Allergic responses induced by goat milk αS1-casein in a murine model of gastrointestinal atopy.

Up to 3% of young children develop milk allergy and this may influence the development of immune-mediated diseases in later life. One protein that has been associated with allergic reactions to ruminant milk is α(S1)-casein (CN). Studies suggest that goat milk with low levels of α(S1)-CN may reduce allergenicity of milk, but the dose response to α(S1)-CN has not been confirmed. In this study, we examined the immune response to varying levels of goat α(S1)-CN in a mouse model of gastrointestinal allergy. BALB/c mice (aged 5 wk) were given intraperitoneal injections with α(S1)-CN and aluminum as adjuvant at 1 and 3 wk to sensitize mice to the antigen. In wk 5, groups of fasting mice (n=8/group) were challenged 4 times on alternate days by intragastric gavage with saline or 2, 10, or 20mg of α(S1)-CN. Serum levels of specific IgE, IgG(1), and IgG(2a) antibodies and mouse mast cell protease-I were determined. Interleukin-4, IL-10, and IFN-γ responses to 48-h activation with antigen were measured in cultured splenocytes. We determined that mice sensitized with α(S1)-CN had higher titers of specific IgG(1) and IgE antibodies compared with controls; however, groups challenged with differing doses of α(S1)-CN did not differ. The group challenged with the highest dose of α(S1)-CN had a 10-fold increase in mouse mast cell protease-I compared with the group challenged with saline. Both IL-4 and IL-10 were produced in a dose-dependent manner by cultured splenocytes incubated with α(S1)-CN. Overall, α(S1)-CN stimulated the production of cytokines associated with allergic disease in a dose-dependent manner. Thus, milk with lower levels of α(S1)-CN should contribute to a lesser antigenic burden.

Effects on adhesion molecule expression and lymphocytes in the bovine mammary gland following intra-mammary immunisation.

Changes to adhesion molecule expression and lymphocyte populations were evaluated in alveolar mammary tissue collected from cows following an immunisation protocol that involved intra-mammary inoculation to induce an IgA response in mammary secretions. The right quarters of the udder were immunised; the left side acted as a control. Antibody titres in secretions showed that at least two animals responded with antigen-specific IgA. Numbers of T-lymphocytes were 4-fold higher in immunised glands compared with controls (P<0.05). IgA-, IgM- and IgG-positive cell numbers were significantly higher (P<0.01) in immunised glands compared with controls in three of the four cows. No mucosal addressin molecule-1 (MAdCAM-1), vascular cell-adhesion molecule-1 (VCAM-1) or peripheral node addressin (PNAd) protein expression was detected on smaller venules that stained positively for von Willebrand factor in alveolar mammary tissues, from either immunised or control glands. Both VCAM-1 and PNAd were detected on smaller venules in supramammary lymph nodes, however, there was no significant difference between immunised and control glands. Quantification of MAdCAM-1 mRNA showed very low expression in both immunised and control alveolar tissue compared with Peyer's patch positive-control tissue. These findings suggest that the bovine mammary gland is capable of a mucosal antibody response; however, MAdCAM-1 is not involved with lymphocyte homing to the mammary gland in this species.

Adhesion molecule expression in the bovine mammary gland.

The bovine mammary gland requires lymphocytes for immune protection of the gland from foreign pathogens and, in addition, to transfer immune protection to the neonate via colostrum and milk. The process of homing primed lymphocytes to tissues is mediated by the interaction of cell-adhesion molecules displayed on the surface of lymphocytes and counter receptors displayed on the vascular endothelium. This study was conducted to identify the cell-adhesion molecules involved in homing lymphocytes to the bovine mammary gland at four different physiological stages; pregnant, colostral, lactation and involution. The expression and distribution of adhesion molecules in alveolar tissues and supramammary lymph nodes from the mammary glands of healthy cows was determined in situ by immunohistochemical analysis and compared with bovine Peyer's patch, used as a typical mucosal-associated lymphoid tissue and positive control. The mucosal addressin molecule, MAdCAM-1, was not detected in bovine mammary tissues at any of the four different physiological stages. Absence of MAdCAM-1 expression was verified by quantitative real-time RT-PCR analysis. Transcription levels of MAdCAM-1 mRNA were found to be more then 5 x 10(3)-fold lower in mammary alveolar tissues compared with bovine Peyer's patch tissues. In contrast to MAdCAM-1, phase-dependent protein expression of VCAM-1 was detected in both mammary alveolar tissues and the supramammary lymph nodes, with the highest expression observed in colostral phase cows. The protein expression in mammary alveolar tissues was limited to larger venules, although in colostral phase cows, VCAM-1 was also detected around the alveoli perimeter. In the supramammary lymph node, VCAM-1 protein was observed on both small and large venules. PNAd was detected in supramammary lymph nodes at all physiological stages of the mammary gland; however, it was not found in mammary alveolar tissues. Lymphocytes expressing beta7 were not detected in mammary tissues and lymphocytes expressing CD62L were only observed in the supramammary lymph nodes. Overall the data suggest that MAdCAM-1 and VCAM-1 are not involved in homing lymphocytes to the bovine mammary gland; whereas, VCAM-1 and PNAd may have this role in the supramammary lymph node.

