8 results on '"Stefano F. Liparoto"'
Search Results
2. Development and validation of a flow cytometric method to evaluate phagocytosis of pHrodo™ BioParticles® by granulocytes in multiple species
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Julia Lefor, Katherine E. Lich, Stefano F. Liparoto, Yi Qun Xiao, and Andreea Neaga
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Male ,Cytochalasin D ,Phagocytosis ,Immunology ,Inflammation ,Immunotoxicology ,Granulocyte ,Biology ,Mice ,Immune system ,Escherichia coli ,medicine ,Animals ,Immunology and Allergy ,Enzyme Inhibitors ,Nucleic Acid Synthesis Inhibitors ,Mice, Inbred BALB C ,Innate immune system ,Dose-Response Relationship, Drug ,Reproducibility of Results ,Flow Cytometry ,Acquired immune system ,Mice, Inbred C57BL ,Macaca fascicularis ,medicine.anatomical_structure ,Female ,Macrolides ,medicine.symptom ,Ex vivo ,Granulocytes - Abstract
Phagocytosis plays a pivotal and essential role in host immune defense, both as a focal constituent of the innate immune response and a bridging element linking innate and adaptive immunity. Phagocytosis has been demonstrated to be critical in development, tissue remodeling, wound healing and resolution of inflammation through clearance of foreign organisms, apoptotic cells and the production of anti-inflammatory mediators. During pre-clinical investigations, therapeutic drug candidates may alter host resistance to infectious agents by modulating the immune system. The assessment of phagocytic function can be a critical parameter of immunotoxicology for this adverse effect. Utilizing pH-sensitive pHrodo™ BioParticles®, a flow cytometric phagocytosis method was developed and validated in rodent and non-human primate (NHP) species under rigorous GLP compliant procedures. Using species-specific granulocyte markers as well as appropriate temperature and pharmacologic controls, we have developed an ex vivo assay to measure phagocytic function. The method has been optimized to utilize minimal sample volume of whole blood. The assay represents a rapid and reliable tool that can be implemented to evaluate the immunotoxic and immunomodulatory effects of therapeutic candidates.
- Published
- 2013
3. Solution assembly of the pseudo-high affinity and intermediate affinity interleukin-2 receptor complexes
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Michael J. Nemeth, Zining Wu, Thomas M. Laue, Thomas L. Ciardelli, Byron Goldstein, and Stefano F. Liparoto
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Receptor complex ,Protein Conformation ,Molecular Sequence Data ,B-cell receptor ,Receptors, Interleukin-2 ,Interleukin-17 receptor ,Biology ,Interleukin-13 receptor ,Binding, Competitive ,Biochemistry ,Recombinant Proteins ,Interleukin 10 receptor, alpha subunit ,Solutions ,Radioligand Assay ,Biopolymers ,Biophysics ,Amino Acid Sequence ,GABBR1 ,Receptor ,Ultracentrifugation ,Molecular Biology ,Protease-activated receptor 2 ,Research Article - Abstract
The high affinity interleukin-2 receptor is composed of three cell surface subunits, IL-2Ralpha, IL-2Rbeta, and IL-2Rgamma. Functional forms of the IL-2 receptor exist, however, that enlist only two of the three subunits. On activated T-cells, the alpha- and beta-subunits combine as a preformed heterodimer (the pseudo-high affinity receptor) that serves to capture IL-2. On a subpopulation of natural killer cells, the beta- and gamma-subunits interact in a ligand-dependent manner to form the intermediate affinity receptor site. Previously, we have demonstrated the feasibility of employing coiled-coil molecular recognition for the solution assembly of a heteromeric IL-2 receptor complex. In that study, although the receptor was functional, the coiled-coil complex was a trimer rather than the desired heterodimer. We have now redesigned the hydrophobic heptad sequences of the coiled-coils to generate soluble forms of both the pseudo-high affinity and the intermediate affinity heterodimeric IL-2 receptors. The properties of these complexes were examined and their relevance to the physiological IL-2 receptor mechanism is discussed.
