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PubMedDate not supplied

Improved in vitro gene transfer mediated by fluorinated lipoplexes in the presence of a bile salt surfactant.

BACKGROUND: Progress in the field of gene transfer with non-viral vectors requires systems that allow efficient gene expression in the presence of biological fluids such as pulmonary surfactants, for gene transfer to the respiratory epithelium for cystic fibrosis gene therapy, or bile salts (which contain powerful anionic detergents), for gene transfer to the biliary epithelium for gene therapy of the hepatobiliary disease associated with cystic fibrosis (CF). We have performed a comparative analysis of the disintegration and DNA accessibility of fluorinated and conventional lipoplexes, and their in vitro transfection potential in the presence of a powerful biliary surfactant. METHODS: The disintegration and DNA accessibility of conventional and fluorinated cationic lipoplexes and their in vitro transfection efficiency of human lung carcinoma epithelial A549 cells were studied in the presence of various concentrations of sodium taurocholate (STC), an anionic bile salt detergent. The conventional and fluorinated lipoplexes were formulated from Transfectam" (or DOGS) and from fluorinated lipospermines, analogs of DOGS, respectively, and a luciferase reporter plasmid. The fluorinated lipids used in the present study were selected for their different degrees of fluorination in order to investigate the impact on stability and transfection. The effects of the detergent on lipoplex integrity were examined by evaluating the ability of the lipospermines to prevent, in the presence of the surfactant, ethidium bromide (BET) intercalation into the plasmid (fluorescence monitoring). RESULTS: Fluorinated cationic lipoplexes exhibited greater stability than DOGS lipoplexes with respect to STC lytic activity. Indeed, while the DOGS lipoplexes were fully disintegrated at a [STC]/[lipid] molar ratio of 1,320, all the DNA intercalation sites of the most fluorinated lipoplexes investigated became accessible to BET for a two-fold higher [STC]/[lipid] molar ratio. A higher transfection potential in the presence of the detergent was also shown for the fluorinated lipoplexes as compared with the DOGS preparation. At a 10 mM concentration of STC and at a [STC]/[lipid] molar ratio of 264, lipofection when mediated by DOGS was fully inhibited while the detergent had no inhibitory effects on the lipofection mediated by the fluorinated DF4C11-GS [spermine-5-carboxyglycine N,N-di-11-(F-butyl)-undecylamide] or DF6E11-GS [spermine-5-carboxyglycine N,N-di-[11-(F-hexyl)-undec-10-enyl]amide] lipospermines. A higher detergent concentration (up to 17.5 mM) and a higher [STC]/[lipid] ratio (up to 462) were necessary to inhibit lipofection by the fluorinated formulations. Overall, the lipoplex stability and transfection potential in the presence of the detergent was found to improve with increasing degrees of fluorination of the lipospermines. CONCLUSIONS: The present work shows improved stability of, and higher lipofection levels with, fluorinated lipoplexes in the presence of surfactants. The results confirm the very promising potential of fluorinated lipoplexes as gene transfer vectors. These compounds constitute a very attractive alternative to their more conventional homologs. The correlation found between the degree of fluorination of the lipoplexes, their stability and their lipofection levels suggests that enhanced lipophobic and hydrophobic properties protect them against disintegration and, consequently, prevents DNA from being degraded and from interacting with lipophilic and hydrophilic biocompounds responsible for lipofection inhibition.

Bile Acids and Salts↗
PubMed1995

Immunochemical features of a macromolecule of Treponema denticola.

In this study the extraction and the immunochemical features of a lipopolysaccharide-like (LPSL) macromolecule of T. denticola strains 35405, 35404, 33521 and 11 were investigated. The yield of LPSL molecule ranged between 0.5-0.9% of the cell dry weight, it possessed Limulus amebocyte lysate clotting activity, and it contained glucosamine, phosphate, heptose, glucose, small amounts of KDO, myristic and beta hydroxy myristic acid. Sera obtained from healthy individuals (ADA type I) periodontitis, from 3-8 month old infants, or the mouse monoclonal antibody, diluted 1:2, against T. pallidum did not react with the LPSL antigens of T. denticola strains 35405, 35404, 33521, and 11. Sera from patients with ADA type III-IV periodontitis were reactive with two 8-14 kDa bands even at serum dilutions of 1:2000. Sera from patients with ADA type II periodontitis showed good antibody response to the 8-14 kDa band at a dilution of 1:50, but were weekly reactive, or nonreactive at serum dilutions of 1:200. This study indicates that extraction of a lipopolysaccharide-like macromolecule is feasible from the assay spirochetes, and this macromolecule may be used as an antigen for the diagnosis of ADA types II-IV periodontitis.

