Immunologic studies on hamsters infected with Entamoeba histolytica.

The serologic and cell-mediated immune responses of hamsters exposed to 2 strains of Entamoeba histolytica (HM-1 and HM-19) were evaluated by a series of in vitro tests. The pathogenicity of the 2 strains was evaluated in terms of their ability to produce liver abscesses and spleen enlargement. Antibody response was evaluated by the indirect hemagglutination test. The cellular immune response was assayed by increased DNA synthesis by lymphocytes and migration inhibition of macrophages.     

Fluorescence-based viability assay for studies of reactive drug intermediates

Studies of drug toxicity, toxicologic structure-function relationships, screening of idiosyncratic drug reactions, and a variety of cytotoxic events and cellular functions in immunology and cell biology require the sensitive and rapid processing of often large numbers of cell samples. This report describes the development of a high-sensitivity, high-throughput viability assay based on (a) the carboxyfluorescein derivative 2'-7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) as a vital dye, (b) instrumentation capable of processing multiple small (less than 100 cells) samples, and (c) a 96-well unidirectional vacuum filtration plate. Double staining of cultured peripheral blood mononuclear cells with BCECF and propidium iodide (PI) showed no overlap between PI+ (nonviable) and BCECF+ (viable) cells by flow cytometric analysis. Optimal conditions were developed for dye loading and minimizing physical cell damage and fluorescence quench during the assay procedure. The ratio of BCECF fluorescence to internal standard fluorescent particles was linear from 40 to greater than 20,000 cells with a signal:noise ratio of approximately 3 at 40 cells/well. Sulfamethoxazole hydroxylamine (SMX-HA) was used as a model toxic drug metabolite to explore the validity of the BCECF procedure. SMX-HA, but not its parent compound sulfamethoxazole, resulted in a dose dependent loss of cellular fluorescence and the parallel accumulation of PI+ nonviable cells. When compared to the currently used tetrazolium dye reduction viability assay, the BCECF method was 3-fold more sensitive, greater than 10-fold faster, and required 1/10-1/100 the cell numbers.     

Antibodies against neuroactive amino acids and neuropeptides. I. A new two-step procedure for their conjugation to carrier proteins and the production of an anti-Met-enkephalin antibody reactive with glutaraldehyde-fixed tissues.

We developed a new two-step procedure to couple haptens to bovine serum albumin (BSA) via glutaraldehyde (GA). After activation of BSA with excess GA and removal of unreacted GA, the hapten was bound to the activated protein in a second step. This two-step procedure is easy to use, the desired molecular ratio of coupled hapten to protein is conveniently adjusted, and no visible precipitation of the conjugate is detected. Using a low peptide concentration, nearly 50% of the inserted haptens are bound to the protein, and unbound expensive peptide can be recovered after Sephadex chromatography. Antisera to neuroactive amino acids (GABA, glycine, and glutamate) and neuropeptides (Met-enkephalin) were prepared by immunization of rabbits with these conjugates. Immunological analysis of immune sera by dot-blot and ELISA techniques and subsequent removal of crossreactivities by solid-phase adsorption yielded monospecific antibodies, which were further purified by affinity chromatography. The immunocytochemical specificities of these purified antibodies were verified in adjacent sections of GA-fixed rat spinal cord. Pre-embedding staining with anti-Met-enkephalin in combination with post-embedding staining for amino acids such as GABA allowed double staining of the two antigens in a single semi-thin section.     

Influence of stereotactic hypothalamotomy on alcohol and drug addiction.

2 or 3 years follow-up of 13 hypothalamotomy patients with alcohol and drug addiction reveals that the addicted patients regained their self-control, the severe preoperative social deterioration was removed, and the tendency to social stabilisation was clearly in evidence.     

Immunocytochemical detection of ricin. I. Preliminary immunofluorescence studies

Summary Ricin injected into rat musclein vivo can be localized within a few hors using routine immunofluorescence techniques on formalin-fixed tissues. However, the level of sensitivity decreases with increase in size of animal injected, time after administration and decrease in dose given. These findings are discussed in relation to the known chemistry and subcellular mode of action of ricin.     

Production of specific antibodies to contractile proteins, and their use in immunofluorescence microscopy. I. Antibodies to smooth and striated chicken muscle myosins.

Antibodies prepared against actomyosins can be shown to behave similarly, if not identically to more recently prepared antibodies against highly purified myosins. Details of the purification of the antigens, and of the production of antibodies to chick myosins from smooth gizzard muscle and from striated pectoral muscle are given. The antibody specificity appears to be directed against the heavy chains of the myosin molecules, since these antibodies specifically inhibit the myosin ATPase reaction, and since in situ staining of myosin polypeptide chains on an SDS gel using the antibodies in indirect fluorescence shows staining only in the heavy band region. Use of the antibodies in immunofluorescence microscopy suggest that the antibodies are tissue, but not species, specific. Example of their use in staining tissue sections are shown.     

Influence of obstetricians' attitudes on their use of prenatal diagnosis for the detection of Down's syndrome.

Practising obstetricians were surveyed to determine the relation between their referral patterns and their knowledge and attitudes concerning prenatal diagnosis by amniocentesis for women aged 35 years and over. Although 82% had referred at least one eligible patient for prenatal diagnosis during the past year, almost none had used the available services for all appropriate patients. There was a statistically significant trend for increased referral as correct knowledge of the risks and accuracy of prenatal diagnosis increased. Moreover, a discriminant function combining risk and accuracy estimates, type and size of practice, and language distinguished the referrers from the nonreferrers (P = 0.0002), although there was considerable overlap between the physicians classified according to a high, moderate or low rate of referral. The data suggest that while knowledge and practice characteristics can distinguish obstetricians who refer patients for prenatal diagnosis from those who never do so, the frequency of referral may involve other factors, such as how physicians accept innovation and perceive risks.     

Rheological studies on mixed phospholipid sols and their interaction with penicillins I. Continuous shear viscometry

The effects of sonication and ageing of phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) sols have been examined. Ageing was found to significantly alter the nature of the flow curves obtained for sonicated mixtures; only after 48 hr was reproducibility obtained. Rheological studies on aged mixed sols indicated behaviour similar to that previously reported. Addition of urea and guanidine hydrochloride in concentrations up to 8 M indicated hydrophobic interaction between PC and LPC.

Addition of Penicillin G and Ampicillin to mixed PC/LPC sols indicated that Penicillin G interacted to a greater degree than Ampicillin; pronounced Penicillin G interaction was observed in a mixed sol containing 60% PC, 40% LPC, a system where the asymmetry of the aggregates is at a minimum. Ampicillin appeared to interact to a greater degree with mixtures containing more LPC than PC. Differences in hydrophobic interaction were indicated by use of urea and guanidine hydrochloride, which is of interest in the light of present knowledge regarding the in vivo activity of these antibiotics.