A. Guercio

Production of canine mesenchymal stem cells from adipose tissue and their application in dogs with chronic osteoarthritis of the humeroradial joints.

Autologous AD‐MSC [adipose‐derived MSC (mesenchymal stem cell)] therapy involves harvesting fat from the patient by isolating the stem and regenerative cells and administering the cells back to the patient. This study evaluated the production of canine AD‐MSCs and their possible application in cellular therapy for dogs. To assess whether cellular therapy can replace drug therapy, the clinical effect of a single intra‐articular injection of AD‐MSCs was evaluated on 4 dogs with lameness associated with OA (osteoarthritis) of the humeroradial joints. MSCs were readily isolated from adult dog adipose tissue, and their ability to form colony and differentiate into various phenotypes was confirmed. AD‐MSCs expressed OCT4, NANOG and SOX2 at the mRNA level, pluripotency markers usually ascribed to embryonic stem cells. The results suggest the stemness of the cells isolated from canine fat, and good quality control made them available for both experimental and clinical use. Follow‐up studies to evaluate the effects of AD‐MSC therapy showed that OA of the elbow joints improved with time, indicating significant potential for clinical use in the treatment of lameness, particularly when administered before the injury becomes severe.

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 time-dependent 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.

Canine mesenchymal stem cells (MSCs): characterization in relation to donor age and adipose tissue-harvesting site.

Adipose tissue as a stem cell source is ubiquitously available and has several advantages compared to other sources, for example it is easily accessible in large quantities with minimal invasive harvesting procedure, and isolation of adipose‐derived mesenchymal stem cells (MSCs) yields a high amount of stem cells, essential for stem cell‐based therapies and tissue engineering. We have explored the effect of donor age, and the anatomical origin of the adipose tissue on several aspects of MSCs in dogs, such as cell yield, proliferative ability, multi‐differentiation potential, colony‐forming capacity, stemness marker expression. We also assessed the effect of cell passaging on the MSCs stemness. We found that the anatomical origin of the adipose tissue and the age of donors have effects only on the proliferative capacity of the MSCs. Moreover, cells show a progressive loss of the stemness characteristics with passages. Cell therapies need a suitable number of cells to use in clinical applications. Characterization of MSCs at different passages, allowed us to demonstrate that, under our culture conditions, the best quantitative and qualitative characteristics are obtained at early passages. Adult MSCs are of particular interest for the therapeutic approach to musculoskeletal diseases, and the dog provides an excellent preclinical model for the development of new approaches in regenerative medicine that might be applied to humans.

Extended Genetic Diversity of Bovine Viral Diarrhea Virus and Frequency of Genotypes and Subtypes in Cattle in Italy between 1995 and 2013

Genetic typing of bovine viral diarrhea virus (BVDV) has distinguished BVDV-1 and BVDV-2 species and an emerging putative third species (HoBi-like virus), recently detected in southern Italy, signaling the occurrence of natural infection in Europe. Recognizing the need to update the data on BVDV genetic variability in Italy for mounting local and European alerts, a wide collection of 5′ UTR sequences (n = 371) was selected to identify the frequency of genotypes and subtypes at the herd level. BVDV-1 had the highest frequency, followed by sporadic BVDV-2. No novel HoBi-like viruses were identified. Four distribution patterns of BVDV-1 subtypes were observed: highly prevalent subtypes with a wide temporal-spatial distribution (1b and 1e), low prevalent subtypes with a widespread geographic distribution (1a, 1d, 1g, 1h, and 1k) or a restricted geographic distribution (1f), and sporadic subtypes detected only in single herds (1c, 1j, and 1l). BVDV-1c, k, and l are reported for the first time in Italy. A unique genetic variant was detected in the majority of herds, but cocirculation of genetic variants was also observed. Northern Italy ranked first for BVDV introduction, prevalence, and dispersion. Nevertheless, the presence of sporadic variants in other restricted areas suggests the risk of different routes of BVDV introduction.

Cetacean strandings in Italy: an unusual mortality event along the Tyrrhenian Sea coast in 2013

An unusual mortality event involving cetaceans, mainly striped dolphins Stenella coeruleoalba (Meyen, 1833), occurred along the Tyrrhenian Sea coast of Italy during the first 3 mo of 2013. Based on post-mortem analyses carried out according to body condition on 66 dolphins (54% of stranded animals), several hypotheses to explain the causes of this mortality event were proposed. Although no definitive conclusions can be drawn, dolphin morbillivirus was deemed the most likely cause, although other infectious agents (including Photobacterium damselae damselae and herpesvirus) or environmental factors may also have contributed to this recent mortality event.

