Fibrinolytic-deficiencies predispose hosts to septicemia from a catheter-associated UTI.

Catheter-associated urinary tract infections (CAUTIs) are amongst the most common nosocomial infections worldwide and are difficult to treat partly due to development of multidrug-resistance from CAUTI-related pathogens. Importantly, CAUTI often leads to secondary bloodstream infections and death. A major challenge is to predict when patients will develop CAUTIs and which populations are at-risk for bloodstream infections. Catheter-induced inflammation promotes fibrinogen (Fg) and fibrin accumulation in the bladder which are exploited as a biofilm formation platform by CAUTI pathogens. Using our established mouse model of CAUTI, here we identified that host populations exhibiting either genetic or acquired fibrinolytic-deficiencies, inducing fibrin deposition in the catheterized bladder, are predisposed to severe CAUTI and septicemia by diverse uropathogens in mono- and poly-microbial infections. Furthermore, here we found that Enterococcus faecalis, a prevalent CAUTI pathogen, uses the secreted protease, SprE, to induce fibrin accumulation and create a niche ideal for growth, biofilm formation, and persistence during CAUTI.

Fibrinolytic-deficiencies predispose hosts to septicemia from a catheter-associated UTI.

Catheter-associated urinary tract infections (CAUTIs) are amongst the most common nosocomial infections worldwide and are difficult to treat due to multi-drug resistance development among the CAUTI-related pathogens. Importantly, CAUTI often leads to secondary bloodstream infections and death. A major challenge is to predict when patients will develop CAUTIs and which populations are at-risk for bloodstream infections. Catheter-induced inflammation promotes fibrinogen (Fg) and fibrin accumulation in the bladder which are exploited as a biofilm formation platform by CAUTI pathogens. Using our established mouse model of CAUTI, we identified that host populations exhibiting either genetic or acquired fibrinolytic-deficiencies, inducing fibrin deposition in the catheterized bladder, are predisposed to severe CAUTI and septicemia by diverse uropathogens in mono- and poly-microbial infections. Furthermore, we found that E. faecalis, a prevalent CAUTI pathogen, uses the secreted protease, SprE, to induce fibrin accumulation and create a niche ideal for growth, biofilm formation, and persistence during CAUTI.

Altered Smooth Muscle Cell Histone Acetylome by the SPHK2/S1P Axis Promotes Pulmonary Hypertension.

BACKGROUND: Epigenetic regulation of vascular remodeling in pulmonary hypertension (PH) is poorly understood. Transcription regulating, histone acetylation code alters chromatin accessibility to promote transcriptional activation. Our goal was to identify upstream mechanisms that disrupt epigenetic equilibrium in PH. METHODS: Human pulmonary artery smooth muscle cells (PASMCs), human idiopathic pulmonary arterial hypertension (iPAH):human PASMCs, iPAH lung tissue, failed donor lung tissue, human pulmonary microvascular endothelial cells, iPAH:PASMC and non-iPAH:PASMC RNA-seq databases, NanoString nCounter, and cleavage under targets and release using nuclease were utilized to investigate histone acetylation, hyperacetylation targets, protein and gene expression, sphingolipid activation, cell proliferation, and gene target identification. SPHK2 (sphingosine kinase 2) knockout was compared with control C57BL/6NJ mice after 3 weeks of hypoxia and assessed for indices of PH. RESULTS: We identified that Human PASMCs are vulnerable to the transcription-promoting epigenetic mediator histone acetylation resulting in alterations in transcription machinery and confirmed its pathological existence in PH:PASMC cells. We report that SPHK2 is elevated as much as 20-fold in iPAH lung tissue and is elevated in iPAH:PASMC cells. During PH pathogenesis, nuclear SPHK2 activates nuclear bioactive lipid S1P (sphingosine 1-phosphate) catalyzing enzyme and mediates transcription regulating histone H3K9 acetylation (acetyl histone H3 lysine 9 [Ac-H3K9]) through EMAP (endothelial monocyte activating polypeptide) II. In iPAH lungs, we identified a 4-fold elevation of the reversible epigenetic transcription modulator Ac-H3K9:H3 ratio. Loss of SPHK2 inhibited hypoxic-induced PH and Ac-H3K9 in mice. We discovered that pulmonary vascular endothelial cells are a priming factor of the EMAP II/SPHK2/S1P axis that alters the acetylome with a specificity for PASMC, through hyperacetylation of histone H3K9. Using cleavage under targets and release using nuclease, we further show that EMAP II-mediated SPHK2 has the potential to modify the local transcription machinery of pluripotency factor KLF4 (Krüppel-like factor 4) by hyperacetylating KLF4 Cis-regulatory elements while deletion and targeted inhibition of SPHK2 rescues transcription altering Ac-H3K9. CONCLUSIONS: SPHK2 expression and its activation of the reversible histone H3K9 acetylation in human pulmonary artery smooth muscle cell represent new therapeutic targets that could mitigate PH vascular remodeling.

