Quantification of gold nanoparticle cell uptake under controlled biological conditions and adequate resolution

Aim: We examined cellular uptake mechanisms of fluorescently labeled polymer-coated gold nanoparticles (NPs) under different biological conditions by two quantitative, microscopic approaches. Materials & methods: Uptake mechanisms were evaluated using endocytotic inhibitors that were tested for specificity and cytotoxicity. Cellular uptake of gold NPs was analyzed either by laser scanning microscopy or transmission electron microscopy, and quantified by means of stereology using cells from the same experiment. Results: Optimal inhibitor conditions were only achieved with chlorpromazine (clathrin-mediated endocytosis) and methyl-β-cyclodextrin (caveolin-mediated endocytosis). A significant methyl-β-cyclodextrin-mediated inhibition (63-69%) and chlorpromazine-mediated increase (43-98%) of intracellular NPs was demonstrated with both imaging techniques, suggesting a predominant uptake via caveolin-medicated endocytois. Transmission electron microscopy imaging revealed more than 95% of NPs localized in intracellular vesicles and approximately 150-times more NP events/cell were detected than by laser scanning microscopy. Conclusion: We emphasize the importance of studying NP-cell interactions under controlled experimental conditions and at adequate microscopic resolution in combination with stereology. Original submitted 10 July 2012; Revised submitted 23 January 2013.

Quantification of nanoparticles at the single-cell level: an overview about state-of-the-art techniques and their limitations.

With the increasing production and use of engineered nanoparticles it is crucial that their interaction with biological systems is understood. Due to the small size of nanoparticles, their identification and localization within single cells is extremely challenging. Therefore, various cutting-edge techniques are required to detect and to quantify metals, metal oxides, magnetic, fluorescent, as well as electron-dense nanoparticles. Several techniques will be discussed in detail, such as inductively coupled plasma atomic emission spectroscopy, flow cytometry, laser scanning microscopy combined with digital image restoration, as well as quantitative analysis by means of stereology on transmission electron microscopy images. An overview will be given regarding the advantages of those visualization/quantification systems, including a thorough discussion about limitations and pitfalls.

Paracrine effects of mesenchymal stem cells enhance vascular regeneration in ischemic murine skin

New theories on the regeneration of ischemic vasculature have emerged indicating a pivotal role of adult stem cells. The aim of this study was to investigate homing and hemodynamic effects of circulating bone marrow-derived mesenchymal stem cells (MSCs) in a critically ischemic murine skin flap model. Bone marrow-derived mesenchymal stem cells (Lin(-)CD105(+)) were harvested from GFP(+)-donor mice and transferred to wildtype C57BL/6 mice. Animals receiving GFP(+)-fibroblasts served as a control group. Laser scanning confocal microscopy and intravital fluorescence microscopy were used for morphological analysis, monitoring and quantitative assessment of the stem cell homing and microhemodynamics over two weeks. Immunohistochemical staining was performed for GFP, eNOS, iNOS, VEGF. Tissue viability was analyzed by TUNEL-assay. We were able to visualize perivascular homing of MSCs in vivo. After 4 days, MSCs aligned along the vascular wall without undergoing endothelial or smooth muscle cell differentiation during the observation period. The gradual increase in arterial vascular resistance observed in the control group was abolished after MSC administration (P<0.01). At capillary level, a strong angiogenic response was found from day 7 onwards. Functional capillary density was raised in the MSC group to 197% compared to 132% in the control group (P<0.01). Paracrine expression of VEGF and iNOS, but not eNOS could be shown in the MSC group but not in the controls. In conclusion, we demonstrated that circulating bone marrow-derived MSCs home to perivascular sites in critically ischemic tissue, exhibits paracrine function and augment microhemodynamics. These effects were mediated through arteriogenesis and angiogenesis, which contributed to vascular regeneration.

