Layersome: development and optimization of stable liposomes as drug delivery system.

This paper describes the development of stable drug delivery systems named layersomes. The layersomes are conventional liposomes coated with one or multiple layers of biocompatible polyelectrolytes in order to stabilise their structure. The formulation strategy is based on an alternative coating procedure of positive poly(lysine) (pLL) and negative poly(glutamic acid) (pGA) polypeptides on initially charged small unilamellar liposomes (SUVs). The size distribution and the zeta potential of the final entity depend on the number of polyelectrolyte layers and the charge of the last coating layer. Morphological studies were achieved by flux cytometry and cryo electron microscopy. Release studies of encapsulated hydrophilic 5(6)-carboxyfluorescein (5,6CF) in the presence of Triton or ethanol showed an increased membrane resistance of the layersomes compared to classical SUVs. Finally, encapsulation of piroxicam (PX) was performed with success.

The role of iron and aluminium in digestion and odor formation.

The role of iron and aluminium in determining volatile solids reduction and odors from anaerobically digested, dewatered sludge cakes was evaluated from data collected from a variety of wastewater treatment plants. It was found that volatile solids reduction generally increased as the iron content of the sludge increased. It was also observed that odors increased with increasing iron. No correlation with aluminium or divalent cations was found. Based on these data it appears that the volatile solids reduction by anaerobic digestion is not useful for predicting the odors from anaerobically digested sludges.

Neo-mannosylated liposomes: synthesis and interaction with mouse Kupffer cells and resident peritoneal macrophages.

In order to target liposomes to cells expressing at their surface mannose receptors, e.g. mouse Kupffer cells and peritoneal macrophages, we have developed a new synthetic strategy which allows a chemically well defined preparation of neo-mannosylated vesicles. alpha-D-Thiomannopyranoside residues, substituted with a hydrophilic spacer arm and functionalized with a sulfhydryl group, were covalently coupled to preformed large unilamellar vesicles containing 4-(p-maleimidophenyl)butyryl phosphatidylethanolamine. Liposomes, containing 15 mol% of mannosyl residues, were specifically aggregated with concanavalin A; this aggregation could be reversed by an excess of free methyl alpha-D-mannopyranoside indicating that the surface ligands were freely accessible to the lectin. The neo-mannosylated liposomes presented in vitro an increased binding to cells possessing alpha-D-mannose specific binding sites. At 37 degrees C a specific binding, up to 9-fold compared to control vesicles, was observed. These neo-mannosylated vesicles represent attractive tools for targeting bio-active molecules to macrophage-associated diseases.

Targeting of liposomes by covalent coupling with ecto-NAD+-glycohydrolase ligands.

We have tested the feasibility of targeting liposomes via interaction with specific ecto-enzymes, i.e., enzymes which have their active site oriented to the external surface of the cell. 3,4-Dimethylpyridine adenine dinucleotide, a competitive inhibitor of ecto-NAD+-glycohydrolase, was substituted at N6 with a hydrophilic spacer arm, functionalized with a sulfhydryl group, and covalently linked to performed liposomes containing 4-(p-maleimidophenyl)butyryl phosphatidylethanolamine. We show that compared to control vesicles, the binding of the conjugated liposomes was greatly increased (up to 5-fold) to cells presenting ecto-NAD+-glycohydrolase activity (Swiss 3T3 fibroblasts, mouse peritoneal macrophages); in contrast, no specific binding was detected with hepatoma tissue culture cells, which lack this enzyme. Specific binding was found to depend on the ligand/lipid molar ratio of the vesicles and on the length of the arm. High concentrations of free 3,4-dimethylpyridine adenine dinucleotide virtually abolished the specific binding to cells of the targeted liposomes. Analysis of binding revealed that the ligand conjugated to the liposomes presented a functional affinity for 3T3 fibroblasts 15-fold superior to that of the free ligand.

Efficacy and safety of an olive oil-based intravenous fat emulsion in adult patients on home parenteral nutrition.

BACKGROUND: The most frequently used intravenous lipid emulsions are composed of 100% long chain triacylglycerols from soybean oil or of 50% long chain triacylglycerols-50% medium chain triacylglycerols. A newer emulsion, ClinOleic 20% containing 80% olive oil and 20% soybean oil, was suggested to reduce lipid peroxidation and immune function impairment. AIM: To assess ClinOleic 20%'s efficacy, safety and effect upon systemic inflammatory parameters in adults on home parenteral nutrition. METHODS: In stable home parenteral nutrition patients, the initial intravenous lipid emulsion was changed for ClinOleic 20%. Nutritional status, clinical and biological tolerance, and systemic inflammatory markers were analysed before and after 1 and 3 months of home parenteral nutrition, with ClinOleic 20% as intravenous lipid emulsion. RESULTS: Clinical and biological nutritional markers and inflammatory parameters did not differ between day 0 and month +3. There was no essential fatty acids deficiency. No side-effects were reported. Three of five patients presenting with migraine during home parenteral nutrition infusion at day 0 felt consistently better at month +3. CONCLUSIONS: ClinOleic 20% is safe and efficient in adult home parenteral nutrition. It maintains normal essential fatty acids status and did not influence inflammatory parameters. In contrast to studies in preterm infants or paediatric patients, no effect on vitamin E concentration or lipid peroxidation was observed.

