Valutazione degli effetti del dimetilamino-partenolide (DMAPT) in vivo in xenotrapianti di cellule di carcinoma mammario MDA-MB231

Obiettivo degli studi condotti in questa tesi è stato quello di valutare l’efficacia del Partenolide in vivo in xenotrapianti ottenuti da cellule di carcinoma mammario triplo negativo MDA-MB231 in topi immunodeficienti. Lo studio è stato condotto impiegando il dimetilamino-partenolide (DMAPT), un analogo del partenolide più solubile. I risultati delle ricerche hanno dimostrato che la somministrazione di DMAPT a topi immunodeficienti portatori di xenotrapianti di cellule MDA-MB231 determina una significativa inibizione della crescita dei tumori e un incremento della sopravvivenza degli animali. Analisi immunoistochimiche condotte su preparati istologici ottenuti dai tumori hanno anche dimostrato che il trattamento con DMAPT determina una marcata riduzione delle metastasi polmonari, nonché una riduzione dei livelli di fattori angiogenetici, quali il VEGF, e invasivi, quali le metalloproteasi 2 e 9. Il DMAPT, inoltre, determina una riduzione dei livelli di p65, una componente del fattore di trascrizione NF-kB, responsabile dell’attivazione trascrizionale di VEGF e delle metalloproteasi, ed un incremento dei livelli di JNK fosforilato, un fattore correlato con l’induzione di stress ossidativo. Complessivamente i dati prodotti in questo studio costituiscono un’incoraggiante evidenza del possibile impiego del DMAPT nel prossimo futuro nella terapia del carcinoma mammario triplo negativo

An innovative method for the detection of contaminant viral genome in cell cultures

The use of cell cultures involves different fields of biology, from diagnosis to research. Moreover, technologies based on animal cells represent a useful tool to the development of biological products for the prophylaxis and therapy in humans and animals. Therefore, it is necessary to perform quality controls, including virological tests. Several tests performed in research laboratories are able to discriminate one or more viral species, but it is not possible to demonstrate the presence of contaminant viral genome with one non-specific method. The aim of this work consisted on the realization of a biomolecular method able to detect and to identify by sequencing extraneous viral genome in cell cultures of animal and human origin in the absence of any specific information about the virus

Quality controls for cell cultures: identification of interspecies cross-contamination by PCR-RFLP analysis of the cytochrome b gene

Cross-contaminations of a cell line with cells of different species represent a potential risk in laboratories handling human and animal cells. Therefore, it is necessary to control such contaminations. Tests based on mitochondrial DNA (mtDNA) are used in forensic analysis, phylogenetic studies and in food authentication. However, the use of mtDNA in quality controls of cell cultures is recent. Mitochondrial sequence differences of closely related animal species are five- to tenfold higher than those of nuclear genes. On the contrary, intraspecies variation in mitochondrial sequences is low in most animal species. Moreover, each cell contains 100–10.000 mitochondrial genomes. The amount of mtDNA is greater than nuclear DNA, so that mtDNA can be analyzed also from small or partially degraded samples. In the present study, a method based on a PCR-Restriction Fragment Length Polymorphism (RFLP) analysis of the mitochondrial cytochrome b gene was used (2). This gene has some stable sequences which are recognized from universal primers and some variable sequences used for animal species identification by PCR-RFLP method

Viral Encephalopathy and Retinopathy (VER) in Mediterranean wild and farmed fish species : the experience of the ‘Istituto Zooprofilattico Sperimentale’ in Sicily

Betanodavirus infection is widespread in a broad spectrum of fish species worldwide. In Italy, it is responsible for outbreaks of Viral Encephalo-Retinopathy (VER) that causes mortality and economic losses in sea fish farming. The infection is also widespread in the wild and not only in managed systems, where there are generally no observed clinical manifestations.peer-reviewe

