Biomedical subjects

M E Klegerman

Publications and source records attributed to M E Klegerman.

At least 19 recordsLinked to original sources

PubMed2001

Improving ultrasound reflectivity and stability of echogenic liposomal dispersions for use as targeted ultrasound contrast agents.

Targeted echogenic liposome dispersions for ultrasonic enhancement of vasoactive and pathological components of endothelium and atherosclerosis have recently been developed. The component lipids required for acoustic and targeting properties include phosphatidylcholine, phosphatidylethanolamine phosphatidylglycerol (PG), and cholesterol (CH), initially in a 60:8:2:30 mol % ratio. Component lipids, lyophilization, sugars, and freezing conditions were varied to optimize acoustic ultrasound reflectivity and acoustic stability. Echogenic liposome dispersions were made by using the dehydration-rehydration process. The lipid concentrations were varied (CH in the range 1 to 40 mol % and PG from 1 to 16 mol %). Variations in type and concentration of sugars were examined. The effect of freezing conditions and re-lyophilization was examined. Ultrasound reflectivity was assessed by using a 20-MHz intravascular ultrasound catheter and computer-assisted videodensitometry. Ultrasound reflectivity was optimized at a CH concentration of 10 mol %; PG concentration variation had essentially no effect on initial values of echogenicity. Optimal acoustic stability was observed with concentrations of 10-15 mol % CH and with a PG concentration greater than 4 mol %. Preparations made with 0.2 M mannitol were more ultrasound reflective than those made with lactose, trehalose, and sucrose. Re-lyophilization and freezing temperatures below -20 degrees C increased ultrasound reflectivity. We optimized the ultrasound properties of echogenic liposomal dispersions, the conditions of which provide some insight into the underlying lipid structures responsible. The preparations developed are now more stable and acoustically reflective than our previous preparations. This advances the development of echogenic lipid dispersions as targeted ultrasound contrast agents for use in general ultrasound as well as cardiovascular imaging.

Carbohydrates↗

PubMed2000

Development of echogenic, plasmid-incorporated, tissue-targeted cationic liposomes that can be used for directed gene delivery.

RATIONALE AND OBJECTIVES: Echogenic antibody-conjugated anionic liposomes have been developed that allow directed tissue targeting and acoustic enhancement. These are not efficient for gene delivery. A cationic formulation that allows directed gene delivery while retaining acoustic properties may provide more efficient transfection. METHODS: Cationic liposomes were prepared and acoustic reflectivity was determined. Anti-fibrinogen-conjugated liposomes were laid on fibrin-coated slides and adherence was quantified using fluorescence techniques. Liposomes were combined with a reporter gene and plated on cell cultures. Human umbilical vein endothelial cells were stimulated to upregulate intercellular adhesion molecule-1 (ICAM-1) and were treated with anti-ICAM-1-conjugated liposomes, and gene expression was quantified. RESULTS: Cationic liposomes retained their acoustic reflectivity and demonstrated specific adherence to fibrin under flow conditions. Significant transfection of human umbilical vein endothelial cells was demonstrated, with higher gene expression seen with specific antibody-conjugated liposomes. CONCLUSIONS: Novel acoustic cationic liposomes have been developed that can be antibody conjugated for site-specific adherence and directed cell modification. This presents exciting potential for a vector that allows tissue enhancement and targeted gene delivery.

Animals↗

PubMed1997

In vitro targeting of antibody-conjugated echogenic liposomes for site-specific ultrasonic image enhancement.

