Profiling antibody responses by multiparametric analysis of primary B cells.
AuthorsStory CM, Papa E, Hu CC, Ronan JL, Herlihy K, Ploegh HL, Love JC,
JournalProc Natl Acad Sci U S A
PubMed ID19004776
'Determining the efficacy of a vaccine generally relies on measuring neutralizing antibodies in sera. This measure cannot elucidate the mechanisms responsible for the development of immunological memory at the cellular level, however. Quantitative profiles that detail the cellular origin, extent, and diversity of the humoral (antibody-based) immune response would improve ... More
Intercellular spreading of Porphyromonas gingivalis infection in primary gingival epithelial cells.
AuthorsYilmaz O, Verbeke P, Lamont RJ, Ojcius DM
JournalInfect Immun
PubMed ID16369027
'Porphyromonas gingivalis, an important periodontal pathogen, is an effective colonizer of oral tissues. The organism successfully invades, multiplies in, and survives for extended periods in primary gingival epithelial cells (GECs). It is unknown whether P. gingivalis resides in the cytoplasm of infected cells throughout the infection or can spread to ... More
T cell receptor engagement leads to the recruitment of IBP, a novel guanine nucleotide exchange factor, to the immunological synapse.
AuthorsGupta S, Fanzo JC, Hu C, Cox D, Jang SY, Lee AE, Greenberg S, Pernis AB
JournalJ Biol Chem
PubMed ID12923183
'Reorganization of the actin cytoskeleton is crucial to the formation and function of the immunological synapse. Rho GTPases are critical mediators of cytoskeletal reorganization, and their activity at the synapse is likely to be stringently regulated. Guanine nucleotide exchange factors (GEFs) belonging to the Dbl family of proteins represent one ... More
Automated image analysis of live/dead staining of the fungus Aureobasidium pullulans on microscope slides and leaf surfaces.
AuthorsNelson CD, Spear RN, Andrews JH
JournalBiotechniques
PubMed ID11056819
'An image analysis program and protocol for the identification and enumeration of live versus dead cells of the yeast-like fungus Aureobasidium pullulans was developed for both populations on microscope slides and leaf surfaces. Live cells took up CellTracker Blue, while nonviable cells stained with DEAD Red. Image analysis macro programs ... More
CX3CR1-fractalkine expression regulates cellular mechanisms involved in adhesion, migration, and survival of human prostate cancer cells.
AuthorsShulby SA, Dolloff NG, Stearns ME, Meucci O, Fatatis A
JournalCancer Res
PubMed ID15256432
'Chemokines and their receptors might be involved in the selection of specific organs by metastatic cancer cells. For instance, the CXCR4-SDF-1alpha pair regulates adhesion and migration of breast as well as prostate cancer cells to metastatic sites. In this study, we present the first evidence for the expression of CX3CR1--the ... More
Quantum dots for tracking dendritic cells and priming an immune response in vitro and in vivo.
AuthorsSen D, Deerinck TJ, Ellisman MH, Parker I, Cahalan MD,
JournalPLoS ONE
PubMed ID18820727
'Dendritic cells (DCs) play a key role in initiating adaptive immune response by presenting antigen to T cells in lymphoid organs. Here, we investigate the potential of quantum dots (QDs) as fluorescent nanoparticles for in vitro and in vivo imaging of DCs, and as a particle-based antigen-delivery system to enhance ... More
Targeting microspheres and cells to polyethylene glycol-modified biological surfaces.
AuthorsDeglau TE, Johnson JD, Villanueva FS, Wagner WR
JournalJ Biomed Mater Res A
PubMed ID17177289
'It has previously been demonstrated that damaged arterial tissue can be acutely modified with protein-reactive polyethylene glycol (PEG) to block undesirable platelet deposition. This concept might be expanded by employing PEG-biotin and its strong interaction with avidin for site-specific targeted delivery. Toward this end, cultured endothelial cells (ECs) were surface ... More
Multiple inhibitory ligands induce impaired T-cell immunologic synapse function in chronic lymphocytic leukemia that can be blocked with lenalidomide: establishing a reversible immune evasion mechanism in human cancer.