Adult↗
PubMed1980

Terminal changes in mice experimentally infected with Borrelia duttoni.

The terminal changes following Borrelia duttoni infection in mice consisted of disseminated intravascular coagulation with associated depression in temperature, and in white cell and platelet counts with the development of a positive limulus lysate test. Apart from thrombosis and haemorrhage, similar changes were found in mice given cyclophosphamide prior to infection. The results suggest that neither intravascular coagulation nor immune complex formation are major factors in causing death in borreliosis.

Animals↗
PubMed2006

Design of a minimized cyclic tetrapeptide that neutralizes bacterial endotoxins.

Septic shock is a leading cause of mortality in intensive care patients, and no specific drugs are as yet available for its treatment. Therefore, new leads are required in order to increase the number of active molecules that may develop into efficacious and safe LPS-neutralizing molecules during pre-clinical stages. We used peptides, derived from the binding regions of known LPS-binding proteins, as scaffolds to introduce modifications at the amino acid level. Structure-activity relationship studies have shown that these modifications generate highly active peptides. Thus, from a bioactive peptide with an initial 16 amino acid residues, a tetrapeptide sequence was determined. After inserting this sequence in a Cys cyclic peptide, it showed the same biological activity as the parent peptide. This sequence could provide the basis for the design of small molecules with LPS-binding properties.

Amino Acid Sequence↗
PubMed1995

Biotinylated lipopolysaccharide binds to endotoxin receptor in endothelial and monocytic cells.

Endotoxin or lipopolysaccharide (LPS), a major cell surface component of gram-negative bacteria, which could bind to different cell types when released into the bloodstream, plays a central role in the pathogenesis of septic shock syndromes. We have established a biotinylation procedure for labeling purified LPS molecules from Salmonella minnesota R595 and Escherichia coli J5 bacteria. The biotin group was conjugated to the bacterial LPS either by chemical oxidation of the LPS carbohydrate moiety (inner core region), followed by reduction with biotin-LC-hydrazide (biotinamido hexanoyl hydrazide), or by photoactivatable cross-linking with biotin-LC-ASA [1-(4-azidosalicylamido-)-6(biotinamido)-hexane], which was randomly attached to the carbohydrate and fatty acid (lipid A) groups of the LPS. Both labeled products retained biological activity (or endotoxicity) as evidenced by coagulation of the Limulus amoebocyte lysate. To determine its ability to bind avidin/streptavidin which in turn could be conjugated with enzymatic and fluorescent probes, the biotinylated LPS was used in enzyme immunoassay, Western blot, and flow cytometry. These assays were also used to analyze the binding of LPS ligand to its counterreceptor(s) on whole cell surface, membrane fragments, and in detergent lysates from human endothelial and monocytic cells. The described biotinylated LPS probes can be applied in a wide variety of techniques in receptor biochemistry, immunohistochemistry, and molecular cell biology.

Avidin↗
PubMed1998

Proteinase K digestion of proteins improves detection of bacterial endotoxins by the Limulus amebocyte lysate assay: application for endotoxin removal from cationic proteins.

Cationic proteins, such as lysozyme, ribonuclease A, and human IgG, impaired the detection of endotoxins with the Limulus amebocyte lysate assay (LAL assay) through formation of endotoxin-protein complexes, demonstrating pronounced masking of endotoxins. Methods, such as phenol extraction, dilution heating, and perchloric acid treatment failed to demask the endotoxins. Also, digestion with trypsin, chymotrypsin, or pronase recovered only 10 to 20% of the applied endotoxins. However, endotoxin recoveries up to 100% were obtained with proteinase K digestion of the samples prior to the LAL assay. This method was then applied to examine the impact of endotoxin masking on endotoxin removal from protein solutions by selective adsorption on membrane adsorbers. It was found that poly-L-lysine and poly(ethyleneimine) as endotoxin-selective ligands were able to pull endotoxins off the proteins studied, thereby guaranteeing successful decontamination.

Adsorption↗
PubMed1983

A novel micromethod for the Limulus amebocyte lysate (LAL) assay for endotoxin, based on hydrostatic pressure.

