Towards a system analysis of the early steps of infectious endocarditis pathogenesis

L'endocardite infectieuse (EI) est une maladie potentiellement mortelle qui doit être prévenue dans toute la mesure du possible. Au cours de ces dernières 50 années, les recommandations Américaines et Européennes pour la prophylaxie de PEI proposaient aux patients à risques de prendre un antibiotique, préventif avant de subir une intervention médico-chirurgicale susceptible d'induire une bactériémie transitoire. Cependant, des études épidémiologiques récentes ont montré que la plupart des EI survenaient en dehors de tous actes médico-chirurgicaux, et indépendamment de la prise ou non de prophylaxie antibiotique . L'EI pourrait donc survenir suite à la cumulation de bactériémies spontanées de faibles intensités, associées à des activités de la vie courante telle que le brossage dentaire pour le streptocoques, ou à partir de tissus colonisés ou de cathéters infectés pour les staphylocoques. En conséquence, les recommandations internationales pour la prophylaxie de PEI ont été revues et proposent une diminution drastique de l'utilisation d'antibiotiques. Cependant, le risque d'EI représenté par le cumul de bactériémies de faibles intensités n'a pas été démontré expérimentalement. Nous avons développé un nouveau modèle d'EI expérimentale induite par une inoculation en continu d'une faible quantité de bactéries, simulant le cumul de bactériémies de faibles intensités chez l'homme, et comparé l'infection de Streptococcus gordonii et de Staphylococcus aureus dans ce modèle avec celle du modèle d'IE induite par une bactériémie brève, mais de forte intensité. Nous avons démontré, après injection d'une quantité égale de bactéries, que le nombre de végétations infectées était similaire dans les deux types d'inoculations. Ces résultats expérimentaux ont confirmé l'hypothèse qu'une exposition cumulée à des bactériémies de faibles intensités, en dehors d'une procédure médico-chirurgicale, représentait un risque pour le développement d'une El, comme le suggéraient les études épidémiologiques. En plus, ces résultats ont validé les nouvelles recommandations pour la prophylaxie de l'El, limitant drastiquement l'utilisation d'antibiotiques. Cependant, ces nouvelles recommandations laissent une grande partie (> 90%) de cas potentiels d'EI sans alternatives de préventions, et des nouvelles stratégies prophylactiques doivent être investiguées. Le nouveau modèle d'EI expérimentale représente un modèle réaliste pour étudier des nouvelles mesures prophylactiques potentielles appliquées à des expositions cumulées de bactériémies de faible nombre. Dans un contexte de bactériémies spontanées répétitives, les antibiotiques ne peuvent pas résoudre le problème de la prévention de l'EI. Nous avons donc étudié la une alternative de prévention par l'utilisation d'agents antiplaquettaires. La logique derrière cette approche était basée sur le fait que les plaquettes sont des composants clés dans la formation des végétations cardiaques, et le fait que les bactéries capables d'interagir avec les plaquettes sont plus enclines à induire une El. Les agents antiplaquettaires utilisés ont été l'aspirine (inhibiteur du COX1), la ticlopidine (inhibiteur du P2Y12, le récepteur de l'ADP), et l'eptifibatide et Pabciximab, deux inhibiteurs du GPIIb/IIIa, le récepteur plaquettaire pour le fibrinogène. Les anticoagulants étaient le dabigatran etexilate, inhibant lathrombine et l'acenocumarol, un antagoniste de la vitamine K. L'aspirine, la ticlopidine ou l'eptifibatide seuls n'ont pas permis de prévenir l'infection valvulaire (> 75% animaux infectés). En revanche, la combinaison d'aspirine et de ticlopidine, aussi bien que l'abciximab, ont protégé 45% - 88% des animaux de l'EI par S. gordonii et par S. aureus. L'antithrombotique