New in vitro and in vivo models to evaluate antibiotic efficacy in Staphylococcus aureus prosthetic vascular graft infection.

OBJECTIVE: Prosthetic vascular graft infection (PVGI) is an emerging disease, mostly caused by staphylococci, with limited data regarding efficacy of current antistaphylococcal agents. We aimed to assess the efficacy of different antibiotic regimens. METHODS: Six different strains of MSSA and MRSA were used. We compared results of minimal biofilm inhibitory and eradicating concentrations (MBICs and MBECs) obtained with a Calgary Biofilm Pin Lid Device (CBPD) with those yielded by an original Dacron(®)-related minimal inhibitory and eradicating concentration measure model. We then used a murine model of Staphylococcus aureus vascular prosthetic material infection to evaluate efficacy of different antibiotic regimens: vancomycin and daptomycin combined or not with rifampicin for MRSA and the same groups with cloxacillin and cloxacillin combined with rifampicin for MSSA. RESULTS: We demonstrated that classical measures of MBICs and MBECs obtained with a CPBD could overestimate the decrease in antibiotic susceptibility in material-related infections and that the nature of the support used might influence the measure of biofilm susceptibility, since results yielded by our Dacron(®)-related minimal eradicating assay were lower than those found with a plastic device. In our in vivo model, we showed that daptomycin was significantly more bactericidal than comparators for some strains of MRSA or MSSA but not for all. For the majority of strains, it was as efficient as comparators. The addition of rifampicin to daptomycin did not enhance daptomycin efficacy. CONCLUSIONS: Despite the heterogeneity of results according to bacterial strains, these innovative models represent an option to better evaluate the in vitro efficacy of antibiotics on Dacron(®)-related biofilm S. aureus infections, and to screen different antibiotic regimens in a mouse model of PVGIs.

Evidence of oxidative stress and mitochondrial respiratory chain dysfunction in an in vitro model of sepsis-induced kidney injury.

To investigate the role of oxidative stress and/or mitochondrial impairment in the occurrence of acute kidney injury (AKI) during sepsis, we developed a sepsis-induced in vitro model using proximal tubular epithelial cells exposed to a bacterial endotoxin (lipopolysaccharide, LPS). This investigation has provided key features on the relationship between oxidative stress and mitochondrial respiratory chain activity defects. LPS treatment resulted in an increase in the expression of inducible nitric oxide synthase (iNOS) and NADPH oxidase 4 (NOX-4), suggesting the cytosolic overexpression of nitric oxide and superoxide anion, the primary reactive nitrogen species (RNS) and reactive oxygen species (ROS). This oxidant state seemed to interrupt mitochondrial oxidative phosphorylation by reducing cytochrome c oxidase activity. As a consequence, disruptions in the electron transport and the proton pumping across the mitochondrial inner membrane occurred, leading to a decrease of the mitochondrial membrane potential, a release of apoptotic-inducing factors and a depletion of adenosine triphosphate. Interestingly, after being targeted by RNS and ROS, mitochondria became in turn producer of ROS, thus contributing to increase the mitochondrial dysfunction. The role of oxidants in mitochondrial dysfunction was further confirmed by the use of iNOS inhibitors or antioxidants that preserve cytochrome c oxidase activity and prevent mitochondrial membrane potential dissipation. These results suggest that sepsis-induced AKI should not only be regarded as failure of energy status but also as an integrated response, including transcriptional events, ROS signaling, mitochondrial activity and metabolic orientation such as apoptosis.

Correlative time-resolved fluorescence microscopy to assess antibiotic diffusion-reaction in biofilms.

The failure of antibiotics to inactivate in vivo pathogens organized in biofilms has been shown to trigger chronic infections. In addition to mechanisms involving specific genetic or physiological cell properties, antibiotic sorption and/or reaction with biofilm components may lessen the antibiotic bioavailability and consequently decrease their efficiency. To assess locally and accurately the antibiotic diffusion-reaction, we used for the first time a set of advanced fluorescence microscopic tools (fluorescence recovery after photobleaching, fluorescence correlation spectroscopy, and fluorescence lifetime imaging) that offer a spatiotemporal resolution not available with the commonly used time-lapse confocal imaging method. This set of techniques was used to characterize the dynamics of fluorescently labeled vancomycin in biofilms formed by two Staphylococcus aureus human isolates. We demonstrate that, at therapeutic concentrations of vancomycin, the biofilm matrix was not an obstacle to the diffusion-reaction of the antibiotic that can reach all cells through the biostructure.

