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In patients with asthma or chronic obstructive pulmonary disease, rhinovirus (RV) infections can provoke acute worsening of disease, and limited treatment options exist. Viral replication in the host cell induces significant remodeling of intracellular membranes, but few studies have explored this mechanistically or as a therapeutic opportunity. We performed unbiased lipidomic analysis on human bronchial epithelial cells infected over a 6 h period with the RV-A1b strain of RV to determine changes in 493 distinct lipid species. Through pathway and network analysis, we identified temporal changes in the apparent activities of a number of lipid metabolizing and signaling enzymes. In particular, analysis highlighted FA synthesis and ceramide metabolism as potential anti-rhinoviral targets. To validate the importance of these enzymes in viral replication, we explored the effects of commercially available enzyme inhibitors upon RV-A1b infection and replication. Ceranib-1, D609, and C75 were the most potent inhibitors, which confirmed that FAS and ceramidase are potential inhibitory targets in rhinoviral infections. More broadly, this study demonstrates the potential of lipidomics and pathway analysis to identify novel targets to treat human disorders.

Purpose:\ud \ud To define the phenotypic and mutational spectrum of epilepsies related to DEPDC5, NPRL2 and NPRL3 genes encoding the GATOR1 complex, a negative regulator of the mTORC1 pathway.\ud \ud Methods:\ud \ud We analyzed clinical and genetic data of 73 novel probands (familial and sporadic) with epilepsy-related variants in GATOR1-encoding genes and proposed new guidelines for clinical interpretation of GATOR1 variants.\ud \ud Results:\ud \ud The GATOR1 seizure phenotype consisted mostly in focal seizures (e.g., hypermotor or frontal lobe seizures in 50%), with a mean age at onset of 4.4 years, often sleep-related and drug-resistant (54%), and associated with focal cortical dysplasia (20%). Infantile spasms were reported in 10% of the probands. Sudden unexpected death in epilepsy (SUDEP) occurred in 10% of the families. Novel classification framework of all 140 epilepsy-related GATOR1 variants (including the variants of this study) revealed that 68% are loss-of-function pathogenic, 14% are likely pathogenic, 15% are variants of uncertain significance and 3% are likely benign.\ud \ud Conclusion:\ud \ud Our data emphasize the increasingly important role of GATOR1 genes in the pathogenesis of focal epilepsies (>180 probands to date). The GATOR1 phenotypic spectrum ranges from sporadic early-onset epilepsies with cognitive impairment comorbidities to familial focal epilepsies, and SUDEP.

The Dot/Icm type IV secretion system (T4SS) is essential for the pathogenesis of Legionella species and translocates a multitude of effector proteins into host cells. The identification of host cell targets of these effectors is often critical to unravel their roles in controlling the host. Here we describe a method to characterize the protein complexes associated with effectors in infected host cells. To achieve this, Legionella expressing an effector of interest fused to a Bio-tag, a combination of hexahistidine tags and a specific recognition sequence for the biotin ligase BirA, are used to infect host cells expressing BirA, which leads to biotinylation of the translocated effector. Following chemical cross-linking, effector interactomes are isolated by tandem affinity purification employing metal affinity and NeutrAvidin resins and identified by western blotting or mass spectrometry.

Rhinoviruses (RVs) are the pathogens most often responsible for the common cold, and are a frequent cause of exacerbations in asthma, chronic obstructive pulmonary disease and cystic fibrosis. Here we report the discovery of IMP-1088, a picomolar dual inhibitor of the human N-myristoyltransferases NMT1 and NMT2, and use it to demonstrate that pharmacological inhibition of host-cell N-myristoylation rapidly and completely prevents rhinoviral replication without inducing cytotoxicity. The identification of cooperative binding between weak-binding fragments led to rapid inhibitor optimization through fragment reconstruction, structure-guided fragment linking and conformational control over linker geometry. We show that inhibition of the co-translational myristoylation of a specific virus-encoded protein (VP0) by IMP-1088 potently blocks a key step in viral capsid assembly, to deliver a low nanomolar antiviral activity against multiple RV strains, poliovirus and foot and-mouth disease virus, and protection of cells against virus-induced killing, highlighting the potential of host myristoylation as a drug target in picornaviral infections.

Picornavirus replication is known to cause extensive remodeling of Golgi and endoplasmic reticulum membranes, and a number of the host proteins involved in the viral replication complex have been identified, including oxysterol binding protein (OSBP) and phosphatidylinositol 4-kinase III beta (PI4KB). Since both OSBP and PI4KB are substrates for protein kinase D (PKD) and PKD is known to be involved in the control of Golgi membrane vesicular and lipid transport, we hypothesized that PKD played a role in viral replication. We present multiple lines of evidence in support of this hypothesis. First, infection of HeLa cells with human rhinovirus (HRV) induced the phosphorylation of PKD. Second, PKD inhibitors reduced HRV genome replication, protein expression, and titers in a concentration-dependent fashion and also blocked the replication of poliovirus (PV) and foot-and-mouth disease virus (FMDV) in a variety of cells. Third, HRV replication was significantly reduced in HeLa cells overexpressing wild-type and mutant forms of PKD1. Fourth, HRV genome replication was reduced in HAP1 cells in which the PKD1 gene was knocked out by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9. Although we have not identified the molecular mechanism through which PKD regulates viral replication, our data suggest that this is not due to enhanced interferon signaling or an inhibition of clathrin-mediated endocytosis, and PKD inhibitors do not need to be present during viral uptake. Our data show for the first time that targeting PKD with small molecules can inhibit the replication of HRV, PV, and FMDV, and therefore, PKD may represent a novel antiviral target for drug discovery. IMPORTANCE Picornaviruses remain an important family of human and animal pathogens for which we have a very limited arsenal of antiviral agents. HRV is the causative agent of the common cold, which in itself is a relatively trivial infection; however, in asthma and chronic obstructive pulmonary disease (COPD) patients, this virus is a major cause of exacerbations resulting in an increased use of medication, worsening symptoms, and, frequently, hospital admission. Thus, HRV represents a substantial health care and economic burden for which there are no approved therapies. We sought to identify a novel host target as a potential anti-HRV therapy. HRV infection induces the phosphorylation of PKD, and inhibitors of this kinase effectively block HRV replication at an early stage of the viral life cycle. Moreover, PKD inhibitors also block PV and FMDV replication. This is the first description that PKD may represent a target for antiviral drug discovery.

Background The aim of this study was to assess the midterm results of percutaneous angioplasty in patients with critical limb ischemia (CLI) and long tibial occlusions. Methods Between January and September 2011, 34 consecutive patients with patent femoropopliteal artery and 49 segmental tibial occlusions >8 cm were included in our prospective, single-center cohort study. Clinical success (defined as wound healing and survival without major amputation), patency, and freedom from target vessel revascularization (TVR) were examined. Results The median age of the patients was 75 (53–89) years, 74% were diabetic, and 89% of the limbs studied were Rutherford 5 and 6. Median follow-up was 12.5 (1–15) months. The 1-year clinical success rate was 65%, higher among patients with technical success (76% vs. 25%, P = 0.01) and patients with 2 or 3 patent tibial arteries after the procedure (90% vs. 41% in patients with only 1 patent artery, P = 0.003). At 1 year, primary and secondary patency rates were 13% and 32%, respectively (24% and 58% without technical failures). The 1-year freedom-from-TVR rate was 34%. Conclusions Despite high technical failure rates and the need for repeat procedures, percutaneous angioplasty of long tibial occlusions enhances wound healing, especially when integrated into a maximal revascularization approach.

