Synergism of Heat Shock Protein 90 and Histone Deacetylase Inhibitors in Synovial Sarcoma

Current systemic therapies have little curative benefit for synovial sarcoma. Histone deacetylase (HDAC) inhibitors and the heat shock protein 90 (Hsp90) inhibitor 17-AAG have recently been shown to inhibit synovial sarcoma in preclinical models. We tested combinations of 17-AAG with the HDAC inhibitor MS-275 for synergism by proliferation and apoptosis assays. The combination was found to be synergistic at multiple time points in two synovial sarcoma cell lines. Previous studies have shown that HDAC inhibitors not only induce cell death but also activate the survival pathway NF-κB, potentially limiting therapeutic benefit. As 17-AAG inhibits activators of NF-κB, we tested if 17-AAG synergizes with MS-275 through abrogating NF-κB activation. In our assays, adding 17-AAG blocks NF-κB activation by MS-275 and siRNA directed against histone deacetylase 3 (HDAC3) recapitulates the effects of MS-275. Additionally, we find that the NF-κB inhibitor BAY 11-7085 synergizes with MS-275. We conclude that agents inhibiting NF-κB synergize with HDAC inhibitors against synovial sarcoma

Scrub typhus ecology: a systematic review of Orientia in vectors and hosts

Abstract Scrub typhus, caused by Orientia tsutsugamushi, is an important and neglected vector-borne zoonotic disease with an expanding known distribution. The ecology of the disease is complex and poorly understood, impairing discussion of public health interventions. To highlight what we know and the themes of our ignorance, we conducted a systematic review of all studies investigating the pathogen in vectors and non-human hosts. A total of 276 articles in 7 languages were included, with 793 study sites across 30 countries. There was no time restriction for article inclusion, with the oldest published in 1924. Seventy-six potential vector species and 234 vertebrate host species were tested, accounting for over one million trombiculid mites (‘chiggers’) and 83,000 vertebrates. The proportion of O. tsutsugamushi positivity was recorded for different categories of laboratory test and host species. Vector and host collection sites were geocoded and mapped. Ecological data associated with these sites were summarised. A further 145 articles encompassing general themes of scrub typhus ecology were reviewed. These topics range from the life-cycle to transmission, habitats, seasonality and human risks. Important gaps in our understanding are highlighted together with possible tools to begin to unravel these. Many of the data reported are highly variable and inconsistent and minimum data reporting standards are proposed. With more recent reports of human Orientia sp. infection in the Middle East and South America and enormous advances in research technology over recent decades, this comprehensive review provides a detailed summary of work investigating this pathogen in vectors and non-human hosts and updates current understanding of the complex ecology of scrub typhus. A better understanding of scrub typhus ecology has important relevance to ongoing research into improving diagnostics, developing vaccines and identifying useful public health interventions to reduce the burden of the disease.</jats:p

Deuterium nuclear magnetic resonance in model membrane systems : an investigation of the interaction of a synthetic, amphiphilic polypeptide with charged lipids

The theory of the quadrupole interaction in nuclear magnetic resonance spectroscopy and relaxation measurements is presented in detail, with applications to ²H-NMR studies of order and dynamics in bilayers of deuterated lipids. Investigations of lipid-protein interactions in reconstituted membrane systems and intact biological membranes are reviewed. An experimental program is described which uses a synthetic amphiphilic polypeptide, with known geometry and variable length, to isolate questions about the geometrical interpretation of orientational order in lipid-protein interactions. A report is presented of an investigation of the effects of this polypeptide, Lys₂-Gly-Leu₂₀-Lys₂-Ala-amide, on the mixed bilayer system: Dimyristoylphosphatidylcholine Di-per-deuterio-myristoylphosphatidic Acid. The addition of the peptide was found to have little effect (≤5%) on the first and second moments of the distribution of quadrupole splittings in the liquid crystalline phase. Similarly, the spin-lattice relaxation time constants were affected by ≤10% in the liquid crystalline phase. The time constant for the decay of the quadrupole echo decreased dramatically above the phase transition with the addition of peptide, a phenomenon which is explained in terms of the presence of a new slow motion in the lipid-peptide systems. A simple model of the slow motion induced by the peptide is proposed, in which the lipid molecules undergo a rapid exchange between boundary and bulk sites. An effective correlation time is determined from an average over the rotations on each of these sites. Using this model, estimates are made of the change in the second moment brought about by the onset of the rotations, and. of the number of binding sites on the peptide. These estimates are found to be in agreement with independent measurements of the change in the second moment, and the number of binding sites is within the range predicted by simple considerations of charge balance. The change in the lineshape with the variation of the spacing of the pulses in the quadrupole echo experiment was investigated, and it was determined that the transverse relaxation time constants have a slight orientation dependence. It was also determined that the addition of the peptide has no significant effect on the variation of the lineshape. Some experiments which could answer some of the questions raised by these results are suggested.Science, Faculty ofPhysics and Astronomy, Department ofGraduat

The development of automated systems for metaphase location in cytogenetic preparations of human bone marrow

