83 results on '"Howell AR"'
Search Results
2. A Humanized Mouse Model Coupled with Computational Analysis Identifies Potent Glycolipid Agonist of Invariant NKT Cells.
- Author
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Saavedra-Avila NA, Pigni NB, Caldwell DR, Chena-Becerra F, Intano J Jr, Ng TW, Chennamadhavuni D, Porcelli SA, Gascón JA, and Howell AR
- Subjects
- Animals, Humans, Mice, Antigens, CD1d, Galactosylceramides, Glycolipids agonists, Natural Killer T-Cells
- Abstract
Invariant natural killer T (iNKT) cells play an important role in many innate and adaptive immune responses, with potential applications in cancer immunotherapy. The glycolipid KRN7000, an α-galactosylceramide, potently activates iNKT cells but has shown limited anticancer effects in human clinical trials conducted so far. In spite of almost three decades of structure-activity relationship studies, no alternative glycolipid has yet emerged as a superior clinical candidate. One reason for the slow progress in this area is that standard mouse models do not accurately reflect the specific ligand recognition by human iNKT cells and their requirements for activation. Here we evaluated a series of KRN7000 analogues using a recently developed humanized mouse model that expresses a human αTCR chain sequence and human CD1d. In this process, a more stimulatory, previously reported but largely overlooked glycolipid was identified, and its activity was probed and rationalized via molecular simulations.
- Published
- 2024
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3. Lysosomal processing of sulfatide analogs alters target NKT cell specificity and immune responses in cancer.
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Nishio K, Pasquet L, Camara K, DiSapio J, Hsu KS, Kato S, Bloom A, Richardson SK, Welsh JA, Jiang T, Jones JC, Cardell S, Watarai H, Terabe M, Olkhanud PB, Howell AR, and Berzofsky JA
- Subjects
- Humans, Sulfoglycosphingolipids metabolism, Antigens, CD1d genetics, Antigen Presentation, Sulfates metabolism, Natural Killer T-Cells, Neoplasms drug therapy, Neoplasms metabolism
- Abstract
In a structure-function study of sulfatides that typically stimulate type II NKT cells, we made an unexpected discovery. We compared analogs with sphingosine or phytosphingosine chains and 24-carbon acyl chains with 0-1-2 double bonds (C or pC24:0, 24:1, or 24:2). C24:1 and C24:2 sulfatide presented by the CD1d monomer on plastic stimulated type II, not type I, NKT cell hybridomas, as expected. Unexpectedly, when presented by bone marrow-derived DCs (BMDCs), C24:2 reversed specificity to stimulate type I, not type II, NKT cell hybridomas, mimicking the corresponding β-galactosylceramide (βGalCer) without sulfate. C24:2 induced IFN-γ-dependent immunoprotection against CT26 colon cancer lung metastases, skewed the cytokine profile, and activated conventional DC subset 1 cells (cDC1s). This was abrogated by blocking lysosomal processing with bafilomycin A1, or by sulfite blocking of arylsulfatase or deletion of this enyzme that cleaves off sulfate. Thus, C24:2 was unexpectedly processed in BMDCs from a type II to a type I NKT cell-stimulating ligand, promoting tumor immunity. We believe this is the first discovery showing that antigen processing of glycosylceramides alters the specificity for the target cell, reversing the glycolipid's function from stimulating type II NKT cells to stimulating type I NKT cells, thereby introducing protective functional activity in cancer. We also believe our study uncovers a new role for antigen processing that does not involve MHC loading but rather alteration of which type of cell is responding.
- Published
- 2023
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4. 1,6-Dioxo-2-azaspiro[3.4]oct-2-enes and Related Spirocycles: Heterocycles from [3 + 2] Nitrile Oxide Cycloadditions with 2-Methyleneoxetanes, -Thietanes, and -Azetidines.
- Author
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Intano J Jr, Riel LP, Lim J, Robinson JR, and Howell AR
- Abstract
Isoxazolines and 4-membered heterocycles are significant structural motifs in numerous synthetic intermediates and natural products. [3 + 2] Cycloadditions between enol ethers and nitrile oxides have been well studied; however, nitrile oxide cycloadditions with 4-membered heterocycles to give spiroisoxazolines are unreported. Here, we showcase the regio- and diastereoselective [3 + 2] nitrile oxide cycloadditions of 2-methyleneoxetanes, -azetidines, and -thietanes to give an array of 1,6-dioxo-2-azaspiro[3.4]oct-2-enes and related spirocycles. 2D NMR experiments suggested that most of the observed diastereoselectivities were dictated by steric interactions; however, dipolarophiles with H bonding donors reversed the stereochemical outcome. X-ray crystallography confirmed the structural assignments.
- Published
- 2023
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5. A humanized mouse model for in vivo evaluation of invariant Natural Killer T cell responses.
- Author
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Saavedra-Avila NA, Dellabona P, Casorati G, Veerapen N, Besra GS, Howell AR, and Porcelli SA
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- Mice, Humans, Animals, Disease Models, Animal, Glycolipids, Receptors, Antigen, T-Cell metabolism, Cytokines metabolism, Receptors, Chemokine metabolism, Galactosylceramides, Natural Killer T-Cells
- Abstract
Invariant natural killer T (iNKT) cells mediate immune responses when stimulated by glycolipid agonists presented by CD1d. In extensive studies of synthetic analogues of α-galactosyl ceramides, we identified numerous examples of significant differences in the recognition of specific glycolipids in wild type mice versus human iNKT cell clones or PBMC samples. To predict human iNKT cell responses more accurately in a mouse model, we derived a mouse line in which compound genetic modifications were used to express a human-like iNKT cell TCR along with human CD1d in place of the endogenous mouse proteins. Detailed transcriptional and phenotypic profiling demonstrated that these partially humanized mice developed an expanded population of T cells recognizing CD1d-presented glycolipid antigens, among which a subset characterized by expression of chemokine receptor CXCR6 had features characteristic of authentic iNKT cells. Responses to iNKT cell activating glycolipids in these mice generated cytokine production in vitro and in vivo that showed a pattern of fine specificity that closely resembled that of cultured human iNKT cell clones. Anti-tumor responses to variants of α-galactosyl ceramide in VαKI mice also correlated with their potency for stimulating human iNKT cells. This genetically modified mouse line provides a practical model for human presentation and recognition of iNKT cell activators in the context of a normally functioning immune system, and may furnish valuable opportunities for preclinical evaluation of iNKT cell-based therapies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Saavedra-Avila, Dellabona, Casorati, Veerapen, Besra, Howell and Porcelli.)
- Published
- 2022
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6. Chemical proteomic analysis of palmostatin beta-lactone analogs that affect N-Ras palmitoylation.
- Author
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Suciu RM, Luvaga IK, Hazeen A, Weerasooriya C, Richardson SK, Firestone AJ, Shannon K, Howell AR, and Cravatt BF
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- Humans, Lactones metabolism, Lactones pharmacology, Molecular Structure, Propiolactone analysis, Propiolactone metabolism, Propiolactone pharmacology, Sulfones metabolism, Sulfones pharmacology, ras Proteins antagonists & inhibitors, ras Proteins chemistry, Lactones analysis, Propiolactone analogs & derivatives, Proteomics, Sulfones analysis, ras Proteins metabolism
- Abstract
S-Palmitoylation is a reversible post-translational lipid modification that regulates protein trafficking and signaling. The enzymatic depalmitoylation of proteins is inhibited by the beta-lactones Palmostatin M and B, which have been found to target several serine hydrolases. In efforts to better understand the mechanism of action of Palmostatin M, we describe herein the synthesis, chemical proteomic analysis, and functional characterization of analogs of this compound. We identify Palmostatin M analogs that maintain inhibitory activity in N-Ras depalmitoylation assays while displaying complementary reactivity across the serine hydrolase class as measured by activity-based protein profiling. Active Palmostatin M analogs inhibit the recently characterized ABHD17 subfamily of depalmitoylating enzymes, while sparing other candidate depalmitoylases such as LYPLA1 and LYPLA2. These findings improve our understanding of the structure-activity relationship of Palmostatin M and refine the set of serine hydrolase targets relevant to the compound's effects on N-Ras palmitoylation dynamics., (Copyright © 2021. Published by Elsevier Ltd.)
- Published
- 2021
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7. Unusual Transformations of Strain-Heightened Oxetanes.
