The T cell response to major grass allergens is regulated and includes IL-10 production in atopic but not in non-atopic subjects.

BACKGROUND: The incidence of allergic diseases is increasing in industrialized countries and the immunological mechanisms leading to tolerance or allergy are poorly understood. Cytokines with suppressive abilities and CD4(+)CD25(+) regulatory T cells have been suggested to play a central role in allergen-specific responses. The aim was to determine whether major grass allergens induce production of suppressive cytokines in allergic and healthy subjects and to examine the inhibitory effect of these cytokines on allergic responses. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from healthy and grass-allergic donors and stimulated with the major grass allergens Phl p 1 or Phl p 5. The effects of endogenous IL-10 and/or TGF-beta on proliferation and cytokine production were determined by use of blocking antibodies. In addition, the number of CD4(+)CD25(+) T cells and their expression of chemokine receptors were investigated by flow cytometry. RESULTS: Phl p 1 and Phl p 5 induced IL-10 production, which down-regulated proliferation and cytokine production, in PBMC cultures from atopic but not from non-atopic donors. Comparable frequencies of CD4(+)CD25(+) T cells were present in PBMCs in the two groups, but fewer cells from atopic donors were CD4(+)CD25(+)CCR4(+) and more cells were CD4(+)CD25(+)CLA(+) compared to healthy donors. CONCLUSION: Allergen-specific responses of grass allergic patients but not in non-atopic subjects are influenced by regulatory cytokines produced in response to the important allergens. Differences in CD4(+)CD25(+) T cell expression of chemokine receptors in allergic compared to non-atopic donors could suggest that the homing of CD4(+)CD25(+) T cells is important for the regulation of allergen-specific responses.

Exploring abiotic stress on asynchronous protein metabolism in single kernels of wheat studied by NMR spectroscopy and chemometrics.

Extreme climate events are being recognized as important factors in the effects on crop growth and yield. Increased climatic variability leads to more frequent extreme conditions which may result in crops being exposed to more than one extreme event within a growing season. The aim of this study was to examine the implications of different drought treatments on the protein fractions in grains of winter wheat using (1)H nuclear magnetic resonance spectroscopy followed by chemometric analysis. Triticum aestivum L. cv. Vinjett was studied in a semi-field experiment and subjected to drought episodes either at terminal spikelet, during grain-filling or at both stages. Principal component trajectories of the total protein content and the protein fractions of flour as well as the (1)H NMR spectra of single wheat kernels, wheat flour, and wheat methanol extracts were analysed to elucidate the metabolic development during grain-filling. The results from both the (1)H NMR spectra of methanol extracts and the (1)H HR-MAS NMR of single kernels showed that a single drought event during the generative stage had as strong an influence on protein metabolism as two consecutive events of drought. By contrast, a drought event at the vegetative growth stage had little effect on the parameters investigated. For the first time, (1)H HR-MAS NMR spectra of grains taken during grain-filling were analysed by an advanced multiway model. In addition to the results from the chemical protein analysis and the (1)H HR-MAS NMR spectra of single kernels indicating that protein metabolism is influenced by multiple drought events, the (1)H NMR spectra of the methanol extracts of flour from mature grains revealed that the amount of fumaric acid is particularly sensitive to water deficits.

Non-haem iron availability from pork meat: Impact of heat treatments and meat protein dose.

Pork meat heated at 60, 80, 100, and 120°C (1h), raw pork meat, BSA, casein and haemoglobin were examined for their effects on in vitro iron availability measured as Fe(2+)-dialysability, and on iron-reducing capacity following in vitro protein digestion (IVPD-dialysis). The pepsin-digested samples of meat heated at 80, 100, and 120°C resulted in increased in vitro iron availability. Generally, the capacity to reduce Fe(3+) to Fe(2+) was higher in the pepsin digests, whereas Fe(2+) decreased significantly after pepsin+pancreatin digestion and only part of the Fe(2+) was dialysable. Regardless of protein concentration, casein had no effect on in vitro iron availability, while pork meat protein treated at 120°C showed dose dependency reaching a plateau at 50mg protein/ml. In conclusion, the major effects on iron availability in vitro was shown in pepsin digests under conditions mimicking those in the duodenal lumen and heat-treatment of meat at 120°C showed the most pronounced effects.

A nylon ball solid-phase radioimmunoassay for specific antibodies in human sera. Application to measurement of IgG antibodies to pollen allergens.