Evaluation of a new method for the analysis of free catecholamines in plasma using automated sample trace enrichment with dialysis and HPLC.

BACKGROUND: Analysis of urinary free catecholamines was automated recently, but analysis of plasma samples posed special difficulties. The present study was undertaken to evaluate a new method for the automated analysis of plasma catecholamines. METHODS: The procedure is based on an improved sample handling system that includes dialysis and sample clean-up on a strong cation trace-enrichment cartridge. The catecholamines norepinephrine, epinephrine, and dopamine are then separated by reversed-phase ion-pair chromatography and quantified by electrochemical detection. RESULTS: Use of a 740- microL sample is required to give the catecholamine detection limit of 0.05 nmol/L and analytical imprecision (CV) between 1.1% and 9.3%. The assay can be run unattended, although >12 h of analysis time is not recommended without cooling of the autosampler rack. Comparison (n = 68) of the automated cation-exchange clean-up with the well-established manual alumina procedure gave excellent agreement (mean, 3.78 +/- 2.76 and 3.8 +/- 2.89 nmol/L for norepinephrine and 0.99 +/- 1.72 and 1.08 +/- 1.78 nmol/L for epinephrine). Hemodialysis had no clear effect on plasma norepinephrine. Epinephrine concentrations were similar (0.05 < P < 0.1) in chronic renal failure patients (0.24 +/- 0.3 nmol/L; n = 15) and healthy controls (0.5 +/- 0.24 nmol/L; n = 31). Dopamine was not quantified, being usually <0.2 nmol/L. CONCLUSION: The availability of such a fully automated procedure should encourage the more widespread use of plasma catecholamine estimation, e.g., after dialysis, exercise, or trauma/surgery and in the investigation of catecholamine-secreting tumors, particularly in the anuric patient.

Development of fluoroimmunoassays for flecainide.

Antibodies raised in sheep against a flecainide:protein conjugate and fluorescein-labeled drug were used to develop simple fluoroimmunoassays for the measurement of flecainide acetate in serum or plasma. A rapid, nonseparation assay, based on fluorescence polarization was optimized for therapeutic drug monitoring. A separation fluoroimmunoassay, using antibodies covalently linked to magnetizable particles to avoid the need for centrifugation, was also optimized and validated for monitoring flecainide therapy. It is applicable to lipemic, hemolyzed or icteric samples unsuitable for the nonseparation approach and for laboratories with access only to a simple fluorimeter. Finally, the separation fluoroimmunoassay was modified slightly to improve markedly sensitivity for use in pharmacokinetic studies.

Single-reagent polarization fluoroimmunoassay for theophylline in serum.

A polarization fluoroimmunoassay for theophylline was developed employing fluorescein-labeled drug and antiserum precombined in a single reagent. Assay was performed simply by addition of sample to an aliquot of the single reagent, incubation, and determination of fluorescence polarization. Because of the relatively rapid dissociation kinetics of the labeled drug from antibody binding, added unlabeled theophylline caused displacement within a practical time period. The precision, accuracy, and specificity of the simplified single-reagent assay were similar to those obtained by a conventional immunoassay procedure using the same reagents. Results for the assay of patients' serum specimens correlated well with those by an established enzymoimmunoassay.

Direct determination of theophylline in serum by fluoroimmunoassay using highly specific antibodies.

Described is the development of a fluoroimmunoassay for theophylline using a fluorescein labelled derivative of theophylline as tracer and antibodies coupled to magnetizable solid-phase particles. Three approaches are described for the preparation of antibodies for theophylline, of which one produced highly specific, high titre antibodies. The fluoroimmunoassay using these antibodies required a 10 microL sample, reached equilibrium within 5 min, and the results correlated closely with those of an established enzymoimmunoassay method. Potentially interfering endogenous fluorophores from the serum sample were reliably removed at the separation step of the bound and free fractions. There was no significant cross-reactivity with all other structurally related compounds.

Fluoroimmunoassay of digoxin in serum.

A heterogeneous (solid-phase separation) fluoroimmunoassay for digoxin in serum was developed employing antibodies coupled to magnetisable cellulose/iron oxide particles and a fluorescein-labelled digoxin derivative as tracer. Intrinsic fluorophores and other potentially interfering components of serum samples were reliably and completely removed at the separation and wash steps of the assay procedure which were facilitated by magnetic sedimentation. In order to attain adequate sensitivity (detection limit 0.2 micrograms/l (0.26 nmol/l) serum digoxin), a sample volume of 500 microliters was necessary. Assay results for patients' specimens correlated well with those obtained using established charcoal--separation (r = 0.96) and magnetisable solid-phase (r = 0.95) radioimmunoassays. The feasibility of a "stat" adaptation of the fluoroimmunoassay that involved only two standards (0.5 and 4 micrograms/l digoxin) was demonstrated. The stat method would be suitable for the assay of urgent or single specimens.