Optimal perfusion computed tomographic thresholds for ischemic core and penumbra are not time dependent in the clinically relevant time window.

BACKGROUND AND PURPOSE: This study aims to determine whether perfusion computed tomographic (PCT) thresholds for delineating the ischemic core and penumbra are time dependent or time independent in patients presenting with symptoms of acute stroke. METHODS: Two hundred seventeen patients were evaluated in a retrospective, multicenter study. Patients were divided into those with either persistent occlusion or recanalization. All patients received admission PCT and follow-up imaging to determine the final ischemic core, which was then retrospectively matched to the PCT images to identify optimal thresholds for the different PCT parameters. These thresholds were assessed for significant variation over time since symptom onset. RESULTS: In the persistent occlusion group, optimal PCT parameters that did not significantly change with time included absolute mean transit time, relative mean transit time, relative cerebral blood flow, and relative cerebral blood volume when time was restricted to 15 hours after symptom onset. Conversely, the recanalization group showed no significant time variation for any PCT parameter at any time interval. In the persistent occlusion group, the optimal threshold to delineate the total ischemic area was the relative mean transit time at a threshold of 180%. In patients with recanalization, the optimal parameter to predict the ischemic core was relative cerebral blood volume at a threshold of 66%. CONCLUSIONS: Time does not influence the optimal PCT thresholds to delineate the ischemic core and penumbra in the first 15 hours after symptom onset for relative mean transit time and relative cerebral blood volume, the optimal parameters to delineate ischemic core and penumbra.

Antibiotics shaping bacterial genome: deletion of an IS91 flanked virulence determinant upon exposure to suinhibitory antibiotic concentrations

The nucleoid-associated proteins Hha and YdgT repress the expression of the toxin α-hemolysin. An Escherichia coli mutant lacking these proteins overexpresses the toxin α-hemolysin encoded in the multicopy recombinant plasmid pANN202-312R. Unexpectedly, we could observe that this mutant generated clones that no further produced hemolysin (Hly-). Generation of Hly- clones was dependent upon the presence in the culture medium of the antibiotic kanamycin (km), a marker of the hha allele (hha::Tn5). Detailed analysis of different Hly- clones evidenced that recombination between partial IS91 sequences that flank the hly operon had occurred. A fluctuation test evidenced that the presence of km in the culture medium was underlying the generation of these clones. A decrease of the km concentration from 25 mg/l to 12.5 mg/l abolished the appearance of Hly- derivatives. We considered as a working hypothesis that, when producing high levels of the toxin (combination of the hha ydgT mutations with the presence of the multicopy hemolytic plasmid pANN202-312R), the concentration of km of 25 mg/l resulted subinhibitory and stimulated the recombination between adjacent IS91 flanking sequences. To further test this hypothesis, we analyzed the effect of subinhibitory km concentrations in the wild type E. coli strain MG1655 harboring the parental low copy number plasmid pHly152. At a km concentration of 5 mg/l, subinhibitory for strain MG1655 (pHly152), generation of Hly- clones could be readily detected. Similar results were also obtained when, instead of km, ampicillin was used. IS91 is flanking several virulence determinants in different enteric bacterial pathogenic strains from E. coli and Shigella. The results presented here evidence that stress generated by exposure to subinhibitory antibiotic concentrations may result in rearrangements of the bacterial genome. Whereas some of these rearrangements may be deleterious, others may generate genotypes with increased virulence, which may resume infection.

Procedure for human saphenous veins ex vivo perfusion and external reinforcement.

The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal hyperplasia (IH) that eventually leads to vessel occlusion and graft failure. Mechanical forces, particularly low shear stress and high wall tension, are thought to initiate and to sustain these cellular and molecular changes, but their exact contribution remains to be unraveled. To selectively evaluate the role of pressure and shear stress on the biology of IH, an ex vivo perfusion system (EVPS) was created to perfuse segments of human saphenous veins under arterial regimen (high shear stress and high pressure). Further technical innovations allowed the simultaneous perfusion of two segments from the same vein, one reinforced with an external mesh. Veins were harvested using a no-touch technique and immediately transferred to the laboratory for assembly in the EVPS. One segment of the freshly isolated vein was not perfused (control, day 0). The two others segments were perfused for up to 7 days, one being completely sheltered with a 4 mm (diameter) external mesh. The pressure, flow velocity, and pulse rate were continuously monitored and adjusted to mimic the hemodynamic conditions prevailing in the femoral artery. Upon completion of the perfusion, veins were dismounted and used for histological and molecular analysis. Under ex vivo conditions, high pressure perfusion (arterial, mean = 100 mm Hg) is sufficient to generate IH and remodeling of human veins. These alterations are reduced in the presence of an external polyester mesh.

