Biology subjects

McCrae, K.

Publications and source records attributed to McCrae, K..

5 recordsLinked to original sources

bioRxiv2026

Bradykinin Contributes to Vasogenic Edema in Murine Experimental Cerebral Malaria

Cerebral malaria (CM) due to Plasmodium falciparum (Pf) infection is a major cause of death in African children. Bradykinin (BK) is a mediator of vasogenic edema. It could contribute to the pathogenesis of central nervous system malaria in Kenyan children and P. berghei ANKA (PbA) infected C57BL/6J mice with experimental cerebral malaria. Cleaved plasma high molecular weight kininogen (cHK) is a marker for prior BK release. 40% of children with central nervous system malaria had plasma cHK versus 18% of children with uncomplicated malaria. Wild-type PbA-infected mice had circulating plasma cHK, elevated BK levels, and reduced HK and prekallikrein levels. HK null (Kng1-/-), combined BK B1 and B2 receptor null (Bdkrb1-/- / Bdkrb2-/-), BK B2 (Bdkrb2-/-) or BK B1 (Bdkrb1-/-) receptor null mice were protected from neurologic deterioration and brain edema compared to wild-type mice. F12-/-mice were not protected from neurological deterioration. Prekallikrein null (Klkb1-/-), prolylcarboxypeptidase hypomorphs (Prcpgt/gt), and brain endothelial cell conditional knockout of PRCP (Prcpfl/fl Cre) mice had reduced neurologic deterioration and brain edema. Adjuvant plasma kallikrein inhibition combined with artesunate treatment of PbA-infected mice reversed neurologic deterioration and brain edema and prolonged survival relative to artesunate alone. BK-induced vasogenic edema contributes to human and murine CM.

pathology↗

bioRxiv2024

Targeting an Olfactory Receptor Mitigates High Residual Platelet Reactivity and Arterial Thrombosis Through Actin Cytoskeleton Depolymerization

BackgroundDespite antiplatelet therapy, some patients remain at high risk for ischemic events due to medication non-responsiveness or High Residual Platelet Reactivity (HRPR). Our goal was to target an orphan G-Protein-Coupled Receptor (GPCR) on platelets belonging to the olfactory receptor family as a new antithrombotic strategy. MethodsUsing an engineered reporter cell line expressing Olfactory Receptor 2L13 (OR2L13) that was recently identified as an orphan GPCR to limit platelet reactivity, we performed a high-throughput screen (HST) of non-odorant bioactive compounds with counter-screen validation, followed by studies to determine changes in platelet function in healthy subjects and in patients with coronary artery disease and peripheral artery disease. Phospho-proteomics identified key signal transduction pathways, and a variety of ex vivo assays and in vivo functional studies examined the impact of an identified non-odorant compound on platelet signaling, platelet biomechanics, and thrombosis. ResultsWe identified 6 ligands specific for OR2L13 that function as receptor agonists, leading to the suppression of platelet aggregation and -granule exocytosis via P2Y12, PAR1, Thromboxane Receptor (TxR) and glycoprotein VI (GPVI) receptors, suggesting involvement of common downstream mediator. The lead OR2L13 agonist identified phosphorylates heat shock protein 27 (HSP27) and depolymerizes the platelet filamentous actin cytoskeleton which was further confirmed in a clot retraction assay (CCF0054500 clot area 70.6 vs. vehicle clot area 5.2, P<0.0001). This anti-thrombotic effect of the OR2L13 agonist was reversed by a HSP27 inhibitor (clot area 3.6, P<0.0001). In a murine cremaster arterial injury model, platelet accumulation at the injury site was reduced by 88.9% by the lead OR2L13 agonist compared to vehicle (P<0.0003) without altering fibrin generation in vivo, or interfering with the coagulation cascade, and without impairing the protective mechanism of platelet hemostasis. ConclusionWe describe and characterize the first non-olfactory tool to target an olfactory receptor for the purpose of inhibiting platelet activation and thrombosis through downstream HSP27 in a comprehensive investigation using a first-of-its kind platelet inhibitor targeting an orphan platelet GPCR.

physiology↗

bioRxiv2024

Complement Biosensors Identify a Classical Pathway Stimulus in Complement-Mediated Hemolytic Uremic Syndrome

