A requirement for Lim domain binding protein 1 in erythropoiesis.
AuthorsLi L, Lee JY, Gross J, Song SH, Dean A, Love PE,
JournalJ Exp Med
PubMed ID21041453
During erythrocyte development, the nuclear cofactor Lim domain binding protein 1 (Ldb1) functions as a core subunit of multiprotein DNA binding complexes that include the transcription factors Scl and Gata-1 and the Lim-only adapter Lmo2. Scl, Gata-1, and Lmo2 are each required for erythropoiesis, suggesting that Ldb1-nucleated transcription complexes regulate ... More
Multiplex detection of surface molecules on colorectal cancers.
AuthorsEllmark P, Belov L, Huang P, Lee CS, Solomon MJ, Morgan DK, Christopherson RI
JournalProteomics
PubMed ID16485257
'A technique of fluorescence multiplexing is described for analysis of the plasma membrane proteome of colorectal cancer cells from surgically resected specimens, enabling detection and immunophenotyping when the cancer cells are in the minority. A single-cell suspension was prepared from a colorectal tumour, and the mixed population of cells was ... More
CD8 T cell competition for dendritic cells in vivo is an early event in activation.
AuthorsWillis RA, Kappler JW, Marrack PC
JournalProc Natl Acad Sci U S A
PubMed ID16880405
'T cell responses against an antigen are often focused on a small fraction of potentially immunogenic determinants, a phenomenon known as immunodominance. Immunodominance can be established at several stages of antigen presentation, including antigen processing, binding of peptides to MHC, and competition between T cells for dendritic cells (DCs). The ... More
Prostate specific antigen biosensor based on long range surface plasmon-enhanced fluorescence spectroscopy and dextran hydrogel binding matrix.
AuthorsWang Y, Brunsen A, Jonas U, Dostálek J, Knoll W,
JournalAnal Chem
PubMed ID19894697
'A new biosensor based on surface plasmon-enhanced fluorescence spectroscopy (SPFS), which employs long-range surface plasmons (LRSP) and a photo-cross-linkable carboxymethyl dextran (PCDM) hydrogel binding matrix, is reported. LRSPs are surface plasmon modes that propagate along a thin metallic film with orders of magnitude lower damping compared to regular surface plasmons. ... More
Activation-induced deaminase cloning, localization, and protein extraction from young VH-mutant rabbit appendix.
'Studies in mouse, human, and chicken suggest that activation-induced deaminase (AID) is involved in three known processes leading to antibody diversification: somatic hypermutation, gene conversion, and class-switch recombination. Developing rabbit appendix provides a particularly good site for studying all three of these B cell maturation events. We report here successful ... More
Molecular evolution of antibody cross-reactivity for two subtypes of type A botulinum neurotoxin.
AuthorsGarcia-Rodriguez C, Levy R, Arndt JW, Forsyth CM, Razai A, Lou J, Geren I, Stevens RC, Marks JD
JournalNat Biotechnol
PubMed ID17173035
'Broadening antibody specificity without compromising affinity should facilitate detection and neutralization of toxin and viral subtypes. We used yeast display and a co-selection strategy to increase cross-reactivity of a single chain (sc) Fv antibody to botulinum neurotoxin type A (BoNT/A). Starting with a scFv that binds the BoNT/A1 subtype with ... More
Two-color fluorescence analysis of individual virions determines the distribution of the copy number of proteins in herpes simplex virus particles.
AuthorsClarke RW, Monnier N, Li H, Zhou D, Browne H, Klenerman D,
JournalBiophys J
PubMed ID17513380
'We present a single virion method to determine absolute distributions of copy number in the protein composition of viruses and apply it to herpes simplex virus type 1. Using two-color coincidence fluorescence spectroscopy, we determine the virion-to-virion variability in copy numbers of fluorescently labeled tegument and envelope proteins relative to ... More
Single-molecule level analysis of the subunit composition of the T cell receptor on live T cells.
AuthorsJames JR, White SS, Clarke RW, Johansen AM, Dunne PD, Sleep DL, Fitzgerald WJ, Davis SJ, Klenerman D,
JournalProc Natl Acad Sci U S A
PubMed ID17971442
'The T cell receptor (TCR) expressed on most T cells is a protein complex consisting of TCRalphabeta heterodimers that bind antigen and cluster of differentiation (CD) 3epsilondelta, epsilongamma, and zetazeta dimers that initiate signaling. A long-standing controversy concerns whether there is one, or more than one, alphabeta heterodimer per complex. ... More
Antibody to Langerin/CD207 localizes large numbers of CD8alpha+ dendritic cells to the marginal zone of mouse spleen.
