JoVE Visualize What is visualize?
Stop Reading. Start Watching.
Advanced Search
Stop Reading. Start Watching.
Regular Search
Find video protocols related to scientific articles indexed in Pubmed.
Enzymatic 'stripping' and degradation of PEGylated carbon nanotubes.
Nanoscale
PUBLISHED: 11-07-2014
Show Abstract
Hide Abstract
Single-walled carbon nanotubes (SWCNTs) coated or functionalized with PEG chains of different molecular weight were assessed for their propensity to undergo biodegradation under in vitro conditions using recombinant myeloperoxidase (MPO) or ex vivo using freshly isolated primary human neutrophils. Our findings suggest that under natural conditions, a combined process of 'stripping' (i.e., defunctionalization) and biodegradation of PEG-SWCNTs might occur and that PEG-SWCNTs are a promising - and degradable - nanomedicine vector.
Related JoVE Video
Mechanism-based genotoxicity screening of metal oxide nanoparticles using the ToxTracker panel of reporter cell lines.
Part Fibre Toxicol
PUBLISHED: 09-02-2014
Show Abstract
Hide Abstract
The rapid expansion of manufacturing and use of nano-sized materials fuels the demand for fast and reliable assays to identify their potential hazardous properties and underlying mechanisms. The ToxTracker assay is a recently developed mechanism-based reporter assay based on mouse embryonic stem (mES) cells that uses GFP-tagged biomarkers for detection of DNA damage, oxidative stress and general cellular stress upon exposure. Here, we evaluated the ability of the ToxTracker assay to identify the hazardous properties and underlying mechanisms of a panel of metal oxide- and silver nanoparticles (NPs) as well as additional non-metallic materials (diesel, carbon nanotubes and quartz).
Related JoVE Video
Graphene oxide attenuates Th2-type immune responses, but augments airway remodeling and hyperresponsiveness in a murine model of asthma.
ACS Nano
PUBLISHED: 05-23-2014
Show Abstract
Hide Abstract
Several lines of evidence indicate that exposure to nanoparticles (NPs) is able to modify airway immune responses, thus facilitating the development of respiratory diseases. Graphene oxide (GO) is a promising carbonaceous nanomaterial with unique physicochemical properties, envisioned for a multitude of medical and industrial applications. In this paper, we determined how exposure to GO modulates the allergic pulmonary response. Using a murine model of ovalbumin (OVA)-induced asthma, we revealed that GO, given at the sensitization stage, augmented airway hyperresponsiveness and airway remodeling in the form of goblet cell hyperplasia and smooth muscle hypertrophy. At the same time, the levels of the cytokines IL-4, IL-5, and IL-13 were reduced in broncho-alveolar lavage (BAL) fluid in GO-exposed mice. Exposure to GO during sensitization with OVA decreased eosinophil accumulation and increased recruitment of macrophages in BAL fluid. In line with the cytokine profiles, sensitization with OVA in the presence of GO stimulated the production of OVA-specific IgG2a and down-regulated the levels of IgE and IgG1. Moreover, exposure to GO increased the macrophage production of the mammalian chitinases, CHI3L1 and AMCase, whose expression is associated with asthma. Finally, molecular modeling has suggested that GO may directly interact with chitinase, affecting AMCase activity, which has been directly proven in our studies. Thus, these data show that GO exposure attenuates Th2 immune response in a model of OVA-induced asthma, but leads to potentiation of airway remodeling and hyperresponsiveness, with the induction of mammalian chitinases.
Related JoVE Video
Extracellular entrapment and degradation of single-walled carbon nanotubes.
Nanoscale
PUBLISHED: 05-20-2014
Show Abstract
Hide Abstract
Neutrophils extrude neutrophil extracellular traps (NETs) consisting of a network of chromatin decorated with antimicrobial proteins to enable non-phagocytic killing of microorganisms. Here, utilizing a model of ex vivo activated human neutrophils, we present evidence of entrapment and degradation of carboxylated single-walled carbon nanotubes (SWCNTs) in NETs. The degradation of SWCNTs was catalyzed by myeloperoxidase (MPO) present in purified NETs and the reaction was facilitated by the addition of H2O2 and NaBr. These results show that SWCNTs can undergo acellular, MPO-mediated biodegradation and imply that the immune system may deploy similar strategies to rid the body of offending microorganisms and engineered nanomaterials.
Related JoVE Video
Exploring the anti-apoptotic role of HAX-1 versus BCL-XL in cytokine-dependent bone marrow-derived cells from mice.
FEBS Lett.
PUBLISHED: 05-07-2014
Show Abstract
Hide Abstract
HS-1-associated protein X-1 (HAX-1) is a multi-functional protein that has been implicated in the regulation of apoptosis, cell motility and calcium homeostasis. In the present study, we set out to assess the postulated functional resemblance of HAX-1 to the BCL-2 family of anti-apoptotic proteins using non-transformed, cytokine-dependent murine bone marrow cells as a model system. BCL-X(L), but not HAX-1 protected against cytokine withdrawal-induced apoptosis while HAX-1 and BCL-X(L) significantly reduced thapsigargin-triggered (calcium-dependent) apoptosis. The data argue in favor of cell type- and stimulus-specific roles of HAX-1 in regulation of cell survival.
Related JoVE Video
Classification framework for graphene-based materials.
Angew. Chem. Int. Ed. Engl.
PUBLISHED: 03-14-2014
Show Abstract
Hide Abstract
Graphing graphene: Because the naming of graphene-based materials (GBMs) has led to confusion and inconsistency, a classification approach is necessary. Three physical-chemical properties of GBMs have been defined by the GRAPHENE Flagship Project of the European Union for the unequivocal classification of these materials (see grid).
Related JoVE Video
Size-dependent cytotoxicity of silver nanoparticles in human lung cells: the role of cellular uptake, agglomeration and Ag release.
Part Fibre Toxicol
PUBLISHED: 02-10-2014
Show Abstract
Hide Abstract
Silver nanoparticles (AgNPs) are currently one of the most manufactured nanomaterials. A wide range of toxicity studies have been performed on various AgNPs, but these studies report a high variation in toxicity and often lack proper particle characterization. The aim of this study was to investigate size- and coating-dependent toxicity of thoroughly characterized AgNPs following exposure of human lung cells and to explore the mechanisms of toxicity.
Related JoVE Video
Targeted uptake of folic acid-functionalized iron oxide nanoparticles by ovarian cancer cells in the presence but not in the absence of serum.
Nanomedicine
PUBLISHED: 01-12-2014
Show Abstract
Hide Abstract
Targeted delivery of nanoparticles to cells or tissues of interest is arguably the "holy grail" of nanomedicine. Using primary human macrophages and ovarian cancer cells, we evaluated the biocompatibility and specific targeting of folic acid (FA)-conjugated iron oxide nanoparticles with organic [poly(ethylene glycol), PEG] or inorganic (SiO2) intermediate surface coatings. Reduction of folate receptor-? expression using specific siRNA resulted in a significant decrease in cellular uptake of the SiO2-coated nanoparticles, but did not affect uptake of PEG-coated nanoparticles. Notably, specific (i.e. FA-dependent) uptake was observed only in the presence of serum proteins. The strategy presented here for receptor-mediated uptake of nanoparticles with pre-defined surface chemistry may enable targeting of nanoparticles for therapeutic and imaging applications.
Related JoVE Video
Macrophage clearance of neutrophil extracellular traps is a silent process.
J. Immunol.
PUBLISHED: 07-31-2013
Show Abstract
Hide Abstract
Neutrophil extracellular traps (NETs) facilitate the extracellular killing of pathogens. However, in recent years, excessive NET formation has been implicated in several pathological conditions. Indeed, NETs that are not removed from tissues or from the circulation might serve to trigger autoimmune responses. We observed that physiological amounts of DNase I do not suffice to completely degrade NETs in vitro, suggesting that additional mechanisms are required for the removal of these extracellular structures. We show in this article that human monocyte-derived macrophages are able to engulf NETs in a cytochalasin D-dependent manner, indicating that this is an active, endocytic process. Furthermore, preprocessing of NETs by DNase I facilitated their clearance by macrophages. In addition, both recombinant C1q and endogenous C1q derived from human serum were found to opsonize NETs, and this facilitated NET clearance. Upon internalization, NETs were apparently degraded in lysosomes, as treatment with chloroquine led to accumulation of extranuclear DNA in human monocyte-derived macrophages. Finally, uptake of NETs alone did not induce proinflammatory cytokine secretion, whereas LPS-induced production of IL-1?, IL-6, and TNF-? was promoted by the uptake of NETs. In summary, we show that macrophages are capable of clearance of NETs and that this occurs in an immunologically silent manner.
Related JoVE Video
Oxidative stress and dermal toxicity of iron oxide nanoparticles in vitro.
Cell Biochem. Biophys.
PUBLISHED: 06-15-2013
Show Abstract
Hide Abstract
A number of commercially available metal/metal oxide nanoparticles (NPs) such as superparamagnetic iron oxide (SPION) are utilized by the medical field for a wide variety of applications. These NPs may able to induce dermal toxicity via their physical nature and reactive surface properties. We hypothesize that SPION may be toxic to skin via the ability of particles to be internalized and thereby initiate oxidative stress, inducing redox-sensitive transcription factors affecting/leading to inflammation. Due to the skins susceptibility to UV radiation, it is also of importance to address the combined effect of UVB and NPs co-exposure. To test this hypothesis, the effects of dextran-coated SPION of different sizes (15-50 nm) and manufacturers (MicroMod, Rostock-Warnemunde, Germany and KTH-Royal Institute of Technology, Stockholm, Sweden) were evaluated in two cell lines: normal human epidermal keratinocytes (HEK) and murine epidermal cells (JB6 P(+)). HEK cells exposed to 20 nm (KTH and MicroMod) had a decrease in viability, while the 15 and 50 nm particles were not cytotoxic. HEK cells were also capable of internalizing the KTH particles (15 and 20 nm) but not the MicroMod SPION (20 and 50 nm). IL-8 and IL-6 were also elevated in HEK cells following exposure to SPION. Exposure of JB6 P(+) cells to all SPIONs evaluated resulted in activation of AP-1. Exposure to SPION alone was not sufficient to induce NF-?B activation; however, co-exposure with UVB resulted in significant NF-?B induction in cells exposed to 15 and 20 nm KTH SPION and 50 nm MicroMod particles. Pre-exposure of JB6 P(+) cells to UVB followed by NPs induced a significant depletion of glutathione, release of cytokines, and cell damage as assessed by release of lactate dehydrogenase. Altogether, these data indicate that co-exposure to UVB and SPIONs was associated with induction of oxidative stress and release of inflammatory mediators. These results verify the need to thoroughly evaluate the adverse effects of UVB when evaluating dermal toxicity of engineered NPs on skin.
