Combination Of Flow Cytometry Research Articles

Eicosanoid Profiling in an Orthotopic Model of Lung Cancer Progression by Mass Spectrometry Demonstrates Selective Production of Leukotrienes by Inflammatory Cells of the Microenvironment

Eicosanoids are bioactive lipid mediators derived from arachidonic acid1 (AA), which is released by cytosolic phospholipase A2 (cPLA2). AA is metabolized through three major pathways, cyclooxygenase (COX), lipoxygenase (LO) and cytochrome P450, to produce a family of eicosanoids, which individually have been shown to have pro- or anti-tumorigenic activities in cancer. However, cancer progression likely depends on complex changes in multiple eicosanoids produced by cancer cells and by tumor microenvironment and a systematic examination of the spectrum of eicosanoids in cancer has not been performed. We used liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) to quantitate eicosanoids produced during lung tumor progression in an orthotopic immunocompetent mouse model of lung cancer, in which Lewis lung carcinoma (LLC) cells are injected into lungs of syngeneic mice. The presence of tumor increased products of both the cyclooxygenase and the lipoxygenase pathways in a time-dependent fashion. Comparing tumors grown in cPLA2 knockout vs wild-type mice, we demonstrated that prostaglandins (PGE2, PGD2 and PGF2a) were produced by both cancer cells and the tumor microenvironment (TME), but leukotriene (LTB4, LTC4, LTD4, LTE4) production required cPLA2 expression in the TME. Using flow cytometry, we recovered tumor-associated neutrophils and 2 types of tumor-associated macrophages from tumor-bearing lungs and we defined their distinct eicosanoid profiles by LC/MS/MS. The combination of flow cytometry and LC/MS/MS unravels the complexity of eicosanoid production in lung cancer and provides a rationale to develop therapeutic strategies that target select cell populations to inhibit specific classes of eicosanoids.

Aneuploidy underlies a multicellular phenotypic switch

Although microorganisms are traditionally used to investigate unicellular processes, the yeast Saccharomyces cerevisiae has the ability to form colonies with highly complex, multicellular structures. Colonies with the "fluffy" morphology have properties reminiscent of bacterial biofilms and are easily distinguished from the "smooth" colonies typically formed by laboratory strains. We have identified strains that are able to reversibly toggle between the fluffy and smooth colony-forming states. Using a combination of flow cytometry and high-throughput restriction-site associated DNA tag sequencing, we show that this switch is correlated with a change in chromosomal copy number. Furthermore, the gain of a single chromosome is sufficient to switch a strain from the fluffy to the smooth state, and its subsequent loss to revert the strain back to the fluffy state. Because copy number imbalance of six of the 16 S. cerevisiae chromosomes and even a single gene can modulate the switch, our results support the hypothesis that the state switch is produced by dosage-sensitive genes, rather than a general response to altered DNA content. These findings add a complex, multicellular phenotype to the list of molecular and cellular traits known to be altered by aneuploidy and suggest that chromosome missegregation can provide a quick, heritable, and reversible mechanism by which organisms can toggle between phenotypes.

AB0036 Il-20 targets local tissue inflammation as opposed to systemic inflammation

BackgroundInterleukin-20 (IL-20) is a proinflammatory cytokine that has been implicated in the pathogenesis of autoimmune disease. In a Phase 2a clinical trial in rheumatoid arthritis, a monoclonal antibody neutralizing the...

A novel Bruton’s tyrosine kinase gene (BTK) invariant splice site mutation in a Malaysian family with X-linked agammaglobulinemia

X-linked agammaglobulinemia (XLA) is a rare genetic disorder caused by mutations in the Bruton's tyrosine kinase (BTK) gene. These mutations cause defects in early B cell development. A patient with no circulating B cells and low serum immunoglobulin isotypes was studied as were his mother and sister. Monocyte BTK protein expression was evaluated by flow cytometry. The mutation was determined using PCR and followed by sequencing. Flow cytometry showed the patient lacked BTK protein expression in his monocytes while the mother and sister had 62% and 40% of the monocytes showing BTK protein expressions respectively. The patient had a novel base substitution in the first nucleotide of intron 9 in the BTK gene, and the mutation was IVS9+1G<C. This mutation resulted in exon 9 skipping. This defect rendered the patient susceptible to asthma, failure to thrive, recurrent pyogenic infections, otitis media and bronchopneumonia. His mother and sister were heterozygous for this mutation. The combination of flow cytometry and genetic study is necessary in the diagnosis of X-linked agammaglobulinemia and may be used for subsequent genetic counseling, carrier detection and prenatal diagnosis.