Fluorescence correlation spectroscopy to study diffusion and reaction of bacteriophages inside biofilms.

In the natural environment, most of the phages that target bacteria are thought to exist in biofilm ecosystems. The purpose of this study was to gain a clearer understanding of the reactivity of these viral particles when they come into contact with bacteria embedded in biofilms. Experimentally, we quantified lactococcal c2 phage diffusion and reaction through model biofilms using in situ fluorescence correlation spectroscopy with two-photon excitation. Correlation curves for fluorescently labeled c2 phage in nonreacting Stenotrophomonas maltophilia biofilms indicated that extracellular polymeric substances did not provide significant resistance to phage penetration and diffusion, even though penetration and diffusion were sometimes restricted because of the noncontractile tail of the viral particle. Fluctuations in the fluorescence intensity of the labeled phage were detected throughout the thickness of biofilms formed by c2-sensitive and c2-resistant strains of Lactococcus lactis but could never be correlated with time, revealing that the phage was immobile. This finding confirmed that recognition binding receptors for the viral particles were present on the resistant bacterial cell wall. Taken together, our results suggest that biofilms may act as "active" phage reservoirs that can entrap and amplify viral particles and protect them from harsh environments.

Three-dimensional time-resolved fluorescence imaging by multifocal multiphoton microscopy for a photosensitizer study in living cells.

Two-photon fluorescence microscopy is widely applied to biology and medicine to study both the structure and dynamic processes in living cells. The main issue is the slow acquisition rate due to the point scanning approach limiting the multimodal detection (x, y, z, t). To extend the performances of this powerful technique, we present a time-resolved multifocal multiphoton microscope (MMM) based on laser amplitude splitting. An array of 8 x 8 foci is created on the sample that gives a direct insight of the fluorescence localization. Four-dimensional (4D) imaging is obtained by combining simultaneous foci scanning, time-gated detection, and z displacement. We illustrate time-resolved MMM capabilities for 4D imaging of a photosensitizer inside living colon cancer cells. The aim of this study is to have a better understanding of the photophysical processes implied in the photosensitizer reactivity.

Enhanced fluorescence cell imaging with metal-coated slides.

Fluorescence labeling is the prevailing imaging technique in cell biology research. When they involve statistical investigations on a large number of cells, experimental studies require both low magnification to get a reliable statistical population and high contrast to achieve accurate diagnosis on the nature of the cells' perturbation. Because microscope objectives of low magnification generally yield low collection efficiency, such studies are limited by the fluorescence signal weakness. To overcome this technological bottleneck, we proposed a new method based on metal-coated substrates that enhance the fluorescence process and improve collection efficiency in epifluorescence observation and that can be directly used with a common microscope setup. We developed a model based on the dipole approximation with the aim of simulating the optical behavior of a fluorophore on such a substrate and revealing the different mechanisms responsible for fluorescence enhancement. The presence of a reflective surface modifies both excitation and emission processes and additionally reshapes fluorescence emission lobes. From both theoretical and experimental results, we found the fluorescence signal emitted by a molecular cyanine 3 dye layer to be amplified by a factor approximately 30 when fluorophores are separated by a proper distance from the substrate. We then adapted our model to the case of homogeneously stained micrometer-sized objects and demonstrated mean signal amplification by a factor approximately 4. Finally, we applied our method to fluorescence imaging of dog kidney cells and verified experimentally the simulated results.

Photochemistry of 2,6-diisopropylphenol (propofol).