Background Subintimal recanalization allows for the treatment of percutaneously extended occlusive lesions. The aim of this study was to evaluate the feasibility and short- and mid-term results of subintimal recanalization in the treatment of femoropopliteal occlusive lesions in patients with critical ischemia. Methods Between January 2009 and December 2010, consecutive patients with critical ischemia presenting with femoropopliteal arterial occlusion were included in this study. These patients underwent subintimal recanalization, and all patients had clinical and ultrasound follow-up. Results Sixty-six procedures were performed on 66 patients (mean age, 79 ± 10 yrs). All patients were American Society of Anesthesiologists classification 3 or 4. Thirty-two patients had diabetes and 27 had chronic renal insufficiency. More than two-thirds (76%) of the lesions had a Trans-Atlantic Inter-Society Classification of C or D. The mean occlusion length was 13.5 ± 7 cm. The rate of technical success was 85%. The peroperative complication rate was 4.5%. There were no cases of operative mortality. Occlusion length was the only predictive factor of restenosis ( P = 0.049). At 1 year, primary and secondary patency rates were 56% and 70%, respectively, for a 92% rate of limb salvage. Conclusion The subintimal recanalization technique is feasible and minimally invasive. At 1 year postprocedure, the primary patency is poor, but this technique is associated with a high rate of limb salvage. It is suitable for the treatment of critical ischemia, taking into account the often precarious clinical backgrounds of these patients.

Summary Objective To obtain perspective on epilepsy in patients referred to tertiary centers in France, and describe etiology, epilepsy syndromes, and identify factors of drug resistance and comorbidities. Methods We performed a cross-sectional analysis of the characteristics of 5,794 pediatric and adult patients with epilepsy included in a collaborative database in France between 2007 and 2013. Comparisons between groups used Student's t-test or Fisher's exact test for binary or categorical variables. Factors associated with drug resistance and intellectual disability were evaluated in multi-adjusted logistic regression models. Results Mean age at inclusion was 17.9 years; children accounted for 67%. Epilepsy was unclassified in 20% of patients, and etiology was unknown in 65%, including those with idiopathic epilepsies. Etiologies differed significantly in adult- when compared to pediatric-onset epilepsy; however, among focal structural epilepsies, mesial temporal lobe epilepsy with hippocampal sclerosis began as often in the pediatric as in adult age range. Drug resistance concerned 53% of 4,210 patients evaluable for seizure control and was highest in progressive myoclonic epilepsy (89%), metabolic diseases (84%), focal cortical dysplasia (70%), other cortical malformations (69%), and mesial temporal lobe epilepsy with hippocampal sclerosis (67%). Fifty-nine percent of patients with focal structural epilepsy and 69% with epileptic encephalopathies were drug resistant; however, 40–50% of patients with West syndrome and epileptic encephalopathy with continuous spike-and-waves during sleep were seizure-free. Ages at onset in infancy and in young adults shared the highest risk of drug resistance. Epilepsy onset in infancy comprised the highest risk of intellectual disability, whereas specific cognitive impairment affected 36% of children with idiopathic focal epilepsy. Significance Our study provides a snapshot on epilepsy in patients referred to tertiary centers and discloses needs for diagnosis and treatment. Large databases help identify patients with rare conditions that could benefit from specific prospective studies.

Legionella pneumophila is a ubiquitous inhabitant of environmental water reservoirs. The bacteria infect a wide variety of protozoa and, after accidental inhalation, human alveolar macrophages, which can lead to severe pneumonia. The capability to thrive in phagocytic hosts is dependent on the Dot/Icm type IV secretion system (T4SS), which translocates multiple effector proteins into the host cell. In this study, we determined the draft genome sequence of L. pneumophila strain 130b (Wadsworth). We found that the 130b genome encodes a unique set of T4SSs, namely, the Dot/Icm T4SS, a Trb-1-like T4SS, and two Lvh T4SS gene clusters. Sequence analysis substantiated that a core set of 107 Dot/Icm T4SS effectors was conserved among the sequenced L. pneumophila strains Philadelphia-1, Lens, Paris, Corby, Alcoy, and 130b. We also identified new effector candidates and validated the translocation of 10 novel Dot/Icm T4SS effectors that are not present in L. pneumophila strain Philadelphia-1. We examined the prevalence of the new effector genes among 87 environmental and clinical L. pneumophila isolates. Five of the new effectors were identified in 34 to 62% of the isolates, while less than 15% of the strains tested positive for the other five genes. Collectively, our data show that the core set of conserved Dot/Icm T4SS effector proteins is supplemented by a variable repertoire of accessory effectors that may partly account for differences in the virulences and prevalences of particular L. pneumophila strains.

Clostridium difficile is a leading cause of nosocomial infections, causing a spectrum of diseases ranging from diarrhoea to pseudomembranous colitis triggered by a range of virulence factors including C. difficile toxins A (TcdA) and B (TcdB). TcdA and TcdB are monoglucosyltransferases that irreversibly glycosylate small Rho GTPases, inhibiting their ability to interact with their effectors, guanine nucleotide exchange factors, and membrane partners, leading to disruption of downstream signalling pathways and cell death. In addition, TcdB targets the mitochondria, inducing the intrinsic apoptotic pathway resulting in TcdB-mediated apoptosis. Modulation of apoptosis is a common strategy used by infectious agents. Recently, we have shown that the enteropathogenic Escherichia coli (EPEC) type III secretion system effector NleH has a broad-range anti-apoptotic activity. In this study we examined the effects of NleH on cells challenged with TcdB. During infection with wild-type EPEC, NleH inhibited TcdB-induced apoptosis at both low and high toxin concentrations. Transfected nleH1 alone was sufficient to block TcdB-induced cell rounding, nuclear condensation, mitochondrial swelling and lysis, and activation of caspase-3. These results show that NleH acts via a global anti-apoptotic pathway.

Attaching and effacing (A/E) lesions and actin polymerization, the hallmark of enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC) and Citrobacter rodentium (CR) infections, are dependent on the effector Tir. Phosphorylation of Tir(EPEC/CR) Y474/1 leads to recruitment of Nck and neural Wiskott-Aldrich syndrome protein (N-WASP) and strong actin polymerization in cultured cells. Tir(EPEC/CR) also contains an Asn-Pro-Tyr (NPY(454/1)) motif, which triggers weak actin polymerization. In EHEC the NPY(458) actin polymerization pathway is amplified by TccP/EspF(U), which is recruited to Tir via IRSp53 and/or insulin receptor tyrosine kinase substrate (IRTKS). Here we used C. rodentium to investigate the different Tir signalling pathways in vivo. Following infection with wild-type C. rodentium IRTKS, but not IRSp53, was recruited to the bacterial attachment sites. Similar results were seen after infection of human ileal explants with EHEC. Mutating Y471 or Y451 in Tir(CR) abolished recruitment of Nck and IRTKS respectively, but did not affect recruitment of N-WASP or A/E lesion formation. This suggests that despite their crucial role in actin polymerization in cultured cells the Tir:Nck and Tir:IRTKS pathways are not essential for N-WASP recruitment or A/E lesion formation in vivo. Importantly, wild-type C. rodentium out-competed the tir tyrosine mutants during mixed infections. These results uncouple the Tir:Nck and Tir:IRTKS pathways from A/E lesion formation in vivo but assign them an important in vivo role.

