Biofilm production by multiresistant Corynebacterium striatumassociated with nosocomial outbreak

Corynebacterium striatum is a potentially pathogenic microorganism that causes nosocomial outbreaks. However, little is known about its virulence factors that may contribute to healthcare-associated infections (HAIs). We investigated the biofilm production on abiotic surfaces of multidrug-resistant (MDR) and multidrug-susceptible (MDS) strains of C. striatum of pulsed-field gel electrophoresis types I-MDR, II-MDR, III-MDS and IV-MDS isolated during a nosocomial outbreak in Rio de Janeiro, Brazil. The results showed that C. striatum was able to adhere to hydrophilic and hydrophobic abiotic surfaces. The C. striatum1987/I-MDR strain, predominantly isolated from patients undergoing endotracheal intubation procedures, showed the greatest ability to adhere to all surfaces. C. striatumbound fibrinogen to its surface, which contributed to biofilm formation. Scanning electron microscopy showed the production of mature biofilms on polyurethane catheters by all pulsotypes. In conclusion, biofilm production may contribute to the establishment of HAIs caused by C. striatum.

Fab antibodies capable of blocking T cells by competitive binding have the identical specificity but a higher affinity to the MHC-peptide-complex than the T cell receptor.

In transplant rejection, graft versus host or autoimmune diseases T cells are mediating the pathophysiological processes. Compared to unspecific pharmacological immune suppression specific inhibition of those T cells, that are involved in the disease, would be an alternative and attractive approach. T cells are activated after their T cell receptor (TCR) recognizes an antigenic peptide displayed by the Major Histocompatibility Complex (MHC). Molecules that interact with MHC-peptide-complexes in a specific fashion should block T cells with identical specificity. Using the model of the SSX2 (103-111)/HLA-A*0201 complex we investigated a panel of MHC-peptide-specific Fab antibodies for their capacity blocking specific T cell clones. Like TCRs all Fab antibodies reacted with the MHC complex only when the SSX2 (103-111) peptide was displayed. By introducing single amino acid mutations in the HLA-A*0201 heavy chain we identified the K66 residue as the most critical binding similar to that of TCRs. However, some Fab antibodies did not inhibit the reactivity of a specific T cell clone against peptide pulsed, artificial targets, nor cells displaying the peptide after endogenous processing. Measurements of binding kinetics revealed that only those Fab antibodies were capable of blocking T cells that interacted with an affinity in the nanomolar range. Fab antibodies binding like TCRs with affinities on the lower micromolar range did not inhibit T cell reactivity. These results indicate that molecules that block T cells by competitive binding with the TCR must have the same specificity but higher affinity for the MHC-peptide-complex than the TCR.

Four main virotypes among extended-spectrum-β-lactamase-producing isolates of Escherichia coli O25b:H4-B2-ST131: bacterial, epidemiological, and clinical characteristics.

A total of 1,021 extended-spectrum-β-lactamase-producing Escherichia coli (ESBLEC) isolates obtained in 2006 during a Spanish national survey conducted in 44 hospitals were analyzed for the presence of the O25b:H4-B2-ST131 (sequence type 131) clonal group. Overall, 195 (19%) O25b-ST131 isolates were detected, with prevalence rates ranging from 0% to 52% per hospital. Molecular characterization of 130 representative O25b-ST131 isolates showed that 96 (74%) were positive for CTX-M-15, 15 (12%) for CTX-M-14, 9 (7%) for SHV-12, 6 (5%) for CTX-M-9, 5 (4%) for CTX-M-32, and 1 (0.7%) each for CTX-M-3 and the new ESBL enzyme CTX-M-103. The 130 O25b-ST131 isolates exhibited relatively high virulence scores (mean, 14.4 virulence genes). Although the virulence profiles of the O25b-ST131 isolates were fairly homogeneous, they could be classified into four main virotypes based on the presence or absence of four distinctive virulence genes: virotypes A (22%) (afa FM955459 positive, iroN negative, ibeA negative, sat positive or negative), B (31%) (afa FM955459 negative, iroN positive, ibeA negative, sat positive or negative), C (32%) (afa FM955459 negative, iroN negative, ibeA negative, sat positive), and D (13%) (afa FM955459 negative, iroN positive or negative, ibeA positive, sat positive or negative). The four virotypes were also identified in other countries, with virotype C being overrepresented internationally. Correspondingly, an analysis of XbaI macrorestriction profiles revealed four major clusters, which were largely virotype specific. Certain epidemiological and clinical features corresponded with the virotype. Statistically significant virotype-specific associations included, for virotype B, older age and a lower frequency of infection (versus colonization), for virotype C, a higher frequency of infection, and for virotype D, younger age and community-acquired infections. In isolates of the O25b:H4-B2-ST131 clonal group, these findings uniquely define four main virotypes, which are internationally distributed, correspond with pulsed-field gel electrophoresis (PFGE) profiles, and exhibit distinctive clinical-epidemiological associations.