Characterization of surface markers of continuously growing murine resident macrophages.

Conditions have been described which allow an in vitro indefinite multiplication of differentiated murine macrophages (Lombard et al: Biol Cell 53, 219, 1985). R. and MAY-1 cell lines, which were obtained, respectively, from mouse (Balb/c) spleen and resident peritoneal macrophages, have been further characterized. They present at their surface, besides the Mac-1 antigen and Fc-receptor, a mannose receptor which was characterized for its binding properties. This receptor is responsive for a specific phagocytosis of mannosylated particles, i.e., mannosylated latex beads or oil droplets containing mannosylated bovine serum albumin. Moreover, R and MAY-1 cells present an ectoenzyme profile (NAD+ glycohydrolase and nucleotide pyrophosphatase) similar to those of the corresponding resident macrophages.

Both mannose and beta-glucan receptors are involved in phagocytosis of unopsonized, heat-killed Saccharomyces cerevisiae by murine macrophages.

We studied the involvement of lectin-like receptors in phagocytosis of unopsonized heat-killed yeast (Saccharomyces cerevisiae) by murine macrophage-like cell lines and murine peritoneal resident macrophages. For this purpose we used a technique that allowed us to discriminate ingested and adsorbed heat-killed yeast. The internalization can be partly inhibited by soluble polyosides such as laminarin (beta-glucan) or alpha-mannan. However, when they were used together (0.4 mg/ml alpha-mannan and 0.4 mg/ml laminarin), almost complete inhibition of phagocytosis was obtained. These observations suggest that phagocytosis of unopsonized heat-killed yeast by murine macrophage-like cell lines as well as murine peritoneal resident macrophages is mediated by both mannose and beta-glucan receptors. The respective activity of these two types of receptors is a function of in vitro cell differentiation. To achieve maximal phagocytosis of unopsonized heat-killed yeast, coexpression of both mannose and beta-glucan receptors is required.

Mycophenolic acid suppresses protein N-linked glycosylation in human monocytes and their adhesion to endothelial cells and to some substrates.

Mycophenolic acid (MPA) is the active part of the corresponding morpholinoethyl ester pro-drug Mycophenolate Mofetil. MPA, an inhibitor of IMP dehydrogenase, depletes GTP and thereby suppresses transfer of mannose and fucose to proteins. Treatment of human monocytes with a clinically attainable concentration of MPA (10 microM) decreases their attachment to endothelial cells and to laminin, but not to type I collagen or fibronectin. Our results not only elucidate a major role of mannose/fucose residues in homing of monocytes on activated endothelium but also explain in part the beneficial effects of MPA in rheumatoid arthritis and organ graft rejection.

Cochlear function and transgene expression in the guinea pig cochlea, using adenovirus- and adeno-associated virus-directed gene transfer.

Development of a viral vector that can infect hair cells of the cochlea without producing viral-associated ototoxic effects is crucial for utilizing gene replacement therapy as a treatment for certain forms of hereditary deafness. In the present study, cochlear function was monitored using distortion-product otoacoustic emissions (DPOAEs) in guinea pigs that received infusions of either (E1(-), E3(-)) adenovirus, or adeno-associated virus (AAV), directly into the scala tympani. Replication-deficient (E1(-), E3(-)) adenovirus-directed gene transfer, using the cytomegalovirus (CMV) promoter, drove transgene expression to inner hair cells and pillar cells of the cochlea. AAV transduction was tested with several promoters, such as platelet-derived growth factor (PDGF), neuron-specific enolase (NSE), and elongation factor 1alpha (EF-1alpha) promoters; which drove transgene expression to cochlear blood vessels, nerve fibers, and certain spiral limbus cells, respectively. AAV transgene expression was visualized by green fluorescent protein immunostaining. Immunocytochemistry to heparan sulfate confirmed the absence of proteoglycans in guinea pig hair cells, indicating that the receptor for AAV was not present on these cells. However, the heparan sulfate proteoglycan expression pattern mimicked the AAV transduction pattern. An overall finding was that cochlear function was not altered throughout the infection period using AAV titers as high as 5 x 10(8) IP/infused cochlea. In contrast, cochlear function was severely compromised by 8 days postinfection with adenoviral titers of 5 x 10(8) PFU/infused cochlea, and outer hair cells were eliminated. Thus, cochlear hair cells are amenable to in vivo gene transfer using a replication-deficient (E1(-), E3(-)) adenovirus. However, replication-defective or gutted adenovirus vectors must be employed to overcome the ototoxic effects of (E1(-), E3(-)) adenovirus vectors.