Generation and characterization of a plasminogen-binding group A streptococcal M-protein/streptokinase-sensitive mouse line.

BACKGROUND: Streptococcus pyogenes (GAS) is a human bacterial pathogen that generates various mild to severe diseases. Worldwide, there are approximately 700 million cases of GAS infections per year. In some strains of GAS, the surface-resident M-protein, plasminogen-binding group A streptococcal M-protein (PAM), binds directly to human host plasminogen (hPg), where it is activated to plasmin through a mechanism involving a Pg/bacterial streptokinase (SK) complex as well as endogenous activators. Binding to Pg and its activation are dictated by selected sequences within the human host Pg protein, making it difficult to generate animal models to study this pathogen. OBJECTIVES: To develop a murine model for studying GAS infection by minimally modifying mouse Pg to enhance the affinity to bacterial PAM and sensitivity to GAS-derived SK. METHODS: We used a targeting vector that contained a mouse albumin-promoter and mouse/human hybrid plasminogen cDNA targeted to the Rosa26 locus. Characterization of the mouse strain consisted of both gross and histological techniques and determination of the effects of the modified Pg protein through surface plasmon resonance measurements, Pg activation analyses, and mouse survival post-GAS infection. RESULTS: We generated a mouse line expressing a chimeric Pg protein consisting of 2 amino acid substitutions in the heavy chain of Pg and a complete replacement of the mouse Pg light chain with the human Pg light chain. CONCLUSION: This protein demonstrated an enhanced affinity for bacterial PAM and sensitivity to activation by the Pg-SK complex, making the murine host susceptible to the pathogenic effects of GAS.

Streptolysin S targets the sodium-bicarbonate cotransporter NBCn1 to induce inflammation and cytotoxicity in human keratinocytes during Group A Streptococcal infection.

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 ribosomally-produced 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-κB 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 in numerous context-dependent ways to promote host cell cytotoxicity and increase disease severity. Studies to elucidate additional host targets of SLS have the potential to impact the development of therapeutics for severe GAS infections.

Group A Streptococcus-Induced Activation of Human Plasminogen Is Required for Keratinocyte Wound Retraction and Rapid Clot Dissolution.

Invasive outcomes of Group A Streptococcus (GAS) infections that involve damage to skin and other tissues are initiated when these bacteria colonize and disseminate via an open wound to gain access to blood and deeper tissues. Two critical GAS virulence factors, Plasminogen-Associated M-Protein (PAM) and streptokinase (SK), work in concert to bind and activate host human plasminogen (hPg) in order to create a localized proteolytic environment that alters wound-site architecture. Using a wound scratch assay with immortalized epithelial cells, real-time live imaging (RTLI) was used to examine dynamic effects of hPg activation by a PAM-containing skin-trophic GAS isolate (AP53R+S-) during the course of infection. RTLI of these wound models revealed that retraction of the epithelial wound required both GAS and hPg. Isogenic AP53R+S- mutants lacking SK or PAM highly attenuated the time course of retraction of the keratinocyte wound. We also found that relocalization of integrin ß1 from the membrane to the cytoplasm occurred during the wound retraction event. We devised a combined in situ-based cellular model of fibrin clot-in epithelial wound to visualize the progress of GAS pathogenesis by RTLI. Our findings showed GAS AP53R+S- hierarchically dissolved the fibrin clot prior to the retraction of keratinocyte monolayers at the leading edge of the wound. Overall, our studies reveal that localized activation of hPg by AP53R+S- via SK and PAM during infection plays a critical role in dissemination of bacteria at the wound site through both rapid dissolution of the fibrin clot and retraction of the keratinocyte wound layer.

The Streptococcal Protease SpeB Antagonizes the Biofilms of the Human Pathogen Staphylococcus aureus USA300 through Cleavage of the Staphylococcal SdrC Protein.