Related JoVE Video
Ovarian failure in HAX1-deficient patients: is there a gender-specific difference in pubertal development in severe congenital neutropenia or Kostmann disease?
Acta Paediatr.
PUBLISHED: 05-22-2013
Show Abstract
Hide Abstract
Severe congenital neutropenia (SCN) is a rare disorder of myelopoiesis characterized by neutropenia, recurrent bacterial infections and a maturation arrest of the myelopoiesis in the bone marrow. Homozygous mutations in the HAX1 gene were described in patients with autosomal recessive SCN or Kostmann disease. Some of these patients display neurological disease. We noted, during the course of clinical management of patients with Kostmann disease, insufficient pubertal development in female patients, but not in our male patients. The study objective was to provide a detailed account of this phenotype and its possible relation to HAX1 mutations.
Related JoVE Video
Pro-apoptotic effects of the novel proteasome inhibitor b-AP15 on multiple myeloma cells and natural killer cells.
Exp. Hematol.
PUBLISHED: 03-08-2013
Show Abstract
Hide Abstract
The small molecule b-AP15 is a novel inhibitor of proteasome deubiquitination and recent studies have shown that b-AP15 displays anti-tumor activity in several pre-clinical solid tumor models. Here, we show that b-AP15 triggers time- and dose-dependent apoptosis of the human multiple myeloma (MM) cell lines, RPMI8226 and U266, as determined by phosphatidylserine exposure; apoptosis was dependent on caspase activation and was partially cathepsin D-dependent. Furthermore, b-AP15 triggered processing of pro-caspase-3 and cleavage of poly (ADP-ribose) polymerase (PARP) in MM cells. b-AP15 also induced caspase-independent apoptosis in primary human natural killer (NK) cells. We also demonstrate that b-AP15 induces activation of the mitochondrial apoptosis pathway in MM cells, with activation of the pro-apoptotic protein Bax and a pronounced loss of the mitochondrial transmembrane potential. The latter events, however, appeared largely independent of caspase activation. Our data suggest that proteasome deubiquitinase inhibitors may have potential for treatment of multiple myeloma patients.
Related JoVE Video
Biodegradation of single-walled carbon nanotubes by eosinophil peroxidase.
Small
PUBLISHED: 02-27-2013
Show Abstract
Hide Abstract
Eosinophil peroxidase (EPO) is one of the major oxidant-producing enzymes during inflammatory states in the human lung. The degradation of single-walled carbon nanotubes (SWCNTs) upon incubation with human EPO and H?O? is reported. Biodegradation of SWCNTs is higher in the presence of NaBr, but neither EPO alone nor H?O? alone caused the degradation of nanotubes. Molecular modeling reveals two binding sites for SWCNTs on EPO, one located at the proximal side (same side as the catalytic site) and the other on the distal side of EPO. The oxidized groups on SWCNTs in both cases are stabilized by electrostatic interactions with positively charged residues. Biodegradation of SWCNTs can also be executed in an ex vivo culture system using primary murine eosinophils stimulated to undergo degranulation. Biodegradation is proven by a range of methods including transmission electron microscopy, UV-visible-NIR spectroscopy, Raman spectroscopy, and confocal Raman imaging. Thus, human EPO (in vitro) and ex vivo activated eosinophils mediate biodegradation of SWCNTs: an observation that is relevant to pulmonary responses to these materials.
Related JoVE Video
Mechanisms of carbon nanotube-induced toxicity: Focus on pulmonary inflammation.
Adv. Drug Deliv. Rev.
PUBLISHED: 02-23-2013
Show Abstract
Hide Abstract
Carbon nanotubes have gained tremendous interest in a wide range of applications due to their unique physical, chemical, and electronic properties. Needless to say, close attention to the potential toxicity of carbon nanotubes is of paramount importance. Numerous studies have linked exposure of carbon nanotubes to the induction of inflammation, a complex protective response to harmful stimuli including pathogens, damaged or dying cells, and other irritants. However, inflammation is a double-edged sword as chronic inflammation can lead to destruction of tissues thus compromising the homeostasis of the organism. Here, we provide an overview of the process of inflammation, the key cells and the soluble mediators involved, and discuss research on carbon nanotubes and inflammation, including recent studies on the activation of the so-called inflammasome complex in macrophages resulting in secretion of pro-inflammatory cytokines. Moreover, recent work has shown that inflammatory cells i.e. neutrophils and eosinophils are capable of enzymatic degradation of carbon nanotubes, with mitigation of the pro-inflammatory and pro-fibrotic effects of nanotubes thus underscoring that inflammation is both good and bad.
Related JoVE Video
Dexamethasone differentially regulates Bcl-2 family proteins in human proliferative chondrocytes: Role of pro-apoptotic Bid.
Toxicol. Lett.
PUBLISHED: 02-21-2013
Show Abstract
Hide Abstract
Glucocorticoids (GCs) are widely used to treat inflammatory diseases and cancers. A multitude of undesired side effects have been reported in GC-treated patients including decreased linear bone growth. We have previously reported that GCs activate the caspase cascade and trigger Bax-mediated mitochondrial apoptosis in growth plate chondrocytes causing growth retardation in young mice. To further explore the role of mitochondrial apoptosis in GC-induced bone growth retardation, a number of pro- and anti-apoptotic proteins were studied in ex vivo cultures of human growth plate cartilage and human HCS-2/8 proliferative chondrocytes exposed to dexamethasone. Dexamethasone was found to increase the pro-apoptotic proteins Bcl-xS, Bad, and Bak as well as the proteolysis of Bid. Anti-Bid small interfering RNA partially rescued the chondrocytes from dexamethasone-induced apoptosis. Taken together, our data suggest that GC treatment differentially regulates Bcl-2 family member proteins to facilitate mitochondrial apoptosis in proliferative chondrocytes thereby contributing to GC-induced bone growth impairment. Prevention of this imbalance between pro- and anti-apoptotic Bcl-2 family proteins may provide a new strategy to protect from adverse effects of GCs on bone growth.
Related JoVE Video
Bridge over troubled waters: understanding the synthetic and biological identities of engineered nanomaterials.
Wiley Interdiscip Rev Nanomed Nanobiotechnol
PUBLISHED: 01-17-2013
Show Abstract
Hide Abstract
Engineered nanomaterials offer exciting opportunities for smart drug delivery and in vivo imaging of disease processes, as well as in regenerative medicine. The ability to manipulate matter at the nanoscale enables many new properties that are both desirable and exploitable, but the same properties could also give rise to unexpected toxicities that may adversely affect human health. Understanding the physicochemical properties that drive toxicological outcomes is a formidable challenge as it is not trivial to separate and, hence, to pinpoint individual material characteristics of nanomaterials. In addition, nanomaterials that interact with biological systems are likely to acquire a surface corona of biomolecules that may dictate their biological behavior. Indeed, we propose that it is the combination of material-intrinsic properties (the synthetic identity) and context-dependent properties determined, in part, by the bio-corona of a given biological compartment (the biological identity) that will determine the interactions of engineered nanomaterials with cells and tissues and subsequent outcomes. The delineation of these entwined identities of engineered nanomaterials constitutes the bridge between nanotoxicological research and nanomedicine.
Related JoVE Video
Gene expression profiling of immune-competent human cells exposed to engineered zinc oxide or titanium dioxide nanoparticles.
PLoS ONE
PUBLISHED: 01-01-2013
Show Abstract
Hide Abstract
A comprehensive in vitro assessment of two commercial metal oxide nanoparticles, TiO2 and ZnO, was performed using human monocyte-derived macrophages (HMDM), monocyte-derived dendritic cells (MDDC), and Jurkat T cell leukemia-derived cell line. TiO2 nanoparticles were found to be non-toxic whereas ZnO nanoparticles caused dose-dependent cell death. Subsequently, global gene expression profiling was performed to identify transcriptional response underlying the cytotoxicity caused by ZnO nanoparticles. Analysis was done with doses 1 µg/ml and 10 µg/ml after 6 and 24 h of exposure. Interestingly, 2703 genes were significantly differentially expressed in HMDM upon exposure to 10 µg/ml ZnO nanoparticles, while in MDDCs only 12 genes were affected. In Jurkat cells, 980 genes were differentially expressed. It is noteworthy that only the gene expression of metallothioneins was upregulated in all the three cell types and a notable proportion of the genes were regulated in a cell type-specific manner. Gene ontology analysis revealed that the top biological processes disturbed in HMDM and Jurkat cells were regulating cell death and growth. In addition, genes controlling immune system development were affected. Using a panel of modified ZnO nanoparticles, we obtained an additional support that the cellular response to ZnO nanoparticles is largely dependent on particle dissolution and show that the ligand used to modify ZnO nanoparticles modulates Zn(2+) leaching. Overall, the study provides an extensive resource of transcriptional markers for mediating ZnO nanoparticle-induced toxicity for further mechanistic studies, and demonstrates the value of assessing nanoparticle responses through a combined transcriptomics and bioinformatics approach.
Related JoVE Video
NanoMiner - integrative human transcriptomics data resource for nanoparticle research.
