Magnetically Driven Bioreactors as new Tools in Drug Delivery

The pharmacological properties of many drugs can be improved by drug delivery systems able to drive therapeutic agents to target regions. The use of carriers, in fact, may reduce possible cytotoxic effects of drugs and increase their bioavailability at the site of action, thus improving the efficacy and the safety of treatments. Therefore, we have developed an erythrocyte-based drug delivery system (erythro-magneto-HA virosome), which has the potential to be magnetically guided to specific sites and to fuse with target cells. These engineered erythrocytes have demonstrated in previous work a very high in vitro capability to release anticancer drugs directly inside target cells. Because the erythro-magneto-HA virosomes (EMHVs) proved to be promising carriers, we decided to investigate in more details the effectiveness and safety of this erythrocyte-based drug delivery system. We evaluated the ability of the EMHVs to be specifically localized in vivo to desired sites by means of an external magnetic field and to protect an anticancer drug such as 5-Aza-2\u27-deoxycytidine from degradation. Additionally we have assessed the ability of the EMHVs to act as bioreactors and to convert the pro-drug 5-Aza-2\u27-deoxycytidine into an active drug. Finally, we have studied the interaction of the EMHVs with the host immune system. The pro-drug 5-Aza-2\u27-deoxycytidine has short half-life when systemically injected and needs to be phosphorylated to become an active drug. We found that when inside the engineered erythrocytes it is protected by degradation and is transformed in its active form thus becoming readily available for uptake by the targeted cells. Moreover, we have observed that the EMHVs used didn\u27t cause either a cell-mediated or a humoral immune response in host mice having the same haplotype of the donors. These findings suggest that erythro-magneto-HA virosomes are a safe and useful drug delivery system that may offer numerous advantages for several clinical application

The role of extracellular vesicles and interleukin-8 in regulating and mediating neutrophil-dependent cancer drug resistance

Tumor drug resistance is a multifactorial and heterogenous condition that poses a serious burden in clinical oncology. Given the increasing incidence of resistant tumors, further understanding of the mechanisms that make tumor cells able to escape anticancer drug effects is pivotal for developing new effective treatments. Neutrophils constitute a considerable proportion of tumor infiltrated immune cells, and studies have linked elevated neutrophil counts with poor prognosis. Tumor-associated neutrophils (TANs) can acquire in fact immunoregulatory capabilities, thus regulating tumor progression and resistance, or response to therapy. In this review, we will describe TANs’ actions in the tumor microenvironment, with emphasis on the analysis of the role of interleukin-8 (IL-8) and extracellular vesicles (EVs) as crucial modulators and mediators of TANs biology and function in tumors. We will then discuss the main mechanisms through which TANs can induce drug resistance, finally reporting emerging therapeutic approaches that target these mechanisms and can thus be potentially used to reduce or overcome neutrophil-mediated tumor drug resistance

Prostaglandin E2 Stimulates the Expansion of Regulatory Hematopoietic Stem and Progenitor Cells in Type 1 Diabetes

Hematopoietic stem and progenitor cells (HSPCs) are multipotent stem cells that have been harnessed as a curative therapy for patients with hematological malignancies. Notably, the discovery that HSPCs are endowed with immunoregulatory properties suggests that HSPC-based therapeutic approaches may be used to treat autoimmune diseases. Indeed, infusion with HSPCs has shown promising results in the treatment of type 1 diabetes (T1D) and remains the only “experimental therapy” that has achieved a satisfactory rate of remission (nearly 60%) in T1D. Patients with newly diagnosed T1D have been successfully reverted to normoglycemia by administration of autologous HSPCs in association with a non-myeloablative immunosuppressive regimen. However, this approach is hampered by a high incidence of adverse effects linked to immunosuppression. Herein, we report that while the use of autologous HSPCs is capable of improving C-peptide production in patients with T1D, ex vivo modulation of HSPCs with prostaglandins (PGs) increases their immunoregulatory properties by upregulating expression of the immune checkpoint-signaling molecule PD-L1. Surprisingly, CXCR4 was upregulated as well, which could enhance HSPC trafficking toward the inflamed pancreatic zone. When tested in murine and human in vitro autoimmune assays, PG-modulated HSPCs were shown to abrogate the autoreactive T cell response. The use of PG-modulated HSPCs may thus provide an attractive and novel treatment of autoimmune diabetes

