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2-terminus. Peptides, dissolved in FCA (1 mg/ml) and emulsified in a 1:1 dilution with phosphate-buffered saline, were injected twice into 6-week-old BALB/c mice (50 μg of peptide per mouse) as described (8).
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note
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The GCG (Wisonsin Package) LOOKUP program was used to create a LIST FILE of bacterial and viral sequences from the PIR protein database. The list file was then searched with the GCG program FINDPATTERNS for the sequence pattern MAXXXST.
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BALB/c mice were immunized twice with ChTR1 in FCA (10), and hearts were removed 21 days after the initial immunization. Immunoperoxidase staining of histological heart sections was performed with the following antibodies to rat IgG (all purchased from Pharmingen): anti-CD11b, anti-CD4, anti-CD8α, and anti-CD45R/ B220. Binding of primary antibodies was detected as described (76). Percentages of infiltrating cells were calculated from a minimum of 1000 infiltrating cells counted on representative heart sections.
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37
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0345178451
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Supplemental web material for Fig. 2 (analysis of splenic cell populations) is available at www. sciencemag.org/feature/data/984504.shl.
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38
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0344748308
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note
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Splenocytes were harvested 21 days after the initial immunization with FCA, ChTR1, or M7Aα, and subpopulations were analyzed by fluorescence-activated cell sorting. Specific antibodies to rat IgG (all pur-chased from Pharmingen) were as follows: anti-CD11b, anti-CD4, anti-CD8α, and anti-CD45R/B220.
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39
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0345178450
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note
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3H] thymidine. It should be noted that 10 days after the inital immunization, T cell reactivity is already observed, that is, at a time when no histopathological signs of myocarditis are present.
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40
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0344748306
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note
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+ phenotype.
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42
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note
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Twenty-one days after the initial immunization (10), serum was collected and specific antibody production was determined by enzyme-linked immunosorbent assay (ELISA) (6). Briefly, 96-well plates were coated with 2 μg of peptide per well. Diluted mouse sera (1:100) were allowed to bind to the plates, washed, and binding was detected with horseradish peroxidase-conjugated antibody to mouse IgC (Sigma, #A-3673). Substrate (ABTS, Sigma, #A-1888) conversion was detected measuring absorbance at 405 nm.
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CpG motive-containing synthetic oligodeoxynucleotides (ODNs) were derived either from C trachomatis DNA (CpG 1) or from previously reported bacterial DNA sequences (CpGs 2 and 3) (24). ODNs were phosphorothioate modified to increase their in vivo stability. ODNs (30 μg in 100 μl of 0.15 mM NaCl buffer) were administered ip at the time of the immunizations. BALB/c mice were subcutaneously immunized twice at a 7-day interval with the M7Aα peptide (50 μg per mouse) in a 1:1 emulsion with mineral oil [Freund's incomplete adjuvant (FlA)] (8, 10). Twenty-one days after the initial immunization hearts were analyzed for the presence and severity of myocarditis.
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We thank M. Saunders for critical reading of the manuscript; P. S. Ohashi, T. W. Mak, Y. Y. Kong, A. Oliveirados-Santos, Q. Liu, C. Pummerer, T. Sasaki, P. Liu, and I. Kozieradzki for helpful comments; and the Amgen Protein Synthesis group and C. Richardson for providing peptides and advice. K.B. is a Fellow of the Heart and Stroke Foundation of Canada. J.M.P. is supported by the Medical Research Council of Canada.
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