Role of rifampin against Propionibacterium acnes biofilm in vitro and in an experimental foreign-body infection model.

Propionibacterium acnes is an important cause of orthopedic-implant-associated infections, for which the optimal treatment has not yet been determined. We investigated the activity of rifampin, alone and in combination, against planktonic and biofilm P. acnes in vitro and in a foreign-body infection model. The MIC and the minimal bactericidal concentration (MBC) were 0.007 and 4 μg/ml for rifampin, 1 and 4 μg/ml for daptomycin, 1 and 8 μg/ml for vancomycin, 1 and 2 μg/ml for levofloxacin, 0.03 and 16 μg/ml for penicillin G, 0.125 and 512 μg/ml for clindamycin, and 0.25 and 32 μg/ml for ceftriaxone. The P. acnes minimal biofilm eradication concentration (MBEC) was 16 μg/ml for rifampin; 32 μg/ml for penicillin G; 64 μg/ml for daptomycin and ceftriaxone; and ≥128 μg/ml for levofloxacin, vancomycin, and clindamycin. In the animal model, implants were infected by injection of 10⁹ CFU P. acnes in cages. Antimicrobial activity on P. acnes was investigated in the cage fluid (planktonic form) and on explanted cages (biofilm form). The cure rates were 4% for daptomycin, 17% for vancomycin, 0% for levofloxacin, and 36% for rifampin. Rifampin cured 63% of the infected cages in combination with daptomycin, 46% with vancomycin, and 25% with levofloxacin. While all tested antimicrobials showed good activity against planktonic P. acnes, for eradication of biofilms, rifampin was needed. In combination with rifampin, daptomycin showed higher cure rates than with vancomycin in this foreign-body infection model.

Alkaline phosphatase activity as a tool for assessing nutritional conditions in the seagrass Posidonia oceanica.(L.) Delile

The effects of experimental phosphorus enrichments on alkaline phosphatase activity (APA) of the seagrass Posidonia oceanica (L.) Delile were tested. Short-term additions (as phosphate, 12 hours, in the laboratory) decreased APA by 18-2896, depending on the plant part considered (roots, young leaves or old leaves). The values of APA after this treatment were well correlated with interna1 phosphorus pools (as P concentration in plant tissues). Long-term additions (as phosphate, added to the sediment, 1 month in situ) decreased APA by 40-75%, also depending on the plant part. We conclude that alkaline phosphatase activity is a good indicator of P deficiency in this seagrass. We used this indicator to assess the P-nutritional status in a series of meadows in the NW Mediterranean, finding a high geographical variability, but correlations between APA and basic features of the meadows (carbonate content of the sediment, organic content of the sediment, shoot density, etc.) were not significant. Consequently, phosphorus deficiency does not seem to be directly related to these descriptors.

Blocking hypoxia-induced autophagy in tumors restores cytotoxic T-cell activity and promotes regression.

The relationship between hypoxic stress, autophagy, and specific cell-mediated cytotoxicity remains unknown. This study shows that hypoxia-induced resistance of lung tumor to cytolytic T lymphocyte (CTL)-mediated lysis is associated with autophagy induction in target cells. In turn, this correlates with STAT3 phosphorylation on tyrosine 705 residue (pSTAT3) and HIF-1α accumulation. Inhibition of autophagy by siRNA targeting of either beclin1 or Atg5 resulted in impairment of pSTAT3 and restoration of hypoxic tumor cell susceptibility to CTL-mediated lysis. Furthermore, inhibition of pSTAT3 in hypoxic Atg5 or beclin1-targeted tumor cells was found to be associated with the inhibition Src kinase (pSrc). Autophagy-induced pSTAT3 and pSrc regulation seemed to involve the ubiquitin proteasome system and p62/SQSTM1. In vivo experiments using B16-F10 melanoma tumor cells indicated that depletion of beclin1 resulted in an inhibition of B16-F10 tumor growth and increased tumor apoptosis. Moreover, in vivo inhibition of autophagy by hydroxychloroquine in B16-F10 tumor-bearing mice and mice vaccinated with tyrosinase-related protein-2 peptide dramatically increased tumor growth inhibition. Collectively, this study establishes a novel functional link between hypoxia-induced autophagy and the regulation of antigen-specific T-cell lysis and points to a major role of autophagy in the control of in vivo tumor growth.

