Institut de Neurobiologie de la Méditerranée

Antibodies targeting CTLA-4 have been successfully used as cancer immunotherapy. We find that the antitumor effects of CTLA-4 blockade depend on distinct Bacteroides species. In mice and patients, T cell responses specific for B. thetaiotaomicron or B. fragilis were associated with the efficacy of CTLA-4 blockade. Tumors in antibiotic-treated or germ-free mice did not respond to CTLA blockade. This defect was overcome by gavage with B. fragilis, by immunization with B. fragilis polysaccharides, or by adoptive transfer of B. fragilis-specific T cells. Fecal microbial transplantation from humans to mice confirmed that treatment of melanoma patients with antibodies against CTLA-4 favored the outgrowth of B. fragilis with anticancer properties. This study reveals a key role for Bacteroidales in the immunostimulatory effects of CTLA-4 blockade.

BACKGROUND: In patients undergoing mechanical ventilation for the acute respiratory distress syndrome (ARDS), neuromuscular blocking agents may improve oxygenation and decrease ventilator-induced lung injury but may also cause muscle weakness. We evaluated clinical outcomes after 2 days of therapy with neuromuscular blocking agents in patients with early, severe ARDS. METHODS: In this multicenter, double-blind trial, 340 patients presenting to the intensive care unit (ICU) with an onset of severe ARDS within the previous 48 hours were randomly assigned to receive, for 48 hours, either cisatracurium besylate (178 patients) or placebo (162 patients). Severe ARDS was defined as a ratio of the partial pressure of arterial oxygen (PaO2) to the fraction of inspired oxygen (FIO2) of less than 150, with a positive end-expiratory pressure of 5 cm or more of water and a tidal volume of 6 to 8 ml per kilogram of predicted body weight. The primary outcome was the proportion of patients who died either before hospital discharge or within 90 days after study enrollment (i.e., the 90-day in-hospital mortality rate), adjusted for predefined covariates and baseline differences between groups with the use of a Cox model. RESULTS: The hazard ratio for death at 90 days in the cisatracurium group, as compared with the placebo group, was 0.68 (95% confidence interval [CI], 0.48 to 0.98; P=0.04), after adjustment for both the baseline PaO2:FIO2 and plateau pressure and the Simplified Acute Physiology II score. The crude 90-day mortality was 31.6% (95% CI, 25.2 to 38.8) in the cisatracurium group and 40.7% (95% CI, 33.5 to 48.4) in the placebo group (P=0.08). Mortality at 28 days was 23.7% (95% CI, 18.1 to 30.5) with cisatracurium and 33.3% (95% CI, 26.5 to 40.9) with placebo (P=0.05). The rate of ICU-acquired paresis did not differ significantly between the two groups. CONCLUSIONS: In patients with severe ARDS, early administration of a neuromuscular blocking agent improved the adjusted 90-day survival and increased the time off the ventilator without increasing muscle weakness. (Funded by Assistance Publique-Hôpitaux de Marseille and the Programme Hospitalier de Recherche Clinique Régional 2004-26 of the French Ministry of Health; ClinicalTrials.gov number, NCT00299650.)

Cyclophosphamide is one of several clinically important cancer drugs whose therapeutic efficacy is due in part to their ability to stimulate antitumor immune responses. Studying mouse models, we demonstrate that cyclophosphamide alters the composition of microbiota in the small intestine and induces the translocation of selected species of Gram-positive bacteria into secondary lymphoid organs. There, these bacteria stimulate the generation of a specific subset of "pathogenic" T helper 17 (pT(H)17) cells and memory T(H)1 immune responses. Tumor-bearing mice that were germ-free or that had been treated with antibiotics to kill Gram-positive bacteria showed a reduction in pT(H)17 responses, and their tumors were resistant to cyclophosphamide. Adoptive transfer of pT(H)17 cells partially restored the antitumor efficacy of cyclophosphamide. These results suggest that the gut microbiota help shape the anticancer immune response.

