The evaluation of the immunomodulating properties of ERA-63 a pharmaceutical with estrogenic activity.

This paper describes studies performed with ERA-63 a low molecular weight pharmaceutical with intended immunomodulatory effects. Since this compound was also known to have estrogenic activity a non-conventional approach was taken in order to differentiate between estrogenic and non-estrogenic-induced immunomodulatory effects. EE was included not only for qualitative comparison (hazard identification) between immunomodulatory effects but also, in case of similar effects, to facilitate the extrapolation of the findings in the rat to anticipated effects in humans. After 28 days of treatment with dosages ranging from pharmacological up to clearly toxic levels for both compounds the immunotoxic potential was assessed by performing a T cell-dependent antibody response and a host resistance assay in rats. Selected ERA-63 dose levels (0.167-0.2, 1.67-2 and 16.7-20mg/kg) were expected to have comparable estrogenic activity to respective EE dose levels (0.05, 0.5 and 5mg/kg). General toxicity parameters reflecting estrogenic activity (i.e. decreased body- and organ weights of thymus and testis, and increased bilirubin and GGT levels) confirmed the comparable estrogenic activity for both compounds at the dose levels tested. Together with the comparable estrogen-related immune suppression (i.e. decreases in specific antibody responses and an increased susceptibility for Listeria monocytogenes infects) for both compounds, this indicates that available clinical data for EE facilitates the human risk assessment of ERA-63.

An oral sensitization model in Brown Norway rats to screen for potential allergenicity of food proteins.

We developed an oral sensitization protocol for food proteins for the rat. Young Brown Norway (BN) rats were exposed to 1 mg ovalbumin (OVA) by daily gavage dosing for 42 days without the use of an adjuvant. OVA-specific IgE and IgG responses were determined by ELISA. On an oral challenge with OVA some clinical symptoms of food allergy-like effects on the respiratory system, blood pressure, and permeability of the gastrointestinal barrier were studied. In addition, BN rats were orally exposed to a total hen egg white protein (HEW) extract and cow's milk (CM) and the specificities of induced antibody responses were compared with the specificities of antibodies in sera from egg- and milk-allergic patients using immunoblotting. Animals orally exposed to the allergens developed specific IgE and IgG antibodies which recognized the same proteins compared with antibodies from egg- or CM-allergic patients. Among the various clinical symptoms of food allergy, gut permeability was increased after an oral challenge. In addition, some animals demonstrated a temporary decrease in breathing frequency or systolic blood pressure. The results obtained show that the Brown Norway rat is a suitable animal model for inducing specific IgG and IgE responses on daily intragastric dosing of OVA without the use of an adjuvant. Moreover, local immune-mediated effects on oral challenge are observed. The observation that enterally exposed BN rats and food-allergic patients demonstrate antibody responses to a comparable selection of proteins on exposure to different protein mixtures (HEW and CM) further supports the suitability of the BN rat as an animal model for food allergy research and for the study of the allergenicity of (novel) food proteins.

Intestinal T lymphocytes of different rat strains in immunotoxicity.

In order to study the intestinal mucosal immune cells, with emphasis on single T lymphocytes, an inventory was made of single and organized lymphocytes in the epithelium and lamina propria of the small intestines of untreated Wistar, Fischer 344, and Lewis rats. The single and organized lymphocytes were examined microscopically. In addition, the single lymphocytes in the epithelium (IEL) and lamina propria (LPL) were analyzed by flow cytometry. Next, the use of flow cytometry analysis was explored to detect changes in the IEL T-lymphocyte population in subacute oral studies with the immunomodulating agents azathioprine and hexachlorobenzene. Untreated random-bred Wistar rats exhibited a large interindividual variability in IEL composition, while the variability was small in inbred Fischer 344 and Lewis rats. The explorative study with the 2 model immunomodulating compounds demonstrated that hexachlorobenzene increased the number of intraepithelial T lymphocytes with CD8+ phenotype at the cost of T cells with CD4+ phenotype in Lewis rats. Azathioprine did not induce distinct effects on the percentages of IEL. The data indicate that the intraepithelial lymphocytes in the intestines are a potential target for orally administered immunomodulating compounds and should therefore receive more attention in toxicologic pathology studies.

Identification and partial characterization of multiple major allergens in peanut proteins.