The principle of the radioallergosorbent test (RAST) has been used to measure IgG antibodies to timothy grass pollen allergens in sera from desensitized allergic subjects. 125I-labeled goat anti-human IgG was used as detector protein. Non-specific binding was eliminated by use of a non-porous nylon ball an antigen carrier and by use of a special buffer with high ionic strength and pH, containing 1% bovine gamma globulin and 5% normal rabbit serum as 'balance proteins'. At dilution 1:80 non-specific binding was only 0.28% and the binding ratio for a high-liter serum was about 10. By inhibition experiments the assay was demonstrated to be specific for IgG antibodies to timothy grass pollen. The results obtained with this assay correlated statistically significantly with those found th a double -antibody method (rs equal 0.68, n equal 20, t equal 3.93, P less than 0.001). Serum dilution curves were parallel, indicating that the assay is in allergen excess. The within-assay coefficient of variation ranged from 3.9 to 7.6%; the between-assay coefficient of variation from 8.4 to 19.5%. The assay is very simple to perform, requiring no centrifugation. The allergen-coated balls are stable for at least 3 months. The assay should be applicable to measurement of IgG antibodies and IgG subclass antibodies to any protein antigen of interest.

A sensitive and reproducible method for the determination of subnanogram quantities of immunoglobulin E (IgE).

An immunoradiometric assay for immunoglobulin E (IgE) using high adsorption polystyrene test tubes as the solid phase (the 'Maxisorp' assay) is described. The sensitivity of the method was found to be 50 pg IgE/ml and the within-assay reproducibility was 3-7%. The accuracy was estimated by means of a comparison between the Maxisorp assay and paper radioimmunosorbent test (PRIST) and a correlation coefficient of 0.98 (P less than 0.001) was obtained. We conclude that the Maxisorp assay is a fast and reliable assay for IgE determination in cord blood, cell culture supernatants and other highly diluted IgE preparations.

Inhibition of 5-HT uptake into neurons and platelets in mice treated chronically with chlorimipramine and femoxetine.

A single treatment with 5-HT uptake inhibitors potentiates the hypermotility in mice produced by the MAO-inhibitor nialamide. The effect of nialamide on motility was studied in mice after 4 weeks of feeding with a normal diet and diets containing various concentrations of the 5-HT uptake inhibitors chlorimipramine and femoxetine. Chronic treatment with the two substances enhanced the motor effects of nialamide about equally, which indicates a preservation of the neuronal 5-HT uptake inhibition during such treatment. The effect of chlorimipramine and femoxetine was obtained at plasma levels equivalent to or lower than the steady-state plasma concentrations found in patients treated with two 5-HT uptake inhibitors. Determination of decreased blood 5-HT after the 4 weeks of treatment was used as an in vivo test for inhibition of 5-HT uptake into platelets. Femoxetine was a much weaker depletor of blood 5-HT than chlorimipramine. These results indicate that blockade of neuronal 5-HT uptake is obtained at lower doses of femoxetine than blockade of 5-HT uptake into platelets. In contrast, chlorimipramine presumably inhibits 5-HT uptake into neurons and platelets at about the same dose.

Identification of wheat varieties using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry and an artificial neural network

A novel tool for variety identification of wheat (Triticum aestivum L.) has been developed: an artificial neural network (ANN) is used to classify the gliadin fraction analysed by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS). The robustness of this novel method with respect to various experimental parameters has been tested. The results can be summarised: (i) With this approach 97% of the wheat varieties can be classified correctly with a corresponding correlation coefficient of 1.0, (ii) The method is fast since the time of extracting gliadins from flour can be reduced to 20 min without significant decrease in overall performance, (iii) The storage of flour or extracts under standard conditions does not influence the classification ability (i. e. the generalisation ability) of the method, and (iv) The classification obtained is not influenced by the identity of the operator making the analysis. This study demonstrates that a combination of an ANN and MALDI-TOFMS analysis of the gliadin fraction provides a fast and reliable tool for the variety identification of wheat. Copyright 1999 John Wiley & Sons, Ltd.

Spectroscopic investigations on the highly purified lactoperoxidase Fe(III)-heme catalytic site.