dabigatran etexilate à protégé 75% des rats contre l'EI par S. aureus, mais pas (< 30% de protection) par S. gordonii. L'acenocoumarol n'a pas eu d'effet sur aucun des deux organismes. En général, ces résultats suggèrent un possible rôle pour les antiplaquettaires et du dabigatran etexilate dans la prophylaxie de l'EI dans un contexte de bactériémies récurrentes de faibles intensités. Cependant, l'effet bénéfique des antiplaquettaires doit être soupesé avec le risque d'hémorragie inhérent à ces molécules, et le fait que les plaquettes jouent un important rôle dans les défenses de l'hôte contre les infections endovasculaires. En plus, le double effet bénéfique du dabigatran etexilate devrait être revu chez les patients porteurs de valves prothétiques, qui ont besoin d'une anticoagulation à vie, et chez lesquels l'EI à S. aureus est associée avec une mortalité de près de 50%. Comme l'approche avec des antiplaquettaires et des antithrombotiques pourrait avoir des limites, une autre stratégie prophylactique pourrait être la vaccination contre des adhésines de surfaces des pathogènes. Chez S. aureus, la protéine de liaison au fibrinogène, ou dumping factor A (ClfA), et la protéine de liaison à la fibronectine (FnbpA) sont des facteurs de virulence nécessaires à l'initiation et l'évolution de PEI. Elles représentent donc des cibles potentielles pour le développement de vaccins contre cette infection. Récemment, des nombreuses publications ont décrit que la bactérie Lactococcus lactis pouvait être utilisée comme vecteur pour la diffusion d'antigènes bactériens in vivo, et que cette approche pourrait être une stratégie de vaccination contre les infections bactériennes. Nous avons exploré l'effet de l'immunisation par des recombinant de L. lactis exprimant le ClfA, la FnbpA, ou le ClfA ensemble avec et une forme tronquée de la FnbpA (Fnbp, comprenant seulement le domaine de liaison à la fibronectine mais sans le domaine A de liaison au fibrinogène [L. lactis ClfA/Fnbp]), dans la prophylaxie de PIE expérimentale à S. aureus. L. lactis ClfA a été utilisés comme agent d'immunisation contre la souche S. aureus Newman (qui a particularité de n'exprimer que le ClfA, mais pas la FnbpA). L. lactis ClfA, L. lactis FnbpA, et L. lactis ClfA/Fnbp, ont été utilisé comme agents d'immunisation contre une souche isolée d'une IE, S. aureus P8 (exprimant ClfA et FnbpA). L'immunisation avec L. lactis ClfA a généré des anticorps anti-ClfA fonctionnels, capables de bloquer la liaison de S. aureus Newman au fibrinogène in vitro et protéger 13/19 (69%) animaux d'une El due à S. aureus Newman (P < 0.05 comparée aux contrôles). L'immunisation avec L. lactis ClfA, L. lactis FnbpA, ou L. lactis ClfA/Fnbp, a généré des anticorps contre chacun de ces antigènes. Cependant, ils n'ont pas permis de bloquer l'adhésion de S. aureus P8 au fibrinogène et à la fibronectine in vitro. De plus, l'immunisation avec L. lactis ClfA ou L. lactis FnbpA s'est avérée inefficace in vivo (< 10% d'animaux protégés d'une El) et l'immunisation avec L. lactis ClfA/Fnbp a fourni une protection limitée de l'EI (8/23 animaux protégés; P < 0.05 comparée aux contrôles) après inoculation avec S. aureus P8. Dans l'ensemble, ces résultats indiquent que L. lactis est un système efficace pour la présentation d'antigènes in vivo et potentiellement utile pour la prévention de PEI à S. aureus. Cependant, le répertoire de protéines de surface de S. aureus capable d'évoquer une panoplie d'anticorps efficace reste à déterminer.. En résumé, notre étude a démontré expérimentalement, pour la première fois, qu'une bactériémie répétée de faible intensité, simulant la bactériémie ayant lieu, par exemple, lors des activités de la vie quotidienne, est induire un taux d'EI expérimentale similaire à celle induite par une bactériémie de haute intensité suite