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The use of tailored particle-based adjuvants constitutes a promising way to enhance antigen-specific humoral and cellular immune responses. However, a thorough understanding of the ...mechanisms underlying their adjuvanticity is crucial to generate more effective vaccines. We studied the ability of chitosan-aluminum nanoparticles (CH-Al NPs), which combine the immunostimulatory effects of chitosan and aluminum salts, to promote dendritic cell activation, assess their impact on innate and adaptive immune responses, and compare the results to those reported for conventional chitosan particles (CH-Na NPs). All tested CH-NP formulations were capable of modulating cytokine secretion by dendritic cells. CH-Al NPs promoted NLRP3 inflammasome activation, enhancing the release of IL-1β without significantly inhibiting Th1 and Th17 cell-polarizing cytokines, IL-12p70 or IL-23, and induced DC maturation, but did not promote pro-inflammatory cytokine production on their own.
In vivo results showed that mice injected with CH-Al NPs generated a local inflammatory response comparable to that elicited by the vaccine adjuvant alum. Importantly, after subcutaneous immunization with CH-Al NPs combined with the hepatitis B surface antigen (HBsAg), mice developed antigen-specific IgG titers in serum, nasal and vaginal washes.
Overall, our results established CH-Al NPs as a potential adjuvant to enhance both innate and adaptive immune responses.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
The generation of strong pathogen-specific immune responses at mucosal surfaces where hepatitis B virus (HBV) transmission can occur is still a major challenge. Therefore, new vaccines are urgently ...needed in order to overcome the limitations of existing parenteral ones. Recent studies show that this may be achieved by intranasal immunization. Chitosan has gained attention as a nonviral gene delivery system; however, its use in vivo is limited due to low transfection efficiency mostly related to strong interaction between the negatively charged DNA and the positively charged chitosan. We hypothesize that the adsorption of negatively charged human serum albumin (HSA) onto the surface of the chitosan particles would facilitate the intracellular release of DNA, enhancing transfection activity. Here, we demonstrate that a robust systemic immune response was induced after vaccination using HSA-loaded chitosan nanoparticle/DNA (HSA-CH NP/DNA) complexes. Furthermore, intranasal immunization with HSA-CH NP/DNA complexes induced HBV specific IgA in nasal and vaginal secretions; no systemic or mucosal responses were detected after immunization with DNA alone. Overall, our results show that chitosan-based DNA complexes elicited both humoral and mucosal immune response, making them an interesting and valuable gene delivery system for nasal vaccination against HBV.
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IJS, KILJ, NUK, PNG, UL, UM
MicroRNAs (miRNAs) are an abundant class of small noncoding RNA molecules that play an important role in the regulation of gene expression at the posttranscriptional level. Due to their ability to ...simultaneously modulate the fate of different genes, these molecules are particularly well suited to act as key regulators during immune cell differentiation and activation, and their dysfunction can contribute to pathological conditions associated with neuroinflammation. Recent studies have addressed the role of miRNAs in the differentiation of progenitor cells into microglia and in the activation process, aiming at clarifying the origin of adult microglia cells and the contribution of the central nervous system (CNS) environment to microglia phenotype, in health and disease. Altered expression of several miRNAs has been associated with Alzheimer’s disease, multiple sclerosis, and ischemic injury, hence strongly advocating the use of these small molecules as disease markers and new therapeutic targets. This review summarizes the recent advances in the field of miRNA-mediated regulation of microglia development and activation. We discuss the role of specific miRNAs in the maintenance and switching of microglia activation states and illustrate the potential of this class of nucleic acids both as biomarkers of inflammation and new therapeutic tools for the modulation of microglia behavior in the CNS.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
