SUMMARYGrave's disease (GD) is characterized by pathogenic autoantibodies to the human thyrotropin receptor (hTSH-R), and is frequently associated with a lymphocytic infiltrate of the thyroid gland. In attempts to establish a murine model of GD, we and others have previously shown that immunization of mice with recombinant preparations of the hTSH-R ectodomain induces high titres of specific antibodies, which, however, are not pathogenic, nor is the response accompanied by the development of thyroiditis. Since earlier reports identified the serological immunodominant determinants within the N-and C-terminal regions of hTSH-R ectodomain, we reasoned that immunization of mice with truncated fragments of ectodomain lacking these dominant regions might result in skewing of the response to other determinants of the molecule, with consequent induction of immunopathological features present in GD. We show here that multiple challenge of BALB/c mice with an amino acid fragment of residues 43-282 generates antibodies directed at hTSH-R peptides 37-56, 157-176, 217-236 and 232-251. This reactivity pattern is distinct from that induced previously with the whole ectodomain of hTSH-R in BALB/c animals. Thyroid function remained unaffected in these mice, suggesting that pathogenic antibodies were not being induced. Interestingly, some animals developed lymphocytic infiltration of the thyroid gland, clearly indicating the presence of pathogenic T cell determinants within the 43-282 fragment. Challenge with the related fragment 43-316 produced the same pattern of serological response to the synthetic peptides as fragment 43-282, but was not accompanied by thyroiditis. The results demonstrate: (i) the presence of thyroiditogenic determinants within hTSH-R, and (ii) that these pathogenic determinants are likely to be cryptic, as their effect is exhibited only when the hierarchy of immunodominance within hTSH-R is drastically altered.
20110 Background: The 5-year survival rate of Dukes’ B colon cancer patients is approximately 75%. In our earlier genome-wide measurements of gene expression we have identified a 23-gene signature that sub-classifies Dukes’ B tumors and may provide better means of risk assessment on an individual basis for these colon cancer patients. The aim of this study is to validate this gene signature in an independent and more diverse group of patients, and further develop this prognostic signature into a clinical feasible test using formalin-fixed paraffin-embedded (FFPE) tissue samples. Methods: Using Affymetrix U133a GeneChip we analyzed the expression of the 23 genes in total RNA of frozen tumor samples from 123 Dukes’ B patients who did not receive adjuvant systemic treatment. Furthermore, we developed a quantitative RT-PCR assay for this gene signature in order to perform the test with standard clinical FFPE samples. Results: In the independent validation set of 123 patients, the gene signature proved to be informative in identifying patients who would develop distant metastasis (hazard ratio, HR 2.56; 95% confidence interval CI, 1.01–6.48), even when corrected for the traditional prognostic factors in multivariate analysis (HR, 2.73; 95% CI, 0.97–7.73). The RT-PCR assay developed for this gene signature was also validated in an independent set of 114 patients as a strong prognostic factor for the development of distant recurrence (HR, 6.38; 95% CI, 2.88–14.2) in univariate analysis and in multivariate analysis (HR, 13.3; 95% CI, 5.13–34.4). Conclusions: Our data provide not only a validation of the pre-defined prognostic gene signature for Dukes’ B colon cancer patients but also a clear feasibility of testing the gene signature using RT-PCR with standard FFPE specimens. The ability of such a test to identify patients that have an unfavorable outcome demonstrates potential clinical importance that could lead clinicians to choose a more aggressive therapeutic option for the high-risk patients. [Table: see text]
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