à une intervention médicale. Dans ce contexte, où l'utilisation d'antibiotiques est pas raisonnable, nous avons aussi montré que d'autres mesures prophylactiques, comme l'utilisation d'agents antiplaquettaires ou antithrombotiques, ou la vaccination utilisant L. lactis comme vecteur d'antigènes bactériens, sont des alternatives prometteuses qui méritent d'être étudiées plus avant. Thesis Summary Infective endocarditis (IE) is a life-threatening disease that should be prevented whenever possible. Over the last 50 years, guidelines for IE prophylaxis proposed the use of antibiotics in patients undergoing dental or medico-surgical procedures that might induce high, but transient bacteremia. However, recent epidemiological studies indicate that IE occurs independently of medico-surgical procedures and the fact that patients had taken antibiotic prophylaxis or not, i.e., by cumulative exposure to random low-grade bacteremia, associated with daily activities (e.g. tooth brushing) in the case of oral streptococci, or with a colonized site or infected device in the case of staphylococci. Accordingly, the most recent American and European guidelines for IE prophylaxis were revisited and updated to drastically restrain antibiotic use. Nevertheless, the relative risk of IE represented by such cumulative low-grade bacteremia had never been demonstrated experimentally. We developed a new model of experimental IE due to continuous inoculation of low-grade bacteremia, mimicking repeated low-grade bacteremia in humans, and compared the infectivity of Streptococcus gordonii and Staphylococcus aureus in this model to that in the model producing brief, high-level bacteremia. We demonstrated that, after injection of identical bacterial numbers, the rate of infected vegetations was similar in both types of challenge. These experimental results support the hypothesis that cumulative exposure to low-grade bacteremia, outside the context of procedure-related bacteremia, represents a genuine risk of IE, as suggested by human epidemiological studies. In addition, they validate the newer guidelines for IE prophylaxis, which drastic limit the procedures in which antibiotic prophylaxis is indicated. Nevertheless, these refreshed guidelines leave the vast majority (> 90%) of potential IE cases without alternative propositions of prevention, and novel strategies must be considered to propose effective alternative and "global" measures to prevent IE initiation. The more realistic experimental model of IE induced by low-grade bacteremia provides an accurate experimental setting to study new preventive measures applying to cumulative exposure to low bacterial numbers. Since in a context of spontaneous low-grade bacteremia antibiotics are unlikely to solve the problem of IE prevention, we addressed the role of antiplatelet and anticoagulant agents for the prophylaxis of experimental IE induced by S. gordonii and S. aureus. The logic of this approach was based on the fact that platelets are key players in vegetation formation and vegetation enlargement, and on the fact that bacteria capable of interacting with platelets are more prone to induce IE. Antiplatelet agents included the COX1 inhibitor aspirin, the inhibitor of the ADP receptor P2Y12 ticlopidine, and two inhibitors of the platelet fibrinogen receptor GPIIb/IIIa, eptifibatide and abciximab. Anticoagulants included the thrombin inhibitor dabigatran etexilate and the vitamin K antagonist acenocoumarol. Aspirin, ticlopidine or eptifibatide alone failed to prevent aortic infection (> 75% infected animals). In contrast, the combination of aspirin with ticlopidine, as well as abciximab, protected 45% to 88% of animals against IE due to S. gordonii and S. aureus. The antithrombin dabigatran etexilate protected 75% of rats against IE due to S. aureus, but