Papers by Deborah Donahue
Blood, Nov 16, 2005
In Clinical sepsis and human endotoxin studies, administration of the recombinant human form of t... more In Clinical sepsis and human endotoxin studies, administration of the recombinant human form of the anticoagulant activated protein C (rhaPC) demonstrates benefit in parameters of coagulation, inflammation, shock, and sepsis survival. Utilizing transgenic mice deficient in the endothelial protein C receptor, (EPCR), we investigated whether provision of rhaPC improved the hypotensive response and survival subsequent to administration of a lethal dose of endotoxin (LPS). A novel central venous infusion protocol for rhaPC was developed using low expressing EPCR mice (EPCRδ/δ surface expression <10% of WT EPCR) which were generated in our center. WT C57Bl/6 (>F8) mice were used as controls. An aortic arch continuous blood pressure (B.P.) monitor and right internal jugular vein catheter were placed. The continuous infusion dosing of rhaPC (Eli Lilly) was approximately 1.8 μg/g/hr for 20 hr through the central venous catheter (N=3 per group). The control group received saline. Bolus LPS (10 μg/g i.p.) was administered concomitant to rhaPC. Continuous B.P. measurements were obtained until death or for up to 72 hr. RhaPC kinetic parameters (N=4 per group) collected from WT citrate-benzamidine plasma samples were determined by monoclonal antibody capture assays. In initial kinetic studies plasma levels of rhaPC in WT mice given LPS achieved steady state concentrations (90 ng/mL at 4 hr) similar to that found in human studies (45 ng/mL at 2hr). RhaPC dosing produced no allergic reaction or overt bleeding. The subsequent 72 hour B.P. study achieved the goal of suppressing endotoxin-induced hypotension in both WT and EPCRδ/δ mice. The two LPS/rhaPC-treated groups were able to overcome the hypotensive stress and survive longer than the two LPS-only treated groups. RhaPC treated EPCRδ/δ mice were able to recover blood pressure to the same or greater degree than the rhaPC treated WT mice, suggesting either enhanced cell signaling potential by less cell surface EPCR or an alternate aPC receptor. The administered volume of 30 μL/hr (0.6…
Journal of Bacteriology, Mar 23, 2020
Streptococcus pyogenes, or Group A Streptococcus (GAS) is both a pathogen and asymptomatic coloni... more Streptococcus pyogenes, or Group A Streptococcus (GAS) is both a pathogen and asymptomatic colonizer of human hosts, and produces a large number of surfaceexpressed and secreted factors that contribute to a variety of infection outcomes. The GAS-secreted cysteine protease SpeB has been well studied for its effects on the human host; however, despite its broad proteolytic activity, studies on how this factor is utilized in polymicrobial environments are lacking. Here, we utilized various forms of SpeB protease to evaluate antimicrobial and anti-biofilm properties against the clinically important human colonizer Staphylococcus aureus, which occupies similar niches to GAS. For our investigation, we used a skin-trophic GAS strain, AP53CovS+, and its isogenic ΔspeB mutant to compare the production and activity of native SpeB protease. We also generated active and inactive forms of recombinant purified SpeB for functional studies. We demonstrate that SpeB exhibits potent biofilm disruption activity at multiple stages of S. aureus biofilm formation. We hypothesized that the surface expressed adhesin SdrC in S. aureus was cleaved by SpeB, which contributed to the observed biofilm disruption. Indeed, we found that SpeB cleaved recombinant SdrC in vitro and in the context of the full S. aureus biofilm. Our results suggest an understudied role for the broadly proteolytic SpeB as an important factor for GAS colonization and competition with other microorganisms in its niche. 3 Importance Streptococcus pyogenes (GAS) causes a range of disease in humans ranging from mild to severe, and produces many virulence factors in order to be a successful pathogen. One factor produced by many GAS strains is the protease SpeB, which has been studied for its ability to cleave and degrade human proteins, important in GAS pathogenesis. An understudied role for SpeB is the manner in which its broad proteolytic activity affects other microorganisms that co-occupy similar niches to GAS. The significance of the research reported herein is the demonstration that SpeB can degrade the biofilms of the human pathogen Staphylococcus aureus, which has important implications as to how SpeB may be utilized by GAS to successfully compete in a polymicrobial environment.
