip bioassay on adults. The larval mortality was evaluated by dip bioassay on treated leaves. The activity of P450-dependent monooxygenases, a relevant enzyme system involved in insecticide resistance of C. pyri, was also determined in adults by 7-ethoxycoumarin O-deethylation (ECOD assay). Tests on treated eggs and on larvae showed no significant differences in LC50 and LC90, although these values were always lower in individuals collected from organic farms in comparison with all other farms. Tests on overwintering adults revealed differences among populations, probably more related to collection time than to field pest control strategies. Unexpectedly, the ECOD assay on adults showed a slightly higher cytochrome P450 monooxygenase activity in the population undergoing organic control in comparison to others. Our results indicate that egg spray is the most reliable bioassay to verify data of open-field applications. Apparently, no resistance to abamectin has yet been developed by C. pyri in Emilia-Romagna.

Ultrastructural Morphology of the Antenna and Maxillary Palp of Sarcophaga tibialis (Diptera: Sarcophagidae)

Abstract A scanning electron microscopy investigation of the antenna and maxillary palp of the adult of Sarcophaga tibialis Macquart (Diptera: Sarcophagidae), a species of medical, veterinary, and forensic relevance, is presented for the first time. Adults of both sexes used in this study were obtained from larvae collected in a case of traumatic myiasis in a domestic cat in northern Italy. The antenna of S. tibialis is that typical of cyclorrhaphan Diptera, consisting of three segments: the scape, the pedicel, and the postpedicel, bearing the arista. The scape is covered by microtrichia and has a row of long chaetic sensilla. The pedicel is also covered by microtrichia and has three types of chaetic sensilla and a cluster of setiferous plaques. Trichoid, styloconic, clavate, and basiconic sensilla are distributed among the microtrichia on the postpedicel. Invaginated basiconic-like sensilla and olfactory pits are also present, the latter ones more numerous in the female. Our results are compared with those obtained for other calyptrate flies, mainly in the family Sarcophagidae. The data obtained may represent a basis for electrophysiological studies on the sensorial activity of the species related to the search for food sources, mates, and suitable larviposition sites, and for comparative morphological studies with other Diptera.

Preliminary evaluation of insecticide resistance in a strain of Musca domestica (Diptera: Muscidae) from an intensive chicken farm of Northern Italy.

The house fly, Musca domestica L., a relevant sanitary pest, is mainly controlled by insecticides. However, an inappropriate use of chemicals may induce resistance, treatment efficacy decline and environmental damages. We evaluated toxicity of some organophosphates, pyrethroids, spinosad and neonicotinoids by topical applications on adults of a M. domestica strain (OcRo) collected from an intensive chicken farm in Northern Italy, in comparison to a susceptible strain (s-DBF). The OcRo strain exhibited higher levels of resistance (RR50) to four pesticides in comparison to s-DBF. Spinosad and imidacloprid had very low RR50, thus were still efficient in OcRo control. We also tested resistance to pesticides in OcRo after topical applications of synergistic compounds. Hydrolases were involved in phosphorganic detoxification and cytochrome P450 monoxygenases in that of pyrethroids. These results indicate that OcRo strain is now multiresistant to organophosphates and pyrethroids, and this should be considered for an environmentally safe pest management.