failed (< 30% protection) against S. gordonii. Acenocoumarol had no effect against any bacteria. Overall, these results suggest a possible role for antiplatelet agents and dabigatran etexilate in the prophylaxis of IE in humans in a context of recurrent low- grade bacteremia. However, the potential beneficial effect of antiplatelet agents should be balanced against the risk of bleeding and the fact that platelets play an important role in the host defenses against intravascular infections. In addition, the potential dual benefit of dabigatran etexilate might be revisited in patients with prosthetic valves, who require life-long anticoagulation and in whom S. aureus IE is associated with high mortality rate. Because the antiplatelet and anticoagulant approach might be limited in the context of S. aureus bacteremia, other prophylactic strategies for the prevention of S. aureus IE, like vaccination with anti-adhesion proteins was tested. The S. aureus surface proteins fibrinogen-binding protein clumping-factor A (ClfA) and the fibronectin-binding protein A (FnbpA) are critical virulence factors for the initiation and development of IE. Thus, they represent key targets for vaccine development against this disease. Recently, numerous reports have described that the harmless bacteria Lactococcus lactis can be used as a bacterial vector for the efficient delivery of antigens in vivo, and that this approach is a promising vaccination strategy against bacterial infections. We therefore explored the immunization capacity of non- living recombinant L. lactis ClfA, L. lactis FnbpA, or L. lactis expressing ClfA together with Fnbp (a truncated form of FnbpA with only the fibronectin-binding domain but lacking the fibrinogen-binding domain A [L. lactis ClfA/Fnbp]), to protect against S. aureus experimental IE. L. lactis ClfA was used as immunization agent against the laboratory strain S. aureus Newman (expressing ClfA, but lacking FnbpA). L. lactis ClfA, L. lactis FnbpA, as well as L. lactis ClfA/Fnbp, were used as immunization agents against the endocarditis isolate S. aureus P8 (expressing both ClfA and FnbpA). Immunization with L. lactis ClfA produced anti-ClfA functional antibodies, which were able to block the binding of S. aureus Newman to fibrinogen in vitro and protect 13/19 (69%) animals from IE due to S. aureus Newman (P < 0.05 compared to controls). Immunization with L. lactis ClfA, L. lactis FnbpA or L. lactis ClfA/Fnbp, produced antibodies against each antigen. However, they were not sufficient to block S. aureus P8 binding to fibrinogen and fibronectin in vitro. Moreover, immunization with L. lactis ClfA or L. lactis FnbpA was ineffective (< 10% protected animals) and immunization with L. lactis ClfA/Fnbp conferred limited protection from IE (8/23 protected animals; P < 0.05 compared to controls) after challenge with S. aureus P8. Together, these results indicate that L. lactis is an efficient delivering antigen system potentially useful for preventing S. aureus IE. They also demonstrate that expressing multiple antigens in L. lactis, yet to be elucidated, will be necessary to prevent IE due to clinical S. aureus strains fully equipped with virulence determinants. In summary, our study has demonstrated experimentally, for the first time, the hypothesis that low-grade bacteremia, mimicking bacteremia occurring outside of a clinical intervention, is equally prone to induce experimental IE as high-grade bacteremia following medico-surgical procedures. In this context, where the use of antibiotics for the prophylaxis of IE is limited, we showed that other prophylactic measures, like the use of antiplatelets, anticoagulants, or vaccination employing L. lactis as delivery vector of bacterial antigens, are reasonable alternatives that warrant to be further investigated.

