Confocal microscopy of FM4-64 as a tool for analysing endocytosis and vesicle trafficking in living fungal hyphae.

Confocal microscopy of amphiphilic styryl dyes has been used to investigate endocytosis and vesicle trafficking in living fungal hyphae. Hyphae were treated with FM4-64, FM1-43 or TMA-DPH, three of the most commonly used membrane-selective dyes reported as markers of endocytosis. All three dyes were rapidly internalized within hyphae. FM4-64 was found best for imaging the dynamic changes in size, morphology and position of the apical vesicle cluster within growing hyphal tips because of its staining pattern, greater photostability and low cytotoxicity. FM4-64 was taken up into both the apical and subapical compartments of living hyphae in a time-dependent manner. The pattern of stain distribution was broadly similar in a range of fungal species tested (Aspergillus nidulans, Botrytis cinerea, Magnaporthe grisea, Neurospora crassa, Phycomyces blakesleeanus, Puccinia graminis, Rhizoctonia solani, Sclerotinia sclerotiorum and Trichoderma viride). With time, FM4-64 was internalized from the plasma membrane appearing in structures corresponding to putative endosomes, the apical vesicle cluster, the vacuolar membrane and mitochondria. These observations are consistent with dye internalization by endocytosis. A speculative model of the vesicle trafficking network within growing hyphae is presented.

Brown Norway rat model of food allergy: effect of plant components on the development of oral sensitization.

The Brown Norway (BN) rat was examined as a model for investigating factors that influence the development of food allergy. An antigen dose-response curve for the production of antigen-specific reaginic antibody (IgE) induced through the oral route was determined. Animals were dosed orally with 1.0, 2.5, 5.0, 7.5, 10.0 and 12.0 mg ovalbumin/ml (0.5 ml/100 g twice a week for 6 wk). To promote IgE production the adjuvant carrageenan was administered once a week by the i.p. route. The effect on oral sensitization of 1.5 mg Gypsophila sp. saponin/ml administered together with the antigen on oral sensitization was examined in animals treated with 2.5, 6.0 or 10.0 mg ovalbumin/ml. The number of animals producing antigen specific reaginic antibody in response to 2.5 mg ovalbumin/ml was significantly increased (P < 0.01) in the group that received saponin with 2.5 mg ovalbumin/ml. These studies indicate that the BN rat is a sensitive model for the investigation of allergic reactions to food and has the potential to determine the impact of other dietary factors on the development of oral sensitization.

Assessment [correction of Asessment] of the brown Norway rat as a suitable model for the investigation of food allergy.

We have investigated the potential of the inbred Brown Norway (BN) rat as a model for food allergy using two different antigens, ovalbumin (OA) and semi-skimmed milk (SSM). The use of milk-free diet prior to and during exposure to SSM was a key factor in the induction of sensitisation to milk proteins. Investigation of dose received and timing of administration identified a sensitisation regimen using 500 micrograms SSM injected i.p. together with 1 mg CGN (adjuvant) on days 0 and 7 as the optimum conditions for induction of reaginic antibody production. In this model milk proteins were less allergenic than OA as the amount of SSM required to induce sensitivity was 20-fold greater. Examination of antigen-specificity of the IgG and reaginic antibody responses to a range of proteins, present in SSM, showed that the BN rats were capable of recognising a similar profile of allergens as those recognised by milk sensitive humans. Lactoferrin which is present in low concentrations in milk proved as allergenic as the major proteins in milk, the caseins and beta-lactoglobulin. These studies have identified conditions for induction of sensitisation to milk proteins, and have shown the antibody specificity of the response to be similar to that in man. This suggests that the BN rat could provide the basis of a model for the investigation of allergic reactions to food.

Adjuvant properties of polysaccharides: effect of iota-carrageenan, pectic acid, pectin, dextran and dextran sulphate on the humoral immune response in the rat.

