The molecular diversity of receptors in human blood vessels remains largely unexplored. We developed a selection method in which peptides that home to specific vascular beds are identified after administration of a peptide library. Here we report the first in vivo screening of a peptide library in a patient. We surveyed 47,160 motifs that localized to different organs. This large-scale screening indicates that the tissue distribution of circulating peptides is nonrandom. High-throughput analysis of the motifs revealed similarities to ligands for differentially expressed cell-surface proteins, and a candidate ligand-receptor pair was validated. These data represent a step toward the construction of a molecular map of human vasculature and may have broad implications for the development of targeted therapies.

BackgroundCombinatorial phage display has been used in the last 20 years in the identification of protein-ligands and protein-protein interactions, uncovering relevant molecular recognition events. Rate-limiting steps of combinatorial phage display library selection are (i) the counting of transducing units and (ii) the sequencing of the encoded displayed ligands. Here, we adapted emerging genomic technologies to minimize such challenges.Methodology/Principal FindingsWe gained efficiency by applying in tandem real-time PCR for rapid quantification to enable bacteria-free phage display library screening, and added phage DNA next-generation sequencing for large-scale ligand analysis, reporting a fully integrated set of high-throughput quantitative and analytical tools. The approach is far less labor-intensive and allows rigorous quantification; for medical applications, including selections in patients, it also represents an advance for quantitative distribution analysis and ligand identification of hundreds of thousands of targeted particles from patient-derived biopsy or autopsy in a longer timeframe post library administration. Additional advantages over current methods include increased sensitivity, less variability, enhanced linearity, scalability, and accuracy at much lower cost. Sequences obtained by qPhage plus pyrosequencing were similar to a dataset produced from conventional Sanger-sequenced transducing-units (TU), with no biases due to GC content, codon usage, and amino acid or peptide frequency. These tools allow phage display selection and ligand analysis at >1,000-fold faster rate, and reduce costs ∼250-fold for generating 106 ligand sequences.Conclusions/SignificanceOur analyses demonstrates that whereas this approach correlates with the traditional colony-counting, it is also capable of a much larger sampling, allowing a faster, less expensive, more accurate and consistent analysis of phage enrichment. Overall, qPhage plus pyrosequencing is superior to TU-counting plus Sanger sequencing and is proposed as the method of choice over a broad range of phage display applications in vitro, in cells, and in vivo.

Molecules differentially expressed in blood vessels among organs or between damaged and normal tissues, are attractive therapy targets; however, their identification within the human vasculature is challenging. Here we screened a peptide library in cancer patients to uncover ligand-receptors common or specific to certain vascular beds. Surveying ∼2.35 × 10 6 motifs recovered from biopsies yielded a nonrandom distribution, indicating that systemic tissue targeting is feasible. High-throughput analysis by similarity search, protein arrays, and affinity chromatography revealed four native ligand-receptors, three of which were previously unrecognized. Two are shared among multiple tissues (integrin α4/annexin A4 and cathepsin B/apolipoprotein E3) and the other two have a restricted and specific distribution in normal tissue (prohibitin/ annexin A2 in white adipose tissue) or cancer (RAGE/leukocyte proteinase-3 in bone metastases). These findings provide vascular molecular markers for biotechnology and medical applications. To discover or analyze functional ligand-receptor interactions in blood vessels under disease conditions, we have used combinatorial screenings based on phage display, which has enabled the targeted delivery of agents to specific vascular beds (8,9). This approach allows the selection of homing peptides to specific organs in vivo after systemic administration of random peptide libraries (10, 11). We have isolated ligand peptides and identified their tissue-specific receptors in rodents and in a patient, and have developed a ligand-receptor in prostate cancer (12, 13) that serves as the basis for an ongoing first-in-man trial.Over the past few years we have developed a tripartite approach to enable serial combinatorial selections to humans. First, we established an ethical framework to ensure respectful research in patients who were brain-dead or whose families decided to terminate life support (14,15). Second, we adapted techniques that were validated in rodents (16) to enable synchronous selection of ligands to multiple organs. Third, we integrated genomic tools, in which recovery of 10 6 peptides is ∼1;000-fold faster and ∼250-fold cheaper (17). Here, these quantitative and qualitative methods enabled refined combinatorial selections in patients and identified unique ligand-receptors.

BACKGROUND.Patients with cancer who have thrombocytopenia may experience acute coronary syndromes (ACS), and the use of aspirin (ASA) poses an increased risk of bleeding. The purpose of this study was to test the hypothesis that the benefit of ASA therapy in the treatment of ACS would extend to cancer patients with thrombocytopenia and outweigh the risks of severe bleeding.METHODS.The records of all cancer patients diagnosed with an ACS in 2001 and referred for cardiology consultation were reviewed. Patients were divided into 2 groups on the basis of platelet count, >100 cells k/μL and ≤100 cells k/μL. Data were collected on the use of ASA therapy, bleeding complications, and survival rates. The authors assessed group differences by using the Wilcoxon rank sum test or 2‐tailed Fisher exact test, as appropriate. Univariate and multivariate logistic regression models were used to assess factors potentially associated with 7‐day survival.RESULTS.In cancer patients with ACS and thrombocytopenia, those who did not receive ASA had a 7‐day survival rate of 6% compared with 90% in those who did receive ASA (P < .0001). There were no severe bleeding complications. Patients with a platelet count (>100 cells k/μL) who received ASA had a 7‐day survival rate of 88% compared with 45% in those who did not receive ASA (P = .0096).CONCLUSIONS.Therapy with ASA was associated with a significantly improved 7‐day survival after ACS in cancer patients, with or without thrombocytopenia, and not associated with more severe bleeding. Cancer 2007;109:621–627. © 2006 American Cancer Society.

SummaryA patient in the right lateral position underwent left nephrectomy, after which he was placed supine for insertion of The diagnosis of intra-operative pneumothorax canThe peak inspiratory pressure was increased slightly from occasionally be difficult. We report a case where a decrease 14-16 cmH,O in the supine position to 20-22 cmH,O in electrocardiogram (ECG) amplitude was the first sign of when he was in the lateral position. The ECG demonthe event. This change occurred 25 min before a rapid strated sinus rhythm and was similar to the pre-induction deterioration in the patient's clinical condition necessitated ECG (Fig. I). Following nephrectomy he was moved to the urgent treatment.supine position in preparation for the A-V shunt placement. Re-assessment of airway and monitors showed no changes. Ten minutes later, during the preparation of the left upper arm, the lead I1 ECG tracing was noted to be dramatically reduced in amplitude (Fig. 2) when compared to the pre-induction tracing (Fig. I). Re-examination of the Case historyA 55-year-old male patient with progressive renal insufficiency and a left renal mass presented for left nephrectomy and arteriovenous (A-V) shunt placement. His past medical history included well-controlled hypertension, Crohn's disease, progressive renal insufficiency, and a history of cigarette smoking. Medications included atenolol and sulfasalazine. He underwent a n uncomplicated induction of anaesthesia and tracheal intubation. A radial arterial line was placed after induction of anaesthesia and he was positioned in the right lateral decubitus position for surgery. He remained haemodynamically stable during the nephrectomy with a mean arterial pressure (MAP) of 75-85mmHg, and an oxygen saturation (Spo,) of 98%.