Complement-mediated hemolytic uremic syndrome (CM-HUS) is a thrombotic microangiopathy characterized by germline variants or acquired antibodies to complement proteins and regulators. Building upon our prior experience with the modified Ham (mHam) assay for ex vivo diagnosis of complementopathies, we have developed an array of cell-based complement "biosensors by selective removal of complement regulatory proteins (CD55 and CD59, CD46, or a combination thereof) in an autonomously bioluminescent HEK293 cell line. These biosensors can be used as a sensitive method for diagnosing CM-HUS and monitoring therapeutic complement blockade. Using specific complement pathway inhibitors, this model identifies IgM-driven classical pathway stimulus during both acute disease and in many patients during clinical remission. This provides a potential explanation for ~50% of CM-HUS patients who lack an alternative pathway "driving" variant and suggests at least a subset of CM-HUS is characterized by a breakdown of IgM immunologic tolerance. Key PointsO_LICM-HUS has a CP stimulus driven by polyreactive IgM, addressing the mystery of why 40% of CM-HUS lack complement specific variants C_LIO_LIComplement biosensors and the bioluminescent mHam can be used to aid in diagnosis of CM-HUS and monitor complement inhibitor therapy C_LI

immunology↗

bioRxiv2021

Lymphocyte-specific Protein Tyrosine Kinase Regulates Homologous Recombination (HR) DNA Repair and Targeting Enhances PARPi Utility in HR Proficient Ovarian Cancer

Poly-ADP Ribose Polymerase (PARP) targeted therapy is clinically approved for the treatment of homologous recombination (HR) repair deficient tumors. The remarkable success in treatment of HR repair deficient cancers has not translated to HR-proficient cancer. Our studies identify the mechanism of non-receptor lymphocyte-specific protein tyrosine kinase (LCK) in HR repair in endometrioid epithelial ovarian cancer (eEOC). LCK expression is induced and activation in the nucleus in response to DNA damage insult. LCK inhibition attenuates expression of RAD51, BRCA1, and BRCA2 proteins necessary for HR-mediated DNA repair, sufficient to suppress RAD51 foci formation, and augments {gamma}H2AX foci formation. Mechanistically, DNA damage leads to direct interaction of LCK with RAD51 and BRCA1 in a kinase dependent manner. Attenuation of LCK sensitized HR-proficient eEOC cells to PARP inhibitor in cell culture and pre-clinical mouse studies. These findings identify a new mechanism for expanding utility of PARP inhibitors in HR proficient ovarian cancer. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/433791v2_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1715c60org.highwire.dtl.DTLVardef@167cb5forg.highwire.dtl.DTLVardef@158d807org.highwire.dtl.DTLVardef@1ced201_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2020

X-ray and solution structures of human beta-2 glycoprotein I reveal a new mechanism of autoantibody recognition

Venous and arterial thromboses in patients suffering from the autoimmune disorder Antiphospholipid Syndrome (APS) are caused by the presence of antiphospholipid antibodies (aPL). Emerging evidence indicates that autoantibodies targeting the epitope R39-R43 in the N-terminal domain, Domain I (DI), of {beta}2-glycoprotein I ({beta}2GPI) are among the most pathogenic aPL in patients with APS. How such autoantibodies engage {beta}2GPI at the molecular level remains incompletely understood. Here, we have used X-ray crystallography, single-molecule FRET, and small-angle X-ray scattering to demonstrate that, in the free form, under physiological pH and salt concentrations, human recombinant {beta}2GPI adopts an elongated, flexible conformation in which DI is exposed to the solvent, thus available for autoantibody recognition. Consistent with this structural model, binding and mutagenesis studies revealed that the elongated form interacts with a pathogenic anti-DI antibody in solution, without the need of phospholipids. Furthermore, complex formation was affected neither by the neighboring domains, nor by the presence of the linkers, nor by the glycosylations. Since the pathogenic autoantibody requires residues R39 and R43 for optimal binding, these findings challenge longstanding postulates in the field envisioning {beta}2GPI adopting immunologic inert conformations featuring inaccessibility of the epitope R39-R43 in DI and support an alternative model whereby the preferential binding of anti-DI antibodies towards phospholipid-bound {beta}2GPI arises from the ability of the pre-existing elongated form to bind to the membranes and then oligomerize, processes that are likely to be supported by protein conformational changes. Interfering with these steps may limit the pathogenic effects of anti-DI antibodies in APS patients. SignificanceIn the autoimmune disorder called Antiphospholipid Syndrome (APS), the presence of autoantibodies targeting the plasma glycoprotein beta-2 glycoprotein I ({beta}2GPI) is associated with arterial and venous thrombosis as well as pregnancy complications. Understanding how {beta}2GPI becomes immunogenic and how autoantibodies in complex with {beta}2GPI cause the blood to clot remains a top priority in the field. By elucidating the structural architecture of {beta}2GPI free in solution, our studies challenge longstanding postulates in the field and shed new light on the pathogenic mechanisms of APS that may help the development of new diagnostics and therapeutic approaches.

biochemistry↗