CD133+ brain tumor-initiating cells are dependent on STAT3 signaling to drive medulloblastoma recurrence.

Medulloblastoma (MB), the most common malignant paediatric brain tumor, is currently treated using a combination of surgery, craniospinal radiotherapy and chemotherapy. Owing to MB stem cells (MBSCs), a subset of MB patients remains untreatable despite standard therapy. CD133 is used to identify MBSCs although its functional role in tumorigenesis has yet to be determined. In this work, we showed enrichment of CD133 in Group 3 MB is associated with increased rate of metastasis and poor clinical outcome. The signal transducers and activators of transcription-3 (STAT3) pathway are selectively activated in CD133+ MBSCs and promote tumorigenesis through regulation of c-MYC, a key genetic driver of Group 3 MB. We screened compound libraries for STAT3 inhibitors and treatment with the selected STAT3 inhibitors resulted in tumor size reduction in vivo. We propose that inhibition of STAT3 signaling in MBSCs may represent a potential therapeutic strategy to treat patients with recurrent MB.

Description of an original integron encompassing blaVIM-2, qnrVC1 and genes encoding bacterial group II intron proteins in Pseudomonas aeruginosa.

OBJECTIVES: A burn unit of a hospital in Tunis underwent an endemic situation caused by imipenem-resistant Pseudomonas aeruginosa. For nine non-repetitive isolates of a clonal VIM-2-producing strain, the blaVIM-2 genetic background was characterized and the associated qnrVC1 gene molecularly analysed. METHODS: The imipenem resistance mechanism was investigated by phenotypic and molecular tests, and resistance transfer was studied by conjugation and transformation experiments. The integron's structure was characterized by sequencing, and qnrVC1 expression was explored after cloning experiments. RESULTS: The nine VIM-2-producing strains were collected from eight patients and one environmental sample. All transfer assays failed, suggesting a chromosomal location of blaVIM-2. This latter was found to be part of a class 1 integron of ∼7500 bp, which also contains blaOXA-2, aadA1 and two copies of the aadB, arr-6 and qnrVC1 genes. qnrVC1 exhibited higher homology with the chromosomally encoded qnr genes of Vibrionaceae than with plasmid-mediated qnr genes of Enterobacteriaceae. The qnrVC1 gene cassette possesses a promoter allowing its expression, and it conferred decreased fluoroquinolone susceptibility to Escherichia coli. Additionally, on the same integron, genes encoding an uncommon group IIC-attC intron were detected. CONCLUSIONS: A VIM-2-producing P. aeruginosa outbreak led us to characterize an integron harbouring a qnrVC1 cassette and a new group IIC-attC intron. This is the first known description of a qnr determinant in a P. aeruginosa strain. Its presence conferred a low level of resistance to quinolones in E. coli, which might favour the emergence of highly resistant mutants.

Predicting pathogen-specific CD8 T cell immune responses from a modeling approach.

The primary CD8 T cell immune response constitutes a major mechanism to fight an infection by intra-cellular pathogens. We aim at assessing whether pathogen-specific dynamical parameters of the CD8 T cell response can be identified, based on measurements of CD8 T cell counts, using a modeling approach. We generated experimental data consisting in CD8 T cell counts kinetics during the response to three different live intra-cellular pathogens: two viruses (influenza, vaccinia) injected intranasally, and one bacteria (Listeria monocytogenes) injected intravenously. All pathogens harbor the same antigen (NP68), but differ in their interaction with the host. In parallel, we developed a mathematical model describing the evolution of CD8 T cell counts and pathogen amount during an immune response. This model is characterized by 9 parameters and includes relevant feedback controls. The model outputs were compared with the three data series and an exhaustive estimation of the parameter values was performed. By focusing on the ability of the model to fit experimental data and to produce a CD8 T cell population mainly composed of memory cells at the end of the response, critical parameters were identified. We show that a small number of parameters (2-4) define the main features of the CD8 T cell immune response and are characteristic of a given pathogen. Among these parameters, two are related to the effector CD8 T cell mediated control of cell and pathogen death. The parameter associated with memory cell death is shown to play no relevant role during the main phases of the CD8 T cell response, yet it becomes essential when looking at the predictions of the model several months after the infection.

