PubMed · 10459380
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J Pepping. 1999-08-15. Creatine.. https://doi.org/10.1093/ajhp%2F56.16.1608
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Intracellular cytokine staining and flow cytometry can be used to measure T-cell responses to defined antigens. Although CD8+ T-cell responses to soluble proteins are inefficiently detected by this approach, peptides can be used as antigens. Using overlapping peptides spanning an entire protein sequence, CD8+ T-cell responses can be detected to multiple epitopes, regardless of HLA type. In this study, overlapping peptide mixes of various lengths were compared and 15 amino acid peptides with 11 amino acid overlaps were found to stimulate both CD4+ and CD8+ T-cell responses. Such peptide mixes stimulated CD4+ T-cell responses equivalent to those observed with whole recombinant protein, while simultaneously stimulating CD8+ T-cell responses much higher than those observed with whole protein. Although 8-12 amino acid peptides produced the highest level of CD8+ T-cell responses, 15 amino acid peptides were still very effective. Peptides that were 20 amino acids in length, however, did not stimulate strong CD8+ T-cell responses at the same peptide dose. The cytokine responses to individual epitopes added up approximately to the response to the entire mix, demonstrating that large mixes can detect responses in a quantitative fashion. Unlike whole protein antigens, peptide mixes were effective at stimulating responses in both cryopreserved PBMC and blood stored for 24 h at room temperature. Thus, overlapping 15 amino acid peptide mixes may facilitate the analysis of antigen-specific CD4+ and CD8+ T-cell responses by cytokine flow cytometry, using clinical specimens that include shipped blood or cryopreserved PBMC.
We consider a two- and a three-stage hierarchical design containing the effects of k clusters with n units per cluster. In the two-stage model, the conditional distribution of the discrete response Y(i) is assumed to be independent binomial with mean n(straight theta)i (I=1,....k). The success probabilities, straight theta(i)'s, are assumed exchangeable across the k clusters, each arising from a beta distribution. In the three-stage model, the parameters in the beta distribution are assumed to have independent gamma distributions. The size of each cluster, n, is determined for functions of straight theta(i). Lengths of central posterior intervals are computed for various functions of the straight theta(i)'s using Markov chain Monte Carlo and Monte Carlo simulations. Several prior distributions are characterized and tables are provided for n with given k. Methods for sample size calculations under the two- and three-stage models are illustrated and compared for the design of a multi-institutional study to evaluate the appropriateness of discharge planning rates for a cohort of patients with congestive heart failure.
Clinical studies in which a major objective is to produce Kaplan-Meier estimates of survival probabilities should be designed to produce those estimates with a desired prespecified precision as measured by their standard errors. By considering the Peto and Greenwood formulae for the estimated standard error of the Kaplan-Meier estimate and replacing their constituents with expected values based on the study's design parameters, formulae for projected standard errors can be produced. These formulae are shown, through simulations, to be quite accurate.