Journal of Thrombosis and Haemostasis, Jun 1, 2023
Frontiers in Cellular and Infection Microbiology, Nov 1, 2022
Group A Streptococcus (GAS, Streptococcus pyogenes) is a Gram-positive human pathogen that employ... more Group A Streptococcus (GAS, Streptococcus pyogenes) is a Gram-positive human pathogen that employs several secreted and surface-bound virulence factors to manipulate its environment, allowing it to cause a variety of disease outcomes. One such virulence factor is Streptolysin S (SLS), a ribosomallyproduced peptide toxin that undergoes extensive post-translational modifications. The activity of SLS has been studied for over 100 years owing to its rapid and potent ability to lyse red blood cells, and the toxin has been shown to play a major role in GAS virulence in vivo. We have previously demonstrated that SLS induces hemolysis by targeting the chloride-bicarbonate exchanger Band 3 in erythrocytes, indicating that SLS is capable of targeting host proteins to promote cell lysis. However, the possibility that SLS has additional protein targets in other cell types, such as keratinocytes, has not been explored. Here, we use bioinformatics analysis and chemical inhibition studies to demonstrate that SLS targets the electroneutral sodium-bicarbonate cotransporter NBCn1 in keratinocytes during GAS infection. SLS induces NF-kB activation and host cytotoxicity in human keratinocytes, and these processes can be mitigated by treating keratinocytes with the sodium-bicarbonate cotransport inhibitor S0859. Furthermore, treating keratinocytes with SLS disrupts the ability of host cells to regulate their intracellular pH, and this can be monitored in real time using the pH-sensitive dye pHrodo Red AM in live imaging studies. These results demonstrate that SLS is a multifunctional bacterial toxin that GAS uses Frontiers in Cellular and Infection Microbiology frontiersin.org 01
Frontiers in Cardiovascular Medicine, Jun 10, 2021
Scientific reports, Jan 23, 2017
Plasminogen activator inhibitor-1 (PAI-1) is known to protect mice against cardiac fibrosis. It h... more Plasminogen activator inhibitor-1 (PAI-1) is known to protect mice against cardiac fibrosis. It has been speculated that PAI-1 may regulate cardiac fibrosis by inactivating urokinase-type plasminogen activator (uPA) and ultimately plasmin (Pm) generation. However, the in vivo role of PAI-1 in inactivating uPA and limiting the generation of Pm during cardiac fibrosis remains to be established. The objective of this study was to determine if the cardioprotective effect of PAI-1 is mediated through its ability to directly regulate urokinase -mediated activation of plasminogen (Pg). An Angiotensin II (AngII)-aldosterone (Ald) infusion mouse model of hypertension was utilised in this study. Four weeks after AngII-Ald infusion, PAI-1-deficient (PAI-1(-/-)) mice developed severe cardiac fibrosis. However, a marked reduction in cardiac fibrosis was observed in PAI-1(-/-)/uPA(-/-) double knockout mice that was associated with reduced inflammation, lower expression levels of TGF-β and proteas...
The development of antibiotic resistance and the resulting emergence of multidrug-resistant bacte... more The development of antibiotic resistance and the resulting emergence of multidrug-resistant bacteria has become one of the main threats in the public health system, commonly leading to nosocomial infections. Many researchers have turned their focus to developing alternative classes of antibacterial systems based on various nanomaterials. We have developed an antibiotic-free nanoparticle system, inspired by naturally occurring bacteriophages, to fight antibiotic-resistant bacteria. Our phage-mimicking nanoparticles (PhaNPs) display structural mimicry of protein-turret distribution on the head structure of bacteriophages. By mimicking phages, we are able to take advantage of their evolutionary constant shape and their high antibacterial activity while avoiding immune reactions of the human body, potentially caused by phages. We describe the synthesis of hierarchically arranged core-shell nanoparticles, with a silica core conjugated with silver-coated gold nanospheres. Improving on our...