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An J, Riel LP, and Howell AR
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- Models, Molecular, Molecular Structure, Stereoisomerism, Ethers, Cyclic chemistry, Ethers, Cyclic metabolism
- Abstract
Oxetanes are important motifs for drug discovery and are valuable templates in organic synthesis. Much of their use as synthetic intermediates exploits their inherent strain, often resulting in chain extensions at the expense of the heterocycle. Modifications on the carbon alpha to the oxygen of oxetanes, such as the C═O of β-lactones, extend the modes of reactivity. Nevertheless, the outcomes are still largely predictable. On the other hand, other alpha modifications, such as a ═CH
2 , a spiro-oxiranyl moiety, or a spiro-cyclopropyl group, increase strain and open pathways not available to simple oxetanes or β-lactones. Methods in generating 2-methyleneoxetanes, 1,5-dioxaspiro[3.2]hexanes, and 4-oxaspiro[2.3]hexanes have been developed by us and others. To date, reactions of these systems have sometimes been predictable, but often the outcomes have been unexpected. This has provided fertile ground for thinking about what controls reactivity and what other reaction pathways might be accessible to these strain-heightened oxetanes.This Account summarizes the published literature on the most straightforward approaches to 2-methyleneoxetanes, dioxaspirohexanes, and oxaspirohexanes and on their reactivity. In contrast to simple oxetanes, reactions of 2-methyleneoxetanes with nucleophiles at C4 release an enolate rather than an alkoxide. Also, 2-methyleneoxetanes can be converted to homopropargyl alcohols or undergo a silicon accelerated isomerization/electrocyclic ring opening, processes accessible only because of the exocyclic double bond. In addition, oxetane oxocarbenium ions, derived from protonation of the enol ether, can react with nucleophiles to provide 2,2-disubstituted oxetanes. Oxaspirohexanes are readily prepared by Simmons-Smith cyclopropanation of 2-methyleneoxetanes. These unusual systems undergo a variety of substituent dependent rearrangements in the presence of the Lewis acid BF3 ·Et2 O. In addition, upon treatment with Zeise's dimer, oxaspirohexanes are transformed to synthetically useful 3-methylenetetrahydrofurans. Dioxaspirohexanes are easily accessed by dimethyldioxirane oxidation of 2-methyleneoxetanes. Predictably, dioxaspirohexanes react with many nucleophiles to give α-functionalized-β'-hydroxy ketones. Unexpectedly, 2,2-disubstituted oxetanes can also be selectively produced. This latter pathway has led to further unusual transformations, illuminating computational studies, and novel routes to biologically relevant molecules.- Published
- 2021
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8. ABHD17 regulation of plasma membrane palmitoylation and N-Ras-dependent cancer growth.
- Author
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Remsberg JR, Suciu RM, Zambetti NA, Hanigan TW, Firestone AJ, Inguva A, Long A, Ngo N, Lum KM, Henry CL, Richardson SK, Predovic M, Huang B, Dix MM, Howell AR, Niphakis MJ, Shannon K, and Cravatt BF
- Subjects
- Cell Proliferation, Cells, Cultured, Humans, Leukemia, Myeloid, Acute pathology, Leukemia, Promyelocytic, Acute pathology, Lipoylation, Microsomes, Liver chemistry, Microsomes, Liver metabolism, Molecular Structure, Cell Membrane metabolism, Hydrolases metabolism, Leukemia, Myeloid, Acute metabolism, Leukemia, Promyelocytic, Acute metabolism, ras Proteins metabolism
- Abstract
Multiple Ras proteins, including N-Ras, depend on a palmitoylation/depalmitoylation cycle to regulate their subcellular trafficking and oncogenicity. General lipase inhibitors such as Palmostatin M (Palm M) block N-Ras depalmitoylation, but lack specificity and target several enzymes displaying depalmitoylase activity. Here, we describe ABD957, a potent and selective covalent inhibitor of the ABHD17 family of depalmitoylases, and show that this compound impairs N-Ras depalmitoylation in human acute myeloid leukemia (AML) cells. ABD957 produced partial effects on N-Ras palmitoylation compared with Palm M, but was much more selective across the proteome, reflecting a plasma membrane-delineated action on dynamically palmitoylated proteins. Finally, ABD957 impaired N-Ras signaling and the growth of NRAS-mutant AML cells in a manner that synergizes with MAP kinase kinase (MEK) inhibition. Our findings uncover a surprisingly restricted role for ABHD17 enzymes as regulators of the N-Ras palmitoylation cycle and suggest that ABHD17 inhibitors may have value as targeted therapies for NRAS-mutant cancers., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)
- Published
- 2021
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9. Easily accessible non-aromatic heterocycles with handles: 4-bromo-2,3-dihydrofurans from 1,2-dibromohomoallylic alcohols.
- Author
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An J, Intano J Jr, Richard A, Kim T, Gascón JA, and Howell AR
- Abstract
The first general preparation of 4-bromo-2,3-dihydrofurans is reported. These non-aromatic heterocycles containing a useful coupling handle are accessed via Cu-catalyzed intramolecular cyclization of 1,2-dibromohomoallylic alcohols, which are themselves available in just two steps from aromatic and aliphatic aldehydes and ketones. Molecular dynamics simulations using the simple substrates and key geometric parameters provide a rationale for the selectivities observed. The synthetic utility of the 4-bromodihydrofurans is also demonstrated., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)
- Published
- 2021
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10. α-Methylene-β-Lactone Scaffold for Developing Chemical Probes at the Two Ends of the Selectivity Spectrum.
- Author
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Wang L, Riel LP, Bajrami B, Deng B, Howell AR, and Yao X
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- Humans, Lactones chemistry, Molecular Probes chemistry, Molecular Structure, Orlistat analysis, Proteomics, Sesquiterpenes analysis, Tandem Mass Spectrometry, Lactones chemical synthesis, Molecular Probes chemical synthesis
- Abstract
The utilities of an α-methylene-β-lactone (MeLac) moiety as a warhead composed of multiple electrophilic sites are reported. We demonstrate that a MeLac-alkyne not only reacts with diverse proteins as a broadly reactive measurement probe, but also recruits reduced endogenous glutathione (GSH) to assemble a selective chemical probe of GSH-β-lactone (GSH-Lac)-alkyne in live cells. Tandem mass spectrometry reveals that MeLac reacts with nucleophilic cysteine, serine, lysine, threonine, and tyrosine residues, through either Michael or acyl addition. A peptide-centric proteomics platform demonstrates that the proteomic selectivity profiles of orlistat and parthenolide, which have distinct reactivities, are measurable by MeLac-alkyne as a high-coverage probe. The GSH-Lac-alkyne selectively probes the glutathione S-transferase P responsible for multidrug resistance. The assembly of the GSH-Lac probe exemplifies a modular and scalable route to develop selective probes with different recognizing moieties., (© 2020 Wiley-VCH GmbH.)
- Published
- 2021
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11. Amide-Linked C4″-Saccharide Modification of KRN7000 Provides Potent Stimulation of Human Invariant NKT Cells and Anti-Tumor Immunity in a Humanized Mouse Model.
- Author
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Saavedra-Avila NA, Keshipeddy S, Guberman-Pfeffer MJ, Pérez-Gallegos A, Saini NK, Schäfer C, Carreño LJ, Gascón JA, Porcelli SA, and Howell AR
- Subjects
- Animals, Galactosylceramides pharmacology, Glycolipids pharmacology, Humans, Killer Cells, Natural immunology, Mice, Models, Animal, Amides chemistry, Galactosylceramides chemistry, Killer Cells, Natural drug effects, Neoplasms immunology, Sugars chemistry
- Abstract
Activation of invariant natural killer T (iNKT) cells by α-galactosylceramides (α-GalCers) stimulates strong immune responses and potent anti-tumor immunity. Numerous modifications of the glycolipid structure have been assessed to derive activating ligands for these T cells with altered and potentially advantageous properties in the induction of immune responses. Here, we synthesized variants of the prototypical α-GalCer, KRN7000, with amide-linked phenyl alkane substitutions on the C4″-position of the galactose ring. We show that these variants have weak iNKT cell stimulating activity in mouse models but substantially greater activity for human iNKT cells. The most active of the C4″-amides in our study showed strong anti-tumor effects in a partially humanized mouse model for iNKT cell responses. In silico analysis suggested that the tether length and degree of flexibility of the amide substituent affected the recognition by iNKT cell antigen receptors of the C4″-amide substituted glycolipids in complex with their antigen presenting molecule CD1d. Our findings establish the use of stable C4″-amide linked additions to the sugar moiety for further exploration of the immunological effects of structural modifications of iNKT cell activating glycolipids and highlight the critical need for more accurate animal models to assess these compounds for immunotherapeutic potential in humans.
- Published
- 2020
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12. A single-domain bispecific antibody targeting CD1d and the NKT T-cell receptor induces a potent antitumor response.
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Lameris R, Shahine A, Pellicci DG, Uldrich AP, Gras S, Le Nours J, Groen RWJ, Vree J, Reddiex SJJ, Quiñones-Parra SM, Richardson SK, Howell AR, Zweegman S, Godfrey DI, de Gruijl TD, Rossjohn J, and van der Vliet HJ
- Subjects
- Antigens, CD1d chemistry, Humans, Receptors, Antigen, T-Cell chemistry
- Abstract
Antibody-mediated modulation of major histocompatibility complex (MHC) molecules, or MHC class I-like molecules, could constitute an effective immunotherapeutic approach. We describe how single-domain antibodies (VHH), specific for the human MHC class I-like molecule CD1d, can modulate the function of CD1d-restricted T cells and how one VHH (1D12) specifically induced strong type I natural killer T (NKT) cell activation. The crystal structure of the VHH1D12-CD1d(α-GalCer)-NKT T-cell receptor (TCR) complex revealed that VHH1D12 simultaneously contacted CD1d and the type I NKT TCR, thereby stabilizing this interaction through intrinsic bispecificity. This led to greatly enhanced type I NKT cell-mediated antitumor activity in in vitro, including multiple myeloma and acute myeloid leukemia patient-derived bone marrow samples, and in vivo models. Our findings underscore the versatility of VHH molecules in targeting composite epitopes, in this case consisting of a complexed monomorphic antigen-presenting molecule and an invariant TCR, and represent a generalizable antitumor approach., (© 2020. The Author(s), under exclusive licence to Springer Nature America, Inc.)
- Published
- 2020
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13. Immunomodulatory sphingosine-1-phosphates as plasma biomarkers of Alzheimer's disease and vascular cognitive impairment.