PLoS ONE
PUBLISHED: 01-01-2013
Show Abstract
Hide Abstract
The potential impact of nanoparticles on the environment and on human health has attracted considerable interest worldwide. The amount of transcriptomics data, in which tissues and cell lines are exposed to nanoparticles, increases year by year. In addition to the importance of the original findings, this data can have value in broader context when combined with other previously acquired and published results. In order to facilitate the efficient usage of the data, we have developed the NanoMiner web resource (http://nanominer.cs.tut.fi/), which contains 404 human transcriptome samples exposed to various types of nanoparticles. All the samples in NanoMiner have been annotated, preprocessed and normalized using standard methods that ensure the quality of the data analyses and enable the users to utilize the database systematically across the different experimental setups and platforms. With NanoMiner it is possible to 1) search and plot the expression profiles of one or several genes of interest, 2) cluster the samples within the datasets, 3) find differentially expressed genes in various nanoparticle studies, 4) detect the nanoparticles causing differential expression of selected genes, 5) analyze enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene Ontology (GO) terms for the detected genes and 6) search the expression values and differential expressions of the genes belonging to a specific KEGG pathway or Gene Ontology. In sum, NanoMiner database is a valuable collection of microarray data which can be also used as a data repository for future analyses.
Related JoVE Video
Morphological properties of nanoporous folic acid materials and in vitro assessment of their biocompatibility.
Nanomedicine (Lond)
PUBLISHED: 11-23-2011
Show Abstract
Hide Abstract
Mesoporous silica-based particles are of potential interest for the development of novel therapeutic targeted delivery vehicles. Their ability to load and release large quantities of active pharmaceutical products with varying properties, combining controlled and targeted release functions make them unique amongst nanotechnology-based carrier systems.
Related JoVE Video
Stability and biocompatibility of a library of polyester dendrimers in comparison to polyamidoamine dendrimers.
Biomaterials
PUBLISHED: 11-07-2011
Show Abstract
Hide Abstract
Dendrimers can be designed for several biomedical applications due to their well-defined structure, functionality and dimensions. The present study focused on the in vitro biocompatibility evaluation of a library of aliphatic polyester dendrimers based on 2,2-bis(methylol)propionic acid (bis-MPA) with an overall diameter of 0.5-2 nm. In addition, dendrimers with two different chemical surfaces (neutral with hydroxyl end group and anionic with carboxylic end group) and dendrons corresponding to the structural fragments of the dendrimers were evaluated. Commercial polyamidoamine dendrimers (PAMAM) with cationic (amine) or neutral (hydroxyl) end group were also included for comparison. Cell viability studies were conducted in human cervical cancer (HeLa) and acute monocytic leukemia cells (THP.1) differentiated into macrophage-like cells as well as in primary human monocyte-derived macrophages. Excellent biocompatibility was observed for the entire hydroxyl functional bis-MPA dendrimer library, whereas the cationic, but not the neutral PAMAM exerted dose-dependent cytotoxicity in cell lines and primary macrophages. Studies to evaluate material stability as a function of pH, temperature, and time, demonstrated that the stability of the 4th generation hydroxyl functional bis-MPA dendrimer increased at acidic pH. Taken together, bis-MPA dendrimers are degradable and non-cytotoxic to human cell lines and primary cells.
Related JoVE Video
Mapping the surface adsorption forces of nanomaterials in biological systems.
ACS Nano
PUBLISHED: 10-27-2011
Show Abstract
Hide Abstract
The biological surface adsorption index (BSAI) is a novel approach to characterize surface adsorption energy of nanomaterials that is the primary force behind nanoparticle aggregation, protein corona formation, and other complex interactions of nanomaterials within biological systems. Five quantitative nanodescriptors were obtained to represent the surface adsorption forces (hydrophobicity, hydrogen bond, polarity/polarizability, and lone-pair electrons) of the nanomaterial interaction with biological components. We have mapped the surface adsorption forces over 16 different nanomaterials. When the five-dimensional information of the nanodescriptors was reduced to two dimensions, the 16 nanomaterials were classified into distinct clusters according their surface adsorption properties. BSAI nanodescriptors are intrinsic properties of nanomaterials useful for quantitative structure-activity relationship (QSAR) model development. This is the first success in quantitative characterization of the surface adsorption forces of nanomaterials in biological conditions, which could open a quantitative avenue in predictive nanomedicine development, risk assessment, and safety evaluation of nanomaterials.
Related JoVE Video
Familial hemophagocytic lymphohistiocytosis type 3 (FHL3) caused by deep intronic mutation and inversion in UNC13D.
Blood
PUBLISHED: 09-19-2011
Show Abstract
Hide Abstract
Familial hemophagocytic lymphohistiocytosis (FHL) is an autosomal recessive, often-fatal hyperinflammatory disorder. Mutations in PRF1, UNC13D, STX11, and STXBP2 are causative of FHL2, 3, 4, and 5, respectively. In a majority of suspected FHL patients from Northern Europe, sequencing of exons and splice sites of such genes required for lymphocyte cytotoxicity revealed no or only monoallelic UNC13D mutations. Here, in 21 patients, we describe 2 pathogenic, noncoding aberrations of UNC13D. The first is a point mutation localized in an evolutionarily conserved region of intron 1. This mutation selectively impairs UNC13D transcription in lymphocytes, abolishing Munc13-4 expression. The second is a 253-kb inversion straddling UNC13D, affecting the 3-end of the transcript and likewise abolishing Munc13-4 expression. Carriership of the intron 1 mutation was found in patients across Europe, whereas carriership of the inversion was limited to Northern Europe. Notably, the latter aberration represents the first description of an autosomal recessive human disease caused by an inversion. These findings implicate an intronic sequence in cell-type specific expression of Munc13-4 and signify variations outside exons and splice sites as a common cause of FHL3. Based on these data, we propose a strategy for targeted sequencing of evolutionary conserved noncoding regions for the diagnosis of primary immunodeficiencies.
Related JoVE Video
Global phospholipidomics analysis reveals selective pulmonary peroxidation profiles upon inhalation of single-walled carbon nanotubes.
ACS Nano
PUBLISHED: 08-04-2011
Show Abstract
Hide Abstract
It is commonly believed that nanomaterials cause nonspecific oxidative damage. Our mass spectrometry-based oxidative lipidomics analysis of all major phospholipid classes revealed highly selective patterns of pulmonary peroxidation after inhalation exposure of mice to single-walled carbon nanotubes. No oxidized molecular species were found in the two most abundant phospholipid classes: phosphatidylcholine and phosphatidylethanolamine. Peroxidation products were identified in three relatively minor classes of anionic phospholipids, cardiolipin, phosphatidylserine, and phosphatidylinositol, whereby oxygenation of polyunsaturated fatty acid residues also showed unusual substrate specificity. This nonrandom peroxidation coincided with the accumulation of apoptotic cells in the lung. A similar selective phospholipid peroxidation profile was detected upon incubation of a mixture of total lung lipids with H(2)O(2)/cytochrome c known to catalyze cardiolipin and phosphatidylserine peroxidation in apoptotic cells. The characterized specific phospholipid peroxidation signaling pathways indicate new approaches to the development of mitochondria-targeted regulators of cardiolipin peroxidation to protect against deleterious effects of pro-apoptotic effects of single-walled carbon nanotubes in the lung.
Related JoVE Video
Water-soluble superparamagnetic magnetite nanoparticles with biocompatible coating for enhanced magnetic resonance imaging.
ACS Nano
PUBLISHED: 08-02-2011
Show Abstract
Hide Abstract
Ultrasmall superparamagnetic Fe(3)O(4) nanoparticles (USIRONs) were synthesized by a novel, easily scalable chemical reduction of colloidal iron hydroxide under hydrothermal conditions. The average crystallite size (5.1 ± 0.5 nm) and good crystallinity of the samples were supported by HR-TEM analysis and the saturation magnetization value (47 emu g(-1)). Vitamin C, used as a chemical reducing agent, also served as a capping agent in the oxidized form (dehydroascorbic acid, DHAA) to impart nanoparticles with exceptional solubility and stability in water, PBS buffer, and cell culture medium. Detailed physicochemical analysis of the USIRON suspensions provided insight into the magnetic ordering phenomena within the colloid, arising from the formation of uniform clusters displaying a hydrodynamic size of 41 nm. Phantom experiments on the contrast agent (clinical 3 T MRI scanner) revealed an enhanced r(2)/r(1) ratio of 36.4 (r(1)= 5 s(-1) mM(-1) and r(2)= 182 s(-1) mM(-1)) when compared to the clinically approved agents. The potential of the DHAA-Fe(3)O(4) nanoparticles as negative contrast agents for MRI with optimal hydrodynamic size for extended blood circulation times was confirmed by strong contrast observed in T(2)- and T(2)*-weighted images. The cell tests performed with primary human immune-competent cells confirmed the excellent biocompatibility of USIRONs.
Related JoVE Video
Differentiation-promoting culture of competent and noncompetent keratinocytes identifies biomarkers for head and neck cancer.
Am. J. Pathol.
PUBLISHED: 07-20-2011
Show Abstract
Hide Abstract
Aberrant contact-inhibited proliferation and differentiation induction couple with tumor severity, albeit with an imprecise association with prognosis. Assessment of contact inhibition and differentiation-promoting culture in this study of normal and immortalized oral keratinocytes (NOK and SVpgC2a, respectively) demonstrated elevated cloning ability and saturation density in the immortalized versus normal state, including consistent absence of differentiated morphological features. Transcriptomic analysis implicated 48 gene ontology categories, 8 molecular networks, and 10 key regulator genes in confluency-induced differentiation of NOK, all of which remained nonregulated in SVpgC2a. The SVpgC2a versus NOK transcriptome enriched 52 gene ontology categories altogether, 18 molecular networks, and 39 key regulator genes, several of which were associated with epithelial-mesenchymal transition. Assessment of the previously described gene sets relative to training data sets of head and neck squamous cell carcinoma samples, one including data on tumor differentiation and patient outcome and one present in the Human Gene Expression Map, identified four genes with association to poor survival (COX7A1, MFAP5, MPDU1, and POLD1). This gene set predicted poor outcome in an independent data set of 71 head and neck squamous cell carcinomas. The present study defines, for the first time to our knowledge, the broad gene spectrum that couples to induction, and loss, of oral keratinocyte differentiation. Bioinformatics assessments of the results relative to clinical data generated novel differentiation-related tumor biomarkers relevant to patient outcome.
Related JoVE Video
Direct effects of carbon nanotubes on dendritic cells induce immune suppression upon pulmonary exposure.
