po 040 development of a tunable form of interferon alpha for in vivo cancer gene therapy

Introduction The immune system is a double-edge sword in cancer. On the one hand, it exerts immunosurveillance to eradicate transformed cells that occasionally appear in the body; on the other hand, cancer cells can recruit immune cells endowed with pro-tumorigenic activity. Our lab previously developed a strategy for targeted gene-based delivery of interferon alpha (IFNa) to tumours by tumour infiltrating monocytes/macrophages, which induces robust anti-cancer responses in several experimental models without inducing strong IFN responses in normal tissues as compared to systemic administration of recombinant IFNa. Whereas a sustained output could ensure long-term protection from tumour recurrence, it may raise concerns for long-term side effects, especially in case of cancer eradication.To overcome this issue, we are developing inducible strategies to control the amount of IFNa secreted in the tumour microenvironment. Material and methods By fusing a destabilising domain (DD) to a protein of interest (POI) the former can confer its instability to the latter. This destabilisation can be rescued in a reversible and dose dependent manner with the addition of a small molecule specifically binding to the DD. To apply this technology to our strategy we have designed and in vitro tested different fusion proteins of IFNa (DD-IFNa). We also developed improved DD-IFNa with the addition of flexible and/or cleavable linkers and selected them for their capacity to be stabilised in a dose dependent manner in presence of their specific ligand in vitro . Results and discussions Through this approach, we have identified effective fusion proteins with low basal activity and high fold induction upon ligand treatment. These novel tunable forms of IFNa are functional and their specific activity are comparable to the wild type cytokine in inducing IFN responsive genes. Based on these promising in vitro results we are now translating these new platforms in vivo to test their efficacy in inducing anti-tumour responses in melanoma, colon and glioma models of cancer. Conclusion In the perspective of clinical translation our approach can be used in the future to switch on/off the levels of IFNa in a tunable and personalised fashion for cancer eradication

Position sensitivity in 3 × 3 LaBr3:Ce scintillators

The position sensitivity in a 33 LaBr 3:Ce crystal has been simulated using collimated beams of medium and high energy -rays. The simulations have been done using the GEANT4 libraries which allow to follow both the gamma ray interaction in the detector and the scintillation light up to the photocathode. In the simulations the crystal has been coupled to an ideal PSPMT and/or a shielded PMT. The results indicate that in the 3 3 crystal with darkened surfaces it should be possible to localize the first interaction point of a 662 keV gamma ray within 1 cm, in the case of diffusive surfaces the position sensitivity deteriorates but it is not destroyed. As expected, the position sensitivity improves as the -ray energy increases. The measurements generally confirm the results given by simulations. \ua9 2010 IEEE

ABCD Meeting \u201cOrganelle Biogenesis and Signal Transduction\u201d

An ubiquitin-proteasome-endocytic pathway but not autophagy orchestrates ErbB2 internalization and cleavage in HSP90-inhibited breast cancer cell

71\ub0 CONGRESSO NAZIONALE SIAI, Societ\ue0 di Anatomia e Istologia, Taormina, 20 - 22 Settembre 2017