AuthorsRamsay AG, Clear AJ, Fatah R, Gribben JG,
JournalBlood
PubMed ID22547582
'Cancer immune evasion is an emerging hallmark of disease progression. We have demonstrated previously that impaired actin polymerization at the T-cell immunologic synapse is a global immune dysfunction in chronic lymphocytic leukemia (CLL). Direct contact with tumor cells induces defective actin polarization at the synapse in previously healthy T cells, ... More
Surfactant protein A enhances alveolar macrophage phagocytosis of apoptotic neutrophils.
AuthorsSchagat TL, Wofford JA, Wright JR
JournalJ Immunol
PubMed ID11160338
'Surfactant protein A (SP-A) is an innate immune molecule that binds foreign organisms that invade the lungs and targets them for phagocytic clearance by the resident pulmonary phagocyte, the alveolar macrophage (AM). We hypothesized that SP-A binds to and enhances macrophage uptake of other nonself particles, specifically apoptotic polymorphonuclear neutrophils ... More
Heterogeneous breast tumoroids: An in vitro assay for investigating cellular heterogeneity and drug delivery.
'Breast tumors are typically heterogeneous and contain diverse subpopulations of tumor cells with differing phenotypic properties. Planar cultures of cancer cell lines are not viable models of investigation of cell-cell and cell-matrix interactions during tumor development. This article presents an in vitro coculture-based 3-dimensional heterogeneous breast tumor model that can ... More
Model study detecting breast cancer cells in peripheral blood mononuclear cells at frequencies as low as 10(-7).
AuthorsGross HJ, Verwer B, Houck D, Hoffman RA, Recktenwald D
JournalProc Natl Acad Sci U S A
PubMed ID7831325
'A flow cytometric assay was developed to detect rare cancer cells in blood and bone marrow. Multiple markers; each identified by a separate color of immunofluorescence (yellow and two shades of red), are used to reliably identify the cancer cells. Blood or bone marrow cells, which are not of interest ... More
T cell anergy is reversed by active Ras and is regulated by diacylglycerol kinase-alpha.
AuthorsZha Y, Marks R, Ho AW, Peterson AC, Janardhan S, Brown I, Praveen K, Stang S, Stone JC, Gajewski TF
JournalNat Immunol
PubMed ID17028589
'T cell anergy has been correlated with defective signaling by the GTPase Ras, but causal and mechanistic data linking defective Ras activity with T cell anergy are lacking. Here we used adenoviral transduction to genetically manipulate nonproliferating T cells and show that active Ras restored interleukin 2 production and mitogen-activated ... More
Identification of the mstE gene encoding a glucose-inducible, low affinity glucose transporter in Aspergillus nidulans.
AuthorsForment JV, Flipphi M, Ramón D, Ventura L, Maccabe AP
JournalJ Biol Chem
PubMed ID16418173
'The mstE gene encoding a low affinity glucose transporter active during the germination of Aspergillus nidulans conidia on glucose medium has been identified. mstE expression also occurs in hyphae, is induced in the presence of other repressing carbon sources besides glucose, and is dependent on the function of the transcriptional ... More
Retention of fluorescent probes during aldehyde-free anhydrous freeze-substitution.
AuthorsHyde GJ, Davies DS, Cole L, Ashford AE
JournalJ Microsc
PubMed ID12753094
'Fluorescent probes are widely used for microscopy of live-cell processes, but few such probes can also be used with classically fixed or otherwise immobilized material, and none has been used without aldehyde fixation, which can introduce artefacts of structure and probe localization. Here we show that the fluorescence patterns in ... More
Fabrication of cell-containing hydrogel microstructures inside microfluidic devices that can be used as cell-based biosensors.
AuthorsKoh WG, Pishko MV
JournalAnal Bioanal Chem
PubMed ID16847626
'This paper describes microfluidic systems containing immobilized hydrogel-encapsulated mammalian cells that can be used as cell-based biosensors. Mammalian cells were encapsulated in three-dimensional poly(ethylene glycol)(PEG) hydrogel microstructures which were photolithographically polymerized in microfluidic devices and grown under static culture conditions. The encapsulated cells remained viable for a week and were ... More
Nanotubular highways for intercellular organelle transport.