ACS Nano
PUBLISHED: 06-15-2011
Show Abstract
Hide Abstract
Pharyngeal aspiration of single-walled carbon nanotubes (SWCNTs) caused inflammation, pulmonary damage, and an altered cytokine network in the lung. Local inflammatory response in vivo was accompanied by modified systemic immunity as documented by decreased proliferation of splenic T cells. Preincubation of nai?ve T cells in vitro with SWCNT-treated dendritic cells reduced proliferation of T cells. Our data suggest that in vivo exposure to SWCNT modifies systemic immunity by modulating dendritic cell function.
Related JoVE Video
Solution-engineered palladium nanoparticles: model for health effect studies of automotive particulate pollution.
ACS Nano
PUBLISHED: 06-13-2011
Show Abstract
Hide Abstract
Palladium (Pd) nanoparticles are recognized as components of airborne automotive pollution produced by abrasion of catalyst materials in the car exhaust system. Here we produced dispersions of hydrophilic spherical Pd nanoparticles (Pd-NP) of uniform shape and size (10.4 ± 2.7 nm) in one step by Bradleys reaction (solvothermal decomposition in an alcohol or ketone solvent) as a model particle for experimental studies of the Pd particles in air pollution. The same approach provided mixtures of Pd-NP and nanoparticles of non-redox-active metal oxides, such as Al(2)O(3). Particle aggregation in applied media was studied by DLS and nanoparticle tracking analysis. The putative health effects of the produced Pd nanoparticles and nanocomposite mixtures were evaluated in vitro, using human primary bronchial epithelial cells (PBEC) and a human alveolar carcinoma cell line (A549). Viability of these cells was tracked by vital dye exclusion, and apoptosis was also assessed. In addition, we monitored the release of IL-8 and PGE(2) in response to noncytotoxic doses of the nanoparticles. Our studies demonstrate cellular uptake of Pd nanoparticles only in PBEC, as determined by TEM, with pronounced and dose-dependent effects on cellular secretion of soluble biomarkers in both cell types and a decreased responsiveness of human epithelial cells to the pro-inflammatory cytokine TNF-?. When cells were incubated with higher doses of the Pd nanoparticles, apoptosis induction and caspase activation were apparent in PBEC but not in A549 cells. These studies demonstrate the feasibility of using engineered Pd nanoparticles to assess the health effects of airborne automotive pollution.
Related JoVE Video
Late-onset neutropenia following rituximab therapy in rheumatic diseases: association with B lymphocyte depletion and infections.
Arthritis Rheum.
PUBLISHED: 05-12-2011
Show Abstract
Hide Abstract
Late-onset neutropenia following rituximab therapy is a well-recognized side effect in lymphoma patients, but only a few cases of late-onset neutropenia have been reported in patients with autoimmune disorders. The purpose of this study was to define the incidence, clinical features, and some of the underlying mechanisms of late-onset neutropenia in relation to rituximab use in several rheumatic diseases.
Related JoVE Video
Efficient internalization of silica-coated iron oxide nanoparticles of different sizes by primary human macrophages and dendritic cells.
Toxicol. Appl. Pharmacol.
PUBLISHED: 03-04-2011
Show Abstract
Hide Abstract
Engineered nanoparticles are being considered for a wide range of biomedical applications, from magnetic resonance imaging to "smart" drug delivery systems. The development of novel nanomaterials for biomedical applications must be accompanied by careful scrutiny of their biocompatibility. In this regard, particular attention should be paid to the possible interactions between nanoparticles and cells of the immune system, our primary defense system against foreign invasion. On the other hand, labeling of immune cells serves as an ideal tool for visualization, diagnosis or treatment of inflammatory processes, which requires the efficient internalization of the nanoparticles into the cells of interest. Here, we compare novel monodispersed silica-coated iron oxide nanoparticles with commercially available dextran-coated iron oxide nanoparticles. The silica-coated iron oxide nanoparticles displayed excellent magnetic properties. Furthermore, they were non-toxic to primary human monocyte-derived macrophages at all doses tested whereas dose-dependent toxicity of the smaller silica-coated nanoparticles (30nm and 50nm) was observed for primary monocyte-derived dendritic cells, but not for the similarly small dextran-coated iron oxide nanoparticles. No macrophage or dendritic cell secretion of pro-inflammatory cytokines was observed upon administration of nanoparticles. The silica-coated iron oxide nanoparticles were taken up to a significantly higher degree when compared to the dextran-coated nanoparticles, irrespective of size. Cellular internalization of the silica-coated nanoparticles was through an active, actin cytoskeleton-dependent process. We conclude that these novel silica-coated iron oxide nanoparticles are promising materials for medical imaging, cell tracking and other biomedical applications.
Related JoVE Video
In search of the Holy Grail: Folate-targeted nanoparticles for cancer therapy.
Biochem. Pharmacol.
PUBLISHED: 01-29-2011
Show Abstract
Hide Abstract
Targeted drug therapy or "smart" drug delivery, potentially combined with simultaneous imaging modalities to monitor the delivery of drugs to specific tissues, is arguably the "holy grail" of pharmacology. Therapeutic approaches that exploit nanoparticles to deliver drugs selectively to cancer cells are currently considered one of the most promising avenues in the area of cancer therapeutics and imaging. The potential to deliver active chemotherapeutic drugs in the vicinity or directly within specific tumors via receptor mediated pathways, and to image tumors through the use of nanoparticles has been conceptually and experimentally shown for several classes of nanoparticles. Nanoparticles functionalized with the vitamin folic acid are of particular interest as a variety of malignant tumors are known to overexpress the folate receptor(s). Indeed, several nanoparticle architectures with improved retention time, administration route, biocompatibility, absorption, and clearance are being proposed and are in late stage clinical development. This commentary highlights some of the most important concepts related to nanoparticles and folate-mediated drug delivery and imaging in cancer research.
Related JoVE Video
Nanotoxicology: no small matter.
Nanoscale
PUBLISHED: 09-27-2010
Show Abstract
Hide Abstract
Engineered nanomaterials i.e. materials deliberately manufactured on a nanoscale offer exciting new opportunities in technology and medicine. However, the increasing use of nanomaterials in society also raises concerns as to their possible adverse effects on human health and the environment. This review considers the potential application of high-throughput screening approaches to assess hazards of engineered nanomaterials. The disciplinary identity of toxicology is also discussed as attention shifts towards nanoscale objects.
Related JoVE Video
Toxicology of engineered nanomaterials: focus on biocompatibility, biodistribution and biodegradation.
Biochim. Biophys. Acta
PUBLISHED: 04-12-2010
Show Abstract
Hide Abstract
It is widely believed that engineered nanomaterials will be increasingly used in biomedical applications. However, before these novel materials can be safely applied in a clinical setting, their biocompatibility, biodistribution and biodegradation needs to be carefully assessed.
Related JoVE Video
The proteasome inhibitor bortezomib disrupts tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression and natural killer (NK) cell killing of TRAIL receptor-positive multiple myeloma cells.
Mol. Immunol.
PUBLISHED: 03-14-2010
Show Abstract
Hide Abstract
Bortezomib, a potent 26S proteasome inhibitor, is approved for the treatment of multiple myeloma (MM) and clinical trials are under way to evaluate its efficacy in other malignant diseases. However, cytotoxic effects of bortezomib on immune-competent cells have also been observed. In this study, we show that bortezomib downregulates cell surface expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on primary human interleukin (IL)-2-activated natural killer (NK) cells. Pharmacological inhibition of the transcription factor, NF-kappaB also profoundly decreased TRAIL expression, suggesting that NF-kappaB is involved in the regulation of TRAIL expression in activated human NK cells. Furthermore, perforin-independent killing of the human MM cell lines RPMI8226 and U266 by NK cells was markedly suppressed following bortezomib treatment. In addition, blocking cell surface-bound TRAIL with a TRAIL antibody impaired NK cell-mediated lysis of the TRAIL-sensitive MM cell line, RPMI8226. In conclusion, the proteasome is likely to be involved in the regulation of TRAIL expression in primary human IL-2-activated NK cells. Proteasome inhibition by bortezomib disrupts TRAIL expression and TRAIL dependent and/or independent pathway-mediated killing of myeloma cells, suggesting that bortezomib may potentially hamper NK-dependent immunosurveillance against tumors in patients treated with this drug.
Related JoVE Video
Carbon nanotubes degraded by neutrophil myeloperoxidase induce less pulmonary inflammation.
Nat Nanotechnol
PUBLISHED: 02-17-2010
Show Abstract
Hide Abstract
We have shown previously that single-walled carbon nanotubes can be catalytically biodegraded over several weeks by the plant-derived enzyme, horseradish peroxidase. However, whether peroxidase intermediates generated inside human cells or biofluids are involved in the biodegradation of carbon nanotubes has not been explored. Here, we show that hypochlorite and reactive radical intermediates of the human neutrophil enzyme myeloperoxidase catalyse the biodegradation of single-walled carbon nanotubes in vitro, in neutrophils and to a lesser degree in macrophages. Molecular modelling suggests that interactions of basic amino acids of the enzyme with the carboxyls on the carbon nanotubes position the nanotubes near the catalytic site. Importantly, the biodegraded nanotubes do not generate an inflammatory response when aspirated into the lungs of mice. Our findings suggest that the extent to which carbon nanotubes are biodegraded may be a major determinant of the scale and severity of the associated inflammatory responses in exposed individuals.
Related JoVE Video
Programmed cell clearance: molecular regulation of the elimination of apoptotic cell corpses and its role in the resolution of inflammation.
Biochem. Biophys. Res. Commun.
PUBLISHED: 02-08-2010
Show Abstract
Hide Abstract
Programmed cell clearance is a physiological process of elimination of apoptotic cell corpses. Recent studies have disclosed several ligand-receptor interactions that dictate the recognition or non-recognition of cells by macrophages and other phagocytes. The externalization of the anionic phospholipid, phosphatidylserine is effectively recognized by specific receptors on professional phagocytes and facilitates the clearance of apoptotic cells. Macrophage disposal of cells at sites of inflammation is believed to play an important role in the resolution of the inflammatory process, and recent studies have suggested a role for the NADPH oxidase in the process of macrophage elimination of activated neutrophils. The present review will focus on the molecular regulation of programmed cell clearance, and discuss the role of cell elimination in the resolution of inflammation.
Related JoVE Video
Close encounters of the small kind: adverse effects of man-made materials interfacing with the nano-cosmos of biological systems.
Annu. Rev. Pharmacol. Toxicol.