Cytogenetic evaluation of human bone marrow cells is one of the principal sources of diagnostic and prognostic information in the evaluation of the myeloid leukemias. In the majority of cases, these diseases are characterized by non-random chromosomal changes in the cells of the malignant clone. The chromosomal abnormalities are present only in the leukemic cells, which are distributed along with normal cells in the bone marrow and throughout the circulation. The objective of this thesis was to test the hypothesis that suitable criteria could be established for automated metaphase detection using human bone marrow preparations. This involved computerized, low resolution scanning of a specimen slide, and the measurement of object features which allowed metaphases to be adequately distinguished from nuclei and debris. Two approaches were investigated. The first used a line-scanning system, in which microscope slides were scanned line by line with a linear CCD detector, and focussing was performed automatically. Eighteen signal features were measured for each detected object. Three group discriminant function analysis was performed on objects from a large number of slides from both types of preparations, in order to distinguish metaphases from nuclei and debris. The second method evaluated the use of a frame scanning system. Objects were detected in a frame-by-frame scan of microscope slides, using a two dimensional CD camera. Feature measurements were performed for all objects within a specified area range, and three group discriminant function analysis was performed on data from a large number of slides. In both approaches, the performance of the discriminant functions was evaluated on independent samples collected from a number of patients, in order to determine the operational error rates of the systems. The sensitivity of the line scan system for metaphase detection was 86%, compared to 92% fror the frame scannning system, while the specificity was 84% for the line scan system, and 86% for the frame scanning system. The frame scan system was shown to be useful for determining the mitotic index of cells cultured for varying periods of time prior to fixation. Four patients with AML were examined, and the results of the analysis show that the mitotic indices could be determined in this way to an accuracy of approximately 5%. The mitotic indices differed as a function of time for different patients.Science, Faculty ofPhysics and Astronomy, Department ofGraduat

Confocal 3D DNA cytometry: assessment of required coefficient of variation by computer simulation

BACKGROUND: Confocal Laser Scanning Microscopy (CLSM) provides the opportunity to perform 3D DNA content measurements on intact cells in thick histological sections. So far, sample size has been limited by the time consuming nature of the technology. Since the power of DNA histograms to resolve different stemlines depends on both the sample size and the coefficient of variation (CV) of histogram peaks, interpretation of 3D CLSM DNA histograms might be hampered by both a small sample size and a large CV. The aim of this study was to analyze the required CV for 3D CLSM DNA histograms given a realistic sample size. METHODS: By computer simulation, virtual histograms were composed for sample sizes of 20000, 10000, 5000, 1000, and 273 cells and CVs of 30, 25, 20, 15, 10 and 5%. By visual inspection, the histogram quality with respect to resolution of G0/1 and G2/M peaks of a diploid stemline was assessed. RESULTS: As expected, the interpretability of DNA histograms deteriorated with decreasing sample sizes and higher CVs. For CVs of 15% and lower, a clearly bimodal peak pattern with well distinguishable G0/1 and G2/M peaks were still seen at a sample size of 273 cells, which is our current average sample size with 3D CLSM DNA cytometry. CONCLUSIONS: For unambiguous interpretation of DNA histograms obtained using 3D CLSM, a CV of at most 15% is tolerable at currently achievable sample sizes. To resolve smaller near diploid stemlines, a CV of 10% or better should be aimed at. With currently available 3D imaging technology, this CV is achievable

Malignancy-Associated Changes in Breast Tissue Detected by Image Cytometry

In several tissues, nuclear differences have been described in normal‐appearing cells from patients with invasive carcinomas compared to cases without invasive carcinoma, a phenomenon known as malignancy‐associated changes (MACs). The aim of this study was to determine the presence of malignancy‐associated changes in breast tissue

Confocal DNA cytometry: a contour-based segmentation algorithm for automated three-dimensional image segmentation

BACKGROUND: Confocal laser scanning microscopy (CLSM) presents the opportunity to perform three-dimensional (3D) DNA content measurements on intact cells in thick histological sections. So far, these measurements have been performed manually, which is quite time-consuming. METHODS: In this study, an intuitive contour-based segmentation algorithm for automatic 3D CLSM image cytometry of nuclei in thick histological sections is presented. To evaluate the segmentation algorithm, we measured the DNA content and volume of human liver and breast cancer nuclei in 3D CLSM images. RESULTS: A high percentage of nuclei could be segmented fully automatically (e.g., human liver, 92%). Comparison with (time-consuming) interactive measurements on the same CLSM images showed that the results were well correlated (liver, r = 1.00; breast, r = 0.92). CONCLUSIONS: Automatic 3D CLSM image cytometry enables measurement of volume and DNA content of large numbers of nuclei in thick histological sections within an acceptable time. This makes large-scale studies feasible, whereby the advantages of CLSM can be exploited fully. The intuitive modular segmentation algorithm presented in this study detects and separates overlapping objects, also in two-dimensional (2D) space. Therefore, this algorithm may also be suitable for other applications

Discrimination between benign and malignant prostate tissue using chromatin texture analysis in 3-D by confocal laser scanning microscopy

BACKGROUND: Analysis of chromatin texture may improve both the diagnosis and the assessment of the prognosis of prostate cancer. Confocal laser scanning microscopy (CLSM) allows performing measurements in nuclei reconstructed in 3-D. The aim of this study was to evaluate the clinical usefulness of 3-D texture analysis of prostate tissue. METHODS: Image stacks of eight prostate cancer sections were obtained by CLSM of both benign and malignant areas. Texture feature values were computed for individual nuclei. The discriminative power of the texture features was established by receiver operating characteristic (ROC) analysis and linear discriminant analysis (LDA). RESULTS: Texture features were identified that could discriminate between benign and malignant nuclei. LDA correctly classified 89% of the nuclei of the pooled set of benign and malignant nuclei. CONCLUSIONS: 3-D nuclear texture features allow discrimination of most benign and malignant prostate nuclei. We estimate that the classification rates can be increased by improving the image quality