Biomedical subjects

J Lagacé

Publications and source records attributed to J Lagacé.

At least 19 recordsLinked to original sources

PubMed2002

Differential behaviour of fluid liposomes toward mammalian epithelial cells and bacteria: restriction of fusion to bacteria.

Previous work demonstrated that fluid liposomes developed in our laboratory are able to fuse with bacterial outer membranes. This fusion improved the penetration and activity of liposome-encapsulated antibiotics and antisense oligonucleotides into the bacterial cells. Because it is anticipated that fluid liposome encapsulated antibiotics will be administered by aerosols to patients with chronic pulmonary infections or cystic fibrosis (CF), we conducted comparative studies in E. coli, P. aeruginosa and human lung epithelial cells using lipid-mixing assays to investigate the possibility that fluid liposomes might fuse with surrounding epithelial cells. After a 2 h incubation at 4 and 37 degrees C, no fusion between fluid liposomes and human lung epithelial cells was observed, whereas mean levels of 71 and 37% of fusion were observed at 37 degrees C with E. coli and P. aeruginosa cells, respectively. No fusion was observed at 4 degrees C in any cells. A kinetic study where temperature was gradually increased from 7 to 37 degrees C indicated that the fusion process in the two bacteria starts between 28 and 31 degrees C with a mean fusion rate of 0.60%/min at 31 degrees C to reach 1.18%/min at 37 degrees C. The present work suggests that it is unlikely that fluid liposomes fuse with host cells lining the human respiratory tract and further elucidates the fusogenic properties of fluid liposomes with respect to prokaryotes and eukaryotes.

Bacteria↗

PubMed2001

Encapsulation of DNA in negatively charged liposomes and inhibition of bacterial gene expression with fluid liposome-encapsulated antisense oligonucleotides.

Antisense therapy for the treatment of bacterial infections is a very attractive alternative to overcome drug resistance problems. However, the penetration of antisense oligonucleotides into bacterial cells is a major huddle that has delayed research and application in this field. In the first part of this study, we defined efficient conditions to encapsulate plasmid DNA and antisense oligonucleotides in a fluid negatively charged liposome. Subsequently, we evaluated the potential of liposome-encapsulated antisense oligonucleotides to penetrate the bacterial outer membrane and to inhibit gene expression in bacteria. It was found that 48.9+/-12% and 43.5+/-4% of the purified plasmid DNA and antisense oligonucleotides were respectively encapsulated in the liposomes. Using fluorescence-activated cell sorting analysis, it was shown, after subtraction of the fluorescence values due to the aggregation phenomenon measured at 4 degrees C, that about 57% of bacterial cells had integrated the encapsulated antisense oligonucleotides whereas values for free antisenses were negligible. The uptake of the encapsulated anti-lacZ antisense oligonucleotides resulted in a 42% reduction of beta-galactosidase compared to 9% and 6% for the encapsulated mismatch antisense oligonucleotides and the free antisense oligonucleotides respectively. This work shows that it is possible to encapsulate relatively large quantities of negatively charged molecules in negative fluid liposomes and suggests that fluid liposomes could be used to deliver nucleic acids in bacteria to inhibit essential bacterial genes.

DNA↗

PubMed2001

Development of a rapid and sensitive test for identification of major pathogens in bovine mastitis by PCR.

Bovine mastitis is the most important source of loss for the dairy industry. A rapid and specific test for the detection of the main pathogens of bovine mastitis is not actually available. Molecular probes reacting in PCR with bacterial DNA from bovine milk, providing direct and rapid detection of Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus parauberis, and Streptococcus uberis, have been developed. Two sets of specific primers were designed for each of these microorganisms and appeared to discriminate close phylogenic bacterial species (e.g., S. agalactiae and S. dysgalactiae). In addition, two sets of universal primers were designed to react as positive controls with all major pathogens of bovine mastitis. The sensitivities of the test using S. aureus DNA extracted from milk with and without a pre-PCR enzymatic lysis step of bacterial cells were compared. The detection limit of the assay was 3.125 x 10(2) CFU/ml of milk when S. aureus DNA was extracted with the pre-PCR enzymatic step compared to 5 x 10(3) CFU/ml of milk in the absence of the pre-PCR enzymatic step. This latter threshold of sensitivity is still compatible with its use as an efficient tool of diagnosis in bovine mastitis, allowing the elimination of expensive reagents. The two PCR tests avoid cumbersome and lengthy cultivation steps, can be performed within hours, and are sensitive, specific, and reliable for the direct detection in milk of the six most prevalent bacteria causing bovine mastitis.