Background Viral load in whole blood is the main virological marker for assessing HHV-6 infection and is used as an indication to begin antiviral therapy. Results are usually expressed as the number of genomic equivalent copies (gec) per mL of blood, although HHV-6 DNA in blood is mainly localized in lymphocytes and polymorphonuclear leukocytes. Objectives Since leukocyte counts vary in immunocompromised patients, especially in stem cell transplant recipients, the aim of this study was to compare HHV-6 load expressed as gec per mL with load expressed as gec per million cells (mc), using quantitative real-time PCR for HHV-6 and cell DNA. Study design 194 blood samples from 101 patients were analyzed. Leukocyte count was obtained for 142 samples. Results The two modes of expression were incompletely correlated ( p R 2 = 0.732). To understand this relative discrepancy, samples were classified according to hematological criteria (normal leukocyte count, leukopenia, agranulocytosis, lymphopenia). The expression modes were correlated in all cases except for agranulocytosis ( p = 0.21; R 2 = 0.087). Moreover, the median of ratio between gec per mc and gec per mL ranged from 0.5 when leukocyte count was normal, to 8.2 in cases of agranulocytosis. HHV-6 load follow-up suggested that in agranulocytosis expressing results as gec per mc tended to provide a more representative result. Conclusions The different expression of HHV-6 load in whole blood, as either gec per mL or gec per mc resulted in different estimations of infection in the case of agranulocytosis. In this situation, the latter mode of expression is preferred.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important human pathogen that colonizes the gut mucosa via attaching and effacing (A/E) lesions; A/E lesion formation in vivo and ex vivo is dependent on the type III secretion system (T3SS) effector Tir. Infection of cultured cells by EHEC leads to induction of localized actin polymerization, which is dependent on Tir and a second T3SS effector protein, TccP, also known as EspF U . Recently, cortactin was shown to bind both the N terminus of Tir and TccP via its SH3 domain and to play a role in EHEC-triggered actin polymerization in vitro. In this study, we investigated the recruitment of cortactin to the site of EHEC adhesion during infection of in vitro-cultured cells and mucosal surfaces ex vivo (using human terminal ileal in vitro organ cultures [IVOC]). We have shown that cortactin is recruited to the site of EHEC adhesion in vitro downstream of TccP and N-WASP. Deletion of the entire N terminus of Tir or replacing the N-terminal polyproline region with alanines did not abrogate actin polymerization or cortactin recruitment. In contrast, recruitment of cortactin to the site of EHEC adhesion in IVOC is TccP independent. These results imply that cortactin is recruited to the site of EHEC adhesion in vitro and ex vivo by different mechanisms and suggest that cortactin might have a role during EHEC infection of mucosal surfaces.

Objective To evaluate TNFα antagonist continuation rates in patients with rheumatoid arthritis (RA), ankylosing spondylitis (AS), or psoriatic arthritis (PsA). Methods We retrospectively reviewed the charts of patients treated with etanercept, infliximab, or adalimumab at our teaching hospital. Drug continuation was evaluated using Kaplan–Meier survival curves. The logrank test was used to compare continuation rates. Results We identified 442 patients who were prescribed 571 TNFα antagonist treatments between August 1999 and June 2005. Among them, 304 had RA, 92 AS, and 46 PsA. In the RA group, continuation rates were high with etanercept ( n = 157; 87% after 12 months and 68% after 24 months) and adalimumab ( n = 43, 83% and 66%) but significantly lower with infliximab ( n = 104, 68% and 46%; P = 0.0001 vs. etanercept and P = 0.01 vs. adalimumab). In the AS group, in contrast, infliximab ( n = 53) showed significantly higher continuation rates (89% and 83%) than did etanercept ( n = 39; 76% after 12 months: P = 0.03). Overall continuation rates were higher in AS than in RA ( P = 0.01). Conclusion Continuation was better with etanercept than with infliximab in patients with RA, whereas the opposite was noted in patients with AS.

The aim of this study was to identify prospectively the prevalence of unnecessary or inappropriate fluoroquinolone prescriptions in our teaching hospital and to identify the contributing factors. 55% of the 110 prescriptions evaluated were unnecessary or inappropriate. Ward and combination therapies were significantly associated with misuse.

Legionella pneumophila, the causative agent of Legionnaire's disease, uses its type IV secretion system to translocate over 300 effector proteins into host cells. These effectors subvert host cell signaling pathways to ensure bacterial proliferation. Despite their importance for pathogenesis, the roles of most of the effectors are yet to be characterized. Key to understanding the function of effectors is the identification of host proteins they bind during infection. We previously developed a novel tandem-affinity purification (TAP) approach using hexahistidine and BirA-specific biotinylation tags for isolating translocated effector complexes from infected cells whose composition were subsequently deciphered by mass spectrometry. Here we further advanced the workflow for the TAP approach and determined the infection-dependent interactomes of the effectors SidM and LidA, which were previously reported to promiscuously bind multiple Rab GTPases in vitro. In this study we defined a stringent subset of Rab GTPases targeted by SidM and LidA during infection, comprising of Rab1A, 1B, 6, and 10; in addition, LidA targets Rab14 and 18. Taken together, this study illustrates the power of this approach to profile the intracellular interactomes of bacterial effectors during infection.

The replication of picornaviruses has been described to cause fragmentation of the Golgi apparatus that blocks the secretory pathway. The inhibition of major histocompatibility complex class I upregulation and cytokine, chemokine and interferon secretion may have important implications for host defense. Previous studies have shown that disruption of the secretory pathway can be replicated by expression of individual nonstructural proteins; however the situation with different serotypes of human rhinovirus (HRV) is unclear. The expression of 3A protein from HRV14 or HRV2 did not cause Golgi apparatus disruption or a block in secretion, whereas other studies showed that infection of cells with HRV1A did cause Golgi apparatus disruption which was replicated by the expression of 3A. HRV16 is the serotype most widely used in clinical HRV challenge studies; consequently, to address the issue of Golgi apparatus disruption for HRV16, we have systematically and quantitatively examined the effect of HRV16 on both Golgi apparatus fragmentation and protein secretion in HeLa cells. First, we expressed each individual nonstructural protein and examined their cellular localization and their disruption of endoplasmic reticulum and Golgi apparatus architecture. We quantified their effects on the secretory pathway by measuring secretion of the reporter protein Gaussia luciferase. Finally, we examined the same outcomes following infection of cells with live virus. We demonstrate that expression of HRV16 3A and 3AB and, to a lesser extent, 2B caused dispersal of the Golgi structure, and these three nonstructural proteins also inhibited protein secretion. The infection of cells with HRV16 also caused significant Golgi apparatus dispersal; however, this did not result in the inhibition of protein secretion. IMPORTANCE The ability of replicating picornaviruses to influence the function of the secretory pathway has important implications for host defense. However, there appear to be differences between different members of the family and inconsistent results when comparing infection with live virus to expression of individual nonstructural proteins. We demonstrate that individual nonstructural HRV16 proteins, when expressed in HeLa cells, can both fragment the Golgi apparatus and block secretion, whereas viral infection fragments the Golgi apparatus without blocking secretion. This has major implications for how we interpret mechanistic evidence derived from the expression of single viral proteins.

