Identification of Potential Urinary Metabolite Biomarkers of Pseudomonas aeruginosa Ventilator-Associated Pneumonia.

Introduction: Ventilator-associated pneumonia (VAP) caused by Pseudomonas aeruginosa is a major cause of morbidity and mortality in hospital intensive care units (ICU). Rapid identification of P. aeruginosa-derived markers in easily accessible patients' samples can enable an early detection of P. aeruginosa VAP (VAP-PA), thereby stewarding antibiotic use and improving clinical outcomes. Methods: Metabolites were analysed using liquid chromatography-mass spectrometry (LC-MS) in prospectively collected urine samples from mechanically ventilated patients admitted to the Antwerp University Hospital ICU. Patients were followed from the start of mechanical ventilation (n = 100 patients) till the time of clinical diagnosis of VAP (n = 13). Patients (n = 8) in whom diagnosis of VAP was further confirmed by culturing respiratory samples and urine samples were studied for semi-quantitative metabolomics. Results: We first show that multivariate analyses highly discriminated VAP-PA from VAP-non-PA as well as from the pre-infection groups (R 2 = .97 and .98, respectively). A further univariate analysis identified 58 metabolites that were significantly elevated or uniquely present in VAP-PA compared to the VAP-non-PA and pre-infection groups (P < .05). These comprised both a known metabolite of histidine as well as a novel nicotine metabolite. Most interestingly, we identified 3 metabolites that were not only highly upregulated for, but were also highly specific to, VAP-PA, as these metabolites were completely absent in all pre-infection timepoints and in VAP-non-PA group. Conclusions: Considerable differences exist between urine metabolites in VAP-PA compared to VAP due to other bacterial aetiologies as well to non-VAP (pre-infection) timepoints. The unique urinary metabolic biomarkers we describe here, if further validated, could serve as highly specific diagnostic biomarkers of VAP-PA.

Aging-related modifications to G protein-coupled receptor signaling diversity.

Aging is a highly complex molecular process, affecting nearly all tissue systems in humans and is the highest risk factor in developing neurodegenerative disorders such as Alzheimer's and Parkinson's disease, cardiovascular disease and Type 2 diabetes mellitus. The intense complexity of the aging process creates an incentive to develop more specific drugs that attenuate or even reverse some of the features of premature aging. As our current pharmacopeia is dominated by therapeutics that target members of the G protein-coupled receptor (GPCR) superfamily it may be prudent to search for effective anti-aging therapeutics in this fertile domain. Since the first demonstration of GPCR-based ß-arrestin signaling, it has become clear that an enhanced appreciation of GPCR signaling diversity may facilitate the creation of therapeutics with selective signaling activities. Such 'biased' ligand signaling profiles can be effectively investigated using both standard molecular biological techniques as well as high-dimensionality data analyses. Through a more nuanced appreciation of the quantitative nature across the multiple dimensions of signaling bias that drugs possess, researchers may be able to further refine the efficacy of GPCR modulators to impact the complex aberrations that constitute the aging process. Identifying novel effector profiles could expand the effective pharmacopeia and assist in the design of precision medicines. This review discusses potential non-G protein effectors, and specifically their potential therapeutic suitability in aging and age-related disorders.

Mechanical Ventilation Impairs IL-17 Cytokine Family Expression in Ventilator-Associated Pneumonia.

Mechanical ventilation (MV) is the primary risk factor for the development of ventilator-associated pneumonia (VAP). Besides inducing a pro-inflammatory T-helper (Th)-1 cytokine response, MV also induces an anti-inflammatory Th2 cytokine response, marked by increased IL-4 secretion and reduced bacterial phagocytic capacity of rodent lung macrophages. Since IL-4 is known to downregulate both Th1 and Th17 cytokines, the latter is important in mediating mucosal immunity and combating bacterial and fungal growth, we studied and showed here in a rat model of MV that Th17 cytokines (IL-17A, IL-17F, and IL-22) were significantly upregulated in the lung as a response to different MV strategies currently utilized in clinic. To study whether the increased IL-4 levels are associated with downregulation of the anti-bacterial Th17 cytokines, we subsequently challenged mechanically ventilated rats with an intratracheal inoculation of Pseudomonas aeruginosa (VAP model) and showed a dramatic downregulation of IL-17A, IL-17F, and IL-22, compared to animals receiving the same bacterial burden without MV. For the studied Th1 cytokines (IFN, TNF, IL-6, and IL-1), only IFN showed a significant decrease as a consequence of bacterial infection in mechanically ventilated rats. We further studied IL-17A, the most studied IL-17 family member, in intensive care unit (ICU) pneumonia patients and showed that VAP patients had significantly lower levels of IL-17A in the endotracheal aspirate compared to patients entering ICU with pre-existing pneumonia. These translational data, obtained both in animal models and in humans, suggest that a deficient anti-bacterial Th17 response in the lung during MV is associated with VAP development.

