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Biomedical subjects

B A Baldo

Publications and source records attributed to B A Baldo.

At least 19 recordsLinked to original sources
PubMed1992

Measurement of PAF in blood by radioimmunoassay. Examination of interfering factors and problems involved in accurate quantification.

A recently developed radioimmunoassay for PAF was applied to blood extracts with the aims of defining and overcoming problems that lead to erroneous results and establishing optimum conditions for the accurate determination of PAF levels. The high lipid content of blood was found to interfere with the assay, and it appeared that phosphatidylcholine and/or sphingomyelin might be among the lipids responsible. Interference was eliminated by either dilution or preparative TLC of blood extract prior to RIA although dilution is unlikely to be generally useful due to the low amounts of PAF normally present in human blood. Lipid extraction of whole blood followed by preparative TLC proved to be necessary in the preparation of samples prior to performance of the RIA. The problems encountered in the measurement of PAF levels in blood by RIA highlight the importance of determining the correct method of sample preparation for any tissue/fluid prior to its inclusion in the assay.

Chromatography, Thin Layer↗
PubMed1992

Olive (Olea europea) and privet (Ligustrum vulgare) pollen allergens. Identification and cross-reactivity with grass pollen proteins.

Protein blotting studies showed that three olive pollen components with mol. wts approximately 18-19, 20 and 40 kD can be considered to be major allergens. For privet pollen, the highest recognition frequencies were for allergens of mol. wts approximately 20, approximately 19, approximately 40 and approximately 70 kD. When results with the 62 subjects examined were separated into groups corresponding to their geographical locations, viz. Italy, France and Australia, subjects sensitized to olive, but not other pollens (some Italian subjects), were found to show higher frequencies of recognition of major olive allergens than subjects sensitized to olive pollen via cross-reacting allergens from unrelated pollen sources (the Australian and French subjects). Blotting, adsorption and elution and inhibition studies clearly demonstrated allergenic cross-reactivity (that is, antigenic cross-reactivity detected by IgE antibodies) between olive, privet, ryegrass (Lolium perenne) and couch grass (Bermuda grass: Cynodon dactylon) pollen components. As with our previous findings with birch pollen, we conclude that the presence of pollen-reactive IgE antibodies may not necessarily be a true reflection of the sensitizing pollen species.

Allergens↗
PubMed1992

Persistence of allergy to anaesthetic drugs.

Intradermal testing and RIA testing for specific IgE antibodies to neuromuscular blocking drugs (NMBDs) were performed in patients referred to an Anaesthetic Allergy Clinic. Six patients were initially investigated four to 29 years after clinical anaphylaxis during anaesthesia and two of these patients and sixteen others were investigated by intradermal testing on two occasions at least four years apart. Seven patients had RIA tests for NMBD-specific IgE antibodies on two occasions at the time of skin testing. In all but two patients the evidence for drug-specific antibodies persisted 4-29 years after the reactions. In one patient all tests became negative and in another the skin test became negative but the positive RIA persisted. Evidence of antibodies to NMBDs persisted in 21 of 22 patients who had had anaphylactic reactions to these drugs during anaesthesia. In the absence of evidence of allergy diminishing with time in the majority of patients it would seem wise to avoid drugs responsible for reactions for the rest of the patient's life.

Alcuronium↗
PubMed1991

Stability of platelet activating factor (PAF) in human saliva. Quantitation by radioimmunoassay.

Platelet activating factor (PAF) is thought to mediate many inflammatory processes and its involvement in health and disease may be clarified by examining PAF levels in human secretions. The known presence of PAF, the ease of obtaining samples and the relative stability of PAF in saliva, makes this fluid a preferred source for examination of PAF in health and disease. The activity of PAF-acetylhydrolase (the PAF degrading enzyme) in saliva was 1,000-fold lower than that found in human plasma. Extraction of saliva with chloroform/methanol/water resulted in 70-90% recovery of PAF. Using the radioimmunoassay (RIA), PAF levels in the range 0.5-21 ng/ml were found in normal human salivas. These values were significantly higher than those reported from bioassay studies based on washed platelets. The validity of the RIA was checked by isolating and quantitating the PAF fraction from whole saliva extract, and by treatment of the extracts with the enzyme phospholipase A2. Direct comparison of salivary PAF levels, determined by both platelet aggregation (PA) and RIA confirmed our original finding that values obtained were lower using the bioassay method. Furthermore, these bioassay values compared favourably with those in the literature. Investigations revealed the presence of a substance(s) in saliva which inhibited PAF-induced platelet aggregation but which did not affect the radioimmunoassay.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1991

Synthesis of a PAF immunogen and production of PAF-specific antibodies.

