A Single Hospital-Wide Antibiogram is Insufficient to Account for Differences in Antibiotic Resistance Patterns Across Multiple ICUs.

BACKGROUND: Infection is a common cause of mortality within intensive care units (ICUs). Antibiotic resistance patterns and culture data are used to create antibiograms. Knowledge of antibiograms facilitates guiding empiric therapies and reduces mortality. Most major hospitals utilize data collection to create hospital-wide antibiograms. Previous studies have shown significant differences in susceptibility patterns between hospital wards and ICUs. We hypothesize that institutional or combined ICU antibiograms are inadequate to account for differences in susceptibility for patients in individual ICUs. METHODS: Culture and susceptibility data were reviewed over a 1-year period for 13 bacteria in the following ICUs: Surgical/Trauma, Medical, Neuroscience, Burn, and Emergency department. Antibiotic management decisions are made by individual teams. RESULTS: Nine species had sufficient data for inclusion into an All-ICU antibiogram. E coli and S aureus were the most common isolates. Seven species had significant differences in susceptibility patterns between ICUs. E cloacae showed higher rates of resistance to multiple antibiotics in the STICU than other ICUs. P aeruginosa susceptibility rates in the NSICU and BICU were 88% and 92%, respectively, compared to 60% and 55% in the STICU and MICU. Cephalosporins and Aztreonam had reduced efficacy against E coli in the NSICU, however remain effective in other ICUs. CONCLUSIONS: The results of this study show that different ICUs do have variability in antibiotic susceptibility patterns within a single hospital. While this only represents a single institution, it shows that the use of hospital-wide antibiograms is inadequate for creating empiric antibiotic protocols within individual ICUs.

Amyloid-ß and caspase-1 are indicators of sepsis and organ injury.

Background: Sepsis is a life-threatening condition that results from a dysregulated host response to infection, leading to organ dysfunction. Despite the prevalence and associated socioeconomic costs, treatment of sepsis remains limited to antibiotics and supportive care, and a majority of intensive care unit (ICU) survivors develop long-term cognitive complications post-discharge. The present study identifies a novel regulatory relationship between amyloid-ß (Aß) and the inflammasome-caspase-1 axis as key innate immune mediators that define sepsis outcomes. Methods: Medical ICU patients and healthy individuals were consented for blood and clinical data collection. Plasma cytokine, caspase-1 and Aß levels were measured. Data were compared against indices of multiorgan injury and other clinical parameters. Additionally, recombinant proteins were tested in vitro to examine the effect of caspase-1 on a functional hallmark of Aß, namely aggregation. Results: Plasma caspase-1 levels displayed the best predictive value in discriminating ICU patients with sepsis from non-infected ICU patients (area under the receiver operating characteristic curve=0.7080). Plasma caspase-1 and the Aß isoform Aßx-40 showed a significant positive correlation and Aßx-40 associated with organ injury. Additionally, Aß plasma levels continued to rise from time of ICU admission to 7 days post-admission. In silico, Aß harbours a predicted caspase-1 cleavage site, and in vitro studies demonstrated that caspase-1 cleaved Aß to inhibit its auto-aggregation, suggesting a novel regulatory relationship. Conclusions: Aßx-40 and caspase-1 are potentially useful early indicators of sepsis and its attendant organ injury. Additionally, Aßx-40 has emerged as a potential culprit in the ensuing development of post-ICU syndrome.

Tau and Aß42 in lavage fluid of pneumonia patients are associated with end-organ dysfunction: A prospective exploratory study.