High levels of LDL-cholesterol rescue the neonatal mortality associated with afibrinogenemia in mice
Proceedings of the National Academy of Sciences, 2011
The protein C (PC) pathway is a well-characterized coagulation system. Endothelial PC receptors a... more The protein C (PC) pathway is a well-characterized coagulation system. Endothelial PC receptors and thrombomodulin mediate the conversion of PC to its activated form, a potent anticoagulant and anti-inflammatory molecule. Here we show that the PC pathway is expressed on intestinal epithelial cells. The epithelial expression of PC and endothelial PC receptor is down-regulated In patients with inflammatory bowel disease. PC −/− /PC(Tg) mice, expressing only 3% of WT PC, developed spontaneous intestinal inflammation and were prone to severe experimental colitis. These mice also demonstrated spontaneous elevated production of inflammatory cytokines and increased intestinal permeability. Structural analysis of epithelial tight junction molecules revealed that lack of PC leads to decreased JAM-A and claudin-3 expression and an altered pattern of ZO-1 expression. In vitro, treatment of epithelial cells with activated PC led to protection of tight junction disruption induced by TNF-α, and i...
Journal of Trauma and Acute Care Surgery, 2014
BACKGROUND-Coagulopathy in traumatic brain injury (CTBI) is a well-established phenomenon, but it... more BACKGROUND-Coagulopathy in traumatic brain injury (CTBI) is a well-established phenomenon, but its mechanism is poorly understood. Various studies implicate protein C activation related to the global insult of hemorrhagic shock or brain tissue factor release with resultant platelet dysfunction and depletion of coagulation factors. We hypothesized that the platelet dysfunction of CTBI is a distinct phenomenon from the coagulopathy following hemorrhagic shock. METHODS-We used thrombelastography with platelet mapping as a measure of platelet function, assessing the degree of inhibition of the adenosine diphosphate (ADP) and arachidonic acid (AA) receptor pathways. First, we studied the early effect of TBI on platelet inhibition by performing thrombelastography with platelet mapping on rats. We then conducted an analysis of admission blood samples from trauma patients with isolated head injury (n = 70). Patients in shock or on clopidogrel or aspirin were excluded. RESULTS-In rats, ADP receptor inhibition at 15 minutes after injury was 77.6% ± 6.7% versus 39.0% ± 5.3% for controls (p < 0.0001). Humans with severe TBI (Glasgow Coma Scale [GCS] score ≤ 8) showed an increase in ADP receptor inhibition at 93.1% (interquartile range [IQR],
Biomedicines
The fibrinolytic system has been implicated in the genesis and progression of atherosclerosis. It... more The fibrinolytic system has been implicated in the genesis and progression of atherosclerosis. It has been reported that a plasminogen (Pg) deficiency (Plg−/−) exacerbates the progression of atherosclerosis in Apoe−/− mice. However, the manner in which Plg functions in a low-density lipoprotein-cholesterol (LDL-C)-driven model has not been evaluated. To characterize the effect of Pg in an LDL-C-driven model, mice with a triple deficiency of the LDL-receptor (LDLr), along with the active component (apobec1) of the apolipoprotein B editosome complex, and Pg (L−/−/A−/−/Plg−/−), were generated. Atherosclerotic plaque formation was severely retarded in the absence of Pg. In vitro studies demonstrated that LDL uptake by macrophages was enhanced by plasmin (Pm), whereas circulating levels of LDL were enhanced, relative to L−/−/A−/− mice, and VLDL synthesis was suppressed. These results indicated that clearance of lipoproteins in the absence of LDLr may be regulated by Pg/Pm. Conclusions: T...