PUBLISHED: 01-09-2010
Show Abstract
Hide Abstract
Engineered nanomaterials have unique physico-chemical properties that make them promising for many technological and biomedical applications, including tissue regeneration, drug and gene delivery, and in vivo monitoring of disease processes. However, with the burgeoning capabilities to manipulate structures at the nano-scale, intentional as well as unintentional human exposures to engineered nanomaterials are set to increase. Nanotoxicology is an emerging discipline focused on understanding the properties of engineered nanomaterials and their interactions with biological systems, and may be viewed as the study of the undesirable interference between man-made nanomaterials and cellular nanostructures or nanomachines. In this review, we discuss recognition of engineered nanomaterials by the immune system, our primary defense system against foreign invasion. Moreover, as oxidative stress is believed to be one of the major deleterious consequences of exposure to nanomaterials, we explore triggering of pro- and antioxidant pathways as well as biomarkers of oxidative stress. Finally, we highlight in vivo studies of the toxicological outcomes of engineered nanomaterials, including carbon nanotubes, with an emphasis on inflammation and genotoxic responses.
Related JoVE Video
The ins and outs of phospholipid asymmetry in the plasma membrane: roles in health and disease.
Crit. Rev. Biochem. Mol. Biol.
PUBLISHED: 09-29-2009
Show Abstract
Hide Abstract
A common feature of all eukaryotic membranes is the non-random distribution of different lipid species in the lipid bilayer (lipid asymmetry). Lipid asymmetry provides the two sides of the plasma membrane with different biophysical properties and influences numerous cellular functions. Alteration of lipid asymmetry plays a prominent role during cell fusion, activation of the coagulation cascade, and recognition and removal of apoptotic cell corpses by macrophages (programmed cell clearance). Here we discuss the origin and maintenance of phospholipid asymmetry, based on recent studies in mammalian systems as well as in Caenhorhabditis elegans and other model organisms, along with emerging evidence for a conserved role of mitochondria in the loss of lipid asymmetry during apoptosis. The functional significance of lipid asymmetry and its disruption during health and disease is also discussed.
Related JoVE Video
N(alpha)-tosyl-L-phenylalanine chloromethyl ketone induces caspase-dependent apoptosis in transformed human B cell lines with transcriptional down-regulation of anti-apoptotic HS1-associated protein X-1.
J. Biol. Chem.
PUBLISHED: 08-13-2009
Show Abstract
Hide Abstract
N(alpha)-tosyl-L-phenylalanine chloromethylketone (TPCK) has been widely used to investigate signal transduction pathways that are involved in gene expression and cell survival/cell death. However, contradictory effects of TPCK on apoptosis have been reported, and the underlying signaling events leading to TPCK-induced promotion or prevention of apoptosis are not fully understood. Here, we show that TPCK induces caspase-dependent apoptosis in Epstein-Barr virus (EBV)-transformed human B cell lines with release of pro-apoptotic proteins from mitochondria. TPCK treatment also results in down-regulation of the anti-apoptotic proteins, cIAP1, cIAP2, and HAX-1, and caspase-dependent cleavage of the anti-apoptotic proteins, Bcl-2 and XIAP. Quantitative PCR analysis confirmed that the TPCK-induced down-regulation of HAX-1 occurred at the transcriptional level, and experiments using the specific pharmacological inhibitor, Bay 11-7082, suggested that HAX-1 expression is subject to regulation by the transcription factor, NF-kappaB. B cell lines derived from patients with homozygous HAX1 mutations were more sensitive to TPCK-induced apoptosis when compared with normal donor cell lines. Furthermore, N-acetylcysteine effectively blocked TPCK-induced apoptosis in EBV-transformed B cell lines and prevented the down-regulation or cleavage of anti-apoptotic proteins. Taken together, our studies demonstrate that TPCK induces apoptosis in human B cell lines and exerts multiple effects on pro- and anti-apoptotic factors.
Related JoVE Video
Involvement of a functional NADPH oxidase in neutrophils and macrophages during programmed cell clearance: implications for chronic granulomatous disease.
Am. J. Physiol., Cell Physiol.
PUBLISHED: 07-01-2009
Show Abstract
Hide Abstract
Resolution of inflammation requires clearance of activated neutrophils by macrophages in a manner that prevents injury to adjacent tissues. Surface changes, including phosphatidylserine (PS) exposure, may target neutrophils for phagocytosis. In this study, we show that externalization of PS is defective in phorbol myristate acetate (PMA)-activated neutrophils obtained from chronic granulomatous disease (CGD) patients with mutations in components of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Moreover, activated neutrophils from CGD patients failed to undergo clearance upon cocultivation with macrophages from normal donors. In line with these results, treatment of donor neutrophils with diphenyleneiodonium (DPI), an inhibitor of NADPH oxidase, blocked PMA-induced PS oxidation and externalization and prevented their engulfment by macrophages. Furthermore, primary macrophages from CGD patients or human gp91(phox)-deficient PLB-985 cells differentiated into macrophage-like cells were defective for engulfment of apoptotic target cells. Pretreatment of normal macrophages with DPI also suppressed the subsequent ingestion of PS-positive target cells. Together, these data demonstrate that NADPH oxidase plays an important role in the process of macrophage disposal of target cells (programmed cell clearance). Thus we speculate that the lack of a functional NADPH oxidase results in impaired neutrophil clearance and the exaggerated inflammation that is characteristic for CGD.
Related JoVE Video
Single-walled carbon nanotubes impair human macrophage engulfment of apoptotic cell corpses.
Inhal Toxicol
PUBLISHED: 06-30-2009
Show Abstract
Hide Abstract
Single-walled carbon nanotubes (SWCNT) are being produced in increasing quantities and the application of these materials in a large number of new technologies and consumer products necessitates studies of their potential impact on human health and the environment. To determine whether SWCNT affect viability or function of macrophages, important components of the innate immune system, we performed in vitro studies using primary human monocyte-derived macrophages (HMDM). Our findings show that SWCNT with a low content of metal impurities do not exert direct cytotoxic effects on HMDM. However, SWCNT suppressed chemotaxis of primary human monocytes in a standard chemotaxis assay. Moreover, macrophage engulfment of apoptotic target cells was significantly impaired following pre-incubation of HMDM with SWCNT at non-cytotoxic concentrations. These results are in line with previous studies showing that ultrafine carbon particles and carbon nanotubes may impair alveolar macrophage ingestion of microorganisms, and suggest that tissue homeostasis may be compromised by SWCNT due to suppressive effects on macrophages.
Related JoVE Video
Compound heterozygous HAX1 mutations in a Swedish patient with severe congenital neutropenia and no neurodevelopmental abnormalities.
Pediatr Blood Cancer
PUBLISHED: 06-06-2009
Show Abstract
Hide Abstract
Kostmann disease or severe congenital neutropenia (SCN) is an autosomal recessive disorder of neutrophil production. Homozygous HAX1 mutations were recently identified in SCN patients belonging to the original family in northern Sweden described by Kostmann. Moreover, recent studies have suggested an association between neurological dysfunction and HAX1 deficiency. Here we describe a patient with a compound heterozygous HAX1 mutation consisting of a nonsense mutation (c.568C > T, p.Glu190X) and a frame-shift mutation (c.91delG, p.Glu31LysfsX54) resulting in a premature stop codon. The patient has a history of neutropenia and a propensity for infections, but has shown no signs of neurodevelopmental abnormalities.
Related JoVE Video
Induction of caspase- and reactive oxygen species-independent phosphatidylserine externalization in primary human neutrophils: role in macrophage recognition and engulfment.
J. Leukoc. Biol.
PUBLISHED: 04-07-2009
Show Abstract
Hide Abstract
Macrophage recognition and disposal of neutrophils are important steps in the resolution of inflammation. Externalization of phosphatidylserine (PS) on the cell surface serves as a common recognition signal for macrophages and is associated with the apoptosis program in neutrophils. Here, we report that macrophage-differentiated PLB-985 cells induce rapid, caspase-independent PS externalization in human neutrophils. A similar degree of PS externalization was seen when neutrophils were cocultured with gp91(phox)-deficient PLB-985 macrophages, thus demonstrating that macrophage-induced PS externalization was NADPH oxidase-independent. Macrophage-induced PS externalization required cell-to-cell contact and kinase activation and was shown to correlate with neutrophil degranulation. Of note, the degree of engulfment of such PS-positive neutrophils by activated human monocyte-derived macrophages was considerably lower than for neutrophils undergoing constitutive apoptosis, indicating that PS externalization alone is not sufficient for macrophage disposal of neutrophils. However, addition of recombinant milk fat globule epidermal growth factor 8, a PS-binding protein, restored engulfment of the macrophage-cocultured target cells. Finally, neutrophils undergoing spontaneous apoptosis but not macrophage-cocultured neutrophils displayed surface expression and release of annexin I, and the addition of N-t-Boc-Phe-D-Leu-Phe-D-Leu-Phe (Boc1), a formyl peptide receptor/lipoxin receptor antagonist, suppressed clearance of apoptotic neutrophils. Conditioned medium from apoptotic neutrophils also promoted the engulfment of macrophage-cocultured neutrophils, and Boc1 blocked this process. Taken together, these studies highlight a novel pathway of PS externalization in primary human neutrophils and also provide evidence for an auxiliary function of annexin I in macrophage clearance of neutrophils.
Related JoVE Video
HAX-1: a multifunctional protein with emerging roles in human disease.
Biochim. Biophys. Acta
PUBLISHED: 03-02-2009
Show Abstract
Hide Abstract
HS-1-associated protein X-1 (HAX-1) was identified more than 10 years ago as a novel protein with ubiquitous tissue expression and a predominantly mitochondrial localization at the subcellular level. Recent studies have shown that homozygous mutations in the HAX1 gene are associated with autosomal recessive forms of severe congenital neutropenia (also known as Kostmann disease), and results from studies in mice and men are beginning to unravel a prominent role for HAX-1 in apoptosis signaling not only in the hematopoietic compartment, but also in the central nervous system. Moreover, several different cellular and viral binding partners of HAX-1 have been identified thus pointing toward a complex and multifunctional role of this protein. HAX-1 has also been shown to bind to the 3 untranslated regions of certain mRNAs and could therefore contribute to the regulation of transport and/or stability of such transcripts. The present review discusses the emerging and divergent roles of HAX-1, including its involvement in cell migration, apoptosis signaling, and mRNA surveillance. The importance of HAX-1 in human disease is also highlighted and outstanding questions that remain to be addressed are identified.