Age-related changes in the bimanual advantage and in brain oscillatory activity during tapping movements suggest a decline in processing sensory reafference

Deficits in the processing of sensory reafferences have been suggested as accounting for age-related decline in motor coordination. Whether sensory reafferences are accurately processed can be assessed based on the bimanual advantage in tapping: because of tapping with an additional hand increases kinesthetic reafferences, bimanual tapping is characterized by a reduced inter-tap interval variability than unimanual tapping. A suppression of the bimanual advantage would thus indicate a deficit in sensory reafference. We tested whether elderly indeed show a reduced bimanual advantage by measuring unimanual (UM) and bimanual (BM) self-paced tapping performance in groups of young (n = 29) and old (n = 27) healthy adults. Electroencephalogram was recorded to assess the underlying patterns of oscillatory activity, a neurophysiological mechanism advanced to support the integration of sensory reafferences. Behaviorally, there was a significant interaction between the factors tapping condition and age group at the level of the inter-tap interval variability, driven by a lower variability in BM than UM tapping in the young, but not in the elderly group. This result indicates that in self-paced tapping, the bimanual advantage is absent in elderly. Electrophysiological results revealed an interaction between tapping condition and age group on low beta band (14âeuro"20 Hz) activity. Beta activity varied depending on the tapping condition in the elderly but not in the young group. Source estimations localized this effect within left superior parietal and left occipital areas. We interpret our results in terms of engagement of different mechanisms in the elderly depending on the tapping mode: a âeuro~kinestheticâeuro? mechanism for UM and a âeuro~visual imageryâeuro? mechanism for BM tapping movement.

Comparative assessement of intimal hyperplasia development after 14 days in two different experimental settings : tissue culture versus ex vivo continuous perfusion of human saphenous vein

