Luminescent properties of ZrO2:Tb nanoparticles for applications in neuroscience

In this paper a new generation of non-toxic nanoparticles based on the zirconium oxide doped with 0.5%Tb and co-doped by the range of 0–70% with Y was evaluated for the use as a fluorescent biomarker of neuronal trafficking. The ZrO₂:Tb nanoparticles were created by microwave driven hydrothermal method. Influence of the yttrium content and thermal processing on the Tb³⁺ related luminescence emission was discussed. The higher intensities were achieved, when host was cubic and for the nanoparticles with 33 nm. Presence of yttrium was associated with the energy coupling of the host and dopant, wide excitation band is present at 309 and 322 nm before and after calcination respectively.For the experiment on living primary neurons, nanoparticles doped with 0.5%Tb and 7%Y were chosen based on their luminescence emission intensity. Recently transfer of the nanoparticles through the barriers in the organism including blood–brain barrier following their alimentary absorption was confirmed (Godlewski and Godlewski, 2012). This raised the possibility of the nanoparticle application as a tool in the neuroscience, and the question of potential mechanisms of nanoparticle turnover in neurons. Concentration of 0.001 mg/ml of ZrO₂:0.5%Tb 7%Y in growth medium was added to the primary murine culture medium, and the intracellular trafficking of nanoparticles was observed following 15 min pre-incubation period. ZrO₂:0.5%Tb 7%Y nanoparticles were dynamically absorbed by the neurons and the dynamic passage of transport vesicles containing ZrO₂:0.5%Tb 7%Y nanoparticles was observed along the neuronal processes and in between two neighbouring neurons. Reassuming, the ZrO₂:0.5%Tb 7%Y nanoparticles proved to be biocompatible and a valid tool to assess intracellular trafficking dynamics in the neurobiology.     

Lactoperoxidase-mediated degradation of single-walled carbon nanotubes in the presence of pulmonary surfactant

Carbon nanotubes (CNTs) may elicit inflammatory responses following pulmonary exposure. Conversely, enzymatic biodegradation of CNTs by inflammatory cells has also been reported. The aim of this study was to study the degradation of oxidized single-walled CNTs (ox-SWCNTs) by lactoperoxidase (LPO), a secreted peroxidase present in the airways, and whether pulmonary surfactant affects this biodegradation. To this end, ox-SWCNTs were incubated in vitro with recombinant bovine LPO + H₂O₂ + NaSCN in the presence and absence of porcine lung surfactant (Curosurf®) and biodegradation was monitored using UV–Vis–NIR spectroscopy, Raman spectroscopy, and scanning electron microscopy. The interaction of recombinant LPO with bundles of ox-SWCNTs was confirmed by atomic force microscopy. Cell-free biodegradation of ox-SWCNTs was also observed ex vivo in murine bronchoalveolar lavage fluid in the presence of H₂O₂ + NaSCN. Our study provides evidence for biodegradation of ox-SWCNTs with a lung surfactant ‘bio-corona’ and expands the repertoire of mammalian peroxidases capable of biodegradation of ox-SWCNTs. These findings are relevant to inhalation exposure to these materials, as LPO serves as an important component of the airway defense system.     

The Effect of Electroporation of a Lyotroic Liquid Crystal Genistein-Based Formulation in the Recovery of Murine Melanoma Lesions

A lamellar lyotropic liquid crystal genistein-based formulation (LLC-Gen) was prepared in order to increase the aqueous solubility of the lipophilic phytocompound genistein. The formulation was applied locally, in a murine model of melanoma, with or without electroporation. The results demonstrated that, when the formulation was applied by electroporation, the tumors appeared later. During the 21 days of the experiment, the LLC-Gen formulation decreased the tumor volume, the amount of melanin and the degree of erythema, but when electroporation was applied, all these parameters indicated a better prognosis even (lower tumor volume, amount of melanin and degree of erythema). Although hematoxylin–eosin (HE) staining confirmed the above events, application of the LLC-Gen formulation by electroporation did not lead to a significant effect in terms of the serum concentrations of the protein S100B and serum neuron specific enolase (NSE), or the tissue expression of the platelet-derived growth factor receptor β (PDGFRβ) antibody.     

Lipopolysaccharide influences on the toxicity of oxidised multiwalled carbon nanotubes to murine splenocytes in vitro

This experimental study evaluated the influence of the presence of lipopolysaccharide (LPS), a common bacterial endotoxin, on the nitric acid oxidised multiwalled carbon nanotubes (Ox-MWCNT) during the assessment of in vitro toxicity to splenocytes, an immune system cell isolated from BALB/c mice. The concentration of LPS was determined by Limulus Amebocyte assay and this endotoxin was removed from Ox-MWCNT by using three cycles of autoclave. Splenocytes were cultured in RPMI 1640 media with 1.0, 5.0 or 10 ng/mL of Ox-MWCNT. The results showed that the presence of LPS on Ox-MWCNT did not affect the growth of splenocytes in vitro. However, the absence of LPS decreased the splenocytes viability significantly.     