Streptococcus pyogenes, or group A Streptococcus (GAS), is both a pathogen and an asymptomatic colonizer of human hosts and produces a large number of surface-expressed 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 its antimicrobial and antibiofilm properties against the clinically important human colonizer Staphylococcus aureus, which occupies niches similar to those of GAS. For our investigation, we used a skin-tropic 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.IMPORTANCEStreptococcus pyogenes (GAS) causes a range of diseases 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, an important factor in GAS pathogenesis. An understudied aspect of SpeB is the manner in which its broad proteolytic activity affects other microorganisms that co-occupy niches similar to that of 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 for how SpeB may be utilized by GAS to successfully compete in a polymicrobial environment.

Neutralization of Streptolysin S-Dependent and Independent Inflammatory Cytokine IL-1ß Activity Reduces Pathology During Early Group A Streptococcal Skin Infection.

The bacterial pathogen Group A Streptococcus (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-1ß was found to be markedly upregulated in the presence of SLS, and further investigation revealed that this cytokine contributes to cytotoxicity in human keratinocytes during infection. Subcutaneous infection studies were performed in mice to address the physiological impact of increased IL-1ß production. These studies demonstrated that IL-1ß is produced during GAS skin infection in an SLS-dependent manner. Furthermore, inhibition of this cytokine and the upstream kinases and other signaling mediators that drive its production reduced SLS-mediated lesion formation early in the infection process. Together, our findings indicate that pharmacological inhibition of this inflammatory axis holds promise as a therapeutic strategy to reduce tissue destruction during severe invasive Group A Streptococcal infections.

A deficiency of the GluN2C subunit of the N-methyl-D-aspartate receptor is neuroprotective in a mouse model of ischemic stroke.

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), GluN2C-/- male mice displayed similar volumes of infarct as wild-type (WT) mice. However, GluN2C-/- mice showed decreased cerebral edema and an enhanced rate of neurological recovery compared to WT mice. The ischemic penumbra of GluN2C-/- mice showed fewer cytoarchitectural deficits and decreased tauopathy relative to WT mice. These neuroprotective changes in GluN2C-/- mice also corresponded with decreased expression of Fyn kinase and decreased phosphorylation of GluN2B subunit at Tyr1336. Lastly, a GluN2C deficiency modified the NMDAR/pro-survival signaling axis, as shown by increased levels of nuclear CREB(P-Ser133). Thus, the GluN2C subunit enhances ischemic stroke pathology by promoting neuronal dysfunction in the penumbra region.

Plasminogen Activator Inhibitor-1 Protects Mice Against Cardiac Fibrosis by Inhibiting Urokinase-type Plasminogen Activator-mediated Plasminogen Activation.

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-ß and proteases associated with tissue remodeling, and diminished Smad2 signaling. Moreover, total ablation of cardiac fibrosis was observed in PAI-1-/- mice that express inactive plasmin (Pm) but normal levels of zymogen Pg (PAI-1-/-/PgS743A/S743A). Our findings indicate that PAI-1 protects mice from hypertension-induced cardiac fibrosis by inhibiting the generation of active Pm.

Activation of band 3 mediates group A Streptococcus streptolysin S-based beta-haemolysis.

Streptococcus pyogenes, or group A Streptococcus (GAS), is a human bacterial pathogen that can manifest as a range of diseases from pharyngitis and impetigo to severe outcomes such as necrotizing fasciitis and toxic shock syndrome. GAS disease remains a global health burden with cases estimated at over 700 million annually and over half a million deaths due to severe infections(1). For over 100 years, a clinical hallmark of diagnosis has been the appearance of complete (beta) haemolysis when grown in the presence of blood. This activity is due to the production of a small peptide toxin by GAS known as streptolysin S. Although it has been widely held that streptolysin S exerts its lytic activity through membrane disruption, its exact mode of action has remained unknown. Here, we show, using high-resolution live cell imaging, that streptolysin S induces a dramatic osmotic change in red blood cells, leading to cell lysis. This osmotic change was characterized by the rapid influx of Cl(-) ions into the red blood cells through SLS-mediated disruption of the major erythrocyte anion exchange protein, band 3. Chemical inhibition of band 3 function significantly reduced the haemolytic activity of streptolysin S, and dramatically reduced the pathology in an in vivo skin model of GAS infection. These results provide key insights into the mechanism of streptolysin S-mediated haemolysis and have implications for the development of treatments against GAS.

Early coagulation events induce acute lung injury in a rat model of blunt traumatic brain injury.