BACKGROUND: Peanuts are a major cause of food allergies both in children as in adults which can induce an anaphylactic shock. The identification and characterization of peanut allergens could lead to more insight into the mechanism and contribute to the improvement of diagnostic tests and treatment for peanut allergy. OBJECTIVE: In the present study, the peanut protein-specific immunoglobulin concentrations as well as their recognition of the various peanut proteins or protein subunits was determined in the plasma of peanut-allergic (PA) and non-allergic (NA) individuals. Moreover, two peanut allergens were characterized in more detail to confirm them as the earlier described Ara h1 and Ara h2. METHODS: The presence of Ig-binding sites in peanut proteins was studied by immunoblotting assays whereas the concentrations of peanut-specific Ig was determined by ELISA. RESULTS: Peanut proteins were found to contain multiple binding sites for immunoglobulins. Of these proteins, six were recognized by peanut-specific IgE present in more than 50% of the plasma samples of the PA group. Their molecular weights were approximately 44, 40, 33, 21, 20 and 18 kDa. The last three protein bands were recognized by peanut-specific IgE present in more than 70% of the PA plasma samples and were thought to contain Ara h2. This allergen as well as another protein that was thought to be Ara h1, which was not recognized by the majority of the patients' IgE-containing plasma samples, were isolated and the N terminal amino acid sequence was determined. Peanut protein-specific IgA, IgM, IgG and IgG-subclasses showed a more diverse recognition pattern of peanut protein in the PA group compared to the NA group. No differences were found in the plasma concentrations of peanut protein-specific immunoglobulins of the various classes between the PA and NA group. CONCLUSIONS: From the present study, we conclude that peanuts contain multiple allergens, of which six can be described as major allergens, Ara h2 included. In our population Ara h1 is not a major allergen. The recognition of peanut proteins by immunoglobulins is more diverse in PA individuals compared with NA individuals which, however, is not substantiated in the concentrations of peanut-specific immunoglobulins in plasma, other than IgE.

Oral sensitization to food proteins: a Brown Norway rat model.

BACKGROUND: Although several in vivo antigenicity assays using parenteral immunization are operational, no adequate enteral sensitization models are available to study food allergy and allergenicity of food proteins. OBJECTIVE: This paper describes the development of an enteral model for food allergy research in the Brown Norway (BN) rat. METHODS: The animals were exposed to ovalbumin either ad libitum via the drinking water (0.002 to 20 mg/mL) continuously for 6 weeks or by gavage (1 mg/mL per rat). Gavage dosing was performed either daily, twice a week, once a week or once every 2 weeks during a period of 6 weeks. No adjuvants were used during the sensitization studies. RESULTS: After intra-gastric administration of ovalbumin once or twice a week or once every two weeks, no or only a very low frequency of ovalbumin-specific antibody responses were detected. Daily intra-gastric dosing with ovalbumin resulted in antigen-specific IgG as well as IgE responses in almost all animals tested. Upon ad libitum exposure, ovalbumin-specific IgG but no ovalbumin-specific IgE was detected. The cellular response was examined by determination of delayed-type hypersensitivity (DTH) reactions in the animals dosed by daily gavage and in the ad libitum exposed rats. Both sensitization protocols sensitized for DTH. The response was most pronounced in ad libitum exposed rats at day 28 of exposure. CONCLUSIONS: These studies show that the BN rat may provide a suitable animal model for inducing specific IgG and IgE responses as well as specific T-cell mediated hypersensitivity (DTH) to ovalbumin upon exposure via the enteral route without the use of adjuvants.

A new method to measure iron absorption from the enrichment of 57Fe and 58Fe in young erythroid cells.

Iron absorption can be measured by the incorporation of stable iron isotopes into erythrocytes, 14 days after isotope administration. The disadvantage of this method is the high dose of isotopes needed to obtain a sufficient enrichment. Therefore, in this study cell fractions rich in young erythroid cells were prepared by using a density separation method. From 10 women blood was taken 4, 5, and 7 days after oral and intravenous administration of 57Fe and 58Fe. In these cell fractions and in whole blood taken 14 days after isotope administration, isotope enrichment was measured and absorption calculated. Absorption calculated from the isotope enrichment in the reticulocyte-rich cell fractions (12.2 +/- SEM 3.7%) was not significantly different from absorption based on whole-blood values (13.0 +/- 3.3%). Because a threefold higher isotope enrichment was found in the cell fractions, the required dose of stable isotopes can be reduced to one-third of the dose used in the traditional method without loss of sensitivity.

