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1.
Clin Transl Immunology ; 10(3): e1260, 2021.
Article in English | MEDLINE | ID: mdl-33732459

ABSTRACT

OBJECTIVES: A major COVID-19 vaccine strategy is to induce antibodies that prevent interaction between the Spike protein's receptor-binding domain (RBD) and angiotensin-converting enzyme 2 (ACE2). These vaccines will also induce T-cell responses. However, concerns were raised that aberrant vaccine-induced immune responses may exacerbate disease. We aimed to identify minimal epitopes on the RBD that would induce antibody responses that block the interaction of the RBD and ACE2 as a strategy leading to an effective vaccine with reduced risk of inducing immunopathology. METHODS: We procured a series of overlapping 20-amino acid peptides spanning the RBD and asked which were recognised by plasma from COVID-19 convalescent patients. Identified epitopes were conjugated to diphtheria-toxoid and used to vaccinate mice. Immune sera were tested for binding to the RBD and for their ability to block the interaction of the RBD and ACE2. RESULTS: Seven putative vaccine epitopes were identified. Memory B-cells (MBCs) specific for one of the epitopes were identified in the blood of convalescent patients. When used to vaccinate mice, six induced antibodies that bound recRBD and three induced antibodies that could partially block the interaction of the RBD and ACE2. However, when the sera were combined in pairs, we observed significantly enhanced inhibition of binding of RBD to ACE2. Two of the peptides were located in the main regions of the RBD known to contact ACE2. Of significant importance to vaccine development, two of the peptides were in regions that are invariant in the UK and South African strains. CONCLUSION: COVID-19 convalescent patients have SARS-CoV-2-specific antibodies and MBCs, the specificities of which can be defined with short peptides. Epitope-specific antibodies synergistically block RBD-ACE2 interaction.

2.
Cancer Chemother Pharmacol ; 87(5): 673-679, 2021 05.
Article in English | MEDLINE | ID: mdl-33585999

ABSTRACT

PURPOSE: Preclinical studies support the JAK2-STAT3 signaling pathway as a key driver in CD44+ CD24- "stem-cell-like" breast cancer cells. Ruxolitinib is an orally bioavailable JAK1/2 inhibitor. We aimed to identify the recommended phase 2 dose (RP2D) of ruxolitinib in combination with paclitaxel in patients with HER2-negative metastatic breast cancer (MBC). METHODS: Eligible patients had HER2-negative MBC and had received ≤ 3 chemotherapy regimens for advanced disease. Patients received oral ruxolitinib (10-25 mg bid) in a 3 + 3 dose escalation design in combination with weekly paclitaxel 80 mg/m2 in a 3-week cycle. The primary objective was to determine the maximum tolerated dose (MTD) and the RP2D. RESULTS: Nineteen patients received protocol therapy (mean age 52 years). Eight (42%) had triple-negative breast cancer and 11 (58%) had hormone receptor-positive disease; 12 (63%) had visceral disease. Ten (53%) patients had not received prior treatment for advanced disease. Patients received a median number of 5 cycles of combination therapy (range 1-12) and five patients continued single-agent ruxolitinib. The MTD of ruxolitinib was 25 mg bid when combined with paclitaxel, and the RP2D for the combination was 15 mg bid. Thirteen (68%) patients required dose reductions or holds. Most frequent toxicities reported of any grade were neutropenia (50%) and anemia (33%). There were no grade 4/5 toxicities attributed to study drug. Four (21%) patients had PR, 12 (63%) had SD and three (16%) had PD as their best response. CONCLUSION: The combination of ruxolitinib and weekly paclitaxel was well tolerated with evidence of clinical activity. Further analysis of this combination is ongoing (NCT02041429). TRIAL REGISTRATION: NCT02041429. Date of registration: January 22, 2014.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Breast Neoplasms/drug therapy , Janus Kinase Inhibitors/administration & dosage , Paclitaxel/administration & dosage , Pyrazoles/administration & dosage , Adult , Aged , Breast Neoplasms/chemistry , Breast Neoplasms/pathology , Female , Humans , Middle Aged , Neoplasm Metastasis , Nitriles , Paclitaxel/adverse effects , Pyrazoles/adverse effects , Pyrimidines , Receptor, ErbB-2/analysis
3.
Breast Cancer Res ; 21(1): 43, 2019 03 21.
Article in English | MEDLINE | ID: mdl-30898150