Usefulness of double locus sequence typing (DLST) for regional and international epidemiological surveillance of methicilin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of nosocomial infections worldwide. To differentiate reliably among S. aureus isolates, we recently developed double locus sequence typing (DLST) based on the analysis of partial sequences of clfB and spa genes. In the present study, we evaluated the usefulness of DLST for epidemiological investigations of MRSA by routinely typing 1242 strains isolated in Western Switzerland. Additionally, particular local and international collections were typed by pulsed field gel electrophoresis (PFGE) and DLST to check the compatibility of DLST with the results obtained by PFGE, and for international comparisons. Using DLST, we identified the major MRSA clones of Western Switzerland, and demonstrated the close relationship between local and international clones. The congruence of 88% between the major PFGE and DLST clones indicated that our results obtained by DLST were compatible with earlier results obtained by PFGE. DLST could thus easily be incorporated in a routine surveillance procedure. In addition, the unambiguous definition of DLST types makes this method more suitable than PFGE for long-term epidemiological surveillance. Finally, the comparison of the results obtained by DLST, multilocus sequence typing, PFGE, Staphylococcal cassette chromosome mec typing and the detection of Panton-Valentine leukocidin genes indicated that no typing scheme should be used on its own. It is only the combination of data from different methods that gives the best chance of describing precisely the epidemiology and phylogeny of MRSA.

Caracterització radiològica i dosimètrica ambiental d'una planta de producció de fosfat bicàlcic

The production of dicalcium phosphate are included in the list of industries classified as NORM in Euratom 29/96. The aim of this study is to determine the con-centrations of specific flows and their variability over time of 226Ra, 210Pb and 210Po in the inputs and out-puts of the production process. Also classified areas of the plant and the workers according to the radio-logical risk and radiation protection measures have been proposed. The results show that the rock phos-phate has a high specific activity of the 226Rn, 210Po and 210Pb in secular equilibrium (1500-2000 Bq • kg-1) but the outputs of the process will distort the secu-lar equilibrium. The only shortfall is the flow balance of 226Ra, which accumulates in the process. The dis-tribution of the dose in the plant concentrates on the area of reactor tanks and slop pipes as regards exter-nal irradiation dose and the grinding zone, the area of packaging and loading area so respects dose inhaled. We propose a signaling areas, cleaning and replace-ment of old equipment in the facilities and radiological safety of the maintenance staff.

Establishment of animal models to analyze the kinetics and distribution of human tumor antigen-specific CD8⁺ T cells.

Many patients develop tumor antigen-specific T cell responses detectable in peripheral blood mononuclear cells (PBMCs) following cancer vaccine. However, measurable tumor regression is observed in a limited number of patients receiving cancer vaccines. There is a need to re-evaluate systemically the immune responses induced by cancer vaccines. Here, we established animal models targeting two human cancer/testis antigens, NY-ESO-1 and MAGE-A4. Cytotoxic T lymphocyte (CTL) epitopes of these antigens were investigated by immunizing BALB/c mice with plasmids encoding the entire sequences of NY-ESO-1 or MAGE-A4. CD8(+) T cells specific for NY-ESO-1 or MAGE-A4 were able to be detected by ELISPOT assays using antigen presenting cells pulsed with overlapping peptides covering the whole protein, indicating the high immunogenicity of these antigens in mice. Truncation of these peptides revealed that NY-ESO-1-specific CD8(+) T cells recognized D(d)-restricted 8mer peptides, NY-ESO-181-88. MAGE-A4-specific CD8(+) T cells recognized D(d)-restricted 9mer peptides, MAGE-A4265-273. MHC/peptide tetramers allowed us to analyze the kinetics and distribution of the antigen-specific immune responses, and we found that stronger antigen-specific CD8(+) T cell responses were required for more effective anti-tumor activity. Taken together, these animal models are valuable for evaluation of immune responses and optimization of the efficacy of cancer vaccines.