BACKGROUND: Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry accurately identifies both selected bacteria and bacteria in select clinical situations. It has not been evaluated for routine use in the clinic. METHODS: We prospectively analyzed routine MALDI-TOF mass spectrometry identification in parallel with conventional phenotypic identification of bacteria regardless of phylum or source of isolation. Discrepancies were resolved by 16S ribosomal RNA and rpoB gene sequence-based molecular identification. Colonies (4 spots per isolate directly deposited on the MALDI-TOF plate) were analyzed using an Autoflex II Bruker Daltonik mass spectrometer. Peptidic spectra were compared with the Bruker BioTyper database, version 2.0, and the identification score was noted. Delays and costs of identification were measured. RESULTS: Of 1660 bacterial isolates analyzed, 95.4% were correctly identified by MALDI-TOF mass spectrometry; 84.1% were identified at the species level, and 11.3% were identified at the genus level. In most cases, absence of identification (2.8% of isolates) and erroneous identification (1.7% of isolates) were due to improper database entries. Accurate MALDI-TOF mass spectrometry identification was significantly correlated with having 10 reference spectra in the database (P=.01). The mean time required for MALDI-TOF mass spectrometry identification of 1 isolate was 6 minutes for an estimated 22%-32% cost of current methods of identification. CONCLUSIONS: MALDI-TOF mass spectrometry is a cost-effective, accurate method for routine identification of bacterial isolates in <1 h using a database comprising > or =10 reference spectra per bacterial species and a 1.9 identification score (Brucker system). It may replace Gram staining and biochemical identification in the near future.

Q fever is a zoonosis with a worldwide distribution with the exception of New Zealand. The disease is caused by Coxiella burnetii, a strictly intracellular, gram-negative bacterium. Many species of mammals, birds, and ticks are reservoirs of C. burnetii in nature. C. burnetii infection is most often latent in animals, with persistent shedding of bacteria into the environment. However, in females intermittent high-level shedding occurs at the time of parturition, with millions of bacteria being released per gram of placenta. Humans are usually infected by contaminated aerosols from domestic animals, particularly after contact with parturient females and their birth products. Although often asymptomatic, Q fever may manifest in humans as an acute disease (mainly as a self-limited febrile illness, pneumonia, or hepatitis) or as a chronic disease (mainly endocarditis), especially in patients with previous valvulopathy and to a lesser extent in immunocompromised hosts and in pregnant women. Specific diagnosis of Q fever remains based upon serology. Immunoglobulin M (IgM) and IgG antiphase II antibodies are detected 2 to 3 weeks after infection with C. burnetii, whereas the presence of IgG antiphase I C. burnetii antibodies at titers of >/=1:800 by microimmunofluorescence is indicative of chronic Q fever. The tetracyclines are still considered the mainstay of antibiotic therapy of acute Q fever, whereas antibiotic combinations administered over prolonged periods are necessary to prevent relapses in Q fever endocarditis patients. Although the protective role of Q fever vaccination with whole-cell extracts has been established, the population which should be primarily vaccinated remains to be clearly identified. Vaccination should probably be considered in the population at high risk for Q fever endocarditis.

PURPOSE: Focal cortical dysplasias (FCD) are localized regions of malformed cerebral cortex and are very frequently associated with epilepsy in both children and adults. A broad spectrum of histopathology has been included in the diagnosis of FCD. An ILAE task force proposes an international consensus classification system to better characterize specific clinicopathological FCD entities. METHODS: Thirty-two Task Force members have reevaluated available data on electroclinical presentation, imaging, neuropathological examination of surgical specimens as well as postsurgical outcome. KEY FINDINGS: The ILAE Task Force proposes a three-tiered classification system. FCD Type I refers to isolated lesions, which present either as radial (FCD Type Ia) or tangential (FCD Type Ib) dyslamination of the neocortex, microscopically identified in one or multiple lobes. FCD Type II is an isolated lesion characterized by cortical dyslamination and dysmorphic neurons without (Type IIa) or with balloon cells (Type IIb). Hence, the major change since a prior classification represents the introduction of FCD Type III, which occurs in combination with hippocampal sclerosis (FCD Type IIIa), or with epilepsy-associated tumors (FCD Type IIIb). FCD Type IIIc is found adjacent to vascular malformations, whereas FCD Type IIId can be diagnosed in association with epileptogenic lesions acquired in early life (i.e., traumatic injury, ischemic injury or encephalitis). SIGNIFICANCE: This three-tiered classification system will be an important basis to evaluate imaging, electroclinical features, and postsurgical seizure control as well as to explore underlying molecular pathomechanisms in FCD.

