Evaluation of RAS Mutational Status in Liquid Biopsy to Monitor Disease Progression in Metastatic Colorectal Cancer Patients.

In this study we evaluated both~ K- and N-RAS mutations in plasma samples from patients with metastatic colorectal cancer by means of the BEAMing technology, and we assessed their diagnostic performance compared to RAS analyses performed on tissue. The sensitivity of BEAMing in identifying KRAS mutations was of 89.5%, with a fair specificity. The agreement with tissue analysis was moderate. The sensitivity for NRAS was high with a good specificity, and the agreement between tissue analysis and BEAMing was fair. Interestingly, significantly higher mutant allele fraction (MAF) levels were detected in patients with G2 tumors, liver metastases, and in those who did not receive surgery. NRAS MAF level was significantly higher in patients with mucinous adenocarcinoma and for those with lung metastases. A sharp increase in the MAF values was observed in patients who moved towards disease progression. More strikingly, molecular progression always anticipated the radiological one in these patients. These observations pave the way to the possibility of using liquid biopsy to monitor patients during treatment, and to enable oncologists to anticipate interventions compared to radiological analyses. This will allow time to be saved and ensure a better management of metastatic patients in the near future.

Stromal-like Wilms tumor cells induce human Natural Killer cell degranulation and display immunomodulatory properties towards NK cells.

The similarity of stromal-like Wilms tumor (str-WT) cells with mesenchymal stem cells (MSC), suggests their relevant role in the interplay with immune cells in the tumor microenvironment. We investigated the interaction between str-WT cells and NK cells. We observed that str-WT cells expressed some major ligands for activating and inhibitory NK cell receptors. Moreover, they expressed inhibitory checkpoint molecules involved in the negative regulation of anti-tumor immune response. The analysis of the interaction between str-WT cells and NK lymphocytes revealed that activated NK cells could efficiently degranulate upon interaction with str-WT cells. On the other hand, str-WT cells could exert potent inhibitory effects on cytokine-induced activation of NK cell proliferation and phenotype, which were mediated by the production of IDO and PGE2 inhibitory factors. Our data provide insight into the molecular interactions between str-WT cells and NK lymphocytes that may result in different outcomes possibly occurring in the WT microenvironment.

Characterization of MDCK cells and evaluation of their ability to respond to infectious and non-infectious stressors.

The Madin-Darby Canine Kidney (MDCK) cell line is widely used as epithelial cell model in studies ranging from viral infection to environmental pollutants, and vaccines production. However, little is known about basal expression of genes involved in innate immunity, and the ability to respond to infectious and non-infectious stressors. Therefore, the aims of our study were to evaluate the basal level of expression of pivotal genes in the innate immune response and cell cycle regulation, as well as to evaluate the ability of this cell line to respond to infectious or non-infectious stressors. As surmised in our working hypothesis, we demonstrated the constitutive expression of genes involved in the innate immune response and cell defense alike, including TLRs, Interleukins, Myd88, p65/NF-kB and p53. Moreover, we described the ability of this cell line to respond to LPS and cadmium (Cd2+) in terms of gene expression and cytokine release. These data confirm the possibility of using this cell line as a model in studies of host/pathogen interaction and response to non-infectious stressors.

Yersinia enterocolitica-specific modulation of innate immune responses in jejunal epithelial cells.

Gut is often subject to infection by different pathogens like Y. enterocolitica. To date, biotypes (BTs) 1A have been considered as non-pathogenic, because they do not express plasmid of virulence pYV; however, BTs 1A strains present other chromosomic virulence genes and recent studies suggest an implication of this microorganism in reactive arthritis. Although many studies highlighted the molecular basis of pathogenesis of Ye infection, scanty data are available about several environmental BTs 1A strains, often isolated in cases of foodborne disease but not included in pathogenicity studies. The aim of our work was to verify the ability of different Ye 1A strains to adhere and penetrate IPEC-J2 cells and to modulate intestinal innate immunity. Our results showed that all strains under study were able to adhere and penetrate enterocytes, causing inflammatory responses. Indeed, adhesion and invasion of enterocytes is an essential step in Ye pathogenesis (Fàbrega and Vila, 2012). Moreover, our data suggest the possible involvement of strains Ye2/O:9 in reactive arthritis, due to their ability (i) to penetrate enterocytes as pathogenic Ye1/O:8 strains do, and (ii) to increase IL-6, IL-8, IL-12 and IL-18 release. Lastly, our results confirm that IPEC-J2 cells are a very good model to evaluate host-pathogen interaction, and indicate IL-8, TNF-α, TLRs1 and 4 as possible markers of the ability of Ye strains to penetrate enterocytes. Moreover, we showed that Ye strains differently affect the host's innate immune responses.