Lack of tumor recognition by hTERT peptide 540-548-specific CD8(+) T cells from melanoma patients reveals inefficient antigen processing.

Telomerase is a ribonucleoprotein complex responsible for the maintenance of the length of the telomeres during cell division, which is active in germ-line cells as well as in the vast majority of tumors but not in most normal tissues. The wide expression of the human telomerase catalytic subunit (hTERT) in tumors makes it an interesting candidate vaccine for cancer. hTERT-derived peptide 540-548 (hTERT(540)) has been recently shown to be recognized in an HLA-A*0201-restricted fashion by T cell lines derived from peptide-stimulated peripheral blood mononuclear cells (PBMC) from healthy donors. As a first step to the inclusion of this peptide in immunotherapy clinical trials, it is crucial to assess hTERT(540)-specific T cell reactivity in cancer patients as well as the ability of hTERT-specific CD8(+) T lymphocytes to recognize and lyse hTERT-expressing target cells. Here, we have analyzed the CD8(+) T cell response to peptide hTERT(540) in HLA-A*0201 melanoma patients by using fluorescent HLA-A*0201/hTERT(540) peptide tetramers. HLA-A*0201/hTERT(540) tetramer(+) CD8(+) T cells were readily detected in peptide-stimulated PBMC from a significant proportion of patients and could be isolated by tetramer-guided cell sorting. hTERT(540)-specific CD8(+) T cells were able to specifically recognize HLA-A*0201 cells either pulsed with peptide or transiently transfected with a minigene encoding the minimal epitope. In contrast, they failed to recognize hTERT-expressing HLA-A*0201(+) target cells. Furthermore, in vitro proteasome digestion studies revealed inadequate hTERT processing. Altogether, these results raise questions on the use of hTERT(540) peptide for cancer immunotherapy.

Effect of biomagnetic therapy versus physiotherapy for treatment of knee osteoarthritis: a randomized controlled trial.

OBJECTIVE: To assess the effectiveness of pulsed signal therapy in the treatment of knee osteoarthritis (Kellgren II or III). METHODS: A randomized, double-blind controlled clinical trial. The first 95 patients sent to the clinic with knee osteo-arthritis were selected and randomized into treatment with pulsed signal therapy or conventional physiotherapy. Assessment included recording of usual demographic data, pertinent history, baseline medication and radiographs. Clinical evaluation was made at baseline, 6 weeks and 6 months after the end of treatment by the same blinded doctor. At each follow-up time, the patient was asked to complete a visual analogue pain scale and a Lequesne score. The doctor recorded the degree of pain on motion and the ability to move the affected knee. RESULTS: Both treatments resulted in significant improvements in pain and physical function. A statistical difference was observed only for activities of daily living, where the physiotherapy was more efficient (p<0.03). The cost of treatment with pulsed signal therapy was significantly higher, double the treatment cost of conventional physiotherapy. CONCLUSION: Like physiotherapy, pulsed signal therapy has improved the clinical state of treated patients but with no significant statistical difference. Pulsed signal therapy is, however, more expensive.

Degradation of ZAP-70 following antigenic stimulation in human T lymphocytes: role of calpain proteolytic pathway.