Evolution of an incompatibility group IncA/C plasmid harboring blaCMY-16 and qnrA6 genes and its transfer through three clones of Providencia stuartii during a two-year outbreak in a Tunisian burn unit.

During a 2-year period in 2005 and 2006, 64 multidrug-resistant Providencia stuartii isolates, including 58 strains from 58 patients and 6 strains obtained from the same tracheal aspirator, were collected in a burn unit of a Tunisian hospital. They divided into four antibiotypes (ATB1 to ATB4) and three SmaI pulsotypes (PsA to PsC), including 49 strains belonging to clone PsA (48 of ATB1 and 1 of ATB4), 11 strains to clone PsB (7 of ATB2 and 4 of ATB3), and 4 strains to clone PsC (ATB3). All strains, except for the PsA/ATB4 isolate, were highly resistant to broad-spectrum cephalosporins due to the production of the plasmid-mediated CMY-16 ß-lactamase. In addition, the 15 strains of ATB2 and ATB3 exhibited decreased quinolone susceptibility associated with QnrA6. Most strains (ATB1 and ATB3) were gentamicin resistant, related to an AAC(6')-Ib' enzyme. All these genes were located on a conjugative plasmid belonging to the incompatibility group IncA/C(2) of 195, 175, or 100 kb. Despite differences in size and in number of resistance determinants, they derived from the same plasmid, as demonstrated by similar profiles in plasmid restriction analysis and strictly homologous sequences of repAIncA/C(2), unusual antibiotic resistance genes (e.g., aphA-6), and their genetic environments. Further investigation suggested that deletions, acquisition of the ISCR1 insertion sequence, and integron cassette mobility accounted for these variations. Thus, this outbreak was due to both the spread of three clonal strains and the dissemination of a single IncA/C(2) plasmid which underwent a remarkable evolution during the epidemic period.

Nationwide survey of extended-spectrum {beta}-lactamase-producing Enterobacteriaceae in the French community setting.

OBJECTIVES: The aim of this study was to assess the prevalence of the extended-spectrum beta-lactamase (ESBL)-producing enterobacteria (ESBLE) in the French community, during a 2006 survey. METHODS: All enterobacteria isolated from urine samples of patients, exhibiting a decreased susceptibility to broad-spectrum cephalosporins, were analysed for their beta-lactamase content (synergy test, isoelectrofocusing, conjugation transfer, PCR amplification and/or cloning experiments and sequencing). Additional co-resistances were investigated by PCR, sequencing and/or cloning. Epidemiological relationship was studied by PFGE for all species and, in addition, for Escherichia coli by the determination of the phylogenetic group, multilocus sequence type (ST) and O25b antigen. Characteristics of CTX-M-producing E. coli carriers were compared with other ESBLE carriers. RESULTS: Seventy-two ESBLE were collected from 71 patients. Most of them expressed a CTX-M enzyme (n = 42, comprising 40 E. coli), with a predominance of CTX-M-15 (n = 24); 10 CTX-M-15-producing E. coli belonged to the same clone (phylogroup B2, ST131, serotype O25b). The 30 remaining strains possessed a TEM- or SHV-type ESBL. In addition, three strains presented unusual co-resistances such as DHA-1 (n = 2), QnrB4 and ArmA. Risk factors for ESBLE acquisition were substantially less frequent when the ESBL was of the CTX-M type, except for prior antimicrobial therapy. Eighteen percent of the patients were considered to have true community-acquired ESBLE; most of them harboured a CTX-M-producing E. coli. CONCLUSIONS: This first nationwide study reports an ESBLE prevalence of 1.1% in the French community setting in 2006, mainly related to the presence of CTX-M-producing E. coli strains; furthermore, unusual co-resistances rarely found in the community setting were occasionally observed, which may threaten future emergence.

