Cell death induced by the application of alternating magnetic fields to nanoparticle-loaded dendritic cells

In this work, the capability of primary, monocyte-derived dendritic cells (DCs) to uptake iron oxide magnetic nanoparticles (MNPs) is assessed and a strategy to induce selective cell death in these MNP-loaded DCs using external alternating magnetic fields (AMFs) is reported. No significant decrease in the cell viability of MNP-loaded DCs, compared to the control samples, was observed after five days of culture. The amount of MNPs incorporated into the cytoplasm was measured by magnetometry, which confirmed that 1 to 5 pg of the particles were uploaded per cell. The intracellular distribution of these MNPs, assessed by transmission electron microscopy, was found to be primarily inside the endosomic structures. These cells were then subjected to an AMF for 30 min, and the viability of the blank DCs (i.e., without MNPs), which were used as control samples, remained essentially unaffected. However, a remarkable decrease of viability from approximately 90% to 2-5% of DCs previously loaded with MNPs was observed after the same 30 min exposure to an AMF. The same results were obtained using MNPs having either positive (NH2+) or negative (COOH-) surface functional groups. In spite of the massive cell death induced by application of AMF to MNP-loaded DCs, the amount of incorporated magnetic particles did not raise the temperature of the cell culture. Clear morphological changes at the cell structure after magnetic field application were observed using scanning electron microscopy. Therefore, local damage produced by the MNPs could be the main mechanism for the selective cell death of MNP-loaded DCs under an AMF. Based on the ability of these cells to evade the reticuloendothelial system, these complexes combined with an AMF should be considered as a potentially powerful tool for tumour therapy.Comment: In Press. 33 pages, 11 figure

Application of magnetically induced hyperthermia on the model protozoan Crithidia fasciculata as a potential therapy against parasitic infections

Magnetic hyperthermia is currently an EU-approved clinical therapy against tumor cells that uses magnetic nanoparticles under a time varying magnetic field (TVMF). The same basic principle seems promising against trypanosomatids causing Chagas disease and sleeping sickness, since therapeutic drugs available display severe side effects and drug-resistant strains. However, no applications of this strategy against protozoan-induced diseases have been reported so far. In the present study, Crithidia fasciculata, a widely used model for therapeutic strategies against pathogenic trypanosomatids, was targeted with Fe_{3}O_{4} magnetic nanoparticles (MNPs) in order to remotely provoke cell death using TVMFs. The MNPs with average sizes of d approx. 30 nm were synthesized using a precipitation of FeSO_{4}4 in basic medium. The MNPs were added to Crithidia fasciculata choanomastigotes in exponential phase and incubated overnight. The amount of uploaded MNPs per cell was determined by magnetic measurements. Cell viability using the MTT colorimetric assay and flow cytometry showed that the MNPs were incorporated by the cells with no noticeable cell-toxicity effects. When a TVMF (f = 249 kHz, H = 13 kA/m) was applied to MNP-bearing cells, massive cell death was induced via a non-apoptotic mechanism. No effects were observed by applying a TVMF on control (without loaded MNPs) cells. No macroscopic rise in temperature was observed in the extracellular medium during the experiments. Scanning Electron Microscopy showed morphological changes after TVMF experiments. These data indicate (as a proof of principle) that intracellular hyperthermia is a suitable technology to induce the specific death of protozoan parasites bearing MNPs. These findings expand the possibilities for new therapeutic strategies that combat parasitic infections.Comment: 9 pages, four supplementary video file

A lower bound on CNF encodings of the at-most-one constraint

Constraint "at most one" is a basic cardinality constraint which requires that at most one of its $n$ boolean inputs is set to $1$. This constraint is widely used when translating a problem into a conjunctive normal form (CNF) and we investigate its CNF encodings suitable for this purpose. An encoding differs from a CNF representation of a function in that it can use auxiliary variables. We are especially interested in propagation complete encodings which have the property that unit propagation is strong enough to enforce consistency on input variables. We show a lower bound on the number of clauses in any propagation complete encoding of the "at most one" constraint. The lower bound almost matches the size of the best known encodings. We also study an important case of 2-CNF encodings where we show a slightly better lower bound. The lower bound holds also for a related "exactly one" constraint.Comment: 38 pages, version 3 is significantly reorganized in order to improve readabilit