E. coli a‐haemolysin leads to ATP release and P2‐dependent calcium influx in human erythrocytes

The E. coli toxin á‐haemolysin (HlyA) lyses erythrocytes by inserting as pores in the membrane. Recently, we demonstrated that HlyA‐induced haemolysis is a sequential process with an initial volume reduction that precedes swelling and lysis. The initial volume reduction is triggered by an abrupt increase in [Ca2+]i.Moreover, we have shown that HlyA‐induced haemolysis requires extracellular ATP and P2 receptor activation. The present study investigates the mechanism for ATP release and [Ca2+]i increase that precede the volume changes in erythrocytes. By the luciferin/luciferase assay, we show that HlyA leads to ATP release within 5 minutes after toxin is added. ATP release was independent of pannexin‐ and volume‐dependent anion channels; two suggested ATP exit pathways. Thus, ATP may simply be released via the toxin pore. Through a combination of flow cytometry and live cell imaging, we show that the [Ca2+]i response consists of an abrupt increase followed by at slower, sustained increment. P2 antagonists do not interfere with the initial acute [Ca2+]i increase, but significantly reduces the slow phase of the HlyA‐induced influx of [Ca2+]i and cause a marked delay in the swelling of the cells. We suggest that ATP may be released via the toxin pore, which activates P2 receptors in the erythrocyte membrane. The influx of cations through P2 receptors contributes to net ion influx and thereby cell swelling and lysis.

Activation of WIP1 Phosphatase by HTLV-1 Tax Mitigates the Cellular Response to DNA Damage

Genomic instability stemming from dysregulation of cell cycle checkpoints and DNA damage response (DDR) is a common feature of many cancers. The cancer adult T cell leukemia (ATL) can occur in individuals infected with human T cell leukemia virus type 1 (HTLV-1), and ATL cells contain extensive chromosomal abnormalities, suggesting that they have defects in the recognition or repair of DNA damage. Since Tax is the transforming protein encoded by HTLV-1, we asked whether Tax can affect cell cycle checkpoints and the DDR. Using a combination of flow cytometry and DNA repair assays we showed that Tax-expressing cells exit G1 phase and initiate DNA replication prematurely following damage. Reduced phosphorylation of H2AX (γH2AX) and RPA2, phosphoproteins that are essential to properly initiate the DDR, was also observed in Tax-expressing cells. To determine the cause of decreased DDR protein phosphorylation in Tax-expressing cells, we examined the cellular phosphatase, WIP1, which is known to dephosphorylate γH2AX. We found that Tax can interact with Wip1 in vivo and in vitro, and that Tax-expressing cells display elevated levels of Wip1 mRNA. In vitro phosphatase assays showed that Tax can enhance Wip1 activity on a γH2AX peptide target by 2-fold. Thus, loss of γH2AX in vivo could be due, in part, to increased expression and activity of WIP1 in the presence of Tax. siRNA knockdown of WIP1 in Tax-expressing cells rescued γH2AX in response to damage, confirming the role of WIP1 in the DDR. These studies demonstrate that Tax can disengage the G1/S checkpoint by enhancing WIP1 activity, resulting in reduced DDR. Premature G1 exit of Tax-expressing cells in the presence of DNA lesions creates an environment that tolerates incorporation of random mutations into the host genome.

Membrane potential mediates the cellular binding of nanoparticles.

The use of nanoparticles for cellular therapeutic or sensing applications requires nanoparticles to bind, or adhere, to the cell surface. While nanoparticle parameters such as size, shape, charge, and composition are important factors in cellular binding, the cell itself must also be considered. All cells have an electrical potential across the plasma membrane driven by an ion gradient. Under standard conditions the ion gradient will result in a -10 to -100 mV potential across the membrane with a net negative charge on the cytosolic face. Using a combination of flow cytometry and fluorescence microscopy experiments and dissipative particle dynamics simulations, we have found that a decrease in membrane potential leads to decreased cellular binding of anionic nanoparticles. The decreased cellular binding of anionic nanoparticles is a general phenomenon, independent of depolarization method, nanoparticle composition, and cell type. Increased membrane potential reverses this trend resulting in increased binding of anionic nanoparticles. The cellular binding of cationic nanoparticles is minimally affected by membrane potential due to the interaction of cationic nanoparticles with cell surface proteins. The influence of membrane potential on the cellular binding of nanoparticles is especially important when considering the use of nanoparticles in the treatment or detection of diseases, such as cancer, in which the membrane potential is decreased.

