DNA Vaccines: Developing New Strategies against Cancer

Due to their rapid and widespread development, DNA vaccines have entered into a variety of human clinical trials for vaccines against various diseases including cancer. Evidence that DNA vaccines are well tolerated and have an excellent safety profile proved to be of advantage as many clinical trials combines the first phase with the second, saving both time and money. It is clear from the results obtained in clinical trials that such DNA vaccines require much improvement in antigen expression and delivery methods to make them sufficiently effective in the clinic. Similarly, it is clear that additional strategies are required to activate effective immunity against poorly immunogenic tumor antigens. Engineering vaccine design for manipulating antigen presentation and processing pathways is one of the most important aspects that can be easily handled in the DNA vaccine technology. Several approaches have been investigated including DNA vaccine engineering, co-delivery of immunomodulatory molecules, safe routes of administration, prime-boost regimen and strategies to break the immunosuppressive networks mechanisms adopted by malignant cells to prevent immune cell function. Combined or single strategies to enhance the efficacy and immunogenicity of DNA vaccines are applied in completed and ongoing clinical trials, where the safety and tolerability of the DNA platform are substantiated. In this review on DNA vaccines, salient aspects on this topic going from basic research to the clinic are evaluated. Some representative DNA cancer vaccine studies are also discussed

Strategies for successful vaccination against hepatocellular carcinoma.

Current therapies against hepatocellular carcinoma (HCC) are not curative in the majority of patients. In the past, immunotherapy approaches aimed to non-specifically stimulate immune response were quite ineffective. New treatments based on stimulation of specific anti-tumor immune response are currently proposed and appear more promising. Tumor-specific antigens identified in HCC demonstrated immunogenicity both in preclinical and clinical trials. Effectiveness in animal studies raised interest in the clinical applicability of non-specific adoptive immunotherapy that prevented disease recurrence after tumor resection. Dendritic cell (DC)-based tumor vaccines achieved encouraging results, and cellular vaccines based on DCs have already entered clinical trials. Preventive and therapeutic DNA vaccination have been proposed, all based on tumor-associated antigens (TAAs), either modified or not, an example being alpha-fetoprotein (AFP). The concomitant expression of co-stimulatory molecules and cytokines was used to increase tumor immunogenicity. Syngeneic or nude mice models indicated that immunotherapy for HCC could stimulate an anti-tumor T-cell response leading to clinical benefit devoid of significant toxicity. The use of DNA-based vaccination raises exciting possibilities in preventing HCC in high-risk individuals such as those with cirrhosis. Novel immunotherapy strategies may contribute in the future to prevention and treatment of HCC

Inverting sediment bedforms for evaluating the hazard of dilute pyroclastic density currents in the field

Pyroclastic density currents are ground hugging gas-particle flows associated to explosive volcanic eruptions and moving down a volcano's slope, causing devastation and deaths. Because of the hostile nature they cannot be analyzed directly and most of their fluid dynamic behavior is reconstructed by the deposits left in the geological record, which frequently show peculiar structures such as ripples and dune bedforms. Here, a set of equations is simplified to link flow behavior to particle motion and deposition. This allows to construct a phase diagram by which impact parameters of dilute pyroclastic density currents, representing important factors of hazard, can be calculated by inverting bedforms wavelength and grain size, without the need of more complex models that require extensive work in the laboratory

Antimicrobial, antioxidant and anti-tyrosinase properties of extracts of the Mediterranean parasitic plant Cytinus hypocistis

Background: Cytinus is an endophytic parasitic plant occurring in South Africa, Madagascar, and in the Mediterranean region. We have extracted the inflorescences (the only visible part of the plant, emerging from the host roots at the time of blossom) of Cytinus hypocistis collected in Sardinia, Italy, and explored the antimicrobial, antioxidant, anti-tyrosinase, and cytotoxic activities of the extracts. Methods: Extracts from C. hypocistis were prepared using increasing polarity solvents: cyclohexane, ethanol, and water. Phenolic composition were determined through spectrophotometric assays, and antioxidant activity with both electron-transfer and hydrogen-atom assays. Nine different bacterial strains, including clinical isolate methicillin-resistant Staphylococcus aureus, were used in agar diffusion method. Cytotoxicity was tested using against the B16F10 melanoma cell line. Results: While cyclohexane extracts where biologically inactive, ethanolic and aqueous extracts displayed an intriguing activity against several Gram-positive bacterial strains, including methicillin-resistant S. aureus, and against the Gram-negative Acinetobacter baumanii. Compared to the conventional antibiotics like cloxacillin, ampicillin, and oxytetracycline, C. hypocistis extracts were less active in absolute terms, but displayed a wider spectrum (notably, cloxacillin and ampicillin were inactive against methicillin-resistant S. aureus). The ethanolic extract of C. hypocistis was found to be particularly rich in polyphenols, in most part hydrolysable tannins. The antioxidant activity of extracts, tested with several methodologies, resulted to be particularly high in the case of ethanolic extracts, in accordance with the composition in phenolics. In detail, ethanol extracts presented about a twofold higher activity than the water sample when tested through the oxygen radical absorbance capacity-pyrogallol red (ORAC-PYR) assay. Cytotoxicity analysis against the B16F10 melanoma cell line showed that both extracts have not significant cytotoxic effect, even at the highest dose (1000 μg/mL). Tests showed that ethanolic extracts also had the greatest tyrosinase inhibition activity, indicating that C. hypocistis-derived substances could find application in food formulations as anti-browning agents. Conclusions: Overall, these results point to the need of further studies on C. hypocistis extracts, aimed at isolating and fully characterizing its biologically active compounds. © 2015 Zucca et al