Seroprevalencija virusa hepatitisa E u krava s ruralnih područja južne Italije

Hepatitis E virus (HEV) is considered to be an emerging zoonotic disease, which causes numerous cases of hepatitis and deaths worldwide each year. Pigs are a host reservoir of HEV, but numerous other wild and domestic species can be infected. The aim of this study was to carry out serological screening of anti-HEV antibodies in cattle in the Sicily region (Southern Italy). Between April and December 2018, 231 serum samples were collected from cows and analysed with enzyme-linked immunosorbent assay (ELISA), for detection of HEV antibodies (IgG). The overall prevalence of anti-HEV antibodies was 36.36% (84/231; 95% CI 30-43). There were statistically significant differences in the HEV seroprevalence in animals of different ages (12 months: 39.30%; >24 months: 54.10%) (P0.05). This study shows that further investigation of HEV in cattle is required to understand better the epidemiology in farm animals and the potential zoonotic risks for humans. This is the first report of HEV seroprevalence for cows in Italy, which provides baseline data for further studies and for control of HEV infection in cattle.Virus hepatitisa E (HEV) smatra se emergentnom zoonozom koja uzrokuje brojne slučajeve hepatitisa i uginuća širom svijeta svake godine. Svinja je rezervoar HEV-a, no mogu se zaraziti i brojne druge divlje i domaće vrste. Cilj ovog istraživanja bio je provesti serološki probir anti-HEV protutijela u goveda na Siciliji (južna Italija). Od travnja do prosinca 2018. prikupljen je ukupno 231 uzorak seruma krava i analiziran testom ELISA za detekciju protutijela na HEV (IgG). Prevalencija anti-HEV protutijela bila je 36,36 % (84/231; 95 % CI 30 – 43). Pronađena je statistički znakovita razlika (P 12 mjeseci: 39,30 %; > 24 mjeseci: 54,10 %). Nije zabilježena statistički znakovita razlika (P> 0,05) s obzirom na spol (31,37 % u mužjaka i 37,38 % u ženki). Rezultati ukazuju na potrebu daljnjih istraživanja HEV-a u goveda kako bi se bolje razumjela epidemiologija u farmskih životinja i potencijalan zoonotski rizik za ljude. Ovo je prvo izvješće o seroprevalenciji HEV-a u krava u Italiji, kojim se pružaju referentni podaci za daljnja istraživanja i kontrolu infekcije HEV-om u goveda

Molecular characterization of human enteric viruses in food, water samples, and surface swabs in Sicily

Objectives: Enteric viruses are responsible for foodborne and waterborne infections affecting a large number of people. Data on food and water viral contamination in the south of Italy (Sicily) are scarce and fragmentary. The aim of this study was to evaluate the presence of viral contamination in food, water samples, and surface swabs collected in Sicily Methods: The survey was conducted on 108 shellfish, 23 water samples (seawater, pipe water, and torrent water), 52 vegetables, one peach and 17 berries, 11 gastronomic preparations containing fish products and/or raw vegetables, and 28 surface swabs. Hepatitis A virus (HAV), genogroup GI, GII, and GIV norovirus (NoV), enterovirus (EV), rotavirus (RoV), hepatitis E virus (HEV), adenovirus (AdV), and bocavirus (BoV) were detected by nested (RT) PCR, real-time PCR, and sequence analysis. Results: The most frequently detected viruses in shellfish were HAV (13%), NoV (18.5%), and EV (7.4%). Bocavirus was found in 3.7%, HEV in 0.9%, and AdV in 1.9% of the molluscs. Of the 23 water samples, 21.7% were positive for GII NoV and 4.3% for RoV and HEV genotype 3. Of the 70 vegetable samples, 2.9% were positive for NoV GI (GI.5 and GI.6), 2.9% for EV, and 1.4% for HEV. In the gastronomic preparations, only one EV (9%) was detected. No enteric viruses were detected in the berries, fruit, or swabs analyzed. Conclusions: Molecular surveillance of water and food samples clearly demonstrated that human pathogenic viruses are widely found in aquatic environments and on vegetables, and confirmed the role of vegetables and bivalve molluscs as the main reservoirs. Keywords: Enteric virus, PCR, Genotyping, Shellfish, Waters, Vegetable

Parthenolide generates reactive oxygen species and autophagy in MDA-MB231 cells. A soluble parthenolide analogue inhibits tumour growth and metastasis in a xenograft model of breast cancer.