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Chua XY, Chai YL, Chew WS, Chong JR, Ang HL, Xiang P, Camara K, Howell AR, Torta F, Wenk MR, Hilal S, Venketasubramanian N, Chen CP, Herr DR, and Lai MKP
- Subjects
- Biomarkers, Humans, Immunomodulation, Longitudinal Studies, Phosphates, Sphingosine, Alzheimer Disease, Cognitive Dysfunction
- Abstract
Background: There has been ongoing research impetus to uncover novel blood-based diagnostic and prognostic biomarkers for Alzheimer's disease (AD), vascular dementia (VaD), and related cerebrovascular disease (CEVD)-associated conditions within the spectrum of vascular cognitive impairment (VCI). Sphingosine-1-phosphates (S1Ps) are signaling lipids which act on the S1PR family of cognate G-protein-coupled receptors and have been shown to modulate neuroinflammation, a process known to be involved in both neurodegenerative and cerebrovascular diseases. However, the status of peripheral S1P in AD and VCI is at present unclear., Methods: We obtained baseline bloods from individuals recruited into an ongoing longitudinal cohort study who had normal cognition (N = 80); cognitive impairment, no dementia (N = 160); AD (N = 113); or VaD (N = 31), along with neuroimaging assessments of cerebrovascular diseases. Plasma samples were processed for the measurements of major S1P species: d16:1, d17:1, d18:0, and d18:1, along with pro-inflammatory cytokines interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF). Furthermore, in vitro effects of S1Ps on cytokine expression were also studied in an astrocytoma cell line and in rodent primary astrocytes., Results: Of the S1Ps species measured, only d16:1 S1P was significantly reduced in the plasma of VaD, but not AD, patients, while the d18:1 to d16:1 ratios were increased in all cognitive subgroups (CIND, AD, and VaD). Furthermore, d18:1 to d16:1 ratios correlated with levels of IL-6, IL-8, and TNF. In both primary astrocytes and an astroglial cell line, treatment with d16:1 or d18:1 S1P resulted in the upregulation of mRNA transcripts of pro-inflammatory cytokines, with d18:1 showing a stronger effect than d16:1. Interestingly, co-treatment assays showed that the addition of d16:1 reduced the extent of d18:1-mediated gene expression, indicating that d16:1 may function to "fine-tune" the pro-inflammatory effects of d18:1., Conclusion: Taken together, our data suggest that plasma d16:1 S1P may be useful as a diagnostic marker for VCI, while the d18:1 to d16:1 S1P ratio is an index of dysregulated S1P-mediated immunomodulation leading to chronic inflammation-associated neurodegeneration and cerebrovascular damage.
- Published
- 2020
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14. Accuracy, precision, and error in age estimation of Florida manatees using growth layer groups in earbones.
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Lonati GL, Howell AR, Hostetler JA, Schueller P, de Wit M, Bassett BL, Deutsch CJ, and Ward-Geiger LI
- Abstract
Ages of Florida manatees ( Trichechus manatus latirostris ) can be estimated by counting annual growth layer groups (GLGs) in the periotic dome portion of the tympanoperiotic complex of their earbones. The Florida Fish and Wildlife Conservation Commission manages an archive of more than 8,700 Florida manatee earbones collected from salvaged carcasses from 1989 to 2017. Our goal was to comprehensively evaluate techniques used to estimate age, given this large sample size and changes to processing protocols and earbone readers over time. We developed new standards for estimating ages from earbones, involving two independent readers to obtain measurements of within- and between-reader precision. To quantify accuracy, precision, and error, 111 earbones from manatees with approximately known ages (first known as calves: "KAC") and 69 earbones from manatees with minimum known ages ("MKA," based on photo-identification sighting histories) were processed, and their ages were estimated. There was greater precision within readers (coefficient of variation, CV : 2.4-8.5%) than between readers ( CV : 13.1-13.3%). The median of age estimates fell within the true age range for 63.1% of KAC cases and was at least the sighting duration for 75.0% of MKA cases. Age estimates were generally unbiased, as indicated by an average raw error ± SD of -0.05 ± 3.05 years for the KAC group. The absolute error (i.e., absolute value of raw error) of the KAC data set averaged 1.75 ± 2.50 years. Accuracy decreased and error increased with increasing known age, especially for animals over 15 years old, whose ages were mostly underestimated due to increasing levels of resorption (the process of bone turnover that obscures GLGs). Understanding the degree of uncertainty in age estimates will help us assess the utility of age data in manatee population models. We emphasize the importance of standardizing and routinely reviewing age estimation and processing protocols to ensure that age data remain consistent and reliable.
- Published
- 2019
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15. Mrp1 is involved in lipid presentation and iNKT cell activation by Streptococcus pneumoniae.
- Author
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Chandra S, Gray J, Kiosses WB, Khurana A, Hitomi K, Crosby CM, Chawla A, Fu Z, Zhao M, Veerapen N, Richardson SK, Porcelli SA, Besra G, Howell AR, Sharma S, Peters B, and Kronenberg M
- Subjects
- ATP Binding Cassette Transporter, Subfamily B, Member 1 deficiency, ATP Binding Cassette Transporter, Subfamily B, Member 1 metabolism, Animals, Antigens, CD1d immunology, Cell Line, Endosomes metabolism, Gene Regulatory Networks, Lysosomes metabolism, Macrophages metabolism, Membrane Microdomains metabolism, Mice, Inbred BALB C, Mice, Knockout, RNA, Small Interfering metabolism, Antigen Presentation immunology, Lipids immunology, Lymphocyte Activation immunology, Natural Killer T-Cells immunology, Streptococcus pneumoniae immunology
- Abstract
Invariant natural killer T cells (iNKT cells) are activated by lipid antigens presented by CD1d, but the pathway leading to lipid antigen presentation remains incompletely characterized. Here we show a whole-genome siRNA screen to elucidate the CD1d presentation pathway. A majority of gene knockdowns that diminish antigen presentation reduced formation of glycolipid-CD1d complexes on the cell surface, including members of the HOPS and ESCRT complexes, genes affecting cytoskeletal rearrangement, and ABC family transporters. We validated the role in vivo for the multidrug resistance protein 1 (Mrp1) in CD1d antigen presentation. Mrp1 deficiency reduces surface clustering of CD1d, which decreased iNKT cell activation. Infected Mrp1 knockout mice show decreased iNKT cell responses to antigens from Streptococcus pneumoniae and were associated with increased mortality. Our results highlight the unique cellular events involved in lipid antigen presentation and show how modification of this pathway can lead to lethal infection.
- Published
- 2018
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16. Heterogeneous Catalytic Oxidation of Amides to Imides by Manganese Oxides.
- Author
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Biswas S, Khanna HS, Nizami QA, Caldwell DR, Cavanaugh KT, Howell AR, Raman S, Suib SL, and Nandi P
- Abstract
Herein, we report a one-step peroxide mediated heterogeneous catalytic oxidation of amides to imides utilizing a series of manganese oxides. Among them, Cs/Mn
2 O3 was found to be the most active catalyst for the selective partial oxidation of N-benzylbenzamide to diphenyl imide. We have been able to apply an optimized oxidation method to other aromatic substrates. The feasibility of using air as an oxidant, the heterogeneous nature, inexpensive catalytic materials, respectable turnover numbers, and chemoselectivity to imides make this methodology an attractive choice for functional group transformations of amides to imides.- Published
- 2018
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17. Dual Modifications of α-Galactosylceramide Synergize to Promote Activation of Human Invariant Natural Killer T Cells and Stimulate Anti-tumor Immunity.
- Author
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Chennamadhavuni D, Saavedra-Avila NA, Carreño LJ, Guberman-Pfeffer MJ, Arora P, Yongqing T, Pryce R, Koay HF, Godfrey DI, Keshipeddy S, Richardson SK, Sundararaj S, Lo JH, Wen X, Gascón JA, Yuan W, Rossjohn J, Le Nours J, Porcelli SA, and Howell AR
- Published
- 2018
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18. Rh-Catalyzed Conjugate Addition of Aryl and Alkenyl Boronic Acids to α-Methylene-β-lactones: Stereoselective Synthesis of trans-3,4-Disubstituted β-Lactones.
- Author
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Malapit CA, Luvaga IK, Caldwell DR, Schipper NK, and Howell AR
- Subjects
- Lactones, Molecular Structure, Stereoisomerism, Boronic Acids chemistry, Rhodium chemistry
- Abstract
A one-step preparation of 3,4-disubstituted β-lactones through Rh-catalyzed conjugate addition of aryl or alkenyl boronic acids to α-methylene-β-lactones is described. The operationally simple, stereoselective transformation provides a broad range of β-lactones from individual α-methylene-β-lactone templates. This methodology allowed for a direct, final-step C-3 diversification of nocardiolactone, an antimicrobial natural product.
- Published
- 2017
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19. Contact sensitizers trigger human CD1-autoreactive T-cell responses.