ABSTRACT

BACKGROUND: The oncogenic receptor tyrosine kinase (RTK) ERBB2 is known to dimerize with other EGFR family members, particularly ERBB3, through which it potently activates PI3K signalling. Antibody-mediated inhibition of this ERBB2/ERBB3/PI3K axis has been a cornerstone of treatment for ERBB2-amplified breast cancer patients for two decades. However, the lack of response and the rapid onset of relapse in many patients now question the assumption that the ERBB2/ERBB3 heterodimer is the sole relevant effector target of these therapies. METHODS: Through a systematic protein-protein interaction screen, we have identified and validated alternative RTKs that interact with ERBB2. Using quantitative readouts of signalling pathway activation and cell proliferation, we have examined their influence upon the mechanism of trastuzumab- and pertuzumab-mediated inhibition of cell growth in ERBB2-amplified breast cancer cell lines and a patient-derived xenograft model. RESULTS: We now demonstrate that inactivation of ERBB3/PI3K by these therapeutic antibodies is insufficient to inhibit the growth of ERBB2-amplified breast cancer cells. Instead, we show extensive promiscuity between ERBB2 and an array of RTKs from outside of the EGFR family. Paradoxically, pertuzumab also acts as an artificial ligand to promote ERBB2 activation and ERK signalling, through allosteric activation by a subset of these non-canonical RTKs. However, this unexpected activation mechanism also increases the sensitivity of the receptor network to the ERBB2 kinase inhibitor lapatinib, which in combination with pertuzumab, displays a synergistic effect in single-agent resistant cell lines and PDX models. CONCLUSIONS: The interaction of ERBB2 with a number of non-canonical RTKs activates a compensatory signalling response following treatment with pertuzumab, although a counter-intuitive combination of ERBB2 antibody therapy and a kinase inhibitor can overcome this innate therapeutic resistance.


Subject(s)
Breast Neoplasms/metabolism , Drug Resistance, Neoplasm , Protein Kinase Inhibitors/pharmacology , Protein Multimerization , Receptor, ErbB-2/chemistry , Receptor, ErbB-2/metabolism , Animals , Antibodies, Monoclonal, Humanized/pharmacology , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Cell Line, Tumor , Disease Models, Animal , Dose-Response Relationship, Drug , Female , Fluorescent Antibody Technique , Humans , Immunohistochemistry , Mice , Phosphorylation , Receptor, ErbB-2/antagonists & inhibitors , Signal Transduction/drug effects , Trastuzumab/pharmacology , Xenograft Model Antitumor Assays
4.
Plant Cell Environ ; 42(1): 188-197, 2019 01.
Article in English | MEDLINE | ID: mdl-29722016

ABSTRACT

Legumes form root nodules to house beneficial nitrogen-fixing rhizobia bacteria. However, nodulation is resource demanding; hence, legumes evolved a systemic signalling mechanism called autoregulation of nodulation (AON) to control nodule numbers. AON begins with the production of CLE peptides in the root, which are predicted to be glycosylated, transported to the shoot, and perceived. We synthesized variants of nodulation-suppressing CLE peptides to test their activity using petiole feeding to introduce CLE peptides into the shoot. Hydroxylated, monoarabinosylated, and triarabinosylated variants of soybean GmRIC1a and GmRIC2a were chemically synthesized and fed into recipient Pisum sativum (pea) plants, which were used due to the availability of key AON pathway mutants unavailable in soybean. Triarabinosylated GmRIC1a and GmRIC2a suppressed nodulation of wild-type pea, whereas no other peptide variant tested had this ability. Suppression also occurred in the supernodulating hydroxyproline O-arabinosyltransferase mutant, Psnod3, but not in the supernodulating receptor mutants, Pssym29, and to some extent, Pssym28. During our study, bioinformatic resources for pea became available and our analyses identified 40 CLE peptide-encoding genes, including orthologues of nodulation-suppressive CLE peptides. Collectively, we demonstrated that soybean nodulation-suppressive CLE peptides can function interspecifically in the AON pathway of pea and require arabinosylation for their activity.