Triple-negative breast cancers (TNBCs) are clinically aggressive forms associated with a poor prognosis. We evaluated the cytotoxic effect exerted on triple-negative MDA-MB231 breast cancer cells both by parthenolide and its soluble analogue dimethylamino parthenolide (DMAPT) and explored the underlying molecular mechanism. The drugs induced a dose- and timedependent decrement in cell viability, which was not prevented by the caspase inhibitor z-VAD-fmk. In particular in the first hours of treatment (1–3 h), parthenolide and DMAPT strongly stimulated reactive oxygen species (ROS) generation. The drugs induced production of superoxide anion by activating NADPH oxidase. ROS generation caused depletion of thiol groups and glutathione, activation of c-Jun N-terminal kinase (JNK) and downregulation of nuclear factor kB (NF-kB). During this first phase, parthenolide and DMAPT also stimulated autophagic process, as suggested by the enhanced expression of beclin-1, the conversion of microtubule-associated protein light chain 3-I (LC3-I) to LC3-II and the increase in the number of cells positive to monodansylcadaverine. Finally, the drugs increased RIP-1 expression. This effect was accompanied by a decrement of pro-caspase 8, while its cleaved form was not detected and the expression of c-FLIPS markedly increased. Prolonging the treatment (5–20 h) ROS generation favoured dissipation of mitochondrial membrane potential and the appearance of necrotic events, as suggested by the increased number of cells positive to propidium iodide staining. The administration of DMAPT in nude mice bearing xenografts of MDA-MB231 cells resulted in a significant inhibition of tumour growth, an increment of animal survival and a marked reduction of the lung area invaded by metastasis. Immunohistochemistry data revealed that treatment with DMAPT reduced the levels of NF-kB, metalloproteinase-2 and -9 and vascular endothelial growth factor, while induced upregulation of phosphorylated JNK. Taken together, our data suggest a possible use of parthenolide for the treatment of TNBCs

The Biobank of the ‘Istituto Zooprofilattico Sperimentale’ of Sicily: an important resource in medical research for safe and quality storage of biological specimens

The Mediterraean Biobank has been recently launched at the ‘Istituto Zooprofilattico Sperimentale’ (IZS) of Sicily in Palermo. The main aim of the Biobank is to collect and store, under standardised conditions and in a centralised system, several types of certified animal and zoonotic biological resources such as bacterial and viral strains, parasites, nucleic acids, positive/negative sera, cell cultures and tissues, in accordance to quality System procedures as regulated by UNI CEI EN ISO/IEC 17025:2005.peer-reviewe

Evaluation of the in vitro and in vivo antineoplastic effects of Parthenolide on MDA-MB231 breast cancer cells

Triple-negative breast cancer refers to an aggressive subtype of breast cancer in which the tumor cells lack receptors for estrogen, progesterone and the HER2 protein on their surfaces. This type of breast cancer does not respond to treatments such as hormone therapy, like tamoxifen and aromatase inhibitors, or drugs that target HER2, like Herceptin. It is important, therefore, the identification of new selective drugs for the treatment of these tumors. Parthenolide (PN), a sesquiterpene lactone extracted from the medical plant Tanacetum parthenium, exerts anticancer activity on several tumor cell lines in culture, acting through diverse molecular mechanisms. Our previous studies have shown that the PN exerts strong cytotoxic effects on MG63 osteosarcoma and SK-Mel28 melanoma cells, through a caspase-independent mechanism which is associated with production of oxidative stress. Recently, we have undertaken a study in order to investigate the antineoplastic activity of PN on MDA-MB231 cells, a triple-negative breast cancer cell line. Our results demonstrated that this compound reduced the viability of MDA-MB231 cells in a dose- and time- dependent manner. This effect was not prevented by the addition of z-VAD-fmk, a general inhibitor of caspase, thus suggesting a caspase-independent cell death. Time-course experiments provided evidence that the cytotoxic effect of PN occurs in two different phases. In the first phase of treatment (8h) cells resulted positive to monodansylcadaverine (MDC), a fluorochrome that binds to autophagic vacuoles. Prolonging the treatment (16h) MDC-positive cells lowered, and an increase of PI-positive population was found, suggesting the appearance of necrotic events. The study of the mode of PN action provided evidence that treatment with parthenolide induces ROS generation, activation of JNK and inhibition of NF-kB activity. All these effects were prevented by the addition of NAC, thus suggesting the involvement of oxidative stress. The antineoplastic activity of PN has been also assayed in vivo employing diamminoparthenolide (DMAPT), a soluble analogue of PN. Nude mice bearing breast carcinoma MDA-MB231 xenografts were treated daily with DMAPT (50 mg/Kg). It was observed that DMAPT increased survival of MDA-MB231 xenograft-bearing nude mice as well as reduced MDA-MB231 xenografts tumor growth. Moreover, immunohistochemical studies showed that DMAPT was able to decrease the expression of MMP-2, MMP-9 and VEGF, all factors involved in metastatic events. These data suggest a possible use of parthenolide for the treatment of triple negative breast cancers

Valutazione di alcuni indici di stress correlati al differente tipo di management in bovini affetti da Diarrea Virale Bovina/Malattia delle Mucose (BVD-MD) e Theileriosi

Convegno virtuale - "Risultati dell'attività di ricerca dell'IZS Sicilia" , 14 settembre 2020, Istituto Zooprofilattico Sperimentale della Sicilia, Palerm