Animals↗

PubMed2001

Trafficking of surface-linked and encapsulated liposomal antigens in macrophages: an immunocytochemical study.

Liposomal antigens are potent adjuvants of humoral and cell-mediated immunity. Although this property requires as an essential condition a physical association between the antigen and the phospholipid vehicle, the nature of the association, i.e., encapsulation or surface linkage, markedly influences the outcome of the elicited response. Available evidence suggests that macrophages are involved in this fine tuning of the immune response in a manner that is not yet clearly established. It is postulated that this might be related to their capacity to interact differently with surface-linked and encapsulated formulations. Using conalbumin as a model antigen, we address the question by analyzing the movements of encapsulated and surface-linked antigen as well as those of MHC-II molecules in macrophages in a pulse-chase immunoelectron microscopic study carried out over a 24-hr period. The antigen was followed using a polyclonal serum specifically raised against fragmented conalbumin (fCA) that allows the detection of processed antigen and of some MHC-peptide complexes. The results indicate that, in macrophages, the two liposomal formulations affect macrophage morphology in distinct ways and circulate through the various subcellular compartments with different kinetics. On the basis of the overall results, we conclude that surface-linked antigen gains access less readily to the endogenous presentation pathway than encapsulated antigen but can favor a more sustained activation of the immune system through its production of exosome-like structures and its more thorough utilization of the MHC-II pathway.

Animals↗

PubMed2000

Demonstration of intracellular Staphylococcus aureus in bovine mastitis alveolar cells and macrophages isolated from naturally infected cow milk.

Numerous in vitro studies have demonstrated that Staphylococcus aureus may be internalized and survive in a bovine mammary epithelial cell line. We report here the presence of internalized and living S. aureus in alveolar cells and macrophages in milk samples of bovine mastitis. We used fluorochrome labeled monoclonal antibodies, specifically recognizing surface cell markers of bovine alveolar cells and macrophages, to isolate these two types of cells using fluorescence activated cell sorting. Extracellular bacteria and DNA were previously eliminated to exclude possible contamination. In order to detect intracellular bacterial DNA inside the isolated cells, we used PCR amplification of bacterial DNA and the PCR products were analyzed by Southern blot with a specific probe for Staphylococcus. The results showed the presence of Staphylococcus DNA inside the two isolated populations of cells, confirming that S. aureus could penetrate alveolar cells and macrophages. The demonstration of the presence of intracellular living S. aureus was determined by bacteriological culture of positive samples plated onto blood agar plates and by its further identification. Our results showed for the first time that living S. aureus and its DNA are present in both alveolar cells and macrophages in chronically infected cow milk.

Animals↗

PubMed2000

Demonstration of a fusion mechanism between a fluid bactericidal liposomal formulation and bacterial cells.

It was previously demonstrated that fluid liposomal-encapsulated tobramycin, named Fluidosomes, was successful in eradicating mucoid Pseudomonas aeruginosa in an animal model of chronic pulmonary infection, whereas free antibiotic did not reduce colony-forming unit (CFU) counts (C. Beaulac et al., Antimicrob. Agents Chemother. 40 (1996) 665-669; C. Beaulac et al., J. Antimicrob. Chemother. 41 (1998) 35-41). These liposomes were also shown to be bactericidal in in vitro tests against strong resistant P. aeruginosa 64 microg/ml). The time needed to reach the maximal fusion rate was about 5 h for the resistant strain comparatively to much shorter time for the sensitive strain. The specific characteristics of Fluidosomes could help overcome bacterial resistance related to permeability barrier and even enzymatic hydrolysis considering the importance of synergy in the whole process of antibiotic resistance.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1999

Evaluation of the pulmonary and systemic immunogenicity of Fluidosomes, a fluid liposomal-tobramycin formulation for the treatment of chronic infections in lungs.