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Betts RJ, Perkovic A, Mahapatra S, Del Bufalo A, Camara K, Howell AR, Martinozzi Teissier S, De Libero G, and Mori L
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- Acrolein analogs & derivatives, Acrolein pharmacology, Antigen Presentation, Benzoquinones pharmacology, Cell Line, Dendritic Cells immunology, Dinitrochlorobenzene pharmacology, Eugenol analogs & derivatives, Eugenol pharmacology, Humans, Lipids immunology, Lymphocyte Activation, Monocytes drug effects, Resorcinols pharmacology, Skin immunology, Antigens, CD1 immunology, Dermatitis, Contact immunology, Natural Killer T-Cells immunology, T-Lymphocytes immunology
- Abstract
Allergic contact dermatitis is a primarily T-cell-mediated inflammatory skin disease induced by exposure to small molecular-weight haptens, which covalently bind to proteins. The abundance of cutaneous T cells that recognize CD1a antigen-presenting molecules raises the possibility that MHC-independent antigen presentation may be relevant in some hapten-driven immune responses. Here we examine the ability of contact sensitizers to influence CD1-restricted immunity. Exposure of human antigen-presenting cells such as monocyte-derived dendritic cells and THP-1 cells to the prototypical contact sensitizer dinitrochlorobenzene potentiated the response of CD1a- and CD1d-autoreactive T cells, which released a vast array of cytokines in a CD1- and TCR-dependent manner. The potentiating effects of dinitrochlorobenzene depended upon newly synthesized CD1 molecules and the presence of endogenous stimulatory lipids. Further examination of a broad panel of contact sensitizers revealed 1,4-benzoquinone, resorcinol, isoeugenol, and cinnamaldehyde to activate the same type of CD1-restricted responses. These findings provide a basis for the antigen-specific activation of skin-associated CD1-restricted T cells by small molecules and may have implications for contact sensitizer-induced inflammatory skin diseases., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2017
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20. Scaling Proteome-Wide Reactions of Activity-Based Probes.
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Li S, Diego-Limpin PA, Bajrami B, Keshipeddy S, Lam YW, Deng B, Farrokhi V, McShane AJ, Nemati R, Howell AR, and Yao X
- Subjects
- HT29 Cells, Humans, Molecular Structure, Lactones chemistry, Molecular Probes chemistry, Proteome analysis, Threonine chemistry
- Abstract
Unified analysis of complex reactions of an activity-based probe with proteins in a proteome remains an unsolved challenge. We propose a power expression, rate = k
obs [Probe]α , for scaling the progress of proteome-wide reactions and use the scaling factor (0 ≤ α ≤ 1) as an apparent, partial order with respect to the probe to measure the "enzyme-likeness" for a protein in reaction acceleration. Thus, α reports the intrinsic reactivity of the protein with the probe. When α = 0, the involved protein expedites the reaction to the maximal degree; when α = 1, the protein reacts with the probe via an unaccelerated, bimolecular reaction. The selectivity (β) of the probe reacting with two proteins is calculated as a ratio of conversion factors (kobs values) for corresponding power equations. A combination of α and β provides a tiered system for quantitatively assessing the probe efficacy; an ideal probe exhibits high reactivity with its protein targets (low in α) and is highly selective (high in β) in forming the probe-protein adducts. The scaling analysis was demonstrated using proteome-wide reactions of HT-29 cell lysates with a model probe of threonine β-lactone.- Published
- 2017
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21. Rhodium-Catalyzed Addition of Aryl Boronic Acids to 2,2-Disubstituted Malononitriles.
- Author
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Malapit CA, Caldwell DR, Luvaga IK, Reeves JT, Volchkov I, Gonnella NC, Han ZS, Busacca CA, Howell AR, and Senanayake CH
- Abstract
β-Ketonitriles bearing a quaternary carbon at the 2-position were prepared through Rh-catalyzed addition of aryl boronic acids to 2,2-disubstituted malononitriles. In contrast to the previously described transnitrilative cyanation of aryl boronic acids with dialkylmalononitriles, the present reaction avoids retro-Thorpe collapse of the intermediate addition product through the use of a milder base. The reaction was amenable to a variety of aryl boronic acids and disubstituted malononitriles, providing a diverse array of β-ketonitriles. The products could be further derivatized to valuable chiral α,α-disubstituted-β-aminonitriles through addition reactions to the corresponding N-tert-butanesulfinyl imines., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2017
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22. 1,4-Dicarbofunctionalization of 4-Fluoroaryl Grignard and Lithium Reagents with Disubstituted Malononitriles.
- Author
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Malapit CA, Luvaga IK, Reeves JT, Volchkov I, Busacca CA, Howell AR, and Senanayake CH
- Abstract
An efficient one-pot 1,4-dicarbofunctionalization of 4-fluoroaryl Grignard or lithium reagents with 2,2-disubstituted malononitriles is described. The reaction proceeds by sequential transnitrilation and S
N Ar reactions. Commercial Grignard solutions, Grignard reagents prepared in situ by halogen/magnesium exchange with i-PrMgCl, or aryllithium reagents prepared in situ by bromine/lithium exchange with n-BuLi are compatible with the reaction conditions. Moreover, 2,2-disubstituted malononitriles of diverse structures are accommodated. The reaction provides a unique approach to 1,4-dicarbofunctionalization of activated arenes in a tandem, one-pot transformation.- Published
- 2017
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23. Pd-Catalyzed Acyl C-O Bond Activation for Selective Ring-Opening of α-Methylene-β-lactones with Amines.
- Author
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Malapit CA, Caldwell DR, Sassu N, Milbin S, and Howell AR
- Subjects
- Catalysis, Kinetics, Molecular Structure, Stereoisomerism, Amides chemistry, Amines chemistry, Lactones chemistry, Palladium chemistry
- Abstract
A Pd-catalyzed ring-opening of β-lactones with various types of amines (primary, secondary, and aryl) to provide β-hydroxy amides with excellent selectivity toward acyl C-O bond cleavage is reported. The utility of this protocol is demonstrated in an asymmetric kinetic resolution providing enantioenriched α-methylene-β-lactones.
- Published
- 2017
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- View/download PDF
24. Atypical natural killer T-cell receptor recognition of CD1d-lipid antigens.
- Author
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Le Nours J, Praveena T, Pellicci DG, Gherardin NA, Ross FJ, Lim RT, Besra GS, Keshipeddy S, Richardson SK, Howell AR, Gras S, Godfrey DI, Rossjohn J, and Uldrich AP
- Subjects
- Crystallography, X-Ray, Gangliosides immunology, Glucosylceramides immunology, Humans, Lipids immunology, Molecular Docking Simulation, Receptors, Antigen, T-Cell, alpha-beta metabolism, Surface Plasmon Resonance, Antigens, CD1d immunology, Galactosylceramides immunology, Natural Killer T-Cells immunology, Receptors, Antigen, T-Cell, alpha-beta immunology
- Abstract
Crucial to Natural Killer T (NKT) cell function is the interaction between their T-cell receptor (TCR) and CD1d-antigen complex. However, the diversity of the NKT cell repertoire and the ensuing interactions with CD1d-antigen remain unclear. We describe an atypical population of CD1d-α-galactosylceramide (α-GalCer)-reactive human NKT cells that differ markedly from the prototypical TRAV10-TRAJ18-TRBV25-1(+) type I NKT cell repertoire. These cells express a range of TCR α- and β-chains that show differential recognition of glycolipid antigens. Two atypical NKT TCRs (TRAV21-TRAJ8-TRBV7-8 and TRAV12-3-TRAJ27-TRBV6-5) bind orthogonally over the A'-pocket of CD1d, adopting distinct docking modes that contrast with the docking mode of all type I NKT TCR-CD1d-antigen complexes. Moreover, the interactions with α-GalCer differ between the type I and these atypical NKT TCRs. Accordingly, diverse NKT TCR repertoire usage manifests in varied docking strategies and specificities towards CD1d-α-GalCer and related antigens, thus providing far greater scope for diverse glycolipid antigen recognition.
- Published
- 2016
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25. Rhodium-Catalyzed Transnitrilation of Aryl Boronic Acids with Dimethylmalononitrile.
- Author
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Malapit CA, Reeves JT, Busacca CA, Howell AR, and Senanayake CH
- Abstract
An efficient transnitrilation of aryl boronic acids with dimethylmalononitrile (DMMN) is described. This rhodium-catalyzed electrophilic cyanation presents a novel approach to prepare aryl nitriles by using a carbon-bound cyanating reagent which undergoes cross-coupling with the aryl boronic acid. The reaction expands the degree of functional-group compatibility exhibited by the transnitrilation of aryl Grignard and aryllithium reagents. A variety of aryl boronic acid derivatives and dialkylmalononitriles were amenable to the transnitrilation., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2016
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26. Selective Conditions Are Required for the Induction of Invariant NKT Cell Hyporesponsiveness by Antigenic Stimulation.
- Author
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Wingender G, Birkholz AM, Sag D, Farber E, Chitale S, Howell AR, and Kronenberg M
- Subjects
- Animals, B-Lymphocytes cytology, B-Lymphocytes immunology, Cytokines immunology, Dendritic Cells cytology, Dendritic Cells immunology, Mice, Mice, Knockout, Natural Killer T-Cells cytology, Th1 Cells cytology, Th1 Cells immunology, Th2 Cells cytology, Th2 Cells immunology, Antigens immunology, Galactosylceramides immunology, Natural Killer T-Cells immunology
- Abstract
Activation of invariant (i)NKT cells with the model Ag α-galactosylceramide induces rapid production of multiple cytokines, impacting a wide variety of different immune reactions. In contrast, following secondary activation with α-galactosylceramide, the behavior of iNKT cells is altered for months, with the production of most cytokines being strongly reduced. The requirements for the induction of this hyporesponsive state, however, remain poorly defined. In this study, we show that Th1-biasing iNKT cell Ags could induce iNKT cell hyporesponsiveness, as long as a minimum antigenic affinity was reached. In contrast, the Th2-biasing Ag OCH did not induce a hyporesponsive state, nor did cytokine-driven iNKT cell activation by LPS or infections. Furthermore, although dendritic cells and B cells have been reported to be essential for iNKT cell stimulation, neither dendritic cells nor B cells were required to induce iNKT cell hyporesponsiveness. Therefore, our data indicate that whereas some bone marrow-derived cells could induce iNKT cell hyporesponsiveness, selective conditions, dependent on the structure and potency of the Ag, were required to induce hyporesponsiveness., (Copyright © 2015 by The American Association of Immunologists, Inc.)