Acute lung injury (ALI) and systemic coagulopathy are serious complications of traumatic brain injury (TBI) that frequently lead to poor clinical outcomes. Although the release of tissue factor (TF), a potent initiator of the extrinsic pathway of coagulation, from the injured brain is thought to play a key role in coagulopathy after TBI, its function in ALI following TBI remains unclear. In this study, we investigated whether the systemic appearance of TF correlated with the ensuing coagulopathy that follows TBI in ALI using an anesthetized rat blunt trauma TBI model. Blood and lung samples were obtained after TBI. Compared with controls, pulmonary edema and increased pulmonary permeability were observed as early as 5 min after TBI without evidence of norepinephrine involvement. Systemic TF increased at 5 min and then diminished 60 min after TBI. Lung injury and alveolar hemorrhaging were also observed as early as 5 min after TBI. A biphasic elevation of TF was observed in the lungs after TBI, and TF-positive microparticles (MPs) were detected in the alveolar spaces. Fibrin(ogen) deposition was also observed in the lungs within 60 min after TBI. Additionally, preadministration of a direct thrombin inhibitor, Refludan, attenuated lung injuries, thus implicating thrombin as a direct participant in ALI after TBI. The results from this study demonstrated that enhanced systemic TF may be an initiator of coagulation activation that contributes to ALI after TBI.

Genomic Characterization of a Pattern D Streptococcus pyogenes emm53 Isolate Reveals a Genetic Rationale for Invasive Skin Tropicity.

UNLABELLED: The genome of an invasive skin-tropic strain (AP53) of serotype M53 group A Streptococcus pyogenes (GAS) is composed of a circular chromosome of 1,860,554 bp and carries genetic markers for infection at skin locales, viz, emm gene family pattern D and FCT type 3. Through genome-scale comparisons of AP53 with other GAS genomes, we identified 596 candidate single-nucleotide polymorphisms (SNPs) that reveal a potential genetic basis for skin tropism. The genome of AP53 differed by ∼30 point mutations from a noninvasive pattern D serotype M53 strain (Alab49), 4 of which are located in virulence genes. One pseudogene, yielding an inactive sensor kinase (CovS(-)) of the two-component transcriptional regulator CovRS, a major determinant for invasiveness, severely attenuated the expression of the secreted cysteine protease SpeB and enhanced the expression of the hyaluronic acid capsule compared to the isogenic noninvasive AP53/CovS(+) strain. The collagen-binding protein transcript sclB differed in the number of 5'-pentanucleotide repeats in the signal peptides of AP53 and Alab49 (9 versus 15), translating into different lengths of their signal peptides, which nonetheless maintained a full-length translatable coding frame. Furthermore, GAS strain AP53 acquired two phages that are absent in Alab49. One such phage (ΦAP53.2) contains the known virulence factor superantigen exotoxin gene tandem speK-slaA Overall, we conclude that this bacterium has evolved in multiple ways, including mutational variations of regulatory genes, short-tandem-repeat polymorphisms, large-scale genomic alterations, and acquisition of phages, all of which may be involved in shaping the adaptation of GAS in specific infectious environments and contribute to its enhanced virulence. IMPORTANCE: Infectious strains of S. pyogenes (GAS) are classified by their serotypes, relating to the surface M protein, the emm-like subfamily pattern, and their tropicity toward the nasopharynx and/or skin. It is generally agreed that M proteins from pattern D strains, which also directly bind human host plasminogen, are skin tropic. We have sequenced and characterized the genome of an invasive pattern D GAS strain (AP53) in comparison to a very similar strain (Alab49) that is noninvasive and developed a genomic rationale as to possible reasons for the skin tropicity of these two strains and the greater invasiveness of AP53.

Direct Host Plasminogen Binding to Bacterial Surface M-protein in Pattern D Strains of Streptococcus pyogenes Is Required for Activation by Its Natural Coinherited SK2b Protein.