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The use of particulate adjuvants offers an interesting possibility to enhance and modulate the immune responses elicited by vaccines. Aluminium salts have been extensively used as ...vaccine adjuvants, but they lack the capacity to induce a strong cellular and mucosal immune response. Taking this into consideration, in this study we designed a new antigen delivery system combining aluminium salts with chitosan. Chitosan-aluminium nanoparticles (CH-Al NPs) exhibited a mean diameter of 280nm and a positive surface charge. The newly developed CH-Al NPs are more stable at physiological environment than classical CH NPs, showing no cytotoxic effects and revealing potential as a delivery system for a wide range of model antigens. In vivo studies showed that mice immunized with hepatitis B surface antigen (HBsAg)-containing CH NPs display high anti-HBsAg IgG titers in the serum, as well as the highest antigen-specific IgG on vaginal washes. Furthermore, in contrast to mice receiving antigen alone, mice immunized with the particulate adjuvant were able to elicit IgG2c antibody titers and exhibited higher antigen-specific IFN-γ levels in splenocytes. In conclusion, we established that CH-Al NPs, combining two immunostimulants to enhance both humoral and cellular immune responses, are a safe and promising system for antigen delivery. Our findings point towards their potential in future vaccination approaches.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Excitotoxicity is one of the main features responsible for neuronal cell death after acute brain injury and in several neurodegenerative disorders, for which only few therapeutic options are ...currently available. In this work, RNA interference was employed to identify and validate a potential target for successful treatment of excitotoxic brain injury, the transcription factor c-Jun. The nuclear translocation of c-Jun and its upregulation are early events following glutamate-induced excitotoxic damage in primary neuronal cultures. We present evidence for the efficient knockdown of this transcription factor using a non-viral vector consisting of cationic liposomes associated to transferrin (Tf-lipoplexes). Tf-lipoplexes were able to deliver anti-c-Jun siRNAs to neuronal cells in culture, resulting in efficient silencing of c-Jun mRNA and protein and in a significant decrease of cell death following glutamate-induced damage or oxygen–glucose deprivation. This formulation also leads to a significant c-Jun knockdown in the mouse hippocampus
in vivo, resulting in the attenuation of both neuronal death and inflammation following kainic acid-mediated lesion of this region. Furthermore, a strong reduction of seizure activity and cytokine production was observed in animals treated with anti-c-Jun siRNAs. These findings demonstrate the efficient delivery of therapeutic siRNAs to the brain by Tf-lipoplexes and validate c-Jun as a promising therapeutic target in neurodegenerative disorders involving excitotoxic lesions.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Despite recent advances in conventional therapeutic approaches for cancer, the frequently observed acquired drug resistance and toxic side effects have limited their clinical application. The main ...goal of this work was to investigate the combined antitumoral effect of vinblastine with HSV-Tk/GCV “suicide” gene therapy mediated by human serum albumin (HSA)-associated lipoplexes, in mammary adenocarcinoma cells (TSA cells). Our results show that, among the different lipoplex formulations tested, HSA-associated complexes prepared from EPOPC:Chol liposomes, at the (4/1) (+/−) charge ratio, was the most efficient to mediate gene delivery, even in the presence of serum. The simultaneous addition of vinblastine and HSA-EPOPC:Chol/DNA (+/−) (4/1) lipoplexes to TSA cells improved transgene expression more than 10 times. When combined with the HSV-Tk/GCV “suicide” gene therapy mediated by HSA-EPOPC:Chol/DNA (+/−) (4/1) lipoplexes, vinblastine induced a great enhancement in the antitumoral activity in TSA cells. Most importantly, this combined strategy resulted in a significant synergistic effect, thus allowing the use of a much lower dose of the drug to achieve the same therapeutic effect. Overall, our results indicate that this approach has the potential to overcome some major limitations of conventional chemotherapy, and may therefore constitute a promising strategy for future applications in antitumoral therapy.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive and mortal cancer, characterized by a set of known mutations, invasive features, and aberrant microRNA expression that have been ...associated with hallmark malignant properties of PDAC. The lack of effective PDAC treatment options prompted us to investigate whether microRNAs would constitute promising therapeutic targets toward the generation of a gene therapy approach with clinical significance for this disease. In this work, we show that the developed human serum albumin-1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine:cholesterol/anti-microRNA oligonucleotides (+/-) (4/1) nanosystem exhibits the ability to efficiently deliver anti-microRNA oligonucleotides targeting the overexpressed microRNAs miR-21, miR-221, miR-222, and miR-10 in PDCA cells, promoting an almost complete abolishment of microRNA expression. Silencing of these microRNAs resulted in a significant increase in the levels of their targets. Moreover, the combination of microRNA silencing, namely miR-21, with low amounts of the chemotherapeutic drug sunitinib resulted in a strong and synergistic antitumor effect, showing that this combined strategy could be of great importance for therapeutic application in PDAC.
