Developments in cardiovascular ultrasound. Part 2: Arterial applications

Many of the changes resulting from arterial disease can be measured, using Doppler ultrasound for measurement of blood velocity and B-scan imaging for measurement of tissue structure and composition. Wall thickness, the degree of arterial narrowing and plaque volume can be measured using B-scan imaging, and 3D ultrasound can be used to improve the accuracy of measurements of plaque volume and for improved visualisation of complex arterial geometries. Measurement of the dynamic properties of the arterial wall permits estimation of wall elasticity and plaque motion. From the Doppler signal, measurements of blood velocity are used to estimate the degree of arterial narrowing and volumetric flow, although measurement errors can be large. Wall shear stress can be estimated by measuring the velocity gradient at the vessel wall. The problems of inadequate spatial resolution and interference from overlying tissue are largely removed when intravascular systems are used, and these have superior capability in the assessment of arterial structure and tissue composition. However, measurement of quantities relating to blood flow is more difficult using the intravascular approach, as the indwelling cather disturbs the blood flow pattern, and currently, assessment of flow and vessel cross-section are not performed at the same site.     

Negative selection by endogenous antigen and superantigen occurs at multiple thymic sites

The site of negative selection in the thymus has been inferredfrom a range of different experiments. Analysis of thymic deletionof Vß5⁺, V_ß11⁺ or Vß17a⁺ cellsH-2E transgenic mice led to the theory that negative selectionoccurs predominantly in the medulla (specifically, through presentationby medullary dendritic cells). Other experiments investigatedwhether transgenic TCR are deleted at the double-positive (DP)or single-positive stage following encounter with peptide ligand:by flow cytometric analysis deletion is generally found to occurat the DP thymocyte stage and as these cells are found predominantlyin the cortex, it has been inferred that this is the key siteof negative selection. The visualization of apoptotic thymocytesin situ has recently been reported for specific examples ofnegative selection. Using a panel of TCR transgenic lines inwhich negative selection occurs at different stages of thymocytedevelopment, we have used TUNEL staining to analyse the anatomicalsites of thymocyte apoptosis. For the first time we have beenable to compare directly the sites of deletion induced by theendogenous cognate peptides or by endogenous superantigen. Weshow that generalization from the medullary deletion of V_ß5⁺,V_ß11⁺ or V_ß17a⁺ cells by the endogenoussuperantigens Mtv 8 and 9 and from limited examples of corticaldeletion by exogenous peptide administered to TCR transgenicmice is over-simplified. Apoptotic thymocytes in mice lackingMtv superantigens are indeed localized in the cortex. However,when deletion is induced by cognate self peptide, apoptosiscan occur in the cortex, the medulla or at the junction betweenthe two.     

An antigen processing polymorphism revealed by HLA-B8-restricted cytotoxic T lymphocytes which does not correlate with TAP gene polymorphism

In previous studies of antigen presentation through HLA-B27, we identified a healthy person whose lymphoblastoid cells do not present three B27-restricted viral epitopes to specific cytotoxic T lymphocytes (CTL), despite adequate cell surface expression of HLA-B2702 of normal sequence. Similar findings were observed in all members of his family sharing the HLA-A3-B2702 haplotype. The original donor, NW, carries HLA-B8 on his other class I haplotype, which his daughter, HW, has inherited. We now report a failure to present an HLA-B8-restricted epitope from influenza nucleoprotein following viral infection of NW cells, although exogenous added peptide is still presented normally. However, cells from HW, which do not carry the A3-B2702 haplotype, present the expected epitope after viral infection. Another B8-restricted epitope, from human immunodeficiency virus-gag, is presented equally well by both cell lines when infected with gag-vaccinia. This antigen processing phenotype does not correlate with any of the known human TAP-1 and TAP-2 polymorphisms.     

Major histocompatibility complex status in breast carcinogenesis and relationship to apoptosis

Major histocompatibility complex (MHC) molecules are of central importance in regulating the immune response against tumors. In this study we used immunohistochemistry to study human leukocyte antigen (HLA) class I and II antigen expression in normal breast tissues and benign, preneoplastic, primary, and metastatic breast lesions using antibodies against beta-2-microglobulin (beta2-m), heavy-chain, and HLA-DR antigens. Whereas all normal tissues and benign lesions were positive for beta2-m and HLA-A, -B, and -C antigens, total loss of HLA class I antigens was found in 37% (11 of 30) of in situ carcinomas, in 43% (56 of 131) of the primary tumors, and in 70% (31 of 45) of the lymph node metastases. HLA-DR was also underexpressed in breast cancer cells; thus 20% (6 of 30) of in situ carcinomas, 15% of invasive carcinomas (20 of 131), and only 1 metastatic case were positive for this antigen. Both HLA class I and II antigen expression were more frequently down-regulated in metastatic lesions than in primary breast lesions (P <0.05), and a tendency toward a simultaneous defective expression of HLA class I and II antigens was observed in primary carcinomas (P = 0.07). However, no correlation was found between the expression of any of the aforementioned molecules and pathological parameters or survival. Interestingly, HLA class I expression was expressed more frequently in tissues with high apoptotic activity and was significantly associated with the expression of the proapoptotic bax gene (P = 0.02), and was inversely associated with expression of the antiapoptotic bcl-2 gene (P = 0.03). We conclude that alterations in HLA class I and II antigen expression are early events in breast carcinogenesis and play significant roles in metastatic progression. In addition, their expression is correlated with apoptosis-regulating proteins, which may influence the cytotoxicity of T cells against HLA class I-specific tumor antigens.     

