Revelation of different nanoparticle-uptake behavior in two standard cell lines NIH/3T3 and A549 by flow cytometry and time-lapse imaging

The uptake of nanomaterials into different cell types is a central pharmacological issue for the determination of nanotoxicity as well as for the development of drug delivery strategies. Most responses of the cells depend on their intracellular interactions with nanoparticles (NPs). Uptake behavior can be precisely investigated in vitro, with sensitive high throughput methods such as flow cytometry. In this study, we investigated two different standard cell lines, human lung carcinoma (A549) and mouse fibroblast (NIH/3T3) cells, regarding their uptake behavior of titanium dioxide NPs. Cells were incubated with different concentrations of TiO2 NPs and samples were taken at certain time points to compare the uptake kinetics of both cell lines. Samples were analyzed with the help of flow cytometry by studying changes in the side and forward scattering signal. To additionally enable a detection via fluorescence, NPs were labeled with the fluorescent dye fluorescein isothiocyanate (FITC) and propidium iodide (PI). We found that NIH/3T3 cells take up the studied NPs more efficiently than A549 cells. These findings were supported by time-lapse microscopic imaging of the cells incubated with TiO2 NPs. Our results confirm that the uptake behavior of individual cell types has to be considered before interpreting any results of nanomaterial studies.DFG/FOR/2180State of Lower Saxony, German

Cytotoxicity of titanium and silicon dioxide nanoparticles

Different TiO 2 and SiO 2 nanoparticles have been tested concerning their toxicity on selected mammalian cell lines. Various powders and suspensions, all of which consist of titanium or silicon dioxide nanoparticles have been examined. These particles differ in the crystal structure, the size and the BET-surface area. There was also a classification in fixed particles and in particles easily accessible in solution. With focus on the possible adsorption of the nanoparticles into the human organism, via skin and via respiratory tract, the effects on fibroblasts (NIH-3T3) and on a human lung adenocarcinoma epithelial cell line were examined. Additionally, the particles were tested with HEP-G2 cells, which are often used as model cell line for biocompatibility tests, and PC-12 cells, a rat adrenal pheochromocytoma cell line. The viability of the cells was examined by the MTT-test. The viability results were found to partly depend on the type of cells used. The experimental results show that the adhesion of the cells on the different powders strongly depends on the type of cell lines as well as on the type of powder. It was found that the lower viability of some cells on the powder coatings is not only caused by a cytotoxicity effect of the powders, but is also due to a lower adhesion of the cells on the particle surfaces. Furthermore, it could be shown that the physical properties of the powders cannot be easily correlated to any observed biological effect. While some powders show a significant suppression of the cell growth, others with similar physical properties indicate no toxic effect

A Microfluidic-Based Tyrosine Kinase and Phosphatase Assay

Background: Inappropriate prescribing is a well-known clinical problem in nursing home residents, but few interventions have focused on reducing inappropriate medication use. Objective: To examine successful discontinuation of inappropriate medication use and to improve prescribing in nursing home residents. Design: Pragmatic cluster randomized controlled trial, with clustering by elder care physicians and their wards. Setting: 59 Dutch nursing home wards for long-term care. Patients: Residents with a life expectancy greater than 4 weeks who consented to treatment with medication. Intervention: Multidisciplinary Multistep Medication Review (3MR) consisting of an assessment of the patient perspective, medical history, critical appraisal of medications, a meeting between the treating elder care physician and the pharmacist, and implementation of medication changes. Measurements: Successful discontinuation of use of at least 1 inappropriate drug (that is, without relapse or severe withdrawal symptoms) and clinical outcomes (neuropsychiatric symptoms, cognitive function, and quality of life) after 4 months of follow-up. Results: Nineteen elder care physicians (33 wards) performed the 3MR, and 16 elder care physicians (26 wards) followed standard procedures. A total of 426 nursing home residents (233 in the intervention group and 193 in the control group) were followed for an average of 144 days (SD, 21). In an analysis of all participants, use of at least 1 inappropriate medication was successfully discontinued for 91 (39.1%) residents in the intervention group versus 57 (29.5%) in the control group (adjusted relative risk, 1.37 [95% CI, 1.02 to 1.75]). Clinical outcomes did not deteriorate between baseline and follow-up. Limitations: The 3MR was done only once. Some withdrawal symptoms or relapses may have been missed. Conclusion: The 3MR is effective in discontinuing inappropriate medication use in frail nursing home residents without a decline in their well-being. Primary Funding Source: Netherlands Organisation for Health Research and Development

Altered MRP is associated with multidrug resistance and reduced drug accumulation in human SW-1573 cells.