Development of a flow-fluorescence in situ hybridization protocol for the analysis of microbial communities in anaerobic fermentation liquor

BackgroundThe production of bio-methane from renewable raw material is of high interest because of the increasing scarcity of fossil fuels. The process of biomethanation is based on the inter- and intraspecific metabolic activity of a highly diverse and dynamic microbial community. The community structure of the microbial biocenosis varies between different biogas reactors and the knowledge about these microbial communities is still fragmentary. However, up to now no approaches are available allowing a fast and reliable access to the microbial community structure. Hence, the aim of this study was to originate a Flow-FISH protocol, namely a combination of flow cytometry and fluorescence in situ hybridization, for the analysis of the metabolically active microorganisms in biogas reactor samples. With respect to the heterogenic texture of biogas reactor samples and to collect all cells including those of cell aggregates and biofilms the development of a preceding purification procedure was indispensable.ResultsSix different purification procedures with in total 29 modifications were tested. The optimized purification procedure combines the use of the detergent sodium hexametaphosphate with ultrasonic treatment and a final filtration step. By this treatment, the detachment of microbial cells from particles as well as the disbandment of cell aggregates was obtained at minimized cell loss. A Flow-FISH protocol was developed avoiding dehydration and minimizing centrifugation steps. In the exemplary application of this protocol on pure cultures as well as biogas reactor samples high hybridization rates were achieved for commonly established domain specific oligonucleotide probes enabling the specific detection of metabolically active bacteria and archaea. Cross hybridization and autofluorescence effects could be excluded by the use of a nonsense probe and negative controls, respectively.ConclusionsThe approach described in this study enables for the first time the analysis of the metabolically active fraction of the microbial communities within biogas reactors by Flow-FISH.

Counterpoint: Cancer Stem Cells: Nonbelievers' Viewpoint

Several studies have identified a subset of cancer cells, designated as tumor-initiating cells or cancer stem cells (CSCs),3 with the ability for self-renewal and differentiation into distinct cell lineages. In recent years, the hypothesis has emerged, and gained great momentum, that tumors are hierarchically arranged, with CSCs being the principal drivers of tumor growth for proliferation, resistance to chemotherapy, and metastasis (1–3). A combination of flow cytometry and xenotransplantation techniques led to the identification of leukemia-initiating cells (CD34+CD38−) and breast cancer–initiating cells (CD44+CD24−/low) and provided the scientific basis for the CSC hypothesis (3–5). Needless to say, if CSCs exist, their attributes need to be studied, and specific therapies directed against these cells will have to be developed. Although there are some excellent studies suggesting the presence of CSCs, several controversial issues remain. The development of drug resistance and disease recurrence and metastasis could simply be consequences of tumor heterogeneity and plasticity. The latter characteristics also could result in cells having distinct characteristics, including sensitivity to therapy. What factors (i.e., clonal evolution, CSCs, or tumor-microenvironment niche) contribute to adverse outcomes remains an important question. The answer to this question is undoubtedly going to be complex and may vary in different tumors. One of the concerns with the CSC hypothesis is that the methods used to identify these cells are limited. The presence of CSCs is usually inferred on the basis of one of the following characteristics: ( a ) flow cytometry results indicating the presence of “specific” cell surface markers; ( b ) clonogenic assay findings such as mammosphere formation; and ( c ) ability of cells to give rise to metastases in xenotransplantation models (6). Several cell surface markers have been associated with CSC activity. These tend to differ across different studies, with minor …

Temperature-dependent activation of differential apoptotic pathways during cryoablation in a human prostate cancer model