BACKGROUND: Carotid stenting is less invasive than endarterectomy, but it is unclear whether it is as safe in patients with symptomatic carotid-artery stenosis. METHODS: We conducted a multicenter, randomized, noninferiority trial to compare stenting with endarterectomy in patients with a symptomatic carotid stenosis of at least 60%. The primary end point was the incidence of any stroke or death within 30 days after treatment. RESULTS: The trial was stopped prematurely after the inclusion of 527 patients for reasons of both safety and futility. The 30-day incidence of any stroke or death was 3.9% after endarterectomy (95% confidence interval [CI], 2.0 to 7.2) and 9.6% after stenting (95% CI, 6.4 to 14.0); the relative risk of any stroke or death after stenting as compared with endarterectomy was 2.5 (95% CI, 1.2 to 5.1). The 30-day incidence of disabling stroke or death was 1.5% after endarterectomy (95% CI, 0.5 to 4.2) and 3.4% after stenting (95% CI, 1.7 to 6.7); the relative risk was 2.2 (95% CI, 0.7 to 7.2). At 6 months, the incidence of any stroke or death was 6.1% after endarterectomy and 11.7% after stenting (P=0.02). There were more major local complications after stenting and more systemic complications (mainly pulmonary) after endarterectomy, but the differences were not significant. Cranial-nerve injury was more common after endarterectomy than after stenting. CONCLUSIONS: In this study of patients with symptomatic carotid stenosis of 60% or more, the rates of death and stroke at 1 and 6 months were lower with endarterectomy than with stenting. (ClinicalTrials.gov number, NCT00190398 [ClinicalTrials.gov].).

Developing networks follow common rules to shift from silent cells to coactive networks that operate via thousands of synapses. This review deals with some of these rules and in particular those concerning the crucial role of the neurotransmitter gamma-aminobuytric acid (GABA), which operates primarily via chloride-permeable GABA(A) receptor channels. In all developing animal species and brain structures investigated, neurons have a higher intracellular chloride concentration at an early stage leading to an efflux of chloride and excitatory actions of GABA in immature neurons. This triggers sodium spikes, activates voltage-gated calcium channels, and acts in synergy with NMDA channels by removing the voltage-dependent magnesium block. GABA signaling is also established before glutamatergic transmission, suggesting that GABA is the principal excitatory transmitter during early development. In fact, even before synapse formation, GABA signaling can modulate the cell cycle and migration. The consequence of these rules is that developing networks generate primitive patterns of network activity, notably the giant depolarizing potentials (GDPs), largely through the excitatory actions of GABA and its synergistic interactions with glutamate signaling. These early types of network activity are likely required for neurons to fire together and thus to "wire together" so that functional units within cortical networks are formed. In addition, depolarizing GABA has a strong impact on synaptic plasticity and pathological insults, notably seizures of the immature brain. In conclusion, it is suggested that an evolutionary preserved role for excitatory GABA in immature cells provides an important mechanism in the formation of synapses and activity in neuronal networks.

Ticks are currently considered to be second only to mosquitoes as vectors of human infectious diseases in the world. Each tick species has preferred environmental conditions and biotopes that determine the geographic distribution of the ticks and, consequently, the risk areas for tickborne diseases. This is particularly the case when ticks are vectors and reservoirs of the pathogens. Since the identification of Borrelia burgdorferi as the agent of Lyme disease in 1982, 15 ixodid-borne bacterial pathogens have been described throughout the world, including 8 rickettsiae, 3 ehrlichiae, and 4 species of the Borrelia burgdorferi complex. This article reviews and illustrate various aspects of the biology of ticks and the tickborne bacterial diseases (rickettsioses, ehrlichioses, Lyme disease, relapsing fever borrelioses, tularemia, Q fever), particularly those regarded as emerging diseases. Methods are described for the detection and isolation of bacteria from ticks and advice is given on how tick bites may be prevented and how clinicians should deal with patients who have been bitten by ticks.