Ca(2+) release from the sarcoplasmic reticulum activated by the low affinity Ca(2+) chelator TPEN in ventricular myocytes

The Ca(2+) content of the sarcoplasmic reticulum (SR) of cardiac myocytes is thought to play a role in the regulation and termination of SR Ca(2+) release through the ryanodine receptors (RyRs). Experimentally altering the amount of Ca(2+) within the SR with the membrane-permeant low affinity Ca(2+) chelator TPEN could improve our understanding of the mechanism(s) by which SR Ca(2+) content and SR Ca(2+) depletion can influence Ca(2+) release sensitivity and termination. We applied laser-scanning confocal microscopy to examine SR Ca(2+) release in freshly isolated ventricular myocytes loaded with fluo-3, while simultaneously recording membrane currents using the whole-cell patch-clamp technique. Following application of TPEN, local spontaneous Ca(2+) releases increased in frequency and developed into cell-wide Ca(2+) waves. SR Ca(2+) load after TPEN application was found to be reduced to about 60% of control. Isolated cardiac RyRs reconstituted into lipid bilayers exhibited a two-fold increase of their open probability. At the low concentration used (20-40muM), TPEN did not significantly inhibit the SR-Ca(2+)-ATPase in SR vesicles. These results indicate that TPEN, traditionally used as a low affinity Ca(2+) chelator in intracellular Ca(2+) stores, may also act directly on the RyRs inducing an increase in their open probability. This in turn results in an increased Ca(2+) leak from the SR leading to its Ca(2+) depletion. Lowering of SR Ca(2+) content may be a mechanism underlying the recently reported cardioprotective and antiarrhythmic features of TPEN.

Size-dependent accumulation of particles in lysosomes modulates dendritic cell function through impaired antigen degradation.

INTRODUCTION Nanosized particles may enable therapeutic modulation of immune responses by targeting dendritic cell (DC) networks in accessible organs such as the lung. To date, however, the effects of nanoparticles on DC function and downstream immune responses remain poorly understood. METHODS Bone marrow-derived DCs (BMDCs) were exposed in vitro to 20 or 1,000 nm polystyrene (PS) particles. Particle uptake kinetics, cell surface marker expression, soluble protein antigen uptake and degradation, as well as in vitro CD4(+) T-cell proliferation and cytokine production were analyzed by flow cytometry. In addition, co-localization of particles within the lysosomal compartment, lysosomal permeability, and endoplasmic reticulum stress were analyzed. RESULTS The frequency of PS particle-positive CD11c(+)/CD11b(+) BMDCs reached an early plateau after 20 minutes and was significantly higher for 20 nm than for 1,000 nm PS particles at all time-points analyzed. PS particles did not alter cell viability or modify expression of the surface markers CD11b, CD11c, MHC class II, CD40, and CD86. Although particle exposure did not modulate antigen uptake, 20 nm PS particles decreased the capacity of BMDCs to degrade soluble antigen, without affecting their ability to induce antigen-specific CD4(+) T-cell proliferation. Co-localization studies between PS particles and lysosomes using laser scanning confocal microscopy detected a significantly higher frequency of co-localized 20 nm particles as compared with their 1,000 nm counterparts. Neither size of PS particle caused lysosomal leakage, expression of endoplasmic reticulum stress gene markers, or changes in cytokines profiles. CONCLUSION These data indicate that although supposedly inert PS nanoparticles did not induce DC activation or alteration in CD4(+) T-cell stimulating capacity, 20 nm (but not 1,000 nm) PS particles may reduce antigen degradation through interference in the lysosomal compartment. These findings emphasize the importance of performing in-depth analysis of DC function when developing novel approaches for immune modulation with nanoparticles.

Imaging liver-stage malaria parasites

Plasmodium parasites, the causative agents of malaria, first invade and develop within hepatocytes before infecting red blood cells and causing symptomatic disease. Because of the low infection rates in vitro and in vivo, the liver stage of Plasmodium infection is not very amenable to biochemical assays, but the large size of the parasite at this stage in comparison with Plasmodium blood stages makes it accessible to microscopic analysis. A variety of imaging techniques has been used to this aim, ranging from electron microscopy to widefield epifluorescence and laser scanning confocal microscopy. High-speed live video microscopy of fluorescent parasites in particular has radically changed our view on key events in Plasmodium liver-stage development. This includes the fate of motile sporozoites inoculated by Anopheles mosquitoes as well as the transport of merozoites within merosomes from the liver tissue into the blood vessel. It is safe to predict that in the near future the application of the latest microscopy techniques in Plasmodium research will bring important insights and allow us spectacular views of parasites during their development in the liver.