Résumé de l'article : L'hyperplasie intimale est un processus de remodelage vasculaire ubiquitaire après une lésion, pouvant menacer la perméabilité de tout type de reconstruction vasculaire. Les mécanismes physiopathologiques impliqués dans le développement de l'hyperplasie intimale ne sont que partiellement élucidés. Il est par conséquent nécessaire d'effectuer des recherches complémentaires afin d'en améliorer la compréhension et ainsi permettre l'élaboration de nouvelles stratégies thérapeutiques médicamenteuses. La culture de veines en milieu statique permet le développement de l'hyperplasie intimale. Ce modèle maintient la viabilité tissulaire, comme décrit précédemment dans d'autres études, mais empêche l'analyse des paramètres hémodynamiques. La mise au point d'un modèle de perfusion in vitro permettant la perfusion de segments vasculaires représente une approche expérimentale intégrant les différents facteurs hémodynamiques. Le système de perfusion (Ex Vivo Vein Support System) que nous avons élaboré conserve l'intégrité pariétale ainsi que les propriétés vasomotrices des veines pour une durée de 14 jours. Cette étude démontre que les deux modèles permettent le développement de l'hyperplasie intimale. Toutefois, les propriétés vasomotrices ainsi que l'influence des paramètres hémodynamiques ne peuvent être analysées que par l'utilisation du système de perfusion. Ce dernier a permis de perfuser des vaisseaux humains sans contamination bactérienne tout en maintenant l'intégrité cellulaire. Ce modèle de perfusion se rapproche plus des conditions hémodynamiques rencontrées in vivo que le modèle statique. Abstract : Background. Intimal hyperplasia (IH) is a vascular remodeling process which often leads to failure of arterial bypass or hemodialysis access. Experimental and clinical work have provided insight in IH development; however, further studies under precise con-trolled conditions are required to improve therapeutic strategies to inhibit IH development. Ex vivo perfusion of human vessel segments under standardized hemodynamic conditions may provide an adequate experimental approach for this purpose. Therefore, chronically perfused venous segments were studied and compared to traditional static culture procedures with regard to functional and histomorphologic characteristics as well as gene expression. Materials and methods. Static vein culture allowing high tissue viability was performed as previously described. Ex vivo vein support system (EVVSS) was performed using a vein support system consisting of an incubator with a perfusion chamber and a pump. EVVSS allows vessel perfusion under continuous flow while maintaining controlled hemodynamic conditions. Each human saphenous vein was divided in two parts, one cultured in a Pyrex dish and the other part perfused in EVVSS for 14 days. Testing of vasomotion, histomorphometry, expression of CD 31, Factor VIII, MIB 1, α-actin, and PAI-1 were determined before and after 14 days of either experimental conditions. Results, Human venous segments cultured under traditional or perfused conditions exhibited similar IH after 14 days as shown by histomorphometry. Smooth-muscle cell ( SMC) was preserved after chronic perfusion. Although integrity of both endothelial and smooth-muscle cells appears to be maintained in both culture conditions as confirmed by CD31, factor VIII and α-actin expression, a few smooth-muscle cells in the media stained positive for factor VIII. Cell-proliferation marker MIB-1 was also detected in the two settings and PAI-1 mRNA expression and activity increased significantly after 14 days of culture and perfusion. Conclusion. This study demonstrates the feasibility to chronically perfuse human vessels under sterile conditions with preservation of cellular integrity and vascular contractility. To gain insights into the mechanisms leading to IH, it will now be possible to study vascular remodeling not only under static conditions but also in hemodynamic environment mimicking as closely as possible the flow conditions encountered in reconstructive vascular surgery.

Proinflammatory activity of cell-wall constituents from gram-positive bacteria.

Innate immunity reacts to conserved bacterial molecules. The outermost lipopolysaccharide (LPS) of Gram-negative organisms is highly inflammatory. It activates responsive cells via specific CD14 and toll-like receptor-4 (TLR4) surface receptor and co-receptors. Gram-positive bacteria do not contain LPS, but carry surface teichoic acids, lipoteichoic acids and peptidoglycan instead. Among these, the thick peptidoglycan is the most conserved. It also triggers cytokine release via CD14, but uses the TLR2 co-receptor instead of TLR4 used by LPS. Moreover, whole peptidoglycan is 1000-fold less active than LPS in a weight-to-weight ratio. This suggests either that it is not important for inflammation, or that only part of it is reactive while the rest acts as ballast. Biochemical dissection of Staphylococcus aureus and Streptococcus pneumoniae cell walls indicates that the second assumption is correct. Long, soluble peptidoglycan chains (approximately 125 kDa) are poorly active. Hydrolysing these chains to their minimal unit (2 sugars and a stem peptide) completely abrogates inflammation. Enzymatic dissection of the pneumococcal wall generated a mixture of highly active fragments, constituted of trimeric stem peptides, and poorly active fragments, constituted of simple monomers and dimers or highly polymerized structures. Hence, the optimal constraint for activation might be 3 cross-linked stem peptides. The importance of structural constraint was demonstrated in additional studies. For example, replacing the first L-alanine in the stem peptide with a D-alanine totally abrogated inflammation in experimental meningitis. Likewise, modifying the D-alanine decorations of lipoteichoic acids with L-alanine, or deacylating them from their diacylglycerol lipid anchor also decreased the inflammatory response. Thus, although considered as a broad-spectrum pattern-recognizing system, innate immunity can detect very subtle differences in Gram-positive walls. This high specificity underlines the importance of using well-characterized microbial material in investigating the system.

Activities of fosfomycin and rifampin on planktonic and adherent Enterococcus faecalis strains in an experimental foreign-body infection model.