RNF168 promotes noncanonical K27 ubiquitination to signal DNA damage

Ubiquitination regulates numerous cellular processes by generating a versatile communication system based on eight structurally and functionally different chains linked through distinct residues. Except for K48 and K63, the biological relevance of different linkages is largely unclear. Here, we show that RNF168 ubiquitin ligase promotes noncanonical K27-linked ubiquitination both in vivo and in vitro. We demonstrate that residue K27 of ubiquitin (UbK27) is required for RNF168-dependent chromatin ubiquitination, by targeting histones H2A/H2A.X, and that it is the major ubiquitin-based modification marking chromatin upon DNA damage. Indeed, UbK27 is strictly required for the proper activation of the DNA damage response (DDR) and is directly recognized by crucial DDR mediators, namely 53BP1, Rap80, RNF168, and RNF169. Mutation of UbK27 has dramatic consequences on DDR activation, preventing the recruitment of 53BP1 and BRCA1 to DDR foci. Similarly to the DDR, atypical ubiquitin chains could play unanticipated roles in other crucial ubiquitin-mediated biological processes.ISSN:2666-3864ISSN:2211-124

Human papillomavirus type 16 E6 and E7 oncoproteins interact with the nuclear p53-binding protein 1 in an in vitro reconstructed 3D epithelium: new insights for the virus-induced DNA damage response

Abstract Background Despite vaccination and screening measures, anogenital cancer, mainly promoted by HPV16 oncoproteins, still represents the fourth tumor and the second cause of death among women. Cell replication fidelity is the result of the host DNA damage response (DDR). Unlike many DNA viruses that promote their life cycle through the DDR inactivation, HR-HPVs encourage cells proliferation despite the DDR turned on. Why and how it occurs has been only partially elucidated. During HPV16 infection, E6 links and degrades p53 via the binding to the E6AP LXXLL sequence; unfortunately, E6 direct role in the DDR response has not clearly identified yet. Similarly, E7 increases DDR by competing with E2F1-pRb interaction, thus leading to the inactivation of pRb, and promotion, E2F1 mediated, of DDR genes translation, by binding to the pRb-like proteins CBP/p300 and p107, that also harbour LXXLL sequence, and via the interaction and activation of several DDR proteins. Methods To gain information regarding E6 and E7 contribution in DDR activation, we produced an in vitro 3D HPV16-E6E7 infected epithelium, already consolidated study model for HPVs, and validated it by assessing H&E staining and BrdU, HPV16 DNA, E6E7 proteins and γH2A.X/53BP1 double-strand break (DSBs) sensors expression; then we made an immuno-colocalization of E6 and E7 with cyclin E2 and B1. Since 53BP1, like E6 and E7, also binds p53 and pRb, we supposed their possible direct binding. To explore this hypothesis, we performed a double immunofluorescence of E6 and E7 with 53BP1, a sequence analysis of 53BP1 within its BRCT2 domain and then an in situ PLA within CaSki, E6E7HPV16 NHEKs and the 3D model. Results The in vitro epithelium resembled the histology and the events typical of in vivo infected tissues. E6E7HPV16 were both expressed in basal and differentiated strata and induced H2A.X phosphorylation and 53BP1 increment into nuclear foci. After highlighting E6 and E7 co-expression with 53BP1 and a LKVLL sequence within the 53BP1 BRCT2 domain, we demonstrated the bindings via the PLA technique. Conclusions Our results reinforce E6 and E7 role in cellular function control providing potentially new insights into the activity of this tumor virus

TGF-\u3b22 and TGF-\u3b23 from cultured \u3b2-amyloid-treated or 3xTg-AD-derived astrocytes may mediate astrocyte-neuron communication