The hallmark of enteropathogenic Escherichia coli (EPEC) infection is the formation of actin-rich pedestal-like structures, which are generated following phosphorylation of the bacterial effector Tir by cellular Src and Abl family tyrosine kinases. This leads to recruitment of the Nck–WIP–N-WASP complex that triggers Arp2/3-dependent actin polymerization in the host cell. The same phosphorylation-mediated signalling network is also assembled downstream of the Vaccinia virus protein A36 and the phagocytic Fc-gamma receptor FcγRIIa. Here we report that the EPEC type-III secretion system effector EspJ inhibits autophosphorylation of Src and phosphorylation of the Src substrates Tir and FcγRIIa. Consistent with this, EspJ inhibits actin polymerization downstream of EPEC, Vaccinia virus and opsonized red blood cells. We identify EspJ as a unique adenosine diphosphate (ADP) ribosyltransferase that directly inhibits Src kinase by simultaneous amidation and ADP ribosylation of the conserved kinase-domain residue, Src E310, resulting in glutamine-ADP ribose., Non-receptor tyrosine kinases such as Src play fundamental roles in host–pathogen interactions and phagocytosis. Here, Young et al. show that an enteropathogenic Escherichia coli (EPEC) protein, EspJ, inhibits Src activity by simultaneous amidation and ADP ribosylation of a conserved residue on the kinase.

Legionella pneumophila, the causative agent of Legionnaires’ disease, uses the Dot/Icm type IV secretion system (T4SS) to translocate more than 300 effectors into host cells, where they subvert host cell signaling. The function and host cell targets of most effectors remain unknown. PieE is a 69-kDa Dot/Icm effector containing three coiled-coil (CC) regions and 2 transmembrane (TM) helices followed by a fourth CC region. Here, we report that PieE dimerized by an interaction between CC3 and CC4. We found that ectopically expressed PieE localized to the endoplasmic reticulum (ER) and induced the formation of organized smooth ER, while following infection PieE localized to the Legionella-containing vacuole (LCV). To identify the physiological targets of PieE during infection, we established a new purification method for which we created an A549 cell line stably expressing the Escherichia coli biotin ligase BirA and infected the cells with L. pneumophila expressing PieE fused to a BirA-specific biotinylation site and a hexahistidine tag. Following tandem Ni2+ nitrilotriacetic acid (NTA) and streptavidin affinity chromatography, the effector-target complexes were analyzed by mass spectrometry. This revealed interactions of PieE with multiple host cell proteins, including the Rab GTPases 1a, 1b, 2a, 5c, 6a, 7, and 10. Binding of the Rab GTPases, which was validated by yeast two-hybrid binding assays, was mediated by the PieE CC1 and CC2. In summary, using a novel, highly specific strategy to purify effector complexes from infected cells, which is widely applicable to other pathogens, we identified PieE as a multidomain LCV protein with promiscuous Rab GTPase-binding capacity., IMPORTANCE The respiratory pathogen Legionella pneumophila uses the Dot/Icm type IV secretion system to translocate more than 300 effector proteins into host cells. The function of most effectors in infection remains unknown. One of the bottlenecks for their characterization is the identification of target proteins. Frequently used in vitro approaches are not applicable to all effectors and suffer from high rates of false positives or missed interactions, as they are not performed in the context of an infection. Here, we determine key functional domains of the effector PieE and describe a new method to identify host cell targets under physiological infection conditions. Our approach, which is applicable to other pathogens, uncovered the interaction of PieE with several proteins involved in membrane trafficking, in particular Rab GTPases, revealing new details of the Legionella infection strategy and demonstrating the potential of this method to greatly advance our understanding of the molecular basis of infection.

BACKGROUND To assess subclavian vein (SCV) patency and long-term functional outcomes following surgical decompression of the thoracic outlet (SDTO) for Paget-Schroetter Syndrome (PSS). METHODS Between January 1978 and January 2013, we identified 33 patients with PSS who underwent SDTO. Demographic, clinical and radiological data were extracted from electronic databases and patient records. All patients were invited to update their follow-up data during dedicated outpatient visits between October and December 2013. Outcome measures included long-term SCV patency and clinical success rates during follow-up. Clinical success was defined as the combined absence of functional symptoms and patient's ability to maintain normal professional activities at final follow-up. The QuickDASH score was also determined. RESULTS The study population comprised 17 men and 16 women (mean age 34 years; range: 14-53 years) with PSS. Diagnosis was reached by venography (29 cases) or duplex scan (4 cases). SDTO was performed via the transaxillary route (25 cases) or using the combined supra-infraclavicular approach (8 cases). The procedure was carried out within 10 days in 13 patients (early-group), and between 30 to 120 days in the remaining 20 patients (late-group). The former had SCV recanalization obtained actively by thrombolysis (3 cases), thrombectomy (9 cases) or endovenectomy followed by patch venoplasty (1 case). The latter were maintained under chronic oral anticoagulation to allow SCV recanalization. There was neither postoperative death nor major bleeding complications. At a median follow-up of 240 months, 11 SCV remained patent in the early group, while in the other there was 3 re-occlusions, 4 residual stenoses and 5 chronic SCV occlusions. Clinical success was achieved in 73% of patients for the whole cohort, but was significantly better in patients operated on in the early stages (100% vs. 55%; P=0.005). The mean Quick Disabilities of the Arm, Shoulder, and Hand Score was 3.5 (95% CI: 1.5-5.4) in the early-group and 17.3 (95% CI: 8.4-26.2) in the late-group (P=0.01). CONCLUSIONS Our data shows that long-term functional outcomes and SCV patency remained better in PSS patients who underwent early SDTO and active SCV recanalization techniques.

The chemokine receptor CCR4 has at least two natural agonist ligands, MDC (CCL22) and TARC (CCL17) which bind to the same orthosteric site with a similar affinity. Both ligands are known to evoke chemotaxis of CCR4-bearing T cells and also elicit CCR4 receptor internalization. A series of small molecule allosteric antagonists have been described which displace the agonist ligand, and inhibit chemotaxis. The aim of this study was to determine which cellular coupling pathways are involved in internalization, and if antagonists binding to the CCR4 receptor could themselves evoke receptor internalization. CCL22 binding coupled CCR4 efficiently to β-arrestin and stimulated GTPγS binding however CCL17 did not couple to β-arrestin and only partially stimulated GTPγS binding. CCL22 potently induced internalization of almost all cell surface CCR4, while CCL17 showed only weak effects. We describe four small molecule antagonists that were demonstrated to bind to two distinct allosteric sites on the CCR4 receptor, and while both classes inhibited agonist ligand binding and chemotaxis, one of the allosteric sites also evoked receptor internalization. Furthermore, we also characterize an N-terminally truncated version of CCL22 which acts as a competitive antagonist at the orthosteric site, and surprisingly also evokes receptor internalization without demonstrating any agonist activity. Collectively this study demonstrates that orthosteric and allosteric antagonists of the CCR4 receptor are capable of evoking receptor internalization, providing a novel strategy for drug discovery against this class of target.

The karyophilic properties of the human immunodeficiency virus, type I (HIV-1) pre-integration complex (PIC) allow the virus to infect non-dividing cells. To better understand the mechanisms responsible for nuclear translocation of the PIC, we investigated nuclear import of HIV-1 integrase (IN), a PIC-associated viral enzyme involved in the integration of the viral genome in the host cell DNA. Accumulation of HIV-1 IN into nuclei of digitonin-permeabilized cells does not result from passive diffusion but rather from an active transport that occurs through the nuclear pore complexes. HIV-1 IN is imported by a saturable mechanism, implying that a limiting cellular factor is responsible for this process. Although IN has been previously proposed to contain classical basic nuclear localization signals, we found that nuclear accumulation of IN does not involve karyopherins alpha, beta1, and beta2-mediated pathways. Neither the non-hydrolyzable GTP analog, guanosine 5'-O-(thiotriphosphate), nor the GTP hydrolysis-deficient Ran mutant, RanQ69L, significantly affects nuclear import of IN, which depends instead on ATP hydrolysis. Therefore these results support the idea that IN import is not mediated by members of the karyopherin beta family. More generally, in vitro nuclear import of IN does not require addition of cytosolic factors, suggesting that cellular factor(s) involved in this active but atypical pathway process probably remain associated with the nuclear compartment or the nuclear pore complexes from permeabilized cells.