Critical to the continual improvement of cryoablation efficacy is deciphering the biochemical responses of cells to low-temperature exposure. The identification of delayed-onset cell death has allowed for the manipulation of cellular responses through the regulation of apoptosis. We hypothesized that in addition to delayed apoptotic events associated with mild subfreezing temperatures (10 to -25 °C), cells exposed to ultra-low temperatures (<-30 °C) may undergo rapid, early-onset apoptosis. Human prostate cancer model and cells (PC-3) were exposed to temperatures of -60, -30 and -15 °C to simulate a cryoablative procedure. Using a combination of flow-cytometry, fluorescent microscopy and western blot analyses, samples were assessed at various times post thaw to identify the presence, levels and the pathways involved in cell death. Exposure to temperatures <-30 °C yielded a significant apoptotic population within 30 min of thawing, peaking at 90 min (~40%), and by 6 h, only necrosis was observed. In samples only reaching temperatures >-30 °C, apoptosis was not noted until 6-24 h post thaw, with the levels of apoptosis reaching ~10% (-15 °C) and ~25% (-30 °C) at 6 h post thaw. Further, it was found that early-onset apoptosis progressed through a membrane-mediated mechanism, whereas delayed apoptosis progressed through a mitochondrial path. These data demonstrate the impact of apoptotic continuum, whereby the more severe cryogenic stress activated the extrinsic, membrane-regulated pathway, whereas less severe freezing activated the intrinsic, mitochondrial-mediated path. The rapid induction and progression of apoptosis at ultra-low temperatures provides an explanation as to why such results have not previously been identified following freezing. Ultimately, an understanding of the events and signaling pathways involved in triggering apoptosis following freezing may provide a path for selective induction of the rapid-onset and delayed programmed cell death pathways in an effort to improve the overall cryoablation efficacy.

How many dendritic cells are required to initiate a T-cell response?

AbstractT-cell activation in lymph nodes relies on encounters with antigen (Ag)–bearing dendritic cells (DCs) but the number of DCs required to initiate an immune response is unknown. Here we have used a combination of flow cytometry, 2-photon imaging, and computational modeling to quantify the probability of T cell–DC encounters. We calculated that the chance for a T cell residing 24 hours in a murine popliteal lymph nodes to interact with a DC was 87, 587, and 997 in the presence of 10, 100, and 1000 Ag-bearing DCs, respectively. Our results reveal the existence of a threshold in DC numbers below which T-cell responses fail to be elicited for probabilistic reasons. In mice and probably humans, we estimate that a minimum of 85 DCs are required to initiate a T-cell response when starting from precursor frequency of 10−6. Our results have implications for the rational design of DC-based vaccines.

Dissecting T Cell Contraction In Vivo Using a Genetically Encoded Reporter of Apoptosis

Contraction is a critical phase of immunity whereby the vast majority of effector T cells die by apoptosis, sparing a population of long-lived memory cells. Where, when, and why contraction occurs has been difficult to address directly due in large part to the rapid clearance of apoptotic T cells in vivo. To circumvent this issue, we introduced a genetically encoded reporter for caspase-3 activity into naive T cells to identify cells entering the contraction phase. Using two-photon imaging, we found that caspase-3 activity in T cells was maximal at the peak of the response and was associated with loss of motility followed minutes later by cell death. We demonstrated that contraction is a widespread process occurring uniformly in all organs tested and targeting phenotypically diverse T cells. Importantly, we identified a critical window of time during which antigen encounters act to antagonize T cell apoptosis, supporting a causal link between antigen clearance and T cell contraction. Our results offer insight into a poorly explored phase of immunity and provide a versatile methodology to study apoptosis during the development or function of a variety of immune cells in vivo.

Clathrin-mediated Endocytosis and Subsequent Endo-Lysosomal Trafficking of Adeno-associated Virus/Phage

Adeno-associated virus/phage (AAVP) is a gene delivery vector constructed as a hybrid between adeno-associated virus and filamentous phage. Tumor targeting following systemic administration has previously been demonstrated in several in vivo cancer models, with tumor specificity achieved through display of an α(v) integrin-targeting ligand on the capsid. However, high titers of AAVP are required for transduction of large numbers of mammalian cells. This study is the first to investigate the mechanisms involved in entry and intracellular trafficking of AAVP. Using a combination of flow cytometry, confocal, and electron microscopy techniques, together with pharmacological agents, RNAi and dominant negative mutants, we have demonstrated that targeted AAVP endocytosis is both dynamin and clathrin-dependent. Following entry, the majority of AAVP particles are sequestered by the endosomal-lysosomal degradative pathway. Finally, we have demonstrated that disruption of this pathway leads to improved transgene expression by AAVP, thus demonstrating that escape from the late endosomes/lysosomes is a critical step for improving gene delivery by AAVP. These findings have important implications for the rational design of improved AAVP and RGD-targeted vectors.