- Published
- 2015
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27. Recent Applications of Oxetanes in the Synthesis of Heterocyclic Compounds.
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Malapit CA and Howell AR
- Abstract
Oxetanes are valuable intermediates in organic synthesis, and strategic manipulations of this strained heterocycle continue to emerge. In this Synopsis, recent, distinct approaches to construct heterocyclic systems using oxetanes are described. These include ring expansion, ring opening, and C-2 functionalization.
- Published
- 2015
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28. The alpha and omega of galactosylceramides in T cell immune function.
- Author
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Birkholz AM, Howell AR, and Kronenberg M
- Published
- 2015
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- View/download PDF
29. A Solvent-free Approach to Glycosyl Amides: Towards the Synthesis of α- N -Galactosyl Ceramides.
- Author
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Chennamadhavuni D and Howell AR
- Abstract
A new, simple and efficient method for the synthesis of both α- and β-glycosyl amides using solvent-free conditions is described. This method involves the coupling of glycosyl amines with the p -nitrophenol esters of lipids as a key step.
- Published
- 2015
- Full Text
- View/download PDF
30. Pt-catalyzed rearrangement of oxaspirohexanes to 3-methylenetetrahydrofurans: scope and mechanism.
- Author
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Malapit CA, Chitale SM, Thakur MS, Taboada R, and Howell AR
- Subjects
- Catalysis, Furans chemistry, Oxidation-Reduction, Carbon Radioisotopes chemistry, Cyclopropanes chemistry, Furans chemical synthesis, Platinum Compounds chemical synthesis, Platinum Compounds chemistry, Spiro Compounds chemistry
- Abstract
A novel Pt-catalyzed rearrangement of oxaspirohexanes to 3-methylenetetrahydrofurans is reported. Mechanistic studies by (13)C-labeling experiments confirm oxidative addition of Pt(II) regioselectively to the least substituted carbon-carbon bond of the cyclopropane to form a platinacyclobutane intermediate. To our knowledge, this is the first alkoxy-substituted platinacyclobutane that has been observed spectroscopically. The scope and a proposed mechanism of this new Pt-catalyzed transformation are described.
- Published
- 2015
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- View/download PDF
31. Immunomodulatory lysophosphatidylserines are regulated by ABHD16A and ABHD12 interplay.
- Author
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Kamat SS, Camara K, Parsons WH, Chen DH, Dix MM, Bird TD, Howell AR, and Cravatt BF
- Subjects
- Animals, Brain enzymology, Brain immunology, Brain metabolism, Cell Line, Cytokines immunology, Cytokines metabolism, Humans, Immunologic Factors immunology, Lysophospholipids immunology, Macrophages enzymology, Macrophages immunology, Macrophages metabolism, Male, Mice, Knockout, Mutation, Phospholipases antagonists & inhibitors, Immunologic Factors metabolism, Lysophospholipids metabolism, Monoacylglycerol Lipases genetics, Phospholipases genetics
- Abstract
Lysophosphatidylserines (lyso-PSs) are a class of signaling lipids that regulate immunological and neurological processes. The metabolism of lyso-PSs remains poorly understood in vivo. Recently, we determined that ABHD12 is a major brain lyso-PS lipase, implicating lyso-PSs in the neurological disease polyneuropathy, hearing loss, ataxia, retinitis pigmentosa and cataract (PHARC), which is caused by null mutations in the ABHD12 gene. Here, we couple activity-based profiling with pharmacological and genetic methods to annotate the poorly characterized enzyme ABHD16A as a phosphatidylserine (PS) lipase that generates lyso-PS in mammalian systems. We describe a small-molecule inhibitor of ABHD16A that depletes lyso-PSs from cells, including lymphoblasts derived from subjects with PHARC. In mouse macrophages, disruption of ABHD12 and ABHD16A respectively increases and decreases both lyso-PSs and lipopolysaccharide-induced cytokine production. Finally, Abhd16a(-/-) mice have decreased brain lyso-PSs, which runs counter to the elevation in lyso-PS in Abhd12(-/-) mice. Our findings illuminate an ABHD16A-ABHD12 axis that dynamically regulates lyso-PS metabolism in vivo, designating these enzymes as potential targets for treating neuroimmunological disorders.
- Published
- 2015
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- View/download PDF
32. Combining cross-metathesis and activity-based protein profiling: new β-lactone motifs for targeting serine hydrolases.
- Author
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Camara K, Kamat SS, Lasota CC, Cravatt BF, and Howell AR
- Subjects
- Animals, Binding, Competitive, Brain enzymology, Chromatography, Liquid, Colonic Neoplasms enzymology, Humans, Mice, Molecular Structure, Serine Endopeptidases metabolism, Serine Proteinase Inhibitors chemistry, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Stereoisomerism, Structure-Activity Relationship, Tumor Cells, Cultured, Brain drug effects, Colonic Neoplasms drug therapy, Lactones chemistry, Lactones pharmacology, Proteome analysis, Serine Endopeptidases chemistry, Serine Proteinase Inhibitors pharmacology
- Abstract
β-Lactones are a privileged structural motif as enzyme inhibitors and chemical probes, particularly for the inhibition of enzymes from the serine hydrolase class. Herein, we demonstrate that cross-metathesis (CM) of α-methylene-β-lactones offers rapid access to structurally diverse, previously unexplored β-lactones. Combining this approach with competitive activity-based protein profiling (ABPP) identified lead β-lactone inhibitors/probes for several serine hydrolases, including disease-associated enzymes and enzymes of uncharacterized function. The structural diversity afforded by the α-methylene-β-lactone scaffold thus expands the landscape of serine hydrolases that can be targeted by small-molecule inhibitors and should further the functional characterization of enzymes from this class through the optimization of target-selective probes., (Copyright © 2014 Elsevier Ltd. All rights reserved.)
- Published
- 2015
- Full Text
- View/download PDF
33. The molecular bases of δ/αβ T cell-mediated antigen recognition.
- Author
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Pellicci DG, Uldrich AP, Le Nours J, Ross F, Chabrol E, Eckle SB, de Boer R, Lim RT, McPherson K, Besra G, Howell AR, Moretta L, McCluskey J, Heemskerk MH, Gras S, Rossjohn J, and Godfrey DI
- Subjects
- Amino Acid Sequence, Clone Cells, Galactosylceramides immunology, Humans, Jurkat Cells, Lipids immunology, Lymphocyte Activation immunology, Models, Molecular, Molecular Sequence Data, Peptides chemistry, Peptides immunology, Receptors, Antigen, T-Cell, alpha-beta chemistry, Receptors, Antigen, T-Cell, gamma-delta chemistry, Antigens, CD1d immunology, Receptors, Antigen, T-Cell, alpha-beta immunology, Receptors, Antigen, T-Cell, gamma-delta immunology, T-Lymphocytes immunology
- Abstract
αβ and γδ T cells are disparate T cell lineages that can respond to distinct antigens (Ags) via the use of the αβ and γδ T cell Ag receptors (TCRs), respectively. Here we characterize a population of human T cells, which we term δ/αβ T cells, expressing TCRs comprised of a TCR-δ variable gene (Vδ1) fused to joining α and constant α domains, paired with an array of TCR-β chains. We demonstrate that these cells, which represent ∼50% of all Vδ1(+) human T cells, can recognize peptide- and lipid-based Ags presented by human leukocyte antigen (HLA) and CD1d, respectively. Similar to type I natural killer T (NKT) cells, CD1d-lipid Ag-reactive δ/αβ T cells recognized α-galactosylceramide (α-GalCer); however, their fine specificity for other lipid Ags presented by CD1d, such as α-glucosylceramide, was distinct from type I NKT cells. Thus, δ/αβTCRs contribute new patterns of Ag specificity to the human immune system. Furthermore, we provide the molecular bases of how δ/αβTCRs bind to their targets, with the Vδ1-encoded region providing a major contribution to δ/αβTCR binding. Our findings highlight how components from αβ and γδTCR gene loci can recombine to confer Ag specificity, thus expanding our understanding of T cell biology and TCR diversity., (© 2014 Pellicci et al.)
- Published
- 2014
- Full Text
- View/download PDF
34. Synthesis of a 2"-deoxy-β-GalCer.
- Author
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Thakur MS, Khurana A, Kronenberg M, and Howell AR
- Subjects
- Animals, Antigens, CD1d metabolism, Galactosylceramides chemistry, Galactosylceramides pharmacology, Interleukin-2 biosynthesis, Mice, Natural Killer T-Cells drug effects, Natural Killer T-Cells metabolism, Galactosylceramides chemical synthesis
- Abstract
Structural studies of ternary complexes of CD1d/glycosyl ceramides/iNKT cells and CD1d/sulfatide/sulfatide reactive Type II NKT cells have shown how the polar moieties on the glycolipids interact with both the antigen presenting protein (CD1d) and the T cell receptors. However, these structures alone do not reveal the relative importance of these interactions. This study focuses on the synthesis of the previously unknown 2"-deoxy-β-galactosyl ceramide 2. This glycolipid is also evaluated for its ability to stimulate iNKT cells and sulfatide-reactive Type II NKT cells.
- Published
- 2014
- Full Text
- View/download PDF
35. Silicon acceleration of a tandem alkene isomerization/electrocyclic ring-opening of 2-methyleneoxetanes to α,β-unsaturated methylketones.