Oligomerization and DNA binding of Ler, a master regulator of pathogenicity of enterohemorrhagic and enteropathogenic Escherichia coli

Ler is a DNA-binding, oligomerizable protein that regulates pathogenicity islands in enterohemorrhagic and enteropathogenic Escherichia coli strains. Ler counteracts the transcriptional silencing effect of H-NS, another oligomerizable nucleoid-associated protein. We studied the oligomerization of Ler in the absence and presence of DNA by atomic force microscopy. Ler forms compact particles with a multimodal size distribution corresponding to multiples of 35 units of Ler. DNA wraps around Ler particles that contain more than 1516 Ler monomers. The resulting shortening of the DNA contour length is in agreement with previous measurements of the length of DNA protected by Ler in footprinting assays. We propose that the repetition unit corresponds to the number of monomers per turn of a tight helical Ler oligomer. While the repressor (H-NS) and anti-repressor (Ler) have similar DNA-binding domains, their oligomerization domains are unrelated. We suggest that the different oligomerization behavior of the two proteins explains the opposite results of their interaction with the same or proximal regions of DNA.

Laparoscopic surgery for coeliac artery compression syndrome: current management and technical aspects.

Objectives: The study aims to assess the feasibility and midterm outcome of trans-peritoneal laparoscopy for coeliac artery compression syndrome (CACS).Design: Retrospective chart review involving four European vascular surgery departments and two surgical teams.Materials and methods: charts for patients who underwent laparoscopy for symptomatic CACS between December 2003 and November 2009 were reviewed. Preoperative computed tomography (CT) angiography and postoperative duplex scan and/or CT angiography were performed.Results: Eleven consecutive patients (nine women) with a median age of 52 years (interquartile range: 42.5-59 years) underwent trans-peritoneal laparoscopy for CACS. All patients had a history of postprandial abdominal pain; weight loss exceeded 10% of the body mass in eight cases. Preoperative CT angiography revealed coeliac trunk stenosis >70% in all cases. One patient had additional aortitis and inferior mesenteric artery occlusion, while another patient presented with an occluded superior mesenteric artery. Two conversions occurred (one difficult dissection and one aorto-hepatic bypass needed for incomplete release of CACS). The median blood loss was 195 ml (range: 50-900 ml) and median operative time was 80 min (interquartile range: 65-162.5 years). Symptoms improved immediately in 10/11 patients (no residual stenosis) while one remained unchanged despite a residual stenosis treated by a percutaneous angioplasty. Symptoms reappeared in one patient due to coeliac axis occlusion. The mean follow-up period was 35 +/- 23 months (range: 12-78 months).Conclusion: Our study demonstrates that trans-peritoneal laparoscopy for treating median arcuate ligament syndrome is safe and feasible. Additional patients and a longer follow-up are needed for long-term assessment of this laparoscopic technique. (C) 2011 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved.

Successful combined minimally invasive direct coronary artery bypass and transapical aortic valve implantation.

Transapical aortic valve implantation is indicated in high-risk patients with aortic stenosis and peripheral vascular disease requiring aortic valve replacement. Minimally invasive direct coronary artery bypass grafting is also a valid, minimally invasive option for myocardial revascularization in patients with critical stenosis on the anterior descending coronary artery. Both procedures are performed through a left minithoracotomy, without cardiopulmonary bypass, aortic cross-clamping, and cardioplegic arrest. We describe a successful combined transapical aortic valve implantation and minimally invasive direct coronary bypass in a high-risk patient with left anterior descending coronary artery occlusion and severe aortic valve stenosis.

Critical role for the alpha-1B adrenergic receptor at the sympathetic neuroeffector junction.

