Kinetics of clonazepam in relation to electroencephalographic and clinical effects.

The aim of this study was to see if the immediate EEG and clinical response to an intravenous dose of clonazepam was predictive for the effect of oral clonazepam maintenance therapy. Four children with petit mal epilepsy were given clonazepam intravenously during continuous EEG recording. Clonazepam plasma concentrations were determined repeatedly with a high performance liquid chromatographic method using a reversed phase system. The day after the intravenous dose the patients were given oral therapy with clonazepam. Repeated long-term EEG recordings were made and plasma concentrations of clonazepam were determined. There was no clinically satisfactory effect of clonazepam during oral maintenance treatment in three of the children who responded well to the intravenous dose of clonazepam. Thus, the immediate response to intravenous clonazepam was not a good predictor of the long-term effects in our patients.

Role of cell-associated pathogen metabolism in infection of tracheal explants by Mycoplasma pneumoniae.

The effect of environmental conditions on the relative pathogenicity of Mycoplasma pneumoniae for hamster tracheal explants was investigated. Organisms from the early stages of the growth cycle (e.g., day 1 to 2) were more effective in the induction of ciliostasis than were older cultures. Both the degree of ciliostasis and the speed of onset were affected. The type of explant culture medium also affected pathogenic potential. M. pneumoniae infection produced significantly greater ciliostasis and cytonecrosis in a "permissive" medium, i.e., one capable of supporting mycoplasma metabolism and replication, than in a "nonpermissive" medium. However, no adenine protection effect could be detected under permissive conditions, though it was quite striking when a nonpermissive medium was used as the post-infection explant medium. This suggests that the cell damage noted under permissive conditions may result from processes distinct from those operative in the actual host-parasite cellular interaction.

Ciliated respiratory epithelial monolayers: new model for Mycoplasma pneumoniae infection.

Hamster respiratory epithelial cells were cultured in a monolayer format, and 20% of the cells were ciliated. Mycoplasma pneumoniae attached to the epithelial cells in a neuraminidase-specific fashion and induced ciliostasis and cytonecrosis.

Role of adenine in pathogenesis of Mycoplasma pneumoniae infections of tracheal epithelium.

Adenine sulfate and several related compounds were evaluated for their ability to retard the cytotoxic effect which normally accompanies M. pneumoniae infections of hamster tracheal explants. Adenine sulfate, at the 0.01 mM level, was found to exert a significant protective effect. Little or no ciliostasis or loss of cell viability was detectable when organ cultures were infected with 10(7) CFU of virulent M. pneumoniae in the presence of the adenine supplement. Mycoplasmas grew in broth and on plastic surfaces in the presence of adenine, and no significant diminution of growth rate or cell yield was detectable. Organisms adhered to the tracheal epithelial surface regardless of the presence or absence of adenine. When explants were incubated in the presence of 14C-(8)-adenine, rinsed, and then infected with M. pneumoniae, the adenine label could be recovered from the mycoplasmas 20 h after the infection. These data are compatible with the known nucleic acid requirement of mycoplasmas and with a model which ascribes a role for purine/pyrimidine competition and/or depletion in the infective process.

Differences in the attachment of Mycoplasma pneumoniae cells and membranes to tracheal epithelium.

Hamster trachea organ cultures were exposed to isolated membranes of Mycoplasma pneumoniae, PI 1428. Attachment, monitored by the uptake of tritiated membranes, was relatively insensitive to neuraminidase pretreatment, unlike the attachment of viable cells. Membrane attachment was optimal when explants were incubated with 50 to 100 micrograms of membrane protein per ml in minimal essential medium broth while gently being rotated (1 rpm) in a roller apparatus for 90 to 120 min at 37 degrees C. Saturation of the receptor sites with viable cells failed to inhibit subsequent membrane attachment. Induction of squamous metaplasia by extended cultivation of tracheal explants in a vitamin A-free medium reduced the content of ciliated cells without significantly affecting total cell viability, but did not alter the attachment of M. pneumoniae membranes. Collectively, the data indicate that the mechanism of attachment of M. pneumoniae membranes to respiratory epithelium is distinct from the receptor site-mediated attachment of M. pneumoniae cells.