The photochemistry of the anaesthetic agent propofol (PPF) was investigated in three different solvents of quite different polarity (cyclohexane, methanol and phosphate buffer pH 7) by means of nanosecond laser flash photolysis and absorption spectroscopy. GC-MS spectrometry measurements of PPF in cyclohexane have revealed the formation of two major products upon low intensity UV continuous irradiation of PPF in aerated solution: the diphenol derivative of PPF and 2,6-diisopropyl-p-benzoquinone (PPFQ). Only the diphenol compound was obtained in anaerobic solution. PPF phenoxyl radical (PPF ) generation has been assigned as the original step leading to the formation of both the diphenol compound and PPFQ in cyclohexane as revealed by laser flash photolysis at 266 nm and by electron paramagnetic resonance spectroscopy as well. Investigation of PPF by nanosecond flash photolysis at 266 nm in the other solvents revealed the occurrence of different photochemical processes depending on the nature and the polarity of the solvent. A reaction scheme is proposed in order to discuss the mechanism of reaction of PPF in all media.

Diode-pumped passively mode-locked Nd:YVO4 laser at 914 nm.

We demonstrate, for the first time, to our knowledge, a diode-pumped passively mode-locked Nd:YVO4 laser, operating on the 4F(3/2)-4I(9/2) transition of the neodymium ion at 914 nm. We obtained 8.8 ps pulses at approximately 914 nm at a repetition rate of 94 MHz, and an averaged output power of 87 mW by using a semiconductor saturable absorber mirror.

Reactivity towards singlet oxygen of propofol inside liposomes and neuronal cells.

Singlet oxygen (1O2), a reactive oxygen species, has been found to be implicated in many cellular events and pathological disorders. Herein, we investigated the reactivity of 1O2 towards the anaesthetic agent propofol (PPF) encapsulated within DMPC liposomes. By time resolved luminescence, the rate constant of 1O2 quenching by PPF was evaluated, depending on the location of the sensitizer, with following values: 1.35+/-0.05x10(7) M(-1) s(-1) for deuteroporphyrin (as embedded source) and 0.8+/-0.04x10(7) M(-1) s(-1) for uroporphyrin (as external source), respectively. The nature of the oxidation product, resulting from the reaction of 1O2 with PPF, was determined using absorption and HPLC techniques. Finally, the in vitro protective effect of PPF towards the 1O2-induced neuronal cell toxicity was evaluated in terms of cell viability.

[Twelve years' experience with Carbomedics bileaflet valves]. / Douze ans d'expérience avec la prothèse valvulaire a ailettes Carbomedics. Influence de l'age des patients.

The authors report long-term results of a cohort of patients who underwent valve replacement with a Carbomedics bileaflet mechanical prosthesis. The influence of patient age on the results was examined. Three hundred and ninety patients were operated between 1988 and 2000. Complete follow-up was available in 98.5% of cases. For aortic valve replacement, the total experience was 1,061 person-years. The hospital mortality was 2.3%. Global actuarial 12 year survival was 74 +/- 7%. The actuarial probability of absence of thrombo-embolic complications at 12 years was 95 +/- 3%, and of absence of haemorrhagic complications 89 +/- 4%. For mitral valve replacement, the total experience was 610 person-years. The hospital mortality was 3.4%. The global actuarial 12 year survival was 78 +/- 5%. The actuarial probability of absence of thrombo-embolic complications at 12 years was 79 +/- 9% and of absence of haemorrhagic complications 82 +/- 5%. The results of survival and prosthesis-related complications were comparable to those reported in the literature with second generation mechanical prostheses, with the exception of thromboses of the mitral valve for which a higher linear rate was observed. Multivariate analysis showed an increased (x 2.3) risk of haemorrhage after 60 years of age irrespective of the site of implantation of the prosthesis. In patients over 60 years of age, the risks of long-term anticoagulant therapy should raise questions concerning the modality of follow-up and the indications. The use of biological prostheses is a possible alternative.

Fluorescence-lifetime imaging with a multifocal two-photon microscope.

Two-photon microscopy is a powerful tool for imaging of cells or tissues. However, it presents the drawback of being a laser-scanning technique that involves a long acquisition time for fluorescence-lifetime imaging. Thus it is commonly limited to intensity images that give only indications of the location of fluorophores but do not identify the physicochemical properties and interactions between cells' components. To protect biological samples from experiments that are too long and to provide a more comprehensive spectroscopic tool we have developed a time-resolved multifocal multiphoton microscope. This setup allows us to speed up the acquisition while retaining the possibility of measuring both intensity and lifetime images of the sample.