Related JoVE Video
Proteasome inhibition induces apoptosis in primary human natural killer cells and suppresses NKp46-mediated cytotoxicity.
Haematologica
PUBLISHED: 02-19-2009
Show Abstract
Hide Abstract
Bortezomib is a selective and potent inhibitor of the proteasome and has prominent effects in vitro and in vivo against tumors. Very recently, cytotoxic effects of bortezomib on immune-competent cells such as T cells and dendritic cells were also revealed. The aim of the study was to investigate the effects of this agent on natural killer cell survival and function.
Related JoVE Video
Phosphatidylserine targets single-walled carbon nanotubes to professional phagocytes in vitro and in vivo.
PLoS ONE
PUBLISHED: 02-09-2009
Show Abstract
Hide Abstract
Broad applications of single-walled carbon nanotubes (SWCNT) dictate the necessity to better understand their health effects. Poor recognition of non-functionalized SWCNT by phagocytes is prohibitive towards controlling their biological action. We report that SWCNT coating with a phospholipid "eat-me" signal, phosphatidylserine (PS), makes them recognizable in vitro by different phagocytic cells - murine RAW264.7 macrophages, primary monocyte-derived human macrophages, dendritic cells, and rat brain microglia. Macrophage uptake of PS-coated nanotubes was suppressed by the PS-binding protein, Annexin V, and endocytosis inhibitors, and changed the pattern of pro- and anti-inflammatory cytokine secretion. Loading of PS-coated SWCNT with pro-apoptotic cargo (cytochrome c) allowed for the targeted killing of RAW264.7 macrophages. In vivo aspiration of PS-coated SWCNT stimulated their uptake by lung alveolar macrophages in mice. Thus, PS-coating can be utilized for targeted delivery of SWCNT with specified cargoes into professional phagocytes, hence for therapeutic regulation of specific populations of immune-competent cells.
Related JoVE Video
Efficient internalization of mesoporous silica particles of different sizes by primary human macrophages without impairment of macrophage clearance of apoptotic or antibody-opsonized target cells.
Toxicol. Appl. Pharmacol.
PUBLISHED: 01-28-2009
Show Abstract
Hide Abstract
Macrophage recognition and ingestion of apoptotic cell corpses, a process referred to as programmed cell clearance, is of considerable importance for the maintenance of tissue homeostasis and in the resolution of inflammation. Moreover, macrophages are the first line of defense against microorganisms and other foreign materials including particles. However, there is sparse information on the mode of uptake of engineered nanomaterials by primary macrophages. In this study, mesoporous silica particles with cubic pore geometries and covalently fluorescein-grafted particles were synthesized through a novel route, and their interactions with primary human monocyte-derived macrophages were assessed. Efficient and active internalization of mesoporous silica particles of different sizes was observed by transmission electron microscopic and flow cytometric analysis and studies using pharmacological inhibitors suggested that uptake occurred through a process of endocytosis. Moreover, uptake of silica particles was independent of serum factors. The silica particles with very high surface areas due to their porous structure did not impair cell viability or function of macrophages, including the ingestion of different classes of apoptotic or opsonized target cells. The current findings are relevant to the development of mesoporous materials for drug delivery and other biomedical applications.
Related JoVE Video
Better safe than sorry: Understanding the toxicological properties of inorganic nanoparticles manufactured for biomedical applications.
Adv. Drug Deliv. Rev.
PUBLISHED: 01-10-2009
Show Abstract
Hide Abstract
The development of nanoparticles for biomedical applications including medical imaging and drug delivery is currently undergoing a dramatic expansion. However, as the range of nanoparticle types and applications increases, it is also clear that the potential toxicities of these novel materials and the properties driving such toxic responses must also be understood. Indeed, a detailed assessment of the factors that influence the biocompatibility and/or toxicity of nanoparticles is crucial for the safe and sustainable development of the emerging nanotechnologies. This review summarizes some of the recent developments in the field of nanomedicine with particular emphasis on inorganic nanoparticles for drug delivery. The synthesis routes, physico-chemical characteristics, and cytotoxic properties of inorganic nanoparticles are thus explored and lessons learned from the toxicological investigation of three common types of engineered nanomaterials of titania, gold, and mesoporous silica are discussed. Emphasis is placed on the recognition versus non-recognition of engineered nanomaterials by the immune system, the primary surveillance system against microorganisms and particles, which, in turn, is intimately linked to the issue of targeted drug delivery using such nanomaterials as carrier systems.
Related JoVE Video
Applying quantitative structure-activity relationship approaches to nanotoxicology: current status and future potential.
Toxicology
Show Abstract
Hide Abstract
The potential (eco)toxicological hazard posed by engineered nanoparticles is a major scientific and societal concern since several industrial sectors (e.g. electronics, biomedicine, and cosmetics) are exploiting the innovative properties of nanostructures resulting in their large-scale production. Many consumer products contain nanomaterials and, given their complex life-cycle, it is essential to anticipate their (eco)toxicological properties in a fast and inexpensive way in order to mitigate adverse effects on human health and the environment. In this context, the application of the structure-toxicity paradigm to nanomaterials represents a promising approach. Indeed, according to this paradigm, it is possible to predict toxicological effects induced by chemicals on the basis of their structural similarity with chemicals for which toxicological endpoints have been previously measured. These structure-toxicity relationships can be quantitative or qualitative in nature and they can predict toxicological effects directly from the physicochemical properties of the entities (e.g. nanoparticles) of interest. Therefore, this approach can aid in prioritizing resources in toxicological investigations while reducing the ethical and monetary costs that are related to animal testing. The purpose of this review is to provide a summary of recent key advances in the field of QSAR modelling of nanomaterial toxicity, to identify the major gaps in research required to accelerate the use of quantitative structure-activity relationship (QSAR) methods, and to provide a roadmap for future research needed to achieve QSAR models useful for regulatory purposes.
Related JoVE Video
Interactions of engineered nanoparticles with organs protected by internal biological barriers.
Small
Show Abstract
Hide Abstract
Engineered nanomaterials may exert adverse effects on human health which, in turn, may be linked to their propensity to cross biological barriers in the body. Here, available evidence is discussed, based on in vivo studies for interactions of commercially relevant nanoparticles with critical internal barriers. The internal barriers in focus in this review are the blood-brain barrier, protecting the brain, the blood-testis barrier, protecting the male germ line, and the placenta, protecting the developing fetus. The route of exposure (pulmonary, gastro-intestinal, intravenous, intraperitoneal, dermal), and, hence, the portal of entry of nanoparticles into the body, is of critical importance. Different physico-chemical properties, not only size, may determine the ability of nanoparticles to breach biological barriers; the situation is further compounded by the formation of a so-called corona of biomolecules on the surfaces of nanoparticles, the composition of which may vary depending on the route of exposure and the translocation of nanoparticles from one biological compartment to another. The relevance of nanoparticle interactions with internal biological barriers for their impact on the organs protected by these barriers is also discussed.
Related JoVE Video
Requirement of apoptotic protease-activating factor-1 for bortezomib-induced apoptosis but not for Fas-mediated apoptosis in human leukemic cells.
Mol. Pharmacol.
Show Abstract
Hide Abstract
Bortezomib is a highly selective inhibitor of the 26S proteasome and has been approved for clinical use in the treatment of relapsing and refractory multiple myeloma and mantle cell lymphoma. Clinical trials are also underway to assess the role of bortezomib in several other human malignancies, including leukemia. However, the mechanism(s) by which bortezomib acts remain to be fully understood. Here, we studied the molecular requirements of bortezomib-induced apoptosis using the human T-cell leukemic Jurkat cells stably transfected with or without shRNA against apoptotic protease-activating factor-1 (Apaf-1). The Apaf-1-deficient Jurkat T cells were resistant to bortezomib-induced apoptosis, as assessed by caspase-3 activity, poly(ADP-ribose) polymerase cleavage, phosphatidylserine externalization, and hypodiploid DNA content. In contrast, Apaf-1-deficient cells were sensitive to Fas-induced apoptosis. Bortezomib induced an upregulation of the pro-apoptotic protein Noxa, loss of mitochondrial transmembrane potential, and release of cytochrome c in cells expressing or not expressing Apaf-1. Transient silencing of Apaf-1 expression in RPMI 8402 T-cell leukemic cells also diminished bortezomib-induced apoptosis. Fas-associated death domain (FADD)-deficient Jurkat cells were resistant to Fas-mediated apoptosis yet remained sensitive to bortezomib. Our results show that bortezomib induces apoptosis by regulating pathways that are mechanistically different from those activated upon death receptor ligation. Furthermore, in silico analyses of public transcriptomics databases indicated elevated Apaf-1 expression in several hematologic malignancies, including acute lymphoblastic and myeloid leukemia. We also noted variable Apaf-1 expression in a panel of samples from patients with acute lymphoblastic leukemia. Our results suggest that the expression of Apaf-1 may be predictive of the response to proteasome inhibition.
Related JoVE Video
AIF and Scythe (Bat3) regulate phosphatidylserine exposure and macrophage clearance of cells undergoing Fas (APO-1)-mediated apoptosis.
PLoS ONE
Show Abstract
Hide Abstract
Phosphatidylserine (PS) exposure on the cell surface has been considered a characteristic feature of apoptosis and serves as a molecular cue for engulfment of dying cells by phagocytes. However, the mechanism of PS exposure is still not fully elucidated. Here we show that the cytosolic release from mitochondria of apoptosis-inducing factor (AIF) is required for PS exposure during death receptor-induced apoptosis and for efficient clearance of cell corpses by primary human macrophages. Fas-triggered PS exposure was significantly reduced upon siRNA-mediated silencing of AIF expression and by inhibition of the cytosolic translocation of AIF. In addition, AIF localizes to the plasma membrane upon Fas ligation and promotes activation of phospholipid scrambling activity. Finally, cytosolic stabilization of AIF through interaction with Scythe is shown to be involved in apoptotic PS exposure. Taken together, our results suggest an essential role for AIF and its binding partner Scythe in the pathway leading to apoptotic corpse clearance.