A micromethod for performing the Limulus amebocyte lysate (LAL) assay for the presence of endotoxin is described which requires only about five percent of the standard amount of LAL reagent. The method is based upon measuring an increased amount of hydrostatic pressure required to cause a gelled LAL sample to flow from a capillary tube. The method is simple, rapid and correlates well with the results of the standard LAL assay procedure.

Animals↗
PubMed1994

Production of purified alginates suitable for use in immunoisolated transplantation.

Alginate is used as a matrix for immunoisolation of cells and tissues in vivo. We have demonstrated previously that commercial alginates contain various fractions of mitogenic impurities and that they can be removed by free flow electrophoresis. The use of purified material is a necessity in order to reveal the parameters that control biocompatibility of the implanted material (such as stability, size, surface charge and curvature, etc.). In this study, we present a protocol for the chemical purification of alginates on a large-scale. Beads made from alginates purified by this multi-step chemical extraction procedure did not induce a significant foreign body reaction when implanted for 3 weeks either intraperitoneally or beneath the kidney capsule of Lewis or non-diabetic BB/Gi rats.

Alginates↗
PubMed1987

Immunochemical and partial chemical characterization of fractions of membrane-bound smooth lipopolysaccharide-protein complex from Brucella abortus.

Smooth lipopolysaccharide (sLPS) of Brucella abortus was prepared and fractionated by a modification of the procedures of Moreno et al. (J. Bac. 138:361-369, 1979). Washed B. abortus cells were disrupted by 21 freeze-quick thaw cycles with ultrasonication to separate the non-membrane-bound material. Ultrasonicated bacteria were used for preparation of membrane-bound sLPS (approximately f5, the main crude sLPS fraction described by Moreno et al.). Phenol extraction was repeated 3 times and then washed with H2O 10 times to remove most of the chromogen, polysaccharides and nucleic acids, eliminating the need for enzyme treatment as described previously. The membrane-bound sLPS was fractionated into 3 to 5 groups according to the extent of dialysis and centrifugation, these fractions required only 80 ng for positive ELISA, about 0.2 ng for positive Limulus lysate tests, and reacted well with precipitating antibodies in the serum from a strain 2308 infected cow. They had marked differences in precipitin curves and chemical composition. The protein content varied from 16% to 42% as determined by dye binding test and 17 to 60% by Lowry phenol method using bovine serum albumin as the standard, which implies that the proteins associated with LPS may also play important roles in the complex for the immunochemical interactions and the heterogeneity of B. abortus lipopolysaccharide protein complex. As compared with previous reports, a higher yield of sLPS, ranging from 3.6% to 7.7% of dried bacteria, was obtained. Group f5A, which had a standard bell shaped curve in the precipitin assay, is one of the major fractions in all three strains (1119.3, 19 and 2308). The amount of other subfractions obtained varied with batches or strains of B. abortus. These results provide a new profile of the immunochemical reactivities and the heterogeneity on B. abortus smooth membrane-bound endotoxins.

Antigen-Antibody Complex↗
PubMed1993

Effects of medium composition on murine and human blastocyst formation and hatching rate.

PURPOSE: Murine two-cell embryos (n = 5573) were cultured for 96 hr in human tubal fluid (HTF) medium (n = 2709) or alpha modification of minimum essential medium (MEM; n = 2864) through the hatched blastocyst stage from mid-1990 to mid-1991. An additional 373 embryos were cultured in MEM or HTF with 0, 1, 5, or 10 ng/ml E. coli endotoxin. A total of 17 patients had supernumerary embryos simultaneously cultured in HTF (n = 48) or MEM (n = 61). Additionally, pregnancy rates were compared for July to December 1990, when MEM was used as growth medium, and for July to December 1989, when HTF was used. RESULTS: Blastocyst formation was higher (P < 0.001) for murine embryos cultured in MEM (blasts = 95%) compared to HTF (blasts = 70%). When cultured with endotoxin, blastocyst formation was higher (P < 0.01) for embryos cultured in MEM compared with HTF for controls and at each endotoxin level. No difference in human blastocyst development was observed in HTF and MEM. However, more MEM-cultured blastocysts were cryopreserved (P < 0.05). There also was a lower spontaneous abortion rate and a higher multiple gestation rate when embryos were cultured in MEM. CONCLUSION: Thus, MEM may result in healthier blastocyst development, especially when culture conditions are substandard, although this is not an acceptable substitution for meticulous technique.

Animals↗