Indications of coronary artery bypass graft in 2003.

UNLABELLED: During the last 3 decades, coronary artery bypass grafting (CABG) emerged, was developed and has progressed. Additionally, recent surgical innovations have been introduced aimed at reducing the trauma without deviating from the efficiency of conventional procedure. At the same time significant evolution in preventive, medical treatment and percutaneous procedures has been observed. The introduction of new treatment has improved the result in medically treated patients. Percutaneous procedures emerged and were developed. New technologies and advances available to adjunctive medical therapies have appeared and have impacted the effectiveness initially established for percutaneous coronary intervention (PCI). Larger use of stent permits a significant decrease in restenosis and the introduction of coated stent will probably improve the RESULTS: Indication is a moving field. Continuing improvement in medical treatment, technical procedure and development of less invasive surgery modifies the place of each treatment and continuing evaluation and comparison are necessary. Introduction of new treatment, aimed at treating ischemic cardiomyopathy like transmyocardial revascularization, cell transplantation or gene therapy will probably modify indications in the future.

Heterogeneity of diffusion inside microbial biofilms determined by fluorescence correlation spectroscopy under two-photon excitation.

Fluorescence correlation spectroscopy (FCS) under two-photon excitation was applied successfully to characterize the penetration and diffusion capabilities of fluorescent probes (latex beads and fluorescein isothiocyanate-dextran) of different size and electrical charge in two models of monomicrobial biofilms with low (Lactococcus lactis biofilm) or high (Stenotrophonas maltophilia biofilm) contents of extracellular polymeric substance (EPS). FCS measurements performed on each biofilm can show deviation from Brownian diffusion, depending on the local structure of the biofilm and the fluorophore size. In this case, we fitted the data to an anomalous diffusion model and determined apparent diffusion coefficients, which can be 50 times smaller than the values in aqueous solutions. This result was interpreted as steric hindrance of the diffusion of the fluorescent particles within the biofilm that can lead to a total inhibition as observed particularly in the mushroom-like structure of the S. maltophilia biofilm. Alternatively, mechanisms for the absence of FCS signal behavior were related to attractive electrostatic interactions between cationic particles and negatively charged bacteria or to specific interactions between dextrans and EPS of the biofilm matrix.

Fifteen-year experience with the mitral Carpentier-Edwards PERIMOUNT pericardial bioprosthesis.

BACKGROUND: This multicenter study concerning the mitral PERIMOUNT valve previously reported clinical results at 12 years; this report updates the performance to 15 years postoperatively. METHODS: The 435 patients (mean age 60.7+/-11.6 years; 41.1% male) underwent implantation with the PERIMOUNT valve between 1984 and 1989 at seven institutions. Follow-up was complete for 96.1% of the cohort. The mean follow-up was 8.1+/-4.4 years (range 0 to 15.4 years) for a total of 3492 patient-years. RESULTS: There were 34 (7.8%) operative deaths, one (0.2%) valve related. The late mortality rate was 5.3%/patient-year (2.2%/patient-year valve related). At 14 years, the overall actuarial survival rate was 37.1%+/-3.3% (63.1%+/-4.4% valve related). Actuarial freedom from complications at 14 years was as follows: thromboembolism, 83.8%+/-3.2% (1.1%/patient-year); hemorrhage, 86.6%+/-3.2% (1.1%/patient-year); and explant due to structural valve deterioration (SVD), 68.8%+/-4.7%. Actual freedom from explant due to SVD was 83.4%+/-2.3%. Rates of structural failure decreased with increasing age at implant. CONCLUSIONS: The Carpentier-Edwards PERIMOUNT Pericardial Bioprosthesis is a reliable choice for a tissue valve in the mitral position, especially in patients more than 60 years of age.

Inhibitory activity of antibodies against the human atrial 5-HT(4)receptor.