T cell activation by the specific Ag results in dramatic changes of the T cell phenotype that include a rapid and profound down-regulation and degradation of triggered TCRs. In this work, we investigated the fate of the TCR-associated ZAP-70 kinase in Ag-stimulated T cells. T cells stimulated by peptide-pulsed APCs undergo an Ag dose-dependent decrease of the total cellular content of ZAP-70, as detected by FACS analysis and confocal microscopy on fixed and permeabilized T cell-APC conjugates and by Western blot on total cell lysates. The time course of ZAP-70 consumption overlaps with that of zeta-chain degradation, indicating that ZAP-70 is degraded in parallel with TCR internalization and degradation. Pharmacological activation of protein kinase C (PKC) does not induce ZAP-70 degradation, which, on the contrary, requires activation of protein tyrosine kinases. Two lines of evidence indicate that the Ca2+-dependent cysteine protease calpain plays a major role in initiating ZAP-70 degradation: 1) treatment of T cells with cell-permeating inhibitors of calpain markedly reduces ZAP-70 degradation; 2) ZAP-70 is cleaved in vitro by calpain. Our results show that, in the course of T cell-APC cognate interaction, ZAP-70 is rapidly degraded via a calpain-dependent mechanism.

A nonapeptide encoded by human gene MAGE-1 is recognized on HLA-A1 by cytolytic T lymphocytes directed against tumor antigen MZ2-E.

We have reported the identification of human gene MAGE-1, which directs the expression of an antigen recognized on a melanoma by autologous cytolytic T lymphocytes (CTL). We show here that CTL directed against this antigen, which was named MZ2-E, recognize a nonapeptide encoded by the third exon of gene MAGE-1. The CTL also recognize this peptide when it is presented by mouse cells transfected with an HLA-A1 gene, confirming the association of antigen MZ2-E with the HLA-A1 molecule. Other members of the MAGE gene family do not code for the same peptide, suggesting that only MAGE-1 produces the antigen recognized by the anti-MZ2-E CTL. Our results open the possibility of immunizing HLA-A1 patients whose tumor expresses MAGE-1 either with the antigenic peptide or with autologous antigen-presenting cells pulsed with the peptide.

Feasibility and electromagnetic compatibility study of the ClearPEM front-end electronics for simultaneous PET-MR imaging

In this work we present a first feasibility study of the ClearPEM technology for simultaneous PET-MR imaging. The mutual electromagnetic interference (EMI) effects between both systems were evaluated on a 7 T magnet by characterizing the response behavior of the ClearPEM detectors and front-end electronics to pulsed RF power and switched magnetic field gradients; and by analyzing the MR system performance degradation from noise pickup into the RF receiver chain, and from magnetic susceptibility artifacts caused by PET front-end materials.

The discriminatory power of MALDI-TOF mass spectrometry to differentiate between isogenic teicoplanin-susceptible and teicoplanin-resistant strains of methicillin-resistant Staphylococcus aureus.

To explore the discriminatory power of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for detecting subtle differences in isogenic isolates, we tested isogenic strains of Staphylococcus aureus differing in their expression of resistance to methicillin or teicoplanin. More important changes in MALDI-TOF MS spectra were found with strains differing in methicillin than in teicoplanin resistance. In comparison, very minor or no changes were recorded in pulsed-field gel electrophoresis profiles or peptidoglycan muropeptide digest patterns of these strains, respectively. MALDI-TOF MS might be useful to detect subtle strain-specific differences in ionizable components released from bacterial surfaces and not from their peptidoglycan network.

Photodamage to Oxygen Evolving Complex - An Initial Event in Photoinhibition of Photosystem II.