The alpha-1 adrenergic receptors (alpha(1)ARs) are critical in sympathetically mediated vasoconstriction. The specific role of each alpha(1)AR subtype in regulating vasoconstriction remains highly controversial. Limited pharmacological studies suggest that differential alpha(1)AR responses may be the result of differential activation of junctional versus extrajunctional receptors. We tested the hypothesis that the alpha(1B)AR subtype is critical in mediating sympathetic junctional neurotransmission. We measured in vivo integrated cardiovascular responses to a hypotensive stimulus (induced via transient bilateral carotid occlusion [TBCO]) in alpha(1B)AR knockout (KO) mice and their wild-type (WT) littermates. In WT mice, after dissection of the carotid arteries and denervation of aortic baroreceptor buffering nerves, TBCO produced significant pressor and positive inotropic effects. Both responses were markedly attenuated in alpha(1B)AR KO mice (change systolic blood pressure 46+/-8 versus 11+/-2 mm Hg; percentage change in the end-systolic pressure-volume relationship [ESPVR] 36+/-7% versus 12+/-2%; WT versus KO; P<0.003). In vitro alpha(1)AR mesenteric microvascular contractile responses to endogenous norepinephrine (NE; elicited by electrical field stimulation 10 Hz) was markedly depressed in alpha(1B)AR KO mice compared with WT (12.4+/-1.7% versus 21.5+/-1.2%; P<0.001). In contrast, responses to exogenous NE were similar in alpha(1B)AR KO and WT mice (22.4+/-7.3% versus 33.4+/-4.3%; NS). Collectively, these results demonstrate a critical role for the alpha(1B)AR in baroreceptor-mediated adrenergic signaling at the vascular neuroeffector junction. Moreover, alpha(1B)ARs modulate inotropic responses to baroreceptor activation. The critical role for alpha(1B)AR in neuroeffector regulation of vascular tone and myocardial contractility has profound clinical implications for designing therapies for orthostatic intolerance.

Optimization and regeneration kinetics of lymphatic-specific photodynamic therapy in the mouse dermis.

Lymphatic vessels transport fluid, antigens, and immune cells to the lymph nodes to orchestrate adaptive immunity and maintain peripheral tolerance. Lymphangiogenesis has been associated with inflammation, cancer metastasis, autoimmunity, tolerance and transplant rejection, and thus, targeted lymphatic ablation is a potential therapeutic strategy for treating or preventing such events. Here we define conditions that lead to specific and local closure of the lymphatic vasculature using photodynamic therapy (PDT). Lymphatic-specific PDT was performed by irradiation of the photosensitizer verteporfin that effectively accumulates within collecting lymphatic vessels after local intradermal injection. We found that anti-lymphatic PDT induced necrosis of endothelial cells and pericytes, which preceded the functional occlusion of lymphatic collectors. This was specific to lymphatic vessels at low verteporfin dose, while higher doses also affected local blood vessels. In contrast, light dose (fluence) did not affect blood vessel perfusion, but did affect regeneration time of occluded lymphatic vessels. Lymphatic vessels eventually regenerated by recanalization of blocked collectors, with a characteristic hyperplasia of peri-lymphatic smooth muscle cells. The restoration of lymphatic function occurred with minimal remodeling of non-lymphatic tissue. Thus, anti-lymphatic PDT allows control of lymphatic ablation and regeneration by alteration of light fluence and photosensitizer dose.

Thrombin-induced ischemic tolerance is prevented by inhibiting c-jun N-terminal kinase.

We have studied ischemic tolerance induced by the serine protease thrombin in two different models of experimental ischemia. In organotypic hippocampal slice cultures, we demonstrate that incubation with low doses of thrombin protects neurons against a subsequent severe oxygen and glucose deprivation. L-JNKI1, a highly specific c-jun N-terminal kinase (JNK) inhibitor, and a second specific JNK inhibitor, SP600125, prevented thrombin preconditioning (TPC). We also show that the exposure to thrombin increases the level of phosphorylated c-jun, the major substrate of JNK. TPC, in vivo, leads to significantly smaller lesion sizes after a 30-min middle cerebral artery occlusion (MCAo), and the preconditioned mice were better off in the three tests used to evaluate functional recovery. In accordance with in vitro results, TPC in vivo was prevented by administration of L-JNKI1, supporting a role for JNK in TPC. These results, from two different TPC models and with two distinct JNK inhibitors, show that JNK is likely to be involved in TPC.

