[Cerebral trunk abscess due to Neisseria meningitidis in a 28-year-old immunocompetent patient]. / Abcès du tronc cérébral àNeisseria meningitidis chez une patiente de 28ans, immunocompétente.

Meningococcal disease can manifest in very different clinical situations. Meningococcal meningitis and fulminant meningococcemia (or purpura fulminans) are the most common forms of these infections, but many other manifestations can be seen including septic arthritis, pericarditis, pleurisy and conjunctivitis. Brain abscesses have also been described; they are a rare complication of meningococcal disease. We report the case of a 28-year-old immunocompetent patient who developed meningococcal infection associated with brain abscesses and oligo-arthritis.

Application of Ultrasound for Targeted Nanotherapy of Malignant Tumors.

The article describes the study of targeted chemotherapeutic intervention on solid tumors by means of ultrasound and doxorubicin- or paclitaxel-loaded perfluoropentane nanoemulsions. Nanodroplets of the emulsions accumulated in a tumor by passive targeting. Under the action of a tumor-directed therapeutic ultrasound, the nanodroplets converted into vapor microbubbles. In vivo, the nanodroplets strongly retained the loaded drugs; yet, under ultrasound-mediated vaporization they released the drugs into the tumor tissue, thereby implementing effective targeting into the tumor. The tumors subjected to this treatment regressed effectively; however, after some time they recurred. The recurring tumors were more resistant to the repeated therapy than the primary ones. At present, the causes of of the resistance development and methods for its elimination are unclear and they are under investigation.

Combined cancer therapy by micellar-encapsulated drug and ultrasound.

A new modality of drug targeting to tumors that is under development in our lab is based on the drug encapsulation in polymeric micelles followed by a localized release at the tumor site triggered by focused ultrasound. The rationale behind this approach is that drug encapsulation in micelles decreases systemic concentration of drug and provides for a passive drug targeting to tumors via the enhanced penetration and retention (EPR) effect, thus, reducing unwanted drug interactions with healthy tissues. In addition, polymeric micelles sensitize multidrug resistant (MDR) cells to the action of drugs. Upon the accumulation of drug-loaded micelles at the tumor site, ultrasonic irradiation of the tumor is used to provide for the effective intracellular drug uptake. Ultrasound releases drug from micelles and enhances the intracellular uptake of both released and encapsulated drug. An important advantage of ultrasound is that it is noninvasive, can penetrate deep into the interior of the body, can be focused and carefully controlled. The results of the in vitro application of this technique for delivering anthracyclin drugs to ovarian carcinoma A2780 drug-sensitive and MDR cells are described.

Ultrasound-triggered drug targeting of tumors in vitro and in vivo.

The new modality of drug targeting of tumors that we are currently developing is based on drug encapsulation in polymeric micelles, followed by the localized release at the tumor site triggered by focused ultrasound. The rationale behind this approach is that drug encapsulation in micelles decreases systemic concentration of drug, diminishes intracellular drug uptake by normal cells, and provides passive drug targeting of tumors, thus reducing unwanted drug interactions with healthy tissues. Ultrasound irradiation is used to release drug from micelles at the tumor site and to enhance the intracellular drug uptake by tumor cells. An important advantage of ultrasound is that it is noninvasive, can penetrate deep into the interior of the body, can be focused and carefully controlled. Here we describe factors involved in the ultrasound interaction with viable cells in the absence and presence of drug carriers and anti-cancer drugs. We present in vivo effects of 1 MHz ultrasound on drug biodistribution, intratumoral distribution, and survival rates of immuno-compromised athymic nu/nu mice bearing ovarian carcinoma tumors.

Mechanism of the ultrasonic activation of micellar drug delivery.

