Reactivity was dependant on an alkaline phosphatase-based ELISA while described in Materials and methods

Reactivity was dependant on an alkaline phosphatase-based ELISA while described in Materials and methods. transfer of p2340-primed lymph node cells. P2340 is the 1st Tg peptide found to be pathogenic in low as well as high responder mouse strains and thus will allow us to investigate mechanisms of EAT induction inside a genetically resistant sponsor. 0005, 5), dose-dependent and specific proliferation in ethnicities with LNC isolated from all the strains of mice analyzed, as demonstrated in Fig. 1(a, c, e, g). In contrast, LNC from all four strains were unresponsive to the p2652 or undamaged hTg suggesting that p2340 does not comprise dominating T-cell epitope(s). This was further confirmed in three of the four strains, as hTg-primed LNC from H-2k, s and b mice responded to hTg 0005, 5) stimulated by p2340, suggesting that in BALB/c mice p2340 behaves like a subdominant epitope(s) (Fig. 1f). Open in a separate window Number 1 Proliferative LNC reactions (SI) to p2340 (?), p2652 (?) and hTg (^). CBA/J, SJL/J, Lep BALB/c, C57BL/6 mice were s.c. immunized with 100 nmol of p2340 (a, c, e and g) or 100 g of hTg (b, d, f and h) and LNC were collected 9 or 10 days later on and cultured in triplicates in the presence or absence of the above antigens at different concentrations for 3 days. [3H]-methyl thymidine was added during the Monoisobutyl phthalic acid last 18 hr of tradition. Background c.p.m. assorted from 1000 to Monoisobutyl phthalic acid 5000. Error bars represent standard deviations for triplicate wells. The results are representative of three self-employed experiments. Characterization of cytokines produced by p2340-primed T-cells To identify the subset of T-cells (type 1 T-helper cells (Th1) or type 2 T-helper cells (Th2)) proliferating and with p2340, into these hosts. Pathology appeared to be organ specific because mononuclear cell infiltrates were not recognized in kidneys or Monoisobutyl phthalic acid livers of these animals. Open in a separate window Number 3 Histological appearance of mononuclear cell infiltration in mouse thyroids after administration of p2340. Normal gland: I.I. = 0 (a), Interstitial build up of inflammatory cells: I.I. = 1 (b), One or two foci of inflammatory cells: I.I. = 2 (c), Considerable infiltration 10C40% of total thyroid gland: I.I. = 3 (d). Magnification: 125. Table 1 Induction of EAT by p2340 in the four mouse strains with 9 m of the same peptide. Three days later, LNC were adoptively transferred (AT) into syngeneic recipients (five to seven mice per strain). Thyroid glands were collected 14 days after the transfer and EAT was obtained for I.I. Immunogenicity of p2340 in the B-cell level Analysis by ELISA of serum samples, acquired at the time of thyroid gland removal, showed the generation of high antibody titres specific to p2340 (Fig. 4). The anti-p2340 antibodies from all four strains presented, normally, 50% binding at 1/3375 dilution, while no reactivity was recognized for control p2652 or hTg at any dilution tested (data not demonstrated). The IgG reactions of the four mouse strains did not differ much. This finding demonstrates p2340 bears not only T-cell epitope(s) but also B-cell epitope(s). Open in a separate window Number 4 Mean IgG reactions against p2340 from four to six mice of each strain. CBA/J (?), SJL/J (?), BALB/c (?) and C57BL/6 (?) mice (= 4C6) were immunized with 100 nmol of p2340, as mentioned in Table 1, and bled after 5 weeks. Reactivity was determined by an alkaline phosphatase-based ELISA as explained in Materials and methods. The anti-peptide reactivity of the sera of each mouse strain is definitely offered as the mean.