Kalemia during combined therapy with an angiotensin converting enzyme inhibitor and a potassium-sparing diuretic.

Both angiotensin converting enzyme (ACE) inhibitors and potassium-sparing diuretics tend to increase serum potassium levels. This retrospective study was undertaken to assess whether these two types of agents can nevertheless be combined safely. Twelve hypertensive patients were treated for 1-70 months (mean = 17) with an ACE inhibitor together with a potassium-sparing diuretic (spironolactone, n = 10; amiloride, n = 2). In addition, eight patients also took a thiazide or a loop diuretic. Nine patients had a normal and three a slightly impaired renal function. No clinically relevant hyperkalemia was observed during the course of the study. These data suggest that it is not impossible to combine an ACE inhibitor with a potassium-sparing diuretic, as long as renal function is normal and serum potassium concentration is monitored closely.

AMMAF : Agent Mòbil Monitor d'Alteració de Fitxers

Aquest projecte pretén realitzar un sistema en xarxa que monitoritzi periòdicament l'estat de fitxers clau de cadascun dels computadors de la xarxa emprant el paradigma dels agents mòbils. Es tracta de crear una eina enfocada a la protecció de la xarxa que solucioni problemes típicament associats a les aproximacions existents a les tècniques de la monitorització de fitxers.

Mechanisms of antagonism of Pseudomonas fluorescens EPS62e against Erwinia amylovora, the causal agent of fire blight

Pseudomonas fluorescens EPS62e was selected during a screening procedure for its high efficacy in controlling infections by Erwinia amylovora, the causal agent of fire blight disease, on different plant materials. In field trials carried out in pear trees during bloom, EPS62e colonized flowers until the carrying capacity, providing a moderate efficacy of fire-blight control. The putative mechanisms of EPS62e antagonism against E. amylovora were studied. EPS62e did not produce antimicrobial compounds described in P. fluorescens species and only developed antagonism in King’s B medium, where it produced siderophores. Interaction experiments in culture plate wells including a membrane filter, which physically separated the cultures, confirmed that inhibition of E. amylovora requires cell-to-cell contact. The spectrum of nutrient assimilation indicated that EPS62e used significantly more or different carbon sources than the pathogen. The maximum growth rate and affinity for nutrients in immature fruit extract were higher in EPS62e than in E. amylovora, but the cell yield was similar. The fitness of EPS62e and E. amylovora was studied upon inoculation in immature pear fruit wounds and hypanthia of intact flowers under controlled-environment conditions. When inoculated separately, EPS62e grew faster in flowers, whereas E. amylovora grew faster in fruit wounds because of its rapid spread to adjacent tissues. However, in preventive inoculations of EPS62e, subsequent growth of EPS101 was significantly inhibited. It is concluded that cell-to-cell interference as well as differences in growth potential and the spectrum and efficiency of nutrient use are mechanisms of antagonism of EPS62e against E. amylovora

Phosphatidylethanolamine critically supports internalization of cell-penetrating protein C inhibitor.

Although their contribution remains unclear, lipids may facilitate noncanonical routes of protein internalization into cells such as those used by cell-penetrating proteins. We show that protein C inhibitor (PCI), a serine protease inhibitor (serpin), rapidly transverses the plasma membrane, which persists at low temperatures and enables its nuclear targeting in vitro and in vivo. Cell membrane translocation of PCI necessarily requires phosphatidylethanolamine (PE). In parallel, PCI acts as a lipid transferase for PE. The internalized serpin promotes phagocytosis of bacteria, thus suggesting a function in host defense. Membrane insertion of PCI depends on the conical shape of PE and is associated with the formation of restricted aqueous compartments within the membrane. Gain- and loss-of-function mutations indicate that the transmembrane passage of PCI requires a branched cavity between its helices H and D, which, according to docking studies, precisely accommodates PE. Our findings show that its specific shape enables cell surface PE to drive plasma membrane translocation of cell-penetrating PCI.

L-canavanine, an inhibitor of inducible nitric oxide synthase, improves venous return in endotoxemic rats.