The Dot/Icm type IV secretion system (T4SS) of Legionella pneumophila is crucial for the pathogen to survive in protozoa and cause human disease. Although more than 275 effector proteins are delivered into the host cell by the T4SS, the function of the majority is unknown. Here we have characterized the Dot/Icm effector LtpD. During infection, LtpD localized to the cytoplasmic face of the membrane of the Legionella -containing vacuole (LCV). In A549 lung epithelial cells, ectopically expressed LtpD localized to large vesicular structures that contained markers of endosomal compartments. Systematic analysis of LtpD fragments identified an internal 17-kDa fragment, LtpD 471-626 , which was essential for targeting ectopically expressed LtpD to vesicular structures and for the association of translocated LtpD with the LCV. LtpD 471-626 bound directly to phosphatidylinositol 3-phosphate [PtdIns(3)P] in vitro and colocalized with the PtdIns(3)P markers FYVE and SetA in cotransfected cells. LtpD was also found to bind the host cell enzyme inositol ( myo )-1 (or 4)-monophosphatase 1, an important phosphatase involved in phosphoinositide production. Analysis of the role of LtpD in infection showed that LtpD is involved in bacterial replication in THP-1 macrophages, the larvae of Galleria mellonella , and mouse lungs. Together, these data suggest that LtpD is a novel phosphoinositide-binding L. pneumophila effector that has a role in intracellular bacterial replication.

A new methodology to evaluate the medication-use system based on a risk cartography tool, has been developed. This work has been promoted by the Observatoire du médicament et des dispositifs médicaux stériles et de l'innovation thérapeutique (OMEDIT) from Provence-Alpes-Côte d'Azur (PACA)-Corse regions.This new methodology has been developed with Excel (Microsoft(®)) and has led to the mediEVAL tool. It consists in two categories of Excel files: evaluating Excel files (1 for each job of the medication-use system) and synthesis Excel files which allow to compile a group of evaluating files for a defined area (department, hospital…).mediEVAL is a new tool to evaluate quality and risk management of the entire medication-use system which has to be used by private or public hospitals of PACA and Corsica areas in their appropriate medication-use contract. Then, the OMEDIT can get data to provide an inventory of fixtures of the PACA-Corse area medication-use system situation.

The translocation of effector proteins by the Dot/Icm type IV secretion system is central to the ability of Legionella pneumophila to persist and replicate within eukaryotic cells. The subcellular localization of translocated Dot/Icm proteins in host cells provides insight into their function. Through co-staining with host cell markers, effector proteins may be localized to specific subcellular compartments and membranes, which frequently reflects their host cell target and mechanism of action. In this chapter, we describe protocols to (1) localize effector proteins within cells by ectopic expression using green fluorescent protein fusions and (2) localize effector proteins within infected cells using epitope-tagged effector proteins and immuno-fluorescence microscopy.

Translocation of effector proteins via a type III secretion system (T3SS) is a widespread infection strategy among Gram-negative bacterial pathogens. Each pathogen translocates a particular set of effectors that subvert cell signaling in a way that suits its particular infection cycle. However, as effector unbalance might lead to cytotoxicity, the pathogens must employ mechanisms that regulate the intracellular effector concentration. We present evidence that the effector EspZ controls T3SS effector translocation from enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli. Consistently, an EPEC espZ mutant is highly cytotoxic. Following ectopic expression, we found that EspZ inhibited the formation of actin pedestals as it blocked the translocation of Tir, as well as other effectors, including Map and EspF. Moreover, during infection EspZ inhibited effector translocation following superinfection. Importantly, while EspZ of EHEC O157:H7 had a universal “translocation stop” activity, EspZ of EPEC inhibited effector translocation from typical EPEC strains but not from EHEC O157:H7 or its progenitor, atypical EPEC O55:H7. We found that the N and C termini of EspZ, which contains two transmembrane domains, face the cytosolic leaflet of the plasma membrane at the site of bacterial attachment, while the extracellular loop of EspZ is responsible for its strain-specific activity. These results show that EPEC and EHEC acquired a sophisticated mechanism to regulate the effector translocation., IMPORTANCE Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) are important diarrheal pathogens responsible for significant morbidity and mortality in developing countries and the developed world, respectively. The virulence strategy of EPEC and EHEC revolves around a conserved type III secretion system (T3SS), which translocates bacterial proteins known as effectors directly into host cells. Previous studies have shown that when cells are infected in two waves with EPEC, the first wave inhibits effector translocation by the second wave in a T3SS-dependent manner, although the factor involved was not known. Importantly, we identified EspZ as the effector responsible for blocking protein translocation following a secondary EPEC infection. Interestingly, we found that while EspZ of EHEC can block protein translocation from both EPEC and EHEC strains, EPEC EspZ cannot block translocation from EHEC. These studies show that EPEC and EHEC employ a novel infection strategy to regulate T3SS translocation.

Objectives The objective is to report our results with the arm composite autogenous vascular access (ACAVA) using the great saphenous vein (GSV) and the femoral vein (FV) in tertiary vascular access surgery. Design Retrospective single-centre study. Prospectively collected clinical database. Methods Between August 2009 and March 2011, 17 patients with no suitable upper extremity vein, repeated prosthetic access failure and/or infection underwent the construction of an ACAVA. Outcome measures included the graft patency and complication rates. Results The median follow-up was 25 months (5–32). Thirty-day morbidity affected 10 patients (59%): four wound-healing issues, three lower limb swelling, two early thromboses and one upper limb haematoma. No postoperative death occurred. At 3 months, the primary patency rate was 88% ± 8%. At 6 months, the assisted-primary patency rate was 82.4% ± 9.2%. At 12 months, the secondary patency rate was 81.6% ± 9.6%. Twenty-four secondary interventions were performed. Steal syndrome occurred in one patient following a secondary procedure. Swelling of the lower limb remained in two patients at the end of their follow-up. Three ACAVAs developed irreversible occlusion leading to loss of access. Conclusion With a high rate of postoperative morbidity and re-intervention, the ACAVA is a useful additional technique that should be restricted to difficult cases with limited vascular access options.

We report the case of a child with isolated late-onset epileptic spasms who significantly improved after focal frontal cortectomy. Clusters of axial and limb tonic contractions with head nodding began at 2 years of age. They occurred only during sleep, lasting 15 to 20 minutes and were pharmacoresistant. The child suffered slight mental delay. Interictal EEG showed left frontal spikes in slow sleep. Ictal video-EEG showed patterns of asymmetric spasms. MRI revealed a distinct white matter abnormality in the left frontal superior gyrus, corresponding clearly to localised cortical hypometabolism on FDG-PET and PET-MRI co-recording. SEEG investigation showed that the first spasm of the clusters corresponded to a localised discharge of rapid rhythms from the electrodes placed within the area identified as a lesion by MRI. Discharges then became more diffuse across the left frontal and temporal electrodes throughout the duration of the cluster. A tailored focal frontal resection was performed at 16 years of age. Spasms were very rare during the following three years (Engel class II). This observation illustrates the fact that isolated epileptic spasms can be cured by focal cortical resection despite a lack of clearly localised EEG surface anomalies.