Enterococcal implant-associated infections are difficult to treat because antibiotics generally lack activity against enterococcal biofilms. We investigated fosfomycin, rifampin, and their combinations against planktonic and adherent Enterococcus faecalis (ATCC 19433) in vitro and in a foreign-body infection model. The MIC/MBClog values were 32/>512 μg/ml for fosfomycin, 4/>64 μg/ml for rifampin, 1/2 μg/ml for ampicillin, 2/>256 μg/ml for linezolid, 16/32 μg/ml for gentamicin, 1/>64 μg/ml for vancomycin, and 1/5 μg/ml for daptomycin. In time-kill studies, fosfomycin was bactericidal at 8× and 16× MIC, but regrowth of resistant strains occurred after 24 h. With the exception of gentamicin, no complete inhibition of growth-related heat production was observed with other antimicrobials on early (3 h) or mature (24 h) biofilms. In the animal model, fosfomycin alone or in combination with daptomycin reduced planktonic counts by ≈4 log10 CFU/ml below the levels before treatment. Fosfomycin cleared planktonic bacteria from 74% of cage fluids (i.e., no growth in aspirated fluid) and eradicated biofilm bacteria from 43% of cages (i.e., no growth from removed cages). In combination with gentamicin, fosfomycin cleared 77% and cured 58% of cages; in combination with vancomycin, fosfomycin cleared 33% and cured 18% of cages; in combination with daptomycin, fosfomycin cleared 75% and cured 17% of cages. Rifampin showed no activity on planktonic or adherent E. faecalis, whereas in combination with daptomycin it cured 17% and with fosfomycin it cured 25% of cages. Emergence of fosfomycin resistance was not observed in vivo. In conclusion, fosfomycin showed activity against planktonic and adherent E. faecalis. Its role against enterococcal biofilms should be further investigated, especially in combination with rifampin and/or daptomycin treatment.

The stereochemistry of the amino acid side chain influences the inflammatory potential of muramyl dipeptide in experimental meningitis.

Intrathecal injections of 50 to 100 micro g of (N-acetylmuramyl-L-alanyl-D-isoglutamine) muramyl dipeptide (MDP)/rabbit dose-dependently triggered tumor necrosis factor alpha (TNF-alpha) secretion (12 to 40,000 pg/ml) preceding the influx of leukocytes in the subarachnoid space of rabbits. Intrathecal instillation of heat-killed unencapsulated R6 pneumococci produced a comparable leukocyte influx but only a minimal level of preceding TNF-alpha secretion. The stereochemistry of the first amino acid (L-alanine) of the MDP played a crucial role with regard to its inflammatory potential. Isomers harboring D-alanine in first position did not induce TNF-alpha secretion and influx of leukocytes. This stereospecificity of MDPs was also confirmed by measuring TNF-alpha release from human peripheral mononuclear blood cells stimulated in vitro. These data show that the inflammatory potential of MDPs depends on the stereochemistry of the first amino acid of the peptide side chain and suggest that intact pneumococci and MDPs induce inflammation by different pathways.

Infectivity of Lactobacillus rhamnosus and Lactobacillus paracasei isolates in a rat model of experimental endocarditis.