AuthorsIdoyaga J, Suda N, Suda K, Park CG, Steinman RM,
JournalProc Natl Acad Sci U S A
PubMed ID19168629
Dendritic cells (DCs) are strategically positioned to take up antigens and initiate adaptive immunity. One DC subset expresses CD8alphaalpha in mice and is specialized to capture dying cells and process antigens for MHC class I ... More
Evidence for resonance optical trapping of individual fluorophore-labeled antibodies using single molecule fluorescence spectroscopy.
AuthorsLi H, Zhou D, Browne H, Klenerman D
JournalJ Am Chem Soc
PubMed ID16637638
We report single molecule fluorescence studies of the diffusion of individual multiple fluorophore-labeled antibodies in solution, which show that a trapping potential of about 3.6 k(B)T can be obtained at laser powers below 1 mW with resonant excitation. Individual antibodies can be trapped for up to 140 ms, and bound ... More
Immune complex relay by subcapsular sinus macrophages and noncognate B cells drives antibody affinity maturation.
AuthorsPhan TG, Green JA, Gray EE, Xu Y, Cyster JG,
JournalNat Immunol
PubMed ID19503106
Subcapsular sinus (SCS) macrophages capture antigens from lymph and present them intact for B cell encounter and follicular delivery. However, the properties of SCS macrophages are poorly defined. Here we show SCS macrophage development depended on lymphotoxin-alpha1beta2, and the cells had low lysosomal enzyme expression and retained opsonized antigens on ... More
Antibodies targeting human OX40 expand effector T cells and block inducible and natural regulatory T cell function.
AuthorsVoo KS, Bover L, Harline ML, Vien LT, Facchinetti V, Arima K, Kwak LW, Liu YJ,
Journal
PubMed ID24014877
Current cancer vaccines induce tumor-specific T cell responses without sustained tumor regression because immunosuppressive elements within the tumor induce exhaustion of effector T cells and infiltration of immune-suppressive regulatory T cells (Tregs). Therefore, much effort has been made to generate agonistic Abs targeting members of the TNFR superfamily, such as ... More
Systematic identification of antiprion drugs by high-throughput screening based on scanning for intensely fluorescent targets.
AuthorsBertsch U, Winklhofer KF, Hirschberger T, Bieschke J, Weber P, Hartl FU, Tavan P, Tatzelt J, Kretzschmar HA, Giese A,
JournalJ Virol
PubMed ID15919931
Conformational changes and aggregation of specific proteins are hallmarks of a number of diseases, like Alzheimer's disease, Parkinson's disease, and prion diseases. In the case of prion diseases, the prion protein (PrP), a neuronal glycoprotein, undergoes a conformational change from the normal, mainly alpha-helical conformation to a disease-associated, mainly beta-sheeted ... More
Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen.
AuthorsSmith K, Garman L, Wrammert J, Zheng NY, Capra JD, Ahmed R, Wilson PC,
JournalNat Protoc
PubMed ID19247287
We describe herein a protocol for the production of antigen-specific human monoclonal antibodies (hmAbs). Antibody-secreting cells (ASCs) are isolated from whole blood collected 7 d after vaccination and sorted by flow cytometry into single cell plates. The antibody genes of the ASCs are then amplified by RT-PCR and nested PCR, ... More
A hyperspectral fluorescence system for 3D in vivo optical imaging.
AuthorsZavattini G, Vecchi S, Mitchell G, Weisser U, Leahy RM, Pichler BJ, Smith DJ, Cherry SR
JournalPhys Med Biol
PubMed ID16585843
In vivo optical instruments designed for small animal imaging generally measure the integrated light intensity across a broad band of wavelengths, or make measurements at a small number of selected wavelengths, and primarily use any spectral information to characterize and remove autofluorescence. We have developed a flexible hyperspectral imaging instrument ... More
Modulation of antigen presentation by autoreactive B cell clones specific for GAD65 from a type I diabetic patient.
AuthorsBanga JP, Moore JK, Duhindan N, Madec AM, van Endert PM, Orgiazzi J, Endl J
JournalClin Exp Immunol
PubMed ID14678267
We used a GAD65-specific human B-T cell line cognate system in vitro to investigate the modulation of GAD65 presentation by autoantibody, assessed in a proliferation assay. Generally, if the T cell determinant overlaps or resides within the antibody epitope, effects of presentation are blunted while if they are distant can ... More
Arf gene loss enhances oncogenicity and limits imatinib response in mouse models of Bcr-Abl-induced acute lymphoblastic leukemia.
AuthorsWilliams RT, Roussel MF, Sherr CJ
JournalProc Natl Acad Sci U S A
PubMed ID16618932
Mouse bone marrow cells transduced with retroviral vectors encoding either of two oncogenic Bcr-Abl isoforms (p210(Bcr-Abl) and p185(Bcr-Abl)) induce B cell lympholeukemias when transplanted into lethally irradiated mice. If the activity of the Arf tumor suppressor is compromised, these donor cells initiate a much more highly aggressive and rapidly fatal ... More
A functional RNAi screen for regulators of receptor tyrosine kinase and ERK signalling.