CD14 expression by human mononuclear phagocytes is modulated by Clostridium difficile toxin B.

Toxin B, an exotoxin produced by the anaerobic Gram-positive bacteria Clostridium difficile, is responsible for pseudomembranous colitis in humans. It deeply modifies morphology of cultured cells and enhances their membrane surface area, which suggests a possible alteration of membrane receptor distribution. Since toxin B and bacterial lipopolysaccharide can act synergistically on TNF-alpha production by mononuclear phagocytes, the effect of toxin B on CD14 expression was investigated using flow cytometric analysis. It was shown that monocytes overexpressed CD14 after 5 h of treatment with toxin B. In contrast, after 24 h of treatment, the percentage of CD14 monocytes decreased, although, most frequently, the remaining positive cells expressed high levels of CD14 compared with untreated cells. Macrophages treated for 5 h with toxin B overexpressed CD14, but this effect persisted for at least 24 h. Both the percentage of positive macrophages and the mean level of CD14 per cell were increased. Thus toxin B can modulate expression of CD14 and its modulation depends on the differentiation status and maybe on the activation state, since some individual variations were observed in monocyte response to toxin.

Ovarian carcinoma cells are effectively transfected by polyethylenimine (PEI) derivatives.

As a prerequisite to nonviral gene therapy approaches of ovarian carcinoma, we evaluated the possibility of transfecting established tumor cell lines (SKOV3, IGROV1) as well as primary mesothelial and tumor cells by various polyethylenimine (PEI) derivatives. Several PEI-based vectors were able to effectively transfect these cells, as shown by high luciferase expression levels (10(8) to 10(9) relative light units per milligram of cell protein) that corresponded with 25-50% of green fluorescent protein-positive cells after 24 hours. However, unpredictable differences were observed among the vectors and cell types that a posteriori justified the screening procedure. We also showed that cells that were not transfected after the first experiment remained transfectable in a subsequent transfection experiment to a level similar to that of the initial population. This experiment does not support the emergence of a transfection-resistant cell population and opens the door to multiple therapeutic gene deliveries. Although efficacy and cell targeting still remain to be improved, PEI derivatives appear to be promising molecules for the development of nonviral gene therapy of ovarian carcinoma.

Effect of ammonia on endocytosis and cytokine production by immortalized human microglia and astroglia cells.

Ammonium acetate decreased in a concentration-dependent manner the phagocytic uptake of mannosylated latex microspheres and of yeast by immortalized human microglia (CHME-5) and astroglioma (GL-15) cells. In both cell lines ammonium acetate affected also the secretion of certain cytokines. The most conspicuous effects were the following: in both cell lines ammonium acetate enhanced greatly the secretion of tumor necrosis factor-alpha in the absence of any other stimulus. in the human microglia cells ammonia decreased the constitutive secretion of interleukin-6, but it enhanced the stimulated (interleukin-1 alpha, tumor necrosis factor-alpha, gamma-interferon and gamma-interferon + tumor necrosis factor-alpha) secretion of interleukin-8. In the astroglioma cell line, the stimulated release of tumor necrosis factor-alpha, interleukin-6 and interleukin-8 was diminished by ammonium acetate. The magnitude of the ammonia-effect depended on the stimulating agent (lipopolysaccharide, interleukin-1 alpha, tumor necrosis factor-alpha, gamma-interferon). The results are discussed with regard to their potential importance in the pathogenesis of human diseases with elevated blood and brain ammonia concentrations.

Latex beads phagocytosis capacity and ecto-NAD+ glycohydrolase activity of rat brain microglia cells in vitro.

Conditions are described which allow the preparation in vitro of pure (greater than 95%) microglial cell cultures isolated from newborn rat brain. Such ameboid cells cultivated in vitro can efficiently phagocytize opsonized latex beads and are capable of ingesting more (100-200 beads of 1.1 micron diameter per cell) and larger (6.4 microns) particles than other nerve cells, such as oligodendrocytes and astroglia. The microglial cells also show an important ecto-NAD+ glycohydrolase activity which is characteristic of phagocytic cells. We noted that the phagocytic capacity and ecto-NAD+ glycohydrolase of these cells were correlated and increased notably during the in vitro culture. Microglia cultivated in vitro appear to be a good model to study the activation of phagocytic properties in the central nervous system and corresponding modulation by natural or pharmacological immunomodulators.