Endogenous CCL2 neutralization restricts HIV-1 replication in primary human macrophages by inhibiting viral DNA accumulation

Macrophages are key targets of HIV-1 infection. We have previously described that the expressionof CC chemokine ligand 2 (CCL2) increases during monocyte differentiation to macrophages and it is furtherup-modulated by HIV-1 exposure. Moreover, CCL2 acts as an autocrine factor that promotes viral replication ininfected macrophages. In this study, we dissected the molecular mechanisms by which CCL2 neutralization inhibitsHIV-1 replication in monocyte-derived macrophages (MDM), and the potential involvement of the innate restrictionfactors protein sterile alpha motif (SAM) histidine/aspartic acid (HD) domain containing 1 (SAMHD1) and apolipoproteinB mRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) family members.Results:CCL2 neutralization potently reduced the number of p24 Gag+cells during the course of either productive orsingle cycle infection with HIV-1. In contrast, CCL2 blocking did not modify entry of HIV-1 based Virus Like Particles, thusdemonstrating that the restriction involves post-entry steps of the viral life cycle. Notably, the accumulation of viralDNA, both total, integrated and 2-LTR circles, was strongly impaired by neutralization of CCL2. Looking for correlates ofHIV-1 DNA accumulation inhibition, we found that the antiviral effect of CCL2 neutralization was independent of themodulation of SAMHD1 expression or function. Conversely, a strong and selective induction of APOBEC3A expression,to levels comparable to those of freshly isolated monocytes, was associated with the inhibition of HIV-1 replicationmediated by CCL2 blocking. Interestingly, the CCL2 neutralization mediated increase of APOBEC3A expression was typeI IFN independent. Moreover, the transcriptome analysis of the effect of CCL2 blocking on global gene expressionrevealed that the neutralization of this chemokine resulted in the upmodulation of additional genes involved in thedefence response to viruses.Conclusions:Neutralization of endogenous CCL2 determines a profound restriction of HIV-1 replication in primaryMDM affecting post-entry steps of the viral life cycle with a mechanism independent of SAMHD1. In addition, CCL2blocking is associated with induction of APOBEC3A expression, thus unravelling a novel mechanism which mightcontribute to regulate the expression of innate intracellular viral antagonistsin vivo. Thus, our study may potentially leadto the development of new therapeutic strategies for enhancing innate cellular defences against HIV-1 and protecting macrophages from infection

Neem oil nanoemulsions: characterisation and antioxidant activity

The aim of the present work is to develop nanoemulsions (NEs), nanosized emulsions, manufactured for improving the delivery of active pharmaceutical ingredients. In particular, nanoemulsions composed of Neem seed oil, contain rich bioactive components, and Tween 20 as nonionic surfactant were prepared. A mean droplet size ranging from 10 to 100nm was obtained by modulating the oil/surfactant ratio. Physicochemical characterisation was carried out evaluating size, f-potential, microviscosity, polarity and turbidity of the external shell and morphology, along with stability in simulated cerebrospinal fluid (CSF), activity of Neem oil alone and in NEs, HEp-2 cell interaction and cytotoxicity studies. This study confirms the formation of NEs by Tween 20 and Neem oil at different weight ratios with small and homogenous dimensions. The antioxidant activity of Neem oil alone and in NEs was comparable, whereas its cytotoxicity was strongly reduced when loaded in NEs after interaction with HEp-2 cells

High Doses of Silica Nanoparticles Obtained by Microemulsion and Green Routes Compromise Human Alveolar Cells Morphology and Stiffness Differently