The mechanism of the ultrasonic enhancement of the uptake of cytotoxic drugs, doxorubicin (DOX) and ruboxyl (Rb) by HL-60 cells from Pluronic micelles was studied. DOX and Rb sorption from either PBS or micellar Pluronic solutions is described by Langmuir-type isotherms characteristic of substrates with limited number of sorption centers. The sorption limits for Rb from PBS and Pluronic were considerably higher than those for DOX, presumably due to much higher Rb partitioning into cell membranes. The overall number of drug sorption centers for both drugs decreased in the presence of Pluronic implying the effect of Pluronic on the DNA conformation, which was confirmed by the electron paramagnetic resonance (EPR) experiments using Rb as a spin probe. Ultrasound increased drug uptake by the cells from PBS and Pluronic solutions. The fluorescence microscopy and flow cytometry experiments using fluorescently-labeled Pluronic showed that ultrasound enhanced both the intracellular uptake of Pluronic micelles and Pluronic trafficking into cell nuclei. A scheme is suggested that describes various equilibria controlling drug/cell interactions and effect of ultrasound on these equilibria. Under the action of ultrasound, the equilibrium between the micellar-encapsulated and free drug is shifted in the direction of free drug due to micelle perturbation; the equilibrium between extracellular and internalized drug is shifted to the intracellular drug due to the ultrasound-induced cellular changes that enhance the accessibility of various cellular structures to drug. An important advantage offered by ultrasound is that the same degree of the intracellular drug uptake may be achieved at a substantially lower drug concentration in the incubation medium.

Acoustic activation of drug delivery from polymeric micelles: effect of pulsed ultrasound.

The effect of a continuous wave (CW) and pulsed 20-kHz ultrasound on the Doxorubicin (DOX) uptake by HL-60 cells from the phosphate buffered saline solution (PBS) and Pluronic micellar solutions was studied. Both CW and pulsed ultrasound enhanced DOX uptake from PBS and Pluronic micelles. The main factor that effected drug uptake was ultrasound power density; however, with increasing power, the enhanced drug uptake was accompanied by the extensive cell sonolysis. For PBS, no significant effect of duration of the ultrasound pulse or inter-pulse interval on the drug uptake was observed. For Pluronic micelles, the uptake increased with increasing pulse duration in the range 0.1-2 s, overall sonication time being the same. For 2-s pulses, the uptake was close to that under CW ultrasound. There was no significant effect of the duration of the inter-pulse interval on the drug uptake from Pluronic micelles. The data on the effect of pulse duration on drug uptake suggest that the characteristic times of drug release from micelles and drug uptake by the cells are comparable. The results point to two independent mechanisms controlling acoustic activation of drug uptake from Pluronic micelles. Both mechanisms work in concert. The first one is related to the acoustically-triggered drug release from micelles that results in higher concentration of the free drug in the incubation medium. The second mechanism is based on the perturbation of cell membranes that results in the increased uptake of the micellar-encapsulated drug. The intracellular uptake of Pluronic micelles was confirmed by fluorescence microscopy.

Factors affecting acoustically triggered release of drugs from polymeric micelles.

A custom ultrasonic exposure chamber with real-time fluorescence detection was used to measure acoustically-triggered drug release from Pluronic P-105 micelles under continuous wave (CW) or pulsed ultrasound in the frequency range of 20 to 90 kHz. The measurements were based on the decrease in fluorescence intensity when drug was transferred from the micelle core to the aqueous environment. Two fluorescent drugs were used: doxorubicin (DOX) and its paramagnetic analogue, ruboxyl (Rb). Pluronic P-105 at various concentrations in aqueous solutions was used as a micelle-forming polymer. Drug release was most efficient at 20-kHz ultrasound and dropped with increasing ultrasonic frequency despite much higher power densities. These data suggest an important role of transient cavitation in drug release. The release of DOX was higher than that of Rb due to stronger interaction and deeper insertion of Rb into the core of the micelles. Drug release was higher at lower Pluronic concentrations, which presumably resulted from higher local drug concentrations in the core of Pluronic micelles when the number of micelles was low. At constant frequency, drug release increased with increasing power density. At constant power density and for pulse duration longer than 0.1 s, peak release under pulsed ultrasound was the same as stationary release under CW ultrasound. Released drug was quickly re-encapsulated between the pulses of ultrasound, which suggests that upon leaving the sonicated volume, the non-extravasated and non-internalized drug would circulate in the encapsulated form, thus preventing unwanted drug interactions with normal tissues.