Design of stable magnetic hybrid nanoparticles of Si-entrapped HRP

Hybrid and composite nanoparticles represent an attractive material for enzyme integration due to possible synergic advantages of the structural builders in the properties of the nanobiocatalyst. In this study, we report the synthesis of a new stable hybrid nanobiocatalyst formed by biomimetic silica (Si) nanoparticles entrapping both Horseradish Peroxidase (HRP) (EC 1.11.1.7) and magnetic nanoparticles (MNPs). We have demonstrated that tailoring of the synthetic reagents and post immobilization treatments greatly impacted physical and biocatalytic properties such as an unprecedented ~280 times increase in the half-life time in thermal stability experiments. The optimized nanohybrid biocatalyst that showed superparamagnetic behaviour, was effective in the batch conversion of indole-3-acetic acid, a prodrug used in Direct Enzyme Prodrug Therapy (DEPT). Our system, that was not cytotoxic per se, showed enhanced cytotoxic activity in the presence of the prodrug towards HCT-116, a colorectal cancer cell line. The strategy developed proved to be effective in obtaining a stabilized nanobiocatalyst combining three different organic/inorganic materials with potential in DEPT and other biotechnological applications

Improvement of the Quality Control Plan in the reception of waste glass. Application in Verallia

The objective of the work is the improvement of the current Quality Control Plan in the reception of recycling material (cullet) in a glass factory, to check the conditions of the requirement specifications and, in particular, to reduce the presence of critical contaminants (ceramics, stone, porcelain,…) in the input materia

Triggering antitumoural drug release and gene expression by magnetic hyperthermia

Magnetic nanoparticles (MNPs) are promising tools for a wide array of biomedical applications. One of their most outstanding properties is the ability to generate heat when exposed to alternating magnetic fields, usually exploited in magnetic hyperthermia therapy of cancer. In this contribution, we provide a critical review of the use of MNPs and magnetic hyperthermia as drug release and gene expression triggers for cancer therapy. Several strategies for the release of chemotherapeutic drugs from thermo-responsive matrices are discussed, providing representative examples of their application at different levels (from proof of concept to in vivo applications). The potential of magnetic hyperthermia to promote in situ expression of therapeutic genes using vectors that contain heat-responsive promoters is also reviewed in the context of cancer gene therapy

Biocompatibility Studies of Local Antibiotic-eluting Devices for Orthopaedic Applications

Introduction: Efficient local antibiotic-eluting devices could be an alternative for delivery of locally therapeutic antibiotics into tissues, avoiding bacterial contamination on implanted materials and minimizing side effects. A proper assessment of biocompatibility of the biomaterials used is important to improve safety after implantation. We present cytotoxicological and implantation tests results to evaluate biocompatibility of two drug-eluting systems with potential use in orthopaedic implants. Materials and Methods: Cytotoxicological studies were carried out by evaluating the in-vitro dose-dependent effect of cefazolin and linezolid on fibroblasts, keratinocytes, macrophages and osteoblasts. Cells were incubated with antibiotic concentrations ranging from 0.25 to 1.5 mg/ml. Cellular viability was assessed by the Alamar blue test. Cell cycle and apoptosis were measured by flow cytometry. Short-term implantation tests were performed in an ovine model to assess the local effects of the device. Two implants were used: (1) a macroporous stainless steel reservoir loaded with linezolid and (2) stainless steel pins with orifices drilled in the reservoir wall loaded with cefazolin. Implants were placed in sheep tibias. Tissues were studied by pathological means, determining the local effect and tissue response from the implant. (Ethical committee approval number: PI36/14) Results: Cytotoxic effects of cefazolin and linezolid were only found at 1.5 mg/ml on keratinocytes and osteoblasts, respectively. There were no significant changes on cell cycle and apoptosis at 1.0 mg/ml. Sheep with both antibiotic-loaded implants did not show local or systemic adverse effects. Conclusions: These results showed no potential toxic effects for the designed devices. However, the antibiotic local concentration should not exceed 1.0 mg/ml

