The predictive value of anginal chest pain as an indicator of coronary disease during exercise testing

To determine the significance of anginal chest pain during exercise testing, a series of 302 patients undergoing coronary arteriography with exercise testing was reviewed. Of the 302 patients, 85 had ischemic ECG changes and chest pain (Group I); 87 patients had ischemic ECG changes but no chest pain (Group II); 25 patients had chest pain but no ischemic ECG changes (Group III); 105 patients had neither chest pain nor ischemic ECG changes (Group IV). Coronary artery disease was present in 95 per cent of Group I, 75 per cent of Group II, 72 per cent of Group III, and 28 per cent of Group IV. Of those patients with coronary disease, multiple vessels were involved in 94 per cent of Group I, 51 per cent of Group II, 67 per cent of Group III, and 21 per cent of Group IV. The predictive value for presence and extent of coronary disease showed Group I > Groups II and III > Group IV (p < 0.025). We conclude that (1) anginal chest pain during exercise testing predicts the presence and extent of coronary disease more accurately than its absence; (2) the presence of chest pain even without an ischemic ECG response during exercise testing appears to be as predictive of coronary disease as an ischemic ECG response alone; and (3) the combination of anginal chest pain during exercise testing and an ischemic ECG response is highly predictive of multivessel coronary artery disease.     

Single-cell proteomic chip for profiling intracellular signaling pathways in single tumor cells

We describe a microchip designed to quantify the levels of a dozen cytoplasmic and membrane proteins from single cells. We use the platform to assess protein–protein interactions associated with the EGF-receptor-mediated PI3K signaling pathway. Single-cell sensitivity is achieved by isolating a defined number of cells (n = 0–5) in 2 nL volume chambers, each of which is patterned with two copies of a miniature antibody array. The cells are lysed on-chip, and the levels of released proteins are assayed using the antibody arrays. We investigate three isogenic cell lines representing the cancer glioblastoma multiforme, at the basal level, under EGF stimulation, and under erlotinib inhibition plus EGF stimulation. The measured protein abundances are consistent with previous work, and single-cell analysis uniquely reveals single-cell heterogeneity, and different types and strengths of protein–protein interactions. This platform helps provide a comprehensive picture of altered signal transduction networks in tumor cells and provides insight into the effect of targeted therapies on protein signaling networks.Although signal transduction inhibitors occasionally offer clinical benefit for cancer patients (1), signal flux emanating from oncogenes is often distributed through multiple pathways (2), potentially underlying the failure of most such inhibitors (3). Measuring signal flux through multiple pathways, in response to signal transduction inhibitors, may help uncover network interactions that contribute to therapeutic resistance and that are not predicted by analyzing pathways in isolation (4). The cellular and molecular complexity of a solid tumor microenvironment (5) suggests the need to study signaling in individual cancer cells.Protein–protein interactions within signaling pathways are often elucidated by assessing the levels of relevant pathway proteins in model and tumor-derived cell lines and with various genetic and molecular perturbations. Such interactions, and the implied signaling networks, may also be elucidated via quantitative measurements of multiple pathway-related proteins within single cells (6). At the single-cell level, inhibitory and activating protein–protein relationships, as well as stochastic (single-cell) fluctuations, are revealed. However, most techniques for profiling signaling pathways (7, 8) require large numbers of cells. Single-cell immunostaining (9) is promising, and some flow cytometry (6) techniques are relevant, as discussed below.We describe quantitative, multiplex assays of intracellular signaling proteins from single cancer cells using a platform called the single-cell barcode chip (SCBC). The SCBC is simple in concept: A single or defined number of cells is isolated within an approximately 2 nL volume microchamber that contains an antibody array (10) for the capture and detection of a panel of proteins. The SCBC design (11) permits lysis of each individual trapped cell.Intracellular staining flow cytometry can assay up to 11 phosphoproteins from single cells (6). Our SCBC can profile a similar size panel, but only for approximately 100 single cells per chip. Each protein is assayed twice, yielding some statistical assessment for each experiment. The SCBC is a relatively simple platform and only requires a few hundred cells per assay.We used the SCBC to study signal transduction in glioblastoma multiforme (GBM), a primary malignant brain tumor (12). GBM has been genetically characterized, yet the nature of signaling pathways downstream of key oncogenic mutations, such as epidermal growth factor receptor activating mutation (EGFRvIII) and phosphatase and tensin homolog (PTEN) tumor suppressor gene loss associated with receptor tyrosine kinase (RTK)/PI3K signaling, are incompletely understood (13–15). Single-cell experiments may also help resolve the characteristic heterogeneity of GBM.We interrogated 11 proteins directly or potentially associated with PI3K signaling (see SI Appendix, Methods I) through three isogenic GBM cell lines: U87 (expressing wild-type p53, mutant PTEN, and low levels of wild-type EGFR, no EGFRvIII) (16, 17), U87 EGFRvIII (U87 cells stably expressing EGFRvIII deletion mutant), and U87 EGFRvIII PTEN (U87 cells coexpressing EGFRvIII and PTEN) (18). Fig. 1 diagrams this biology. Each cell line was investigated under conditions of standard cell culture, in response to EGF stimulation, and after erlotinib treatment followed by EGF stimulation. The proteins assayed represented RTKs and proteins signifying activation of PI3K and MAPK signaling. They were (p- denotes phosphorylation) p-Src, p-mammalian target of rapamycin (p-mTOR), p-p70 ribosomal protein S6 kinase (p-p70S6K), p-glycogen synthase kinase-3 (p-GSK-3α/β), p-p38 mitogen activated protein kinase (p-p38α), p-extracellular regulated kinase (p-ERK), p-c-Jun N-terminal kinase (p-JNK2), p-platelet derived growth factor receptor β (p-PDGFRβ), p-vascular endothelial growth factor receptor 2 (p-VEGFR2), tumor protein 53 (P53), and total EGFR.Open in a separate windowFig. 1.The PI3K pathway activated by EGF-stimulated EGFR or by the constitutively activated EGFRvIII. All proteins in light blue with central yellow background were assayed. Orange background proteins were expressed in the cell lines U87 EGFRvIII or U87 EGFRvIII PTEN. The oval, yellow background components are the investigated molecular perturbations.     