Loss of p53 or p73 in human papillomavirus type 38 E6 and E7 transgenic mice partially restores the UV-activated cell cycle checkpoints.

We have previously shown that human keratinocytes expressing E6 and E7 from the cutaneous human papillomavirus (HPV) type 38 have high levels of a specific form of p53, which in turn activate the transcription of DeltaNp73 gene. Expression of HPV38 E6 and E7 in mouse skin also promotes p53 and DeltaNp73 accumulation. Interestingly, keratinocytes of these mice do not undergo cell cycle arrest after skin ultraviolet (UV) irradiation. Here, we provide several lines of evidence that DeltaNp73 expression and lack of the UV response are directly linked. Loss of p53 gene in HPV38 E6/E7 transgenic mice abolished DeltaNp73 expression and partially restored the UV-activated cell cycle checkpoints. Similarly, loss of p73, and consequently DeltaNp73, led to restoration of the p53 pathways. In fact, keratinocytes of p73-/- HPV38 E6/E7 transgenic mice upon UV irradiation express high levels of p21(WAF1) and are cell cycle arrested. Thus, HPV38 E6 and E7, via DeltaNp73 accumulation, are able to alter the regulation of cell cycle checkpoints activated by UV radiation. These data suggest that UV and HPV may cooperate in skin carcinogenesis.

Differential expression of a human endogenous retrovirus E transmembrane envelope glycoprotein in normal, psoriatic and atopic dermatitis human skin.

BACKGROUND: Psoriasis is a common inflammatory skin disease characterized by uncontrolled proliferation of keratinocytes and recruitment of T lymphocytes into the skin. The possible role of human endogenous retroviruses (HERVs) in the induction of psoriasis has been suggested, based upon the previous observations of retrovirus-like particles in psoriasis from skin lesional plaques, urine and stimulated lymphocytes. OBJECTIVES: To investigate the expression of HERV-E transmembrane envelope glycoprotein (HERV-E env) in normal, psoriatic and atopic human skin, and to examine the influence of ultraviolet (UV) B irradiation on HERV-E env expression in normal human epidermal keratinocytes. METHODS: The analysis was performed on both skin biopsies and organotypic skin cultures using immunofluorescence and Western immunoblotting. UVB irradiation (312 nm) of cultured normal human keratinocytes was performed using a dose of 30 mJ cm(-2). RESULTS: Positive staining was observed in most of the psoriatic and atopic skin samples, whereas only 15% of the normal skin samples were faintly positive. In addition, the pattern of expression of HERV-E env differed markedly in psoriasis vs. atopy. By Western blotting analysis, two main proteins of 54 and 57 kDa were detected in extracts of normal skin, normal keratinocyte cultures and reconstructed epidermis from psoriatic and normal punch biopsies. An increased level of expression of these proteins was noted in extracts from psoriatic vs. normal reconstructed epidermis. The overexpression of the 57-kDa protein in normal human cultured keratinocytes was dramatically reduced by UVB irradiation. CONCLUSIONS: These data suggest for the first time that HERV-E env is expressed in normal and pathological human skin. Further studies are now required to elucidate the role of such viral proteins in the pathogenesis of psoriasis.

Antibiotic resistance rates and phenotypes among isolates of Enterobacteriaceae in French extra-hospital practice.