Fluorescent-magnetic hybrid nanoparticles induce a dose-dependent increase in proinflammatory response in lung cells in vitro correlated with intracellular localization

Iron-platinum nanoparticles embedded in a poly(methacrylic acid) (PMA) polymer shell and fluorescently labeled with the dye ATTO 590 (FePt-PMA-ATTO-2%) are investigated in terms of their intracellular localization in lung cells and potential to induce a proinflammatory response dependent on concentration and incubation time. A gold core coated with the same polymer shell (Au-PMA-ATTO-2%) is also included. Using laser scanning and electron microscopy techniques, it is shown that the FePt-PMA-ATTO-2% particles penetrate all three types of cell investigated but to a higher extent in macrophages and dendritic cells than epithelial cells. In both cell types of the defense system but not in epithelial cells, a particle-dose-dependent increase of the cytokine tumor necrosis factor alpha (TNFalpha) is found. By comparing the different nanoparticles and the mere polymer shell, it is shown that the cores combined with the shells are responsible for the induction of proinflammatory effects and not the shells alone. It is concluded that the uptake behavior and the proinflammatory response upon particle exposure are dependent on the time, cell type, and cell culture.

Assessment of melanocytic skin lesions with a high-definition laser Doppler imaging system

Early detection is a major goal in the management of malignant melanoma. Besides clinical assessment many noninvasive technologies such as dermoscopy, digital dermoscopy and in vivo laser scanner microscopy are used as additional methods. Herein we tested a system to assess lesional perfusion as a tool for early melanoma detection.

Canine distemper virus persistence in demyelinating encephalitis by swift intracellular cell-to-cell spread in astrocytes is controlled by the viral attachment protein

The mechanism of viral persistence, the driving force behind the chronic progression of inflammatory demyelination in canine distemper virus (CDV) infection, is associated with non-cytolytic viral cell-to-cell spread. Here, we studied the molecular mechanisms of viral spread of a recombinant fluorescent protein-expressing virulent CDV in primary canine astrocyte cultures. Time-lapse video microscopy documented that CDV spread was very efficient using cell processes contacting remote target cells. Strikingly, CDV transmission to remote cells could occur in less than 6 h, suggesting that a complete viral cycle with production of extracellular free particles was not essential in enabling CDV to spread in glial cells. Titration experiments and electron microscopy confirmed a very low CDV particle production despite higher titers of membrane-associated viruses. Interestingly, confocal laser microscopy and lentivirus transduction indicated expression and functionality of the viral fusion machinery, consisting of the viral fusion (F) and attachment (H) glycoproteins, at the cell surface. Importantly, using a single-cycle infectious recombinant H-knockout, H-complemented virus, we demonstrated that H, and thus potentially the viral fusion complex, was necessary to enable CDV spread. Furthermore, since we could not detect CD150/SLAM expression in brain cells, the presence of a yet non-identified glial receptor for CDV was suggested. Altogether, our findings indicate that persistence in CDV infection results from intracellular cell-to-cell transmission requiring the CDV-H protein. Viral transfer, happening selectively at the tip of astrocytic processes, may help the virus to cover long distances in the astroglial network, "outrunning" the host's immune response in demyelinating plaques, thus continuously eliciting new lesions.

Synthesis and two-photon photolysis of 6-(ortho-nitroveratryl)-caged IP3 in living cells

The synthesis of a photolabile derivative of inositol-1,4,5-trisphosphate (IP3) is described. This new caged second messenger (6-ortho-nitroveratryl)-IP3 (6-NV-IP3) has an extinction coefficient of 5000 M(-1) cm(-1) at 350 nm, and a quantum yield of photolysis of 0.12. Therefore, 6-NV-IP3 is photolyzed with UV light about three times more efficiently than the widely used P(4(5))-1-(2-nitrophenyl)ethyl-caged IP3 (NPE-IP3). 6-NV-IP3 has a two-photon cross-section of about 0.035 GM at 730 nm. This absorbance is sufficiently large for effective two-photon excitation in living cells at modest power levels. Using near-IR light (5 mW, 710 nm, 80 MHz, pulse-width 70 fs), we produced focal bursts of IP3 in HeLa cells, as revealed by laser-scanning confocal imaging of intracellular Ca2+ concentrations. Therefore, 6-NV-IP3 can be used for efficient, subcellular photorelease of IP3, not only in cultured cells but also, potentially, in vivo. It is in the latter situation that two-photon photolysis should reveal its true forte.