OBJECTIVE: To investigate the hemodynamic effects of L-canavanine (an inhibitor of inducible, but not of constitutive, nitric oxide synthase) in endotoxic shock. DESIGN: Controlled, randomized, experimental study. SETTING: Animal laboratory. SUBJECTS: Wistar rats. INTERVENTIONS: Rats were anesthetized with pentobarbital, and hemodynamically monitored. One hour after an intravenous challenge with 5 mg/kg of Escherichia coli endotoxin, the rats were randomized to receive a continuous infusion of either L-canavanine (20 mg/kg/hr; n = 8) or vehicle only (isotonic saline, n = 11). In all animals, the infusion was given over 5 hrs at a rate of 2 mL/kg/hr. These experiments were repeated in additional rats challenged with isotonic saline instead of endotoxin (sham experiments). MEASUREMENTS AND MAIN RESULTS: Arterial blood pressure, heart rate, thermodilution cardiac output, central venous pressure, mean systemic filling pressure, urine output, arterial blood gases, blood lactate concentration, and hematocrit were measured. In sham experiments, hemodynamic stability was maintained throughout and L-canavanine had no detectable effect. Animals challenged with endotoxin and not treated with L-canavanine developed progressive hypotension and low cardiac output. After 6 hrs of endotoxemia, both central venous pressure and mean systemic filling pressure were significantly below their baseline values, indicating relative hypovolemia as the main determinant of reduced cardiac output. In endotoxemic animals treated with L-canavanine, hypotension was less marked, while cardiac output, central venous pressure, and mean systemic filling pressure were maintained throughout the experiment. L-canavanine had no effect on the time-course of hematocrit. L-canavanine significantly increased urine output and reduced the severity of lactic acidosis. CONCLUSIONS: Six hours after an endotoxin challenge in rats, low cardiac output develops, which appears to be primarily related to relative hypovolemia. L-canavanine, a selective inhibitor of the inducible nitric oxide synthase, increases the mean systemic filling pressure, thereby improving venous return, under these conditions.

ROCK Inhibitor Enhances Adhesion and Wound Healing of Human Corneal Endothelial Cells.

Maintenance of corneal transparency is crucial for vision and depends mainly on the endothelium, a non-proliferative monolayer of cells covering the inner part of the cornea. When endothelial cell density falls below a critical threshold, the barrier and "pump" functions of the endothelium are compromised which results in corneal oedema and loss of visual acuity. The conventional treatment for such severe disorder is corneal graft. Unfortunately, there is a worldwide shortage of donor corneas, necessitating amelioration of tissue survival and storage after harvesting. Recently it was reported that the ROCK inhibitor Y-27632 promotes adhesion, inhibits apoptosis, increases the number of proliferating monkey corneal endothelial cells in vitro and enhance corneal endothelial wound healing both in vitro and in vivo in animal models. Using organ culture human cornea (N = 34), the effect of ROCK inhibitor was evaluated in vitro and ex vivo. Toxicity, corneal endothelial cell density, cell proliferation, apoptosis, cell morphometry, adhesion and wound healing process were evaluated by live/dead assay standard cell counting method, EdU labelling, Ki67, Caspase3, Zo-1 and Actin immunostaining. We demonstrated for the first time in human corneal endothelial cells ex vivo and in vitro, that ROCK inhibitor did not induce any toxicity effect and did not alter cell viability. ROCK inhibitor treatment did not induce human corneal endothelial cells proliferation. However, ROCK inhibitor significantly enhanced adhesion and wound healing. The present study shows that the selective ROCK inhibitor Y-27632 has no effect on human corneal endothelial cells proliferative capacities, but alters cellular behaviours. It induces changes in cell shape, increases cell adhesion and enhances wound healing ex vivo and in vitro. Its absence of toxicity, as demonstrated herein, is relevant for its use in human therapy.

Poly-N-Acetyl Glucosamine Fibers Induce Angiogenesis in ADP Inhibitor-Treated Diabetic Mice

Background: It has been previously demonstrated that short-fiber poly-N-acetyl-glucosamine (sNAG) nanofibers specifically interact with platelets, are hemostatic, and stimulate diabetic wound healing by activating angiogenesis, cell proliferation, and reepithelialization. Platelets play a significant physiologic role in wound healing. The influence of altered platelet function by treatment with the ADP inhibitor Clopidogrel (CL) on wound healing and the ability of sNAG to repair wounds in diabetic mice treated with CL were studied.Methods: Dorsal 1 cm2 skin wounds were excised on genetically diabetic 8-week to 12-week-old, Lep/r-db/db male mice, and wound healing kinetics were determined. Microscopic analysis was performed for angiogenesis (PECAM-1) and cell proliferation (Ki67). Mice were either treated with CL (P2Y12 ADP receptor antagonist, CL) or saline solution (NT). CL wounds were also treated with either a single application of topical sNAG (CL-sNAG) or were left untreated (CL-NT).Results: CL treatment did not alter wound healing kinetics, while sNAG induced faster wound closure in CL-treated mice compared with controls. CL treatment of diabetic mice caused an augmentation of cell proliferation and reduced angiogenesis compared with nontreated wounds. However, sNAG reversed the effects of CL on angiogenesis and partially reversed the effect on cell proliferation in the wound beds. The sNAG-treated wounds in CL-treated mice showed higher levels of cell proliferation and not did inhibit angiogenesis.Conclusions: CL treatment of diabetic mice decreased angiogenesis and increased cell proliferation in wounds but did not influence macroscopic wound healing kinetics. sNAG treatment did not inhibit angiogenesis in CL-treated mice and induced faster wound closure; sNAG technology is a promising strategy to facilitate the healing of complex bleeding wounds in CL-treated diabetic patients.

