Effect of nanoliposomes containing Zataria multiflora Boiss. essential oil on gene expression of Shiga toxin 2 in Escherichia coli O157:H7.

AIMS: Enterohaemorrhagic Escherichia coli serotype O157:H7 as a major human pathogen is responsible for food borne outbreaks, bloody diarrhoea, haemorrhagic colitis and haemolytic uraemic syndrome and even death. In this study, the antibacterial activity of the Zataria multiflora essential oil (ZMEO) and nanoliposome-encapsulated ZMEO was evaluated on the pathogenicity of E. coli O157:H7. METHODS AND RESULTS: The minimum inhibitory concentrations (MIC) of essential oil (EO) were determined against the bacterium before and after encapsulation into nanoliposome. Then, the effect of subinhibitory concentrations was evaluated on Shiga toxin 2 (Stx2) production. The effect of free and nanoliposomal EO was also studied on the gene expression of Stx2 by real-time PCR. It was found that inhibitory activity of EO was improved after incorporation into nanoliposomes (P < 0·05). The MIC of free EO against E. coli O157:H7 was 0·03% (v/v), while this value decreased to 0·015%, after encapsulation of EO into nanoliposomes. Furthermore, subinhibitory concentrations of liposomal EO (50 and 75% MIC) had significantly higher inhibitory effect on Stx2 titre than its free form (P < 0·05). Sub-MICs of nanoencapsulated EO also showed a better activity in reduction of Stx2A gene expression than free EO. Using 75% MIC of nanoliposomal EO, the relative transcriptional level of Stx2A gene was decreased from 0·721 to 0·646. CONCLUSIONS: The findings of present study suggest that application of nanoliposomes can improve the antibacterial effect of EOs like ZMEO. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to the enhancement of antimicrobial activity, nanoencapsulation of plant EOs and extracts may increase their commercial application not only in food area but also in the pharmaceutics, cosmetics and health products.

[Secondary prevention of coronary heart disease: results of Euroaspire IV]. / Secundaire preventie van coronaire hartziekte.

OBJECTIVE: Secondary prevention is an important part of cardiovascular risk management. Since 1996, an inventory of cardiovascular risk factors and their treatment has been carried out periodically among patients with coronary heart disease within the framework of the European Action on Secondary Prevention by Intervention to Reduce Events (Euroaspire) project. DESIGN: Retrospective investigation of consecutively hospitalised patients with coronary heart disease. METHOD: Major cardiovascular risk factors and their treatment were investigated using standardised methods in patients who were hospitalised following a first heart infarction or with coronary revascularisation in the Amsterdam and Rijnmond regions of the Netherlands from 2012 to 2013. The investigations were carried out at an average of 18 months after admission. In addition, an oral glucose-tolerance test was carried out in patients without known diabetes. RESULTS: We studied 498 patients. The average BMI was 28 kg/m2, almost 75% had a BMI ≥ 25 kg/m2and 29% had a BMI ≥ 30 kg/m2. The mean cholesterol level was 4.4 mmol/l. Among those included, 16% smoked and 20% had diabetes mellitus; the oral glucose-tolerance test led to a new diabetes-mellitus diagnosis in 1% of the patients without known diabetes. A large majority of those included used antihypertensive agents, and slightly more than half used two or more medications. Despite this, half of the patients were hypertensive. CONCLUSION: As far as cardiovascular risk factors are concerned, smoking has almost halved in the past 20 years. Secondary preventative medication has increased to a stable high level. Blood pressure and overweight continue to be serious points for attention. Treatment of hypertension, in particular, should be improved, for instance by dose increases or combination of hypertensive medications. Routine oral glucose-tolerance tests are not useful in cardiac patients.

[Human babesiosis]. / Babésioses humaines.