Methods to Investigate the Molecular Basis of Progranulin Actions on Brain and Behavior In Vivo Using Knockout Mice.

Currently one of the few molecules that equally excites a neuroscientist, a cancer biologist, an immunologist, and a developmental biologist is progranulin (GRN/Grn)-a pluripotent growth factor that plays key roles in cell survival, proliferation, development, tissue regeneration, inflammation, wound healing, and angiogenesis. However, the molecular pathways associated with GRN signaling involved in these varied physiological processes are not understood. Gene inactivation has been considered as one of the best methods to delineate the biological role of a protein, and gene targeting is a direct means to disrupt a gene's open reading frame and block its expression, for instance, in a mouse. Such a gene knockout animal model also served as an in vivo disease model where loss of gene or its function is thought to be the primary disease mechanism, as is the case with progranulin loss of function in frontotemporal lobar degeneration (FTLD). It is estimated that up to half of the cases of familial, dominant FTLD might be due to GRN haploinsufficiency. To understand the molecular pathways associated with GRN loss, constitutive and conditional progranulin knockout (Grn-/-) mice have also been constructed in several laboratories, including ours. These mice show several disease-characteristic features and suggest that continued studies on the Grn-/- mice would be instructive in the understanding of complex GRN biology in health and disease.

GIT2-A keystone in ageing and age-related disease.

Since its discovery, G protein-coupled receptor kinase-interacting protein 2, GIT2, and its family member, GIT1, have received considerable interest concerning their potential key roles in regulating multiple inter-connected physiological and pathophysiological processes. GIT2 was first identified as a multifunctional protein that is recruited to G protein-coupled receptors (GPCRs) during the process of receptor internalization. Recent findings have demonstrated that perhaps one of the most important effects of GIT2 in physiology concerns its role in controlling multiple aspects of the complex ageing process. Ageing can be considered the most prevalent pathophysiological condition in humans, affecting all tissue systems and acting as a driving force for many common and intractable disorders. The ageing process involves a complex interplay among various deleterious activities that profoundly disrupt the body's ability to cope with damage, thus increasing susceptibility to pathophysiologies such as neurodegeneration, central obesity, osteoporosis, type 2 diabetes mellitus and atherosclerosis. The biological systems that control ageing appear to function as a series of interconnected complex networks. The inter-communication among multiple lower-complexity signaling systems within the global ageing networks is likely coordinated internally by keystones or hubs, which regulate responses to dynamic molecular events through protein-protein interactions with multiple distinct partners. Multiple lines of research have suggested that GIT2 may act as one of these network coordinators in the ageing process. Identifying and targeting keystones, such as GIT2, is thus an important approach in our understanding of, and eventual ability to, medically ameliorate or interdict age-related progressive cellular and tissue damage.

CD8 signaling in microglia/macrophage M1 polarization in a rat model of cerebral ischemia.

Classical or M1 activity of microglia/macrophages has been described in several neurodegenerative and brain inflammatory conditions and has also been linked to expansion of ischemic injury in post-stroke brain. While different pathways of M1 polarization have been suggested to occur in the post-stroke brain, the precise underlying mechanisms remain undefined. Using a transient middle cerebral artery occlusion (MCAO) rat model, we showed a progressive M2 to M1 polarization in the perilesional brain region with M1 cells becoming one of the dominant subsets by day 4 post-stroke. Comparing key receptors involved in M1 polarization (CD8, IFNγR, Clec4, FcγR, TLR3 and TLR4) and their signal transducers (Syk, Stat1, Irf3, and Traf6) at the day 4 time point, we showed a strong upregulation of CD8 along with SYK transducer in dissected perilesional brain tissue. We further showed that CD8 expression in the post-stroke brain was associated with activated (CD68+) macrophages and that progressive accumulation of CD8+CD68+ cells in the post-stroke brain coincided with increased iNOS (M1 marker) and reduced Arg1 (M2 marker) expression on these cells. In vitro ligand-based stimulation of the CD8 receptor caused increased iNOS expression and an enhanced capacity to phagocytose E. coli particles; and interestingly, CD8 stimulation was also able to repolarize IL4-treated M2 cells to an M1 phenotype. Our data suggest that increased CD8 signaling in the post-stroke brain is primarily associated with microglia/macrophages and can independently drive M1 polarization, and that modulation of CD8 signaling could be a potential target to limit secondary post-stroke brain damage.

Mechanical Ventilation Induces Interleukin 4 Secretion in Lungs and Reduces the Phagocytic Capacity of Lung Macrophages.