BACKGROUND: Bacterial pneumonia and sepsis are both common causes of end-organ dysfunction, especially in immunocompromised and critically ill patients. Pre-clinical data demonstrate that bacterial pneumonia and sepsis elicit the production of cytotoxic tau and amyloids from pulmonary endothelial cells, which cause lung and brain injury in naïve animal subjects, independent of the primary infection. The contribution of infection-elicited cytotoxic tau and amyloids to end-organ dysfunction has not been examined in the clinical setting. We hypothesized that cytotoxic tau and amyloids are present in the bronchoalveolar lavage fluid of critically ill patients with bacterial pneumonia and that these tau/amyloids are associated with end-organ dysfunction. METHODS: Bacterial culture-positive and culture-negative mechanically ventilated patients were recruited into a prospective, exploratory observational study. Levels of tau and Aß42 in, and cytotoxicity of, the bronchoalveolar lavage fluid were measured. Cytotoxic tau and amyloid concentrations were examined in comparison with patient clinical characteristics, including measures of end-organ dysfunction. RESULTS: Tau and Aß42 were increased in culture-positive patients (n = 49) compared to culture-negative patients (n = 50), independent of the causative bacterial organism. The mean age of patients was 52.1 ± 16.72 years old in the culture-positive group and 52.78 ± 18.18 years old in the culture-negative group. Males comprised 65.3% of the culture-positive group and 56% of the culture-negative group. Caucasian culture-positive patients had increased tau, boiled tau, and Aß42 compared to both Caucasian and minority culture-negative patients. The increase in cytotoxins was most evident in males of all ages, and their presence was associated with end-organ dysfunction. CONCLUSIONS: Bacterial infection promotes the generation of cytotoxic tau and Aß42 within the lung, and these cytotoxins contribute to end-organ dysfunction among critically ill patients. This work illuminates an unappreciated mechanism of injury in critical illness.

Deleterious effects of plasma-derived cellular debris in a porcine model of hemorrhagic shock.

BACKGROUND: Recent studies identify large quantities of inflammatory cellular debris within Fresh Frozen Plasma (FFP). As FFP is a mainstay of hemorrhagic shock resuscitation, we used a porcine model of hemorrhagic shock and ischemia/reperfusion to investigate the inflammatory potential of plasma-derived cellular debris administered during resuscitation. METHODS: The porcine model of hemorrhagic shock included laparotomy with 35 % hemorrhage (Hem), 45 min of ischemia from supraceliac aortic occlusion with subsequent clamp release (IR), followed by protocolized resuscitation for 6 h. Cellular debris (Debris) was added to the resuscitation phase in three groups. The four groups consisted of Hem + IR (n = 4), Hem + IR + Debris (n = 3), Hem + Debris (n = 3), and IR + Debris (n = 3). A battery of laboratory, physiologic, cytokine, and outcome data were compared between groups. RESULTS: As expected, the Hem + IR group showed severe time dependent decrements in organ function and physiologic parameters. All animals that included both IR and Debris (Hem + IR + Debris or IR + Debris) died prior to the six-hour end point, while all animals in the Hem + IR and Hem + Debris survived. Cytokines measured at 30-60 min after initiation of resuscitation revealed significant differences in IL-18 and IL-1ß between all groups. CONCLUSIONS: Ischemia and reperfusion appear to prime the immune system to the deleterious effects of plasma-derived cellular debris. In the presence of ischemia and reperfusion, this model showed the equivalency of 100 % lethality when resuscitation included quantities of cellular debris at levels routinely administered to trauma patients during transfusion of FFP. A deeper understanding of the immunobiology of FFP-derived cellular debris is critical to optimize resuscitation for hemorrhagic shock.

Human YRNA 4 (HY4) plasma levels are a prognostic indicator of SARS-CoV-2 infection clinical severity.

SARS-CoV-2 infection can result in a range of outcomes from asymptomatic/mild disease to severe COVID-19/fatality. In this study, we investigated the differential expression of small noncoding RNAs (sncRNAs) between patient cohorts defined by disease severity. We collected plasma samples, stratified these based on clinical outcomes, and sequenced their circulating sncRNAs. Excitingly, we found YRNA HY4 displays significant differential expression (p=0.025) between patients experiencing mild and severe disease. In agreement with recent reports identifying plasma YRNAs as indicators of influenza infection severity, our results strongly suggest that circulating HY4 levels represent a powerful prognostic indicator of likely SARS-CoV-2 patient infection outcome.

Gamma secretase activating protein promotes end-organ dysfunction after bacterial pneumonia.