Platelet activating factor (PAF), a naturally occurring phospholipid with many potent physiological and pharmacological activities, is implicated as a mediator of many diseases. An immunoassay for PAF would greatly improve quantitation, and hence PAF-specific antibodies were required. Chemically-reactive analogs of PAF, containing an aldehyde group at the end of the 1-O-alkyl chain (hexyl or dodecyl), were synthesized from readily available materials. During the multi-step synthetic procedure, the aldehyde group was protected as an acetal, which was converted by mild acidic hydrolysis to the aldehyde immediately prior to protein coupling. These analogs were coupled to methylated bovine serum albumin and the resultant conjugates were injected into rabbits. Antibodies to PAF were detected using a solid phase radioimmunoassay based on Protein A-Sepharose. The dodecyl PAF conjugate proved to be the more immunogenic conjugate with more than half of the rabbits producing significant levels of antibodies (at least a 10-fold increase in radioactive uptake over pre-immune levels). Results from solid phase immunoassays employing nitrocellulose discs impregnated with PAF, lysoPAF, lecithin, lysolecithin and 2-O-methyl-lysoPAF indicated that the antibodies recognized only PAF. PAF-specific antibodies were isolated by affinity chromatography using a column of PAF-poly(lysine) conjugated to carboxy-activated polyacrylamide. The antibodies may be employed in a sensitive and specific immunoassay for PAF and for many other studies involving PAF.

Animals↗
PubMed1991

A specific, sensitive and high-capacity immunoassay for PAF.

A specific radioimmunoassay for platelet-activating factor (PAF) sensitive in the range 10-1000 pg (0.02-2 pmoles) has been developed. Detailed quantitative hapten inhibition studies showed specificity for the acetyl group at C-2 of PAF, a requirement for the ether linkage at C-1 and some tolerance for substituents on the choline nitrogen. No significant cross-reactivity was found with phosphatidylcholine and lysophosphatidylcholine or with lysoPAF.

Animals↗
PubMed1991

Quantitation by radioimmunoassay of PAF in human saliva.

Approximately 75% of the PAF present in saliva is recovered on extraction of whole saliva (0.8 vol) with chloroform/methanol/water (2:2:1, v/v/v). PAF levels, determined by our recently developed radioimmunoassay, in saliva extracts ranged from 0.5-21 ng/mL with 59% between 2-6 ng/mL. These figures, for apparently healthy subjects, are higher than previously reported levels obtained by platelet assays. The validity of our radioimmunoassay results was checked by isolating and quantitating the PAF fraction from whole saliva. In addition, when we examined our saliva samples by platelet aggregation, low levels of PAF, comparable with the values found in the literature, were detected. Investigations revealed the presence of a substance(s) which inhibited PAF-induced platelet aggregation but which did not affect the radioimmunoassay.

Animals↗
PubMed1991

Inhibitor(s) of platelet-activating factor (PAF) in human saliva.

Quantitation of platelet-activating factor (PAF) in human saliva samples by radioimmunoassay indicated there was, at times, sufficient PAF present to aggregate platelets. However, in certain samples, we observed little or no aggregation, and furthermore, these samples were found to inhibit aggregation induced by PAF (200 pg). Chromatographic fractionation of pooled saliva increased the PAF activity 4-fold, and the observed inhibitory activity was found to co-migrate with the fatty acids. The inhibitory fraction was found to be active against platelet aggregation induced by arachidonic acid (3.4 nmole) as well as PAF (25 pg), but not thrombin (20 mU). These results indicate the existence of a PAF inhibitor in saliva, which may explain why potentially toxic levels of PAF can occur in the saliva of normal, healthy individuals. These findings also highlight an important advantage of the radioimmunoassay over platelet aggregation for the quantitation of PAF in, at least, some biological fluids.

Animals↗
PubMed1991

Cross-sensitization between cocaine and GBR 12909, a dopamine uptake inhibitor.

In the present experiment, rats which had received repeated injections of 20 mg/kg GBR 12909, a selective dopamine uptake inhibitor, were subjected to challenge doses of GBR 12909 and cocaine which were near or below the threshold for locomotor activation in drug-naive animals. The behavior measured was general motor activity, monitored in photocell testing cages. Results verify earlier findings which showed that rats treated chronically with GBR 12909 display a potentiated locomotor response to low doses of the drug (behavioral sensitization). In addition, it was found that GBR 12909-sensitized rats show a potentiated locomotor response to 5 mg/kg cocaine (cross-sensitization). These results are discussed with regard to various mechanisms which may underlie the sensitization phenomena observed, including possible neurochemical changes at the presynaptic level.

Analysis of Variance↗
PubMed1991

Structural features of allergens large and small with emphasis on recombinant allergens.

The application of recombinant DNA techniques to the study of allergen structure has increased our knowledge of primary structures and B- and T-cell determinants. Thus, knowledge of the molecular bases of isoallergens and allergenic cross-reactivities is about to be rapidly expanded. Findings from the early applications of molecular cloning strategies to the study of some polypeptide allergens, together with a summary of our current knowledge of drug allergenic determinants, are presented here.

Allergens↗
PubMed1991

Identification of jack-jumper ant (Myrmecia pilosula) venom allergens.