Tissue-specific ultrasonic enhancement can be used for the detection and characterization of atherosclerosis. We have previously demonstrated the generation of inherently echogenic (acoustically reflective) liposomes solely by varying lipid composition and controlling the method of production. In this study, echogenic liposomes composed of phosphatidylcholine (PC), 4-(p-maleimidophenyl) butyryl phosphatidylethanolamine (MPB-PE), phosphatidylglycerol (PG), and cholesterol were conjugated to human gamma globulin to determine the effect of antibody conjugation on liposomal acoustic reflectivity. The liposomes remained highly echogenic following antibody conjugation. Echogenic liposomes were also conjugated to rabbit antihuman fibrinogen to study their ability to target fibrin. Antibody-conjugated liposomes were targeted to fibrin-coated filter paper and slides, thrombi made in vitro, and segments of atheroma in an animal model of atherosclerosis. Liposomes were detected by scanning electron microscopy, radiolabeling, and imaging with intravascular ultrasound. Electron microscopy revealed attachment of antibody-conjugated liposomes to fibrin on slides and to the fibrous plaques of the arterial segments, whereas unconjugated liposomes did not attach. Similarly, conjugated liposomes did not attach to normal arteries, indicating their binding to the arterial segment is directed towards a component of the fibrous plaque. Ultrasound imaging of the thrombi demonstrated surface attachment of the acoustic conjugated liposomes. 125I-Labeled liposomes conjugated to rabbit anti-human were targeted to fibrin-coated paper. Counting specifically bound radioactivity showed that > 84% of applied liposomes remained attached to the fibrin after washing with saline. These results demonstrate the potential of acoustically reflective liposomes for site-specific targeting and acoustic enhancement.

Animals↗

PubMed1997

Antineoplastic activity of BCG: location of antineoplastic glycans in the cellular integument of Mycobacterium bovis, BCG vaccine, Connaught substrain.

The polysaccharidic integument surrounding growing cells of attenuated Mycobacterium bovis Bacillus Calmette-Guérin (BCG) vaccine, Connaught substrain, can be removed with non-specific proteases. After 5 weeks incubation at 37 degrees C in Middlebrook 7H-9 medium, the collected cells were incubated with pronase and the integument and cells separated by centrifugation in a Ficoll-Histopaque preparation. After washing and drying, the detached integument accounted for 65% w/w of the original dried cell mass. Like the original cellular material, the detached integument manifested antineoplastic activity against a murine sarcoma model in vivo. Solubilization of the otherwise insoluble integument by boiling in water or by digestion in 8 M urea significantly enhanced activity of the integument itself, by 125 and 1,100 times, respectively. Integument extracts were shown to contain mainly glucose, with smaller quantities of other sugars, consistent with the presence in BCG of high molecular weight glycans, as previously reported. It is suggested that most, if not all, of the antineoplastic activity of BCG can be accounted for by the activity associated with the high-molecular weight polysaccharidic glycans which constitute the cellular integument.

Animals↗

PubMed1996

Development of inherently echogenic liposomes as an ultrasonic contrast agent.

Ultrasonic contrast agents have been developed for improved assessment of blood flow and tissue perfusion. Many of these agents are not inherently acoustically reflective (echogenic), and nearly all are not suitable for tissue specific targeting. The purpose of this study was to develop acoustically reflective liposomes, which are suitable for antibody conjugation, without using gas or any other agent entrapment. Echogenic liposomes were prepared from phosphatidylcholine (PC), phophatidylethanolamine (PE), phosphatidylglycerol (PG), and cholesterol (CH), using a dehydration/rehydration method. The formulation was optimized for higher acoustic reflectivity by varying the lipid composition. Liposomes were imaged with a 20 MHz intravascular ultrasonic imaging catheter. Echogenicity levels were expressed using pixel gray scale. The presence of PE and PG at specific concentrations improved echogenicity due to their effects on liposomal morphology as confirmed by freeze-etch electron microscopy. The acoustic reflectivity of liposomes was retained when liposomes were treated with blood at room temperature and 37 degrees C under in vitro conditions. It was demonstrated that the liposomes were also acoustically reflective in vivo after they were injected into a miniswine model. We have developed echogenic liposomes that are stable and suitable for tissue specific targeting as a novel contrast agent. This new contrast agent can be used for ultrasonic image enhancement and/or treatment of targeted pathologic sites.

Cholesterol↗

PubMed1996

Chemical and ultrastructural investigations of Mycobacterium bovis BCG: implications for the molecular structure of the mycobacterial cell envelope.