Purification of the lactoperoxidase (LPO) major cationic isoenzyme was significantly improved by the use of preparative chromatographic and electrophoretic methods combined with analytical electrophoretic techniques and image processing. A detailed report is given of the experimental procedure. Furthermore, electron paramagnetic resonance has played a fundamental role in evaluating the enzyme purity against lactoferrin and minor LPO isoenzyme components in setting the final steps of the purification. With the aim to completely clarify the Fe(III)-heme high-spin nature of the native LPO, two samples of lactoperoxidase, LPO1 and LPO2 (RZ = 0.95) from farm and commercial milk, respectively, were purified and characterized in particular by electron paramagnetic resonance (EPR) spectroscopy, in comparison with a commercial preparation (LPOs). The LPO1 EPR spectrum, at physiological pH, is clearly indictive of the presence of an iron(III)-heme high-spin catalytic site in the native enzyme. On the contrary, in the LPO2 spectrum a thermal equilibrium between high- and low-spin iron(III)-heme species is present. The low-spin component of the spectrum has been assigned to an LPO-NO2- adduct due to the presence of some nitrite impurities originating either from commercial unpasteurized milk or from external sources. The LPOs EPR spectrum shwos the presence of some spurious lines in the g approximately equal to 6 and 4 regions due to the minor LPO isoenzyme components and to lactoferrin, respectively. The LPO EPR spectra previously reported in the literature contain a variable number of spurious lines in the g approximately equal to 4 and 2 regions as a consequence of lactoferrin impurity and LPO low-spin adducts with endogenous or exogenous anions. Furthermore, the interaction of LPO with its native substrate (the thiocyanate anion), which previously was shown by NMR and EPR (at high substrate concentration) spectroscopies, has been confirmed by EPR at low temperature and low substrate concentration and by optical spectroscopy at room temperature and high substrate concentration as a function of pH. The LPO activity at optimum pH (approximately equal to 4-5) has been measured in phosphate and acetate buffer using as an oxidizable substrate the system dimethylamino benzoic acid 3-methyl-2-benzothiazolinone hydrazone hydrochloride monohydrate (DMAB-MBTH), which was considered a good chromogen for other peroxidases such as HRP and zucchini peroxidases. The LPO vs SCN- activity at optimum pH (approximately equal to 5.5) has been measured in phosphate and acetate buffer.(ABSTRACT TRUNCATED AT 400 WORDS)

Autoimmune type I reactions directed against nuclear components in rheumatoid arthritis.

Basophil histamine release was examined in leucocyte suspensions from patients with rheumatoid arthritis (RA) after challenge of the cells with isolated and sonicated leucocyte nuclei from normal individuals. Most of the patients with active disease responded with significant histamine release, whereas no response was obtained in the inactive patients and in normal controls. A similar pattern was found in the urinary excretion of the main metabolite of histamine, NT-methyl-imidazoleacetic acid, since an increased excretion was observed in most patients with severe disease activity in contrast to patients with moderate and quiescent activity and the control group. These findings strongly indicate an involvement of autoimmune allergic type I reactions in RA. The release of histamine and other mediators from basophils and mast cells may cooperate in the inflammatory reactions and the destruction of the joints in RA.

Quantitative determination of 1,4-methyl-imidazoleacetic acid in urine by high performance liquid chromatography.

A reversed phase ion-pair high performance liquid chromatographic (HPLC) method for quantitative determination of the major histamine metabolite 1,4-methyl-imidazoleacetic acid (1,4-MIAA) in urine is described. The sample handling is minimal as the sample only has to be centrifuged and diluted before analysis, in contrast to earlier gas chromatographic and thin-layer chromatographic methods. The method correlates well with a thin-layer chromatographic determination (r = 0.79, n = 15), and results from analysis of urine from different patients suffering from mastocytosis, chronic urticaria, atopic dermatitis and asthma are presented together with samples from normal individuals. Most of the patients analysed showed increased excretion of 1,4-MIAA compared with the controls. Future applications of the method, including analysis on patients with food allergy and patients with abnormalities in their histamine metabolism, are discussed.

Isolation of patient-related antigens from allergen extracts by means of protein A-sepharose-patient IgG1,2,4 bio-affinity chromatography.

A method is described for bio-affinity purification of patient-specific IgG reactive antigens by using protein A-sepharose allergic patient serum and the corresponding antigen/allergen extract. The optimal conditions for each step have been determined and it is shown that the non-specific binding of both serum proteins and antigens can be kept at very low levels by a high ionic strength. The specificity of the reaction has been shown by biospecific elution of 125I-labelled antigens by non-labelled antigens and also by comparison of the amount of antigen bound by sera from hyposensitized patients with that from non-atopic individuals. It is suggested that the isolated antigen-IgG-immune complexes could be used for a specific and active immunotherapy.