Subject(s)
Arabinose/metabolism , Peas/growth & development , Peptides/metabolism , Plant Root Nodulation , Gene Expression Regulation, Plant , Metabolic Networks and Pathways/physiology , Peas/metabolism , Peptides/physiology , Plant Proteins/metabolism , Plant Proteins/physiology , Root Nodules, Plant/growth & development , Root Nodules, Plant/metabolism
5.
J Org Chem ; 81(6): 2607-11, 2016 Mar 18.
Article in English | MEDLINE | ID: mdl-26907566

ABSTRACT

The pseudaminic acids are a family of 5,7-diamino-3,5,7,9-tetradeoxynonulosonic acids that are functional components of flagellin and pili proteins within clinically relevant Gram-negative bacteria. Herein, we describe the total synthesis of the most common pseudaminic acid, 5,7-diacetylpseudaminic acid, from N-acetylneuraminic acid. The divergent nature of the route reported here provides a robust and versatile means to access other members of the family, together with analogues, for probing the functional role of the pseudaminic acids and pseudaminic acid derived proteins in the future.


Subject(s)
Fimbriae Proteins/chemistry , Flagellin/chemistry , Gram-Negative Bacteria/chemistry , N-Acetylneuraminic Acid/chemistry , Sugar Acids/chemistry , Sugar Acids/chemical synthesis , Glycosylation , Magnetic Resonance Spectroscopy
6.
Am J Physiol Cell Physiol ; 299(4): C854-65, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20631249

ABSTRACT

Platelet endothelial cell adhesion molecule-1 (PECAM-1) has been implicated in endothelial cell motility during angiogenesis. Although there is evidence that SHP-2 plays a role in PECAM-1-dependent cell motility, the molecular basis of the activity of SHP-2 in this process has not been defined. To investigate the requirement of SHP-2 in PECAM-1-dependent cell motility, studies were done in which various constructs of SHP-2 were expressed in cell transfectants expressing PECAM-1. We observed that the levels of PECAM-1 tyrosine phosphorylation and SHP-2 association with PECAM-1 were significantly increased in cells expressing a phosphatase-inactive SHP-2 mutant, suggesting that the level of PECAM-1 tyrosine phosphorylation, and thus SHP-2 binding are regulated in part by bound, catalytically active SHP-2. We subsequently found that expression of PECAM-1 stimulated wound-induced migration and the formation of filopodia (a morphological feature of motile cells). These activities were associated with increased mitogen-activated protein kinase (MAPK) activation and the dephosphorylation of paxillin (an event implicated in the activation of MAPK). The phosphatase-inactive SHP-2 mutant, however, suppressed these PECAM-1-dependent phenomena, whereas the activity of PECAM-1 expressing cells was not altered by expression of wild-type SHP-2 or SHP-2 in which the scaffold/adaptor function had been disabled. Pharmacological inhibition of SHP-2 phosphatase activity also suppressed PECAM-1-dependent motility. Furthermore, PECAM-1 expression also stimulates tube formation, but none of the SHP-2 constructs affected this process. These findings therefore suggest a model for the involvement of SHP-2 in PECAM-1-dependent motility in which SHP-2, recruited by its interaction with PECAM-1, targets paxillin to ultimately activate the MAPK pathway and downstream events required for cell motility.