Astrocytes participate in the development and resolution of neuroinflammation in numerous ways, including the release of cytokines and growth factors. Among many, astrocytes release transforming growth factors beta (TGF-\u3b2) TGF-\u3b21, TGF-\u3b22 and TGF-\u3b23. TGF-\u3b21 is the most studied isoform, while production and release of TGF-\u3b22 and TGF-\u3b23 by astrocytes have been poorly characterized. Here, we report that purified cultures of hippocampal astrocytes produce mainly TGF-\u3b23 followed by TGF-\u3b22 and TGF-\u3b21. Furthermore, astrocytes release principally the active form of TGF-\u3b23 over the other two. Changes in release of TGF-\u3b2 were sensitive to the calcineurin (CaN) inhibitor FK506. Starvation had no effect on TGF-\u3b21 and TGF-\u3b23 while TGF-\u3b22 mRNA was significantly up-regulated in a CaN-dependent manner. We further investigated production and release of astroglial TGF-\u3b2 in Alzheimer's disease-related conditions. Oligomeric \u3b2-amyloid (A\u3b2) down-regulated TGF-\u3b21, while up-regulating TGF-\u3b22 and TGF-\u3b23, in a CaN-dependent manner. In cultured hippocampal astrocytes from 3xTg-AD mice, TGF-\u3b22 and TGF-\u3b23, but not TGF-\u3b21, were up-regulated, and this was CaN-independent. In hippocampal tissues from symptomatic 3xTg-AD mice, TGF-\u3b22 was up-regulated with respect to control mice. Finally, treatment with recombinant TGF-\u3b2s showed that TGF-\u3b22 and TGF-\u3b23 significantly reduced PSD95 protein in cultured hippocampal neurons, and this effect was paralleled by conditioned media from A\u3b2-treated astrocytes or from astrocytes from 3xTg-AD mice. Taken together, our data suggest that TGF-\u3b22 and TGF-\u3b23 are produced by astrocytes in a CaN-dependent manner and should be investigated further in the context of astrocyte-mediated neurodegeneration

Gene expression, proteome and calcium signaling alterations in immortalized hippocampal astrocytes from an Alzheimer\u2019s disease mouse model

Abstract Evidence is rapidly growing regarding a role of astroglial cells in the pathogenesis of Alzheimer’s disease (AD), and the hippocampus is one of the important brain regions affected in AD. While primary astroglial cultures, both from wild-type mice and from rodent models of AD, have been useful for studying astrocyte-specific alterations, the limited cell number and short primary culture lifetime have limited the use of primary hippocampal astrocytes. To overcome these limitations, we have now established immortalized astroglial cell lines from the hippocampus of 3xTg-AD and wild-type control mice (3Tg-iAstro and WT-iAstro, respectively). Both 3Tg-iAstro and WT-iAstro maintain an astroglial phenotype and markers (glutamine synthetase, aldehyde dehydrogenase 1 family member L1 and aquaporin-4) but display proliferative potential until at least passage 25. Furthermore, these cell lines maintain the potassium inward rectifying (Kir) current and present transcriptional and proteomic profiles compatible with primary astrocytes. Importantly, differences between the 3Tg-iAstro and WT-iAstro cell lines in terms of calcium signaling and in terms of transcriptional changes can be re-conducted to the changes previously reported in primary astroglial cells. To illustrate the versatility of this model we performed shotgun mass spectrometry proteomic analysis and found that proteins related to RNA binding and ribosome are differentially expressed in 3Tg-iAstro vs WT-iAstro. In summary, we present here immortalized hippocampal astrocytes from WT and 3xTg-AD mice that might be a useful model to speed up research on the role of astrocytes in AD

Magnetically Driven Bioreactors as new Tools in Drug Delivery Monia Taranta1,

The pharmacological properties of many drugs can be improved by drug delivery systems able to drive therapeutic agents to target regions. The use of carriers, in fact, may reduce possible cytotoxic effects of drugs and increase their bioavailability at the site of action, thus improving the efficacy and the safety of treatments. Therefore, we have developed an erythrocyte-based drug delivery system (erythro-magneto-HA virosome), which has the potential to be magnetically guided to specific sites and to fuse with target cells. These engineered erythrocytes have demonstrated in previous work a very high in vitro capability to release anticancer drugs directly inside target cells. Because the erythro-magneto-HA virosomes (EMHVs) proved to be promising carriers, we decided to investigate in more details the effectiveness and safety of this erythrocyte-based drug delivery system. We evaluated the ability of the EMHVs to be specifically localized in vivo to desired sites by means of an external magnetic field and to protect an anticancer drug such as 5-Aza-2\u27-deoxycytidine from degradation. Additionally we have assessed the ability of the EMHVs to act as bioreactors and to convert the pro-drug 5-Aza-2\u27-deoxycytidine into an active drug. Finally, we have studied the interaction of the EMHVs with the host immune system. The pro-drug 5-Aza-2\u27-deoxycytidine has short half-life when systemically injected and needs to be phosphorylated to become an active drug. We found that when inside the engineered erythrocytes it is protected by degradation and is transformed in its active form thus becoming readily available for uptake by the targeted cells. Moreover, we have observed that the EMHVs used didn\u27t cause either a cell-mediated or a humoral immune response in host mice having the same haplotype of the donors. These findings suggest that erythro-magneto-HA virosomes are a safe and useful drug delivery system that may offer numerous advantages for several clinical application