Babesia is one of the most ubiquitous and widespread blood parasite in the world based on numbers and distribution of species in animals. The clinical presentation may vary according to the incriminated species. In some states of the USA this kind of infection is endemic; the number of cases reported in Europe is inferior but more life-threatening. A better understanding of parasite specificities such as cycle and pathogenicity allowed to suggest treatment guidelines adapted to the different clinical and microbiological situations.

Scedosporium apiospermum mycetoma with bone involvement successfully treated with voriconazole.

Treatment of Scedosporium apiospermum mycetoma usually requires limb amputation. A 49-year-old woman, from Ivory Coast, was diagnosed with Madura foot in 1995. She failed to respond to several treatments including itraconazole, fluconazole and co-trimoxazole, and refused limb amputation. In December 2002 she was admitted to hospital in France with a painful, swollen right leg and foot. She had no fever and C-reactive protein was 120 mg/l. Magnetic resonance imaging (MRI) confirmed the destruction of tarsus bones with a tibia extension. Voriconazole (400 mg/day) treatment was initiated in March 2003; a significant clinical improvement was observed within 4 months as confirmed by C-reactive protein (16 mg/l) and MRI. Voriconazole was maintained for 18 months with good tolerance. Cholestasis appeared after the first month and remained stable. In October 2004 voriconazole was discontinued due to side effects on the liver (alanine aminotransferase 17 times the normal level); MRI showed impressive regression of bone lesions. As of July 2005, the patient remains clinically well. Voriconazole appears to be a promising drug for the treatment of S. apiospermum mycetomas.

[Treatment of a septic patient with acquired haemophilia]. / Traitement d'une hémophilie acquise chez un patient septique.

INTRODUCTION: Acquired haemophilia is a rare bleeding diathesis caused by auto-immune depletion of factor VIII. It is characterised by spontaneous haemorrhagic syndrome, which can be fatal sometimes. EXEGESIS: A 71 year-old man presents in a dysimmunitary context (rheumatoid arthritis complicates by an acquired haemophilia) a septicemia with a methicillin resistant staphylococcus aureus. At the time of the hospitalization, the patient is febrile (39 degrees C). The activated partial thromboplastin time is very much increased, the level of factor VIII is lowered by 7% and the title of the inhibitor to factor VIII amounts to 140 Bethesda unities. An haematoma of the right root thigh is also noted. In that case, the concomitant presence of septicemia makes difficult the use of immunosuppressive therapy usually recommended to decrease auto-antibody's level. For the management of the septicemia, an adapted antibiotherapy (vancomycin then teicoplanin) is organized to J1. To control haemorrhagic risk, immunoglobulins are prescribed from d12 to d16, without immediate results. Then prednisone is introduced. We observe a very fast decrease of the anticoagulant circulating title with a neat improvement of the clinical state, allowing so to realize a draining puncture of the psoas. This invasive investigation required the use of prothrombinic complex concentrates ((Feiba) in the dose of 80 UI/kg two to three times a day). Biopsy does not show infection source. CONCLUSION: The infection delayed the prescription of immunosuppressive therapy and the surgery. Use of corticoids, following 5 days of intravenous polyvalent immunoglobulin, was the good choice. After 7 weeks of hospitalization the patient has recovered a normal haemostasis results, and a good general state.

Acute ethanol exposure induces [Ca2+]i transients, cell swelling and transformation of actin cytoskeleton in astroglial primary cultures.

Acute exposure to 100 mM isotonic ethanol (EtOH) increased intracellular Ca2+ concentration ([Ca2+]i), induced cell swelling, and transformed actin cytoskeleton in astroglial primary cultures from rat cerebral cortex. Fluorometric recordings of fluo-3AM- or fura-2AM-incubated astroglial cells revealed that EtOH induced [Ca2+]i transients in a small population of the cells. Cell swelling was estimated using a new method based on three-dimensional fluorescence imaging in conjunction with image analysis and graphic visualization techniques. The method provides detailed results concerning the reformation of structural shape and specific volume alterations, as well as total proportions between the different states. Astroglial cell swelling was registered and quantified in 7 of 39 cells chosen from 12 different coverslips. EtOH also induced reversible conformational changes in filamentous actin, appearing as increases in ring formations and a more dispersed appearance of the filaments. Filamentous actin was stained with Alexa phalloidin after incubation with EtOH for varied periods. The results presented here suggest that EtOH affects astrocytes in a way that could be of physiological relevance.