The mycobacterial cell wall visualized by transmission electron microscopy (TEM) of thin sections of resin-embedded specimens is generally believed to consist of an electron-dense peptidoglycan, an electron-transparent arabinogalactanmycolate layer and an electron-dense outer layer (OL). In addition, a pseudocapsule known as the 'electron-transparent zone' (ETZ) has been observed after phagocytosis of mycobacteria by macrophages. TEM of thin sections of Mycobacterium bovis BCG, Tice substrain, revealed an OL bilayer, each of which measured 2-4 nm in diameter. The intermediate electron-transparent layer varied from 1 to about 250 nm in diameter and appears to be a previously observed oxygen-dependent amorphous integument that consists of hot water-extractable neutral polysaccharides, especially a recently characterized alpha glucan, comprising about 12% of the dry cell weight. This and other recent studies of BCG have revealed cell-surface features that may provide a better understanding of the outer mycobacterial cell envelope.

Cell Membrane↗

PubMed1995

An anti-neoplastic glycan isolated from Mycobacterium bovis (BCG vaccine).

Tice substrain BCG is used clinically as an immunotherapeutic agent against superficial bladder cancer. A boiling-water extract of this BCG showed anti-tumour activity against a murine S180 sarcoma model and was fractionated into three fractions, A, B and C, by the use of Sephadex LH-20 chromatography. An anti-tumour glucan, PS1A1, was isolated from fraction PS1A with Sephadex G-75. The molecular mass of PS1A1 was between 65 and 87 kDa by Sephadex G-100 chromatography. The structure of PS1A1 was investigated by one- and two-dimensional NMR spectroscopy and methylation analysis and was demonstrated to be primarily 1-->6-alpha-linked glucose units. We postulate that the repeating unit is: [Formula: see text]

Animals↗

PubMed1995

Interaction between fibronectin-bearing surfaces and Bacillus Calmette-Guérin (BCG) or gelatin microparticles.

Gelatin, prepared commercially by degradation of animal collagen, was studied to see whether it had an affinity for fibronectin, which has a known affinity for collagen, and whether gelatin-based drugs could be used to target fibronectin-excreting tumours. Bacillus Calmette-Guérin (BCG) vaccine, an attenuated strain of Mycobacterium bovis, is currently the most effective treatment for superficial transitional cell carcinoma of the bladder. The living cells of the BCG vaccine associate with the fibronectin-bearing surfaces of the tumour. Using a multi-well culture plate technique, gelatin microparticles were shown to be adsorbed onto murine S180 sarcoma cells and this reaction was substantially inhibited by the addition of human plasma fibronectin. The avidities of various BCG substrains and gelatin microparticles for glass-bound fibronectin were measured and the association constants determined. The gelatin microparticles associated with the fibronectin with equal avidity as the BCG cells. The results suggest that this model system may allow the investigation of gelatin-based drug delivery devices capable of targeting fibronectin-bearing surfaces associated with some tumours.

Adsorption↗

PubMed1994

In-vivo and in-vitro targeting of a murine sarcoma by gelatin microparticles loaded with a glycan (PS1).

PS1, a complex polysaccharide derived from Mycobacterium bovis (Bacillus Calmette-Guérin, BCG) with considerable antitumor activity in-vivo, was loaded onto gelatin microparticles (mean diam. 1.45 micron) at a level shown to not produce the burst effect often seen with drug-loaded microparticulate systems. In-vitro dissolution experiments had demonstrated a sustained-release behaviour, with a half-life of approximately 8 h for what is an extremely water-soluble material. These PS1/gelatin systems had no measurable cytotoxicity against an S180 murine sarcoma cell in-vitro although fibronectin-mediated targeting of the microparticles for the tumour cells could be demonstrated. Injection into mice, with the S180 cells, of PS1 solutions or suspensions of PS1-loaded gelatin microparticles resulted in almost identical dose-related suppression for the tumour cell growth. When injected at intervals following injection of the tumour cells, however, for a period of 24-48 h there was a relatively enhanced activity of the formulated PS1, compared with the aqueous solution, after which both formulated and unformulated material became progressively less effective.

Animals↗

PubMed1994

Isolation of a fibronectin-binding tryptic peptide from the antigen 85A protein of Mycobacterium bovis BCG.