- Author
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Farber E, Rudnitskaya A, Keshipeddy S, Lao KS, Gascón JA, and Howell AR
- Abstract
The first rearrangement of 2-methyleneoxetanes to α,β-unsaturated methylketones is reported. It is proposed that when these substrates are heated, the corresponding oxetenes are formed and subsequently undergo electrocyclic ring-opening to methyl vinylketones. In particular, α-silyl-α,β-unsaturated methylketones were isolated in moderate to high yields and with high stereoselectivities. Based on the proposed mechanism, density functional theory explains the differential kinetics and stereoselectivities among substrates.
- Published
- 2013
- Full Text
- View/download PDF
36. Cloning and characterization of a hybridoma secreting a 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-specific monoclonal antibody and recombinant F(ab).
- Author
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Wanczyk H, Barker T, Rood D, Zapata DI, Howell AR, Richardson SK, Zinckgraf J, Marusov GP, Lynes MA, and Silbart LK
- Subjects
- Animals, Binding Sites, Antibody, Carrier Proteins chemistry, Cell Line, Tumor, Cloning, Molecular, Electrophoresis, Polyacrylamide Gel, Enzyme-Linked Immunosorbent Assay, Female, HEK293 Cells, Humans, Mice, Mice, Inbred BALB C, Nicotine pharmacology, Nitrosamines chemistry, Spleen cytology, Antibodies, Monoclonal immunology, Hybridomas metabolism, Immunoglobulin Fab Fragments immunology, Nitrosamines immunology
- Abstract
Smokeless tobacco products have been associated with increased risks of oro-pharyngeal cancers, due in part to the presence of tobacco-specific nitrosamines (TSNAs) such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). These potent carcinogens are formed during tobacco curing and as a result of direct nitrosation reactions that occur in the oral cavity. In the current work we describe the isolation and characterization of a hybridoma secreting a high-affinity, NNK-specific monoclonal antibody. A structurally-related benzoyl derivative was synthesized to facilitate coupling to NNK-carrier proteins, which were characterized for the presence of the N-nitroso group using the Griess reaction, and used to immunize BALB/c mice. Splenocytes from mice bearing NNK-specific antibodies were used to create hybridomas. Out of four, one was selected for subcloning and characterization. Approximately 99% of the monoclonal antibodies from this clone were competitively displaced from plate-bound NNKB conjugates in the presence of free NNK. The affinity of the monoclonal antibody to the NNKB conjugates was Kd = 2.93 nM as determined by surface plasmon resonance. Free nicotine was a poor competitor for the NNKB binding site. The heavy and light chain antibody F(ab) fragments were cloned, sequenced and inserted in tandem into an expression vector, with an FMDV Furin 2A cleavage site between them. Expression in HEK 293 cells revealed a functional F(ab) with similar binding features to that of the parent hybridoma. This study lays the groundwork for synthesizing transgenic tobacco that expresses carcinogen-sequestration properties, thereby rendering it less harmful to consumers.
- Published
- 2013
- Full Text
- View/download PDF
37. Human and mouse type I natural killer T cell antigen receptors exhibit different fine specificities for CD1d-antigen complex.
- Author
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Wun KS, Ross F, Patel O, Besra GS, Porcelli SA, Richardson SK, Keshipeddy S, Howell AR, Godfrey DI, and Rossjohn J
- Subjects
- Amino Acid Motifs, Animals, Antigen Presentation, Antigens chemistry, Crystallography, X-Ray methods, Flow Cytometry methods, Glycolipids chemistry, Humans, Leukocytes, Mononuclear cytology, Lipids chemistry, Mice, Models, Molecular, Molecular Conformation, Natural Killer T-Cells metabolism, Structure-Activity Relationship, Surface Plasmon Resonance, Antigens, CD1d metabolism, Natural Killer T-Cells immunology, Receptors, Antigen metabolism
- Abstract
Human and mouse type I natural killer T (NKT) cells respond to a variety of CD1d-restricted glycolipid antigens (Ags), with their NKT cell antigen receptors (NKT TCRs) exhibiting reciprocal cross-species reactivity that is underpinned by a conserved NKT TCR-CD1d-Ag docking mode. Within this common docking footprint, the NKT TCR recognizes, to varying degrees of affinity, a range of Ags. Presently, it is unclear whether the human NKT TCRs will mirror the generalities underpinning the fine specificity of the mouse NKT TCR-CD1d-Ag interaction. Here, we assessed human NKT TCR recognition against altered glycolipid ligands of α-galactosylceramide (α-GalCer) and have determined the structures of a human NKT TCR in complex with CD1d-4',4″-deoxy-α-GalCer and CD1d-α-GalCer with a shorter, di-unsaturated acyl chain (C20:2). Altered glycolipid ligands with acyl chain modifications did not affect the affinity of the human NKT TCR-CD1d-Ag interaction. Surprisingly, human NKT TCR recognition is more tolerant to modifications at the 4'-OH position in comparison with the 3'-OH position of α-GalCer, which contrasts the fine specificity of the mouse NKT TCR-CD1d-Ag recognition (4'-OH > 3'-OH). The fine specificity differences between human and mouse NKT TCRs was attributable to differing interactions between the respective complementarity-determining region 1α loops and the Ag. Accordingly, germline encoded fine-specificity differences underpin human and mouse type I NKT TCR interactions, which is an important consideration for therapeutic development and NKT cell physiology.
- Published
- 2012
- Full Text
- View/download PDF
38. Toward a formal synthesis of laureatin: unexpected rearrangements involving cyclic ether nucleophiles.
- Author
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Keshipeddy S, Martínez I, Castillo BF 2nd, Morton MD, and Howell AR
- Subjects
- Cyclization, Ethers, Cyclic chemical synthesis, Molecular Structure, Ethers, Cyclic chemistry
- Abstract
Laureatin, a metabolite of the red algae Laurencia nipponica, has shown potent activity as a mosquito larvicide. The two previously published syntheses of laureatin involved an initial preparation of the 8-membered cyclic ether, followed by formation of the oxetane ring. Our strategy was the reverse, i.e., to utilize an oxetane as the framework to construct the larger ring. During this work, attempted N-bromosuccinimide (NBS)-mediated cyclization of oxetane alcohol 17, prepared from readily accessible 2-methyleneoxetane 12, yielded epoxytetrahydrofuran 19 rather than the expected laureatin core. Further derivatization of 19 yielded trans fused bis-tetrahydrofuran 32. The synthesis of 19 and 32, as well as structural and stereochemical elucidation studies, are described.
- Published
- 2012
- Full Text
- View/download PDF
39. Glycolipids that elicit IFN-γ-biased responses from natural killer T cells.
- Author
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Tyznik AJ, Farber E, Girardi E, Birkholz A, Li Y, Chitale S, So R, Arora P, Khurana A, Wang J, Porcelli SA, Zajonc DM, Kronenberg M, and Howell AR
- Subjects
- Animals, Antigen-Presenting Cells immunology, Antigen-Presenting Cells metabolism, Antigens, CD1d chemistry, Antigens, CD1d metabolism, Antineoplastic Agents chemical synthesis, Antineoplastic Agents chemistry, Binding Sites, Cell Line, Crystallography, X-Ray, Galactosylceramides chemical synthesis, Galactosylceramides chemistry, Galactosylceramides pharmacology, Glycolipids chemical synthesis, Humans, Mice, Mice, Inbred C57BL, Natural Killer T-Cells immunology, Protein Structure, Tertiary, Antineoplastic Agents pharmacology, Glycolipids chemistry, Glycolipids pharmacology, Interferon-gamma metabolism, Natural Killer T-Cells drug effects, Natural Killer T-Cells metabolism
- Abstract
Natural killer T (NKT) cells recognize glycolipids presented by CD1d. The first antigen described, α-galactosyl ceramide (αGalCer), is a potential anticancer agent whose activity depends upon IFN-γ secretion. We report two analogs of αGalCer based on a naturally occurring glycosphingolipid, plakoside A. These compounds induce enhanced IFN-γ that correlates with detergent-resistant binding to CD1d and an increased stability of the lipid-CD1d complexes on antigen-presenting cells. Structural analysis on one of the analogs indicates that it is more deeply bound inside the CD1d groove, suggesting tighter lipid-CD1d interactions. To our knowledge, this is the first example in which structural information provides an explanation for the increased lipid-CD1d stability, likely responsible for the Th1 bias. We provide insights into the mechanism of IFN-γ-inducing compounds, and because our compounds activate human NKT cells, they could have therapeutic utility., (Copyright © 2011 Elsevier Ltd. All rights reserved.)
- Published
- 2011
- Full Text
- View/download PDF
40. Access to oxetane-containing psico-nucleosides from 2-methyleneoxetanes: a role for neighboring group participation?
- Author
-
Liang Y, Hnatiuk N, Rowley JM, Whiting BT, Coates GW, Rablen PR, Morton M, and Howell AR
- Subjects
- Adenine chemical synthesis, Butylene Glycols chemistry, Fluorine chemistry, Furans chemical synthesis, Humans, Magnetic Resonance Spectroscopy, Molecular Structure, Stereoisomerism, Adenine analogs & derivatives, Antiviral Agents chemical synthesis, Epoxy Compounds chemistry, Ethers, Cyclic chemistry, Lactones chemistry, Nucleosides chemistry
- Abstract
The first psico-oxetanocin analogue of the powerful antiviral natural product, oxetanocin A, has been readily synthesized from cis-2-butene-1,4-diol. Key 2-methyleneoxetane precursors were derived from β-lactones prepared by the carbonylation of epoxides. F(+)-mediated nucleobase incorporation provided the corresponding nucleosides in good yield but with low diastereoselectivity. Surprisingly, attempted exploitation of anchimeric assistance to increase the selectivity was not fruitful. A range of 2-methyleneoxetane and related 2-methylenetetrahydrofuran substrates was prepared to explore the basis for this. With one exception, these substrates also showed little stereoselectivity in nucleobase incorporation. Computational studies were undertaken to examine if neighboring group participation involving fused [4.2.0] or [4.3.0] intermediates is favorable.