Investigating Immune Responses to the scAAV9-HEXM Gene Therapy Treatment in Tay–Sachs Disease and Sandhoff Disease Mouse Models

GM2 gangliosidosis disorders are a group of neurodegenerative diseases that result from a functional deficiency of the enzyme β-hexosaminidase A (HexA). HexA consists of an α- and β-subunit; a deficiency in either subunit results in Tay–Sachs Disease (TSD) or Sandhoff Disease (SD), respectively. Viral vector gene transfer is viewed as a potential method of treating these diseases. A recently constructed isoenzyme to HexA, called HexM, has the ability to effectively catabolize GM2 gangliosides in vivo. Previous gene transfer studies have revealed that the scAAV9-HEXM treatment can improve survival in the murine SD model. However, it is speculated that this treatment could elicit an immune response to the carrier capsid and “non-self”-expressed transgene. This study was designed to assess the immunocompetence of TSD and SD mice, and test the immune response to the scAAV9-HEXM gene transfer. HexM vector-treated mice developed a significant anti-HexM T cell response and antibody response. This study confirms that TSD and SD mouse models are immunocompetent, and that gene transfer expression can create an immune response in these mice. These mouse models could be utilized for investigating methods of mitigating immune responses to gene transfer-expressed “non-self” proteins, and potentially improve treatment efficacy.     

CARD9 Deficiency Increases Hippocampal Injury Following Acute Neurotropic Picornavirus Infection but Does Not Affect Pathogen Elimination

Neurotropic viruses target the brain and contribute to neurologic diseases. Caspase recruitment domain containing family member 9 (CARD9) controls protective immunity in a variety of infectious disorders. To investigate the effect of CARD9 in neurotropic virus infection, CARD9^−/− and corresponding C57BL/6 wild-type control mice were infected with Theiler’s murine encephalomyelitis virus (TMEV). Brain tissue was analyzed by histology, immunohistochemistry and molecular analyses, and spleens by flow cytometry. To determine the impact of CARD9 deficiency on T cell responses in vitro, antigen presentation assays were utilized. Genetic ablation of CARD9 enhanced early pro-inflammatory cytokine responses and accelerated infiltration of T and B cells in the brain, together with a transient increase in TMEV-infected cells in the hippocampus. CARD9^−/− mice showed an increased loss of neuronal nuclear protein⁺ mature neurons and doublecortin⁺ neuronal precursor cells and an increase in β-amyloid precursor protein⁺ damaged axons in the hippocampus. No effect of CARD9 deficiency was found on the initiation of CD8⁺ T cell responses by flow cytometry and co-culture experiments using virus-exposed dendritic cells or microglia-enriched glial cell mixtures, respectively. The present study indicates that CARD9 is dispensable for the initiation of early antiviral responses and TMEV elimination but may contribute to the modulation of neuroinflammation, thereby reducing hippocampal injury following neurotropic virus infection.     

Fms-Like Tyrosine Kinase 3-Independent Dendritic Cells Are Major Mediators of Th2 Immune Responses in Allergen-Induced Asthmatic Mice

Dendritic cells (DCs) are the main mediators of Th2 immune responses in allergic asthma, and Fms-like tyrosine kinase 3 ligand (Flt3L) is an important growth factor for the development and homeostasis of DCs. This study identified the DC populations that primarily cause the initiation and development of allergic lung inflammation using Fms-like tyrosine kinase 3 (Flt3) knockout (KO) mice with allergen-induced allergic asthma. We observed type 2 allergic lung inflammation with goblet cell hyperplasia in Flt3 KO mice, despite a significant reduction in total DCs, particularly CD103⁺ DCs, which was barely detected. In addition, bone marrow-derived dendritic cells (BMDCs) from Flt3 KO mice directed Th2 immune responses in vitro, and the adoptive transfer of these BMDCs exacerbated allergic asthma with more marked Th2 responses than that of BMDCs from wild-type (WT) mice. Furthermore, we found that Flt3L regulated the in vitro expression of OX40 ligand (OX40L) in DCs, which is correlated with DC phenotype in in vivo models. In conclusion, we revealed that Flt3-independent CD11b⁺ DCs direct Th2 responses with the elevated OX40L and are the primary cause of allergic asthma. Our findings suggest that Flt3 is required to control type 2 allergic inflammation.