Antigen 85A, a major secreted mycobacterial fibronectin-binding protein, was isolated from culture fluids of the Tice substrain BCG vaccine. Tryptic digestion of this protein and passage of the digest through a fibronectin affinity chromatographic column resulted in the identification of a polypeptide fragment of molecular weight < or = 6.5 kD, which had marked fibronectin-binding activity. The identity of a 62-residue polypeptide was deduced from the amino-terminal sequence, indicating that the primary structure may define the integrin-binding capability.

Amino Acid Sequence↗

PubMed1994

Characterization of the antigen 85 complex fibronectin-binding proteins derived from aged culture filtrates of Mycobacterium bovis BCG, Tice substrain.

The antigen 85 complex are major T-cell and B-cell antigens and fibronectin-binding proteins secreted by Mycobacterium tuberculosis, M. leprae and attenuated M. bovis (BCG vaccine). The Ag 85 complex was found to comprise a high proportion of the extracellular protein in filtrates of surface-pellicle cultures of Tice-substrain BCG vaccine, attaining a maximum of 25%. This proportion began to decrease prior to the end of the logarithmic growth phase, about 3 weeks after the start of the culture, mainly due to apparent degradation of the Ag 85 complex. Isolation of the main Ag 85 protein and determination of the first 36 residues of the NH2-terminus showed identity with the 85A protein isolated by others from various mycobacteria. Both the Ag 85A and B components were secreted in nearly constant proportions over a 6-week period. No Ag 85C protein was detected.

Amino Acids↗

PubMed1993

Inhibition of murine sarcoma cell adherence to polystyrene substrata by bacillus Calmette-Guérin: evidence for fibronectin-mediated direct antitumor activity of BCG.

Bacillus Calmette-Guérin (BCG) inhibited adherence of S180 mouse sarcoma cells and WI38 human diploid fibroblasts to the polystyrene substratum of 24-well cluster dishes in a dose-dependent manner. This property was retained by washed or heat-killed bacilli, but not by the vaccine filtrate or by the spent bacterial culture medium. Adhesion of bacilli to nonadherent S180 cells was demonstrated by light and scanning electron microscopy, but was not seen after trypsinization of adherent cells, indicating that bacilli bind to cell-surface adhesins. Preincubation of bacilli with human fibronectin abolished their ability to inhibit S180 adherence, suggesting that the phenomenon may be mediated by interaction of bacilli with cell-surface fibronectin. Fibronectin pretreatment of the bacteria also decreased their inhibition of S180 tumor growth in vivo, indicating that this mechanism may be at least partly responsible for BCG vaccine's observed antineoplastic activity.

Adsorption↗

PubMed1993

Phagocytosis of Mycobacterium bovis BCG organisms by murine S180 sarcoma cells.

Instillation of BCG vaccine directly into the bladder of patients suffering from superficial bladder tumours represents the major clinical application of this material. The mode of action is by no means clearly defined but it is known that the BCG cells need to be in close proximity to the tumours and may be capable of directly targeting the tumour cells through a fibronectin/fibronectin receptor interaction. In addition to cellular adherence there is a local stimulation of macrophages which is known to phagocytose mycobacteria. Use of a murine S180 sarcoma model facilitated the quantitative measurement of antitumour activity of BCG and BCG isolates. Ultrastructural examination of the BCG/sarcoma interaction confirmed that, in fact, these tumour cells do phagocytose and subsequently kill the BCG cells. The relationship between this phenomenon and the subsequent tumour cell death is not currently clear, although a strong tumoricidal agent has recently been isolated from BCG cells.

Animals↗

PubMed1992

Development of IgM antibody to group B Streptococcus type III in human infants.

An ELISA was developed to measure IgM antibody to albumin-coupled native capsular polysaccharide of type III group B streptococcus (GBS). The assay was standardized by two double-label methods that agreed within 33%. In quantitative assays, the range of IgM antibody to type III GBS in the sera of 94 adult pregnant women was 1.2-50.6 micrograms/ml (median, 5.4), while each of 38 cord serum samples contained less than 0.03 micrograms/ml IgM antibody. Neonatal rats were passively immunized with a serially diluted human serum containing 15 micrograms/ml IgM and undetectable IgG antibody to type III GBS. The rats were protected against lethal infection with an IgM antibody concentration of 0.5 micrograms/ml. In single serum samples from 31 healthy infants less than 2 years old and serial specimens from 5 infants with type III GBS infections, specific IgM antibody was detectable by 3 months of age. Levels greater than or equal to 0.5 micrograms/ml were present in all samples from infants greater than 7 months of age. The acquisition of specific IgM antibody is inversely correlated with the age-limited incidence of type III GBS infections in young children.