AuthorsFriedman A, Perrimon N
JournalNature
PubMed ID17086199
Receptor tyrosine kinase (RTK) signalling through extracellular-signal-regulated kinases (ERKs) has pivotal roles during metazoan development, underlying processes as diverse as fate determination, differentiation, proliferation, survival, migration and growth. Abnormal RTK/ERK signalling has been extensively documented to contribute to developmental disorders and disease, most notably in oncogenic transformation by mutant RTKs ... More
Application of spectral imaging microscopy in cytomics and fluorescence resonance energy transfer (FRET) analysis.
AuthorsEcker RC, de Martin R, Steiner GE, Schmid JA
JournalCytometry A
PubMed ID15170596
BACKGROUND: Specific signal detection has been a fundamental issue in fluorescence microscopy. In the context of tissue samples, this problem has been even more pronounced, with respect to spectral overlap and autofluorescence. METHODS: Recent improvements in confocal laser scanning microscopy combine sophisticated hardware to obtain fluorescence emission spectra on a ... More
Prohormone convertase 1/3 is essential for processing of the glucose-dependent insulinotropic polypeptide precursor.
The physiology of the incretin hormones, glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), and their role in type 2 diabetes currently attract great interest. Recently we reported an essential role for prohormone convertase (PC) 1/3 in the cleavage of intestinal proglucagon, resulting in formation of GLP-1, as demonstrated ... More
Synergistic and differential modulation of immune responses by Hsp60 and lipopolysaccharide.
AuthorsOsterloh A, Kalinke U, Weiss S, Fleischer B, Breloer M
JournalJ Biol Chem
PubMed ID17164250
Activation of professional antigen-presenting cells (APC) is a crucial step in the initiation of an efficient immune response. In this study we show that Hsp60 mediates immune stimulation by different mechanisms, dependent and independent of lipopolysaccharide (LPS). We have demonstrated earlier that both, Hsp60 and LPS, increase antigen-specific interferon (IFN) ... More
Selective Engagement of Fc?RIV by a M2e-Specific Single Domain Antibody Construct Protects Against Influenza A Virus Infection.
AuthorsDe Vlieger D, Hoffmann K, Van Molle I, Nerinckx W, Van Hoecke L, Ballegeer M, Creytens S, Remaut H, Hengel H, Schepens B, Saelens X
JournalFront Immunol
PubMed ID31921179
'Lower respiratory tract infections, such as infections caused by influenza A viruses, are a constant threat for public health. Antivirals are indispensable to control disease caused by epidemic as well as pandemic influenza A. We developed a novel anti-influenza A virus approach based on an engineered single-domain antibody (VHH) construct ... More
Electrokinetic Mixing for Improving the Kinetics of an HbA1c Immunoassay.
AuthorsYasun E, Trusty T, Abolhosn RW, Clarke NJ, Mezic I
JournalSci Rep
PubMed ID31882622
'The efficiency of the diagnostic platforms utilizing ELISA technique or immunoassays depends highly on incubation times of the recognition elements or signaling molecules and volume of the patient samples. In conventional immunoassays, long incubation times and excess amounts of the recognition and signaling molecules are used. The technology proposed here ... More
B Cell Receptor Crosslinking Augments Germinal Center B Cell Selection when T Cell Help Is Limiting.
AuthorsTurner JS, Ke F, Grigorova IL
JournalCell Rep
PubMed ID30403996
'Antigen-dependent engagement of germinal center (GC) B cell receptors (BCRs) promotes antigen internalization and presentation for follicular helper T cells. However, whether BCR signaling is critical or synergistic with T cell help for GC B cell selection or differentiation is unclear. Here, by adapting an experimental approach that enables independent delivery of ... More
Observing the Reversible Single Molecule Electrochemistry of Alexa Fluor 647 Dyes by Total Internal Reflection Fluorescence Microscopy.
AuthorsFan S, Webb JEA, Yang Y, Nieves DJ, Gonçales VR, Tran J, Hilzenrat G, Kahram M, Tilley RD, Gaus K, Gooding JJ
JournalAngew Chem Int Ed Engl
PubMed ID31418504
Alexa Fluor 647 is a widely used fluorescent probe for cell bioimaging and super-resolution microscopy. Herein, the reversible fluorescence switching of Alexa Fluor 647 conjugated to bovine serum albumin (BSA) and adsorbed onto indium tin oxide (ITO) electrodes under electrochemical potential control at the level of single protein molecules is ... More