Modeling human osteosarcoma in mice through 3AB‐OS cancer stem cell xenografts

Osteosarcoma is the second leading cause of cancer‐related death for children and young adults. In this study, we have subcutaneously injected—with and without matrigel—athymic mice (Fox1nu/nu) with human osteosarcoma 3AB‐OS pluripotent cancer stem cells (CSCs), which we previously isolated from human osteosarcoma MG63 cells. Engrafted 3AB‐OS cells were highly tumorigenic and matrigel greatly accelerated both tumor engraftment and growth rate. 3AB‐OS CSC xenografts lacked crucial regulators of beta‐catenin levels (E‐cadherin, APC, and GSK‐3beta), and crucial factors to restrain proliferation, resulting therefore in a strong proliferation potential. During the first weeks of engraftment 3AB‐OS‐derived tumors expressed high levels of pAKT, beta1‐integrin and pFAK, nuclear beta‐catenin, c‐Myc, cyclin D2, along with high levels of hyperphosphorylated‐inactive pRb and anti‐apoptotic proteins such as Bcl‐2 and XIAP, and matrigel increased the expression of proliferative markers. Thereafter 3AB‐OS tumor xenografts obtained with matrigel co‐injection showed decreased proliferative potential and AKT levels, and undetectable hyperphosphorylated pRb, whereas beta1‐integrin and pFAK levels still increased. Engrafted tumor cells also showed multilineage commitment with matrigel particularly favoring the mesenchymal lineage. Concomitantly, many blood vessels and muscle fibers appeared in the tumor mass. Our findings suggest that matrigel might regulate 3AB‐OS cell behavior providing adequate cues for transducing proliferation and differentiation signals triggered by pAKT, beta1‐integrin, and pFAK and addressed by pRb protein. Our results provide for the first time a mouse model that recapitulates in vivo crucial features of human osteosarcoma CSCs that could be used to test and predict the efficacy in vivo of novel therapeutic treatments. J. Cell. Biochem. 113: 3380–3392, 2012.

Silk fibroin scaffolds enhance cell commitment of adult rat cardiac progenitor cells

The use of three‐dimensional (3D) cultures may induce cardiac progenitor cells to synthesize their own extracellular matrix (ECM) and sarcomeric proteins to initiate cardiac differentiation. 3D cultures grown on synthetic scaffolds may favour the implantation and survival of stem cells for cell therapy when pharmacological therapies are not efficient in curing cardiovascular diseases and when organ transplantation remains the only treatment able to rescue the patients life. Silk fibroin‐based scaffolds may be used to increase cell affinity to biomaterials and may be chemically modified to improve cell adhesion. In the present study, porous, partially orientated and electrospun nanometric nets were used. Cardiac progenitor cells isolated from adult rats were seeded by capillarity in the 3D structures and cultured inside inserts for 21 days. Under this condition, the cells expressed a high level of sarcomeric and cardiac proteins and synthesized a great quantity of ECM. In particular, partially orientated scaffolds induced the synthesis of titin, which is a fundamental protein in sarcomere assembly. Copyright

Investigation and control of a Norovirus outbreak of probable waterborne transmission through a municipal groundwater system

During March 2011 an outbreak of gastroenteritis occurred in Santo Stefano di Quisquina, Agrigento, Sicily, Italy. Within two weeks 156 cases were identified among the 4,965 people living in the municipality. An epidemiological investigation was conducted to characterize the outbreak and target the control measures. A case was defined as a person developing diarrhea or vomiting during February 27-March 13, 2011. Stool specimens were collected from 12 cases. Norovirus (NoV) genotype GII.4 variant New Orleans 2009 was identified in stool samples from 11 of 12 cases tested (91.7%). Epidemiological investigations suggested a possible association with municipal drinking water consumption. Water samples from the public water system were tested for NoV and a variety of genotypes were detected during the first 3 months of surveillance, including GII.4 strains belonging to different variants from that involved in the gastroenteritis outbreak. Contamination of the well and springs supplying the public water network was eventually thought to be the source of the NoV contamination.

Evidence of zoonotic Poxviridae coinfections in clinically diagnosed papillomas using a newly developed mini-array test.

Our study describes a newly developed mini-array test for the rapid detection of poxviruses in animals and humans. The method is based on detection that combines target nucleic acid amplification by polymerase chain reaction and specific hybridization, using enzyme-linked antibodies, allowing identification of zoonotic orthopoxviruses and parapoxviruses in animal and human biological samples. With 100% specificity, the test rules out the possibility of cross-reactions with viral agents causing look-alike diseases. The assay was employed in the field to investigate the causes of several outbreaks of a malignant proliferative skin disease that affected domestic ruminants in Sicily during 2011–2014. Due to specific aspects of the lesions, the animals were clinically diagnosed with papillomatosis. The mini-array test allowed the identification of coinfections caused by more than 1 viral species belonging to the Parapoxvirus and Orthopoxvirus genera, either in goats or in cattle. Our study suggests that the so-called “papillomatosis” can be the result of multiple infections with epitheliotropic viruses, including zoonotic poxviruses that cannot be properly identified with classical diagnostic techniques.

Genotyping of bovine viral diarrhea viruses (BVDV) isolated from cattle in Sicily

Bovine Viral Diarrhea–Mucosal Disease (BVD–MD) is a widely spread infectious disease that causes important economic losses in farms. Several epidemiological studies indicate a high genetic heterogeneity among Bovine Viral Diarrhea Virus (BVDV) strains circulating in Italy. The aim of this study was to investigate the genotypes of BVDV in Sicily, a region in the South of Italy. For this purpose, 17 BVDV strains collected from cattle breed in Sicily between 2005 and 2008 were genetically typed by sequencing of the 5′-untraslated region (5′-UTR) of the viral genome. In this study, phylogenetic analysis showed that all 17 examined strains were clustered within the BVDV genotype 1. Particularly, 14 of them were clustered with the BVDV-1b subgroup, while the remaining three strains were clustered with the BVDV-1e. Moreover, the restriction analysis indicated a bovine origin for all of the 17 strains typed in this study. These results could be useful to carry out an epidemiological survey and to create vaccines that protect cattle against BVDV different subgroups.