Pneumonia elicits the production of cytotoxic beta amyloid (Aß) that contributes to end-organ dysfunction, yet the mechanism(s) linking infection to activation of the amyloidogenic pathway that produces cytotoxic Aß is unknown. Here, we tested the hypothesis that gamma-secretase activating protein (GSAP), which contributes to the amyloidogenic pathway in the brain, promotes end-organ dysfunction following bacterial pneumonia. First-in-kind Gsap knockout rats were generated. Wild-type and knockout rats possessed similar body weights, organ weights, circulating blood cell counts, arterial blood gases, and cardiac indices at baseline. Intratracheal Pseudomonas aeruginosa infection caused acute lung injury and a hyperdynamic circulatory state. Whereas infection led to arterial hypoxemia in wild-type rats, the alveolar-capillary barrier integrity was preserved in Gsap knockout rats. Infection potentiated myocardial infarction following ischemia-reperfusion injury, and this potentiation was abolished in knockout rats. In the hippocampus, GSAP contributed to both pre- and postsynaptic neurotransmission, increasing the presynaptic action potential recruitment, decreasing neurotransmitter release probability, decreasing the postsynaptic response, and preventing postsynaptic hyperexcitability, resulting in greater early long-term potentiation but reduced late long-term potentiation. Infection abolished early and late long-term potentiation in wild-type rats, whereas the late long-term potentiation was partially preserved in Gsap knockout rats. Furthermore, hippocampi from knockout rats, and both the wild-type and knockout rats following infection, exhibited a GSAP-dependent increase in neurotransmitter release probability and postsynaptic hyperexcitability. These results elucidate an unappreciated role for GSAP in innate immunity and highlight the contribution of GSAP to end-organ dysfunction during infection.NEW & NOTEWORTHY Pneumonia is a common cause of end-organ dysfunction, both during and in the aftermath of infection. In particular, pneumonia is a common cause of lung injury, increased risk of myocardial infarction, and neurocognitive dysfunction, although the mechanisms responsible for such increased risk are unknown. Here, we reveal that gamma-secretase activating protein, which contributes to the amyloidogenic pathway, is important for end-organ dysfunction following infection.

Lethal Caspase-1/4-Dependent Injury Occurs in the First Minutes of Coronary Reperfusion and Requires Calpain Activity.

To study the relationship between caspase-1/4 and reperfusion injury, we measured infarct size (IS) in isolated mouse hearts undergoing 50 min global ischemia/2 h reperfusion. Starting VRT-043198 (VRT) at reperfusion halved IS. The pan-caspase inhibitor emricasan duplicated VRT's protection. IS in caspase-1/4-knockout hearts was similarly reduced, supporting the hypothesis that caspase-1/4 was VRT's only protective target. NLRC4 inflammasomes activate caspase-1. NLRC4 knockout hearts were not protected, eliminating NLRC4 as caspase-1/4's activator. The amount of protection that could be achieved by only suppressing caspase-1/4 activity was limited. In wild-type (WT) hearts, ischemic preconditioning (IPC) was as protective as caspase-1/4 inhibitors. Combining IPC and emricasan in these hearts or preconditioning caspase-1/4-knockout hearts produced an additive IS reduction, indicating that more protection could be achieved by combining treatments. We determined when caspase-1/4 exerted its lethal injury. Starting VRT after 10 min of reperfusion in WT hearts was no longer protective, revealing that caspase-1/4 inflicted its injury within the first 10 min of reperfusion. Ca++ influx at reperfusion might activate caspase-1/4. We tested whether Ca++-dependent soluble adenylyl cyclase (AC10) could be responsible. However, IS in AC10-/- hearts was not different from that in WT control hearts. Ca++-activated calpain has been implicated in reperfusion injury. Calpain could be releasing actin-bound procaspase-1 in cardiomyocytes, which would explain why caspase-1/4-related injury is confined to early reperfusion. The calpain inhibitor calpeptin duplicated emricasan's protection. Unlike IPC, adding calpain to emricasan offered no additional protection, suggesting that caspase-1/4 and calpain may share the same protective target.

Defining Pathogen and Susceptibility Patterns for Early Versus Late Ventilator Associated Pneumonia in Trauma Patients to Guide Empiric Treatment Decisions.