Whole transcriptome approach to evaluate the effect of aluminium hydroxide in ovine encephalon

Aluminium hydroxide adjuvants are crucial for livestock and human vaccines. Few studies have analysed their effect on the central nervous system in vivo. In this work, lambs received three different treatments of parallel subcutaneous inoculations during 16 months with aluminium-containing commercial vaccines, an equivalent dose of aluminium hydroxide or mock injections. Brain samples were sequenced by RNA-seq and miRNA-seq for the expression analysis of mRNAs, long non-coding RNAs and microRNAs and three expression comparisons were made. Although few differentially expressed genes were identified, some dysregulated genes by aluminium hydroxide alone were linked to neurological functions, the lncRNA TUNA among them, or were enriched in mitochondrial energy metabolism related functions. In the same way, the miRNA expression was mainly disrupted by the adjuvant alone treatment. Some differentially expressed miRNAs had been previously linked to neurological diseases, oxidative stress and apoptosis. In brief, in this study aluminium hydroxide alone altered the transcriptome of the encephalon to a higher degree than commercial vaccines that present a milder effect. The expression changes in the animals inoculated with aluminium hydroxide suggest mitochondrial disfunction. Further research is needed to elucidate to which extent these changes could have pathological consequences

Epithelial–Mesenchymal Transition in a Case of Metastatic Thyroid Carcinoma in a Brown Bear (Ursus arctos)

A 20-year-old male brown bear (Ursus arctos) with a 20 × 25 cm necrotic mass adjacent to the trachea was diagnosed as having an anaplastic thyroid carcinoma. Metastases were observed in the lungs and one adrenal gland and, histologically, these had anaplastic and follicular carcinoma patterns, respectively. E-cadherin labelling was observed in the adrenal mass only, while N-cadherin immunolabelling was detected in the thyroid gland and lung masses. Thyroid-specific markers (thyroid transcription factor-1, thyroglobulin) were expressed in the adrenal gland metastasis. This case illustrates an example of a primary epithelial–mesenchymal transition (EMT) enabling metastasis to distant organ sites, followed by a mesenchymal–epithelial transition within the adrenal gland microenvironment, allowing invasion and reacquisition of thyroid epithelial cell features. EMTs help to understand the phenomenon of carcinoma cell plasticity in enabling colonization and growth of metastases

An outbreak of abortions, stillbirths and malformations in a Spanish sheep flock associated with a bovine viral diarrhoea virus 2-contaminated orf vaccine

Bovine viral diarrhoea virus (BVDV) is a pestivirus that affects both cattle and sheep, causing an array of clinical signs, which include abortions and malformations in the offspring. Manufacturing of modified live virus (MLV) vaccines often includes the use of bovine-derived products, which implies a risk of contamination with viable BVDV. Recently, the circulation of a specific strain of BVDV 2b among Spanish sheep flocks, associated with outbreaks of abortions and malformations, and whose origin was not determined, has been observed. On February 2018, a MLV orf vaccine was applied to a 1, 600 highly prolific sheep flock in the Northeast of Spain that included 550 pregnant ewes. In May 2018, during the lambing season, an unusual high rate (72.7%) of abortions, stillbirths, congenital malformations and neurological signs in the offspring was observed. It was estimated that about 1, 000 lambs were lost. Three 1- to 3-day-old affected lambs and a sealed vial of the applied vaccine were studied. Lambs showed variable degrees of central nervous system malformations and presence of pestiviral antigen in the brain. Molecular studies demonstrated the presence of exactly the same BVDV 2b in the tissues of the three lambs and in the orf vaccine, thus pointing to a pestivirus contamination in the applied vaccine as the cause of the outbreak. Interestingly, sequencing at the 5'-untranslated region-(UTR) of the contaminating virus showed a complete match with the virus described in the previously reported outbreaks in Spain, thus indicating that the same contaminated vaccine could have also played a role in those cases. This communication provides a clear example of the effects of the application of this contaminated product in a sheep flock. The information presented here can be of interest in putative future cases of suspected circulation of this or other BVDV strains in ruminants