Immune Evasion and Recognition of the Syphilis Spirochete in Blood and Skin of Secondary Syphilis Patients: Two Immunologically Distinct Compartments

BackgroundThe clinical syndrome associated with secondary syphilis (SS) reflects the propensity of Treponema pallidum (Tp) to escape immune recognition while simultaneously inducing inflammation.MethodsTo better understand the duality of immune evasion and immune recognition in human syphilis, herein we used a combination of flow cytometry, immunohistochemistry (IHC), and transcriptional profiling to study the immune response in the blood and skin of 27 HIV(-) SS patients in relation to spirochetal burdens. Ex vivo opsonophagocytosis assays using human syphilitic sera (HSS) were performed to model spirochete-monocyte/macrophage interactions in vivo.ResultsDespite the presence of low-level spirochetemia, as well as immunophenotypic changes suggestive of monocyte activation, we did not detect systemic cytokine production. SS subjects had substantial decreases in circulating DCs and in IFNγ-producing and cytotoxic NK-cells, along with an emergent CD56−/CD16+ NK-cell subset in blood. Skin lesions, which had visible Tp by IHC and substantial amounts of Tp-DNA, had large numbers of macrophages (CD68+), a relative increase in CD8+ T-cells over CD4+ T-cells and were enriched for CD56+ NK-cells. Skin lesions contained transcripts for cytokines (IFN-γ, TNF-α), chemokines (CCL2, CXCL10), macrophage and DC activation markers (CD40, CD86), Fc-mediated phagocytosis receptors (FcγRI, FcγR3), IFN-β and effector molecules associated with CD8 and NK-cell cytotoxic responses. While HSS promoted uptake of Tp in conjunction with monocyte activation, most spirochetes were not internalized.ConclusionsOur findings support the importance of macrophage driven opsonophagocytosis and cell mediated immunity in treponemal clearance, while suggesting that the balance between phagocytic uptake and evasion is influenced by the relative burdens of bacteria in blood and skin and the presence of Tp subpopulations with differential capacities for binding opsonic antibodies. They also bring to light the extent of the systemic innate and adaptive immunologic abnormalities that define the secondary stage of the disease, which in the skin of patients trends towards a T-cell cytolytic response.

Polyploidy levels of Chinese large-flower chrysanthemum determined by flow cytometry

Flow cytometry was used to determine the ploidy level of 405 Chinese large-flower chrysanthemum ( Chrysanthemum morifolium Ramat.) cultivars. Sixty-three cultivars are triploid, 175 cultivars tetraploid, 32 cultivars pentaploid, 46 cultivars hexaploid and 1 cultivar heptaploid. Forty-eight cultivars were then randomly selected for confirmation by chromosome-counting; the results are in agreement with the classification of ploidy level by flow cytometry. Most cultivars are aneuploid. The high percentage of tetraploid and triploid, instead of hexaploid in previous studies, represents the first evidence of low ploidy in large-flower chrysanthemum, which indicated a wider range of ploidy variation in this population. The results also offer further insights to the possible evolution and the regulation of flower size of this large-flower population. Additionally, the combination of flow cytometry and chromosome-counting is proved to be efficient and necessary for large-scale ploidy screening of chrysanthemum. Keywords: Chrysanthemum, ploidy level, flow cytometry

A Combination of Flow Cytometry and Traditional Screening Using Chemicals to Isolate High Glutathione-Producing Yeast Mutants

Traditional screening using chemicals or flow cytometry (FCM) alone is not sufficient to isolate the high glutathione (GSH)-producing yeast strains used in food production. Therefore, to improve screening efficiency, we investigated a combination of both methods. A mutated Saccharomyces cerevisiae strain was labeled with 5-chloromethylfluorescein diacetate and sorted by FCM according to emitted fluorescence intensity. Moderate GSH (1%-2%)-producing mutants were isolated, whereas high GSH (>2%)-producing mutants were not. Traditional screening using cerulenin resulted in similar findings, but a combination of both methods resulted in a 40% increase in the screening yield of high GSH-producing mutants. An analysis of model strains indicated that the ratio of high GSH-producing cells in a sample affected the FCM results. By combining FCM with traditional screening using chemicals, we succeeded in isolating high GSH-producing mutants from several parental strains.