The effect of consumption of milk fermented by Lactobacillus casei strain Shirota on the intestinal microflora and immune parameters in humans.

OBJECTIVE: To determine the effect of consumption of milk fermented by Lactobacillus casei strain Shirota (L. casei Shirota) on the composition and metabolic activities of the intestinal microflora, and immune parameters in humans. SUBJECTS: Twenty healthy male subjects aged 40-65 years were selected. DESIGN: A placebo-controlled trial was performed in which 10 subjects were randomly assigned to a control and 10 to a treatment group. During the first and last two weeks of the 8-week study the subjects received a strictly controlled diet without fermented products. The same controlled diet was given during the intermediate 4-week test period but then the treatment group received three times daily 100 ml of fermented milk containing 10(9) CFU L. casei Shirota/ml, whereas the same amount of unfermented milk was given to the subjects in the control group. RESULTS: In comparison to the control group, the consumption of L. casei Shirota-fermented milk resulted in an increase of the Lactobacillus count in the faeces in which the administered L. casei Shirota was predominant at the level of 10(7) CFU/g wet faeces. This was associated with a significant increase in Bifidobacterium counts (P < 0.05). Some shifts in the other bacterial species were found, such as a decreased number of Clostridium; however the differences were not statistically different between the treatment and the control groups. The beta-glucuronidase and beta-glucosidase activities per 10(10) bacteria decreased significantly (P < 0.05) at the second week of the 4-week test period with the consumption of L. casei Shirota-fermented milk. Furthermore, the consumption of the fermented milk product resulted in a slight but significant increase in the moisture content of the faecal samples (P < 0.05). No treatment effects were observed for any of the immune parameters measured (including natural killer (NK) cell activity, phagocytosis and cytokine production). CONCLUSIONS: The results suggest that consumption of L. casei Shirota-fermented milk is able to modulate the composition and metabolic activity of the intestinal flora and indicate that L. casei Shirota-fermented milk does not influence the immune system of healthy immunocompetent males.

Local lymph node activation and IgE responses in brown Norway and Wistar rats after dermal application of sensitizing and non-sensitizing chemicals.

The local lymph node assay (LLNA) and the IgE test in the mouse are proposed models for predictive recognition of low molecular weight chemicals causing IgE-mediated allergic airway reactions in man. Since rats are commonly used in routine toxicity studies and a previous study (Arts et al. (1996) Food Chem. Toxicol. 34, 55-62) has shown that several rat strains were found appropriate for the LLNA, the suitability of the rat for the IgE test was examined in the present study. Serum IgE concentrations were examined following topical exposure of Brown Norway (BN) and Wistar rats to each of four chemicals with known diverse sensitization potential in humans: trimellitic anhydride (TMA), a dermal and respiratory sensitizer, dinitrochlorobenzene (DNCB), a dermal sensitizer with no or limited potential to cause respiratory allergy; formaldehyde (FA), a skin irritant and dermal sensitizer with equivocal evidence for respiratory sensitizing potential; methyl salicylate (MS), a skin irritant devoid of sensitizing properties. Of the four tested chemicals, only exposure to TMA resulted in a significant increase in serum IgE concentration and this response was only evoked in the high-IgE-responding BN rat. The latter two chemicals were also tested for lymph node activation, in casu the ear-draining lymph nodes. FA caused a dose-dependent activation of the draining lymph nodes whereas MS was inactive. The results as obtained with TMA, DNCB and MS in the rat are in agreement with human data. The results with FA though, indicate the need for further studies of chemicals that have both irritant and sensitizing properties at about similar concentrations or may act through non-IgE-mediated immune mechanisms.

Food allergen (peanut)-specific TH2 clones generated from the peripheral blood of a patient with peanut allergy.