AuthorsRustom A, Saffrich R, Markovic I, Walther P, Gerdes HH
JournalScience
PubMed ID14963329
Cell-to-cell communication is a crucial prerequisite for the development and maintenance of multicellular organisms. To date, diverse mechanisms of intercellular exchange of information have been documented, including chemical synapses, gap junctions, and plasmodesmata. Here, we describe highly sensitive nanotubular structures formed de novo between cells that create complex networks. These ... More
Uptake of fluorescent dyes associated with the functional expression of the cystic fibrosis transmembrane conductance regulator in epithelial cells.
AuthorsWersto RP, Rosenthal ER, Crystal RG, Spring KR
JournalProc Natl Acad Sci U S A
PubMed ID8577734
Specific mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), the most common autosomal recessive fatal genetic disease of Caucasians, result in the loss of epithelial cell adenosine 3',5'-cyclic-monophosphate (cAMP)-stimulated Cl- conductance. We show that the influx of a fluorescent dye, dihydrorhodamine 6G (dR6G), is increased in cells expressing human ... More
A protease-dependent mechanism for initiating T-dependent B cell responses to large particulate antigens.
AuthorsCatron DM, Pape KA, Fife BT, van Rooijen N, Jenkins MK,
JournalJ Immunol
PubMed ID20208013
Ab production is critical for antimicrobial immunity, and the initial step in this process is the binding of Ag to the BCR. It has been shown that small soluble proteins can directly access the lymph node follicles to reach naive B cells, but virus particles must be translocated into follicles ... More
Microglia cells protect neurons by direct engulfment of invading neutrophil granulocytes: a new mechanism of CNS immune privilege.
AuthorsNeumann J, Sauerzweig S, Rönicke R, Gunzer F, Dinkel K, Ullrich O, Gunzer M, Reymann KG,
JournalJ Neurosci
PubMed ID18524901
Microglial cells maintain the immunological integrity of the healthy brain and can exert protection from traumatic injury. During ischemic tissue damage such as stroke, peripheral immune cells acutely infiltrate the brain and may exacerbate neurodegeneration. Whether and how microglia can protect from this insult is unknown. Polymorphonuclear neutrophils (PMNs) are ... More
Heterogeneity in macrophage phagocytosis of Staphylococcus aureus strains: high-throughput scanning cytometry-based analysis.
AuthorsDeLoid GM, Sulahian TH, Imrich A, Kobzik L,
JournalPLoS One
PubMed ID19593389
Alveolar macrophages (AMs) can phagocytose unopsonized pathogens such as S. aureus via innate immune receptors, such as scavenger receptors (SRs). Cytoskeletal events and signaling pathways involved in phagocytosis of unopsonized bacteria likely govern the fate of ingested pathogens, but are poorly characterized. We have developed a high-throughput scanning cytometry-based assay ... More
Micromechanical control of cell-cell interactions.
AuthorsHui EE, Bhatia SN
JournalProc Natl Acad Sci U S A
PubMed ID17389399
The development and function of living tissues depends largely on interactions between cells that can vary in both time and space; however, temporal control of cell-cell interaction is experimentally challenging. By using a micromachined silicon substrate with moving parts, we demonstrate the dynamic regulation of cell-cell interactions via direct manipulation ... More
Rac1 and Cdc42 regulate hyphal growth and cytokinesis in the dimorphic fungus Ustilago maydis.
AuthorsMahlert M, Leveleki L, Hlubek A, Sandrock B, Bölker M
JournalMol Microbiol
PubMed ID16390450
Small GTP-binding proteins of the highly conserved Rho family act as molecular switches regulating cell signalling, cytoskeletal organization and vesicle trafficking in eukaryotic cells. Here we show that in the dimorphic plant pathogenic fungus Ustilago maydis deletion of either cdc42 or rac1 results in loss of virulence but does not ... More
Transfer of NKG2D and MICB at the cytotoxic NK cell immune synapse correlates with a reduction in NK cell cytotoxic function.