INTRODUCTION: Studies have demonstrated that trauma patients with early-ventilator associated pneumonia (early-VAP, < 7 days) have decreased risk of methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa infections. We hypothesize that routinely using broad-spectrum antibiotics is unnecessary to treat trauma patients with the diagnosis of early-VAP. METHODS: This retrospective cohort study included adult trauma patients with the diagnosis of VAP. The primary outcome was the presence of MRSA and/or P. aeruginosa in patients with early- and late-VAP. Secondary outcomes included the bacterial susceptibility of pathogens to methicillin, ampicillin/sulbactam, ceftriaxone, piperacillin/tazobactam, and cefepime. Intensive care unit (ICU) and hospital length of stay (LOS), ventilator-free days, and in-hospital mortality were also collected. RESULTS: 164 patients met inclusion criteria, and 208 organisms (n = 90 early vs n = 118 late) were identified by respiratory culture. The incidence of MRSA and P. aeruginosa in early-VAP was 7.7% (7/90) and 5.6% (5/90), respectively. The susceptibility of bacteria causing early-VAP to ampicillin/sulbactam and ceftriaxone was 73.3% (66/90) and 83.3% (75/90), respectively. Ventilator-free days at 30 days was similar between groups (P = .649). Patients with late-VAP spent more time in the ICU (P = .040); however, in-hospital mortality was higher in the early-VAP group (P = .012). CONCLUSIONS: Ampicillin/sulbactam or ceftriaxone monotherapy did not provide reliable broad-spectrum coverage for early-VAP in our cohort. These findings highlight the importance of each institution performing a similar analysis to ensure adequate initial treatment of VAP.

Coronavirus Lung Infection Impairs Host Immunity against Secondary Bacterial Infection by Promoting Lysosomal Dysfunction.

Postviral bacterial infections are a major health care challenge in coronavirus infections, including COVID-19; however, the coronavirus-specific mechanisms of increased host susceptibility to secondary infections remain unknown. In humans, coronaviruses, including SARS-CoV-2, infect lung immune cells, including alveolar macrophages, a phenotype poorly replicated in mouse models of SARS-CoV-2. To overcome this, we used a mouse model of native murine ß-coronavirus that infects both immune and structural cells to investigate coronavirus-enhanced susceptibility to bacterial infections. Our data show that coronavirus infection impairs the host ability to clear invading bacterial pathogens and potentiates lung tissue damage in mice. Mechanistically, coronavirus limits the bacterial killing ability of macrophages by impairing lysosomal acidification and fusion with engulfed bacteria. In addition, coronavirus-induced lysosomal dysfunction promotes pyroptotic cell death and the release of IL-1ß. Inhibition of cathepsin B decreased cell death and IL-1ß release and promoted bacterial clearance in mice with postcoronavirus bacterial infection.

ExoU Induces Lung Endothelial Cell Damage and Activates Pro-Inflammatory Caspase-1 during Pseudomonas aeruginosa Infection.

The Gram-negative, opportunistic pathogen Pseudomonas aeruginosa utilizes a type III secretion system to inject exoenzyme effectors into a target host cell. Of the four best-studied exoenzymes, ExoU causes rapid cell damage and death. ExoU is a phospholipase A2 (PLA2) that hydrolyses host cell membranes, and P. aeruginosa strains expressing ExoU are associated with poor outcomes in critically ill patients with pneumonia. While the effects of ExoU on lung epithelial and immune cells are well studied, a role for ExoU in disrupting lung endothelial cell function has only recently emerged. Lung endothelial cells maintain a barrier to fluid and protein flux into tissue and airspaces and regulate inflammation. Herein, we describe a pulmonary microvascular endothelial cell (PMVEC) culture infection model to examine the effects of ExoU. Using characterized P. aeruginosa strains and primary clinical isolates, we show that strains expressing ExoU disrupt PMVEC barrier function by causing substantial PMVEC damage and lysis, in a PLA2-dependent manner. In addition, we show that strains expressing ExoU activate the pro-inflammatory caspase-1, in a PLA2-dependent manner. Considering the important roles for mitochondria and oxidative stress in regulating inflammatory responses, we next examined the effects of ExoU on reactive oxygen species production. Infection of PMVECs with P. aeruginosa strains expressing ExoU triggered a robust oxidative stress compared to strains expressing other exoenzyme effectors. We also provide evidence that, intriguingly, ExoU PLA2 activity was detectable in mitochondria and mitochondria-associated membrane fractions isolated from P. aeruginosa-infected PMVECs. Interestingly, ExoU-mediated activation of caspase-1 was partially inhibited by reactive oxygen species scavengers. Together, these data suggest ExoU exerts pleiotropic effects on PMVEC function during P. aeruginosa infection that may inhibit endothelial barrier and inflammatory functions.

The Pseudomonas aeruginosa Type III Secretion System Exoenzyme Effector ExoU Induces Mitochondrial Damage in a Murine Bone Marrow-Derived Macrophage Infection Model.

Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen that causes nosocomial pneumonia, urinary tract infections, and bacteremia. A hallmark of P. aeruginosa pathogenesis is disruption of host cell function by the type III secretion system (T3SS) and its cognate exoenzyme effectors. The T3SS effector ExoU is phospholipase A2 (PLA2) that targets the host cell plasmalemmal membrane to induce cytolysis and is an important virulence factor that mediates immune avoidance. In addition, ExoU has been shown to subvert the host inflammatory response in a noncytolytic manner. In primary bone marrow-derived macrophages (BMDMs), P. aeruginosa infection is sensed by the nucleotide-binding domain containing leucine-rich repeats-like receptor 4 (NLRC4) inflammasome, which triggers caspase-1 activation and inflammation. ExoU transiently inhibits NLRC4 inflammasome-mediated activation of caspase-1 and its downstream target, interleukin 1ß (IL-1ß), to suppress activation of inflammation. In the present study, we sought to identify additional noncytolytic virulence functions for ExoU and discovered an unexpected association between ExoU, host mitochondria, and NLRC4. We show that infection of BMDMs with P. aeruginosa strains expressing ExoU elicited mitochondrial oxidative stress. In addition, mitochondria and mitochondrion-associated membrane fractions enriched from infected cells exhibited evidence of autophagy activation, indicative of damage. The observation that ExoU elicited mitochondrial stress and damage suggested that ExoU may also associate with mitochondria during infection. Indeed, ExoU phospholipase A2 enzymatic activity was present in enriched mitochondria and mitochondrion-associated membrane fractions isolated from P. aeruginosa-infected BMDMs. Intriguingly, enriched mitochondria and mitochondrion-associated membrane fractions isolated from infected Nlrc4 homozygous knockout BMDMs displayed significantly lower levels of ExoU enzyme activity, suggesting that NLRC4 plays a role in the ExoU-mitochondrion association. These observations prompted us to assay enriched mitochondria and mitochondrion-associated membrane fractions for NLRC4, caspase-1, and IL-1ß. NLRC4 and pro-caspase-1 were detected in enriched mitochondria and mitochondrion-associated membrane fractions isolated from noninfected BMDMs, and active caspase-1 and active IL-1ß were detected in response to P. aeruginosa infection. Interestingly, ExoU inhibited mitochondrion-associated caspase-1 and IL-1ß activation. The implications of ExoU-mediated effects on mitochondria and the NLRC4 inflammasome during P. aeruginosa infection are discussed.

Perspectives on the Pseudomonas aeruginosa Type III Secretion System Effector ExoU and Its Subversion of the Host Innate Immune Response to Infection.

Pseudomonas aeruginosa is an opportunistic, Gram-negative pathogen and an important cause of hospital acquired infections, especially in immunocompromised patients. Highly virulent P. aeruginosa strains use a type III secretion system (T3SS) to inject exoenzyme effectors directly into the cytoplasm of a target host cell. P. aeruginosa strains that express the T3SS effector, ExoU, associate with adverse outcomes in critically ill patients with pneumonia, owing to the ability of ExoU to rapidly damage host cell membranes and subvert the innate immune response to infection. Herein, we review the structure, function, regulation, and virulence characteristics of the T3SS effector ExoU, a highly cytotoxic phospholipase A2 enzyme.

Carbonic Anhydrase IX and Hypoxia Promote Rat Pulmonary Endothelial Cell Survival during Infection.

Low tidal volume ventilation protects the lung in mechanically ventilated patients. The impact of the accompanying permissive hypoxemia and hypercapnia on endothelial cell recovery from injury is poorly understood. CA (carbonic anhydrase) IX is expressed in pulmonary microvascular endothelial cells (PMVECs), where it contributes to CO2 and pH homeostasis, bioenergetics, and angiogenesis. We hypothesized that CA IX is important for PMVEC survival and that CA IX expression and release from PMVECs are increased during infection. Although the plasma concentration of CA IX was unchanged in human and rat pneumonia, there was a trend toward increasing CA IX in the bronchoalveolar fluid of mechanically ventilated critically ill patients with pneumonia and a significant increase in CA IX in the lung tissue lysates of pneumonia rats. To investigate the functional implications of the lung CA IX increase, we generated PMVEC cell lines harboring domain-specific CA IX mutations. By using these cells, we found that infection promotes intracellular (IC) expression, release, and MMP (metalloproteinase)-mediated extracellular cleavage of CA IX in PMVECs. IC domain deletion uniquely impaired CA IX membrane localization. Loss of the CA IX IC domain promoted cell death after infection, suggesting that the IC domain has an important role in PMVEC survival. We also found that hypoxia improves survival, whereas hypercapnia reverses the protective effect of hypoxia, during infection. Thus, we report 1) that CA IX increases in the lungs of pneumonia rats and 2) that the CA IX IC domain and hypoxia promote PMVEC survival during infection.