Mechanical chondroplasty: early metabolic consequences in vitro

PURPOSE: The purpose of this study was to determine the depth of penetration from mechanical chondroplasty and metabolic consequences of this procedure on the remaining articular cartilage. METHODS: Mechanical chondroplasty was performed in vitro on a portion of fresh grade I or II articular cartilage from 8 human knee arthroplasty specimens. Treated and control (untreated) explants (approximately 30 mg) were cut from the cartilage. The explants were divided into 2 groups, day 1 and day 4, placed separately in a 48-well plate containing media, and incubated at 37 degrees C for 24 hours. After the 24-hour incubation, the explants were weighed on day 1 and day 4, and explant media were removed and tested for total proteoglycan synthesis and aggrecan synthesis. At time 0, 2 sets (2.6 mm each) of treated and control cartilage slices were cut with a precision saw. One set was stained for confocal laser microscopy via a cytotoxicity stain to determine cell viability. The second set was stained with H;E to determine depth of penetration. RESULTS: The mean depth of penetration was 252.8 +/- 78 microm. There was no significant difference (P > .25) between total proteoglycan synthesis for control versus treatment groups on day 1 or 4. Aggrecan synthesis was significantly reduced on day 1 when normalized for tissue weight (P = .019) and double-stranded deoxyribonucleic acid (P = .004). On day 4, no significant difference was detected. Confocal laser microscopy did not show cell death below the zone of treatment. CONCLUSIONS: There was no significant metabolic consequence caused by chondroplasty to the remaining articular cartilage, and the zone of injury was limited to the treatment area. CLINICAL RELEVANCE: Mechanical chondroplasty causes no significant metabolic consequences to articular cartilage under these conditions.

Liver repopulation with bone marrow derived cells improves the metabolic disorder in the Gunn rat

BACKGROUND: Reversible ischaemia/reperfusion (I/R) liver injury has been used to induce engraftment and hepatic parenchymal differentiation of exogenous beta2-microglubulin(-)/Thy1(+) bone marrow derived cells. AIM: To test the ability of this method of hepatic parenchymal repopulation, theoretically applicable to clinical practice, to correct the metabolic disorder in a rat model of congenital hyperbilirubinaemia. METHODS AND RESULTS: Analysis by confocal laser microscopy of fluorescence labelled cells and by immunohistochemistry for beta2-microglubulin, 72 hours after intraportal delivery, showed engraftment of infused cells in liver parenchyma of rats with I/R, but not in control animals with non-injured liver. Transplantation of bone marrow derived cells obtained from GFP-transgenic rats into Lewis rats resulted in the presence of up to 20% of GFP positive hepatocytes in I/R liver lobes after one month. The repopulation rate was proportional to the number of transplanted cells. Infusion of GFP negative bone marrow derived cells into GFP positive transgenic rats resulted in the appearance of GFP negative hepatocytes, suggesting that the main mechanism underlying parenchymal repopulation was differentiation rather than cell fusion. Transplantation of wild type bone marrow derived cells into hyperbilirubinaemic Gunn rats with deficient bilirubin conjugation after I/R damage resulted in 30% decrease in serum bilirubin, the appearance of bilirubin conjugates in bile, and the expression of normal UDP-glucuronyltransferase enzyme evaluated by polymerase chain reaction. CONCLUSIONS: I/R injury induced hepatic parenchymal engraftment and differentiation into hepatocyte-like cells of bone marrow derived cells. Transplantation of bone marrow derived cells from non-affected animals resulted in the partial correction of hyperbilirubinaemia in the Gunn rat.