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- 2008
4. Affinity enhancement of an in vivo matured therapeutic antibody using structure-based computational design
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Stefano F. Liparoto, John Eldredge, You Li, Christopher Fitch, Mia Rushe, Alexey Lugovskoy, Karl J. M. Hanf, Matthew Jarpe, Herman W. T. van Vlijmen, Woody Sherman, Kenneth Simon, Louis A. Clark, P. Ann Boriack-Sjodin, Miller Stephan S, and Bethany Friedman
- Subjects
Models, Molecular ,Antigen-Antibody Complex ,Mutant ,Antibody Affinity ,Immunoglobulins ,Mutagenesis (molecular biology technique) ,Crystallography, X-Ray ,Biochemistry ,Article ,Antibodies ,Integrin alpha1beta1 ,Protein–protein interaction ,Affinity maturation ,Structure-Activity Relationship ,Structure–activity relationship ,Molecular Biology ,chemistry.chemical_classification ,Hydrogen bond ,Biomolecule ,Crystallography ,Amino Acid Substitution ,chemistry ,Drug Design ,Biophysics ,Computer-Aided Design ,Binding Sites, Antibody - Abstract
Improving the affinity of a high-affinity protein-protein interaction is a challenging problem that has practical applications in the development of therapeutic biomolecules. We used a combination of structure-based computational methods to optimize the binding affinity of an antibody fragment to the I-domain of the integrin VLA1. Despite the already high affinity of the antibody (Kd approximately 7 nM) and the moderate resolution (2.8 A) of the starting crystal structure, the affinity was increased by an order of magnitude primarily through a decrease in the dissociation rate. We determined the crystal structure of a high-affinity quadruple mutant complex at 2.2 A. The structure shows that the design makes the predicted contacts. Structural evidence and mutagenesis experiments that probe a hydrogen bond network illustrate the importance of satisfying hydrogen bonding requirements while seeking higher-affinity mutations. The large and diverse set of interface mutations allowed refinement of the mutant binding affinity prediction protocol and improvement of the single-mutant success rate. Our results indicate that structure-based computational design can be successfully applied to further improve the binding of high-affinity antibodies.
- Published
- 2006
5. Comparative analyses of a small molecule/enzyme interaction by multiple users of Biacore technology
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Stefano F. Liparoto, Melanie Wong, Eric S. Day, Michael Sekar, Donald B. Bennett, Michelle J. Cannon, Dave Casper, Edward J. Collins, Susanne Nyholm Westin, Inna Gorshkova, Yue Jin Li, Karen M. Worthy, Amy Neurauter, Daulet Satpaev, Gerardo R. Marchesini, Paul J. Cachia, Teresa Chu, Ryan James Darling, Jack Simpson, Daniel Kao, Dax A. Rice, Karlicek Shannon Marie, Robert J. Fisher, Magdalena Bynum, Michael Swanson, David G. Myszka, Eric D. Sullivan, Dianne Hodges, Lindsey R. Roberts, Amy M. Liang, Ashique Rafique, Huawei Qiu, Andrew G. Stephen, Giuseppe A. Papalia, Iva Navratilova, Eric Hommema, Béatrice Luginbühl, Jean Inman, and Stephen J. Ullrich
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Analyte ,binding ,mass-transport ,Immobilized enzyme ,RIKILT - Business Unit Veiligheid & Gezondheid ,Biophysics ,Analytical chemistry ,Carbonic Anhydrase II ,Biochemistry ,Chemical kinetics ,Reaction rate constant ,surface ,Molecular Biology ,Observer Variation ,Chromatography ,plasmon resonance biosensors ,Chemistry ,carbonic-anhydrase ,Enzyme Interaction ,Interaction model ,Cell Biology ,Surface Plasmon Resonance ,kinetic-analysis ,Small molecule ,Research Personnel ,rate constants ,Acetazolamide ,Dissociation constant ,Kinetics ,RIKILT - Business Unit Safety & Health ,Protein Binding - Abstract
