Effective induction of oral anaphylaxis to ovalbumin in mice sensitized by feeding of the antigen with aid of oil emulsion and salicylate.

It is important to evaluate the ability of novel proteins in food crops and products to elicit potentially harmful immunologic responses, including allergic hypersensitivity. We developed a novel mouse model of food allergy involving an oral challenge of a protein antigen after feeding of the antigen in combination with modulating factors often ingested in daily life, namely, dietary oil emulsion and salicylate. In the model, BALB/c mice were sensitized orally for three weeks with ovalbumin (OVA) in linoleic acid/lecithin emulsion, followed immediately by intraperitoneal injection of sodium salicylate. At the end of the sensitization, the incidence of mice positive for serum OVA-specific IgG1 but not IgE had significantly increased in the combined-sensitization group. After the 3-week sensitization, a single or double oral challenge with OVA effectively and significantly caused severe anaphylaxis, as compared with the groups sensitized with OVA in the emulsion or the vehicle alone. Moderate increase of plasma histamine and intestinal abnormality in histology was found only in the combined-sensitization group. Anaphylaxis symptoms in the sensitized mice were induced more by oral challenge than by intravenous challenge, suggesting a critical role for the mucosal system. This is the first model for successful induction of oral anaphylaxis in mice sensitized by feeding of food protein without adjuvant. It will be useful to elucidate the mechanism of food allergy and to detect modulating factors of oral allergy at sensitization using this model, which simulates real life conditions.

Inter-laboratory validation of the modified murine local lymph node assay based on 5-bromo-2'-deoxyuridine incorporation.

The murine local lymph node assay (LLNA) is a well-established alternative to the guinea pig maximization test (GPMT) or Buehler test (BT) for the assessment of the skin sensitizing ability of a drug, cosmetic material, pesticide or industrial chemical. Instead of radioisotope using in this method, Takeyoshi M. et al. (2001) has developed a modified LLNA based on the 5-bromo-2'-deoxyuridine (BrdU) incorporation (LLNA:BrdU-ELISA). The LLNA:BrdU-ELISA is practically identical to the LLNA methodology excluding the use of BrdU, for which a single intraperitoneal injection of BrdU is made on day 4, and colorimetric detection of cell turnover. We conducted the validation study to evaluate the reliability and relevance of LLNA:BrdU-ELISA. The experiment involved 7 laboratories, wherein 10 chemicals were examined under blinded conditions. In this study, 3 chemicals were examined in all laboratories and the remaining 7 were examined in 3 laboratories. The data were expressed as the BrdU incorporation using an ELISA method for each group, and the stimulation index (SI) for each chemical-treated group was determined as the increase in the BrdU incorporation relative to the concurrent vehicle control group. An SI of 2 was set as the cut-off value for exhibiting skin sensitization activity. The results obtained in the experiments conducted for all 10 chemicals were sufficiently consistent with small variations in their SI values. The sensitivity, specificity, and accuracy of LLNA:BrdU-ELISA against those of GPMT/BT were 7/7 (100%), 3/3 (100%), and 10/10 (100%), respectively.

Effects of transmaternal exposure to genistein in Hatano high- and low-avoidance rats.

Hatano high- and low-avoidance (HAA and LAA) rats are separated by breeding from Sprague-Dawley rats by high versus low rates of avoidance responses in a shuttle-box task. In addition, compared to HAA rats, LAA rats show lower running-wheel activity, later sexual maturation, 5-day estrous cycling, lower sperm motility, more pronounced immunological reactions, and are generally less reactive to stress. The present study was designed to compare the effects of transmaternal exposure to genistein on these characteristics between HAA and LAA rats. To this aim, litters from both strains were fostered onto Sprague-Dawley rats receiving genistein by gavage with 5 mg/animal/day from day 17 of pregnancy through day 21 of lactation. Inhibited growth after weaning and reduced uterine weight at weaning were observed in the LAA offspring reared by genistein-treated dams. IgM antibody production in response to sheep red blood cells was significantly decreased in the HAA offspring reared by genistein-treated dams. During restraint stress, the plasma concentration of corticosterone was significantly lower in the LAA offspring reared by genistein-treated dams. Strain-related differences were detected in shuttle-box avoidance performance, running-wheel activity, estrous cycling, and sperm motility. The results demonstrate that transmaternal exposure to genistein potentially affects the immunological and stress responses as well as the post-weaning growth of the offspring. It suggests that a comparative study using Hatano rats would be useful for studying the influence of endocrine active chemicals on the whole body systems.