In previous studies, we have developed a fluid bactericidal liposomal formulation containing tobramycin, called Fluidosomes, which has been shown to be highly bactericidal both in in vitro and in in vivo studies against Pseudomonas aeruginosa and other related and unrelated bacteria. One foreseeable application of these Fluidosomes is the treatment of chronic pulmonary infections in cystic fibrosis patients colonized with P. aeruginosa and other related bacteria. Considering the capacity of some liposomal preparations to play an adjuvant role in vaccines, the non-immunogenicity of Fluidosomes has to be demonstrated. The systemic and local immunogenicity of Fluidosomes were assessed by effectuating repeated intraperitoneal (i.p.) and intratracheal (i.t. ) immunizations in BALB/c mouse. No significant mucosal and serum immune responses against Fluidosomes and/or tobramycin were detected as compared with preimmune sera. These data suggest that Fluidosomes could be administered repeatedly without adverse immune responses to control chronic pulmonary infections in cystic fibrosis.

Animals↗

PubMed1999

Aerosolization of low phase transition temperature liposomal tobramycin as a dry powder in an animal model of chronic pulmonary infection caused by Pseudomonas aeruginosa.

Eradication of mucoid Pseudomonas aeruginosa in an animal model of chronic pulmonary infection has been previously demonstrated following the intratracheal administration of Fluidosomes, a low phase transition temperature (low T(C)) liposomal tobramycin preparation administered in liquid form (Beaulac et al., Antimicrob. Agents Chemother., 40, 665-669, 1996). In the present work, the same liposomal formulation was administered as a dry powder aerosol to an animal model of chronic pulmonary infection in view of a possible clinical development in cystic fibrosis patients. Chronic infection was established by intratracheal administration of 10(5) cfu of a mucoid variant of P. aeruginosa, PA 508, prepared in agar beads. Sixteen hours after one aerosol treatment, the cfu counts performed on lungs (pair) treated with liposomal tobramycin were of 4.31x10(5) cfu/lungs comparatively to 1.32x10(8) and 3.02x10(8) cfu/lungs respectively in untreated and in lungs treated with free antibiotic. Considering the quantity of liposome-tobramycin that has reached the lungs, the results suggest that aerosolization of low T(C) liposomal tobramycin used as a dry powder preparation could be an effective way of treating chronic pulmonary infection caused by Pseudomonas.

Aerosols↗

PubMed1998

Aminoglycoside detection using a universal ELISA binding procedure onto polystyrene microtiter plates in comparison with HPLC analysis and microbiological agar-diffusion assay.

The use of enzyme-linked immunosorbent assay for the detection of aminoglycosides has been hindered due to low molecular weight compound adsorption to solid phases. Here, we describe an enzyme-linked immunosorbent assay based on the treatment of polystyrene microtiter plates with Alcian blue prepared in acetic acid prior to coating with the antibiotic. Whereas no detection of tobramycin was possible on commercially treated or untreated enzyme-linked immunosorbent assay plates, the Alcian blue treatment permitted detection of 0.025 and 0.05 microg ml(-1) of tobramycin respectively using 0.05 and 0.1% of Alcian blue with a coefficient of variation of 1.85 and 7.69%, respectively. Comparative studies of five tobramycin samples of unknown quantity using enzyme-linked immunosorbent assay and high-performance liquid chromatography gave equivalent results while those done via microbiological agar-diffusion assay were an overestimation of the actual quantity. The use of the Alcian blue pretreatment enzyme-linked immunosorbent assay procedure has permitted, in previous studies, the measure of antibodies against synthetic peptides and phospholipids. Subsequently, our demonstration of the sensitivity and reliability of this method in the quantification of tobramycin strongly suggests that the use of Alcian blue pretreatment in enzyme-linked immunosorbent assay can be applied universally to avert molecule immobilization problems on solid phases.