Aging↗

PubMed1992

Origins of BCG surface charge: effect of ionic strength and chemical modifications on zeta potential of Mycobacterium bovis BCG, Tice substrain, cells.

The zeta potential of washed Tice substrain BCG organisms was measured over a range of ionic strengths from I = 0.005 to 0.1 M. No change in the isoelectric point of 3.4-3.7 was evident. Proteolytic enzymes (trypsin/chymotrypsin, pepsin, papain and pronase) and fluorodinitrobenzene abolished the cationic charge, suggesting that this is substantially due to amino groups associated with protein. Neither hot HCI nor cold trichloroacetic acid affected the charge, indicating that ionic groups are not associated with extractable polysaccharides. Methanolysis, treatment with HF and carbodiimide, and cationic detergent (cetyltrimethylammonium bromide) binding indicated that the negative charge was provided by carboxylic acids, phosphoesters and strong acidic groups, possibly sulphates. Standardless quantitative X-ray microanalysis revealed the presence of phosphorus and sulphur on the surface of actively growing BCG colonies.

BCG Vaccine↗

PubMed1991

High-dose inhibition and low-dose enhancement of murine sarcoma growth exhibited by BCG vaccine.

An in vivo assay for BCG anti-cancer efficacy was developed, utilizing subcutaneous injection into CFW Swiss-Webster mice of cultured S180 sarcoma cells and mixed with freeze-dried TiceTM BCG vaccine. A BCG dose of 0.156-1.56 mg dry weight significantly inhibited tumor formation, but a BCG dose of 0.156-1.56 micrograms significantly enhanced tumor growth, evidenced by increased tumor incidence, volume and initial growth rate. These antagonistic activities may contribute to the high variability of BCG anti-cancer efficacy seen in animals and humans and indicate the need to exercise caution when employing even low doses of BCG for cancer immunotherapy.

Animals↗

PubMed1991

Surface morphology of Mycobacterium bovis BCG: relation to mechanisms of cellular aggregation.

Growing colonies of Mycobacterium bovis BCG, Tice and Glaxo substrains, and freshly ball milled and freeze-dried Tice BCG vaccines were examined by scanning and transmission electron microscopy (TEM) and by light microscopy after cytochemical staining. BCG organisms in colonies growing on agar were randomly oriented, despite colony morphology, and nearly completely covered by an amorphous material. Aggregates of organisms in vaccine suspensions were also covered with this material, but single cells were not covered. In TEM, the covering material was visualized between groups of cells as an electron-transparent area surrounded by a thin electron-dense layer. This material appeared to originate in the upper cell wall, between the cell wall skeleton and the outer dense layer. Staining of the covering material indicated the presence of protein, carbohydrate and acidic groups, but not exposed lipids. The covering material was absent from the ventral side of colonies, suggesting that its production is oxygen-dependent. These observations suggest that a mycobacterial exudate, previously observed and implicated as a virulence factor, may also bind the cells together, and accounts for the aggregative properties of the organisms in culture.

BCG Vaccine↗

PubMed1990

Cell mass of Mycobacterium bovis BCG estimated by gas chromatography.

The presence of additives and large cellular aggregates in freeze-dried BCG vaccines precludes accurate measurement of total cell content by traditional methods. The possibility that extraction and quantitation of a cell membrane fatty acid may provide a suitable means of cell mass determination was tested. The palmitic acid methyl ester peak area determined by gas chromatography was directly proportional to the wet weight of freshly grown Tice-, Pasteur-, and Glaxo-substrain BCG, as well as the dry weight of the ampoule contents after removal of soluble material. Extraction of palmitic acid from Tice BCG vaccine was not appreciably affected by lyophilization and the calculated dry cell mass values of freeze-dried vaccine samples correlated well with particle number. This method, therefore, may be useful in measuring BCG cell mass during all stages of vaccine manufacture and storage.

BCG Vaccine↗