To gauge the experimental variability associated with Biacore analysis, 36 different investigators analyzed a small molecule/enzyme interaction under similar conditions. Acetazolamide (222 g/mol) binding to carbonic anhydrase II (CAII; 30,000 Da) was chosen as a model system. Both reagents were stable and their interaction posed a challenge to measure because of the low molecular weight of the analyte and the fast association rate constant. Each investigator created three different density surfaces of CAII and analyzed an identical dilution series of acetazolamide (ranging from 4.1 to 1000 nM). The greatest variability in the results was observed during the enzyme immobilization step since each investigator provided their own surface activating reagents. Variability in the quality of the acetazolamide binding responses was likely a product of how well the investigators’ instruments had been maintained. To determine the reaction kinetics, the responses from the different density surfaces were fit globally to a 1:1 interaction model that included a term for mass transport. The averaged association and dissociation rate constants were 3.1 ± 1.6 × 10 6 M −1 s −1 and 6.7 ± 2.5 × 10 −2 s −1 , respectively, which corresponded to an average equilibrium dissociation constant ( K D ) of 2.6 ± 1.4 × 10 −8 M. The results provide a benchmark of variability in interpreting binding constants from the biosensor and highlight keys areas that should be considered when analyzing small molecule interactions.
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- 2004
6. Analysis of the Role of the Interleukin-2 Receptor γ Chain in Ligand Binding
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Zining Wu, Thomas M. Laue, Stefano F. Liparoto, Thomas L. Ciardelli, Byron Goldstein, and David G. Myszka
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Cell type ,medicine.medical_treatment ,Receptors, Interleukin-2 ,Biosensing Techniques ,Biology ,Ligands ,Ligand (biochemistry) ,Interleukin-13 receptor ,Biochemistry ,Affinities ,Recombinant Proteins ,Kinetics ,Radioligand Assay ,Cytokine ,Tumor Cells, Cultured ,medicine ,Humans ,Interleukin-2 ,Binding site ,Receptor ,Ultracentrifugation ,Biosensor ,Protein Binding - Abstract
Interleukin-2 is the primary T cell growth factor secreted by activated T cells. IL-2 is an alpha-helical cytokine that binds to a multisubunit receptor expressed on the surface of a variety of cell types. IL-2Ralpha, IL-2Rbeta, and IL-2Rgammac receptor subunits expressed on the surface of cells may aggregate to form distinct binding sites of differing affinities. IL-2Rgammac was the last receptor subunit to be identified. It has since been shown to be shared by at least five other cytokine receptors. In this study, we have probed the role of IL-2Rgammac in the assembly of IL-2R complexes and in ligand binding. We demonstrate that in the absence of ligand IL-2Rgammac does not possess detectable affinity for IL-2Ralpha, IL-2Rbeta, or the pseudo-high-affinity binding site composed of preformed IL-2Ralpha/beta. We also demonstrate that IL-2Rgammac possesses an IL-2-dependent affinity for IL-2Rbeta and IL-2Ralpha/beta. We performed a detailed biosensor analysis to examine the interaction of soluble IL-2Rgammac with IL-2-bound IL-2Rbeta and IL-2-bound IL-2Ralpha/beta. The kinetic and equilibrium constants for sIL-2Rgammac binding to these two different liganded complexes were similar, indicating that IL-2Ralpha does not play a role in recruitment of IL-2Rgammac. We also determined that the binding of IL-2 to the isolated IL-2Rgammac was very weak (approximate K(D) = 0.7 mM). The experimental methodologies and principles derived from these studies can be extended to at least five other cytokines that share IL-2Rgammac as a receptor subunit.