Streptokinase (SK), secreted by Group A Streptococcus (GAS), is a single-chain ∼47-kDa protein containing three consecutive primary sequence regions that comprise its α, ß, and γ modules. Phylogenetic analyses of the variable ß-domain sequences from different GAS strains suggest that SKs can be arranged into two clusters, SK1 and SK2, with a subdivision of SK2 into SK2a and SK2b. SK2b is secreted by skin-tropic Pattern D M-protein strains that also express plasminogen (human Pg (hPg)) binding Group A streptococcal M-protein (PAM) as its major cell surface M-protein. SK2a-expressing strains are associated with nasopharynx tropicity, and many of these strains express human fibrinogen (hFg) binding Pattern A-C M-proteins, e.g. M1. PAM interacts with hPg directly, whereas M1 binds to hPg indirectly via M1-bound hFg. Subsequently, SK is secreted by GAS and activates hPg to plasmin (hPm), thus generating a proteolytic surface on GAS that enhances its dissemination. Due to these different modes of hPg/hPm recognition by GAS, full characterizations of the mechanisms of activation of hPg by SK2a and SK2b and their roles in GAS virulence are important topics. To more fully examine these subjects, isogenic chimeric SK- and M-protein-containing GAS strains were generated, and the virulence of these chimeric strains were analyzed in mice. We show that SK and M-protein alterations influenced the virulence of GAS and were associated with the different natures of hPg activation and hPm binding. These studies demonstrate that GAS virulence can be explained by disparate hPg activation by SK2a and SK2b coupled with the coinherited M-proteins of these strains.

Abrogation of plasminogen activator inhibitor-1-vitronectin interaction ameliorates acute kidney injury in murine endotoxemia.

Sepsis-induced acute kidney injury (AKI) contributes to the high mortality and morbidity in patients. Although the pathogenesis of AKI during sepsis is poorly understood, it is well accepted that plasminogen activator inhibitor-1 (PAI-1) and vitronectin (Vn) are involved in AKI. However, the functional cooperation between PAI-1 and Vn in septic AKI has not been completely elucidated. To address this issue, mice were utilized lacking either PAI-1 (PAI-1-/-) or expressing a PAI-1-mutant (PAI-1R101A/Q123K) in which the interaction between PAI-1 and Vn is abrogated, while other functions of PAI-1 are retained. It was found that both PAI-1-/- and PAI-1R101A/Q123K mice are associated with decreased renal dysfunction, apoptosis, inflammation, and ERK activation as compared to wild-type (WT) mice after LPS challenge. Also, PAI-1-/- mice showed attenuated fibrin deposition in the kidneys. Furthermore, a lack of PAI-1 or PAI-1-Vn interaction was found to be associated with an increase in activated Protein C (aPC) in plasma. These results demonstrate that PAI-1, through its interaction with Vn, exerts multiple deleterious mechanisms to induce AKI. Therefore, targeting of the PAI-1-Vn interaction in kidney represents an appealing therapeutic strategy for the treatment of septic AKI by not only altering the fibrinolytic capacity but also regulating PC activity.

Conantokin-G attenuates detrimental effects of NMDAR hyperactivity in an ischemic rat model of stroke.

The neuroprotective activity of conantokin-G (con-G), a naturally occurring antagonist of N-methyl-D-aspartate receptors (NMDAR), was neurologically and histologically compared in the core and peri-infarct regions after ischemia/reperfusion brain injury in male Sprague-Dawley rats. The contralateral regions served as robust internal controls. Intrathecal injection of con-G, post-middle carotid artery occlusion (MCAO), caused a dramatic decrease in brain infarct size and swelling at 4 hr, compared to 26 hr, and significant recovery of neurological deficits was observed at 26 hr. Administration of con-G facilitated neuronal recovery in the peri-infarct regions as observed by decreased neurodegeneration and diminished calcium microdeposits at 4 hr and 26 hr. Intact Microtubule Associated Protein (MAP2) staining and neuronal cytoarchitecture was observed in the peri-infarct regions of con-G treated rats at both timepoints. Con-G restored localization of GluN1 and GluN2B subunits in the neuronal soma, but not that of GluN2A, which was perinuclear in the peri-infarct regions at 4 hr and 26 hr. This suggests that molecular targeting of the GluN2B subunit has potential for reducing detrimental consequences of ischemia. Overall, the data demonstrated that stroke-induced NMDAR excitoxicity is ameliorated by con-G-mediated repair of neurological and neuroarchitectural deficits, as well as by reconstituting neuronal localization of GluN1 and GluN2B subunits in the peri-infarct region of the stroked brain.

Mutations in the control of virulence sensor gene from Streptococcus pyogenes after infection in mice lead to clonal bacterial variants with altered gene regulatory activity and virulence.