During most of the 20th century, the epidemiology of tick-borne rickettsioses could be summarized as the occurrence of a single pathogenic rickettsia on each continent. An element of this paradigm suggested that the many other characterized and noncharacterized rickettsiae isolated from ticks were not pathogenic to humans. In this context, it was considered that relatively few tick-borne rickettsiae caused human disease. This concept was modified extensively from 1984 through 2005 by the identification of at least 11 additional rickettsial species or subspecies that cause tick-borne rickettsioses around the world. Of these agents, seven were initially isolated from ticks, often years or decades before a definitive association with human disease was established. We present here the tick-borne rickettsioses described through 2005 and focus on the epidemiological circumstances that have played a role in the emergence of the newly recognized diseases.

We recently reported the discovery and preliminary characterization of Mimivirus, the largest known virus, with a 400-nanometer particle size comparable to mycoplasma. Mimivirus is a double-stranded DNA virus growing in amoebae. We now present its 1,181,404-base pair genome sequence, consisting of 1262 putative open reading frames, 10% of which exhibit a similarity to proteins of known functions. In addition to exceptional genome size, Mimivirus exhibits many features that distinguish it from other nucleocytoplasmic large DNA viruses. The most unexpected is the presence of numerous genes encoding central protein-translation components, including four amino-acyl transfer RNA synthetases, peptide release factor 1, translation elongation factor EF-TU, and translation initiation factor 1. The genome also exhibits six tRNAs. Other notable features include the presence of both type I and type II topoisomerases, components of all DNA repair pathways, many polysaccharide synthesis enzymes, and one intein-containing gene. The size and complexity of the Mimivirus genome challenge the established frontier between viruses and parasitic cellular organisms. This new sequence data might help shed a new light on the origin of DNA viruses and their role in the early evolution of eukaryotes.

Free-living amoebae feed on bacteria, fungi, and algae. However, some microorganisms have evolved to become resistant to these protists. These amoeba-resistant microorganisms include established pathogens, such as Cryptococcus neoformans, Legionella spp., Chlamydophila pneumoniae, Mycobacterium avium, Listeria monocytogenes, Pseudomonas aeruginosa, and Francisella tularensis, and emerging pathogens, such as Bosea spp., Simkania negevensis, Parachlamydia acanthamoebae, and Legionella-like amoebal pathogens. Some of these amoeba-resistant bacteria (ARB) are lytic for their amoebal host, while others are considered endosymbionts, since a stable host-parasite ratio is maintained. Free-living amoebae represent an important reservoir of ARB and may, while encysted, protect the internalized bacteria from chlorine and other biocides. Free-living amoebae may act as a Trojan horse, bringing hidden ARB within the human "Troy," and may produce vesicles filled with ARB, increasing their transmission potential. Free-living amoebae may also play a role in the selection of virulence traits and in adaptation to survival in macrophages. Thus, intra-amoebal growth was found to enhance virulence, and similar mechanisms seem to be implicated in the survival of ARB in response to both amoebae and macrophages. Moreover, free-living amoebae represent a useful tool for the culture of some intracellular bacteria and new bacterial species that might be potential emerging pathogens.

Hippocampal sclerosis (HS) is the most frequent histopathology encountered in patients with drug-resistant temporal lobe epilepsy (TLE). Over the past decades, various attempts have been made to classify specific patterns of hippocampal neuronal cell loss and correlate subtypes with postsurgical outcome. However, no international consensus about definitions and terminology has been achieved. A task force reviewed previous classification schemes and proposes a system based on semiquantitative hippocampal cell loss patterns that can be applied in any histopathology laboratory. Interobserver and intraobserver agreement studies reached consensus to classify three types in anatomically well-preserved hippocampal specimens: HS International League Against Epilepsy (ILAE) type 1 refers always to severe neuronal cell loss and gliosis predominantly in CA1 and CA4 regions, compared to CA1 predominant neuronal cell loss and gliosis (HS ILAE type 2), or CA4 predominant neuronal cell loss and gliosis (HS ILAE type 3). Surgical hippocampus specimens obtained from patients with TLE may also show normal content of neurons with reactive gliosis only (no-HS). HS ILAE type 1 is more often associated with a history of initial precipitating injuries before age 5 years, with early seizure onset, and favorable postsurgical seizure control. CA1 predominant HS ILAE type 2 and CA4 predominant HS ILAE type 3 have been studied less systematically so far, but some reports point to less favorable outcome, and to differences regarding epilepsy history, including age of seizure onset. The proposed international consensus classification will aid in the characterization of specific clinicopathologic syndromes, and explore variability in imaging and electrophysiology findings, and in postsurgical seizure control.