Monitoring exocytosis from single mast cells by fast voltammetry

We have used fast differential ramp voltammetry with carbon-fibre electrodes to monitor exocytotic secretion in single rat mast cells. The oxidation peak and other aspects of the electrochemical profile of the substance released were similar to those of 5-hydroxytryptamine (5-HT) and the signals were increased by preloading the secretory granules with exogenous 5-HT. Metabolic blockade inhibited both visible degranulation and the electrochemical signal. For comparison, quinacrine, which is fluorescent and accumulates in secretory vesicles, was used as an alternative means of detecting secretion in single cells. The amplitude of the electrochemical signals observed during degranulation correlated well with the loss of quinacrine fluorescence. Both methods were used to record successive rounds of secretion in single mast cells in response to repeated applications of compound 48/80.     

How can the evaluation of genetic tests be enhanced? Lessons learned from the ACCE framework and evaluating genetic tests in the United Kingdom.

Advances in genetic technology are increasing the availability of genetic tests, not only for rare single gene disorders, but also for common diseases such as breast and colo-rectal cancer. Before there can be widespread uptake of these tests, they must be evaluated to confirm the benefits of their use. But how should genetic tests be evaluated, given the speed at which new tests are emerging? One highly influential approach is the analytic validity, clinical validity, clinical utility and ethical, legal and social issues (ACCE) framework, which has provided a benchmark for the evaluation of genetic tests. The approach has been adopted and adapted by the United Kingdom Genetic Testing Network, with the help of the Public Health Genetics Unit in Cambridge, to evaluate new genetic tests for use in the National Health Service. We discuss a number of conceptual, methodological, and practical issues concerning the evaluation of genetic tests, based on lessons learned from applying the ACCE framework and from the UK experience, and make a number of recommendations to further strengthen the evaluation of genetic tests.     

Alcohol use, urinary cortisol, and heart rate variability in apparently healthy men: Evidence for impaired inhibitory control of the HPA axis in heavy drinkers.

Alcoholism and heavy drinking are associated with a number of physiological, behavioral, affective, and cognitive problems. One such problem involves dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis, with alcoholics showing higher basal cortisol levels and reduced inhibitory feedback control. In addition, alcohol consumption is associated with decreased heart rate variability (HRV). In the present study we examined the relationships among alcohol consumption, cortisol excretion, and HRV in 542 apparently healthy men. Men in the top tertile of self-reported alcohol consumption had higher cortisol levels and lower HRV compared to men in the lower two tertiles of alcohol consumption. In addition, the inverse relationship between cortisol and HRV was greatly attenuated in the heavy drinking group even after accounting for a number of potential confounding factors. These results support prior research on the HPA axis dysregulation in alcoholics and suggest impaired inhibitory control of the HPA axis in heavy drinkers. The findings are consistent with the neurovisceral integration model, which links central and peripheral processes, and may provide a comprehensive framework for the future investigation of the complex mix of physiological, behavioral, affective, and cognitive factors which comprise the heavy drinking phenotype.     

Large deformation three-dimensional image registration in image-guided radiation therapy

In this paper, we present and validate a framework, based on deformable image registration, for automatic processing of serial three-dimensional CT images used in image-guided radiation therapy. A major assumption in deformable image registration has been that, if two images are being registered, every point of one image corresponds appropriately to some point in the other. For intra-treatment images of the prostate, however, this assumption is violated by the variable presence of bowel gas. The framework presented here explicitly extends previous deformable image registration algorithms to accommodate such regions in the image for which no correspondence exists. We show how to use our registration technique as a tool for organ segmentation, and present a statistical analysis of this segmentation method, validating it by comparison with multiple human raters. We also show how the deformable registration technique can be used to determine the dosimetric effect of a given plan in the presence of non-rigid tissue motion. In addition to dose accumulation, we describe a method for estimating the biological effects of tissue motion using a linear-quadratic model. This work is described in the context of a prostate treatment protocol, but it is of general applicability.     

Problematic Methods in the Assessment of Scholarly Productivity in Clinical PhD Programs

We review the methods in the ranking of clinical psychology doctoral programs provided by Stewart, Roberts, and Roy (2007). Using our own program as an example, we identify several areas of concern (e.g., authorship credits, criteria applied, faculty attrition). The inaccuracies identified for our program ranking, in combination with methodological concerns highlighted by previous commentaries, suggest that the validity of the rankings can be called into question.     

Optimization of peptide linker length in production of MHC class II/peptide tetrameric complexes increases yield and stability,and allows identification of antigen-specific CD4+T cells in peripheral blood mononuclear cells

Reliable, efficient systems for producing soluble HLA-DR molecules, suitable for multimerization and use as staining reagents, have proved elusive. We found that the addition of a flexible linker between peptide and N terminus of the DRB1*0101-chain (Crawford, F., Kozono, H., White, J., Marrack, P. and Kappler, J., Immunity 1998. 8: 675-682.), results in greater in vitro folding efficiency of Escherichia coli-expressed alpha- and beta-chains, and increases both the yield and stability of the DRA1*0101/DRB1*0101/peptide complexes. Although a 10-amino acid linker functioned efficiently for a 20mer epitope from HIV p24, a longer linker was required to produce a DR1 MHC class II tetramer with the influenza hemagglutinin epitope (HA(306-318)). The DR1-HA tetramer was able to stain positively over 98% of a specific clone (HA 1.7) with only a brief 30-min incubation. The tetrameric complexes detected clone cells diluted into PBMC, with high sensitivity, coupled with low background staining in CD4(+) cells. It was possible to detect antigen-specific CD4(+) T cells within a population of PBMC stimulated with the HA peptide. This demonstrates the potential to monitor CD4(+) T cell responses in peripheral blood in a number of clinical scenarios.