Exoenzyme Y Contributes to End-Organ Dysfunction Caused by Pseudomonas aeruginosa Pneumonia in Critically Ill Patients: An Exploratory Study.

Pseudomonas aeruginosa is an opportunistic pathogen that causes pneumonia in immunocompromised and intensive care unit (ICU) patients. During host infection, P. aeruginosa upregulates the type III secretion system (T3SS), which is used to intoxicate host cells with exoenzyme (Exo) virulence factors. Of the four known Exo virulence factors (U, S, T and Y), ExoU has been shown in prior studies to associate with high mortality rates. Preclinical studies have shown that ExoY is an important edema factor in lung infection caused by P. aeruginosa, although its importance in clinical isolates of P. aeruginosa is unknown. We hypothesized that expression of ExoY would be highly prevalent in clinical isolates and would significantly contribute to patient morbidity secondary to P. aeruginosa pneumonia. A single-center, prospective observational study was conducted at the University of Alabama at Birmingham Hospital. Mechanically ventilated ICU patients with a bronchoalveolar lavage fluid culture positive for P. aeruginosa were included. Enrolled patients were followed from ICU admission to discharge and clinical P. aeruginosa isolates were genotyped for the presence of exoenzyme genes. Ninety-nine patients were enrolled in the study. ExoY was present in 93% of P. aeruginosa clinical isolates. Moreover, ExoY alone (ExoY+/ExoU-) was present in 75% of P. aeruginosa isolates, compared to 2% ExoU alone (ExoY-/ExoU+). We found that bacteria isolated from human samples expressed active ExoY and ExoU, and the presence of ExoY in clinical isolates was associated with end-organ dysfunction. This is the first study we are aware of that demonstrates that ExoY is important in clinical outcomes secondary to nosocomial pneumonia.

Virulent Pseudomonas aeruginosa infection converts antimicrobial amyloids into cytotoxic prions.

Pseudomonas aeruginosa infection elicits the production of cytotoxic amyloids from lung endothelium, yet molecular mechanisms of host-pathogen interaction that underlie the amyloid production are not well understood. We examined the importance of type III secretion system (T3SS) effectors in the production of cytotoxic amyloids. P aeruginosa possessing a functional T3SS and effectors induced the production and release of cytotoxic amyloids from lung endothelium, including beta amyloid, and tau. T3SS effector intoxication was sufficient to generate cytotoxic amyloid release, yet intoxication with exoenzyme Y (ExoY) alone or together with exoenzymes S and T (ExoS/T/Y) generated the most virulent amyloids. Infection with lab and clinical strains engendered cytotoxic amyloids that were capable of being propagated in endothelial cell culture and passed to naïve cells, indicative of a prion strain. Conversely, T3SS-incompetent P aeruginosa infection produced non-cytotoxic amyloids with antimicrobial properties. These findings provide evidence that (1) endothelial intoxication with ExoY is sufficient to elicit self-propagating amyloid cytotoxins during infection, (2) pulmonary endothelium contributes to innate immunity by generating antimicrobial amyloids in response to bacterial infection, and (3) ExoY contributes to the virulence arsenal of P aeruginosa through the subversion of endothelial amyloid host-defense to promote a lung endothelial-derived cytotoxic proteinopathy.

cAMP signaling primes lung endothelial cells to activate caspase-1 during Pseudomonas aeruginosa infection.