Single-cell volume estimation by three-dimensional wide-field microscopy applied to astroglial primary cultures.

Astrocytes, which constitute a prominent part of the number and volume of brain cells, have a high capacity for controlling their volume, and astrocytic swelling is associated with a number of pathological states affecting the CNS. In order to understand the mechanisms for regulating cell volume in astrocytes better, it is of utmost importance to develop technical instrumentation and analysis methods capable of detecting and characterizing dynamic cell shape changes in a quantitative and robust way. For this purpose, a new method was developed to quantify changes in cell volume at the single-cell level. This method is based on three-dimensional (3D) fluorescence imaging obtained by optical sectioning. An automated image acquisition system was developed for the collection of two-dimensional (2D) microscopic images. A deblurring algorithm was implemented in order to restore the originally unfocused image content. Advanced image analysis techniques were applied for accurate and automated determination of cell volume. The sensitivity and reproducibility of the method was evaluated by using fluorescent beads. The techniques were applied to fura-2-labeled astroglial cells in primary culture exposed to hypo- or hyperosmotic stress. The results show that this method is valuable for determining volume changes in cells or parts thereof.

[Recurrent pneumopathy of the middle lobe...]. / Une pneumopathie récidivante du lobe moyen....

We report a case of a persistent middle-lobar pneumonia, which did not respond to antibiotics. Only a second bronchial endoscopy, with a third thoracic densitometry and histopathological results give the final diagnostic of tracheobronchial foreign body. The choking history happened more than 10 months before. The bronchoscopic extraction restablished the patient.

5-Hydroxytryptamine and glutamate modulate velocity and extent of intercellular calcium signalling in hippocampal astroglial cells in primary cultures.

The effects of 5-hydroxytryptamine or glutamate treatment on mechanically induced intercellular calcium waves were studied in gap junction-coupled astroglial cells using rat astroglial-neuronal primary cultures from hippocampus. Imaging software was developed to study amplitude, velocity and extent of wave propagation. Velocity software was designed to find the cell contours automatically and to calculate travelled distance and time-delay of the calcium wave as it propagates from the stimulated cell to all other cells. Propagation analyses were performed to calculate the area of wave propagation. Mechanical stimulation of a single astroglial cell induced an intercellular calcium wave spreading from cell to cell in the astroglial syncytium. When registering the appearances of calcium signals in individual cells along the wave path upon re-stimulation of the same cell, 44.7% of the cells responded with similar calcium signal appearances the second time as the first time. A second wave from the opposite direction resulted in similar calcium signal appearances in 27.3% of the studied cells. Both amplitude and velocity of the calcium signal decreased most prominently in the first part and showed a later flattening out. Treatment with 5-hydroxytryptamine or glutamate for 20-30 s before mechanical stimulation increased the velocity of the calcium waves. 5-Hydroxytryptamine treatment for varying times decreased the propagation area of the calcium waves. In contrast, glutamate treatment increased the propagation area.

Modulation of mechanically induced calcium waves in hippocampal astroglial cells. Inhibitory effects of alpha 1-adrenergic stimulation.