BACKGROUND: Studies of the bacterial communities of the gut microbiota have revealed a shift in the ratio of Firmicutes and Bacteroidetes in obese patients. Determining the variations of microbial communities in feces may be beneficial for the identification of specific profiles in patients with abnormal weights. The roles of the archaeon Methanobrevibacter smithii and Lactobacillus species have not been described in these studies. METHODS AND FINDINGS: We developed an efficient and robust real-time PCR tool that includes a plasmid-based internal control and allows for quantification of the bacterial divisions Bacteroidetes, Firmicutes, and Lactobacillus as well as the methanogen M. smithii. We applied this technique to the feces of 20 obese subjects, 9 patients with anorexia nervosa, and 20 normal-weight healthy controls. Our results confirmed a reduction in the Bacteroidetes community in obese patients (p<0.01). We found a significantly higher Lactobacillus species concentration in obese patients than in lean controls (p=0.0197) or anorexic patients (p=0.0332). The M. smithii concentration was much higher in anorexic patients than in the lean population (p=0.0171). CONCLUSIONS: Lactobacillus species are widely used as growth promoters in the farm industry and are now linked to obesity in humans. The study of the bacterial flora in anorexic patients revealed an increase in M. smithii. This increase might represent an adaptive use of nutrients in this population.

Phlebotomine sandflies transmit pathogens that affect humans and animals worldwide. We review the roles of phlebotomines in the spreading of leishmaniases, sandfly fever, summer meningitis, vesicular stomatitis, Chandipura virus encephalitis and Carrión's disease. Among over 800 species of sandfly recorded, 98 are proven or suspected vectors of human leishmaniases; these include 42 Phlebotomus species in the Old World and 56 Lutzomyia species in the New World (all: Diptera: Psychodidae). Based on incrimination criteria, we provide an updated list of proven or suspected vector species by endemic country where data are available. Increases in sandfly diffusion and density resulting from increases in breeding sites and blood sources, and the interruption of vector control activities contribute to the spreading of leishmaniasis in the settings of human migration, deforestation, urbanization and conflict. In addition, climatic changes can be expected to affect the density and dispersion of sandflies. Phlebovirus infections and diseases are present in large areas of the Old World, especially in the Mediterranean subregion, in which virus diversity has proven to be higher than initially suspected. Vesiculovirus diseases are important to livestock and humans in the southeastern U.S.A. and Latin America, and represent emerging human threats in parts of India. Carrión's disease, formerly restricted to regions of elevated altitude in Peru, Ecuador and Colombia, has shown recent expansion to non-endemic areas of the Amazon basin.

During a study following a pneumonia outbreak in 1992, a microorganism growing in amoebae and resembling a small Gram-positive coccus ([Fig. 1][1]A) was isolated from the water of a cooling tower in Bradford, England. Despite attempts with various extraction protocols and low-stringency polymerase