We have analysed the contribution of several parameters, e.g. drug accumulation, MDR1 P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP) and topoisomerase (topo) II, to drug resistance in a large set of drug-resistant variants of the human non-small-cell lung cancer cell line SW-1573 derived by selection with low concentrations of doxorubicin or vincristine. Selection with either drug nearly always resulted in MDR clones. The resistance of these clones could be explained by reduced drug accumulation and was associated with a decrease rather than an increase in the low MDR1 mRNA level. To test whether a decrease in MDR1 mRNA indirectly affected resistance in these cells, we introduced a MDR1-specific hammerhead ribozyme into wild-type SW-1573 cells. Although this led to a substantial reduction in MDR1 mRNA, it did not result in resistance. In all resistant clones we found an altered form of the multidrug resistance-associated protein (MRP), migrating slightly slower during SDS-polyacrylamide gel electrophoresis than MRP in parental cells. This altered MRP was also present in non-P-gp MDR somatic cell hybrids of the SW-1573 cells, demonstrating a clear linkage with the MDR phenotype. Treatment of crude cellular membrane fractions with N-glycanase, endoglycosidase H or neuraminidase showed that the altered migration of MRP on SDS-PAGE is due to a post-translational modification. There was no detectable difference in sialic acid content. In most but not all doxorubicin-selected clones, this MDR phenotype was accompanied by a reduction in topo II alpha mRNA level. No reduction was found in the clones selected with vincristine. We conclude from these results that selection of the SW-1573 cell line for low levels of doxorubicin or vincristine resistance, predominantly results in MDR with reduced drug accumulation associated with the presence of an altered MRP protein. This mechanism can be accompanied by other resistance mechanisms, such as reduced topo II alpha mRNA in case of doxorubicin selection

Acquired resistance of human T cells to sulfasalazine: stability of the resistant phenotype and sensitivity to non-related DMARDs.

2.5 weeks) resumption of SSZ resistance and ABCG2 expression as in the original CEM/SSZ cells. CEM/SSZ cells displayed diminished sensitivity to the DMARDs leflunomide (5.1-fold) and methotrexate (1.8-fold), were moderately more sensitive (1.6-2.0 fold) to cyclosporin A and chloroquine, and markedly more sensitive (13-fold) to the glucocorticoid dexamethasone as compared with parental CEM cells. CONCLUSION: The drug efflux pump ABCG2 has a major role in conferring resistance to SSZ. The collateral sensitivity of SSZ resistant cells for some other (non-related) DMARDs may provide a further rationale for sequential mono- or combination therapies with distinct DMARDs upon decreased efficacy of SSZ

Amyotrophic lateral sclerosis (ALS)-associated VAPB-P56S inclusions represent an ER quality control compartment

BACKGROUND: Protein aggregation and the formation of intracellular inclusions are a central feature of many neurodegenerative disorders, but precise knowledge about their pathogenic role is lacking in most instances. Here we have characterized inclusions formed in transgenic mice carrying the P56S mutant form of VAPB that causes various motor neuron syndromes including ALS8.RESULTS: Inclusions in motor neurons of VAPB-P56S transgenic mice are characterized by the presence of smooth ER-like tubular profiles, and are immunoreactive for factors that operate in the ER associated degradation (ERAD) pathway, including p97/VCP, Derlin-1, and the ER membrane chaperone BAP31. The presence of these inclusions does not correlate with signs of axonal and neuronal degeneration, and axotomy leads to their gradual disappearance, indicating that they represent reversible structures. Inhibition of the proteasome and knockdown of the ER membrane chaperone BAP31 increased the size of mutant VAPB inclusions in primary neuron cultures, while knockdown of TEB4, an ERAD ubiquitin-protein ligase, reduced their size. Mutant VAPB did not codistribute with mutant forms of seipin that are associated with an autosomal dominant motor neuron disease, and accumulate in a protective ER derived compartment termed ERPO (ER protective organelle) in neurons.CONCLUSIONS: The data indicate that the VAPB-P56S inclusions represent a novel reversible ER quality control compartment that is formed when the amount of mutant VAPB exceeds the capacity of the ERAD pathway and that isolates misfolded and aggregated VAPB from the rest of the ER. The presence of this quality control compartment reveals an additional level of flexibility of neurons to cope with misfolded protein stress in the ER