Circulating tumor cells isolation:the "post-EpCAM era"

Circulating tumor cells （CTCs） represent a submicroscopic fraction detached from a primary tumor and in transit to a secondary site. The prognostic significance of CTCs in metastatic cancer patients was demonstrated for the first time more than ten years ago. To date, it seems clear enough that CTCs are highly heterogeneous and dynamically change their shape. Thus, the inadequacy of epithelial cell adhesion molecule （EpCAM） as universal marker for CTCs detection seems unquestionable and alternative methods able to recognize a broader spectrum of phenotypes are definitely needed. In this review the pleiotropic functions of EpCAM are discussed in detail and the role of the molecule in the biology of CTCs is critically dissected.

Impact of chronic exposure to bevacizumab on EpCAM-based detection of circulating tumor cells

Background： Circulating tumor cells （CTCs） are often undetected through the immunomagnetic epithelial cell adhesion molecule （EpCAM）-based CellSearch~ System in breast and colorectal cancer （CRC） patients treated with bevacizumab （BEV）, where low CTC numbers have been reported even in patients with evidence of progression of disease. To date, the reasons for this discrepancy have not been clarified. This study was carried out to investigate the molecular and phenotypic changes in CRC cells after chronic exposure to BEV in vitro. Methods： The human CRC cell line WiDr was exposed to a clinically relevant dose of BEV for 3 months in vitro. The expression of epithelial and mesenchymal markers and EpCAM isoforms was determined by western blotting and immunofluorescence. To evaluate the impact of EpCAM variant isoforms expression on CTC enumeration by CellSearch, untreated and treated colon cancer cells were spiked into 7.5 mL of blood from a healthy donor and enumerated by CellSearch. Results： Chronic exposure of CRC cell line to BEV induced decreased expression of EpCAM 40 kDa isoform and increased expression EpCAM 42 kDa isoform, together with a decreased expression of cytokeratins （CK）, while no evidence of epithelial to mesenchymal transition （EMT） in treated cells was observed. The recovery rate of cells through CellSearch was gradually reduced in course of treatment with BEV, being 84% , 70% and 40% at l, 2 and 3 months, respectively. Conclusions： We hypothesize that BEV may prevent CellSearch from capturing CTCs through altering EpCAM isoforms.

Functional Characterization of Porcine (<i>Sus scrofa</i>) BCL10

Human BCL10 (hBCL10) protein is a signal transduction molecule originally identified because of its direct involvement in a subset of mucosa-associated lymphoid tissue (MALT) lymphomas, and later recognized as a crucial factor in regulating activation of NF-kB transcription factor following antigen receptor stimulation on lymphocytes. In this study, we characterized the NF-kB inducing activity of porcine BCL10 (pBCL10). pBCL10 oligimerizes, binds to components of the CARMA/ BCL10/MALT1 complex and forms cytoplasmic filaments. Functionally, in human cells pBCL10 is more effective in activating NF-kB compared to hBCL10, possibly due to the lack of carboxy-terminal inhibitory serine residues present in the human protein. Also, depletion experiments carried out through expression of short hairpin RNAs targeting hBCL10 indicate that pBcl10 can functionally replace the human protein and retains its higher NF-kB-inducing property in the absence of hBCL10. Our results contribute useful information on BCL10 protein in pigs, and may help the development of strategies based on the control of the immune response in pigs.

Hereditary gastrointestinal polyposis: Diagnosis, genetic test and risk assessment