Iota-carrageenan can both enhance agglutinating antibody responses and trigger reaginic antibody production against a protein associated antigen in rats. The present study investigates the strain specificity of this phenomenon and compares the adjuvanticity of high and low molecular weight iota-carrageenan with a series of structurally distinct polysaccharides (pectin, pectic acid, dextran and dextran sulphate). Using ovalbumin as the test antigen, high molecular weight iota-carrageenan induced a potent ovalbumin specific reaginic antibody response in PVG and Hooded Lister strain rats, an intermediate response in Sprague Dawley rats and a weak response in DA, AO and F344 strain rats. Further studies in PVG rats revealed that the nature and magnitude of the antibody response induced was influenced by the type of polysaccharide carrier used. Thus, whereas, high molecular weight carrageenan enhanced the agglutinating antibody (agglutinin) response and simultaneously elicited de novo reaginic antibody production to co-administered ovalbumin, low molecular weight carrageenan facilitated reaginic antibody production but had no effect on the agglutinin response. Pectin and dextran had no effect on the agglutinin response and failed to elicit reaginic antibody production. Conversely, pectic acid and dextran sulphate enhanced the agglutinin response and elicited a transient reaginic anti-ovalbumin response.

The in vitro effects of trichothecenes on the immune system.

Since Fusarium-derived trichothecenes have shown immunosuppressive properties in laboratory animals, the possibility that deoxynivalenol (DON) and acetyldeoxynivalenol (AcDON) might affect the in vitro production of interleukins by macrophages or lymphocytes was studied. When the effects of low and high concentrations of DON on lymphocyte proliferation were compared, phytohaemagglutinin-induced proliferation was enhanced at concentrations between 0.005 and 0.5 ng DON/ml whereas 50 or 100 ng/ml caused a decrease in proliferation. In experiments in which lymphocytes were exposed briefly to 90 ng DON/ml, the level of thymidine incorporation was increased to 130% of control levels. Both DON and AcDON were shown to induce interleukin 1 (IL-1) release in peritoneal macrophages by a mode of action similar to that of cycloheximide. In the presence of DON, cellular IL-1 did not decay, and this resulted in a marked release of IL-1 from the cell during the period of exposure to the inhibitor. This suggests that in vivo effects of trichothecenes on the immune system may vary according to the level of exposure.

A comparative study of the pulmonary effects of NO2 in the rat and hamster.

A study of the response of rat and hamster to nitrogen dioxide (NO2) under identical conditions has been undertaken. Exposure to 20 parts/IO6 NO2 for 24 h produced a mild cytotoxic effect on the terminal bronchiole and proximal alveoli in the rat, whereas the hamster developed a moderate to severe bronchiolitis and alveolitis. Electron microscopic examination of tissue sections showed accumulation of surfactant in lamellar bodies of the alveolar type II cell in the rat but not in the hamster, whereas in the hamster Clara cells were observed in mitosis. Increased levels of surfactant isolated from whole lung homogenates by sucrose gradient centrifugation were found in both rat and hamster. In contrast surfactant isolated from bronchiolo-alveolar lavage was increased only in the hamster. The results suggest that caution must be exercised in the choice of animal model in investigations aimed at the understanding of the toxicological effects of nitrogen dioxide in man.

Inhibitory effect of deoxynivalenol, 3-acetyldeoxynivalenol and zearalenone on induction of rat and human lymphocyte proliferation.

Deoxynivalenol (DON), 3-acetyldeoxynivalenol (acetylDON) and zearalenone (Ze) were examined for their in vitro effect on mitogen-induced lymphocyte blastogenesis using rat or human peripheral blood lymphocytes (PBL). A dose-dependent reduction of lymphocyte proliferation was demonstrated for each mycotoxin. However, the inhibitory effect of DON was significantly higher than that of the acetylated compound. Concentrations of 90 ng/ml and 220 ng/ml inhibited rat and human lymphocyte blastogenesis by 50%, respectively, whereas 450 ng/ml and 1060 ng/ml acetylDON were required to produce the same effect. The amount of Ze necessary to inhibit blastogenesis by 50% was 250 times greater than required for DON. There was no evidence of cell death and combinations of DON and Ze did not alter the expected response.

Allergic drug reactions: an in vitro model using a mixed function oxidase complex to demonstrate antibodies with specificity for a practolol metabolite.

Reactive metabolite products of the drug practolol were generated in vitro using the rat liver mixed function oxidase complex. The metabolites were spontaneously coupled to a non-agglutinating rabbit antibody to human O red blood cells, so forming a metabolite-antibody reagent. This reagent was then used to passively sensitize human O red cells which subsequently acted as indicator cells for detecting anti-metabolite antibodies.