Related JoVE Video
MAML1 acts cooperatively with EGR1 to activate EGR1-regulated promoters: implications for nephrogenesis and the development of renal cancer.
PLoS ONE
Show Abstract
Hide Abstract
Mastermind-like 1 (MAML1) is a transcriptional coregulator of activators in various signaling pathways, such as Notch, p53, myocyte enhancer factor 2C (MEF2C) and beta-catenin. In earlier studies, we demonstrated that MAML1 enhanced p300 acetyltransferase activity, which increased the acetylation of Notch by p300. In this study, we show that MAML1 strongly induced acetylation of the transcription factor early growth response-1 (EGR1) by p300, and increased EGR1 protein expression in embryonic kidney cells. EGR1 mRNA transcripts were also upregulated in the presence of MAML1. We show that MAML1 physically interacted with, and acted cooperatively with EGR1 to increase transcriptional activity of the EGR1 and p300 promoters, which both contain EGR1 binding sites. Bioinformatics assessment revealed a correlation between p300, EGR1 and MAML1 copy number and mRNA alterations in renal clear cell carcinoma and p300, EGR1 and MAML1 gene alterations were associated with increased overall survival. Our findings suggest MAML1 may be a component of the transcriptional networks which regulate EGR1 target genes during nephrogenesis and could also have implications for the development of renal cell carcinoma.
Related JoVE Video
Graphene oxide, but not fullerenes, targets immunoproteasomes and suppresses antigen presentation by dendritic cells.
Small
Show Abstract
Hide Abstract
Graphene oxide (GO) and C60 - or C60 -TRIS fullerenes, internalized by murine dendritic cells (DCs), differently affect their abilities to present antigens to T-cells. While C60 -fullerenes stimulate the ovalbumin-specific MHC class I-restricted T-cell response, GO impairs the stimulatory potential of DCs. In contrast to C60 -fullerenes, GO decreases the intracellular levels of LMP7 immunoproteasome subunits required for processing of protein antigens. This is important for the development of DC-based vaccines.
Related JoVE Video
Clear and present danger? Engineered nanoparticles and the immune system.
Swiss Med Wkly
Show Abstract
Hide Abstract
The innate immune system is the first line of defense against microbial invasion and involves the recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors on the surface of phagocytic cells. The immune system also responds to tissue damage, a process that is triggered by so-called danger- or damage-associated molecular patterns (DAMPs) or "alarmins". How do physico-chemical properties e.g., size, shape, surface charge and solubility affect immune interactions of nanoparticles? Does the adsorption of biomolecules onto the surface of nanoparticles dictate subsequent immune responses? Do engineered nanoparticles per se act as "alarmins" or does the bio-corona on nanoparticles convey a new "identity" and allow innocuous nanoparticles to present NAMPs (nanoparticle-associated molecular patterns)? Finally, what are the parameters that determine particle clearance or biodegradation in a living system? Understanding nano-immuno-interactions is critical for the safe application of engineered nanoparticles in medicine.
Related JoVE Video
Programmed cell death: molecular mechanisms and implications for safety assessment of nanomaterials.
Acc. Chem. Res.
Show Abstract
Hide Abstract
Engineered nanomaterials offer numerous and tantalizing opportunities in many sectors of society, including medicine. Needless to say, attention should also be paid to the potential for unexpected hazardous effects of these novel materials. To date, much of the nanotoxicology literature has focused on the assessment of cell viability or cell death using primitive assays for the detection of plasma membrane integrity or mitochondrial function or assessment of cellular morphology. However, when assessing the cytotoxic effects of engineered nanomaterials, researchers need not only to consider whether cells are dead or alive but also to assess which of the numerous, highly specific pathways of cell death might be involved. Moreover, it is important to diagnose cell death based not only on morphological markers but on the assessment and quantification of biochemical alterations specific to each form of cell death. In this Account, we provide a description of the three major forms of programmed cell death in mammalian cells: apoptosis, autophagic cell death, and regulated necrosis, sometimes referred to as necroptosis. Apoptosis can be activated via the extrinsic (death receptor-dependent) or via the intrinsic (mitochondria-dependent) route. Apoptotic cell death may or may not require the activation of cytosolic proteases known as caspases. Autophagy (self-eating) has an important homeostatic role in the cell, mediating the removal of dysfunctional or damaged organelles thereby allowing the recycling of cellular building blocks. However, unrestrained autophagy can kill cells. Studies in recent years have revealed that necrosis that depends on activation of the kinases RIP1 and RIP3 is a major form of programmed cell death with roles in development and immunity. We also discuss recent examples of the impact of engineered nanoparticles on the three different pathways of programmed cell death. For example, acute exposure of cells to carbon nanotubes (CNTs) can induce apoptosis whereas chronic exposure to CNTs may yield an apoptosis-resistant and tumorigenic phenotype in lung epithelial cells. Several reports show that nanoparticles, including polystyrene particles, are routed to the lysosomal compartment and trigger cell death through the destabilization of lysosomal membranes with engagement of the intrinsic apoptosis pathway. In addition, a number of studies have demonstrated that nanomaterials such as CNTs, quantum dots, and gold nanoparticles can affect cellular autophagy. An improved understanding of the complexities of the nanomaterial-induced perturbation of different cell death pathways may allow for a better prediction of the consequences of human exposure.
Related JoVE Video
Incidence of severe congenital neutropenia in Sweden and risk of evolution to myelodysplastic syndrome/leukaemia.
Br. J. Haematol.
Show Abstract
Hide Abstract
Severe congenital neutropenia (SCN) is characterized by low blood neutrophil counts, early bacterial infections, and risk of leukaemia development. As yet, no population-based incidence estimates of SCN have been reported. Children less than 16 years of age with SCN were sought in Sweden during the 20-year period 1987-2006 by a questionnaire to all Swedish Departments of Paediatrics and by reviewing the Swedish Health and Welfare Statistical Databases. Thirty-two patients were diagnosed with congenital neutropenia during this period. All received treatment with recombinant granulocyte-colony stimulating factor (G-CSF). Twenty-one patients were diagnosed as SCN or probable SCN, corresponding to 1·0 per 100,000 live births. Nine (43%) had ELANE mutations, four (19%) HAX1 mutations and eight (38%) were children with disease of unknown genetic aetiology. Four out of 21 patients (19%) developed myelodysplastic syndrome/leukaemia and three (14%) died, all with leukaemia. The cumulative incidence of myelodysplastic syndrome/leukaemia was 31%. The observed incidence of SCN in this population-based study was higher than previously estimated, possibly because genetic testing now can identify SCN cases previously thought to be idiopathic or benign neutropenia. The risk of developing myelodysplastic syndrome/leukaemia is considerable. ELANE mutations are the most commonly identified genetic defects.
Related JoVE Video
Pulmonary exposure to single-walled carbon nanotubes does not affect the early immune response against Toxoplasma gondii.
Part Fibre Toxicol
Show Abstract
Hide Abstract
Single-walled carbon nanotubes (SWCNT) trigger pronounced inflammation and fibrosis in the lungs of mice following administration via pharyngeal aspiration or inhalation. Human exposure to SWCNT in an occupational setting may occur in conjunction with infections and this could yield enhanced or suppressed responses to the offending agent. Here, we studied whether the sequential exposure to SWCNT via pharyngeal aspiration and infection of mice with the ubiquitous intracellular parasite Toxoplasma gondii would impact on the immune response of the host against the parasite.
Related JoVE Video
Mechanisms of carbon nanotube-induced toxicity: focus on oxidative stress.
Toxicol. Appl. Pharmacol.
Show Abstract
Hide Abstract
Nanotechnologies are emerging as highly promising technologies in many sectors in the society. However, the increasing use of engineered nanomaterials also raises concerns about inadvertent exposure to these materials and the potential for adverse effects on human health and the environment. Despite several years of intensive investigations, a common paradigm for the understanding of nanoparticle-induced toxicity remains to be firmly established. Here, the so-called oxidative stress paradigm is scrutinized. Does oxidative stress represent a secondary event resulting inevitably from disruption of biochemical processes and the demise of the cell, or a specific, non-random event that plays a role in the induction of cellular damage e.g. apoptosis? The answer to this question will have important ramifications for the development of strategies for mitigation of adverse effects of nanoparticles. Recent examples of global lipidomics studies of nanoparticle-induced tissue damage are discussed along with proteomics and transcriptomics approaches to achieve a comprehensive understanding of the complex and interrelated molecular changes in cells and tissues exposed to nanoparticles. We also discuss instances of non-oxidative stress-mediated cellular damage resulting from direct physical interference of nanomaterials with cellular structures.
Related JoVE Video
Factoring-in agglomeration of carbon nanotubes and nanofibers for better prediction of their toxicity versus asbestos.
Part Fibre Toxicol
Show Abstract
Hide Abstract
Carbon nanotubes (CNT) and carbon nanofibers (CNF) are allotropes of carbon featuring fibrous morphology. The dimensions and high aspect ratio of CNT and CNF have prompted the comparison with naturally occurring asbestos fibers which are known to be extremely pathogenic. While the toxicity and hazardous outcomes elicited by airborne exposure to single-walled CNT or asbestos have been widely reported, very limited data are currently available describing adverse effects of respirable CNF.
Related JoVE Video
Impaired clearance and enhanced pulmonary inflammatory/fibrotic response to carbon nanotubes in myeloperoxidase-deficient mice.
PLoS ONE
Show Abstract
Hide Abstract
Advancement of biomedical applications of carbonaceous nanomaterials is hampered by their biopersistence and pro-inflammatory action in vivo. Here, we used myeloperoxidase knockout B6.129X1-MPO (MPO k/o) mice and showed that oxidation and clearance of single walled carbon nanotubes (SWCNT) from the lungs of these animals after pharyngeal aspiration was markedly less effective whereas the inflammatory response was more robust than in wild-type C57Bl/6 mice. Our results provide direct evidence for the participation of MPO - one of the key-orchestrators of inflammatory response - in the in vivo pulmonary oxidative biodegradation of SWCNT and suggest new ways to control the biopersistence of nanomaterials through genetic or pharmacological manipulations.