Subject(s)
Cell Movement/physiology , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Animals , Cell Line , Cell Proliferation , Endothelial Cells/cytology , Endothelial Cells/physiology , Enzyme Activation , Extracellular Signal-Regulated MAP Kinases/genetics , Extracellular Signal-Regulated MAP Kinases/metabolism , Humans , Mutagenesis, Site-Directed , Neovascularization, Physiologic , Paxillin/metabolism , Platelet Endothelial Cell Adhesion Molecule-1/genetics , Protein Tyrosine Phosphatase, Non-Receptor Type 11/genetics , Signal Transduction/physiology
7.
Am J Pathol ; 175(2): 903-15, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19574426

ABSTRACT

Platelet endothelial cell adhesion molecule (PECAM)-1 has been previously implicated in endothelial cell migration; additionally, anti-PECAM-1 antibodies have been shown to inhibit in vivo angiogenesis. Studies were therefore performed with PECAM-1-null mice to further define the involvement of PECAM-1 in blood vessel formation. Vascularization of subcutaneous Matrigel implants as well as tumor angiogenesis were both inhibited in PECAM-1-null mice. Reciprocal bone marrow transplants that involved both wild-type and PECAM-1-deficient mice revealed that the impaired angiogenic response resulted from a loss of endothelial, but not leukocyte, PECAM-1. In vitro wound migration and single-cell motility by PECAM-1-null endothelial cells were also compromised. In addition, filopodia formation, a feature of motile cells, was inhibited in PECAM-1-null endothelial cells as well as in human endothelial cells treated with either anti-PECAM-1 antibody or PECAM-1 siRNA. Furthermore, the expression of PECAM-1 promoted filopodia formation and increased the protein expression levels of Cdc42, a Rho GTPase that is known to promote the formation of filopodia. In the developing retinal vasculature, numerous, long filamentous filopodia, emanating from endothelial cells at the tips of angiogenic sprouts, were observed in wild-type animals, but to a lesser extent in the PECAM-1-null mice. Together, these data further establish the involvement of endothelial PECAM-1 in angiogenesis and suggest that, in vivo, PECAM-1 may stimulate endothelial cell motility by promoting the formation of filopodia.


Subject(s)
Neovascularization, Pathologic/physiopathology , Neovascularization, Physiologic/physiology , Platelet Endothelial Cell Adhesion Molecule-1/physiology , Animals , Cell Movement/genetics , Cells, Cultured , Collagen , Drug Combinations , Humans , Laminin , Mice , Neoplasms/blood supply , Neoplasms/pathology , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/pathology , Neovascularization, Physiologic/genetics , Platelet Endothelial Cell Adhesion Molecule-1/genetics , Proteoglycans , Pseudopodia/physiology , Retina/growth & development , Retinal Vessels , cdc42 GTP-Binding Protein/biosynthesis
8.
Am J Physiol Lung Cell Mol Physiol ; 296(6): L1096-103, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19304908

ABSTRACT

Several protocols for the isolation of endothelial cells (ECs) from murine lung have been described in the literature. We, however, encountered a number of problems while using these procedures that prevented us from consistently or reliably obtaining pure populations of ECs from the lungs of mice. By incorporating specific elements from previously published protocols, as well as adding some novel features, we developed a new strategy for isolating ECs from murine lung. In this approach, a suspension of lung cells is initially prepared from the lungs of 7- to 14-day-old mouse pups using procedures that prevent intravascular clotting and leukocyte activation, minimize mechanical trauma to the lung tissue, and limit exposure to the digesting enzymes. The resulting cell suspension is cultured for 2-3 days, trypsinized to produce a suspension of single cells, and then subjected to fluorescence-activated cell sorting using an anti-ICAM-2 antibody. The sorted cells are then plated and split 1:2 at each passage to maintain a high density of the cells. Using this approach, we have been able to isolate pure populations of ECs that were sustainable for extended periods in culture without the emergence of fibroblast overgrowth or the development of senescence. We believe the success of this approach will provide opportunities to take advantage of the large and growing number of knockout and transgenic mouse lines to investigate the endothelial-specific roles of targeted molecules in the pulmonary vasculature.


Subject(s)
Cell Separation/methods , Endothelial Cells/cytology , Flow Cytometry/methods , Lung/cytology , 3T3 Cells , Animals , Cell Line, Tumor , Cell Separation/instrumentation , Fibroblasts/cytology , Melanoma , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Skin Neoplasms
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