In vitro generation of mature, naive antigen-specific CD8(+) T cells with a single T-cell receptor by agonist selection.

Peripheral blood T cells transduced with a tumor-specific T-cell receptor (TCR) face problems of auto-reactivity and lack of efficacy caused by cross-pairing of exogenous and endogenous TCR chains, as well as short term in vivo survival due to activation and growth factor-induced differentiation. We here studied an alternative strategy for the efficient generation of naive CD8(+) T cells with a single TCR. TCR-transduced human postnatal thymus-derived and adult mobilized blood-derived hematopoietic progenitor cells (HPCs) were differentiated to CD4(+)CD8(+) double-positive T cells using OP9-Delta-like 1 (OP9-DL1) cultures. Addition of the agonist peptide induced double positive cells to cross-present the peptide, leading, in the absence of co-stimulation, to cell cycle arrest and differentiation into mature CD8(+) T cells. Comprehensive phenotypic, molecular and functional analysis revealed the generation of naive and resting CD8(+) T cells through a process similar to thymic positive selection. These mature T cells show a near complete inhibition of endogenous TCRA and TCRB rearrangements and express high levels of the introduced multimer-reactive TCR. Upon activation, specific cytokine production and efficient killing of tumor cells were induced. Using this strategy, large numbers of high-avidity tumor-specific naive T cells can be generated from readily available HPCs without TCR chain cross-pairing.

Identification of Stenotrophomonas maltophilia strains isolated from environmental and clinical samples: a rapid and efficient procedure.

AIMS: Aim of the study is to identify accurately Stenotrophomonas maltophilia isolates recovered from environmental and clinical samples. METHODS AND RESULTS: Recovery of Sten. maltophilia-like isolates from soil samples using the vancomycin, imipenem, amphotericin B (VIA) selective agar medium enabled distinction of various morphotype colonies. A set of soil and clinical isolates was tested for species identification using different methods. 16S rDNA analyses showed the dark green with a blue halo morphotype to be typical Sten. maltophilia strains. The API-20NE, Vitek-2 and Biolog phenotypic analyses typically used for the identification of clinical isolates did not perform well on these soil isolates. The species-specific PCR screening targeting Sten. maltophilia 23S rDNA and the multiplex smeD/ggpS PCR, differentiating Sten. maltophilia from Stenotrophomonas rhizophila, were tested for improvement of these identification schemes. The latter multiplex PCR identified all isolates tested in this study, whatever be their origin. CONCLUSIONS: Isolation on VIA medium and confirmation of Sten. maltophilia species membership by smeD PCR is proposed to identify environmental and clinical isolates of Sten. maltophilia. SIGNIFICANCE AND IMPACT OF THE STUDY: The proposed approach enables isolation and identification of Sten. maltophilia from different environments in an easy and rapid way. This approach will be useful to accurately manage studies on the abundance and distribution of Sten. maltophilia in hospital and nonhospital environments.

Study of the time course of the clinical effect of propofol compared with the time course of the predicted effect-site concentration: Performance of three pharmacokinetic-dynamic models.

BACKGROUND: In the ideal pharmacokinetic-dynamic (PK-PD) model for calculating the predicted effect-site concentration of propofol (Ce(PROP)), for any Ce(PROP), the corresponding hypnotic effect should be constant. We compared three PK-PD models (Marsh PK with Shüttler PD, Schnider PK with fixed ke0, and Schnider PK with Minto PD) in their ability to maintain a constant bispectral index (BIS), while using the respective effect-site-controlled target-controlled infusion (TCI) algorithms. METHODS: We randomized 60 patients to Group M (Marsh's model with k(e0)=0.26 min(-1)), Group S1 or Group S2 (Schnider's model with a fixed k(e0)=0.456 min(-1) or a k(e0) adapted to a fixed time-to-peak effect=1.6 min, respectively). All patients received propofol at a constant rate until loss of consciousness. The corresponding Ce(PROP), as calculated by the respective models, was set as a target for effect-site-controlled TCI. We observed BIS for 20 min. We hypothesized that BIS remains constant, if Ce(PROP) remains constant over time. RESULTS: All patients in Group M woke up, one in Group S1 and none in Group S2. In Groups S1 and S2, BIS remained constant after 11 min of constant Ce(PROP), at a more pronounced level of hypnotic drug effect than intended. CONCLUSIONS: Targeting Ce(PROP) at which patients lose consciousness with effect-site-controlled TCI does not translate into an immediate constant effect.

Distribution and genetic diversity of bacterial thiopurine methyltransferases in soils emitting dimethyl selenide.