Antibiotic resistance among members of the family Enterobacteriaceae was prospectively surveyed by eight French private laboratories over a 5-month period in 1999. A total of 2,599 consecutive and nonduplicate strains were collected, mainly (60.9%) from patients in the community. Most strains (82.9%) derived from urine. Escherichia coli was the predominant (73.9%) organism isolated. The overall rates of antibiotic resistance were as follows: amoxicillin, 53.4%; amoxicillin-clavulanic acid, 27.3%; ticarcillin, 44.2%; piperacillin-tazobactam, 3.2%; cephalothin, 29.2%; cefuroxime, 14.7%; cefoxitin, 11.5%; ceftazidime, 3.6%; cefotaxime, 2.8%; cefepime, 0.3%; imipenem, 0.1%; gentamicin (G), 3.8%; tobramycin (T), 5.0%; netilmicin (Nt), 3.7%; amikacin (A), 0.7%; nalidixic acid, 14.3%; ofloxacin, 10.4%; cotrimoxazole, 21.1%; nitrofurantoin, 12.7%; fosfomycin, 5.2%; tetracycline, 50.1%; and colistin, 12.5%. Beta-lactam resistance phenotypes essentially comprised penicillinase production (33.9%), overexpression of chromosomal cephalosporinase (4.6%), and synthesis of inhibitor-resistant TEM/OXA enzymes (1.5%) or extended-spectrum beta-lactamases (1.5%). Aminoglycoside resistance phenotypes consisted of GTNt (93 strains), TNtA (68 strains), GTNtA (14 strains), T (4 strains), GT (3 strains), G (1 strain), and reduced uptake/permeability (3 strains). Most of the nalidixic acid-resistant strains were resistant to ofloxacin (72.8%). Antibiotic resistance rates and phenotypes varied widely according to the bacterial group and the source of the strains. Significantly higher rates were observed in private healthcare centers than in the community, due to a higher proportion of both resistant species and resistant strains. However, multidrug-resistant isolates, including five extended-spectrum beta-lactamase-producing strains, were also recovered from the community.

SHV-16, a beta-lactamase with a pentapeptide duplication in the omega loop.

A clinical isolate of Klebsiella pneumoniae was found to be resistant to ampicillin (MIC of 128 microg/ml), ticarcillin (MIC of 512 microg/ml), and ceftazidime (MIC of 128 microg/ml) and susceptible to all other beta-lactams; a synergistic effect between clavulanate and ceftazidime suggested the presence of an extended-spectrum beta-lactamase (ESBL). Transconjugants in Escherichia coli were obtained at low levels (10(-7) per donor cell) and exhibited a similar beta-lactam resistance pattern (resistant to ampicillin, ticarcillin, and ceftazidime at 64 microg/ml). The ESBL, pI 7.6, was encoded by a large plasmid (>100 kb) which did not carry any other resistance determinant. The ESBL-encoding gene was amplified by PCR using bla(SHV)-specific primers and was sequenced. The deduced amino acid sequence of the SHV-16 ESBL showed that it differed from SHV-1 by only a pentapeptide insertion (163DRWET167) corresponding to a tandem duplication in the omega loop. The implication of the 163a-DRWET163b-DRWET sequence in ceftazidime resistance was confirmed by cloning either bla(SHV-1) or bla(SHV-16) in the same vector, subsequently introduced in the same E. coli strain. Under these isogenic conditions, SHV-16 conferred a 32-fold increase in ceftazidime MIC compared to that with SHV-1. Furthermore, site-directed mutagenesis experiments modifying either E166aA or E166bA revealed that the functional glutamic residue was that located in the first copy of the duplicated sequence. But surprisingly, the second E166b also conferred a low-level resistance to ceftazidime. This work is the first description of a class A enzyme exhibiting an extended substrate specificity due to an insertion instead of a nucleotide substitution(s) in a clinical isolate.

Nosocomial outbreak due to a multiresistant strain of Pseudomonas aeruginosa P12: efficacy of cefepime-amikacin therapy and analysis of beta-lactam resistance.

Over a 3-year period, 67 patients of the Hospital of Pau (Pau, France), including 64 patients hospitalized in the adult intensive care unit (ICU), were colonized and/or infected by strains of Pseudomonas aeruginosa P12, resistant to all potentially active antibiotics except colistin. Most patients were mechanically ventilated and presented respiratory tract infections. Since cefepime and amikacin were the least inactive antibiotics by MIC determination, all ICU patients were treated with this combination, and most of them benefited. Cefepime-amikacin was found highly synergistic in vitro. Ribotyping and arbitrary primer-PCR analysis confirmed the presence of a single clonal isolate. Isoelectrofocusing revealed that the epidemic strain produced large amounts of the chromosomal cephalosporinase and an additional enzyme with a pI of 5.7, corresponding to PSE-1, as demonstrated by PCR and sequencing. Outer membrane protein profiles on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the absence of a ca. 46-kDa protein, likely to be OprD, and increased production of two ca. 49- and 50-kDa proteins, consistent with the outer membrane components of the efflux systems, MexAB-OprM and MexEF-OprN. Thus, we report here a nosocomial outbreak due to multiresistant P. aeruginosa P12 exhibiting at least four mechanisms of beta-lactam resistance, i.e., production of the penicillinase PSE-1, overproduction of the chromosomal cephalosporinase, loss of OprD, and overexpression of efflux systems, associated with a better activity of cefepime than ceftazidime.

