Diagnostic particle agglutination using ultrasound: a new technology to rejuvenate old microbiological methods.
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Microbial antigen in clinical specimens can be detected rapidly by commercial test-card latex agglutination, but poor sensitivity is a potential difficulty. Antigen detection by immuno-agglutination of coated latex micro-particles can be enhanced in comparison with the conventional test-card method in both rate and sensitivity by the application of a non-cavitating ultrasonic standing wave. Antibody-coated micro-particles suspended in the acoustic field are subjected to physical forces that promote the formation of agglutinates by increasing particle-particle contact. This report reviews the application of ultrasound to immuno-agglutination testing with several commercial antibody-coated diagnostic micro-particles. This technique is more sensitive than commercial card-based agglutination tests by a factor of up to 500 for fungal cell-wall antigen, 64 for bacterial polysaccharide and 16 for viral antigen (in buffer). The detection sensitivity of meningococcal capsular polysaccharide in patient serum or CSF has been increased to a stage where serotyping by ultrasound-enhanced agglutination is comparable to that achievable with the PCR, but is available more rapidly. Serum antigen concentration as measured by ultrasonic agglutination has prognostic value. Increasing the sensitivity of antigen detection by increasing the acoustic forces that act on suspended particles is considered. Employing turbidimetry to measure agglutination as part of an integrated ultrasonic system would enable the turnover of large numbers of specimens. Ultrasound-enhanced latex agglutination offers a rapid, economical alternative to molecular diagnostic methods and may be useful in situations where microbiological and molecular methods are impracticable. (+info)
Mycoplasma hominis: growth, reproduction, and isolation of small viable cells.
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Improved methods for studying the growth of Mycoplasma hominis (ATCC 14027) have been developed, involving modified growth conditions and preparation of the organisms under minimally distorting conditions. Cells so prepared from batch cultures show relatively uniform exponential growth and appear to be dividing by binary fission; but pleomorphic forms appear upon further incubation. Similar behavior was demonstrated by another laboratory-adapted strain and by three clinical isolates, and therefore seems characteristic of the species. The pleomorphic populations contain small forms having diameters within the 100- to 250-nm size range reported for "elementary bodies." Such forms were isolated from this strain of M. hominis by sequential filtration using gravity alone, after cell aggregates were dispersed by Pronase treatment. Of the small bodies which traversed membranes of 220-nm pore size, a negligible number grew in liquid or on solid media, suggesting that these were not essential reproductive units in a life cycle, but involution forms due to growth in an altered environment. (+info)
Inhibitory effect of Pseudomonas aeruginosa on the phagocytic and killing activity of rabbit polymorphonuclear leukocytes: mechanisms of action of a polymorphonuclear leukocyte inhibitor.
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The polymorphonuclear leukocyte (PMN) inhibitor isolated from a strain of Pseudomonas aeruginosa which is resistant to the phagocytic and killing activities of rabbit PMN inhibited migration of PMN and engulfment of latex particles by PMN. In studies of the bactericidal metabolism of PMN, the PMN inhibitor did not inhibit the intracellular activity and extracellular release of lysosomal enzymes. However, the PMN inhibitor caused a decrease of Nitro Blue Tetrazolium reduction. The PMN inhibitor had a cytotoxic effect on PMN and inhibited [14C]tyrosine uptake in intact PMN inhibitor had no inhibitory effect on protein synthesis in cell extracts. (+info)
Suggestive evidence for in vivo binding of specific antitumor antibodies of human melanomas.
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Antibodies eluted from homogenates of human melanoma cells reacted against melanoma cells reacted against melanoma antigens in a complement fixation test. Before elution, sonically treated homogenate did not react significantly against autologous serum but, following elution, antigenic activity increased markedly (up to 32-fold). Eluate of one melanoma reacted with the sonically treated residue of other melanomas but not with similarly prepared residues of sarcoma, carcinomas, or normal tissues. Melanoma eluates comtained more IgG than IgA. Traces of IgM were found in two melanoma eluates. Eluates of normal tissues (lung, kidney, and muscle) were devoid of serum proteins and did not react with the soncially treated melanoma residues. These results support the hypothesis that antitumor antibodies are bound to melanoma cells in vivo and that these antigens are cross-reactive. (+info)
Inflammatory activation of neutrophils by Helicobacter pylori; a mechanism insensitive to pertussis toxin.