The receptor tyrosine kinase ERBB2 interacts with HSP90 and is overexpressed in aggressive breast cancers. Therapeutic HSP90 inhibitors, i.e. Geldanamycin (GA), target ERBB2 to degradation. We have previously shown that HSP90 is responsible for the missorting of recycling ERBB2 to degradation compartments. In this study, we used biochemical, immunofluorescence and electron microscopy techniques to demonstrate that in SKBR3 human breast cancer cells, GA strongly induces polyubiquitination and internalization of the full-length p185-ERBB2, and promotes its cleavage, with the formation of a p116-ERBB2 form in EEA1-positive endosomes (EE). p116-ERBB2 corresponds to a non-ubiquitinated, signaling-impaired, membrane-bound fragment, which is readily sorted to lysosomes and degraded. To define the sequence of events leading to p116-ERBB2 degradation, we first blocked the EE maturation/trafficking to late endosomes/lysosomes with wortmannin, and found an increase in GA-dependent formation of p116-ERBB2; we then inhibited the proteasome activity with MG-132 or lactacystin, and observed an efficient block of p185-ERBB2 cleavage, and its accumulation in EE, suggesting that p185-ERBB2 polyubiquitination is necessary for proteasome-dependent p116-ERBB2 generation occurring in EE. As polyubiquitination has also been implicated in autophagy-mediated degradation of ERBB2 under different experimental conditions, we exploited this possibility and demonstrate that GA strongly inhibits early autophagy, and reduces the levels of the autophagy markers atg5-12 and LC3-II, irrespective of GA-induced ERBB2 polyubiquitination, ruling out a GA-dependent autophagic degradation of ERBB2. In conclusion, we propose that HSP90 inhibition fosters ERBB2 polyubiquitination and proteasome-dependent generation of a non-ubiquitinated and inactive p116-ERBB2 form in EE, which is trafficked from altered EE to lysosomes

ABCD 2015, Biennial Congress of the Italian Association of cell biology and differentiation

HSP90 inhibition induces ErbB2 cleavage and internalization simultaneoulsy with alterations in endosomal trafficking/maturation and autophag

Shared decision-making in neonatology: an utopia or an attainable goal?

Medical decision making is sometimes considered as a relatively simple process in which a decision may be made by the physician, by the patient, or by both patient and physician working together. There are three main models of decision making--paternalism, patient informed choice, and shared decision-making (SDM), having each one of these drawbacks and limitations. Historically, the most adopted one was the paternalism (strongly 'Doctor knows best'), where the professional made the decision based on what he/she considered to be as the patient's best interest, not necessarily contemplating patient's will and wishes. Currently, at the antipodes, the patient informed choice, where the patient makes his/her decision based on information received from the physician with no possible interference of professional's own preferences, seems to be the preferred relationship standard. SDM represents an intermediate approach between the two above-mentioned opposite models, being a medical process that involves actively the doctor and the patient who both bring their own facts and preferences to reach an agreement on the decision on if, when and how to treat a disease. This model, being characterized by elements pertaining to both the others, is gaining popularity in several medical and surgical scenarios whenever a competent patient is able to actively participate into the decisional process. On this basis can this model be implemented also in a Neonatology Intensive Care Unit where little patients are--by nature--incompetent, being the diagnostic/therapeutic choices taken by parents? We focused on this complex item considering four possible different scenarios and it seems to us that it could be possible to introduce such an approach, providing that parents' empowerment, a good physician's communication skill and consideration of all cultural, religious, economic, and ethic values of every single actor have been fairly taken into account

Shared decision-making in neonatology: an utopia or an attainable goal?

Medical decision making is sometimes considered as a relatively simple process in which a decision may be made by the physician, by the patient, or by both patient and physician working together. There are three main models of decision making--paternalism, patient informed choice, and shared decision-making (SDM), having each one of these drawbacks and limitations. Historically, the most adopted one was the paternalism (strongly 'Doctor knows best'), where the professional made the decision based on what he/she considered to be as the patient's best interest, not necessarily contemplating patient's will and wishes. Currently, at the antipodes, the patient informed choice, where the patient makes his/her decision based on information received from the physician with no possible interference of professional's own preferences, seems to be the preferred relationship standard. SDM represents an intermediate approach between the two above-mentioned opposite models, being a medical process that involves actively the doctor and the patient who both bring their own facts and preferences to reach an agreement on the decision on if, when and how to treat a disease. This model, being characterized by elements pertaining to both the others, is gaining popularity in several medical and surgical scenarios whenever a competent patient is able to actively participate into the decisional process. On this basis can this model be implemented also in a Neonatology Intensive Care Unit where little patients are--by nature--incompetent, being the diagnostic/therapeutic choices taken by parents? We focused on this complex item considering four possible different scenarios and it seems to us that it could be possible to introduce such an approach, providing that parents' empowerment, a good physician's communication skill and consideration of all cultural, religious, economic, and ethic values of every single actor have been fairly taken into account