Interlaboratory validation of the modified murine local lymph node assay based on adenosine triphosphate measurement.

INTRODUCTION: The murine local lymph node assay (LLNA) is a well-established alternative to the guinea pig maximization test (GPMT) or Buehler test (BT) for the assessment of the skin sensitizing ability of drugs and chemicals. Daicel Chemical Industries Ltd. has developed a modified LLNA based on the adenosine triphosphate (ATP) content (LLNA-DA). We conducted 2 interlaboratory validation studies to evaluate the reliability and relevance of LLNA-DA. METHODS: The experiment involved 17 laboratories, wherein 14 chemicals were examined under blinded conditions. In the first study, 3 chemicals were examined in 10 laboratories and the remaining 9 were examined in 3 laboratories. In the second study, 1 chemical was examined in 7 laboratories and the remaining 4 chemicals were examined in 4 laboratories. The data were expressed as the ATP content for each chemical-treated group, and the stimulation index (SI) for each chemical-treated group was determined as the increase in the ATP content relative to the concurrent vehicle control group. An SI of 3 was set as the cut-off value for exhibiting skin sensitization activity. RESULTS: The results of the first study obtained in the experiments conducted for the 3 chemicals that were examined in all the 10 laboratories and for 5 of the remaining 9 chemicals were sufficiently consistent with small variations in their SI values. The sensitivity, specificity, and accuracy of LLNA-DA against those of GPMT/BT were 7/8 (87.5%), 3/3 (100%), and 10/11 (90.9%), respectively. In the second study, all the 5 chemicals studied demonstrated acceptably small interlaboratory variations. DISCUSSION: In the first study, a large variation was observed for 2 chemicals; in the second study, this variation was small. It was attributed to the application of dimethylsulfoxide as the solvent for the metallic salts. In conclusion, these 2 studies provide good evidence for the reliability of the LLNA-DA.

Immunological characteristics of Hatano high-and low-avoidance rats.

Hatano high- and low-avoidance (HAA and LAA) rats have been genetically selected on the basis of their two-way active avoidance behavior, and have been shown to differ in other behavioral and hormonal parameters. Since close interconnections among the nervous, endocrine and immune systems have been well documented, these two strains might possess differences in aspects of immunological action. In Experiment 1, plasma levels of IgG, IgM, complement 3 (C3), classical pathway hemolytic complement (CH50) and beta(2)-microglobulin were compared between males of the two strains at 5 and 24 weeks of age. Plasma levels of IgG and CH50 were lower in LAA than HAA rats at 5 weeks of age, whereas those differences disappeared at 24 weeks of age. There were no differences between the two strains in plasma levels of IgM, C3 and beta(2)-microglobulin. In Experiment 2, antibody production to sheep red blood cells (SRBC) and mitogen-induced lymphocyte proliferation were compared between 12-week-old males of the two strains. Antibody responses in the PFC assay, plasma anti-SRBC-IgM levels and spleen weights were higher in LAA than HAA rats. LPS-induced lymphocyte proliferation was greater in LAA than HAA rats. It was concluded that HAA rats show earlier development of immunological development, but that antibody production and mitotic response of B lymphocytes may be more pronounced in adult LAA than HAA rats. The strain differences observed in the immunological response may indicate the usefulness of using Hatano rats in studies of behavioral-immunological relationships.