The structure of nervous tissue and of Gram-positive bacteria studied by cryo-electron microscopy of vitreous sections

Résumé La structure, ou l'architecture, des êtres vivants définit le cadre dans lequel la physique de la vie s'accomplit. La connaissance de cette structure dans ses moindres détails est un but essentiel de la biologie. Son étude est toutefois entravée par des limitations techniques. Malgré son potentiel théorique, la microscopie électronique n'atteint pas une résolution atomique lorsqu'elle est appliquée ä la matièxe biologique. Cela est dû en grande partie au fait qu'elle contient beaucoup d'eau qui ne résiste pas au vide du microscope. Elle doit donc être déshydratée avant d'être introduite dans un microscope conventionnel. Des artéfacts d'agrégation en découlent inévitablement. La cryo-microscopie électronique des sections vitreuses (CEMOVIS) a ëté développée afin de résoudre cela. Les spécimens sont vitrifiés, c.-à-d. que leur eau est immobilisée sans cristalliser par le froid. Ils sont ensuite coupés en sections ultrafines et celles-ci sont observées à basse température. Les spécimens sont donc observés sous forme hydratée et non fixée; ils sont proches de leur état natif. Durant longtemps, CEMOVIS était très difficile à exécuter mais ce n'est plus le cas. Durant cette thèse, CEMOVIS a été appliqué à différents spécimens. La synapse du système nerveux central a été étudiée. La présence dans la fente synaptique d'une forte densité de molécules organisées de manière périodique a été démontrée. Des particules luminales ont été trouvées dans Ies microtubules cérébraux. Les microtubules ont servi d'objets-test et ont permis de démontrer que des détails moléculaires de l'ordre du nm sont préservés. La compréhension de la structure de l'enveloppe cellulaire des bactéries Grampositives aété améliorée. Nos observations ont abouti à l'élaboration d'un nouveau modèle hypothétique de la synthèse de la paroi. Nous avons aussi focalisé notre attention sur le nucléoïde bactérien et cela a suscité un modèle de la fonction des différents états structuraux du nucléoïde. En conclusion, cette thèse a démontré que CEMOVIS est une excellente méthode poux étudier la structure d'échantillons biologiques à haute résolution. L'étude de la structure de divers aspects des êtres vivants a évoqué des hypothèses quant à la compréhension de leur fonctionnement. Summary The structure, or the architecture, of living beings defines the framework in which the physics of life takes place. Understanding it in its finest details is an essential goal of biology. Its study is however hampered by technical limitations. Despite its theoretical potential, electron microscopy cannot resolve individual atoms in biological matter. This is in great part due to the fact. that it contains a lot of water that cannot stand the vacuum of the microscope. It must therefore be dehydrated before being introduced in a conventional mìcroscope. Aggregation artefacts unavoidably happen. Cryo-electron microscopy of vitreous sections (CEMOVIS) has been developed to solve this problem. Specimens are vitrified, i.e. they are rapidly cooled and their water is immobilised without crystallising by the cold. They are then. sectioned in ultrathin slices, which are observed at low temperatures. Specimens are therefore observed in hydrated and unfixed form; they are close to their native state. For a long time, CEMOVIS was extremely tedious but this is not the case anymore. During this thesis, CEMOVIS was applied to different specimens. Synapse of central nervous system was studied. A high density of periodically-organised molecules was shown in the synaptic cleft. Luminal particles were found in brain microtubules. Microtubules, used as test specimen, permitted to demonstrate that molecular details of the order of nm .are preserved. The understanding of the structure of cell envelope of Gram-positive bacteria was improved. Our observations led to the elaboration of a new hypothetic model of cell wall synthesis. We also focused our attention on bacterial nucleoids and this also gave rise to a functional model of nucleoid structural states. In conclusion, this thesis demonstrated that CEMOVIS is an excellent method for studying the structure of bìologìcal specimens at high resolution. The study of the structure of various aspects of living beings evoked hypothesis for their functioning.