Photosystem II (PSII) of oxygenic photosynthesis is susceptible to photoinhibition. Photoinhibition is defined as light induced damage resulting in turnover of the D1 protein subunit of the reaction center of PSII. Both visible and ultraviolet (UV) light cause photoinhibition. Photoinhibition induced by UV light damages the oxygen evolving complex (OEC) via absorption of UV photons by the Mn ion(s) of OEC. Under visible light, most of the earlier hypotheses assume that photoinhibition occurs when the rate of photon absorption by PSII antenna exceeds the use of the absorbed energy in photosynthesis. However, photoinhibition occurs at all light intensities with the same efficiency per photon. The aim of my thesis work was to build a model of photoinhibition that fits the experimental features of photoinhibition. I studied the role of electron transfer reactions of PSII in photoinhibition and found that changing the electron transfer rate had only minor influence on photoinhibition if light intensity was kept constant. Furthermore, quenching of antenna excitations protected less efficiently than it would protect if antenna chlorophylls were the only photoreceptors of photoinhibition. To identify photoreceptors of photoinhibition, I measured the action spectrum of photoinhibition. The action spectrum showed resemblance to the absorption spectra of Mn model compounds suggesting that the Mn cluster of OEC acts as a photoreceptor of photoinhibition under visible light, too. The role of Mn in photoinhibition was further supported by experiments showing that during photoinhibition OEC is damaged before electron transfer activity at the acceptor side of PSII is lost. Mn enzymes were found to be photosensitive under visible and UV light indicating that Mn-containing compounds, including OEC, are capable of functioning as photosensitizers both in visible and UV light. The experimental results above led to the Mn hypothesis of the mechanism of continuous-light-induced photoinhibition. According to the Mn hypothesis, excitation of Mn of OEC results in inhibition of electron donation from OEC to the oxidized primary donor P680+ both under UV and visible light. P680 is oxidized by photons absorbed by chlorophyll, and if not reduced by OEC, P680+ may cause harmful oxidation of other PSII components. Photoinhibition was also induced with intense laser pulses and it was found that the photoinhibitory efficiency increased in proportion to the square of pulse intensity suggesting that laser-pulse-induced photoinhibition is a two-photon reaction. I further developed the Mn hypothesis suggesting that the initial event in photoinhibition under both continuous and pulsed light is the same: Mn excitation that leads to the inhibition of electron donation from OEC to P680+. Under laser-pulse-illumination, another Mn-mediated inhibitory photoreaction occurs within the duration of the same pulse, whereas under continuous light, secondary damage is chlorophyll mediated. A mathematical model based on the Mn hypothesis was found to explain photoinhibition under continuous light, under flash illumination and under the combination of these two.

Interplay between T cell receptor binding kinetics and the level of cognate peptide presented by major histocompatibility complexes governs CD8+ T cell responsiveness.

Through a rational design approach, we generated a panel of HLA-A*0201/NY-ESO-1(157-165)-specific T cell receptors (TCR) with increasing affinities of up to 150-fold from the wild-type TCR. Using these TCR variants which extend just beyond the natural affinity range, along with an extreme supraphysiologic one having 1400-fold enhanced affinity, and a low-binding one, we sought to determine the effect of TCR binding properties along with cognate peptide concentration on CD8(+) T cell responsiveness. Major histocompatibility complexes (MHC) expressed on the surface of various antigen presenting cells were peptide-pulsed and used to stimulate human CD8(+) T cells expressing the different TCR via lentiviral transduction. At intermediate peptide concentration we measured maximum cytokine/chemokine secretion, cytotoxicity, and Ca(2+) flux for CD8(+) T cells expressing TCR within a dissociation constant (K(D)) range of ∼1-5 μM. Under these same conditions there was a gradual attenuation in activity for supraphysiologic affinity TCR with K(D) < ∼1 μM, irrespective of CD8 co-engagement and of half-life (t(1/2) = ln 2/k(off)) values. With increased peptide concentration, however, the activity levels of CD8(+) T cells expressing supraphysiologic affinity TCR were gradually restored. Together our data support the productive hit rate model of T cell activation arguing that it is not the absolute number of TCR/pMHC complexes formed at equilibrium, but rather their productive turnover, that controls levels of biological activity. Our findings have important implications for various immunotherapies under development such as adoptive cell transfer of TCR-engineered CD8(+) T cells, as well as for peptide vaccination strategies.

Assessment of microbial community structure changes by amplified ribosomal DNA restriction analysis (ARDRA)

Amplified ribosomal DNA restriction analysis (ARDRA) is a simple method based on restriction endonuclease digestion of the amplified bacterial 16S rDNA. In this study we have evaluated the suitability of this method to detect differences in activated sludge bacterial communities fed on domestic or industrial wastewater, and subject to different operational conditions. The ability of ARDRA to detect these differences has been tested in modified Ludzack-Ettinger (MLE) configurations. Samples from three activated sludge wastewater treatment plants (WWTPs) with the MLE configuration were collected for both oxic and anoxic reactors, and ARDRA patterns using double enzyme digestions AluI+MspI were obtained. A matrix of Dice similarity coefficients was calculated and used to compare these restriction patterns. Differences in the community structure due to influent characteristics and temperature could be observed, but not between the oxic and anoxic reactors of each of the three MLE configurations. Other possible applications of ARDRA for detecting and monitoring changes in activated sludge systems are also discussed