Colorectal cancer (CRC) is the second cause of cancer deaths, with over 1 million new cases estimated every year. Familial adenomatous polyposis, MUTYH-associated polyposis and hamartomatous polyposis are inherited syndromes that account for 2%-5% of all colon cancer. The mutated genes responsible for the vast majority of these disorders, are now known (MLH1, MSH2, MSH6, PMS2, APC, MYH, LKB1, SMAD4, BMPR1A, and PTEN) and specific mutations have been identified. Molecular caracterization of inherited CRCs allows pre-symptomatic diagnosis identifying at-risk individuals and improving cancer surveillance. Adenomatous polyposis includes familial adenomatous polyposis (FAP), attenuated FAP (AFAP), and MUTYH-associated polyposis (MAP). Hamartomatous polyposis comprises Peutz-Jeghers syndrome (PJS), juvenile polyposis syndrome (JPS) and “PTEN hamartoma tumour syndrome” (PHTS). MAP is an autosomal recessive condition, while all other disorders are inherited in an autosomal dominant manner. Differential dyagnosis could be very difficult between syndromes because of their phenotypic variability. Attenuated FAP, MAP and Lynch syndrome could be all associated with fewer numbers of adenomas (3-10 polyps), nevertheless, each syndrome has distinct cancer risks, characteristic clinical features, and separate genetic etiologies. Thus, differential diagnosis is essential for correct management of the specific disease. In our laboratory we set up a methodology for genetic tests of the colorectal polyposis syndrome. In these reviews we summarize the literature data and our experience about diagnosis, genetic tests and cancer risk assesment associated with colorectal polyposis. According to literature data, in our experience, there is a portion of analyzing patients that remain without identified mutation, after molecular screening of the specific gene involved in the pathogenesis of the disease. Since the sensibility of used techniques, such as DHPLC, MLPA and sequencing, is now very high, we suggest that a different approach to molecular diagnosis of polyposis syndromes is necessary. In our laboratory, we are now planning to set up analysis of a larger pannel of genes that could be involved in colorectal poliposis syndromes, using a next generation sequencing techniques. In our opinion, a better characterization of molecular basis of the polyposis syndromes will allow a more efficient cancer prevention.

Conservation of endangered animals: From biotechnologies to digital preservation

In the recent years, the number of endangered animals, both referred to livestock and wild species, has grown enormously. The “livestock” term refers to animals domesticated for producing commodities for man such as food, fiber and draught. Livestock biodiversity is integral to our culture, history, environment, and economy. Thousands of livestock breeds have evolved over time to suit particular environments and farming systems. Conservation and analyses of these genetic resources rely on demographic characterization and correct breeding schemes. In addition, molecular genetic studies allow to identify and monitor the genetic diversity within and across breeds and to reconstruct their evolution history. The conservation of livestock variability is also a crucial element in order to preserve and valorise specific nutritional and nutraceutical properties of animal products. Efficient ex situ and in situ conservation strategies, as well as the creation of bio-banks and specific biotechnological and bioinformatics tools for genetic analyses and digital preservation, are obligatory requirements in order to implement an appropriate action for the conservation of animal biodiversity. The main issues concerning different species are summarised, with particular reference to the livestock biodiversity still existing. Some examples of ex situ conservation strategies, which mainly refer to cryoconservation of semen, ova, embryos or tissues, developed in Italy, are presented, and the different actions in defense of Animal Genetic Resources (AnGR) developed within the European Community are illustrated. Interestingly, the same strategies for biological and digital analyses and preservation of livestock biodiversity can be exported to wild endangered animals in order to plan a correct conservation and repopulation of the species. Furthermore, the European Union has set up the guidelines to safeguard the biodiversity and to combat the extinction of animal species, and has made the protection of biodiversity and ecosystems one of the main objectives of the Sixth Environment Action Programme.

The physiological polyphosphate as a healing biomaterial for chronic wounds:Crucial roles of its antibacterial and unique metabolic energy supplying properties

Insufficient metabolic energy,in the form of adenosine triphosphate(ATP),and bacterial infections are among the main causes for the development of chronic wounds.Previously we showed that the physi-ological inorganic polymer polyphosphate(polyP)massively accelerates wound healing both in animals(diabetic mice)and,when incorporated into mats,in patients with chronic wounds.Here,we focused on a hydrogel-based gel formulation,supplemented with both soluble sodium polyP(Na-polyP)and amor-phous calcium polyP nanoparticles(Ca-polyP-NP).Exposure of human epidermal keratinocytes to the gel caused a significant increase in extracellular ATP level,an effect that was even enhanced when Na-polyP was combined with Ca-polyP-NP.Furthermore,it is shown that the added polyP in the gel is converted into a coacervate,leading to encapsulation and killing of bacteria.The data on human chronic wounds showed that the administration of hydrogel leads to the complete closure of these wounds.Histological analysis of biopsies showed an increased granulation of the wounds and an enhanced microvessel forma-tion.The results indicate that the polyP hydrogel,due to its properties to entrap bacteria and generate metabolic energy,is a very promising formulation for a new therapy for chronic wounds.