Non-degradative intracellular trafficking of highly compacted polymeric DNA nanoparticles

Highly compacted DNA nanoparticles (DNPs) composed of polyethylene glycol linked to a 30-mer of poly-l-lysine via a single cysteine residue (CK30PEG) have previously been shown to provide efficient gene delivery to the brain, eyes and lungs. In this study, we used a combination of flow cytometry, high-resolution live-cell confocal microscopy, and multiple particle tracking (MPT) to investigate the intracellular trafficking of highly compacted CK30PEG DNPs made using two different molecular weights of PEG, CK30PEG10k and CK30PEG5k. We found that PEG MW did not have a major effect on particle morphology nor nanoparticle intracellular transport. CK30PEG10k and CK30PEG5k DNPs both entered human bronchial epithelial (BEAS-2B) cells via a caveolae-mediated pathway, bypassing degradative endolysosomal trafficking. Both nanoparticle formulations were found to rapidly accumulate in the perinuclear region of cells within 2h, 37±19% and 47±8% for CK30PEG10k and CK30PEG5k, respectively. CK30PEG10k and CK30PEG5k DNPs moved within live cells at average velocities of 0.09±0.04μm/s and 0.11±0.04μm/s, respectively, in good agreement with reported values for caveolae. These findings show that highly compacted DNPs employ highly regulated trafficking mechanisms similar to biological pathogens to target specific intracellular compartments.

A rapid multiplex DNA suspension array method for Salmonella typhimurium subtyping using prophage-related markers

In this study we developed a preliminary proof of concept of method for Salmonella typhimurium subtyping using multiplex PCR-based phage locus typing and a multiplex Luminex DNA suspension array for product detection. Thirty markers were selected from prophages ST64B, ST64T, ST104, P22, Gifsy-1, sopEΦ and mostly phage-related AFLP fragments, and organised into two multiplex PCRs of 15 markers each. A two-group DNA suspension array was developed using a combination of flow cytometry and Luminex xMAP® technology. To assess its subtyping capability the method was applied to 438 non-epidemiological related S. typhimurium isolates of 56 phage types. Eighty-one profiles were generated. Isolates were divided into sixteen main prophage marker profile types. There was a strong tendency for isolates with the same phage type to have the same or closely related profiles and for groups of phage types to share the same profile. The discriminatory power of this method expressed as the Simpson's Index of Diversity (D) was 0.954. A panel of 12 selected markers achieved almost the same D value (0.952) as the 30 markers. This new method provides an alternative typing scheme for S. typhimurium epidemiological investigations. The developed array is in a high-throughput format which could easily be semi-automated, making the test fast and economical.

The effect of ageing on phenotype and function of monocyte-derived Langerhans cells

BackgroundWith increasing age the immune system shows functional decline. In the skin this is associated with an increased incidence of epidermal malignancies and infections. Epidermal Langerhans cells (LCs) act as sentinels of the immune system, recognizing, processing and presenting antigen and inducing T-cell responses. Previous investigations have demonstrated a reduction in the number of epidermal LCs in elderly subjects. Moreover, the ability of LCs to migrate in response to tumour necrosis factor (TNF)-α, but not interleukin (IL)-1β, is significantly impaired in the elderly.ObjectivesTo characterize further the changes in LC function that are associated with increasing chronological age, we have evaluated age-related changes in the response of monocyte-derived LCs (mLCs) to IL-1β and TNF-α.MethodsThe phenotype and function of mLCs were compared in six young (≤ 30 years) and six aged (≥ 70 years) healthy individuals using a combination of flow cytometry, cytokine and chemokine array, and a Transwell migration assay.ResultsMonocytes from aged individuals were able to differentiate into LCs. There were no significant differences in expression of activation markers, or in baseline or inducible cytokine secretion, by mLCs derived from aged or young subjects. Furthermore, migration in response to a chemokine ligand, CCL19, was equivalent in both age groups.ConclusionsThese data demonstrate that changes in LC function in the elderly are not associated with changes in systemic dendritic cell phenotype and function. Conditioning of LCs in situ by the epidermal microenvironment is likely to be more important.

Time and space resolved uptake study of silicananoparticles by human cells

A spatio-temporal mapping of the uptake of silica (SiO(2)) nanoparticles of different sizes by lung epithelial cells has been obtained. Based on high control of nanoparticle dispersion in cell media and cell exposure, one obtains reproducible and quantitative time-resolved data using a combination of flow cytometry, fluorescence and electron microscopies. We are thereby able to give a rather detailed account of the journey of SiO(2) nanoparticles from the early events of uptake to their final sub-cellular localization.