DNA damage induced by micellar-delivered doxorubicin and ultrasound: comet assay study.

To minimize adverse side effects of chemotherapy, we have developed a micellar drug carrier that retains hydrophobic drugs, and then releases the drug by ultrasonic stimulation. This study investigated the DNA damage induced by doxorubicin (DOX) delivered to human leukemia (HL-60) cells from pluronic P-105 micelles with and without the application of ultrasound. The comet assay was used to quantify the amount of DNA damage. No significant DNA damage was observed when the cells were treated with 0.1, 1 and 10 wt% P-105 with or without ultrasound (70 kHz, 1.3 W/cm(2)) for 1 h or for up to 3 h in 10 wt% P-105. However, when cells were incubated with 10 microg/ml free DOX for up to 9 h, DNA damage increased with incubation time (P=0.0011). Exposure of cells to the same concentration of DOX in the presence of 10-wt% P-105 showed no significant DNA damage for up to 9 h of incubation. However, when ultrasound was applied, a rapid and significant increase in DNA damage was observed (P=0.0001). The application of ultrasound causes the release of DOX from micelles or causes the HL-60 cells to take up the micelle encapsulated DOX. Our experiments indicated that the combination of DOX, ultrasound and pluronic P105 causes the largest DNA damage to HL-60 cells. We believe that this technique can be used for controlled drug delivery.

Effect of a polymeric surfactant on electron transport in HL-60 cells.

To assess the effect of a polymeric surfactant, Pluronic P-105 on the activity of electron transport chains in the mitochondria of HL-60 cells, the bioreduction rates of two membrane-localized lipophilic spin probes, 16-doxylstearic acid methyl ester (16-DSME) and 5-doxylstearic acid (5-DS), were studied. In addition, the effect of Pluronic on the bioreduction rate of the DNA-intercalating spin-labeled anthracyclin drug, Ruboxyl (Rb) was evaluated. For 16-DSME, the bioreduction kinetics was zero order with regard to the nitroxide concentration, indicating that the rate was controlled by the concentration of the reducing enzyme(s), which depends on the activity of the electron transport chains. The introduction of Pluronic at concentrations higher than 0.01% resulted in the decrease of the 16-DSME bioreduction rate. The data suggested that short-term cell incubation with Pluronic resulted in reduced activity of the electron transport chains in the mitochondria of HL-60 cells. This was corroborated by the results of an MTT assay. For 5-DS, the bioreduction kinetics was first order in the absence of Pluronic, but did not follow any simple kinetic law after a short-term cell incubation with Pluronic. For Rb, the degree of nitroxide bioreduction dropped progressively with increasing Pluronic concentration. Thus, incubating cells with polymeric surfactants modulates the intracellular energy metabolism, which can affect the rates of energy-dependent intracellular processes.

Micellar delivery of doxorubicin and its paramagnetic analog, ruboxyl, to HL-60 cells: effect of micelle structure and ultrasound on the intracellular drug uptake.

The effect of Pluronic P-105 micelle structure and ultrasound on the uptake of two anthracycline drugs, doxorubicin and its paramagnetic analogue, ruboxyl, by HL-60 cells was investigated. Pluronic micellization was studied over the temperature range of 25-42 degrees C using the EPR and fluorescence spectroscopy. In the presence of Pluronic P-105 at concentrations corresponding to unimers (or loose aggregates), drug uptake by HL-60 cells was enhanced, apparently due to the effect of the polymeric surfactant on cell membrane permeability. At Pluronic concentrations corresponding to the formation of dense micelles with hydrophobic cores, drug uptake was substantially decreased. However, insonation with 70 kHz ultrasound enhanced the intracellular uptake of drugs encapsulated in dense Pluronic micelles. These findings may provide for developing a new technique of drug targeting by encapsulating the drug in micelles to prevent unwanted interactions with healthy cells and focusing ultrasound on a tumor to enhance drug uptake at the tumor site.