Dimethyl selenide (DMSe) and dimethyl diselenide (DMDSe) emissions by soil samples spiked with selenite or (methyl)selenocysteine, with or without a supplement of nutrient broth and glucose were measured. DMSe was the main form of volatile Se produced, and was observed for both Se-substrates. DMDSe was only emitted from soils spiked with (methyl)selenocysteine. Two bacterial thiopurine methyltransferases (TPMTs), TPMT-I and TPMT-E, have been reported to be involved in DMSe and DMDSe emissions [J. Bacteriol. 184 (2002) 3146; Appl. Environ. Microbiol. 69 (2003) 3784]. To establish if these TPMTs or other members of their gene family could have contributed to the DMSe emissions observed, the diversity of bTPMT gene (tpm) sequences among the soils of this study was investigated. Total DNAs from these soils were extracted and screened using the tpm PTCF2-PTCR2 consensus primers defined to PCR amplify this gene family. The PCR products obtained from two soils were cloned, analysed by PCR-RFLP, and sequenced. Their analysis showed an important diversity of tpm lineages (around 12) in soils. Phylogenetic analysis of the deduced TPMT sequences of these soils revealed lineages not previously recorded in the databases, sequences closely related or identical to freshwater TPMTs, or sequences encoding TPMTs closely related to those of Pseudomonas fragi TPMT-K, Pseudomonas Hsa.28 TPMT-I, or Colwellia psychrerythraea TPMT-Z. Nested PCRs, allowing detection of about 13 distinct tpm soil and freshwater lineages by PTCF2-PTCR2 PCR screenings, were performed on the soil total DNAs. These PCRs confirmed the sequencing data, and allowed to recover lineages not detected by the cloning strategy. These results indicate that soils, like the freshwater samples, harbour TPMT-I gene sequences but may also have distinct tpm lineages. This study further supports our hypothesis that TPMTs contribute to DMSe soil emissions.

Freshwater selenium-methylating bacterial thiopurine methyltransferases: diversity and molecular phylogeny.

The diversity of bacterial thiopurine methyltransferases (bTPMT) among five natural Se-methylating freshwaters was investigated by polymerase chain reaction (PCR) screenings and sequencings. DNA sequence analyses confirmed the cloned products' identity and revealed a broad diversity of freshwater TPMTs. Neighbour-joining (NJ) phylogenetic analyses combining these sequences, all GenBank entries closely related to these sequences and deduced TPMTs obtained in this work from selected gamma-proteobacteria showed TPMTs to form a distinct radiation, closely related to UbiG methyltransferases. Inside the TPMT phylogenetic cluster, eukaryote sequences diverged early from the bacterial ones, and all the bacterial database entries belonged to a subgroup of gamma-proteobacteria, with an apparent lateral transfer of a particular allele to beta-proteobacteria of Bordetella. The NJ phylogenetic tree revealed 22 bTPMT lineages, 10 of which harboured freshwater sequences. All lineages showed deep and long branches indicative of major genetic drifts outside regions encoding highly conserved domains. Selected residues among these highly variable domains could reflect adaptations for particular ecological niches. PCR lineage-specific primers differentiated Se-methylating freshwaters according to their 'tpm lineage' signatures. Most freshwater tpm alleles were found to be distinct from those available in the databases, but a group of tpm was found encoding TPMTs identical to an Aeromonas veronii TPMT characterized in this work.

Klebsiella pneumoniae capsule expression is necessary for colonization of large intestines of streptomycin-treated mice.

The role of the Klebsiella pneumoniae capsular polysaccharide (K antigen) during colonization of the mouse large intestine was assessed with wild-type K. pneumoniae LM21 and its isogenic capsule-defective mutant. When bacterial strains were fed alone to mice, the capsulated bacteria persisted in the intestinal tract at levels of 10(8) CFU/g of feces while the capsule-defective strain colonized at low levels, 10(4) CFU/g of feces. In mixed-infection experiments, the mutant was rapidly outcompeted by the wild type. In situ hybridization on colonic sections revealed that bacterial cells of both strains were evenly distributed in the mucus layer at day 1 after infection, while at day 20 the wild type remained dispersed and the capsule-defective strain was seen in clusters in the mucus layer. These results suggest that capsular polysaccharide plays an important role in the gut colonization ability of K. pneumoniae.

Characterization of enteroaggregative Escherichia coli isolates.

Forty enteraggregative Escherichia coli (EAggEC) previously characterized by their ability to adhere to HEp-2 cells or/and their hybridization with the 1-kb EAggEC DNA probe were investigated for the presence of adherence factors and heat-stable enterotoxin (EAST1)-encoding genes. Only 45% of the isolates harbored the EAST1-encoding genes as detected by polymerase chain reaction. None of them hybridized with an AAF/II-encoding gene specific DNA probe and 35% (14/40) were positive in a PCR assay using primers specific for aggC, an accessory gene of the AAF/I-encoding operon. Cloning and sequence analysis of the aggA variant from one isolate, EAggEC 457, revealed 68.9% identity between its deduced amino acid sequence and those of the aggA product from the AAF/I-producing reference strain, E. coli 17.2. No major protein subunit was detected at the surface of EAggEC 457 compared to the bacterial surface extract of E. coli 17.2.

Consequences of reduction of Klebsiella pneumoniae capsule expression on interactions of this bacterium with epithelial cells.