Microcirculation in hypertensive patients

Regardless of the mechanisms that initiate the increase in blood pressure, functional and structural changes in the systemic vasculature are the final result of long-standing hypertension. These changes can occur in the macro- but also in the microvasculature. The supply of the tissues with oxygen, nutrients, and metabolites occurs almost exclusively in the microcirculation (which comprises resistance arterioles, capillaries and venules), and an adequate perfusion via the microcirculatory network is essential for the integrity of tissue and organ function. This review focuses on results from clinical studies in hypertensive patients, which have been performed in close cooperation with different clinical groups over the last three decades. Intravital microscopy was used to study skin microcirculation, microcatheters for the analysis of skeletal muscle microcirculation, the slit lamp for conjunctival microcirculation and the laser scanning ophthalmoscope for the measurement of the retinal capillary network. The first changes of the normal microcirculation can be found in about 93% of patients with essential hypertension, long before organ dysfunctions become clinically manifest. The earliest disorders were found in skin capillaries and thereafter in the retina and the skeletal muscle. In general, the disorders in the different areas were clearly correlated. While capillary rarefaction occurred mainly in the retina and the conjunctiva bulbi, in skin capillaries morphological changes were rare. A significant decrease of capillary erythrocyte velocities under resting conditions together with a marked damping of the postischemic hyperemia was found, both correlating with the duration of hypertension or WHO stage or the fundus hypertonicus stage. Also the mean oxygen tension in the skeletal muscle was correlated with the state of the disease. These data show that the microcirculatory disorders in hypertension are systemic and are hallmarks of the long-term complications of hypertension. There is now a large body of evidence that microvascular changes occur very early and may be important in their pathogenesis and progression.

Mechanical Loading Promoted Discogenic Differentiation of Human Mesenchymal Stem Cells Incorporated in 3D-PEG Scaffolds with RhGDF5 and RGD

Hydrogels have been described as ideal scaffolds for cells of 3D tissue constructs and hold strong promises with respect to in vitro 3D-cell-culture, where cells are isolated from native extracellular matrix (ECM). Synthesized polyethyleneglycol (PEG) hydrogels are appealing with regard to potential for cell therapy or as vehicles for drug delivery or even to regenerate tissue with similar hydrogel-like properties such as the nucleus pulposus of the intervertebral disc (IVD). Here, we tested whether incorporation of RGD motive would hinder discogenic differentiation of primary bone marrow-derived human mesenchymal stem cells (hMSCs) but favor proliferation of undifferentiated hMSCs. HMSCs were embedded in +RGD containing or without RGD PEG hydrogel and pre-conditioned with or without growth and differentiation factor-5 (rhGDF-5) for 13 days. Afterwards, all hMSCs-PEG gels were subsequently cyclically loaded (15% strain, 1Hz) for 5 consecutive days in a bioreactor to generate an IVD-like phenotype. Higher metabolic activity (resazurin assay) was found in groups with rhGDF5 in both gel types with and without RGD. Cell viability and morphology measured by confocal laser microscopy and DNA content showed decreased values (~60%) after 18 days of culture. Real-time RT-PCR of an array of 15 key genes suspected to be distinctive for IVD cells revealed moderate response to rhGDF5 and mechanical loading as also shown by histology staining. Preconditioning and mechanical loading showed relatively moderate responses revealed from both RT-PCR and histology although hMSCs were demonstrated to be potent to differentiate into chondrocyte-progenitor cells in micro- mass and 3D alginate bead culture.

3D bloodstain pattern analysis: ballistic reconstruction of the trajectories of blood drops and determination of the centres of origin of the bloodstains

For crime scene investigation in cases of homicide, the pattern of bloodstains at the incident site is of critical importance. The morphology of the bloodstain pattern serves to determine the approximate blood source locations, the minimum number of blows and the positioning of the victim. In the present work, the benefits of the three-dimensional bloodstain pattern analysis, including the ballistic approximation of the trajectories of the blood drops, will be demonstrated using two illustrative cases. The crime scenes were documented in 3D, using the non-contact methods digital photogrammetry, tachymetry and laser scanning. Accurate, true-to-scale 3D models of the crime scenes, including the bloodstain pattern and the traces, were created. For the determination of the areas of origin of the bloodstain pattern, the trajectories of up to 200 well-defined bloodstains were analysed in CAD and photogrammetry software. The ballistic determination of the trajectories was performed using ballistics software. The advantages of this method are the short preparation time on site, the non-contact measurement of the bloodstains and the high accuracy of the bloodstain analysis. It should be expected that this method delivers accurate results regarding the number and position of the areas of origin of bloodstains, in particular the vertical component is determined more precisely than using conventional methods. In both cases relevant forensic conclusions regarding the course of events were enabled by the ballistic bloodstain pattern analysis.