Involvement of inhibitory NKRs in the survival of a subset of memory-phenotype CD8+ T cells.

Inhibitory natural killer receptors (NKRs) such as killer cell immunoglobulin-like receptors (KIRs) in humans and Ly49 molecules in mice are expressed on NK cells and recognize multiple major histocompatibility (MHC) class I proteins. In humans and mice, a subset of CD8+ T cells also expresses NKRs and harbors a memory phenotype. Using mice that are transgenic for KIR2DL3 and its cognate HLA-Cw3 ligand, we show that engagement of inhibitory NKRs selectively drives the in vivo accumulation of a subset of memory-phenotype CD8+ T cells that express the beta chain of the interleukin 2 receptor. In vitro, recognition of MHC class I molecules by inhibitory NKRs on T cells down-regulated activation-induced cell death. These results unveil an MHC class I-dependent pathway that promotes the survival of a subset of memory-phenotype CD8+ T cells and also reveal an unexpected biological function for inhibitory NKRs on T cells.

Prospective survey of colonization and infection caused by SHV-4 producing Klebsiella pneumoniae in a neurosurgical intensive care unit.

The occurrence of extended-spectrum beta-lactamase producing enterobacteria (ESBLE) has been prospectively surveyed in a neurosurgical intensive care unit (ICU). Of the 47 patients examined, 8 were identified as faecal carriers, and 2 of them developed a subsequent urinary tract infection. ESBLE were also detected in the immediate environment of five colonized and/or infected patients. All isolates were Klebsiella pneumoniae of a particular biotype which exhibited a similar antibiotype and produced an SHV-4 type beta-lactamase. However, plasmid profiling and ribotyping revealed that strains isolated from seven patients of hall A were a single epidemic clone, whereas strains isolated from the eighth patient of hall B were different. Comparison between the characteristics of patients who carried an ESBLE during the surveillance period, and control patients who did not, showed that a recent surgery, and the length of ICU stay were significantly associated with the acquisition of ESBLE.

Impact of an urban effluent on antibiotic resistance of riverine Enterobacteriaceae and Aeromonas spp.

In order to evaluate the impact of an urban effluent on antibiotic resistance of freshwater bacterial populations, water samples were collected from the Arga river (Spain), upstream and downstream from the wastewater discharge of the city of Pamplona. Strains of Enterobacteriaceae (representative of the human and animal commensal flora) (110 isolates) and Aeromonas (typically waterborne bacteria) (118 isolates) were selected for antibiotic susceptibility testing. Most of the Aeromonas strains (72%) and many of the Enterobacteriaceae (20%) were resistant to nalidixic acid. Singly nalidixic acid-resistant strains were frequent regardless of the sampling site for Aeromonas, whereas they were more common upstream from the discharge for enterobacteria. The most common resistances to antibiotics other than quinolones were to tetracycline (24.3%) and beta-lactams (20.5%) for Enterobacteriaceae and to tetracycline (27.5%) and co-trimoxazole (26.6%) for Aeromonas. The rates of these antibiotic resistances increased downstream from the discharge at similar degrees for the two bacterial groups; it remained at high levels for enterobacteria but decreased along the 30-km study zone for Aeromonas. Genetic analysis of representative strains demonstrated that these resistances were mostly (enterobacteria) or exclusively (Aeromonas) chromosomally mediated. Moreover, a reference strain of Aeromonas caviae (CIP 7616) could not be transformed with conjugative R plasmids of enterobacteria. Thus, the urban effluent resulted in an increase of the rates of resistance to antibiotics other than quinolones in the riverine bacterial populations, despite limited genetic exchanges between enterobacteria and Aeromonas. Quinolone resistance probably was selected by heavy antibiotic discharges of unknown origin upstream from the urban effluent.