The Mitochondrial Carrier Family (MCF) is a signature group of integral membrane proteins that transport metabolites across the mitochondrial inner membrane in eukaryotes. MCF proteins are characterized by six transmembrane segments that assemble to form a highly-selective channel for metabolite transport. We discovered a novel MCF member, termed Legionella nucleotide carrier Protein (LncP), encoded in the genome of Legionella pneumophila, the causative agent of Legionnaire's disease. LncP was secreted via the bacterial Dot/Icm type IV secretion system into macrophages and assembled in the mitochondrial inner membrane. In a yeast cellular system, LncP induced a dominant-negative phenotype that was rescued by deleting an endogenous ATP carrier. Substrate transport studies on purified LncP reconstituted in liposomes revealed that it catalyzes unidirectional transport and exchange of ATP transport across membranes, thereby supporting a role for LncP as an ATP transporter. A hidden Markov model revealed further MCF proteins in the intracellular pathogens, Legionella longbeachae and Neorickettsia sennetsu, thereby challenging the notion that MCF proteins exist exclusively in eukaryotic organisms., Author Summary Mitochondrial carrier proteins evolved during endosymbiosis to transport substrates across the mitochondrial inner membrane. As such the proteins are associated exclusively with eukaryotic organisms. Despite this, we identified putative mitochondrial carrier proteins in the genomes of different intracellular bacterial pathogens, including Legionella pneumophila, the causative agent of Legionnaire's disease. We named the mitochondrial carrier protein from L. pneumophila LncP and determined that the protein is translocated into host cells during infection by the bacterial Dot/Icm type IV secretion system. From there, LncP accesses the classical mitochondrial import pathway and is incorporated into the mitochondrial inner membrane as an integral membrane protein. Remarkably, LncP crosses five biological membranes to reach its final location. Biochemically, LncP is a unidirectional nucleotide transporter similar to Aac1 in yeast. Although not essential for intracellular replication, the high carriage rate of lncP among isolates of L. pneumophila suggests that the ability of the pathogen to manipulate mitochondrial ATP transport assists survival of the bacteria in an intracellular environment.

The prescription drug for hypertension in children remains difficult because of the lack of pharmacological data validated for this age of life. The Food and Drug Administration (FDA) has recently proposed some antihypertensive regimens orally. The authors found it useful to reproduce a table by adding all the necessary data for its use in France. Here is a list of French medicines and doses applied to children. Similarly, they made a second table for injectable antihypertensive treatment based on American recommendations--but not yet validated by FDA.

Typical enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) employ either Nck, TccP/TccP2, or Nck and TccP/TccP2 pathways to activate the neuronal Wiskott-Aldrich syndrome protein (N-WASP) and to trigger actin polymerization in. cultured cells. This phenotype is used as a marker for the pathogenic potential of EPEC and EHEC strains. in this paper we report that EPEC O125:H6, which represents a large category of strains, lacks the ability to utilize either Nck or TccP/TccP2 and hence triggers actin polymerization in vitro only inefficiently. However, we show that infection of human intestinal biopsies with EPEC O125:H6 results in formation of typical attaching and effacing lesions. Expression of TccP in EPEC O125:H6, which harbors an EHEC O157-like Tir, resulted in efficient actin polymerization in vitro and enhanced colonization of human intestinal in vitro organ cultures with detectable N-WASP and electron-dense material at the site of bacterial adhesion. These results show the existence of a natural category of EPEC that colonizes the gut mucosa using Nck- and TccP-independent mechanisms. Importantly, the results highlight yet again the fact that conclusions made on the basis of in vitro cell culture models cannot be extrapolated wholesale to infection of mucosal surfaces and that the ability to induce actin polymerization on cultured cells should not be used as a definitive marker for EPEC and EHEC virulence. Univ London Imperial Coll Sci Technol & Med, Div Cell & Mol Biol, London SW7 2AZ, England China CDC, Natl Inst Communicable Dis Control & Prevent, State Key Lab Infect Dis Prevent & Control, Beijing, Peoples R China UCL Royal Free Univ Coll Med Sch, Ctr Paediat Gastroenterol, London, England Miyazaki Univ, Frontier Sci Res Ctr, Div Bioenvironm Sci, Miyazaki 8891692, Japan Univ Maryland, Sch Med, Ctr Vaccine Dev, Baltimore, MD 21201 USA Universidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, São Paulo, Brazil Universidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, São Paulo, Brazil Web of Science

A four-year-old boy with ring chromosome 17 presenting with early-onset, pharmacoresistant epilepsy underwent repeated 24-hour video-EEG monitoring and cytogenetic analyses, including fluorescent in situ hybridization with telomeric and locus-specific probes of chromosome 17. Epilepsy was characterized by nocturnal motor seizures and by prolonged diurnal electrical status epilepticus. The 46, XY, r (17) karyotype was observed in the majority of cell lines. Fluorescent in situ hybridization revealed a deletion at the 17p telomere on the ring chromosome, whereas the 17q telomere and the Miller-Dieker lissencephaly locus were undeleted. The epileptic syndrome observed in this case of ring chromosome 17 resembles the one described in the ring chromosome 20 syndrome, raising the question of the specificity and the pathogenesis of ring chromosome epileptic syndromes. [Published with videosequences].

The insertion of a DNA copy of its RNA genome into a chromosome of the host cell is mediated by the viral integrase with the help of mostly uncharacterized cellular cofactors. We have recently described that the transcriptional co-activator LEDGF/p75 strongly interacts with HIV-1 integrase. Here we show that interaction of HIV-1 integrase with LEDGF/p75 is important for viral replication. Using multiple approaches including two-hybrid interaction studies, random and directed mutagenesis, we could demonstrate that HIV-1 virus harboring a single mutation that disrupts integrase-LEDGF/p75 interaction, resulted in defective HIV-1 replication. Furthermore, we found that LEDGF/p75 tethers HIV-1 integrase to chromosomes and that this interaction may be important for the integration process and the replication of HIV-1.

Nuclear import of HIV-1 preintegration complexes (PICs) allows the virus to infect nondividing cells. Integrase (IN), the PIC-associated viral enzyme responsible for the integration of the viral genome into the host cell DNA, displays karyophilic properties and has been proposed to participate to the nuclear import of the PIC. Styrylquinolines (SQs) have been shown to block viral replication at nontoxic concentrations and to inhibit IN 3'-processing activity in vitro by competing with the DNA substrate binding. However, several lines of evidence suggested that SQs could have a postentry, preintegrative antiviral effect in infected cells. To gain new insights on the mechanism of their antiviral activity, SQs were assayed for their ability to affect nuclear import of HIV-1 IN and compared with the effect of a specific strand transfer inhibitor. Using an in vitro transport assay, we have previously shown that IN import is a saturable mechanism, thus showing that a limiting cellular factor is involved in this process. We now demonstrate that SQs specifically and efficiently inhibit in vitro nuclear import of IN without affecting other import pathways, whereas a specific strand transfer inhibitor does not affect IN import. These data suggest that SQs not only inhibit IN-DNA interaction but would also inhibit the interaction between IN and the cellular factor required for its nuclear import.