Among all the inorganic nanomaterials used in commercial products, industry, and medicine, the amorphous silica nanoparticles (SiO2 NPs) appeared to be often tolerated in living organisms. However, despite several toxicity studies, some concerns about the exposure to high doses of SiO2 NPs with different sizes were raised. Then, we used the microemulsion method to obtain stable SiO2 NPs having different sizes (110 nm, 50 nm, and 25 nm). In addition, a new one-pot green synthetic route using leaves extract of Laurus nobilis was performed, obtaining monodispersed ultrasmall SiO2 NPs without the use of dangerous chemicals. The NPs achieved by microemulsion were further functionalized with amino groups making the NPs surface positively charged. Then, high doses of SiO2 NPs (1 mg/mL and 3 mg/mL) achieved from the two routes, having different sizes and surface charges, were used to assess their impact on human alveolar cells (A549), being the best cell model mimicking the inhalation route. Cell viability and caspase-3 induction were analyzed as well as the cellular uptake, obtaining that the smallest (25 nm) and positive-charged NPs were more able to induce cytotoxicity, reaching values of about 60% of cell death. Surprisingly, cells incubated with green SiO2 NPs did not show strong toxicity, and 70% of them remained vital. This result was unusual for ultrasmall nanoobjects, generally highly toxic. The actin reorganization, nuclear morphology alteration, and cell membrane elasticity analyses confirmed the trend achieved from the biological assays. The obtained data demonstrate that the increase in cellular softness, i.e., the decrease in Young's modulus, could be associated with the smaller and positive NPs, recording values of about 3 kPa. On the contrary, green NPs triggered a slight decrease of stiffness values (c.a. 6 kPa) compared to the untreated cells (c.a. 8 kPa). As the softer cells were implicated in cancer progression and metastasization, this evidence strongly supported the idea of a link between the cell elasticity and physicochemical properties of NPs that, in turn, influenced the interaction with the cell membrane. Thus, the green SiO2 NPs compromised cells to a lesser extent than the other SiO2 NPs types. In this scenario, the elasticity evaluation could be an interesting tool to understand the toxicity of NPs with the aim of predicting some pathological phenomena associated with their exposure

Sustainable Synthesis of FITC Chitosan-Capped Gold Nanoparticles for Biomedical Applications

The quest for novel nanoscale materials for different applications necessitates that they are easy to obtain and have excellent physical properties and low toxicity. Moreover, considering the ongoing environmental impact of noxious chemical waste products, it is important to adopt eco-friendly approaches for nanoparticle synthesis. In this work, a natural polymer (medium molecular weight chitosan) derived from chitin was employed as a reducing agent to obtain gold nanoparticles (AuNPs) with a chitosan shell (AuNPs@CS) by a microwave oven. The chitosan is economically viable and cost-competitive in the market showing also nontoxic behavior in the environment and living organisms. The synthesized AuNPs@CS-FITC NPs were fully characterized by spectroscopic and microscopic characterization techniques. The size distribution of NPs was about 15 nm, which is a suitable dimension to use in biomedical applications due to their high tissue penetration, great circulation in blood, and optimal clearance as well as low toxicity. The prepared polymer-capped NPs were further functionalized with a fluorescent molecule, i.e., Fluorescein-5-isothiocyanate (FITC), to perform imaging in the cell. The results highlighted the goodness of the synthesis procedure, as well as the high internalization rate that resulted in an optimal fluorescence intensity. Thus, this work presents a good sustainable/green approach-mediated polymer nanocomposite for various applications in the field of diagnostic imaging

Sustainable Synthesis of FITC Chitosan-Capped Gold Nanoparticles for Biomedical Applications

The quest for novel nanoscale materials for different applications necessitates that they are easy to obtain and have excellent physical properties and low toxicity. Moreover, considering the ongoing environmental impact of noxious chemical waste products, it is important to adopt eco-friendly approaches for nanoparticle synthesis. In this work, a natural polymer (medium molecular weight chitosan) derived from chitin was employed as a reducing agent to obtain gold nanoparticles (AuNPs) with a chitosan shell (AuNPs@CS) by a microwave oven. The chitosan is economically viable and cost-competitive in the market showing also nontoxic behavior in the environment and living organisms. The synthesized AuNPs@CS-FITC NPs were fully characterized by spectroscopic and microscopic characterization techniques. The size distribution of NPs was about 15 nm, which is a suitable dimension to use in biomedical applications due to their high tissue penetration, great circulation in blood, and optimal clearance as well as low toxicity. The prepared polymer-capped NPs were further functionalized with a fluorescent molecule, i.e., Fluorescein-5-isothiocyanate (FITC), to perform imaging in the cell. The results highlighted the goodness of the synthesis procedure, as well as the high internalization rate that resulted in an optimal fluorescence intensity. Thus, this work presents a good sustainable/green approach-mediated polymer nanocomposite for various applications in the field of diagnostic imaging