Acinetobacter baumannii is a species of nonfermentative gram-negative bacteria commonly found in water and soil. This organism was susceptible to most antibiotics in the 1970s. It has now become a major cause of hospital-acquired infections worldwide due to its remarkable propensity to rapidly acquire resistance determinants to a wide range of antibacterial agents. Here we use a comparative genomic approach to identify the complete repertoire of resistance genes exhibited by the multidrug-resistant A. baumannii strain AYE, which is epidemic in France, as well as to investigate the mechanisms of their acquisition by comparison with the fully susceptible A. baumannii strain SDF, which is associated with human body lice. The assembly of the whole shotgun genome sequences of the strains AYE and SDF gave an estimated size of 3.9 and 3.2 Mb, respectively. A. baumannii strain AYE exhibits an 86-kb genomic region termed a resistance island--the largest identified to date--in which 45 resistance genes are clustered. At the homologous location, the SDF strain exhibits a 20 kb-genomic island flanked by transposases but devoid of resistance markers. Such a switching genomic structure might be a hotspot that could explain the rapid acquisition of resistance markers under antimicrobial pressure. Sequence similarity and phylogenetic analyses confirm that most of the resistance genes found in the A. baumannii strain AYE have been recently acquired from bacteria of the genera Pseudomonas, Salmonella, or Escherichia. This study also resulted in the discovery of 19 new putative resistance genes. Whole-genome sequencing appears to be a fast and efficient approach to the exhaustive identification of resistance genes in epidemic infectious agents of clinical significance.

We conducted a longitudinal study with 32 nonmusician children over 9 months to determine 1) whether functional differences between musician and nonmusician children reflect specific predispositions for music or result from musical training and 2) whether musical training improves nonmusical brain functions such as reading and linguistic pitch processing. Event-related brain potentials were recorded while 8-year-old children performed tasks designed to test the hypothesis that musical training improves pitch processing not only in music but also in speech. Following the first testing sessions nonmusician children were pseudorandomly assigned to music or to painting training for 6 months and were tested again after training using the same tests. After musical (but not painting) training, children showed enhanced reading and pitch discrimination abilities in speech. Remarkably, 6 months of musical training thus suffices to significantly improve behavior and to influence the development of neural processes as reflected in specific pattern of brain waves. These results reveal positive transfer from music to speech and highlight the influence of musical training. Finally, they demonstrate brain plasticity in showing that relatively short periods of training have strong consequences on the functional organization of the children's brain.

Antibiotic resistance mechanisms reported in Gram-negative bacteria are causing a worldwide health problem. The continuous dissemination of 'multidrug-resistant' (MDR) bacteria drastically reduces the efficacy of our antibiotic 'arsenal' and consequently increases the frequency of therapeutic failure. In MDR bacteria, the overexpression of efflux pumps that expel structurally unrelated drugs contributes to the reduced susceptibility by decreasing the intracellular concentration of antibiotics. During the last decade, several clinical data have indicated an increasing involvement of efflux pumps in the emergence and dissemination of resistant Gram-negative bacteria. It is necessary to clearly define the molecular, functional and genetic bases of the efflux pump in order to understand the translocation of antibiotic molecules through the efflux transporter. The recent investigation on the efflux pump AcrB at its structural and physiological levels, including the identification of drug affinity sites and kinetic parameters for various antibiotics, may pave the way towards the rational development of an improved new generation of antibacterial agents as well as efflux inhibitors in order to efficiently combat efflux-based resistance mechanisms.

Natural killer (NK) cells are lymphocytes of the innate immune system that are involved in the early defenses against foreign cells, as well as autologous cells undergoing various forms of stress, such as microbial infection or tumor transformation. NK cell activation is controlled by a dynamic balance between complementary and antagonistic pathways that are initiated upon interaction with potential target cells. NK cells express an array of activating cell surface receptors that can trigger cytolytic programs, as well as cytokine or chemokine secretion. Some of these activating cell surface receptors initiate protein tyrosine kinase (PTK)-dependent pathways through noncovalent associations with transmembrane signaling adaptors that harbor intracytoplasmic ITAMs (immunoreceptor tyrosine-based activation motifs). Additional cell surface receptors that are not directly coupled to ITAMs also participate in NK cell activation. These include NKG2D, which is noncovalently associated to the DAP10 transmembrane signaling adaptor, as well as integrins and cytokine receptors. NK cells also express cell surface inhibitory receptors that antagonize activating pathways through protein tyrosine phosphatases (PTPs). These inhibitory cell surface receptors are characterized by intracytoplasmic ITIMs (immunoreceptor tyrosine-based inhibition motifs). The tyrosine-phosphorylation status of several signaling components that are substrates for both PTKs and PTPs is thus key to the propagation of the NK cell effector pathways. Understanding the integration of these multiple signals is central to the understanding and manipulation of NK cell effector signaling pathways.