Although RNAi-based gene silencing holds a great potential for treatment of neurological disorders, its application to the CNS has been restricted by low levels of tissue distribution and cellular ...uptake. In this work we report that cationic lipid-based vectors can enhance siRNA delivery to neurons both
in vitro and
in vivo. DOTAP:Chol liposomes associated with transferrin (Tf) and complexed with siRNAs (Tf-lipoplexes) were delivered to primary cultures of luciferase-expressing cortical neurons. Confocal microscopy studies revealed efficient cellular uptake of Cy3-labelled siRNAs after Tf-lipoplex delivery, which was reduced but not completely inhibited by blocking the Tf-receptor with excess Tf. Gene silencing was also evaluated after delivery of anti-luciferase or anti-c-Jun siRNAs. Our results demonstrate that Tf-lipoplexes achieve up to 50% luciferase and c-Jun knockdown, 48 h after transfection, without significant cytotoxicity. Similar results were observed
in vivo, where a 40% reduction of luciferase activity was found in the striatum of luciferase mice. In addition, fluorescence microscopy studies showed extensive local distribution and internalization of Tf-lipoplex-associated Cy3-siRNAs without tissue toxicity. Overall, our results demonstrate that Tf-lipoplexes can mediate efficient gene silencing in neuronal cells, both
in vitro an
in vivo, which may prove useful in therapeutic approaches to neuronal protection and repair.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Since clinical application of conventional cancer therapies is usually limited by drug resistance and toxic side effects, combination of chemotherapeutic agents with gene therapy appears as an ...attractive therapeutic strategy to overcome these issues. Being selectively expressed in tumor tissues, survivin is a promising target for the development of anticancer strategies aimed at eliminating tumor cells while sparing normal tissues. In this work, we achieved substantial protein knockdown in a number of human cell lines, namely, A549, HeLa and MCF-7 cells which overexpress survivin, after treatment with anti-survivin siRNAs, which was associated with a significant reduction of cell viability, when compared to treatment with control siRNAs. Interestingly, when the survivin-silencing approach was combined with a chemotherapeutic agent, an enhancement of the therapeutic effect was achieved. Treatment with anti-survivin siRNAs resulted in high levels of caspase 3/7 activation, and an enhancement of this effect was observed when survivin silencing was combined with vinblastine. In addition, we showed that for A549 and HeLa cells survivin silencing contributes to the reversion of cell resistance to doxorubicin. Overall, we demonstrate that the combination of a survivin-directed silencing strategy with chemotherapeutic agents constitutes a valuable approach for cancer treatment.
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IJS, KILJ, NUK, PNG, UL, UM
The behaviour of three vanadium(V) systems, namely the pyridinone (V
V-dmpp), the salicylaldehyde (V
V-salDPA) and the pyrimidinone (V
V-MHCPE) complexes, is studied in aqueous solutions, under ...aerobic and physiological conditions using
51V NMR, EPR and UV–Visible (UV–Vis) spectroscopies. The speciations for the V
V-dmpp and V
V-salDPA have been previously reported. In this work, the system V
V-MHCPE is studied by pH-potentiometry and
51V NMR. The results indicate that, at pH ca. 7, the main species present are (V
VO
2)L
2 and (V
VO
2)LH
−1 (L
=
MHCPE
−) and hydrolysis products, similar to those observed in aqueous solutions of V
V-dmpp. The latter species is protonated as the pH decreases, originating (V
VO
2)L and (V
VO
2)LH. All the V
V-species studied are stable in aqueous media with different compositions and at physiological pH, including the cell culture medium. The compounds were screened for their potential cytotoxic activity in two different cell lines. The toxic effects were found to be incubation time and concentration dependent and specific for each compound and type of cells. The HeLa tumor cells seem to be more sensitive to drug effects than the 3T3-L1 fibroblasts. According to the IC
50 values and the results on reversibility to drug effects, the V
V-species resulting from the V
V-MHCPE system show higher toxicity in the tumor cells than in non-tumor cells, which may indicate potential antitumor activity.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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