Antibodies directed against the second extracellular loop of G protein-coupled receptors have been shown to exert "agonist-like" activities. In order to test the hypothesis that this is a general phenomenon, antibodies were raised in rabbits against a synthetic peptide corresponding to the second extracellular loop of the newly sequenced human cardiac 5-HT(4)receptor. The antibodies were affinity-purified and shown to recognize the 5-HT(4)receptor in immunoblots of Chinese hamster ovary (CHO) cells expressing the receptor. The antibodies had no intrinsic effect but could depress the activation of L -type calcium channel induced by serotonin in human atrial cells. This antagonist-like effect was exerted both by intact IgG and by Fab fragments. These results are physiologically important since it has been shown that the 5-HT(4)receptor could be a target for autoantibodies in mothers at risk of giving birth to children with neonatal atrio-ventricular block.

Analysis of the distribution of histologic myocardial lesions during acute cardiac rejection. Experimental study in rodents.

BACKGROUND AND METHOD: Asymmetric distribution of histologic lesions have been reported in grafted hearts that could hamper the interpretation of right ventricular endomyocardial biopsy. Heterotopic heart transplantations were performed in rats (n=59) and guinea pigs (n=20). Grafted hearts were examined by a pathologist who established the degree of cardiac rejection in the four cardiac cavities. RESULTS: Forty cardiac rejections were diagnosed in rats and ten in guinea pigs. An asymmetric distribution of histologic lesions was observed in 34 (68%) rejected hearts with greater lesions in the auricular myocardium than in the ventricular myocardium (n=25, 50%). One (n=18) or two degrees (n=7) differentiated the severity of rejection between atria and ventricles. Cardiac rejection score was significantly greater in atria than in ventricles (3.12+/-0.18 vs. 2.6+/-0.2 (P<0.01) in rats and 2.35+/-0.37 vs. 1.6+/-0.47 (P<0.001) in guinea pigs. There were histologic lesions of rejection in the auricular myocardium in seven cases, although the ventricular myocardium was completely normal. In nine (18 %) other grafted hearts the degree of rejection was equal in the auricular myocardium and ventricular septum but was greater than the degree of rejection noted in the right and left ventricular free walls. CONCLUSION: The distribution of histologic lesions of acute cardiac rejection in rodents was heterogeneous in grafted hearts which exhibited greater lesions in the atria than in ventricles. This should be taken into consideration in the evaluation of new methods of detection of cardiac rejection and in the diagnosis of acute cardiac rejection in humans.

Structural valve deterioration in mitral replacement surgery: comparison of Carpentier-Edwards supra-annular porcine and perimount pericardial bioprostheses.

BACKGROUND: Bioprostheses preserved with glutaraldehyde, both porcine and pericardial, have been available as second-generation prostheses for valve replacement surgery. The performance with regard to structural valve deterioration with the Carpentier-Edwards supra-annular (CE-SAV) porcine bioprosthesis and the Carpentier-Edwards Perimount (CE-P) pericardial bioprosthesis (Baxter Healthcare Corp, Edwards Division, Santa Ana, Calif) was evaluated to determine whether there was a difference in mitral valve replacement. METHODS: The CE-SAV bioprosthesis was implanted in 1266 overall mitral valve replacements (isolated mitral, 1066; mitral in multiple, 200) and the CE-P bioprosthesis in 429 overall mitral valve replacements (isolated mitral, 328; mitral in multiple, 101). The mean age of the CE-SAV population was 64.2 +/- 12.2 years and that of the CE-P population, 60.7 +/- 11.7 years (P =.0001). For the study, structural valve deterioration was diagnosed at reoperation for explantation. RESULTS: The freedom from structural valve deterioration was evaluated to 10 years, and the freedom rates reported are at 10 years. For the overall mitral valve replacement groups, the actuarial freedom from deterioration was significant (P =.0001): CE-P > CE-SAV for 40 years or younger, 80% versus 60%; 41 to 50 years, 91% versus 61%; 51 to 60 years, 84% versus 69%; 61 to 70 years, 95% versus 75%. The older than 70-year group was 100% versus 92% (no significant difference). The actual freedom from structural valve deterioration also demonstrated the same pattern at 10 years: 40 years or younger, CE-P 82% versus CE-SAV 68%; 41 to 50 years, 92% versus 70%; 51 to 60 years, 90% versus 80%; 61 to 70 years, 97% versus 88%; and older than 70 years, 100% versus 97%. The independent risk factors of structural valve deterioration for the overall mitral valve replacement group were age and age groups and prosthesis type (CE-SAV > CE-P). The prosthesis type either in isolated replacement or in multiple replacement was not predictive of structural valve deterioration. The pathology of structural valve deterioration was different: 70% of CE-P failures were due to calcification and 57% of CE-SAV failures were due to combined calcification and leaflet tear. CONCLUSION: The actuarial and actual freedom from structural valve deterioration, diagnosed at reoperation, is greater at 10 years for CE-P than for CE-SAV bioprostheses. The mode of failure is different, and the cause remains obscure. Long-term evaluation is recommended, because the different modes of failure may alter the clinical performance by 15 and 20 years.