Purification of patient-related allergens by means of bioaffinity chromatography on a sepharose anti-IgE patient IgE immunosorbent.

A method is described for bioaffinity chromatographic purification of patient-specific IgE reactive allergens by using sepharose coupled rabbit anti-human IgE antibodies, patient serum and an allergen extract. The conditions for this bioaffinity chromatographic procedure have been optimised, and it was shown that the nonspecific binding of serum proteins and antigens can be kept at very low levels by a high ionic strength. The specificity of the method has been determined by showing that allergens to which the patient did not react were not bound to the affinity matrix. We suggest that the isolated allergens could be used for more specific immunotherapy in the future.

Allergen content in aqueous and hydrophobic extracts from cow hair and dander. Comparison of extracts by means of quantitative immunoelectrophoresis.

Rabbit antibodies were raised separately against an aqueous and a detergent extract of cow hair and dander. It is shown by means of quantitative immunoelectrophoresis that the extracts contain the same major allergens and that these are equally potent in RAST-inhibition experiments. The extraction with a detergent did not lead to the appearance of new allergens.

Histamine release from basophil leukocytes in asthma patients after in vitro provocation with various neuromuscular blocking drugs and intravenous anaesthetic agents.

Basophil histamine release is a relatively new investigation technique, which can be used in the diagnosis of anaphylactoid reactions. Our aim in this investigation was to determine reference values for asthma patients and normal subjects. Blood from eight asthmatic patients and eight normal subjects was tested for histamine release after in vitro provocation with various neuromuscular blocking drugs and intravenous anaesthetic agents. There was significantly higher histamine release for asthmatic patients than for normal subjects, P less than 0.001 (analysis of variance). This had no effect on the calculated reference value (mean +/- 2 s.d.), which was found to be 0-30%.

A modified crossed radioimmunoelectrophoretic method for determination of allergen-specific IgG antibodies. Elimination of non-specific binding by use of F(ab')2-fragments of rabbit antibodies and 125I-labelled protein A.

A crossed radioimmunoelectrophoretic (CRIE) method for detection of specific IgG antibodies in patients' sera against horse hair and dander was developed. The unacceptably high non-specific binding encountered when substituting 125I-labelled antihuman IgG for 125I-labelled antihuman IgE in an ordinary CRIE was eliminated by the combined use of 125I-labelled Protein A as detector, and F(ab')2-fragments of the allergen-specific rabbit antibodies. The low background binding thus obtained makes the method useful for detection of specific IgG in sera where the ratio between specific and non-specific IgG is low. Therefore the method should also be applicable to other antigen/allergen systems.

Nebulization and selective deposition of LTD4 in human lungs.

The process of nebulization and deposition of LTD4 was studied in detail. The concentration of LTD4 in a saline solution decreased by approximately 90% after 2 min of nebulization in a DeVilbiss 35B ultrasonic nebulizer. This decrease was prevented by diluting LTD4 in a phosphate buffer, pH 7.4. Nebulization of tritiated LTD4 in this phosphate buffer did not cause any appreciable deterioration of the leukotriene, as demonstrated by an unchanged ratio between radioactivity and LTD4 concentration in the test solution before and after nebulization as well as in the condensed aerosol. The aerosol generated by the DeVilbiss 35B ultrasonic nebulizer was shown to generate particles with a mass median diameter of 1.3 microns (dry particle size). Interposition of a settling bag reduced the amount of large particles, reducing the mass median diameter to 0.84 microns (dry particle size). Nine healthy volunteers were challenged on separate days with 40 nmol LTD4 or 100 mumol histamine, and the changes in FEV1 and partial flow volume curves initiated at 50% of vital capacity (Vmax30) were measured. A relative diffuse deposition pattern was ensured by inhalation via a settling bag. These results were compared to challenges with a relatively central deposition pattern as ensured by inhalation directly from the nebulizer with brisk inhalation maneuvers. The diffuse deposition pattern caused minimal changes in FEV1 but pronounced effect in Vmax30. The effects of LTD4 and histamine on FEV1 and Vmax30 changed in parallel when the deposition of the mediators was changed to a more central pattern. This indicates that the two mediators do not differ with respect to any selective effects on different parts of the airways.