The discrepant results available in the literature about the presence of hepatitis C virus (HCV) RNA in seminal plasma of men chronically infected by this agent are related, at least in part, to the molecular techniques used and particularly to the wide range of protocols dedicated to RNA extraction. In order to evaluate these protocols and to standardize the method of detection of HCV RNA in this fluid, a panel of coded specimens was tested blindly in 12 French laboratories; it included 14 seminal plasma specimens and four water controls spiked with HCV RNA ranging from 10 to 20,000 IU/ml and two HCV-negative seminal plasma specimens. The extraction step was performed according to methods using either silica beads (NucliSens [Organon Teknika S.A., Fresnes, France]; RNA viral kit [Qiagen, Courtaboeuf, France]) or guanidinium thiocyanate (Amplicor HCV assay; Roche Diagnostics, Meylan, France), preceded or not by a centrifugation of the seminal plasma. For the amplification step, all the laboratories performed the same reverse transcription-PCR technique (Amplicor HCV Cobas assay). The percentage of correct results ranged from 53.3 to 100, the poorest results being obtained when no centrifugation step preceded the Amplicor extraction protocol. The rate of correct results was significantly higher in laboratories using a preliminary centrifugation of the specimen ( P = 0.034 by chi-square test). By contrast, the overall number of correct results was not correlated to the initial volume of sample used for the test. These results allowed us to validate standardized techniques adapted to the performance of this test on a routine basis, especially in men infected with HCV and involved in programs of medically assisted reproduction.

Patients with severe rheumatoid arthritis and resistant to at least three DMARDS can benefit from anti-TNFalpha (tumor necrosis factor) therapy. In some patients, because of inefficacy or adverse events, treatment with one of the two available TNFalpha drugs (etanercept and infliximab) must be stopped. In this study, we explored the results in efficacy and tolerance of switching from one anti-TNFalpha to the other.Between August 1999 and January 2002, we administered one of the two anti TNFalpha drugs to 131 patients: 67 patients received infiximab and 64 etanercept.Among the 67 patients treated with infliximab, 17 patients had to stop treatment. In 8 of them (4 allergies, 2 infections and 2 non responders) the switch from infliximab to etanercept was beneficial for 5 patients, 2 patients did not respond and 1 patient withdrew for personal reasons. Among the 64 patients treated with etanercept, 13 had to stop treatment. In 6 of them (2 adverse events, 4 failures) the switch from etanercept to infliximab was beneficial for 3 patients, 2 did not respond and 1 withdrew because of adverse events.In all, 14 patients with severe rheumatoid arthritis and treated by one of the two TNFalpha drugs (and in whom treatment was stopped because of adverse events or inefficacy) benefited from the switch to the other anti- TNFalpha, with excellent response in 8 out of 14 patients.

Background Chronic erosive oral lichen planus (EOLP) is a severe form of lichen of the buccal mucosa that is often resistant to systemic or topical therapies. Objective To evaluate the efficacy and safety of topical tacrolimus, 0.1 mg per 100 mL of water, in treating EOLP. Design Open-label, prospective, noncomparative study, with 6 months of treatment and 6 months of follow-up. Setting Dermatology department at a university hospital in Nice, France. Patients Ten patients with histologically proved EOLP that was refractory to treatment. Two patients were withdrawn because of noncompliance; findings in 8 were available for evaluation. Interventions Mouthwashes with tacrolimus, 0.1 mg per 100 mL of distilled water, 4 times daily for 6 months. Main Outcome Measures Efficacy was assessed using a calculated score that combined the intensity of spontaneous and meal-triggered pain and the surface area of erosions. Safety assessment included the monitoring of adverse effects, clinical laboratory values, and blood concentrations of tacrolimus. Results Among the 8 patients evaluated, 1 had no improvement and 7 were improved. The mean score decreased from 7.00 at baseline to 5.43 (a 22.43% decrease) at 1 month, 4.14 (a 40.86% decrease) at 2 months, 3.00 (a 57.14% decrease) at 3 months, 2.43 (a 65.29% decrease) at 4 months, 2.57 (a 63.29% decrease) at 5 months, and 3.43 (a 51.00% decrease) at 6 months. A decrease of symptoms was reported by the 7 responding patients as soon as the first month of treatment. No severe adverse effects were observed. All patients had whole-blood concentrations of tacrolimus below the detection limit of the assay (1.5 ng/mL) at all intervals. At 9 months, 6 patients had had a relapse within a mean of 38.6 days. At 12 months, all patients had had a relapse and required treatment with topical corticosteroids or systemic hydroxychloroquine sulfate. Conclusion Results of our study suggest a rapid and important palliating effect of low concentration of topical tacrolimus in distilled water in patients with EOLP.

The HIV-1 genome contains several genes coding for auxiliary proteins, including the small Vpr protein. Vpr affects the integrity of the nuclear envelope and participates in the nuclear translocation of the preintegration complex containing the viral DNA. Here, we show by photobleaching experiments performed on living cells expressing a Vpr-green fluorescent protein fusion that the protein shuttles between the nucleus and the cytoplasm, but a significant fraction is concentrated at the nuclear envelope, supporting the hypothesis that Vpr interacts with components of the nuclear pore complex. An interaction between HIV-1 Vpr and the human nucleoporin CG1 (hCG1) was revealed in the yeast two-hybrid system, and then confirmed both in vitro and in transfected cells. This interaction does not involve the FG repeat domain of hCG1 but rather the N-terminal region of the protein. Using a nuclear import assay based on digitonin-permeabilized cells, we demonstrate that hCG1 participates in the docking of Vpr at the nuclear envelope. This association of Vpr with a component of the nuclear pore complex may contribute to the disruption of the nuclear envelope and to the nuclear import of the viral DNA.

In recent years, efforts have been made in hospitals to improve antibiotic prescription. Most universities organise courses on the subject, which lead to a local university diploma. However, possessing such a diploma does not give entitlement to prescribe. In fact, most doctors prescribe antibiotics and such courses are only of interest to volunteer physicians. While some are very careful, the majority prescribe the drugs as they are rarely toxic. Others are refractory to any information and particularly to any training. Two methods are typically proposed to reduce unjustified prescription. As a result of imposed restrictions, only trained doctors having met the training standards are allowed to prescribe and have to keep to a limited budget. The persuasive method, on the other hand, opens the way for a wide scope of training courses, which are provided by industry; some are said to be biased as they encourage prescription and the risk of selecting resistant mutant bacteria is scarcely documented. This method does not always coincide with the training curricula. The industry is torn between declared objectives such as judicious drug use and prevailing commercial aims. As a result, prescription is not restrained by any objective limit. It should be noted that prescription varies greatly from one hospital to another and within a given hospital between one department and another. Certain departments prescribe much more than others and these (emergency, medical and surgical intensive care, respiratory disease) should be targeted first.

Multiresistant bacteria are regularly isolated in nosocomial infections occurring in intensive care units due to wide use of antibiotics. We evaluated the impact of systematic infectiology consultations on the quality of antibiotic prescriptions in an intensive care unit.Infectiology consultations (3 per week) were initiated mid February 1999. The infectiologist gave oral advice to be implemented (or not) by the intensive care unit according to ongoing therapeutic options. The hospital pharmacy recorded antibiotic use for March and April 1999 for comparison with use recorded in 1998 for a similar period. We retrospectively reviewed the files of patients hospitalized during these periods and who had received antibiotics to determine the modalities of antibiotic use. The 4 antibiotics used for the longest period for each patient were recorded.Thirty-one patients in 1999 and 30 in 1998 were given antibiotics. The SAPS score was similar for the two groups. Mean duration of antibiotic treatment was lower during the March-April 1999 period than during the corresponding period in 1998: 13 +/- 9 days/patient versus 23 +/- 21 days/patient respectively, p = 0.037. In 1998, there were 596 antibiotic-days and in 1999 there were 455 (-24%). The cost of antibiotic therapy in 1998 was 70,342 FrF compared with 56,804 FrF in 1999 (-19%).Infectiology consultation, in association with the opinion of the intensive care physician, is a simple way to limit antibiotic use.