Goal recognition over POMDPs: inferring the intention of a POMDP agent

Plan recognition is the problem of inferring the goals and plans of an agent from partial observations of her behavior. Recently, it has been shown that the problem can be formulated and solved usingplanners, reducing plan recognition to plan generation.In this work, we extend this model-basedapproach to plan recognition to the POMDP setting, where actions are stochastic and states are partially observable. The task is to infer a probability distribution over the possible goals of an agent whose behavior results from a POMDP model. The POMDP model is shared between agent and observer except for the true goal of the agent that is hidden to the observer. The observations are action sequences O that may contain gaps as some or even most of the actions done by the agent may not be observed. We show that the posterior goal distribution P(GjO) can be computed from the value function VG(b) over beliefs b generated by the POMDPplanner for each possible goal G. Some extensionsof the basic framework are discussed, and a numberof experiments are reported.

Use of green fluorescent protein-based reporters to monitor balanced production of antifungal compounds in the biocontrol agent Pseudomonas fluorescens CHA0.

AIMS: To develop reporter constructs based on stable and unstable variants of the green fluorescent protein (GFP) for monitoring balanced production of antifungal compounds that are crucial for the capacity of the root-colonizing Pseudomonas fluorescens strain CHA0 to control plant diseases caused by soil-borne pathogenic fungi. METHODS AND RESULTS: Pseudomonas fluorescens CHA0 produces the three antifungal metabolites 2,4-diacetylphloroglucinol (DAPG), pyoluteorin (PLT) and pyrrolnitrin (PRN). The gfp[mut3] and gfp[AAV] reporter genes were fused to the promoter regions of the DAPG, PLT and PRN biosynthetic genes. The reporter fusions were then used to follow the kinetics of expression of the three antifungal metabolites in a microplate assay. DAPG and PLT were found to display an inverse relationship in which each metabolite activates its own biosynthesis while repressing the synthesis of the other metabolite. PRN appears not to be involved in this balance. However, the microbial and plant phenolic metabolite salicylate was found to interfere with the expression of both DAPG and PLT. CONCLUSIONS: The results obtained provide evidence that P. fluorescens CHA0 may keep the antifungal compounds DAPG and PLT at a fine-tuned balance that can be affected by certain microbial and plant phenolics. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, the present study is the first to use stable and unstable GFP variants to study antibiotic gene expression in a biocontrol pseudomonad. The developed reporter fusions will be a highly valuable tool to study in situ expression of this bacterial biocontrol trait on plant roots, i.e. at the site of pathogen suppression.

Contrast agent-enhanced, free-breathing, three-dimensional coronary magnetic resonance angiography.