AuthorsRoda-Navarro P, Vales-Gomez M, Chisholm SE, Reyburn HT
JournalProc Natl Acad Sci U S A
PubMed ID16849432
Although transfer of membrane proteins has been shown to occur during immune cell interactions, the functional significance of this process is not well understood. Here we describe the intercellular transfer of NKG2D and MHC class I chain-related molecule (MIC) B proteins at the cytotoxic natural killer cell immune synapse (cNK-IS). ... More
A syntaxin homolog encoded by VAM3 mediates down-regulation of a yeast G protein-coupled receptor.
AuthorsStefan CJ, Blumer KJ
JournalJ Biol Chem
PubMed ID9880567
G protein-coupled receptors that transduce signals for many hormones, neurotransmitters, and inflammatory mediators are internalized and subsequently recycled to the plasma membrane, or down-regulated by targeting to lysosomes for degradation. Here we have characterized yeast alpha-factor receptors tagged with green fluorescent protein (Ste2-GFP) and used them to obtain mutants defective ... More
Calpain activity increases in hepatocytes following addition of ATP. Demonstration by a novel fluorescent approach.
AuthorsRosser BG, Powers SP, Gores GJ
JournalJ Biol Chem
PubMed ID8226886
Our aim was to measure calpain protease activity during increases in cytosolic free calcium (Ca2+i) after addition of extracellular ATP. The calpain protease substrate t-butoxycarbonyl-Leu-Met-7-amino-4-chloromethylcoumarin was synthesized. Nonfluorescent t-butoxycarbonyl-Leu-Met-7-amino-4- chloromethyl-coumarin diffuses into the cell where it is conjugated to glutathione forming t-butoxycarbonyl-Leu-Met-7-amino-4-methylcoumarin glutathione conjugate (Boc-Leu-Met-MAC-SG). The nonfluorescent, membrane impermeant Boc-Leu-Met-MAC-SG ... More
Evidence that the transition of HIV-1 gp41 into a six-helix bundle, not the bundle configuration, induces membrane fusion.
Many viral fusion proteins exhibit a six-helix bundle as a core structure. HIV Env-induced fusion was studied to resolve whether membrane merger was due to the transition into the bundle configuration or occurred after bundle formation. Suboptimal temperature was used to arrest fusion at an intermediate stage. When bundle formation ... More
Vital fluorochromes as tracers for fungal growth studies.
AuthorsStewart A, Deacon JW
JournalBiotech Histochem
PubMed ID7578589
Eight fluorescent dyes were tested for staining the spores or mycelia of six fungi and for their translocation into new growth when the preloaded spores or mycelia were incubated on agar coated coverslips. The dyes studied were Cellufluor, Nile red, fluorescein diacetate (FDA), carboxyfluorescein diacetate (CFDA), chloromethylfluorescein diacetate (CMFDA), aminochloromethyl ... More
A new method of quantifying glutathione levels in freshly isolated single superfused rat cardiomyocytes.
AuthorsKing N, Korolchuk S, McGivan JD, Suleiman MS
JournalJ Pharmacol Toxicol Methods
PubMed ID15519908
INTRODUCTION: Glutathione (GSH) is an important antioxidant in the heart whose content changes during cardiac insults. However, there are currently no methods for continuously monitoring free cytoplasmic GSH levels in single isolated and superfused cardiomyocytes exposed to normal and pathological conditions. METHODS: GSH was measured using CellTracker Blue CMAC (Molecular ... More
Evaluation of fluorescent dyes for measuring intracellular glutathione content in primary cultures of human neurons and neuroblastoma SH-SY5Y.
AuthorsSebastià J, Cristòfol R, Martín M, Rodríguez-Farré E, Sanfeliu C
JournalCytometry A
PubMed ID12500301
BACKGROUND: Reduced glutathione (GSH) protects cells against oxidative injury and maintains a range of vital functions. To study GSH content in human neuronal cell cultures, thiol-sensitive fluorescent techniques requiring a small number of cells may be of great value, but their GSH specificity has not been established in these cells. ... More
Dilation of the human immunodeficiency virus-1 envelope glycoprotein fusion pore revealed by the inhibitory action of a synthetic peptide from gp41.