The potential pathogenicity of selected (potentially) probiotic and clinical isolates of Lactobacillus rhamnosus and Lactobacillus paracasei was investigated in a rat model of experimental endocarditis. In addition, adhesion properties of the lactobacilli for fibrinogen, fibronectin, collagen and laminin, as well as the killing activity of the platelet-microbicidal proteins fibrinopeptide A (FP-A) and connective tissue activating peptide 3 (CTAP-3), were assessed. The 90 % infective dose (ID(90)) of the L. rhamnosus endocarditis isolates varied between 10(6) and 10(7) c.f.u., whereas four of the six (potentially) probiotic L. rhamnosus isolates showed an ID(90) that was at least 10-fold higher (10(8) c.f.u.) (P<0.001). In contrast, the two other probiotic L. rhamnosus isolates exhibited an ID(90) (10(6) and 10(7) c.f.u.) comparable to the ID(90) of the clinical isolates of this species investigated (P>0.05). Importantly, these two probiotic isolates shared the same fluorescent amplified fragment length polymorphism cluster type as the clinical isolate showing the lowest ID(90) (10(6) c.f.u.). L. paracasei tended to have a lower infectivity than L. rhamnosus (ID(90) of 10(7) to > or =10(8) c.f.u.). All isolates had comparable bacterial counts in cardiac vegetations (P>0.05). Except for one L. paracasei strain adhering to all substrates, all tested lactobacilli adhered only weakly or not at all. The platelet peptide FP-A did not show any microbicidal activity against the tested lactobacilli, whereas CTAP-3 killed the majority of the isolates. In general, these results indicate that probiotic lactobacilli display a lower infectivity in experimental endocarditis compared with true endocarditis pathogens. However, the difference in infectivity between L. rhamnosus endocarditis and (potentially) probiotic isolates could not be explained by differences in adherence or platelet microbicidal protein susceptibility. Other disease-promoting factors may exist in these organisms and warrant further investigation.

Consequences of gestational stress on GABAergic modulation of respiratory activity in developing newborn pups.

The GABAergic system modulates respiratory activity and undergoes substantial changes during early life. Because this maturation process is sensitive to stress, we tested the hypothesis that gestational stress (GS) alters development of GABAergic modulation of respiratory control in rat pups. The respiratory responses to the selective GABAA receptor agonist muscimol were compared between pups born to dams subjected to GS (bright light and predator odor; 20 min/day from G9 to G19) or maintained under standard (control) conditions. Respiratory activity was measured on 1 and 4 days old pups of both sexes using in vivo (whole body plethysmography) and in vitro (isolated brainstem-spinal cord preparation) approaches. In intact pups, muscimol injection (0.75 mg/kg; i.p.) depressed minute ventilation; this response was less in GS pups, and at P4, muscimol augmented minute ventilation in GS females. Bath application of muscimol (0.01-0.5 μM) onto brainstem preparations decreased inspiratory (C4) burst frequency and amplitude in a dose-dependent manner; the responsiveness decreased with age. However, GS had limited effects on these results. We conclude that the results obtained in vivo are consistent with our hypothesis and show that GS delays maturation of GABAergic modulation of respiratory activity. The differences in the results observed between experimental approaches (in vivo versus in vitro) indicate that the effect of prenatal stress on maturation of GABAergic modulation of respiratory control mainly affects the peripheral/metabolic components of the respiratory control system.

New experimental tools for bioassays with whitefly in laboratory

The objective of this work was to develop an experimental kit for assessments of repellency, deterrence for oviposition, and insecticidal activity on adults of the whitefly Bemisia tabaci biotype B. The kit, which consisted of arenas and nebulizer, was effective for conducting bioassays, and the application of aqueous extracts by inhaler was adequate. The techniques are simple, cheap, and may contribute to research on this insect.

Experimental Results and Evaluation of SmartBox Stimulation Device in a P2P E-learning System

In this paper, we present the experimental results and evaluation of the SmartBox stimulation device in P2P e-learning system which is based on JXTA-Overlay. We also show the design and implementation of the SmartBox environment that is used for stimulating the learners motivation to increase the learning efficiency. The SmartBox is integrated with our P2P system as a useful tool for monitoring and controlling learners¿ activity. We found by experimental results that the SmartBox is an effective way to increase the learner¿s concentration. We also investigated the relation between learner¿s body movement, concentration, and amount of study. From the experimental results, we conclude that the use of SmartBox is an effective way to stimulate the learners in order to continue studying while maintaining the concentration.

Changes in activity of the regulatory glycolytic enzymes and of the pyruvate-dehydrogenase complex during the development of Xenopus laevis.