- Published
- 2011
- Full Text
- View/download PDF
41. Vβ2 natural killer T cell antigen receptor-mediated recognition of CD1d-glycolipid antigen.
- Author
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Patel O, Pellicci DG, Uldrich AP, Sullivan LC, Bhati M, McKnight M, Richardson SK, Howell AR, Mallevaey T, Zhang J, Bedel R, Besra GS, Brooks AG, Kjer-Nielsen L, McCluskey J, Porcelli SA, Gapin L, Rossjohn J, and Godfrey DI
- Subjects
- Animals, Cloning, Molecular, Epitopes genetics, Epitopes immunology, Flow Cytometry, Glycolipids metabolism, Mice, Mutagenesis, Receptors, Antigen, T-Cell, alpha-beta genetics, Receptors, Antigen, T-Cell, alpha-beta metabolism, Surface Plasmon Resonance, Antigens, CD1d immunology, Glycolipids immunology, Receptors, Antigen, T-Cell, alpha-beta immunology
- Abstract
Natural killer T cell antigen receptors (NKT TCRs) recognize lipid-based antigens (Ags) presented by CD1d. Although the TCR α-chain is invariant, NKT TCR Vβ exhibits greater diversity, with one (Vβ11) and three (Vβ8, Vβ7, and Vβ2) Vβ chains in humans and mice, respectively. With the exception of the Vβ2 NKT TCR, NKT TCRs possess canonical tyrosine residues within complementarity determining region (CDR) 2β that are critical for CD1d binding. Thus, how Vβ2 NKT TCR docks with CD1d-Ag was unclear. Despite the absence of the CDR2β-encoded tyrosine residues, we show that the Vβ2 NKT TCR engaged CD1d-Ag in a similar manner and with a comparable affinity and energetic footprint to the manner observed for the Vβ8.2 and Vβ7 NKT TCRs. Accordingly, the germline-encoded regions of the TCR β-chain do not exclusively dictate the innate NKT TCR-CD1d-Ag docking mode. Nevertheless, clear fine specificity differences for the CD1d-Ag existed between the Vβ2 NKT TCR and the Vβ8.2 and Vβ7 NKT TCRs, with the Vβ2 NKT TCR exhibiting greater sensitivity to modifications to the glycolipid Ag. Furthermore, within the Vβ2 NKT TCR-CD1d-αGalCer complex, the CDR2β loop mediated fewer contacts with CD1d, whereas the CDR1β and CDR3β loops contacted CD1d to a much greater extent compared with most Vβ11, Vβ8.2, and Vβ7 NKT TCRs. Accordingly, there is a greater interplay between the germline- and nongermline-encoded loops within the TCR β-chain of the Vβ2 NKT TCR that enables CD1d-Ag ligation.
- Published
- 2011
- Full Text
- View/download PDF
42. A rapid fluorescence-based assay for classification of iNKT cell activating glycolipids.
- Author
-
Arora P, Venkataswamy MM, Baena A, Bricard G, Li Q, Veerapen N, Ndonye R, Park JJ, Lee JH, Seo KC, Howell AR, Chang YT, Illarionov PA, Besra GS, Chung SK, and Porcelli SA
- Subjects
- Animals, Cell Line, Flow Cytometry, Galactosylceramides chemistry, Humans, Mice, Spectrometry, Fluorescence, Time Factors, Drug Evaluation, Preclinical methods, Galactosylceramides pharmacology, Natural Killer T-Cells cytology, Natural Killer T-Cells drug effects
- Abstract
Structural variants of α-galactosylceramide (αGC) that activate invariant natural killer T cells (iNKT cells) are being developed as potential immunomodulatory agents for a variety of applications. Identification of specific forms of these glycolipids that bias responses to favor production of proinflammatory vs anti-inflammatory cytokines is central to current efforts, but this goal has been hampered by the lack of in vitro screening assays that reliably predict the in vivo biological activity of these compounds. Here we describe a fluorescence-based assay to identify functionally distinct αGC analogues. Our assay is based on recent findings showing that presentation of glycolipid antigens by CD1d molecules localized to plasma membrane detergent-resistant microdomains (lipid rafts) is correlated with induction of interferon-γ secretion and Th1-biased cytokine responses. Using an assay that measures lipid raft residency of CD1d molecules loaded with αGC, we screened a library of ∼200 synthetic αGC analogues and identified 19 agonists with potential Th1-biasing activity. Analysis of a subset of these novel candidate Th1 type agonists in vivo in mice confirmed their ability to induce systemic cytokine responses consistent with a Th1 type bias. These results demonstrate the predictive value of this novel in vitro assay for assessing the in vivo functionality of glycolipid agonists and provide the basis for a relatively simple high-throughput assay for identification and functional classification of iNKT cell activating glycolipids., (© 2011 American Chemical Society)
- Published
- 2011
- Full Text
- View/download PDF
43. A molecular basis for the exquisite CD1d-restricted antigen specificity and functional responses of natural killer T cells.
- Author
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Wun KS, Cameron G, Patel O, Pang SS, Pellicci DG, Sullivan LC, Keshipeddy S, Young MH, Uldrich AP, Thakur MS, Richardson SK, Howell AR, Illarionov PA, Brooks AG, Besra GS, McCluskey J, Gapin L, Porcelli SA, Godfrey DI, and Rossjohn J
- Subjects
- Animals, Carbohydrate Sequence, Cell Line, Cell Proliferation, Glycolipids immunology, Mice, Mice, Inbred C57BL, Models, Molecular, Molecular Sequence Data, Natural Killer T-Cells cytology, Receptors, Natural Killer Cell immunology, Antigens, CD1d immunology, Epitopes, Natural Killer T-Cells immunology
- Abstract
Natural killer T (NKT) cells respond to a variety of CD1d-restricted antigens (Ags), although the basis for Ag discrimination by the NKT cell receptor (TCR) is unclear. Here we have described NKT TCR fine specificity against several closely related Ags, termed altered glycolipid ligands (AGLs), which differentially stimulate NKT cells. The structures of five ternary complexes all revealed similar docking. Acyl chain modifications did not affect the interaction, but reduced NKT cell proliferation, indicating an affect on Ag processing or presentation. Conversely, truncation of the phytosphingosine chain caused an induced fit mode of TCR binding that affected TCR affinity. Modifications in the glycosyl head group had a direct impact on the TCR interaction and associated cellular response, with ligand potency reflecting the t(1/2) life of the interaction. Accordingly, we have provided a molecular basis for understanding how modifications in AGLs can result in striking alterations in the cellular response of NKT cells., (Copyright © 2011 Elsevier Inc. All rights reserved.)
- Published
- 2011
- Full Text
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44. Mouse and human iNKT cell agonist β-mannosylceramide reveals a distinct mechanism of tumor immunity.
- Author
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O'Konek JJ, Illarionov P, Khursigara DS, Ambrosino E, Izhak L, Castillo BF 2nd, Raju R, Khalili M, Kim HY, Howell AR, Besra GS, Porcelli SA, Berzofsky JA, and Terabe M
- Subjects
- Animals, Cell Line, Female, Galactosylceramides chemistry, Galactosylceramides immunology, Humans, Mice, Mice, Inbred BALB C, Mice, Inbred C57BL, Molecular Structure, Natural Killer T-Cells cytology, Neoplasm Transplantation, Ceramides chemistry, Ceramides immunology, Immune Tolerance immunology, Natural Killer T-Cells immunology, Neoplasms immunology
- Abstract
Type 1 or invariant NKT (iNKT) cell agonists, epitomized by α-galactosylceramide, protect against cancer largely by IFN-γ-dependent mechanisms. Here we describe what we believe to be a novel IFN-γ-independent mechanism induced by β-mannosylceramide, which also defines a potentially new class of iNKT cell agonist, with an unusual β-linked sugar. Like α-galactosylceramide, β-mannosylceramide directly activates iNKT cells from both mice and humans. In contrast to α-galactosylceramide, protection by β-mannosylceramide was completely dependent on NOS and TNF-α, neither of which was required to achieve protection with α-galactosylceramide. Moreover, at doses too low for either alone to protect, β-mannosylceramide synergized with α-galactosylceramide to protect mice against tumors. These results suggest that treatment with β-mannosylceramide provides a distinct mechanism of tumor protection that may allow efficacy where other agonists have failed. Furthermore, the ability of β-mannosylceramide to synergize with α-galactosylceramide suggests treatment with this class of iNKT agonist may provide protection against tumors in humans.
- Published
- 2011
- Full Text
- View/download PDF
45. Α-galactosylceramide analogs with weak agonist activity for human iNKT cells define new candidate anti-inflammatory agents.