Agar↗

PubMed1996

Measurement of mouse anti-phospholipid antibodies to solid-phase microspheres by both flow cytofluorometry and Alcian blue-pretreated microtitre plates in an ELISA.

Conventional solid-phase immunoassays measuring interactions between anti-phospholipid antibodies and phospholipids are generally characterized by problems of reproducibility and high levels of non-specific binding. Here we describe two immunoassays based on the use of phospholipids in the form of solid-phase microspheres to measure the presence of anti-phospholipid antibodies in sera. Following the production of antibodies in mice against liposomes containing lipid A, we show that flow cytofluorometric analysis provides a reproducible and sensitive way to detect anti-phospholipid antibodies. We also present a sensitive, rapid and reproducible enzyme-linked immunosorbent assay (ELISA) using Alcian blue pretreated microtitre plates and solid-phase microspheres as coating antigen. This ELISA permitted the detection of antibodies to 1/1000 dilution, while untreated plates gave negative results. Such modified ELISA procedures may be applicable to other types of molecule exhibiting solid-phase binding problems e.g. synthetic peptides (J. Immunol. Methods 175 (1994) 131-135).

Alcian Blue↗

PubMed1996

Eradication of mucoid Pseudomonas aeruginosa with fluid liposome-encapsulated tobramycin in an animal model of chronic pulmonary infection.

Despite controversies associated with forms and value of antibiotic therapy for cystic fibrosis patients, antibiotherapy remains a cornerstone in the management of those patients. Locally administered liposome-encapsulated antibiotics may offer advantages over free antibiotics, including sustained concentration of the antibiotic, minimal systemic absorption, reduced toxicity, and increased efficacy. We evaluated the efficacy of free and encapsulated tobramycin in fluid and rigid liposomal formulations administered to rats chronically infected with Pseudomonas aeruginosa. Chronic infection in lungs was established by intratracheal administration of 10(5) CFU of a mucoid variant of P. aeruginosa PA 508 prepared in agar beads. Antibiotic treatments were given intratracheally at time intervals of 16 h. After the last treatment, lung bacterial counts were determined and tobramycin levels in the lungs and kidneys were evaluated by high-performance liquid chromatographic analysis and microbiological assay. Two independent experiments showed that animals treated with encapsulated tobramycin in fluid liposomes had a number of CFU less than the minimal CFU number required to be statistically acceptable compared with > or = 10(6) CFU per pair of lungs for animals treated with encapsulated tobramycin in rigid liposomes, free antibiotic, or liposomes without tobramycin. Tobramycin measured in the lungs at 16 h after the last treatment following the administration of encapsulated antibiotic was still active, and its concentration was > or = 27 micrograms/mg of tissue. Low levels of tobramycin were detected in the kidneys (0.59 to 0.87 micrograms/mg of tissue) after the administration of encapsulated antibiotic, while 5.31 micrograms/mg of tissue was detected in the kidneys following the administration of free antibiotic. These results suggest that the local administration of fluid liposomes with encapsulated tobramycin could greatly improve the management of chronic pulmonary infection in cystic fibrosis patients.

Animals↗

PubMed1995

Production of ScFv antibody fragments following immunization with a phage-displayed fusion protein and analysis of reactivity to surface-exposed epitopes of the protein F of Pseudomonas aeruginosa by cytofluorometry.