The effects of different adrenoceptor agonists were investigated on mechanically induced Ca2+ waves in astroglial cells in astroglial-neuronal mixed cultures from rat hippocampus. In the initial part of the study some properties of the waves were characterized. The results show that the initiation of the Ca2+ waves was not critically dependent on extracellular Ca2+ but both the calcium signal and the propagation area of the calcium wave were significantly reduced when the experiments were performed in Ca2+-free buffer. In addition, using the phospholipase C (PLC) inhibitor U-73122 (1 microM) and the gap junction uncoupler octanol (1 mM), the results showed that the Ca2+ wave propagation required PLC activation and functional gap junctions. Further, the data also showed that the protein kinase C (PKC) activator phorbol-12-myristate-13-acetate (PMA 150 nM) reduced the spreading of the waves. The adrenoceptor agonists isoproterenol (iso; beta), phenylephrine (phe; alpha1) and clonidine (clon; alpha2) were evaluated for their short-term (<30 s) effects on the wave propagation. The propagation area was persistently decreased 1, 3 and 5 min after removal of phe. No effects were observed after incubation with iso or clon. Furthermore, using U-73122 or PMA together with phe, shortly incubated, the experiments showed that PLC was a central regulator in the initial phase of the initiation procedure of wave propagation. However, under these conditions PKC was shown not to be involved. Instead it appeared that PKC exerted its inhibitory action on the Ca2+ waves in a latter phase, after prolonged phe exposure. Taken together, the results show that the propagation of Ca2+ waves between astroglial cells in primary cultures can be inhibited/regulated in two principally different ways which involve a pronounced time component. The results also further point out the adrenergic signaling system as an important mediator of dynamic neuron-astroglial information exchange.

Glutamate induced astroglial swelling--methods and mechanisms.

Glutamate (Glu) plays an important role in the early development of brain injuries caused by ischemia, i.e. stroke, or brain trauma. Glu induces a rapid astroglial swelling which, in turn, deranges the composition of neuroactive substances in the extracellular space. We report that Glu can induce astroglial cell swelling by interaction with metabotropic Glu receptors (mGluRs). Furthermore, the Na(+)-K(+)-2Cl- cotransporter, a Na(+)-K(+) ATPase, and the Na(+)-dependent electrogenic Glu carrier seem to be involved in this Glu-induced astroglial cell swelling. Two methods for studying cell swelling arc described. One is based on variations in the signal emitted by the fluorescent probe fura-2/AM when excited at its isosbestic point. These variations were shown to be directly proportional to variations in intracellular volume. Relative changes in cell volume and intracellular calcium concentration could be detected simultaneously in single astroglial cells. The other method used permits the cell volume to be calculated in relative terms with the aid of image processing techniques.

Plasmid DNA gene therapy: studies with the human interleukin-2 gene in tumor cells in vitro and in the murine B16 melanoma model in vivo.

The plasmid DNA vector pVCL-1102 containing the coding sequence for the human IL-2 gene was evaluated for expression in tumor cells in vitro and in vivo. In vitro transfection of murine B16 tumor cells with pVCL-1102 resulted in the expression of 36,000 IU (5.7 micrograms) of biologically active IL-2/10(6) cells/48 h. In vitro transfection of human tumor lines and primary cultures from human biopsies with pVCL-1102 resulted in the expression of 1,289 to 9345 IU of IL-2/10(6) cells/48 h and 30 to 794 IU of IL-2/10(6) cells/48 h, respectively. In vivo, direct intratumor injection of pVCL-1102 resulted in retention of intact plasmid DNA in the tumor tissue and IL-2 secretion by cell cultures derived from the injected tumors. Formulation of pVCL-1102 with the cationic lipid DMRIE/DOPE inhibited DNA degradation and enhanced in vivo transfection efficiency over plasmid DNA alone. Antitumor activity of the pVCL-1102/DMRIE/DOPE complex was evaluated in a B16 melanoma model in mice. An IL-2-specific effect could not be demonstrated in a subcutaneous model because the intratumor injection of plasmid DNA lacking the IL-2 coding sequence also resulted in a significant reduction in tumor volume. However, an IL-2-specific effect was observed when B16 cells were transfected in vitro prior to implantation into the mouse. Transient transfection of B16 cells with pVCL-1102 rendered the cells less tumorigenic in vivo and produced a significant reduction in tumor volume. These data demonstrate that a plasmid DNA expression vector can be used to deliver the IL-2 gene to tumor cells in vitro and in vivo, resulting in the expression of significant levels of IL-2 protein. These data also illustrate the need for the use of appropriate controls when evaluating the in vivo biological activity of plasmid DNA in murine tumor models.