A phase I pharmacokinetic study of hypoxic abdominal stop-flow perfusion with gemcitabine in patients with advanced pancreatic cancer and refractory malignant ascites

PURPOSE: As no curative treatment for advanced pancreatic and biliary cancer with malignant ascites exists, new modalities possibly improving the response to available chemotherapies must be explored. This phase I study assesses the feasibility, tolerability and pharmacokinetics of a regional treatment of gemcitabine administered in escalating doses by the stop-flow approach to patients with advanced abdominal malignancies (adenocarcinoma of the pancreas, n = 8, and cholangiocarcinoma of the liver, n = 1). EXPERIMENTAL DESIGN: Gemcitabine at 500, 750 and 1,125 mg/m(2) was administered to three patients at each dose level by loco-regional chemotherapy, using hypoxic abdominal stop-flow perfusion. This was achieved by an aorto-caval occlusion by balloon catheters connected to an extracorporeal circuit. Gemcitabine and its main metabolite 2',2'-difluorodeoxyuridine (dFdU) concentrations were measured by high performance liquid chromatography with UV detection in the extracorporeal circuit during the 20 min of stop-flow perfusion, and in peripheral plasma for 420 min. Blood gases were monitored during the stop-flow perfusion and hypoxia was considered stringent if two of the following endpoints were met: pH </= 7.2, pO(2) nadir ratio </=0.70 or pCO(2) peak ratio >/=1.35. The tolerability of this procedure was also assessed. RESULTS: Stringent hypoxia was achieved in four patients. Very high levels of gemcitabine were rapidly reached in the extracorporeal circuit during the 20 min of stop-flow perfusion, with C (max) levels in the abdominal circuit of 246 (+/-37%), 2,039 (+/-77%) and 4,780 (+/-7.3%) mug/ml for the three dose levels 500, 750 and 1,125 mg/m(2), respectively. These C (max) were between 13 (+/-51%) and 290 (+/-12%) times higher than those measured in the peripheral plasma. Similarly, the abdominal exposure to gemcitabine, calculated as AUC(t0-20), was between 5.5 (+/-43%) and 200 (+/-66%)-fold higher than the systemic exposure. Loco-regional exposure to gemcitabine was statistically higher in presence of stringent hypoxia (P < 0.01 for C (max) and AUC(t0-20), both normalised to the gemcitabine dose). Toxicities were acceptable considering the complexity of the procedure and were mostly hepatic; it was not possible to differentiate the respective contributions of systemic and regional exposures. A significant correlation (P < 0.05) was found between systemic C (max) of gemcitabine and the nadir of both leucocytes and neutrophils. CONCLUSIONS: Regional exposure to gemcitabine-the current standard drug for advanced adenocarcinoma of the pancreas-can be markedly enhanced using an optimised hypoxic stop-flow perfusion technique, with acceptable toxicities up to a dose of 1,125 mg/m(2). However, the activity of gemcitabine under hypoxic conditions is not as firmly established as that of other drugs such as mitomycin C, melphalan or tirapazamine. Further studies of this investigational modality, but with bioreductive drugs, are therefore warranted first to evaluate the tolerance in a phase I study and later on to assess whether it does improve the response to chemotherapy.

Acute mesenteric vein thrombosis: factors associated with evolution to chronic mesenteric vein thrombosis

OBJECTIVE. Acute mesenteric venous thrombosis signs at MDCT are well described, but the literature lacks studies assessing their evolution. We aimed to describe the radiologic evolution of isolated acute mesenteric venous thrombosis and associated prognostic factors. MATERIALS AND METHODS. Patients with isolated acute mesenteric venous thrombosis with follow-up for a minimum of 1 month with MDCT were selected. Images at the acute phase and on follow-up were reviewed in consensus reading. For acute mesenteric venous thrombosis, we searched for low-attenuated intraluminal filling defect. For chronic mesenteric venous thrombosis, we searched for vessel stenosis or occlusion associated with collateral mesenteric veins. Treatment, thrombosis risk factor, symptoms, location, and length and diameter of mesenteric venous thrombosis were reported and correlated with evolution over time. RESULTS. Twenty patients (nine women and 11 men; mean age, 52 years) were selected. Four patients recovered without radiologic sequelae, and 16 developed chronic mesenteric venous thrombosis signs. Anticoagulation did not influence recovery (p = 1). Patients with recovery compared with patients with chronic mesenteric venous thrombosis showed more frequent central lesions (p = 0.03). At diagnosis, the thrombosed segment was shorter and larger in the complete radiologic recovery group compared with the chronic mesenteric venous thrombosis signs group: mean length (± SD) 6.25 ± 3.21 cm and 12.81 ± 5.96 cm, respectively (p = 0.01); mean transverse diameter 1.82 ± 0.42 cm and 1.12 ± 0.34 cm, respectively (p = 0.01). Mesenteric fat infiltration at diagnosis was more frequent in the chronic mesenteric venous thrombosis signs group than in the complete recovery group (p = 0.03). CONCLUSION. Most cases of acute mesenteric venous thrombosis evolve toward the chronic form with vein stenosis or occlusion and development of collateral veins. Location, length of mesenteric venous thrombosis, transverse diameter of the vein, and mesenteric fat infiltration at diagnosis are determinant factors for mesenteric venous thrombosis evolution.