Related JoVE Video
Adsorption of surfactant lipids by single-walled carbon nanotubes in mouse lung upon pharyngeal aspiration.
ACS Nano
Show Abstract
Hide Abstract
The pulmonary route represents one of the most important portals of entry for nanoparticles into the body. However, the in vivo interactions of nanoparticles with biomolecules of the lung have not been sufficiently studied. Here, using an established mouse model of pharyngeal aspiration of single-walled carbon nanotubes (SWCNTs), we recovered SWCNTs from the bronchoalveolar lavage fluid (BALf), purified them from possible contamination with lung cells, and examined the composition of phospholipids adsorbed on SWCNTs by liquid chromatography mass spectrometry (LC-MS) analysis. We found that SWCNTs selectively adsorbed two types of the most abundant surfactant phospholipids: phosphatidylcholines (PC) and phosphatidylglycerols (PG). Molecular speciation of these phospholipids was also consistent with pulmonary surfactant. Quantitation of adsorbed lipids by LC-MS along with the structural assessments of phospholipid binding by atomic force microscopy and molecular modeling indicated that the phospholipids (?108 molecules per SWCNT) formed an uninterrupted "coating" whereby the hydrophobic alkyl chains of the phospholipids were adsorbed onto the SWCNT with the polar head groups pointed away from the SWCNT into the aqueous phase. In addition, the presence of surfactant proteins A, B, and D on SWCNTs was determined by LC-MS. Finally, we demonstrated that the presence of this surfactant coating markedly enhanced the in vitro uptake of SWCNTs by macrophages. Taken together, this is the first demonstration of the in vivo adsorption of the surfactant lipids and proteins on SWCNTs in a physiologically relevant animal model.
Related JoVE Video
Ablation of the pro-apoptotic protein Bax protects mice from glucocorticoid-induced bone growth impairment.
PLoS ONE
Show Abstract
Hide Abstract
Dexamethasone (Dexa) is a widely used glucocorticoid to treat inflammatory diseases; however, a multitude of undesired effects have been reported to arise from this treatment including osteoporosis, obesity, and in children decreased longitudinal bone growth. We and others have previously shown that glucocorticoids induce apoptosis in growth plate chondrocytes. Here, we hypothesized that Bax, a pro-apoptotic member of the Bcl-2 family, plays a key role in Dexa-induced chondrocyte apoptosis and bone growth impairment. Indeed, experiments in the human HCS-2/8 chondrocytic cell line demonstrated that silencing of Bax expression using small-interfering (si) RNA efficiently blocked Dexa-induced apoptosis. Furthermore, ablation of Bax in female mice protected against Dexa-induced bone growth impairment. Finally, Bax activation by Dexa was confirmed in human growth plate cartilage specimens cultured ex vivo. Our findings could therefore open the door for new therapeutic approaches to prevent glucocorticoid-induced bone growth impairment through specific targeting of Bax.
Related JoVE Video
Safety assessment of nanomaterials: implications for nanomedicine.
J Control Release
Show Abstract
Hide Abstract
Nanotechnologies offer exciting opportunities for targeted drug delivery which is anticipated to increase the efficacy of the drug and reduce potential side-effects, through the reduction of the dose of the drug in bystander tissues and an increase of the drug at the desired target site. Nevertheless, understanding whether the nano-scale carriers themselves may exert adverse effects is of great importance. The small size may enable nanoparticles to negotiate various biological barriers in the body which could, in turn, give rise to unexpected toxicities. On the other hand, the potential of nanoparticles to cross barriers can also be exploited for drug delivery. Determining the fate of nanoparticles following their therapeutic or diagnostic application is critical: are nanoparticles excreted, or biodegraded, or do they accumulate, potentially leading to harmful long-term effects? The bio-corona of proteins or lipids on the surface of nanoparticles is a key parameter for the understanding of biological interactions of nanoparticles. In the present review, we discuss some of the major challenges related to safety of nanomedicines.
Related JoVE Video
Microsomal glutathione transferase 1 protects against toxicity induced by silica nanoparticles but not by zinc oxide nanoparticles.
ACS Nano
Show Abstract
Hide Abstract
Microsomal glutathione transferase 1 (MGST1) is an antioxidant enzyme located predominantly in the mitochondrial outer membrane and endoplasmic reticulum and has been shown to protect cells from lipid peroxidation induced by a variety of cytostatic drugs and pro-oxidant stimuli. We hypothesized that MGST1 may also protect against nanomaterial-induced cytotoxicity through a specific effect on lipid peroxidation. We evaluated the induction of cytotoxicity and oxidative stress by TiO(2), CeO(2), SiO(2), and ZnO in the human MCF-7 cell line with or without overexpression of MGST1. SiO(2) and ZnO nanoparticles caused dose- and time-dependent toxicity, whereas no obvious cytotoxic effects were induced by nanoparticles of TiO(2) and CeO(2). We also noted pronounced cytotoxicity for three out of four additional SiO(2) nanoparticles tested. Overexpression of MGST1 reversed the cytotoxicity of the main SiO(2) nanoparticles tested and for one of the supplementary SiO(2) nanoparticles but did not protect cells against ZnO-induced cytotoxic effects. The data point toward a role of lipid peroxidation in SiO(2) nanoparticle-induced cell death. For ZnO nanoparticles, rapid dissolution was observed, and the subsequent interaction of Zn(2+) with cellular targets is likely to contribute to the cytotoxic effects. A direct inhibition of MGST1 by Zn(2+) could provide a possible explanation for the lack of protection against ZnO nanoparticles in this model. Our data also showed that SiO(2) nanoparticle-induced cytotoxicity is mitigated in the presence of serum, potentially through masking of reactive surface groups by serum proteins, whereas ZnO nanoparticles were cytotoxic both in the presence and in the absence of serum.
Related JoVE Video
Scythe cleavage during Fas (APO-1)-and staurosporine-mediated apoptosis.
FEBS Lett.
Show Abstract
Hide Abstract
Scythe is a nuclear protein that has been implicated in the apoptotic process in Drosophila melanogaster; however, its role in apoptosis of mammalian cells is not fully elucidated. Here we show that cleavage of Scythe by caspase-3 occurs after activation of both the extrinsic (i.e. Fas/APO-1-mediated) and the intrinsic (i.e. staurosporine-induced) apoptosis pathway. Moreover, this caspase-dependent cleavage correlates with Scythe translocation from the nucleus to the cytosol. We also show that cytosolic re-localization of Scythe is required for Fas/APO-1-triggered phosphatidylserine (PS) exposure, a signal for macrophage clearance of apoptotic cells. Our data suggest that Scythe cleavage may represent a marker for caspase-3 activation and implicate cytosolic re-localization of Scythe in the pathway of PS exposure.
Related JoVE Video
Co-targeting of the PI3K pathway improves the response of BRCA1 deficient breast cancer cells to PARP1 inhibition.
Cancer Lett.
Show Abstract
Hide Abstract
Although pre-clinical and clinical studies on PARP1 inhibitors, alone and in combination with DNA-damaging agents, show promising results, further ways to improve and broaden the scope of application of this therapeutic approach are warranted. To this end, we have investigated the possibility of improving the response of BRCA1 mutant breast cancer cells to PARP1 inhibition by co-targeting the PI3K pathway. Human breast cancer cell lines with or without the expression of BRCA1 and/or PTEN were treated with PARP1 and PI3K inhibitors as single agents and in combination. PARP1 inhibition induced DNA damage conferring a G2/M arrest and decrease in viability, paralleled by the induction of apoptosis. PI3K inhibition alone caused a G1 arrest and decreased cell growth. Most importantly, sequential combination of PARP and PI3K inhibitors interacted synergistically to significantly decrease growth compared to PARP inhibition alone. Global transcriptional profiling revealed that this decrease in growth was associated with down-regulation of macromolecule biosynthesis and the induction of apoptosis. Taken together, these results suggest an improved treatment strategy for BRCA1-mutant and possibly also triple-negative breast cancers with similar molecular defects.
Related JoVE Video
Bortezomib and IL-12 produce synergetic anti-multiple myeloma effects with reduced toxicity to natural killer cells.
Anticancer Drugs
Show Abstract
Hide Abstract
The aim of this study was to examine the hypothesis that a combination of proteasome inhibition by bortezomib and immune therapy with interleukin-12 (IL-12) can produce enhanced antitumor efficacy relative to the effects of either of these agents alone. A mouse xenograft model of myeloma was developed. The mice were randomly divided into saline control (NS), IL-12 (0.4 µg/animal; intraperitoneal), bortezomib (0.75 mg/kg; intravenous), and bortezomib+IL-12 groups. Effects of treatments on tumor growth were assessed by before and after treatment comparisons and group comparisons. The effects of various treatments on the number of peripheral blood lymphocytes and natural killer (NK) cells were assessed by complete blood count and flow cytometry analysis. The cell-killing function of NK cells in splenocytes was evaluated using the lactate dehydrogenase release assay. IL-12 treatment alone produced a mild decrease in tumor volume compared with control (P>0.05). Bortezomib alone resulted in substantial inhibition of tumor growth at varying time points, reaching ?65 and ?60% reduction in tumor volume after 15 and 21 days of therapy, respectively. At the same time points, the combination therapy produced ?75 and ?84% decreases in tumor growth, respectively, which were significantly greater than the reduction produced by bortezomib monotherapy. Tumors resumed growth upon termination of bortezomib treatment at 2 weeks, although the tumor volume was still significantly smaller than that in the time-matched NS and IL-12 animals. This rebound of tumor growth was completely prevented with the combination therapy, and tumor volume continued to decrease throughout the time course. The percentage and total number of NK cells were significantly decreased after bortezomib monotherapy and combination therapy; however, they remained unaltered after IL-12 treatment compared with no treatment. Further, combination therapy significantly restored the bortezomib-induced functional impairment of the cell-killing capability of NK cells, relative to bortezomib alone. We conclude that the bortezomib-IL-12 combination therapy offers superior antitumor efficacy over monotherapy with either bortezomib or IL-12 in a mouse model of myeloma. Restoration of bortezomib-induced functional impairment of NK cells by IL-12 may be a mechanism for the synergetic effects of the two agents. Therefore, a combination of the two agents may represent a more rational therapeutic approach for myeloma.
Related JoVE Video

What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.