Analytical and Bioanalytical Chemistry
The N-methyl-D-aspartate (NMDA) receptor is a crucial mediator of pathological glutamate-driven e... more The N-methyl-D-aspartate (NMDA) receptor is a crucial mediator of pathological glutamate-driven excitotoxicity and subsequent neuronal death in acute ischemic stroke. Although the roles of the NMDAR's composite GluN2A-C subunits have been investigated in this phenomenon, the relative importance of the GluN2D subunit has yet to be evaluated. Herein, GluN2D −/− mice were studied in a model of ischemic stroke using MALDI FT-ICR mass spectrometry imaging to investigate the role of the GluN2D subunit of the NMDA receptor in brain ischemia. GluN2D −/− mice underwent middle cerebral artery occlusion (MCAO) and brain tissue was subsequently harvested, frozen, and cryosectioned. Tissue sections were analyzed via MALDI FT-ICR mass spectrometry imaging. MALDI analyses revealed increases in several calcium-related species, namely vitamin D metabolites, LysoPC, and several PS species, in wild-type mouse brain tissue when compared to wild type. In addition, GluN2D −/ − mice also displayed an increase in PC, as well as a decrease in DG, suggesting reduced free fatty acid release from brain ischemia. These trends indicate that GluN2D −/− mice show enhanced rates of neurorecovery and neuroprotection from ischemic strokes compared to wild-type mice. The cause of neuroprotection may be the result of an increase in PGP in knockout mice, contributing to greater cardiolipin synthesis and decreased sensitivity to apoptotic signals.
Blood
The serine protease, activated Protein C (aPC) plays an important role in the maintenance of vasc... more The serine protease, activated Protein C (aPC) plays an important role in the maintenance of vascular hemostasis. Besides its ability to regulate coagulation and fibrinolysis, aPC also possesses antiinflammatory and antiapoptotic properties. Severe depletion of plasma PC and aPC contributes to sepsis pathogenesis. Although treatment with recombinant aPC benefits a subset of patients with severe sepsis, mechanisms by which aPC improves survival in these patients remain largely unknown. Using mice genetically predisposed to a severe PC deficiency, we initiated studies to further elucidate the mechanistic relationships between very low endogenous PC levels and inflammatory disease pathogenesis. Here, we show for the first time, that novel genetic dosing of PC strongly correlates with survival outcome following endotoxin (LPS) challenge in mice. Our findings provide evidence that very low endogenous levels of PC predispose mice to early onset of disseminated intravascular coagulation, t...
Frontiers in cellular and infection microbiology, 2018
The bacterial pathogen Group A (GAS) has been shown to induce a variety of human diseases ranging... more The bacterial pathogen Group A (GAS) has been shown to induce a variety of human diseases ranging in severity from pharyngitis to toxic shock syndrome and necrotizing fasciitis. GAS produces a powerful peptide toxin known as Streptolysin S (SLS). Though long recognized as a potent cytolysin, recent evidence from our lab has shown that SLS-dependent cytotoxicity is mediated through activation of the pro-inflammatory mediators p38 MAPK and NFκB. These findings led us to hypothesize that activation of p38 MAPK and NFκB signaling drive the production of pro-inflammatory cytokines which, in turn, serve as positive feedback signals to initiate cytotoxicity in infected host cells. To address this hypothesis, we utilized a cytokine array to characterize the SLS-dependent pro-inflammatory cytokine response to GAS infection in human keratinocytes. From these studies, IL-1β was found to be markedly upregulated in the presence of SLS, and further investigation revealed that this cytokine contri...
Biochemical and biophysical research communications, 2018
The N-methyl-D-aspartate receptor (NMDAR) ion channel plays a pivotal role in the pathology of is... more The N-methyl-D-aspartate receptor (NMDAR) ion channel plays a pivotal role in the pathology of ischemic stroke. The functional receptor consists of two GluN1 subunits (a-h) and two GluN2 subunits (A/B/C/D), the expression of which are spatially and temporally regulated in pathological and physiological conditions. While the roles of the GluN2A and GluN2B subunit in ischemic stroke have been well developed, the role of the GluN2C subunit in ischemia is not well understood. Following middle carotid artery occlusion (MCAO), GluN2Cmale mice displayed similar volumes of infarct as wild-type (WT) mice. However, GluN2Cmice showed decreased cerebral edema and an enhanced rate of neurological recovery compared to WT mice. The ischemic penumbra of GluN2Cmice showed fewer cytoarchitectural deficits and decreased tauopathy relative to WT mice. These neuroprotective changes in GluN2Cmice also corresponded with decreased expression of Fyn kinase and decreased phosphorylation of GluN2B subunit at ...
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Papers by Deborah Donahue