Oncolytic Viruses: An Inventory of Shedding Data from Clinical Trials and Elements for the Environmental Risk Assessment.

Attenuated and/or genetically modified oncolytic viruses (OV) gain increasing interest as a promising approach for cancer therapy. Beside the assessment of subject safety, quality and efficacy aspects of medicinal products for human use, genetically modified viruses are also governed by EU regulatory frameworks requiring an environmental risk assessment (ERA). An important element to be assessed as part of the ERA is the incidence of exposure to OV of individuals, other than the trial subjects, and the environment. The evidence-based evaluation of shedding data is considered to be decisive in that context, as it may impact the OV capacity to be transmitted. This is particularly true for OV still able to (conditionally) replicate as opposed to replication-defective viral vectors commonly used in gene therapy or vaccination. To our knowledge, this article presents the most extensive and up-to-date review of shedding data reported with OV employed in clinics. Besides the identification of a topical need for improving the collection of shedding data, this article aims at providing an aid to the design of an appropriate shedding study, thereby relying on and further complementing principles described in existing guidelines issued by European and international institutions.

Oligomers of grape-seed procyanidin extract activate the insulin receptor and key targets of the insulin signaling pathway differently from insulin.

Procyanidins are bioactive flavonoid compounds from fruits and vegetables that possess insulinomimetic properties, decreasing hyperglycaemia in streptozotocin-diabetic rats and stimulating glucose uptake in insulin-sensitive cell lines. Here we show that the oligomeric structures of a grape-seed procyanidin extract (GSPE) interact and induce the autophosphorylation of the insulin receptor in order to stimulate the uptake of glucose. However, their activation differs from insulin activation and results in differences in the downstream signaling. Oligomers of GSPE phosphorylate protein kinase B at Thr308 lower than insulin does, according to the lower insulin receptor activation by procyanidins. On the other hand, they phosphorylate Akt at Ser473 to the same extent as insulin. Moreover, we found that procyanidins phosphorylate p44/p42 and p38 MAPKs much more than insulin does. These results provide further insight into the molecular signaling mechanisms used by procyanidins, pointing to Akt and MAPK proteins as key points for GSPE-activated signaling pathways. Moreover, the differences between GSPE and insulin might help us to understand the wide range of biological effects that procyanidins have.

Insulin increase in MAP kinase phosphorylation is shifted to early time-points by overexpressing APS, while Akt phosphorylation is not influenced.

Upon insulin stimulation, the adaptor protein APS is recruited to the insulin receptor and tyrosine phosphorylated. APS initiates the insulin-induced TC10 cascade which participates to GLUT4 translocation to the plasma membrane. Nevertheless, the molecular mechanism that governs APS and its SH2 and PH domains action on the insulin transduction cascade is not yet fully understood. Here, we show that APS co-immunoprecipitates with the class I PI 3-kinase regulatory subunit p85, through its SH2 domain but that APS does not modulate neither PtdIns(3,4,5)P3 levels nor Akt phosphorylation provoked by insulin. We have confirmed a previously described positive effect of APS overexpression on insulin-induced MAPK phosphorylation upregulation. Consequently, we analyzed the role of SH2 and PH domains of APS in the APS increased MAPK phosphorylation observed upon insulin stimulation and correlated this with the membrane localization of the protein. The effect observed on MAPK phosphorylation requires the intact PH binding domain of APS as well as its SH2 domain.

The docking properties of SHIP2 influence both JIP1 tyrosine phosphorylation and JNK activity.

SHIP2 (SH2-containing inositol polyphosphate 5-phosphatase 2) is an ubiquitously expressed phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P(3)) 5-phosphatase which contains various motifs susceptible to mediate protein-protein interaction. In cell models, evidence has been provided that SHIP2 plays a role in insulin and growth factor signaling, cytoskeletal organization, cell adhesion and migration. Herein we describe the c-Jun NH2-terminal kinase (JNK)-interacting protein 1 (JIP1) as a new protein partner of SHIP2. The interaction between SHIP2 and JIP1 was confirmed in both overexpression systems and native cells. Without modifying the association of JIP1 with the MAPKs in the scaffold complex and with no apparent change of Akt phosphorylation, SHIP2 positively modulated the MLK3/JIP1-mediated JNK1 activation. Moreover, SHIP2 positively regulated the tyrosine phosphorylation of JIP1. This up-regulation was prevented by inhibitors of the Src family and Abl kinases, PP2 and Glivec. The effects of SHIP2 on JNK activity and JIP1 tyrosine phosphorylation were independent of the SHIP2 phosphoinositide 5-phosphatase activity, as similar results were obtained when using a SHIP2 catalytic inactive mutant instead of wild-type SHIP2. Together, these data suggest that by its docking properties, SHIP2 can modulate JIP1-mediated JNK pathway signaling.

The association between the SH2-containing inositol polyphosphate 5-Phosphatase 2 (SHIP2) and the adaptor protein APS has an impact on biochemical properties of both partners.

SHIP2 (SH2-containing inositol polyphosphate 5-phosphatase 2) is a phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P(3)) 5-phosphatase containing various motifs susceptible to mediate protein-protein interaction. In cell models, SHIP2 negatively regulates insulin signalling through its catalytic PtdIns(3,4,5)P(3) 5-phosphatase activity. We have previously reported that SHIP2 interacts with the c-Cbl associated protein (CAP) and c-Cbl, proteins implicated in the insulin cellular response regulating the small G protein TC10. The first steps of the TC10 pathway are the recruitment and tyrosine phosphorylation by the insulin receptor of the adaptor protein with Pleckstrin Homology and Src Homology 2 domains (APS). Herein, we show that SHIP2 can directly interact with APS in 3T3-L1 adipocytes and in transfected CHO-IR cells (Chinese hamster ovary cells stably transfected with the insulin receptor). Upon insulin stimulation, APS and SHIP2 are recruited to cell membranes as seen by immunofluorescence studies, which is consistent with their interaction. We also observed that SHIP2 negatively regulates APS insulin-induced tyrosine phosphorylation and consequently inhibits APS association with c-Cbl. APS, which specifically interacts with SHIP2, but not PTEN, in turn, increases the PtdIns(3,4,5)P(3) 5-phosphatase activity of SHIP2 in an inositol phosphatase assay. Co-transfection of SHIP2 and APS in CHO-IR cells further increases the inhibitory effect of SHIP2 on Akt insulin-induced phosphorylation. Therefore, the interaction between APS and SHIP2 provides to both proteins potential negative regulatory mechanisms to act on the insulin cascade.

Endogenous SHIP2 does not localize in lipid rafts in 3T3-L1 adipocytes.

SH2 domain containing inositol polyphosphate 5-phosphatase (SHIP2) dephosphorylates phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) into phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P(2)). SHIP2 knock-out mice demonstrated that SHIP2 acts as a negative regulator of insulin cascade in vivo. Our two-hybrid study showed that SHIP2 interacts with c-Cbl associated protein (CAP) and c-Cbl, implicated in the insulin signaling. As some proteins implicated in insulin signaling, like insulin receptor, CAP, c-Cbl or TC10, were reported to localize in lipid rafts, we addressed the same question for SHIP2. SHIP2 was detected in the non-raft fraction in CHO-IR, C2C12 myotubes and 3T3-L1 adipocytes except when it is overexpressed in CHO-IR, where we detected SHIP2 in the raft fraction.