One of the missions of the Agence Française due Sang was to set up a haemovigilance system based on a national network of surveillance and alert, for the whole blood chain from blood collection to receivers' follow-up. This system is now operational and contributes with efficacy to the safety of blood transfusions (see: A quoi sert l'hémovigilance? Transfus Clin Biol 1998; 5: 415-21.). The French law No. 98-535 dated July 1st 1998, whose enforcement orders were published in March 1999, is the legal foundation of the system. In this article the national and regional structures of the national system of sanitary safety are described, and the main guidelines of material vigilance, the last element of this system, is presented.

Determine the causes of malaria attacks in subjects who have returned from endemic areas by assessing prescriptions for chemical prophylaxis and compliance.All patients who developed a paroxysmal episode of malaria diagnosed at the University of Nice hospital in 1995 answered specific questions concerning their anti-malaria prophylaxis.Thirty-three patients were hospitalized for paroxysmal episodes of malaria in 1995. In 32 cases (97%) the attack resulted from either the lack of any prophylaxis (17 cases, 52%), inadequate prescription (11 cases, 12%) or poor compliance (4 cases, 12%). The prescribed chemical prophylaxis was not adapted to the chloroquinone-resistant area in 8 cases (24%) and medical recommendations concerning administration rules were inadequate in 3 cases (9%). Only one patient developed a paroxysmal episode despite correct compliance to a chloroquine-resistant zone-adapted well-conducted prescription. The cost of poor prophylaxis in terms of human suffering and financial cost was high for this preventable disease. Four patients had to be hospitalized in the intensive care unit and one died during hospitalization. The cumulative cost of hospitalization for these 33 cases was evaluated at 660,000 FF.Preventive measures for malaria must include better information for physicians on changing recommendations for chemical prophylaxis as well as better information for travelers provided by all those involved in organizing travel to endemic areas.

Bacillus Calmette Guerin (BCG) is an attenuated strain of Mycobacterium bovis that is currently used as a live vaccine for human tuberculosis. Disseminated BCG infection may rarely occur following vaccination of children. In half of the cases, regarded as idiopathic, no well-defined immunodeficiency condition can account for the infection. However, the high rates of parental consanguinity and familial forms and the associated opportunistic infections with Salmonella suggest that these idiopathic BCG infections result from one or several new type(s) of inherited immune disorder(s). As an approach to the description and understanding of this newly described condition, the associated lesions were examined. Samples from 14 patients collected from a French national retrospective study were analysed. Pathological data from 22 cases reported in the world literature were also reviewed. Two types of granuloma were found. The first type (type I, tuberculoid) consisted of well-circumscribed and well-differentiated granulomas, with epithelioid and multinucleated giant cells containing very few acid-fast rods, surrounded by lymphocytes and fibrosis and occasionally with central caseous necrosis. The second type (type II, lepromatous-like) consisted of ill-defined and poorly differentiated granulomas, with few if any giant cells and lymphocytes but widespread macrophages loaded with acid-fast bacilli. Most children displayed a single type of granuloma. One half displayed type 1 lesions and the other half displayed type II lesions. There was a strong correlation between the type of granuloma and the clinical outcome. Tuberculoid lesions were associated with survival, whilst lepromatous-like lesions correlated with death. Correlation of granuloma structure with clinical outcome defines two types of idiopathic disseminated BCG infection. The phenotypic heterogeneity of the course of BCG infection reflects distinct pathogenic mechanisms and probably results from a genotypic heterogeneity of the underlying inherited immune disorder.

The aim of this review is to gather information in favour of the involvement of voltage-dependent ion channels in epileptogenesis. Although, up to now, no study has shown that epilepsy is accompanied by a modification in the activity to these channels, the recently acquired knowledge of their physiology allows to presume would favor their involvement in epileptogenesis. The results from electrophysiological studies are as follows: a persistent sodium current increases neuronal excitability whereas potassium currents have an inhibitory role. In particular, calcium-dependent potassium current are involved in the post-hyperpolarization phases which follows PDS. Calcium currents are also involved in the genesis of the "bursting pacemaker" activity displayed by the neurons presumed to be inducers of the epileptic activity. Biochemical data has shown that as a consequence of epileptic activity, sodium and calcium channels are down regulated. This down-regulation could be a way to reduces neuronal hyperexcitability. Pharmacological data demonstrate the drugs which activate calcium channels or which inhibit potassium channels have a convusilvant effect. On the contrary, agents which block calcium or sodium channels or which properties. Among the latter ones, some antiepileptic drugs can be found. In summary situations which lead to increase in calcium and sodium currents and/or to an inhibition in potassium currents are potentially epileptogenic.

Objectives: To evaluate the prognostic value of cerebral border-zone infarctions (watershed infarctions) on the early postoperative outcomes of patients undergoing carotid endarterectomy (CEA) after acute ischemic stroke (AIS). Methods: Sixty-six (66) patients with symptomatic carotid stenosis (SCS) that underwent ipsilateral CEA after AIS from January 2007 to March 2012 were included in this study. They were divided into two groups according to the topographic patterns of the stroke: group 1, Territorial Cerebral Ischemic Strokes (TCIS) caused by emboli of carotid origin; group 2, cerebral border-zone infarctions (CBZI) related to an SCS associated with hemodynamic impairment. All data was collected in a prospective database and analyzed. Outcome measures included postoperative neurological morbidity and 30-day mortality. Results: Forty-three (43) patients (65.15%) experienced TCIS and were included in group 1, 23 patients (34.85%) had a CBZI and were included in group 2. There were no postoperative deaths. The postoperative neurologic morbidity rate was significantly higher in the CBZI group (22% vs. 2%, p = 0.02). Multivariate analysis demonstrates that CBZI was the only independent predictive factor of neurologic morbidity after CEA for AIS related to an SCS. Furthermore, the risk of postoperative neurologic morbidity remained significantly higher for patients with CBZI after adjustement for age, sex, initial NHISS scores, and associated contralateral carotid occlusion (HR: 0.059, 95% CI 0.004-0.85; p = 0.03). Conclusion: CBZIs, compared to TCIS, were associated with a higher neurological complication rate during the postoperative period after CEA for SCS in cases of AIS. Further studies are required to better define the timing and the best treatment modality for patients with CBZI related to an SCS in order to reduce associated procedural complications.© 2012 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved.

International audience; The "Standards, Options: Recommendations" (SOR) project has been undertaken by the French National Federation of Cancer Centers (FNCLCC) and is now part of the French National Cancer Institute. The project involves the development and updating of evidence-based Clinical Practice Guidelines (CPG) in oncology. In order to answer questions related to venous thromboembolic events (VTE) treatment and to central venous catheter thrombosis (CVCT) management in cancer patients, the SOR elaborated national guidelines, here presented in a short report. It results of a collaborative work with members from three learned societies (<< Société nationale française de médecine interne >> : SNFMI, << Société française de médecine vasculaire >> : SFMV and << Société française d'anesthésie-réanimation >> : SFAR).