In this study we investigated the variations of the maximal activities of the rate-controlling glycolytic enzymes (i.e., hexokinase, HK; phosphofructokinase, PFK; pyruvate kinase, PK) and of the pyruvate-dehydrogenase complex (PDHc) during the early embryogenesis of Xenopus laevis (from cleavage through hatching). All the enzymatic assays, using different coupled reactions, were performed spectrophotometrically on cytosolic and mitochondrial fractions. The maximal HK activity increases markedly from neurulation onwards, PFK activity presents a peak around gastrulation, PK activity remains relatively constant throughout the period studied and the highest PDHc activity is observed during cleavage. The specific activities display the same temporal pattern. Furthermore, in the sequence of reactions by which glucose is degraded to form acetyl-CoA, the maximal activities of PFK and PK are not limiting while those of HK and PDHc could be rate-limiting at relatively late developmental stages (hatching).

Blood-Borne Circadian Signal Stimulates Daily Oscillations in Actin Dynamics and SRF Activity.

In peripheral tissues circadian gene expression can be driven either by local oscillators or by cyclic systemic cues controlled by the master clock in the brain's suprachiasmatic nucleus. In the latter case, systemic signals can activate immediate early transcription factors (IETFs) and thereby control rhythmic transcription. In order to identify IETFs induced by diurnal blood-borne signals, we developed an unbiased experimental strategy, dubbed Synthetic TAndem Repeat PROMoter (STAR-PROM) screening. This technique relies on the observation that most transcription factor binding sites exist at a relatively high frequency in random DNA sequences. Using STAR-PROM we identified serum response factor (SRF) as an IETF responding to oscillating signaling proteins present in human and rodent sera. Our data suggest that in mouse liver SRF is regulated via dramatic diurnal changes of actin dynamics, leading to the rhythmic translocation of the SRF coactivator Myocardin-related transcription factor-B (MRTF-B) into the nucleus.

Quinupristin-dalfopristin combined with beta-lactams for treatment of experimental endocarditis due to Staphylococcus aureus constitutively resistant to macrolide-lincosamide-streptogramin B antibiotics.

Quinupristin-dalfopristin (Q-D) is an injectable streptogramin active against most gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). In experimental endocarditis, however, Q-D was less efficacious against MRSA isolates constitutively resistant to macrolide-lincosamide-streptogram B (C-MLS(B)) than against MLS(B)-susceptible isolates. To circumvent this problem, we used the checkerboard method to screen drug combinations that would increase the efficacy of Q-D against such bacteria. beta-Lactams consistently exhibited additive or synergistic activity with Q-D. Glycopeptides, quinolones, and aminoglycosides were indifferent. No drugs were antagonistic. The positive Q-D-beta-lactam interaction was independent of MLS(B) or beta-lactam resistance. Moreover, addition of Q-D at one-fourth the MIC to flucloxacillin-containing plates decreased the flucloxacillin MIC for MRSA from 500 to 1,000 mg/liter to 30 to 60 mg/liter. Yet, Q-D-beta-lactam combinations were not synergistic in bactericidal tests. Rats with aortic vegetations were infected with two C-MLS(B)-resistant MRSA isolates (isolates AW7 and P8) and were treated for 3 or 5 days with drug dosages simulating the following treatments in humans: (i) Q-D at 7 mg/kg two times a day (b.i.d.) (a relatively low dosage purposely used to help detect positive drug interactions), (ii) cefamandole at constant levels in serum of 30 mg/liter, (iii) cefepime at 2 g b.i.d., (iv) Q-D combined with either cefamandole or cefepime. Any of the drugs used alone resulted in treatment failure. In contrast, Q-D plus either cefamandole or cefepime significantly decreased valve infection compared to the levels of infection for both untreated controls and those that received monotherapy (P &lt; 0.05). Importantly, Q-D prevented the growth of highly beta-lactam-resistant MRSA in vivo. The mechanism of this beneficial drug interaction is unknown. However, Q-D-beta-lactam combinations might be useful for the treatment of complicated infections caused by multiple organisms, including MRSA.