Bioreduction of Tempone and spin-labeled gentamicin by gram-negative bacteria: kinetics and effect of ultrasound.

The primary objective of this study is the investigation of bioreduction kinetics of hydrophilic spin probes, 2,2,6,6, -tetramethyl-4-oxo-piperidinyl-1-oxyl (Tempone), and spin-labeled antibiotic gentamicin by gram-negative bacteria maintained at various oxygen tensions, with emphasis on the effect of probe penetration rate. This information was used to evaluate the effect of ultrasound on the penetration of hydrophilic compounds, including antibiotics, into Pseudomonas aeruginosa and Escherichia coli cells. Penetration of spin-labeled compounds into the cells was assessed by the reduction rate of the nitroxyl moiety measured by EPR. In cell suspensions, both Tempone and spin-labeled gentamicin were localized predominantly in the aqueous phase surrounding the cells. However, a gradual reduction of the probes in contact with the cells indicated that the probes penetrated through the outer membrane and periplasmic space into the cytoplasmic membrane, where the electron transport chains and other metabolic activities of gram-negative bacteria are localized. The kinetics of probe reduction depended on oxygen tension and presence of electron transport chain blockers. It was found that probe penetration rate through the outer cell membrane affected the rate of probe reduction; damaging the permeability barrier by cell incubation with EDTA or by powerful insonation above the cavitation threshold increased the rate of probe reduction. In contrast, insonation below the cavitation threshold did not affect the rate of probe reduction. These findings imply that the recently observed synergistic effect between hydrophilic antibiotics and low frequency ultrasound in killing gram-negative bacteria did not result from the enhanced antibiotic penetration through bacterial cell walls.

Reducing bacterial resistance to antibiotics with ultrasound.

The effect of erythromycin on planktonic cultures of Psedomonas aeruginosa, with and without application of 70 kHz ultrasound, was studied. Ultrasound was applied at levels that had no inhibitory effect on cultures of Ps. aeruginosa. Ultrasound in combination with erythromycin reduced the viability of Ps. aeruginosa by 1-2 orders of magnitude compared with antibiotic alone, even at concentrations below the minimum inhibitory concentration (MIC). Electron-spin resonance studies suggest that ultrasound induces uptake of antibiotic by perturbing or stressing the membrane. This application of ultrasound may be useful for expanding the number of drugs available for treating localized infections by rendering bacteria susceptible to normally ineffective antibiotics.

Intracellular distribution and intracellular dynamics of a spin-labeled analogue of doxorubicin fluorescence and EPR spectroscopy.

Fluorescence spectroscopy revealed a dramatic difference between the intracellular distributions of doxorubicin (DOX) and its paramagnetic analogue, ruboxyl (Rb). Modification of the anthracyclin structure by introduction of a paramagnetic label at position 14 in the DOX molecule resulted in reduced DNA binding and enhanced partitioning into phospholipid membranes, as evidenced by the fluorescence-quenching experiments. Higher partitioning into cell membranes resulted in stronger intracellular fluorescence of Rb. In addition, a paramagnetic label on the Rb molecule provided a unique opportunity for an EPR investigation of the drug's intracellular distribution and dynamics. The observed changes in the EPR spectra of drug-containing cells during their life cycle suggested either a progressive release of the intercalated drug, cell membrane fluidization, or both.

Ultrasonic activated drug delivery from Pluronic P-105 micelles.