Activation of the inflammasome-caspase-1 axis in lung endothelial cells is emerging as a novel arm of the innate immune response to pneumonia and sepsis caused by Pseudomonas aeruginosa. Increased levels of circulating autacoids are hallmarks of pneumonia and sepsis and induce physiological responses via cAMP signaling in targeted cells. However, it is unknown whether cAMP affects other functions, such as P. aeruginosa-induced caspase-1 activation. Herein, we describe the effects of cAMP signaling on caspase-1 activation using a single cell flow cytometry-based assay. P. aeruginosa infection of cultured lung endothelial cells caused caspase-1 activation in a distinct population of cells. Unexpectedly, pharmacological cAMP elevation increased the total number of lung endothelial cells with activated caspase-1. Interestingly, addition of cAMP agonists augmented P. aeruginosa infection of lung endothelial cells as a partial explanation underlying cAMP priming of caspase-1 activation. The cAMP effect(s) appeared to function as a priming signal because addition of cAMP agonists was required either before or early during the onset of infection. However, absolute cAMP levels measured by ELISA were not predictive of cAMP-priming effects. Importantly, inhibition of de novo cAMP synthesis decreased the number of lung endothelial cells with activated caspase-1 during infection. Collectively, our data suggest that lung endothelial cells rely on cAMP signaling to prime caspase-1 activation during P. aeruginosa infection.

Infection-induced endothelial amyloids impair memory.

Patients with nosocomial pneumonia exhibit elevated levels of neurotoxic amyloid and tau proteins in the cerebrospinal fluid (CSF). In vitro studies indicate that pulmonary endothelium infected with clinical isolates of either Pseudomonas aeruginosa, Klebsiella pneumoniae, or Staphylococcus aureus produces and releases cytotoxic amyloid and tau proteins. However, the effects of the pulmonary endothelium-derived amyloid and tau proteins on brain function have not been elucidated. Here, we show that P. aeruginosa infection elicits accumulation of detergent insoluble tau protein in the mouse brain and inhibits synaptic plasticity. Mice receiving endothelium-derived amyloid and tau proteins via intracerebroventricular injection exhibit a learning and memory deficit in object recognition, fear conditioning, and Morris water maze studies. We compared endothelial supernatants obtained after the endothelia were infected with P. aeruginosa possessing an intact [P. aeruginosa isolated from patient 103 (PA103) supernatant] or defective [mutant strain of P. aeruginosa lacking a functional type 3 secretion system needle tip complex (ΔPcrV) supernatant] type 3 secretion system. Whereas the PA103 supernatant impaired working memory, the ΔPcrV supernatant had no effect. Immunodepleting amyloid or tau proteins from the PA103 supernatant with the A11 or T22 antibodies, respectively, overtly rescued working memory. Recordings from hippocampal slices treated with endothelial supernatants or CSF from patients with or without nosocomial pneumonia indicated that endothelium-derived neurotoxins disrupted the postsynaptic synaptic response. Taken together, these results establish a plausible mechanism for the neurologic sequelae consequent to nosocomial bacterial pneumonia.-Balczon, R., Pittet, J.-F., Wagener, B. M., Moser, S. A., Voth, S., Vorhees, C. V., Williams, M. T., Bridges, J. P., Alvarez, D. F., Koloteva, A., Xu, Y., Zha, X.-M., Audia, J. P., Stevens, T., Lin, M. T. Infection-induced endothelial amyloids impair memory.

Ticagrelor Does Not Protect Isolated Rat Hearts, Thus Clouding Its Proposed Cardioprotective Role Through ENT 1 in Heart Tissue.

P2Y12 receptor-blocking drugs given at reperfusion offer protection against myocardial infarction in animal models of transient coronary occlusion. Two recent reports concluded that ticagrelor was more cardioprotective than clopidogrel and attributed this to ticagrelor's unique ability to raise tissue adenosine by blocking the equilibrative nucleoside transporter 1. Indeed, an adenosine receptor blocker attenuated ticagrelor's protection. The related P2Y12 inhibitor cangrelor, which does not block the transporter, protects hearts only when platelets are in the perfusate, while adenosine is known to protect equally in situ blood-perfused and crystalloid-perfused isolated hearts. We, therefore, tested whether ticagrelor liberates a sufficient amount of adenosine to protect a Krebs buffer-perfused isolated rat heart subjected to 40 minutes of global ischemia followed by 2 hours of reperfusion. In untreated hearts, 77.6% ± 4.0% of the ventricle was infarcted as measured by triphenyltetrazolium staining. Ischemically preconditioned hearts had only 32.7% ± 3.6% infarction ( P < .001 vs untreated), indicating that our model could be protected by preconditioning which is known to involve adenosine. Strikingly, hearts treated with 10 µmol/L ticagrelor in the buffer throughout the reperfusion period had 77.5% ± 2.4% infarction comparable to unprotected controls ( P = NS vs untreated). These data strongly suggest that ticagrelor was unable to release sufficient adenosine from the crystalloid-perfused rat heart to protect it against infarction. Our previous studies have found no difference in the anti-infarct potency among clopidogrel, cangrelor, and ticagrelor in open-chest rats and rabbits, and surprisingly adenosine receptor antagonists block protection from all 3 drugs. We have no explanation why ticagrelor is more protective in the pig than clopidogrel but suspect a species or perhaps a treatment schedule difference that may or may not involve adenosine.