For free-breathing, high-resolution, three-dimensional coronary magnetic resonance angiography (MRA), the use of intravascular contrast agents may be helpful for contrast enhancement between coronary blood and myocardium. In six patients, 0.1 mmol/kg of the intravascular contrast agent MS-325/AngioMARK was given intravenously followed by double-oblique, free-breathing, three-dimensional inversion-recovery coronary MRA with real-time navigator gating and motion correction. Contrast-enhanced, three-dimensional coronary MRA images were compared with images obtained with a T2 prepulse (T2Prep) without exogenous contrast. The contrast-enhanced images demonstrated a 69% improvement in the contrast-to-noise ratio (6.6 +/- 1.1 vs. 11.1 +/- 2.5; P < 0.01) compared with the T2Prep approach. By using the intravascular agent, extensive portions (> 80 mm) of the native left and right coronary system could be displayed consistently with sub-millimeter in-plane resolution. The intravascular contrast agent, MS-325/AngioMARK, leads to a considerable enhancement of the blood/muscle contrast for coronary MRA compared with T2Prep techniques. The clinical value of the agent remains to be defined in a larger patient series. J. Magn. Reson. Imaging 1999;10:790-799.

Development, optimization, and validation of novel anti-TEM1/CD248 affinity agent for optical imaging in cancer.

Tumor Endothelial Marker-1 (TEM1/CD248) is a tumor vascular marker with high therapeutic and diagnostic potentials. Immuno-imaging with TEM1-specific antibodies can help to detect cancerous lesions, monitor tumor responses, and select patients that are most likely to benefit from TEM1-targeted therapies. In particular, near infrared(NIR) optical imaging with biomarker-specific antibodies can provide real-time, tomographic information without exposing the subjects to radioactivity. To maximize the theranostic potential of TEM1, we developed a panel of all human, multivalent Fc-fusion proteins based on a previously identified single chain antibody (scFv78) that recognizes both human and mouse TEM1. By characterizing avidity, stability, and pharmacokinectics, we identified one fusion protein, 78Fc, with desirable characteristics for immuno-imaging applications. The biodistribution of radiolabeled 78Fc showed that this antibody had minimal binding to normal organs, which have low expression of TEM1. Next, we developed a 78Fc-based tracer and tested its performance in different TEM1-expressing mouse models. The NIR imaging and tomography results suggest that the 78Fc-NIR tracer performs well in distinguishing mouse- or human-TEM1 expressing tumor grafts from normal organs and control grafts in vivo. From these results we conclude that further development and optimization of 78Fc as a TEM1-targeted imaging agent for use in clinical settings is warranted.

A peptide inhibitor of c-Jun N-terminal kinase for the treatment of endotoxin-induced uveitis.

PURPOSE: To evaluate the effect of XG-102 (formerly D-JNKI1), a TAT-coupled dextrogyre peptide that selectively inhibits the c-Jun N-terminal kinase, in the treatment of endotoxin-induced uveitis (EIU). METHODS: EIU was induced in Lewis rats by LPS injection. XG-102 was administered at the time of LPS challenge. The ocular biodistribution of XG-102 was evaluated using immunodetection at 24 hours after either 20 microg/kg IV (IV) or 0.2 microg/injection intravitreous (IVT) administrations in healthy or uveitic eyes. The effect of XG-102 on EIU was evaluated using clinical scoring, infiltration cell quantification, inducible nitric oxide synthase (iNOS) expression and immunohistochemistry, and cytokines and chemokines kinetics at 6, 24, and 48 hours using multiplex analysis on ocular media. Control EIU eyes received vehicle injection IV or IVT. The effect of XG-102 on c-Jun phosphorylation in EIU was evaluated by Western blot in eye tissues. RESULTS: After IVT injection, XG-102 was internalized in epithelial cells from iris/ciliary body and retina and in glial and microglial cells in both healthy and uveitic eyes. After IV injection, XG-102 was concentrated primarily in inflammatory cells of uveitic eyes. Using both routes of administration, XG-102 significantly inhibited clinical signs of EIU, intraocular cell infiltration, and iNOS expression together with reduced phosphorylation of c-Jun. The anti-inflammatory effect of XG-102 was mediated by iNOS, IFN-gamma, IL-2, and IL-13. CONCLUSIONS: This is the first evidence that interfering with the JNK pathway can reduce intraocular inflammation. Local administration of XG-102, a clinically evaluated peptide, may have potential for treating uveitis.