Prolongation of drug exposure in cerebrospinal fluid by encapsulation into DepoFoam.

Prolonged maintenance of a therapeutic drug concentration in the cerebrospinal fluid is required for optimal treatment of leptomeningeal leukemia or carcinomatosis with cell cycle-specific antimetabolites. The pharmacokinetics of 1-beta-D-arabinofuranosylcytosine (ara-C) encapsulated into DepoFoam (Depo/Ara-C) was studied in six rhesus monkeys after intrathecal injection into the lumbar sac. Following a single 2-mg dose, the Depo/Ara-C concentration decreased biexponentially with initial and terminal half-lives of 14.6 and 156 h, respectively. The free drug concentration remained above the reported minimal cytotoxic level of 0.1 micrograms/ml (0.4 microM) for more than 672 h (28 days). In contrast, the half-life of ara-C following an intralumbar bolus dose of unencapsulated drug in a single animal was 0.74 h. A single intrathecal injection of Depo/Ara-C can maintain a therapeutic drug concentration in the cerebrospinal fluid for a very prolonged period.

Treatment of leptomeningeal metastasis with intraventricular administration of depot cytarabine (DTC 101). A phase I study.

Depo cytarabine (DTC 101 [formerly identified as Depo/Ara-C]) is a slow-releasing, depot formulation in which cytarabine is encapsulated within the aqueous compartments of microscopic (DepoFoam) particles. A phase I trial of DTC 101, given intraventricularly, was conducted in patients with leptomeningeal metastasis. Nine patients were given 1 to 7 cycles of DTC 101 in doses ranging from 25 to 125 mg that were administered via an Ommaya reservoir into the lateral ventricle. The dose-limiting toxic reaction was encephalopathy that occurred at the 125-mg dose level. All toxic episodes but one were transient and reversible, with the total duration of toxicity lasting from 1 to 7 days. The ventricular concentration of free cytarabine released from DTC 101 into cerebrospinal fluid decreased biexponentially with an initial half-life of 7.2 +/- 1.7 (+/- SEM) hours and a terminal half-life of 140 +/- 49 hours. The cerebrospinal fluid was cleared of malignant cells within 3 weeks of initial therapy in five of six cytologically evaluable patients. The duration of response ranged from 2 to more than 14 weeks, with a median of over 11 weeks. In conclusion, DTC 101 appears to be a pharmacologically attractive agent for use against leptomeningeal metastasis. The toxic episodes that occur with this therapy are well tolerated by patients.

Regulation of initial vinblastine influx by P-glycoprotein.

P-glycoprotein (PGP) is an energy-dependent efflux pump that serves to protect cells against the cytotoxicity of many natural product drugs including vinblastine (VBL). In this study we investigated the role of PGP in regulating initial VBL influx. The apparent influx of VBL, measured over the first 20 s, was 2-fold lower in KB-GRC1 cells expressing a transfected mdr1 gene at high level than in non-expressing parental KB-3-1 cells. Inhibition of PGP efflux function with dipyridamole increased the influx rate constant by 4.0-fold in the KB-GRC1 cells but only 2.1-fold in the KB-3-1 cells. Verapamil, another inhibitor of PGP-mediated efflux, increased the initial influx rate constant by 2.7-fold in the KB-GRC1 cells but only 1.4-fold in the KB-3-1 cells. Inhibition of PGP function by depletion of ATP increased influx by 6.8-fold and 2.2-fold in the two cell types, respectively. Mutation of PGP at both ATP binding sites abolished its ability to limit initial influx. Thus, VBL is serving as an efficient substrate for the efflux pump even within the first few seconds of drug exposure, consistent with the hypothesis that PGP may directly efflux drug from the cell membrane.