Antitumoral properties and reduced toxicity of LPS targeted to macrophages via normal or mannosylated liposomes.

Neo-mannosylated liposomes have been prepared by coupling a mannose derivative bearing a hydrophilic spacer arm to preformed large unilamellar liposomes containing 4-(p-maleimidophenyl) butyryl-phosphatidylethanolamine. Lipopolysaccharide (LPS) was encapsulated in normal or neo-mannosylated liposomes; the neo-mannosylated vesicles showed specificity for the in vitro activation to toxicity of macrophages only in the case of differentiated macrophages presenting mannose receptors at their surface. In vivo, LPS entrapped in neo-mannosylated vesicles showed a reduced toxicity for animals hypersensitive to LPS. Moreover targeting of LPS to tissue macrophages with neo-mannosylated liposomes induced regression of experimental solid tumors in mice (EMT6 sarcoma, 3LL carcinoma) and was effective on lung metastases.

Oral versus transdermal selegiline: antidepressant-like activity in rats.

These studies compared the dose-response effects of oral vs. transdermal selegiline on antidepressant-like activity and brain monoamine oxidase (MAO) activities in rats. Rats received selegiline by gavage (0-100 mg/kg) or via transdermal patches (0-4.8 cm2, 0-8.7 mg/kg) daily for 7 days; antidepressant-like activity was determined using the forced-swim test. Following behavioral testing, cerebral cortices were assayed for MAO-A and MAO-B activities. Doses of selegiline that selectively inhibited MAO-B (3 and 10 mg/kg/day by gavage and 0.4 mg/kg/day via patch) did not alter either immobility or latency time. However, the oral administration of 30 or 100 mg/kg/day or the transdermal administration of 8.7 mg/kg/day, doses that led to greater than 70% inhibition of MAO-A, decreased immobility time significantly. The IC50s for inhibition of MAO-A following oral and transdermal administration for 7 days were 19.8 and 1.1 mg/kg, respectively. Results indicate that both oral and transdermal selegiline have antidepressant-like activity as assessed by the forced-swim test, and that transdermal administration, which bypasses first-pass metabolism, allows for using lower doses than oral administration.

Floc structure and the role of cations.

Cations have been found to influence the settling and dewatering characteristics of biological sludges. This research was directed at investigating the role of iron in floc stability of sensitivity to shear and in the response to anaerobic and aerobic digestion. Our data shows that iron may contribute to floc strength and it appears that the deterioration in sludge dewatering during anaerobic digestion is associated with the reduction and solubilization of iron. Further, it is the presence of proteins in solution that contributes to poor dewatering and to the demand for conditioning chemicals. The addition of iron for improving dewatering shows that iron may selectively coagulate solution proteins.

[How to test at once six cytokines in samples as small as 25 microl?]. / Comment doser simultanément par cytométrie en flux six cytokines sécrétées dans seulement 25 microL d'échantillon ?

Inflammatory and regulatory or anti-inflammatory cytokines (TNFalpha, IL-1beta, -6, -8, -10 and -12) regulate both the humoral and cellular immune responses. Cytokines have diverse peripheral and central functions. They are critical mediators of protective host responses, including defense against microbial invasion and tumorigenesis. However, the production of specific proinflammatory cytokines must be tightly regulated and compartmentalized to prevent the overexpression of these molecules that can end in chronic inflammation and tissue injury. Many diseases like autoimmune disease (rheumatoid arthritis, multiple sclerosis, arteriosclerosis, Crohn's disease), neurodegenerative disease (Alzheimer's and Parkinson's disease), tumor invasion and metastasis correlate with a deregulation in cytokine action. Thus, cytokines network provides an attractive and intensely competitive area of potential targets for therapeutic intervention. To monitor such secretion patterns in presence of putative drugs obtained by high throughput screening (HTS) some new techniques recently appeared on the market. We here compared results obtained by CBA (BD Cytometric Bead Array) to IC50 values obtained by classical sandwich Elisa. The complexity and cost of this new method is largely compensated by simultaneous testing of 6 cytokines in only 25 micro L of cell supernatant.

Fluorometric determination of polystyrene latex: application to the measurement of phagosomes and phagocytosis.

Intrinsic fluorescence of polystyrene dissolved in organic solvents such as 1,2-dimethoxyethane was used to develop a sensitive method for the quantification of polystyrene latex beads. This method allows the assay of latex in the microgram range and is one order of magnitude more sensitive than the conventional spectrophotometric method. The fluorometric technique was used in the quantification of phagocytic latex particle uptake by macrophages and in the quantification of isolated phagosomal fractions.