Analysis of pulmonary vascular injury and repair during Pseudomonas aeruginosa infection-induced pneumonia and acute respiratory distress syndrome.

Herein we describe lung vascular injury and repair using a rodent model of Pseudomonas aeruginosa pneumonia-induced acute respiratory distress syndrome (ARDS) during: 1) the exudative phase (48-hour survivors) and 2) the reparative/fibro-proliferative phase (1-week survivors). Pneumonia was induced by intratracheal instillation of P. aeruginosa strain PA103, and lung morphology and pulmonary vascular function were determined subsequently. Pulmonary vascular function was assessed in mechanically ventilated animals in vivo (air dead space, PaO2, and lung mechanics) and lung permeability was determined in isolated perfused lungs ex vivo (vascular filtration coefficient and extravascular lung water). At 48 hours post infection, histological analyses demonstrated capillary endothelial disruption, diffuse alveolar damage, perivascular cuffs, and neutrophil influx into lung parenchyma. Infected animals displayed clinical hallmarks of ARDS, including increased vascular permeability, increased dead space, impaired gas exchange, and decreased lung compliance. Overall, the animal infection model recapitulated the morphological and functional changes typically observed in lungs from patients during the exudative phase of ARDS. At 1 week post infection, there was lung histological and pulmonary vascular functional evidence of repair when compared with 48 hours post infection; however, some parameters were still impaired when compared with uninfected controls. Importantly, lungs displayed increased fibrosis and cellular hyperplasia reminiscent of lungs from patients during the fibro-proliferative phase of ARDS. Control, sham inoculated animals showed normal lung histology and function. These data represent the first comprehensive assessment of lung pathophysiology during the exudative and reparative/fibro-proliferative phases of P. aeruginosa pneumonia-induced ARDS, and position this pre-clinical model for use in interventional studies aimed at advancing clinical care.

Caspase-1 inhibition by VX-765 administered at reperfusion in P2Y12 receptor antagonist-treated rats provides long-term reduction in myocardial infarct size and preservation of ventricular function.

Patients with acute myocardial infarction receive a P2Y12 receptor antagonist prior to reperfusion, a treatment that has reduced, but not eliminated, mortality, or heart failure. We tested whether the caspase-1 inhibitor VX-765 given at reperfusion (a requirement for clinical use) can provide sustained reduction of infarction and long-term preservation of ventricular function in a pre-clinical model of ischemia/reperfusion that had been treated with a P2Y12 receptor antagonist. To address, the hypothesis open-chest rats were subjected to 60-min left coronary artery branch occlusion/120-min reperfusion. Vehicle or inhibitors were administered intravenously immediately before reperfusion. With vehicle only, 60.3 ± 3.8% of the risk zone suffered infarction. Ticagrelor, a P2Y12 antagonist, and VX-765 decreased infarct size to 42.8 ± 3.3 and 29.2 ± 4.9%, respectively. Combining ticagrelor with VX-765 further decreased infarction to 17.5 ± 2.3%. Similar to recent clinical trials, combining ticagrelor and ischemic postconditioning did not result in additional cardioprotection. VX-765 plus another P2Y12 antagonist, cangrelor, also decreased infarction and preserved ventricular function when reperfusion was increased to 3 days. In addition, VX-765 reduced infarction in blood-free, isolated rat hearts indicating at least a portion of injurious caspase-1 activation originates in cardiac tissue. While the pro-drug VX-765 only protected isolated hearts when started prior to ischemia, its active derivative VRT-043198 provided the same amount of protection when started at reperfusion, indicating that even in blood-free hearts, caspase-1 appears to exert its injury only at reperfusion. Moreover, VX-765 decreased circulating IL-1ß, prevented loss of cardiac glycolytic enzymes, preserved mitochondrial complex I activity, and decreased release of lactate dehydrogenase, a marker of pyroptosis. Our results are the first demonstration of a clinical-grade drug given at reperfusion providing additional, sustained infarct size reduction when added to a P2Y12 receptor antagonist.