Pluronic copolymer P-105 at micellar concentration of 10 wt% was found to increase the activity of the anti-cancer drug, doxorubicin (DOX) against HL-60 cells. Despite the enhanced activity, drug uptake by the cells from P-105 micelles (measured by fluorescence) was found to be much lower than that from a molecular solution of DOX (without the surfactant). Ultrasound (US) was applied as a tool to release drug from the micelles. Based on the combination of ultrasound and micellar treatment, doxorubicin exhibited IC50 concentrations of 2.35, 0.9, 1.25, 0.19 microg/ml with respect to DOX, DOX/US, micellar DOX, and micellar DOX/US, respectively. The results suggest that by encapsulating the anti-cancer drug in micellar carriers and focussing ultrasound on the tumor site, a new approach to drug targetting can be developed.

Factors affecting the permeability of Pseudomonas aeruginosa cell walls toward lipophilic compounds: effects of ultrasound and cell age.

The objective of this research was to elucidate the factors effecting the permeability of cell membranes of gram-negative bacteria toward hydrophobic compounds. Ultrasound treatment, cell age, and the phase state of phospholipid membranes were considered. Spin-labeling EPR method was used to quantify the penetration and distribution of a lipophilic spin probe, 16-doxylstearic acid (16-DS), in Pseudomonas aeruginosa cell membranes. This bacterium was chosen because of its reported resistance to the action of hydrophobic antibiotics caused by the low permeability of the outer cell membrane for hydrophobic compounds. EPR spectra were collected from cell pellets and cell lysates. The overall spin probe uptake was measured in 10% SDS-cell lysates. Lysis with 0.6% SDS revealed the fraction of the probe located in membrane sites readily accessible to the surfactant. The results indicated a structural heterogeneity of P. aeruginosa membranes, with the presence of structurally "stronger" and "weaker" sites characterized by different susceptibility to the SDS treatment. The intracellular concentration of 16-DS was higher in insonated cells and increased linearly with the sonication power. EPR spectra indicated that ultrasound enhanced the penetration of the probe into the structurally stronger sites of the inner and outer cell membranes. The effect of ultrasound on the cell membranes was transient in that the initial membrane permeability was restored upon termination of the ultrasound treatment. These results suggest that the resistance of gram-negative bacteria to the action of hydrophobic antibiotics was caused by a low permeability of the outer cell membranes. This resistance may be reduced by the simultaneous application of antibiotic and ultrasound. This hypothesis was confirmed in our experiments with P. aeruginosa exposed to erythromycin.

Phenobarbital in hair and drug monitoring.

Phenobarbital analysis was performed in vertex hair of patients by gas chromatography mass spectrometry (GC/MS). After washing with dichloromethane, about 250 mg were ground to dust in a ball mill. A 50-mg sample was stirred mechanically for 10 min with 3 ml of NH4Cl/HCl buffer (pH 2.0) containing phenobarbital D5. A solid phase extraction was performed (extrelut Merck) and elution was achieved with chloroform/isopropanol/n-heptane (50:17:33; v/v). A full scan (40-240 uma) acquisition was realized by GC/MS with an ion trap (ITD 700 Finnigan) using a DB5-MS chromatographic column. Quantification was achieved by integrating dominants ions (phenobarbital, 204; phenobarbital D5, 209). Compared to serum, hair concentrates phenobarbital during anti-epileptic therapy (average value 36.4 ng/mg, n = 40 vs. 18.7 mg/l, n = 23). A group correlation exists between phenobarbital in hair and phenobarbital in serum, and between phenobarbital in hair and clinic observation in some typical cases. Phenobarbital in hair yields good information over a long period, especially when blood collection has not been made, when clinical disorders are observed on long-term therapeutic observance.

[Upper cervical intradural lipoma. Apropos of a case]. / Lipome intradural cervical haut. A propos d'une observation.

Intradural lipoma is a rare and slow growing tumor which represents 1% of all intra-spinal tumors. This tumor is often associated with dysraphism. It can be isolated without any associated vertebral or cutaneous malformations. Its clinical evolution is extremely slow and progressive. The fatty composition of this type of tumor allows easy detection by CT as well as by MRI. MRI permits: Reliable and direct assessment (in 3 different plans) of the extension of the lesion. An excellent post operative control.