The cluster of virulence sensor (CovS)/responder (CovR) two-component operon (CovRS) regulates ∼15% of the genes of the Group A Streptococcal pyogenes (GAS) genome. Bacterial clones containing inactivating mutations in the covS gene have been isolated from patients with virulent invasive diseases. We report herein an assessment of the nature and types of covS mutations that can occur in both virulent and nonvirulent GAS strains, and assess whether a nonvirulent GAS can attain enhanced virulence through this mechanism. A group of mice were infected with a globally-disseminated clonal M1T1 GAS (isolate 5448), containing wild-type (WT) CovRS (5448/CovR+S+), or less virulent engineered GAS strains, AP53/CovR+S+ and Manfredo M5/CovR+S+. SpeB negative GAS clones from wound sites and/or from bacteria disseminated to the spleen were isolated and the covS gene was subjected to DNA sequence analysis. Numerous examples of inactivating mutations were found in CovS in all regions of the gene. The mutations found included frame-shift insertions and deletions, and in-frame small and large deletions in the gene. Many of the mutations found resulted in early translation termination of CovS. Thus, the covS gene is a genomic mutagenic target that gives GAS enhanced virulence. In cases wherein CovS- was discovered, these clonal variants exhibited high lethality, further suggesting that randomly mutated covS genes occur during the course of infection, and lead to the development of a more invasive infection.

Traumatic brain injury causes platelet adenosine diphosphate and arachidonic acid receptor inhibition independent of hemorrhagic shock in humans and rats.

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], 44.8-98.3%; n = 29) compared with 56.5% (IQR, 35-79.1%; n = 41) in milder TBI and 15.5% (IQR, 13.2-29.1%) in controls (p = 0.0014 and p < 0.0001, respectively). No patient had significant hypotension or acidosis. Parallel trends were noted in AA receptor inhibition. CONCLUSION: Platelet ADP and AA receptor inhibition is a prominent early feature of CTBI in humans and rats and is linked to the severity of brain injury in patients with isolated head trauma. This phenomenon is observed in the absence of hemorrhagic shock or multisystem injury. Thus, TBI alone is shown to be sufficient to induce a profound platelet dysfunction.

Systemic platelet dysfunction is the result of local dysregulated coagulation and platelet activation in the brain in a rat model of isolated traumatic brain injury.

Coagulopathy after severe traumatic brain injury (TBI) has been extensively reported. Clinical studies have identified a strong relationship between diminished platelet-rich thrombus formation, responsiveness to adenosine diphosphate agonism, and severity of TBI. The mechanisms that lead to platelet dysfunction in the acute response to TBI are poorly understood. The development of a rodent model of TBI that mimics the coagulopathy observed clinically has recently been reported. Using immunohistochemical techniques and thromboelastography platelet mapping, the current study demonstrated that the expression of coagulation (tissue factor and fibrin) and platelet activation (P-selectin) markers in the injured brain paralleled the alteration in systemic platelet responsiveness to the agonists, adenosine diphosphate and arachodonic acid. Results of this study demonstrate that local procoagulant changes in the injured brain have profound effects on systemic platelet function.

Probing NMDA receptor GluN2A and GluN2B subunit expression and distribution in cortical neurons.

The spatial distribution of N-methyl-d-aspartate receptor (NMDAR) subunits in layer 5 (L5) neurons of the medial prefrontal cortex (mPFC) is important for integrating input-output signals involved in cognitive functions and motor behavior. In this study, focal laser scanning photostimulation of caged glutamate, slice electrophysiology, and small peptide pharmacology, were used to map the distribution of functional GluN2A and GluN2B subunits of the NMDAR from L5 neurons of wild-type (WT) and GluN2A(-/-) mice. Focal uncaging of glutamate evoked spatially-restricted glutamatergic responses on various dendritic locations of pyramidal neurons in the mPFC. Analyses of the spatial arrangements of the GluN2A and GluN2B subunits were performed by comparing inhibition of glutamatergic responses in the presence of the GluN2A-selective pharmacological antagonist, NVP-AAM077 (NVP), and the GluN2B-selective peptidic antagonist, conantokin-G (con-G). We found that apical and basal expression and distribution of GluN2A and GluN2B were similar in L5 mPFC neurons of WT mice. However, the inhibition of glutamatergic responses by NVP in brain slices of GluN2A(-/-) mice were dramatically decreased, while con-G inhibition remained similar to that observed in WT brain slices. The data obtained show that expression and spatial arrangement of GluN2B subunits is independent of GluN2A in L5 neurons of the mPFC. These findings have important ramifications for NMDAR organization and function in L5 pyramidal neurons of the mPFC, and show that specific populations of NMDARs can be antagonized, while sparing other subgroups of NMDARs, thus preserving selective NMDAR functions, an important therapeutic advantage.