Parésie trochléaire transitoire comme signe neurologique inaugural d'une panartérite noueuse [Transient trochlear nerve palsy as the presenting neurological sign of panarteritis nodosa]

INTRODUCTION: Panarteritis nodosa (PAN) is a systemic vasculitis affecting small and medium-sized arteries. Neuro-ophthalmological complications of PAN are rare but numerous, and may affect the eye, the visual and the oculomotor pathways. Such complications occur mainly in patients previously diagnosed with PAN. OBSERVATION: A 51-year-old woman presented with an isolated right trochlear (IV) palsy, in the setting of headaches and fluctuating fever of unknown etiology. Erythrocyte sedimentation rate was 13 mm and full blood cell count was normal. Previous chest X-ray and blood studies were negative for an infection or inflammation. Orbital and cerebral CT scan was normal. Spontaneous recovery of diplopia ensued over four days. Two days later, paresthesia and sensory paresis of the dorsal portion of the left foot were present. Lumbar puncture revealed 14 leucocytes (76 percent lymphocytes) with elevated proteins, but blood studies and serologies were negative. A diagnosis of undetermined meningo-myelo-radiculoneuritis was made. Because of a possible tick bite six weeks previously the patient was empirically treated with 2 g intravenous ceftriaxone for 3 weeks. Fever rapidly dropped. Six weeks after the onset of diplopia, acute onset of blindness in her right eye, diffuse arthralgias and fever motivated a new hospitalization. There was a central retinal artery occlusion of the right eye. Blood studies now revealed signs of systemic inflammation (ESR 30 mm, CRP 12 mg/L, ANA 1/80, pANCA 1/40, leucocytosis 12.4 G/L, Hb 111 g/L, Ht 33 percent). Biopsy of the left sural nerve revealed arterial fibrinoid necrosis. A diagnosis of PAN was made. CONCLUSIONS: Transient diplopia can be the heralding symptom of a systemic vasculitis such as PAN, giant cell arteritis and Wegener granulomatosis. In this patient the presence of accompanying systemic symptoms raised a suspicion of systemic inflammation, but the absence of serologic and imaging abnormalities precluded a specific diagnosis initially. A few weeks later, the presence of a second ischemic event (retinal) and positive blood studies led to a further diagnostic procedure. Oculomotor and abducens palsies have rarely been reported in association with PAN. We report the first case of trochlear nerve paresis as the inaugural neurological sign of PAN. This case highlights the importance of considering inflammatory systemic disorders in patients with acute diplopia particularly when they are young, lack vascular risk factors or cause, and complain of associated systemic symptoms.

L'angio-CT post-mortem: un nouvel outil diagnostique

L'angio-CT post-mortem est un examen peu invasif qui permet d'investiguer le système vasculaire d'une manière détaillée impossible à obtenir lors d'une autopsie conventionnelle. Le groupe de recherche lausannois sur l'angio-CT a développé un protocole standardisé pour une technique appelée «angio-CT post-mortem en phases multiples», qui permet de réaliser des angio-CT de manière simple et d'améliorer le diagnostic radiologique. De plus, de nouveaux équipements incluant une pompe à perfusion, du matériel prêt à l'emploi et un produit de contraste spécifique ont été développés. L'angio-CT permet la détection d'une source d'hémorragie, d'une malformation du système vasculaire, de lésions d'artériosclérose, d'une occlusion d'un vaisseau et la visualisation de l'anatomie vasculaire.