To increase the possibilities of obtaining antibodies to surface-exposed epitopes of Pseudomonas aeruginosa protein F, we immunized mice with cloned and expressed oprF gene as a gIII-fusion protein displayed on the M13 phage surface. The fusion protein elicited mouse antibodies reacting with the purified protein F at a limit dilution of 1:10,000. Recombinant clones expressing antibody fragments were constructed from the genes of selected B cells of hyperimmunized mouse after a first round of panning against the protein F. Expression of single chain Fv (ScFv) antibody fragments to the protein of P. aeruginosa was detected by ELISA in 20 of 384 clones obtained after the first panning selection. The 20 positive clones recognizing different protein F epitopes as demonstrated by ELISA were assayed by flow cytometry to identify antibody fragments reacting only with surface-exposed epitopes of the protein F on whole bacteria; one of the 20 clones tested showed a level of reactivity compatible with surface-exposed epitope that can lead to ulterior developments in targeting studies.

Animals↗

PubMed1995

IgG subclass responses to Pseudomonas aeruginosa a- and b-type flagellins in patients with cystic fibrosis: a prospective study.

Sera from 20 cystic fibrosis patients, whose lungs were colonised by Pseudomonas aeruginosa, were examined in a 3-5-year prospective study for any relationship between IgG subclass antibody levels to P. aeruginosa a- and b-type flagellins and pulmonary function (FEV1 and radiological score). Patients were divided into two groups according to their pulmonary status: group 1 comprised 11 patients with poor pulmonary status; group 2 comprised nine patients with relatively good pulmonary status. High concentrations of IgG1, IgG2 and IgG3 antibodies to flagellins, particularly to the b-type, were found in most patients. IgG4 reactivity was observed in only a few patients. Comparison of the two groups of patients showed that those with poor pulmonary status (group 1) had a significantly higher concentration (p < 0.05) of IgG3 for two of the three periods studied and of IgG2 for the last period studied. Moreover, IgG3 and IgG1 reactivities to b-type flagellin and IgG3 to a-type flagellin were also increased significantly (p < 0.05) in group 1 patients between the first and the last period studied. These patients also showed a significant (p < 0.05) time-dependent increase in IgG3 and IgG1 antibody concentrations. These data demonstrate that cystic fibrosis patients with poorer pulmonary status have higher IgG3 levels to flagellin than other cystic fibrosis patients. High concentrations of strong opsonic IgG3 and, to a lesser degree, of IgG1 antibodies may increase pulmonary inflammation and induce heightened pulmonary deterioration.

Adolescent↗

PubMed1994

Alcian blue-treated polystyrene microtitre plates for use in an ELISA to measure antibodies against synthetic peptides.

Short peptides often do not bind well to the plastic surface of microtitre wells in solid-phase immunoassays. To improve the reactivity of synthetic peptides coated on the solid phase, we have developed a highly sensitive, rapid and simple ELISA. This ELISA is based on the treatment of microtitre plates with Alcian blue prepared in acetic acid prior to coating the target peptide. With hyperimmune serum, this treatment increases the specific signal in such a manner that the detection of antibodies needs less antigen than with conventional ELISA. Moreover, the Alcian blue treatment was shown to be particularly effective for the detection of monoclonal antibodies against a synthetic peptide derived from the human immunodeficiency type 1 (HIV-1) vpu protein. These monoclonal antibodies that were not detected in conventional ELISA gave a positive signal up to a 1 in 1000 dilution after Alcian blue treatment of microtitre wells. This assay should be applicable to a variety of synthetic peptides and other types of molecules posing problems in assays requiring their immobilization on solid phases.

Alcian Blue↗

PubMed1994

Use of oligonucleotide probes to analyse the homology of the oprF gene among clinical and heterologous immunotype strains of Pseudomonas aeruginosa.

The conservation of oprF gene among 25 clinical Pseudomonas aeruginosa strains and a set of 17 serotype-specific representative strains of the international antigen typing scheme (IATS) was analysed by dot-blotting using five specific oligonucleotide probes. The oligo 1, 2, 3, 4, 5 correspond to five different regions of the oprF gene and hybridized strongly with respectively 88%, 88%, 76%, 94% and 71% of the IATS strains and 88%, 96%, 92%, 88% and 92% of the clinical strains. A parallel study performed with the whole oprF gene showed a lack of specificity of this probe: indeed, the probe hybridized not only with the 42 Pseudomonas aeruginosa strains but also with Escherichia coli and Salmonella minnesota. This study suggests that the gene sequence encoding the protein F is not totally conserved among Pseudomonas aeruginosa strains.