- Author
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Bricard G, Venkataswamy MM, Yu KO, Im JS, Ndonye RM, Howell AR, Veerapen N, Illarionov PA, Besra GS, Li Q, Chang YT, and Porcelli SA
- Subjects
- CD40 Ligand metabolism, Drug Design, Enzyme-Linked Immunosorbent Assay methods, Flow Cytometry methods, Galactosylceramides agonists, Glycolipids chemistry, HeLa Cells, Humans, Interleukin-13 metabolism, Interleukin-4 metabolism, Leukocytes, Mononuclear cytology, Models, Chemical, Th2 Cells metabolism, Anti-Inflammatory Agents pharmacology, Galactosylceramides chemistry, Natural Killer T-Cells drug effects
- Abstract
CD1d-restricted natural killer T cells with invariant T cell receptor α chains (iNKT cells) are a unique lymphocyte subset that responds to recognition of specific lipid and glycolipid antigens. They are conserved between mice and humans and exert various immunoregulatory functions through their rapid secretion of a variety of cytokines and secondary activation of dendritic cells, B cells and NK cells. In the current study, we analyzed the range of functional activation states of human iNKT cells using a library of novel analogs of α-galactosylceramide (αGalCer), the prototypical iNKT cell antigen. Measurement of cytokines secreted by human iNKT cell clones over a wide range of glycolipid concentrations revealed that iNKT cell ligands could be classified into functional groups, correlating with weak versus strong agonistic activity. The findings established a hierarchy for induction of different cytokines, with thresholds for secretion being consistently lowest for IL-13, higher for interferon-γ (IFNγ), and even higher for IL-4. These findings suggested that human iNKT cells can be intrinsically polarized to selective production of IL-13 by maintaining a low level of activation using weak agonists, whereas selective polarization to IL-4 production cannot be achieved through modulating the strength of the activating ligand. In addition, using a newly designed in vitro system to assess the ability of human iNKT cells to transactivate NK cells, we found that robust secondary induction of interferon-γ secretion by NK cells was associated with strong but not weak agonist ligands of iNKT cells. These results indicate that polarization of human iNKT cell responses to Th2-like or anti-inflammatory effects may best be achieved through selective induction of IL-13 and suggest potential discrepancies with findings from mouse models that may be important in designing iNKT cell-based therapies in humans.
- Published
- 2010
- Full Text
- View/download PDF
46. Unexpected cleavage of 2-azido-2-(hydroxymethyl)oxetanes: conformation determines reaction pathway?
- Author
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Farber E, Herget J, Gascón JA, and Howell AR
- Subjects
- Kinetics, Molecular Conformation, Oxidation-Reduction, Quantum Theory, Azides chemistry, Ethers, Cyclic chemistry
- Abstract
An unanticipated cleavage of 2-azido-2-(hydroxymethyl)oxetanes is reported. In attempts to oxidize the title oxetanyl alcohols to the corresponding carboxylic acids with RuO4, cleaved nitriles were formed as the sole isolable products, while a closely related tetrahydrofuran gave solely the expected carboxylic acid. Quantum chemical calculations suggest that the divergent outcomes are governed by conformational differences in the azidoalcohols.
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- 2010
- Full Text
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47. A CD1d-dependent antagonist inhibits the activation of invariant NKT cells and prevents development of allergen-induced airway hyperreactivity.
- Author
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Lombardi V, Stock P, Singh AK, Kerzerho J, Yang W, Sullivan BA, Li X, Shiratsuchi T, Hnatiuk NE, Howell AR, Yu KO, Porcelli SA, Tsuji M, Kronenberg M, Wilson SB, and Akbari O
- Subjects
- Allergens administration & dosage, Animals, Antigens, CD1d metabolism, Binding, Competitive immunology, Cell Line, Disease Models, Animal, Female, Galactosylceramides administration & dosage, Galactosylceramides antagonists & inhibitors, Humans, Immunosuppressive Agents antagonists & inhibitors, Lymphocyte Activation drug effects, Mice, Mice, Inbred BALB C, Ovalbumin administration & dosage, Ovalbumin immunology, Phosphatidylethanolamines administration & dosage, Polyethylene Glycols administration & dosage, Allergens immunology, Antigens, CD1d physiology, Bronchial Hyperreactivity immunology, Bronchial Hyperreactivity prevention & control, Immunosuppressive Agents pharmacology, Lymphocyte Activation immunology, Natural Killer T-Cells immunology, Phosphatidylethanolamines pharmacology, Polyethylene Glycols pharmacology
- Abstract
The prevalence of asthma continues to increase in westernized countries, and optimal treatment remains a significant therapeutic challenge. Recently, CD1d-restricted invariant NKT (iNKT) cells were found to play a critical role in the induction of airway hyperreactivity (AHR) in animal models and are associated with asthma in humans. To test whether iNKT cell-targeted therapy could be used to treat allergen-induced airway disease, mice were sensitized with OVA and treated with di-palmitoyl-phosphatidyl-ethanolamine polyethylene glycol (DPPE-PEG), a CD1d-binding lipid antagonist. A single dose of DPPE-PEG prevented the development of AHR and pulmonary infiltration of lymphocytes upon OVA challenge, but had no effect on the development of OVA-specific Th2 responses. In addition, DPPE-PEG completely prevented the development of AHR after administration of alpha-galactosylceramide (alpha-GalCer) intranasally. Furthermore, we demonstrate that DPPE-PEG acts as antagonist to alpha-GalCer and competes with alpha-GalCer for binding to CD1d. Finally, we show that DPPE-PEG completely inhibits the alpha-GalCer-induced phosphorylation of ERK tyrosine kinase in iNKT cells, suggesting that DPPE-PEG specifically blocks TCR signaling and thus activation of iNKT cells. Because iNKT cells play a critical role in the development of AHR, the inhibition of iNKT activation by DPPE-PEG suggests a novel approach to treat iNKT cell-mediated diseases such as asthma.
- Published
- 2010
- Full Text
- View/download PDF
48. Adaptability of the semi-invariant natural killer T-cell receptor towards structurally diverse CD1d-restricted ligands.
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Florence WC, Xia C, Gordy LE, Chen W, Zhang Y, Scott-Browne J, Kinjo Y, Yu KO, Keshipeddy S, Pellicci DG, Patel O, Kjer-Nielsen L, McCluskey J, Godfrey DI, Rossjohn J, Richardson SK, Porcelli SA, Howell AR, Hayakawa K, Gapin L, Zajonc DM, Wang PG, and Joyce S
- Published
- 2009
- Full Text
- View/download PDF
49. Recognition of lyso-phospholipids by human natural killer T lymphocytes.
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Fox LM, Cox DG, Lockridge JL, Wang X, Chen X, Scharf L, Trott DL, Ndonye RM, Veerapen N, Besra GS, Howell AR, Cook ME, Adams EJ, Hildebrand WH, and Gumperz JE
- Subjects
- Antigen Presentation, Antigen-Presenting Cells immunology, Antigens, CD1d immunology, Autoantigens immunology, Cell Line, Cytokines biosynthesis, Humans, Inflammation immunology, Lymphocyte Activation, Natural Killer T-Cells metabolism, Phosphorylcholine analogs & derivatives, Phosphorylcholine immunology, Signal Transduction, Sphingosine analogs & derivatives, Sphingosine immunology, Lysophosphatidylcholines immunology, Natural Killer T-Cells immunology
- Abstract
Natural killer T (NKT) cells are a subset of T lymphocytes with potent immunoregulatory properties. Recognition of self-antigens presented by CD1d molecules is an important route of NKT cell activation; however, the molecular identity of specific autoantigens that stimulate human NKT cells remains unclear. Here, we have analyzed human NKT cell recognition of CD1d cellular ligands. The most clearly antigenic species was lyso-phosphatidylcholine (LPC). Diacylated phosphatidylcholine and lyso-phosphoglycerols differing in the chemistry of the head group stimulated only weak responses from human NKT cells. However, lyso-sphingomyelin, which shares the phosphocholine head group of LPC, also activated NKT cells. Antigen-presenting cells pulsed with LPC were capable of stimulating increased cytokine responses by NKT cell clones and by freshly isolated peripheral blood lymphocytes. These results demonstrate that human NKT cells recognize cholinated lyso-phospholipids as antigens presented by CD1d. Since these lyso-phospholipids serve as lipid messengers in normal physiological processes and are present at elevated levels during inflammatory responses, these findings point to a novel link between NKT cells and cellular signaling pathways that are associated with human disease pathophysiology., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2009
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50. Synthesis and evaluation of 3''- and 4''-deoxy and -fluoro analogs of the immunostimulatory glycolipid, KRN7000.
- Author
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Raju R, Castillo BF, Richardson SK, Thakur M, Severins R, Kronenberg M, and Howell AR
- Subjects
- Animals, Antigens, CD1d biosynthesis, Drug Design, Galactosylceramides chemistry, Glycolipids chemistry, Humans, Interleukin-2 metabolism, Mice, Models, Chemical, Natural Killer T-Cells, Porifera, Quinoxalines pharmacology, Adjuvants, Immunologic chemical synthesis, Adjuvants, Immunologic pharmacology, Chemistry, Pharmaceutical methods, Galactosylceramides chemical synthesis, Galactosylceramides pharmacology, Hybridomas metabolism, Quinoxalines chemical synthesis
- Abstract
Four 3''- and 4''-deoxy and -fluorogalactosyl ceramides were synthesized, and their ability to stimulate iNKT cells, based on levels of IL-2 production, was assessed in three NKT cell receptor hybridomas. In two of the hybridomas, 1.2 and 2H4, all of the analogs were immunostimulatory, while in the 1.4 hybridoma only the 4''-fluoro analog led to the production of significant levels of IL-2.
- Published
- 2009
- Full Text
- View/download PDF
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