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Chronic active gastritis of the antral mucosa is a characteristic feature of infection with Helicobacter pylori and interactions between bacterial components and inflammatory cells are believed to play an important pathogenic role. Neutrophils stimulated with H. pylori sonicate were demonstrated to release L-selectin (CD62L) expressed on the cellular surface, with a subsequent up-regulation of the beta2-integrins CD11b and CD11c, both in a dose- and time-dependent manner, reaching maximum levels after 45-60 min of stimulation. No changes were observed for the CD11a receptor upon stimulation. The activating properties of H. pylori sonicates on neutrophils were heat-labile and susceptible to protease attack, indicating the protein nature of the activating factor. After size fractionation, the major neutrophil-inducing activity was detected in the high molecular weight fraction exhibiting urease activity. Pertussis toxin was unable to inhibit neutrophil activation by the H. pylori protein(s). We conclude that proteins from H. pylori have a potent inflammatory effect on the surface membrane molecules CD62L, CD11b and CD11c essential for transendothelial migration of neutrophils to areas of inflammation. The neutrophil-activating protein(s) act via a pertussis toxin-insensitive mechanism. (+info)
Preparation and chemical composition of the cell walls of Streptococcus mutans.
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Purified cell walls from Streptococcus mutans strain BHT were prepared without the use of proteolytic enzymes in order to retain all cell wall constituents for chemical analysis. Of four methods employed, the Ribi cell fractionator produced disrupted cell suspensions which could be most thoroughly purified on sucrose gradients. Results of chemical analyses on purified cell walls prepared in this 8.9% glycerol teichoic acid, 33.6% non-peptidoglycan polysaccharide, and 49.9% peptidoglycan. (+info)
Transmission of human papillomavirus type 11 infection by desquamated cornified cells.
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While much is known about the human papillomavirus (HPV) productive cycle, the mechanisms of virion transmission from person to person are poorly understood. The keratinocyte is the target cell of HPV infection. As keratinocytes differentiate, nuclei are lost and the cornified cell envelope develops. Layers of these desquamated cornified cells (DCCs) are continuously shed from the stratum corneum. Release of HPV requires the cornified cell envelope, a normally very durable structure, to break apart, liberating the contents of the cell. In differentiated keratinocytes infected with HPV 11, the cornified cell envelope is abnormally thin and fragile. In this study, DCCs from HPV 11-infected genital epithelium were used to investigate the mechanisms of viral transmission. First, HPV 11-infected tissue was examined for the presence of virions by transmission electron microscopy. Virions were observed in the nuclei of differentiated keratinocytes. In addition, virions were detected in the cytoplasm of DCCs that had undergone nuclear dissolution. Rarely, virions were observed outside of cells. Next, infectivity of intact and ruptured DCCs was tested in an assay performed in the athymic mouse xenograft system. High-titer cesium chloride gradient-purified HPV 11 virions infected 100% of recovered xenografts. Using intact DCCs derived from HPV 11-infected tissue, 62.5% of recovered xenografts were infected. To test the effects of mechanical stress on infectivity, DCCs were ruptured by sonication and used in the infectivity assay. The infectivity rate increased to 90%. We conclude that DCCs serve as vehicles for efficient, concentrated delivery of virions in HPV 11 infection. (+info)
Sugar-based tertiary amino gemini surfactants with a vesicle-to-micelle transition in the endosomal pH range mediate efficient transfection in vitro.
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Novel reduced sugar gemini amphiphiles linked through their tertiary amino head groups via alkyl spacers of 4 or 6 carbons, and with varying (unsaturated) alkyl tail lengths of 12--18, have been synthesized and tested for transfection in vitro in an adherent Chinese hamster ovary cell line (CHO-K1). Transfection efficiencies peaked at 2.7 times that of the commercial standard Lipofectamine Plus/2000 for pure solutions of the compound bearing unsaturated (oleyl) alkyl tails. For those compounds bearing saturated alkyl tails, transfection efficiency peaked at a tail length of 16, at a level similar to Lipofectamine Plus/2000. All of the amphiphiles formed bilayer vesicles at physiological pH. Some of the amino groups at the surface were protonated, and vesicles therefore bore a positive charge. Increased protonation with reduced pH resulted in greatly increased monomer solubility and a morphology change from vesicle to micelle at characteristic pH values, dependent on the tail length. For the compounds promoting high transfection efficiency, this characteristic pH was within the range found in the endosomal compartment (7.4--4.0). Formation of mixed micelles between gemini surfactant and membrane phospholipids at reduced pH may therefore provide a method of endosome rupture and subsequent escape of entrapped DNA, thus discarding the need for extra fusogenic or endosomolytic agents. The positive charge on the vesicles at physiological pH drives the colloidal association with DNA. Small angle X-ray scattering measurements indicate that lamellar aggregates are formed, which have a d spacing of 48--54 A. Preliminary differential scanning calorimetric measurements suggest that reduction of pH causes a disordering of the hydrocarbon region of the DNA-surfactant complex. (+info)