Studies on sperm antigenicity, 5. In vivo and in vitro cellular reactivity in guinea pigs sensitized with fractions of human spermatozoa.

In vitro delayed type hypersensitivity was demonstrated with peritoneal exudate cells from guinea pigs immunized with different preparations of human and guinea pig seminal components emulsified with complete Freund's adjuvant-H37Ra. The seminal components were intact human spermatozoa (HuSp); intact guinea pig spermatozoa (GPSp); human seminal plasma (HuSePlFr); and fractions of human spermatozoa obtained by centrifugation of the homogenate at 5,000 X g (5S30 and 5p30), at 20,000 X g (20S30 and 20p30), and at 144,000 X g (144p120). Cellular sensitivity was demonstrated in vivo by skin testing and in vitro by the macrophage inhibition technique. Peritoneal exudate cells from guinea pigs sensitized with fractions 5p30 and 20p30 elicited a delayed hypersensitivity reaction which could be detected only with intact human spermatozoa. Other human spermatozoal fractions (5S30, 20S30, and 144p120) were weak immunogens. Sensitization of guinea pigs with fractions of human spermatozoa, in addition to causing delayed hypersensitivity reactions, elicited low titers of spermatoxic antibodies. Antibodies to human spermatozoal fractions 5S30, 5p30, 20S30, and 20p30 cross-reacted with intact human spermatozoa and intact guinea pig spermatozoa. It is postulated that the existence of "spermatozoa-specific" coating antigen(s) derived from other components of the reproductive tract might be responsible for human spermatozoal antigenicity.

Purification and characterization of human plasma lecithin:cholesterol acyltransferase.

A highly purified (approximately 12 000-fold) homogeneous preparation of human plasma lecithin:cholesterol acyltransferase (LCAT) with 16% yield was obtained by a combination of density ultracentrifugation, high density lipoprotein affinity column chromatography, hydroxylapatite chromatography, and finally chromatography on anti-apolipoprotein D immunoglobulin-Sepharose columns to remove apolipoprotein D. This enzyme preparation was homogeneous by the following criteria: a single band by polyacrylamide gel electrophoresis in 8 M urea; a single band on sodium dodecyl sulfate gel electrophoresis with an apparent molecular weight of 68 000 +/- 1600; a single protein peak with a molecular weight of 70 000 on a calibrated Sephadex G-100 column. Its amino acid composition was different from human serum albumin and all other apoproteins isolated from lipoprotein fractions.

Studies on sperm antigenicity. 2. In vivo and in vitro cellular reactivity in guinea pigs sensitized to homologous and heterologous spermatozoal autoantigens.

In vitro delayed type hypersensitivity was demonstrated with peritoneal exudate cells from guinea pigs of the Rockefeller and Hartley strains, immunized with different preparations of the guinea pig male reproductive tract (RMT) emulsified in complete Freund's adjuvant-H37Ra. The RMT preparations were purified guinea pig spermatozoal autoantigens S, P, and T; whole guinea pig spermatozoa, and extract from epididymal tissue. The cellular sensitivity in vivo was demonstrated by injecting the proper antigen into the skin of the tested animals and in vitro by the macrophage inhibition technique. Peritoneal exudate cells from guinea pigs sensitized with whole guinea pig spermatozoa cells were inhibited in vitro by the specific antigen, epididymal extract, and autoantigen-T. Autoantigen-S was found to be a weak immunogen. However, the migration of peritoneal exudate cells from guinea pigs sensitized with large amounts of antigen-S was inhibited by whole spermatozoa in vitro. This cross-reactivity revealed the possibility that the immunogenicity of purified autoantigen-S might be connected to its molecular size. According to the immunizing dose of the antigens, testicular lesions of either the aspermatogenic or orchitis type were found in the testes of sensitized guinea pigs. Lesions in the testes of the guinea pigs were not detectable by cross-immunization with heterologous human or rat spermatozoa, although some degree of in vitro cross-reactivity was detected by skin test studies.