Jack-jumper ant venom proteins were electrophoretically separated on SDS-polyacrylamide gels, transferred to nitrocellulose and probed with sera from subjects who had experienced an allergic reaction after being bitten by a jack-jumper ant. Ant venom components that bound IgE antibodies were detected by addition of 125I-anti-human IgE followed by autoradiography. Of the 17 polypeptides resolved by electrophoresis only three, of molecular weights approximately 14 kD, 12 kD and 10 kD, bound IgE antibodies from the panel of 50 sera examined. There was a marked similarity in the binding patterns by individual sera with almost all of the sera recognizing the 14kD and 12 kD components. IgE-binding profiles of separated ant venoms from ants collected in different regions of Australia appeared to be very similar if not identical. Identification of the ant allergens is a necessary prelude to the preparation of standardized venom sac extracts suitable for safe and effective diagnostic and therapeutic use.

Allergens↗
PubMed1991

Cypress (Cupressus sempervirens) pollen allergens: identification by protein blotting and improved detection of specific IgE antibodies.

On the basis of results of an investigation of the effects of different treatments employed, a dialysed and reduced extract of Cupressus sempervirens was separated electrophoretically on sodium dodecylsulphate-polyacrylamide gels before being transferred and then fixed with glutaraldehyde to nitrocellulose membrane. Probing with sera from 91 subjects allergic to C. sempervirens pollen followed by detection of bound IgE antibodies with [125I]-labelled anti-human IgE revealed 17 IgE-binding proteins in the molecular weight range 14-96 kilodaltons (kDa). One component, of molecular weight approximately 42 kDa, reacted with IgE antibodies in the sera of 81.3% of the allergic subjects and, for each of the subjects, this component bound the greatest quantity of IgE. Almost 50% of the sera recognized only the approximately 42 kDa component, reinforcing the conclusion that this component is the major allergen of C. sempervirens pollen. A comparative study employing C. sempervirens pollen allergen discs prepared commercially or in the laboratory showed that values of the uptakes of [125I]-anti-IgE indicating the presence of pollen-reactive IgE antibodies obtained with the latter discs were consistently higher (means 4.5 vs. 0.88), and that false-negative results were obtained when many sera were used with the commercial discs. The results of this study provide an essential basis for the production of standardized, safe and effective C. sempervirens pollen extract applicable to diagnosis and therapy of cypress pollen allergy.

Allergens↗
PubMed1991

Antibodies to platelet-activating factor (PAF) inhibit PAF-induced platelet aggregation.

Platelet-activating factor (PAF) is the most potent platelet agonist known. PAF-induced platelet aggregation was blocked by pre-incubation of PAF with the immunoglobulin fraction from sheep or rabbit anti-PAF anti-serum. Inhibition was specific for PAF and was dependent upon immunoglobulin and PAF concentrations. Antibody-mediated inhibition of PAF-activity may be a valuable method for studying the biological effects of PAF in some systems.

Adenosine Diphosphate↗
PubMed1990

A specific, sensitive radioimmunoassay for platelet-activating factor (PAF).

A specific radioimmunoassay (RIA) has been developed for platelet-activating factor (PAF) and shown to be sensitive over the range 10-1000 pg (0.02-2 pmol). The anti-PAF antibodies showed specificity for the acetyl group at the C2 position of the PAF molecule and exhibited no significant cross-reactivity with lyso-PAF or the naturally occurring lipids including lecithin and lysolecithin. The sensitivity of the RIA was at least as good as the platelet-based assays for PAF but the RIA was simpler to perform, had a higher capacity and did not have the drawback of the inherent variability associated with the bioassays.

Animals↗
PubMed1990

The specificity of the binding of platelet activating factor (PAF) to anti-PAF antibodies.

Quantitative hapten inhibition experiments employing sheep anti-PAF antibodies and selected PAF analogues were undertaken with the aim of defining the antigenic determinant structures complementary to the antibody combining sites. The most important fine structural features for inhibition of antibody to PAF were shown to be an acetyl group at position 2 of the phospholipid glycerol backbone and an ether group at position 1. Of the naturally occurring compounds, C16- and C18:1-PAF proved to be the most potent inhibitors and more active than C18-PAF while phospholipids with a propionyl, butyryl or hexanoyl group at position 2 showed either weak or no inhibitory activity. The 1-acyl, thioether and deoxy analogues proved inactive. Variations in the polar head group of PAF were found to be less critical with, for example, the dimethyl and ethanolamine derivatives retaining some activity. This antibody recognition pattern is very similar to that of the PAF receptor, although the antibodies appear to have a more specific requirement for an acyl linkage at position 2.

Animals↗
PubMed1990

Identification of penicillin allergenic determinants that bind IgE antibodies in the sera of subjects with penicillin allergy.

Much of the literature on penicillin hypersensitivity is devoted to the identification of penicillin antigens rather than allergens. Human IgE-binding determinants on different penicillins have rarely been closely investigated with the view of defining fine structural allergenic features and differences. We have developed radioimmunoassays employing ampicillin, amoxicillin and ticarcillin solid phases for the detection of penicillin-reactive IgE antibodies. Quantitative hapten inhibition studies employed to identify IgE-binding regions on the penicillin molecules revealed a heterogeneous group of allergenic determinants consisting exclusively, or in part, of the alpha-aminobenzyl and benzyl side chain groups and the beta-lactam and thiazolidine rings of the penicillin nucleus.

Adult↗