Identifying naturally occurring peptides bound to HLA class I molecules recognized by HLA-restricted cytotoxic T lymphocytes (CTL) is both relevant and central to the development of effective immunotherapeutic strategies against cancer. Several cancer-related genes have been reported for ovarian cancer, but very few are known to be naturally processed T cell epitopes. In the present study we used mass spectrometry to identify 16 novel HLA-A2-bound peptides from HLA-A2(+) ovarian cancer cell lines. All 16 peptides are derived from source proteins with diverse functions and marked homology to known proteins found in public databases. Synthetic peptide analogues of identified sequences were found to stabilize HLA-A2.1, albeit with varying affinities. The peptides were found to be antigenic in that a primary CD8(+) CTL response could be elicited from normal donor blood. The CTL generated were not only peptide specific, but failed to recognize targets pulsed with control peptides. In addition, recognition of shared HLA-A2-restricted epitopes by these CTL is suggested by their reactivity with a subset of HLA-A2(+) tumor lines and freshly isolated cancer cells or cell lines established from peritoneal ascites. These results were further corroborated by competitive inhibition of lysis of an otherwise susceptible cell line in the presence of cold peptide-pulsed targets. Furthermore, lack of recognition of several HLA-A2(+) control cell lines or cells isolated from normal ovaries suggests that these peptides are cancer related. These findings broaden the list of CTL-defined antigens that could lead to the development of multi-epitope vaccines for the treatment of ovarian cancer.

The thyroid couples two iodotyrosine molecules to produce thyroid hormone at the acceptor site in thyroglobulin, leaving dehydroalanine or pyruvate at the donor position. Previous work has located the acceptors but not the principal iodotyrosine donors. We incorporated [14 C]tyrosine into beef thyroid slices, isolated and iodinated the [ 14 C]thyroglobulin (Tg I), separated its Nterminal ϳ22-kDa hormone-rich peptide, and digested the latter with trypsin and endoproteinase Glu-C (EC 3.4.21.19). Nonlabeled thyroglobulin (Tg II) was isolated from the same glands and processed similarly, without iodination in vitro. Tg I was used to initially recognize pyruvate in peptide fractions, and Tg II was used to then identify its location in the thyroglobulin polypeptide chain. Sequencing of a tryptic peptide by mass spectrometry and Edman degradation showed a cleavage after Val129 . An endoproteinase Glu-C-generated peptide had the predicted molecular mass of a fragment containing residues 130 -146 with Tyr 130 replaced by pyruvate; the identification of this peptide was supported by obtaining the expected shortened fragment after tryptic digestion.14 C-labeled pyruvate was identified in the same fraction as this peptide. We conclude that Tyr 130 is an important donor of the outer iodothyronine ring. Its likely acceptor is Tyr 5 , the most important hormonogenic site of thyroglobulin, because Tyr 5 and Tyr 130 are proximate, because they are the most prominent early iodination sites in this part of thyroglobulin, and because the N-terminal region was previously found capable of forming T 4 by itself.

Identification of Mycobacterium tuberculosis proteins that can provide immunological protection against tuberculosis is essential for the development of a more effective vaccine. To identify new vaccine targets, we have used immunoaffinity chromatography to isolate class I HLA-A*0201-peptide complexes from M. tuberculosis-infected cells and sequenced the isolated peptides by mass spectrometry. From this material, we have identified three peptides derived from a single M. tuberculosis protein that is encoded by the M. tuberculosis Rv0341 gene. Although no known protein encoded by the Rv0341 gene has been described, it is predicted to give rise to a 479-amino-acid protein with a molecular mass of 43.9 kDa. The three peptides identified are all nested and were found to be antigenic, in that they were capable of inducing peptide-specific, CD8؉ T cells from healthy blood donors in vitro and capable of recognizing and lysing M. tuberculosis-infected dendritic cells. This methodology provides a powerful tool for the identification of M. tuberculosis proteins that can be evaluated as potential vaccine candidates.

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OBJECTIVE Meals are a major hurdle to glycemic control in type 1 diabetes (T1D). Our objective was to test a fully automated closed-loop control (CLC) system in the absence of announcement of carbohydrate ingestion among adolescents with T1D, who are known to commonly omit meal announcement. RESEARCH DESIGN AND METHODS Eighteen adolescents with T1D (age 15.6 ± 1.7 years; HbA1c 7.4 ± 1.5%; 9 females/9 males) participated in a randomized crossover clinical trial comparing our legacy hybrid CLC system (Unified Safety System Virginia [USS]-Virginia) with a novel fully automated CLC system (RocketAP) during two 46-h supervised admissions (each with one announced and one unannounced dinner), following 2 weeks of data collection. Primary outcome was the percentage time-in-range 70–180 mg/dL (TIR) following the unannounced meal, with secondary outcomes related to additional continuous glucose monitoring-based metrics. RESULTS Both TIR and time-in-tight-range 70–140 mg/dL (TTR) were significantly higher using RocketAP than using USS-Virginia during the 6 h following the unannounced meal (83% [interquartile range 64–93] vs. 53% [40–71]; P = 0.004 and 49% [41–59] vs. 27% [22–36]; P = 0.002, respectively), primarily driven by reduced time-above-range (TAR >180 mg/dL: 17% [1.3–34] vs. 47% [28–60]), with no increase in time-below-range (TBR <70 mg/dL: 0% median for both). RocketAP also improved control following the announced meal (mean difference TBR: −0.7%, TIR: +7%, TTR: +6%), overall (TIR: +5%, TAR: −5%, TTR: +8%), and overnight (TIR: +7%, TTR: +19%, TAR: −5%). RocketAP delivered less insulin overall (78 ± 23 units vs. 85 ± 20 units, P = 0.01). CONCLUSIONS A new fully automated CLC system with automatic prandial dosing was proven to be safe and feasible and outperformed our legacy USS-Virginia in an adolescent population with and without meal announcement.