BACKGROUND: Increasing evidence indicates a prominent role of allergen-specific TH2 cells, with high IL-4 and IL-5 production and low interferon-gamma production, in the regulation of IgE and eosinophil production in allergic disorders. However, most studies have concentrated on T cells reactive with inhalation allergens, whereas little is known about the properties of food allergen-reactive T cells. OBJECTIVES: In this study we therefore characterized peanut-specific T cells, cloned from a patient with severe peanut allergy. METHODS: Peripheral blood mononuclear cells from patients with peanut allergy and nonallergic individuals were stimulated with crude peanut extract (CPE) to compare the proliferative responses and to select a suitable patient for the cloning of CPE-specific T cells. The resultant panel of CPE-reactive T-lymphocyte clones was serologically phenotyped by flow cytometry and analyzed for cytokine secretion by ELISA. RESULTS: The patients' peripheral blood mononuclear cells showed a dose-dependent proliferation response to CPE, which was significantly higher (p < 0.05) than in peripheral blood mononuclear cells of nonallergic donors. The CPE-specific T-lymphocyte clones generated from the selected patient were all CD4+/CD8- T helper cells with a TH2 cytokine profile, secreting high amounts of IL-4 and IL-5, but little or no interferon-gamma. CONCLUSIONS: This study demonstrates that peanut-specific T cells do occur in the peripheral blood of patients with peanut allergy and suggests an increased frequency of these T cells in patients compared with nonallergic control subjects. The CD4+ phenotype and the TH2 cytokine profile of the CPE-specific T-lymphocyte clones suggest a functional role of allergen-specific TH2 cells in the pathophysiology of food allergy, similar to the function of inhalation allergen-specific TH2 cells.

Effect of beta-carotene on immunological indexes in healthy male smokers.

To evaluate the immunomodulatory effects of beta-carotene we performed a randomized, double-blind trial in healthy male cigarette smokers. Lymphocyte subsets in peripheral blood were assessed by using double labeling with monoclonal antibodies before and after 14 wk beta-carotene (20 mg/d; n = 21) or placebo (n = 24) supplements. In addition we measured the ex vivo phytohemagglutinin and concanavalin A induced lymphocyte proliferation in a separate group (23 placebo, 24 beta-carotene). The beta-carotene and placebo groups were comparable on all initial characteristics. During the intervention plasma concentrations of beta-carotene increased 13-fold in the treatment group whereas retinol concentrations remained constant. Beta-carotene had no effect on lymphocyte subpopulations in peripheral blood. After treatment the beta-carotene group showed 12% higher PHA-induced lymphocyte proliferations than the placebo group (P = 0.02). For ConA induced proliferations no significant difference was observed. These results suggest that supplementary beta-carotene can moderately enhance certain aspects of immune response in healthy male cigarette smokers.

Effects of the colour additive caramel colour III on the immune system: a study with human volunteers.

Administration of the colour additive Caramel Colour III to rats has been associated with decreased numbers of lymphocytes and several other changes in the immune system, as well as in immune function parameters, specifically in animals fed a diet with a relatively low vitamin B6 content. The effects are caused by the imidazole derivative 2-acetyl-4(5)-tetrahydroxybutylimidazole (THI). Caramel Colour III is commonly used in food products such as bakery products, soya-bean sauces, brown sauces, gravies, soup aromas, brown (dehydrated) soups, brown malt caramel blend for various applications, vinegars and beers, and effects in humans on dietary intake cannot be excluded. Elderly male volunteers with a marginal deficit in vitamin B6 were considered a relevant and potentially sensitive group to study possible effects of Caramel Colour III on blood lymphocyte numbers (total and within subsets) or on proliferative responses of lymphocytes to mitogenic stimulation. In addition, several other haematological parameters, as well as serum immunoglobulin levels and immunoglobulin production in vitro by pokeweed mitogen-stimulated mononuclear blood cells were studied. The results of this double-blind intervention study demonstrated that in a selected test group of apparently healthy elderly male volunteers with a biochemically marginally deficient vitamin B6 status, Caramel Colour III containing 23 (commercial sample) or 143 (research sample) ppm THI and administered at the level of the current acceptable daily intake of 200 mg/kg body weight/day for 7 days did not affect any of the factors investigated.

Effects of Caramel Colour III on the number of blood lymphocytes: a human study on Caramel Colour III immunotoxicity and a comparison of the results with data from rat studies.

Administration of the colour additive Ammonia Caramel Colour (Caramel Colour III) to rats has been associated with decreased lymphocyte counts, specifically in rats fed a diet low in vitamin B6. This effect is rapidly reversible and is caused by an imidazole derivative (THI) in Caramel Colour III. In the present paper, the conduct of a human study with Caramel Colour III is outlined and the results of blood lymphocyte counts are presented. No decrease in the number of blood lymphocytes occurred in marginally vitamin B6-deficient humans who consumed Caramel Colour III at the acceptable daily intake level (200 mg/kg body weight/day) for 7 days. These data are discussed in relation to the effects of Caramel Colour III and THI on blood lymphocyte numbers in rats.