High value, cost-conscious care: An international imperative

Health care costs in the United States are the highest in the world, and are continuing to rise at a level that is unsustainable. However, although this problem is more acute in the United States than elsewhere in the world, it is a challenge for all nations to control the costs of health care. The high cost of health care in the U.S. is not accompanied by a higher quality of care, but rather is related in large measure to health system "waste" that does not benefit patients but adds to cost. Representing approximately 30% of dollars spent on health care, this waste includes a significant amount of money spent on overuse and misuse of diagnostic testing, including screening tests. The American College of Physicians, the largest specialty society for physicians in the U.S., representing internal medicine and all of its subspecialties, has embarked upon a High Value, Cost-Conscious Care initiative, aimed at identifying areas of overuse and misuse of care, and leading to development of guidelines, educational materials, and other resources targeted to health care providers, trainees, and the general public. It is incumbent upon physicians, non-physician health care professionals, patients, and other health care stakeholders to address the issue of reducing care that is not appropriate, both to improve the overall quality of care and to reduce the associated unsustainable financial burden to society.     

CLT1 targets angiogenic endothelium through CLIC1 and fibronectin

Angiogenesis is important for tumor growth and metastasis. CLT1 (CGLIIQKNEC), a peptide that binds to tumor interstitial spaces in the presence of fibrin-fibronectin, has structural similarity to the anti-angiogenic β-sheet peptides anastellin and anginex. This similarity is reflected in the ability of CLT1 to form co-aggregates with fibronectin that induce an unfolded protein response and cause autophagic cell death in proliferating endothelial cells. CLT1 cytotoxicity is mediated at least in parts by a novel CLT1 binding protein, Chloride Intracellular Channel 1 (CLIC1), which promotes internalization of CLT1-fibronectin co-aggregates in a mechanism that depends on the LIIQK amino acid sequence of CLT1. LIIQK encompasses amino acid residues relevant for CLT1 binding to CLIC1 and in addition, facilitates the formation of CLT1-fibronectin co-aggregates, which in turn promote translocation of CLIC1 to the endothelial cell surface through ligation of integrin αvβ3. Paralleling the in vitro results, we found that CLT1 co-localizes with CLIC1 and fibronectin in angiogenic blood vessels in vivo, and that CLT1 treatment inhibited angiogenesis and tumor growth. Our findings show that CLT1 is a new anti-angiogenic compound, and its mechanism of action is to form co-aggregates with fibronectin, which bind to angiogenic endothelial cells through integrins, become internalized through CLIC1 and elicit a cytotoxic unfolded protein response. The simple structure and high potency of CLT1 make it a potentially useful compound for anti-angiogenic treatments.     

Circumoral complications in hereditary sensory and autonomic neuropathy — a case of simple lip reconstruction?

We report a case of a 7-year-old Bangladeshi boy who caused himself oral incontinence by self-mutilation. The patient was known to suffer from hereditary sensory and autonomic neuropathy type V. As definitive management, a full dental clearance was performed along with reconstruction of the lower lip with a good functional and aesthetic outcome. He did not experience any adverse effects from the full dental clearance with regard to feeding, nutrition or development. We discuss the dilemma and challenges raised in the management of this patient and highlight the need for a multi-disciplinary specialist input for what appeared to be a simple case of lip reconstruction for a plastic surgeon.