Base Sequence↗

PubMed1994

Pulmonary retention of free and liposome-encapsulated tobramycin after intratracheal administration in uninfected rats and rats infected with Pseudomonas aeruginosa.

The pulmonary residence time of free and liposome-encapsulated tobramycin was studied with uninfected rats and rats infected with Pseudomonas aeruginosa. Chronic infection in lungs was established by intratracheal administration of 10(8) CFU of P. aeruginosa PA 508 prepared in agar beads. After 3 days, a single dose (300 micrograms) of free or liposome-encapsulated tobramycin was given intratracheally to both infected and uninfected rats. At various time intervals (0.25 to 16 h) after drug instillations, the remaining tobramycin was evaluated in blood, lungs, and kidneys by a microbiological assay. Intratracheal instillation of liposome-encapsulated tobramycin resulted in high and sustained levels of tobramycin in lungs of uninfected and infected rats over the 16-h period studied; however, the tobramycin levels were two times higher in uninfected rats. There was no tobramycin detected in the blood or kidneys from these animals. In contrast, the intratracheally instilled free tobramycin was cleared within 3 and 1 h from the lungs of uninfected and infected animals, respectively. These data suggest that the encapsulation of tobramycin in liposomes can result in a significant increase of its residence time within lungs. This study also shows that pulmonary infection was associated with a lowering of tobramycin levels in lungs.

Animals↗

PubMed1992

Quantitative analysis of immunoglobulin G subclass responses to Pseudomonas aeruginosa antigens in cystic fibrosis.

The four subclasses of IgG have different structures, functions and implications in the antibody response. IgG subclass reactions to individual Pseudomonas aeruginosa structural antigens in 22 adolescents and young adults with cystic fibrosis (CF) were studied qualitatively and quantitatively by densitometric analysis of Western blot assays. These patients had been infected by P. aeruginosa for 7 years or longer and were divided into two groups according to their pulmonary status: Group 1 comprised 11 patients with relatively good pulmonary status; Group 2 consisted of 11 patients with poor pulmonary status. There was a relative decrease of IgG1 and a relative increase of IgG2 and, especially, of IgG3 and IgG4 antibodies against P. aeruginosa antigens in the CF patients. Comparison of the two CF patient groups showed a significant increase in the proportion of IgG3 in the Group 2 patients. This could be a potential cause or effect in the deterioration of their pulmonary function. Densitometric analysis of Western blots revealed more than 24 P. aeruginosa antigens and indicated those that were the targets of the isotype antibody response(s) that were apparently most harmful. Thus, there was a significant increase of IgG2 or IgG3 reactivity (or of both) against proteins F, H (H1 and H2), and I in the Group 2 patients. One other striking observation of this study was the high reactivity of IgG4 antibodies to protein H. IgG4 was the major antibody to this protein in seven of the 11 Group 1 patients compared to two of the 11 in Group 2. We hypothesise that IgG4 antibodies may antagonise IgG2 antibodies, helping to preserve stable pulmonary function.

Adolescent↗

PubMed1991

Four epitopes of Pseudomonas aeruginosa elastase defined by monoclonal antibodies.

Monoclonal antibodies against the elastase of Pseudomonas aeruginosa were produced from spleen cells of BALB/c mice primed with purified elastase of P. aeruginosa and P3-X63-Ag8-U1 myeloma cells. The six clones established generated antibodies which reacted with a 33,000-Da peptide and recognized four different elastase epitopes by a competitive binding enzyme-linked immunosorbent assay. The monoclonal antibodies designated as ELA-17 and ELA-42 that recognize two different epitopes reacted by dot-enzyme immunoassay and by Western immunoblotting with all clinical and International Antigen Typing Scheme strains of P. aeruginosa positive for elastase.

Animals↗