Patients receiving mechanical ventilation are at risk of developing ventilator-associated pneumonia. Here, we show that clinically utilized ventilation protocols in rats with 5 mL/kg or 8 mL/kg tidal volumes cause increased interleukin 4 (IL-4) expression, lowered ratio of TH1:TH2 transcriptional factors (Tbet:Gata3), and increased arginase 1-positive (Arg1+) macrophages and eosinophils in lungs. Macrophages from ventilated lungs had reduced ex vivo capacity toward phagocytosing bacteria. Ventilated animals, when further challenged with bacterial pneumonia, continued to show persistence of Arg1+ M2 macrophages as well as an increased bacterial burden compared with spontaneously breathing animals receiving the same bacterial dose. Increased IL-4 expression also occurred in a mouse ventilation model, and abrogation of IL-4 signaling restored lung bacterial burden in an IL-4Rα-/- ventilator-associated pneumonia model. Our data suggest that mechanical ventilation induces an immunosuppressive state in lungs, providing new insight in the development of ventilator-associated pneumonia.

Biofilm-Induced Type 2 Innate Immunity in a Cystic Fibrosis Model of Pseudomonas aeruginosa.

Biofilm-producing strains of Pseudomonas aeruginosa are a major cause of morbidity and mortality in cystic fibrosis (CF) patients. In these patients, increased levels of IL-17 as well as of IL-5 and IL-13 along with arginase (Arg)-positive macrophages have been observed in bronchoalveolar lavage fluid. While IL-17 is a strong proinflammatory cytokine associated with host defense against bacterial and fungal infections and is also elevated in several autoimmune diseases, IL-5/IL-13 and Arg1-positive M2 macrophages are part of the anti-inflammatory type 2 (Th2) immunity. To study whether increased IL-5 and IL-13 levels are related to biofilm formation, which is frequently observed in CF patients colonized by P. aeruginosa, we utilized an agarose bead-embedded P. aeruginosa rat model commonly employed in in vivo biofilm studies. We showed that "sterile" agarose bead instillation in rat notably increased lung transcript levels of IL-5 and IL-13 at two post-instillation study-points, day 1 and day 3. Concurrently, increased infiltration of type 2 innate cells such as eosinophils and Arg1 positive M2 activated macrophages (Arg1+CD68+) was also observed both at day 1 and day 3 while the proportion of M1 activated macrophages (iNOS+CD68+) at these time-points decreased. In contrast, P. aeruginosa-loaded beads caused a drastic elevation of proinflammatory Th1 (IFNγ, TNFα, IL-12a) and antibacterial Th17 (IL-17a, IL-17f, IL-22, IL-23a) cytokines along with a high influx of neutrophils and M1 macrophages, while Th2 cytokines (IL-5 and IL-13) drastically declined at day 1 post-infection. Interestingly, at day 3 post-infection, both Th1 and Th17 cytokines sharply declined and corroborated with decreased M1 and increased M2 macrophages. These data suggest that while IL-17 is linked to episodes of acute exacerbations of infection in CF patients, the increased Th2 cytokines and M2 macrophages observed in these patients are largely due to the biofilm matrix. The data presented here has important implications for clinical management of CF patients.

99mTc-Duramycin SPECT Imaging of Early Tumor Response to Targeted Therapy: A Comparison with 18F-FDG PET.

Molecular imaging of cell death may provide a detailed readout of the cellular response to novel therapies and prognostic information on tumor treatment efficacy, assisting in the design of individualized therapy. We compared the predictive power of cell death imaging using 99mTc-duramycin with the current gold standard 18F-FDG for treatment response evaluation after targeted therapy. Methods: Early therapy response evaluation was assessed by 99mTc-duramycin SPECT and 18F-FDG PET imaging in treatment-sensitive COLO205 and treatment-resistant HT29 human colorectal cancer xenografts 24 h after a single dose of conatumumab or IgG1 control. The specificity of 99mTc-duramycin for apoptosis was assessed using 99mTc-linear duramycin control radiotracer. Radiotracer uptake was validated ex vivo by γ-counting and autoradiography and compared with cleaved caspase-3 (CC3) activation and DNA fragmentation (TdT-mediated dUTP nick-end labeling [TUNEL]). Data were analyzed with the Student t test and Pearson correlation. All statistical tests were 2-sided. Results: COLO205 tumor uptake of 99mTc-duramycin was increased 7-fold from baseline in conatumumab- versus IgG1-treated control mice (P < 0.001), in good correlation with histologic analysis of apoptosis (CC3, r = 0.842, and TUNEL, r = 0.894; P < 0.001). No response was detected in HT29 tumors. No change in 99mTc-linear duramycin uptake could be detected in COLO205 tumors after treatment, indicating specificity of the 99mTc-duramycin tumor signal. 18F-FDG uptake was not significantly increased from baseline in conatumumab- versus IgG1-treated COLO205 and HT29 tumor-bearing mice (P = 0.104 and 0.779, respectively) and did not correlate with immunohistochemical evidence of apoptosis. Conclusion: We have demonstrated that 99mTc-duramycin specifically accumulates in apoptotic tumors in which 18F-FDG was not able to differentiate responding from nonresponding tumors early after treatment. 99mTc-duramycin holds promise as a noninvasive imaging radiotracer for early treatment evaluation in the clinic.