AuthorsMuñoz-Barroso I, Durell S, Sakaguchi K, Appella E, Blumenthal R
JournalJ Cell Biol
PubMed ID9442107
We have monitored fusion between cell pairs consisting of a single human immunodeficiency virus-1 (HIV-1) envelope glycoprotein-expressing cell and a CD4+ target cell, which had been labeled with both a fluorescent lipid in the membrane and a fluorescent solute in the cytosol. We developed a new three-color assay to keep ... More
The evolution of catalytic efficiency and substrate promiscuity in human theta class 1-1 glutathione transferase.
AuthorsGriswold KE, Aiyappan NS, Iverson BL, Georgiou G
JournalJ Mol Biol
PubMed ID17011574
Theta class glutathione transferases (GST) from various species exhibit markedly different catalytic activities in conjugating the tripeptide glutathione (GSH) to a variety of electrophilic substrates. For example, the human theta 1-1 enzyme (hGSTT1-1) is 440-fold less efficient than the rat theta 2-2 enzyme (rGSTT2-2) with the fluorogenic substrate 7-amino-4-chloromethyl coumarin ... More
Spatial organization of signal transduction molecules in the NK cell immune synapses during MHC class I-regulated noncytolytic and cytolytic interactions.
AuthorsVyas YM, Mehta KM, Morgan M, Maniar H, Butros L, Jung S, Burkhardt JK, Dupont B
JournalJ Immunol
PubMed ID11591760
The cytolytic activity of NK cells is tightly regulated by inhibitory receptors specific for MHC class I Ags. We have investigated the composition of signal transduction molecules in the supramolecular activation clusters in the MHC class I-regulated cytolytic and noncytolytic NK cell immune synapses. KIR2DL3-positive NK clones that are specifically ... More
Evaluation of glutathione-sensitive fluorescent dyes in cortical culture.
AuthorsTauskela JS, Hewitt K, Kang LP, Comas T, Gendron T, Hakim A, Hogan M, Durkin J, Morley P
JournalGlia
PubMed ID10797613
The sensitivity of six fluorophores to glutathione (GSH) was evaluated in living rat cortical neuronal/glial mixed cultures during the first 23 days in vitro (DIV). Four of the dyes require glutathione-S-transferase (GST) to form a fluorescent conjugate, potentially conferring specificity for GSH: these included t-butoxycarbonyl-Leu-Met-7-amino-4-chloromethylcoumarin (CMAC), 7-amino-4-chloromethylcoumarin (CMAC-blue), monochlorobimane (MCB), ... More
Vac8 determines phagophore assembly site vacuolar localization during nitrogen starvation-induced autophagy.
Authors
JournalAutophagy
PubMed ID32508216
Vav2 catalysis-dependent pathways contribute to skeletal muscle growth and metabolic homeostasis.
Authors
JournalNat Commun
PubMed ID33199701
Post-translational regulation of autophagy is involved in intra-microbiome suppression of fungal pathogens.
Authors
JournalMicrobiome
PubMed ID34092253
NBR1 is involved in selective pexophagy in filamentous ascomycetes and can be functionally replaced by a tagged version of its human homolog.
Authors
JournalAutophagy
PubMed ID30081713
Overcoming CAR-Mediated CD19 Downmodulation and Leukemia Relapse with T Lymphocytes Secreting Anti-CD19 T-cell Engagers.
Authors
JournalCancer Immunol Res
PubMed ID35362043
Eukaryotic cell biology is temporally coordinated to support the energetic demands of protein homeostasis.
Authors
JournalNat Commun
PubMed ID32943618
Effect of homotypic and heterotypic interaction in 3D on the E-selectin mediated adhesive properties of breast cancer cell lines.
Authors
JournalBiomaterials
PubMed ID22992472
Cellular packing, mechanical stress and the evolution of multicellularity.
Authors
JournalNat Phys
PubMed ID31723354
The Inner Nuclear Membrane Is a Metabolically Active Territory that Generates Nuclear Lipid Droplets.
Authors
JournalCell
PubMed ID29937227
Visualization of T Cell Migration in the Spleen Reveals a Network of Perivascular Pathways that Guide Entry into T Zones.