Effects of T3R alpha 1 and T3R alpha 2 gene deletion on T and B lymphocyte development.

Thyroid hormones bind to several nuclear receptors encoded by T3R alpha and T3R beta genes. There is now accumulating evidence that thyroid hormones act on the immune system. Indeed, mice deficient for thyroid hormones show a reduction in lymphocyte production. However, the mechanisms involved and, in particular, the role of the different thyroid hormone receptors in lymphocyte development have not been investigated. To address that question, we have studied lymphocyte development in mice deficient for the T3R alpha 1 and T3R alpha 2 gene products. A strong decrease in spleen cell numbers was found compared with wild-type littermates, B lymphocytes being more severely affected than T lymphocytes. A significant decrease in splenic macrophage and granulocyte numbers was also found. In bone marrow, a reduction in CD45+/IgM- pro/pre-B cell numbers was found in these mice compared with wild-type littermates. This decrease seems to result from a proliferation defect, as CD45+/IgM- cells incorporate less 5-bromo-2'-deoxyuridine in vivo. To define the origin of the bone marrow development defect, chimeric animals between T3R alpha-/- and Rag1-/- mice were generated. Results indicate that for B cells the control of the population size by T3R alpha 1 and T3R alpha 2 is intrinsic. Altogether, these results show that T3R alpha 1 or T3R alpha 2 gene products are implicated in the control of the B cell pool size.

Characterization at the single-cell level of naive and primed CD8 T cell cytokine responses.

The aim of this study was to characterize differences between naive and primed CD8 T cells. Our results show that (i) naive and primed CD8 T cells display similar activation thresholds, with no direct evidence for a difference in their TCR signals, and (ii) primed cells differ mainly in their capacity to secrete IFN-gamma. A comparison of the two populations at the single-cell level demonstrated that the increased production of IFN-gamma by the primed cell subset is due to a larger proportion of single cells that are able to synthesize this cytokine early following activation. These results indicate that the intrinsic effector capabilities of individual CD8 T cells expressing the same TCR are heterogeneous and that cells with identical antigen specificity but increased effector capacities are generated or selected during the primary response.

Molecular characterization of TEM-59 (IRT-17), a novel inhibitor-resistant TEM-derived beta-lactamase in a clinical isolate of Klebsiella oxytoca.

A clinical isolate of Klebsiella oxytoca (Kox 443) was found to have a low-level resistance to broad-spectrum penicillins (MICs of amoxicillin and ticarcillin, 256 and 32 microg/ml, respectively), without substantial potentiation by 2 microg of clavulanic acid per ml (amoxicillin- and ticarcillin-clavulanate, 128 and 8 microg/ml, respectively), while being fully susceptible to cephalosporins and other beta-lactam antibiotics. These resistances were carried by a ca. 50-kb conjugative plasmid that encodes a single beta-lactamase with a pI of 5.6. Compared to TEM-2, this enzyme exhibited a 3- to 30-fold higher Km and a decreased maximal hydrolysis rate for beta-lactams; higher concentrations of suicide inactivators (5- to 500-fold higher concentrations giving a 50% reduction in hydrolysis) were required for inhibition. Nucleotide sequence analysis revealed identity between the blaTEM gene of Kox 443 and the blaTEM-2 gene, except for a single A-to-G change at position 590, leading to the amino acid change from Ser-130 Gly. This mutation has not been reported previously in the TEM type beta-lactamases produced by clinical strains, and the novel enzyme was called TEM-59 (alternative name IRT-17). This is the first description of an inhibitor-resistant TEM-derived enzyme in the species K. oxytoca.