Severe and early alteration of action potential during acute cardiac rejection in rats.

INTRODUCTION: Alteration of cardiac action potential and its adaptation to heart rate could contribute to cardiac dysfunction and arrhythmias during acute cardiac rejection. METHODS AND RESULTS: Heterotopic heart transplantation was performed in allogeneic and syngeneic rats in which the action potentials of right and left ventricles were measured at 1, 2.5, 3.3, and 5.7 Hz successively using standard microelectrode techniques and compared with nontransplanted hearts. For each frequency, we measured action potential amplitude, action potential duration, transmembrane resting potential, and Vmax. In the right ventricle, at 1 Hz in the presence of rejection (n = 40), a significant increase was observed in action potential duration at 20%, 50%, and 70% repolarization (82.5%, 75.6%, and 70.8%, respectively) and in action potential amplitude (+17.9 mV), and the resting potential was decreased (-5.3 mV). A lack of adaptation of action potential duration to the driving frequency was observed in the rejecting heart group in contrast to controls (n = 20) and nonrejecting hearts (n = 13). Similar results were observed in the left ventricle and surprisingly in the native hearts (n = 11) of recipients with allografted rejecting hearts in the abdominal position. CONCLUSION: Action potential and its adaptation to the driving frequency is considerably altered during acute rejection. A humoral factor could contribute to cardiac dysfunction.

Twelve-year experience with the 19 mm Carpentier-Edwards pericardial aortic valve.

BACKGROUND AND AIMS OF THE STUDY: In patients with small aortic annuli, the choice of prosthesis should be based on hemodynamics and valve durability. The Carpentier-Edwards pericardial valve offers good hemodynamic performance and long-term valve durability. We reviewed our 12-year experience with 121 patients who received a 19 mm prosthesis. METHODS: A total of 121 patients (97 females, 24 males; mean age 73.2 +/- 9.4 years) underwent isolated aortic valve replacement with a 19 mm Carpentier-Edwards pericardial bioprosthesis in our institution between July 1984 and December 1995. Patients were followed up for an average of 4.84 years after surgery; total follow up was 581 patient-years (pt-yr). RESULTS: The operative mortality rate was 3.3% (4/121). At the present time, 77 patients (86%) are in NYHA class I or II with a mean gradient of 18.0 +/- 6.9 mmHg and mean effective orifice area 1.1 +/- 0.23 cm2. There were 25 late deaths. After 12 years the actuarial survival rate was 42 +/- 26%. Eight patients died of valve-related cause (one endocarditis, one structural failure, two thromboembolisms, one anticoagulant-related hemorrhage, three sudden deaths). At 12 years, the actuarial rate of freedom from valve-related death was 61 +/- 37% and from non-sudden valve-related death 88 +/- 11%. Valve-related complications included six thromboembolic episodes (1.0%/pt-yr), one endocarditis (0.17%/pt-yr), two reoperations (0.34%/pt-yr) and two structural valve failures (0.34%/pt-yr). After 12 years, freedom from reoperation was 89 +/- 11%, from valve failure 92 +/- 8%, from thromboembolic episodes 82 +/- 16% and from endocarditis 99 +/- 1%. CONCLUSION: With a low rate of structural valve failure at 12 years and a good clinical performance, the Carpentier-Edwards prosthesis is a reliable alternative for small aortic annuli.