Most Klebsiella pneumoniae clinical isolates are fully encapsulated and adhere in vitro to intestinal cell lines with an aggregative pattern. In this study, the influence of the capsule on interactions with epithelial cells was investigated by creating an isogenic mutant defective in the synthesis of the capsule. Determination of the uronic acid content of bacterial extracts confirmed that the mutant did not produce capsular polysaccharides whereas, with the wild-type strain, the level of encapsulation was growth phase dependent and reached a maximum during the lag and early log phases. Assays performed with different epithelial cell lines, Int-407, A-549, and HEp-2, showed that the capsule-defective mutant demonstrated greater adhesion than did the wild-type strain and that the aggregative pattern was maintained, indicating that the capsule was not related to the adhesion phenotype. In contrast, when the mucus-producing HT-29-MTX cells were used, the encapsulated wild-type strain adhered more strongly than did the capsule-defective mutant. No invasion properties were observed with any of the capsular phenotypes or cell lines used. The K. pneumoniae adhesin CF29K was detected by Western blot analysis and enzyme-linked immunosorbent assay on the surface of transconjugants obtained after transfer of a conjugative plasmid harboring the CF29K-encoding genes into both the wild-type and the capsule-defective strains. The amounts of adhesin detected were greater in the capsule-defective background strain than in the wild-type encapsulated strain and were associated with an increase in the level of adhesion to Caco-2 cells. Moreover, RNA slot blot experiments showed that transcription of the adhesin-encoding gene was markedly increased in the capsule-defective mutant compared to the wild-type encapsulated background. These results suggest (i) that the capsule plays an active role during the initial steps of the pathogenesis by interacting with mucus-producing cells but is subsequently not required for the adhesin-related interaction with the epithelial cell surface and (ii) that the expression of the adhesin is modulated by the presence of a capsule at a transcriptional level.

Inhibitory effect of roxithromycin on adhesion of Klebsiella pneumoniae strains 3051, CF504 and LM21.

The effect of subinhibitory concentrations of roxithromycin on the adhesion of three strains of Klebsiella pneumoniae to Int-407 cells was studied. Adherence was markedly inhibited and the effect was increased when roxithromycin was added to the cell culture medium rather than to the bacterial growth medium. Several assays were performed in order to understand the mechanism by which roxithromycin exerted this inhibitory effect. The greatest effect was obtained when roxithromycin was concentrated in the extracellular compartment; when roxithromycin was concentrated in the intracellular compartment, the inhibitory effect was reduced. The analysis of adhesion factors of bacteria showed that exposure to roxithromycin did not alter their apparent structure or quantity. Roxithromycin appears to interfere in the interaction between bacteria and eukaryotic receptors.

Enteroadherent Escherichia coli and diarrhea in children: a prospective case-control study.

The relative contribution of diarrheagenic Escherichia coli was examined during a 1-year prospective study of hospitalized children in Clermont-Ferrand, France, including 220 case patients (with diarrhea) and 211 matched controls. Fecal isolates were characterized by means of their pattern of adherence to HEp-2 cells and by colony hybridization with DNA probes specific for the six categories of diarrheagenic E. coli. No enteroinvasive or enterotoxigenic E. coli isolates were isolated. Twenty-eight (6.5%) eae-positive isolates and 39 (9%) enteroaggregative E. coli isolates characterized with the aggregative adherence probe and/or by their adherence pattern were detected; they were equally distributed among the patients and the controls. Diffusely adhering E. coli was the predominant pathotype: 30.7% were detected by their adherence pattern and 13.7% were detected with the daaC probe. They were isolated with similar frequencies from the patients and the controls, thereby showing no association with diarrhea. However, daaC-positive strains were significantly associated with a past record of urinary tract infections. These results suggest that the diffusely adhering E. coli organisms isolated in the present study are not true intestinal pathogens but may be regarded as resident colonic strains.

Aggregative adherence of Klebsiella pneumoniae to human intestine-407 cells.

Aggregative adhesion of Klebsiella pneumoniae LM3 to Intestine-407 (Int-407) cells was studied. Adhesive capacities were affected by the bacterial growth phase (with a maximum of adherence obtained during the exponential phase), temperature, multiplicity of infection, and length of incubation with Int-407 cells. Adhesion occurred through a cytochalasin D-sensitive process and was greatly reduced after treatment of Int-407 with cycloheximide, indicating that aggregative adhesion requires active participation of Int-407 cells. Transmission electron microscopy revealed that adherent bacteria were surrounded by a capsule-like material, apparently involved in both bacterium-Int-407 cell and bacterium-bacterium adherence. Examination with a scanning electron microscope showed interactions of intestinal cell microvilli with bacteria and formation in 3 h of a fibrous network within and around the bacterial clusters. We speculate that aggregative adhesion of K. pneumoniae mediated by a capsule-like extracellular material might explain the persistence of these strains inside the host gastrointestinal tract.