- Published
- 2002
7. Increased Endosomal Sorting of Ligand to Recycling Enhances Potency of an Interleukin-2 Analog
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Douglas A. Lauffenburger, Stefano F. Liparoto, Eric M. Fallon, Kathy J. Lee, and Thomas L. Ciardelli
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Interleukin 2 ,Endosome ,media_common.quotation_subject ,Endocytic cycle ,Endosomes ,Biology ,Lymphocyte Activation ,Biochemistry ,Cell Line ,Cell surface receptor ,Heterotrimeric G protein ,medicine ,Humans ,Receptor ,Internalization ,Molecular Biology ,media_common ,Receptors, Interleukin-2 ,Cell Biology ,Hydrogen-Ion Concentration ,Ligand (biochemistry) ,Endocytosis ,Cell biology ,Killer Cells, Natural ,Interleukin-2 ,medicine.drug - Abstract
An interleukin-2 (IL-2) variant containing adjacent point mutations (L18M/L19S, termed 2D1) displaying binding affinity to the heterotrimeric IL-2 receptor similar to that of wild-type IL-2 (WT) had been previously found to surprisingly exhibit increased bioactivity in a peripheral blood lymphocyte proliferation assay. In order to provide an explanatory mechanism for this unexpected potency enhancement, we hypothesize that altered endocytic trafficking of the 2D1 variant might be responsible by increasing the number of ligand-receptor complexes. We demonstrate here that the internalization kinetics of 2D1 via the high affinity IL-2 receptor are equivalent to those of WT but that a significantly increased fraction of internalized 2D1 is sorted to recycling instead of to lysosomal degradation. We further find a reduced pH sensitivity of binding to IL-2 receptor alpha relative to IL-2 receptor beta compared with WT, which could be responsible for the altered sorting behavior of 2D1 in the acidic endosomal compartment. Accordingly, the 2D1 variant displays a half-life 36 h longer than that of IL-2 in T-lymphocyte culture at concentrations equal to the K(D) of the IL-2 receptor. The extended half-life of intact 2D1 provides enhanced mitogenesis as compared with IL-2. In addition, 2D1 stimulates natural killer cells to a lesser degree than IL-2 at equal concentrations. We conclude that this IL-2 variant provides increased mitogenic stimulation that could not be easily predicted from its cell surface receptor binding affinity while minimizing undesired stimulation of natural killer cells. This concept of altering trafficking dynamics may offer a generalizable approach to generating improvements in the pharmacological efficacy of therapeutic cytokines.
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- 2000
8. Biosensor analysis of the interleukin-2 receptor complex
- Author
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Stefano F. Liparoto and Thomas L. Ciardelli
- Subjects
Receptor complex ,Radioligand binding ,Structural Biology ,Binding properties ,Spr biosensor ,Computational biology ,Surface plasmon resonance ,Biology ,Molecular Biology ,Biosensor ,Molecular biology - Abstract
Surface plasmon resonance (SPR) biosensor technology has been a significant addition to the evolution and refinement of methods to study macromolecular interactions. Prior to the advent of SPR, we employed a variety of biochemical and biological techniques to study the interleukin-2/interleukin-2 receptor system (IL-2/IL-2R). By combining site-directed mutagenesis, equilibrium and kinetic radioligand binding, and competitive biological assays, we and others had begun to understand many aspects of the structure-activity relationships of the IL-2/IL-2R system. Due to the complexity of the IL-2R, cell-based assays proved limited in their ability to provide quantitative information on the binding characteristics of subclasses of the IL-2 receptor. SPR technology promised to be a new and powerful approach to the quantitative analysis of complex receptor systems. To demonstrate the feasibility of this technology, we employed Biacore analysis to investigate the ligand binding characteristics of novel, pre-assembled, IL-2R coiled-coil complexes. The results of these studies, although limited by instrumentation and data analysis, clearly established the utility of this method. Subsequently, by incorporating advancements in both of these areas, we have been able to carry out detailed kinetic analyses of the binding properties of individual IL-2R subunits as well as heteromeric complexes on the surface of a biosensor. Therefore, SPR biosensor analysis combined with other established analytical methods has proven to be a powerful tool for the analysis of complex hematopoietic receptor systems. Published in 1999 by John Wiley & Sons, Ltd.
- Published
- 1999
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