Using a semi-structured interview to explore imagery experienced during social anxiety for clients with a diagnosis of psychosis: an exploratory study conducted within an early intervention for psychosis service.

BACKGROUND: Social phobia frequently occurs as a comorbid condition, with high rates reported among people with psychosis. Little is known about the nature of social anxiety in this population or whether current psychological theories apply. AIMS: This paper aims to develop and pilot a suitable measure to explore imagery experienced by participants with comorbid psychosis and social anxiety and to provide preliminary indications as to its nature. METHOD: A semi-structured interview exploring imagery was used with seven participants (adapted from Hackmann, Surawy and Clark, 1998) and the results were analysed using qualitative template analysis. RESULTS: Initial indications from this sample suggest that some participants experience typical social anxiety images, as identified by Hackmann et al. (1998). However, some experience images that appear more threatening, and may be related to residual psychotic paranoia. Image perspective was also explored: typical social anxiety images tended to be seen from an observer perspective, while those that may have been more related to psychosis tended to be seen from a field perspective. CONCLUSIONS: This exploratory study has facilitated the careful adaptation and development of an imagery interview for use in this population and has suggested areas for further research and raised questions around clinical implications.

Segmentation of whole cells and cell nuclei from 3-D optical microscope images using dynamic programming.

Communications between cells in large part drive tissue development and function, as well as disease-related processes such as tumorigenesis. Understanding the mechanistic bases of these processes necessitates quantifying specific molecules in adjacent cells or cell nuclei of intact tissue. However, a major restriction on such analyses is the lack of an efficient method that correctly segments each object (cell or nucleus) from 3-D images of an intact tissue specimen. We report a highly reliable and accurate semi-automatic algorithmic method for segmenting fluorescence-labeled cells or nuclei from 3-D tissue images. Segmentation begins with semi-automatic, 2-D object delineation in a user-selected plane, using dynamic programming (DP) to locate the border with an accumulated intensity per unit length greater that any other possible border around the same object. Then the two surfaces of the object in planes above and below the selected plane are found using an algorithm that combines DP and combinatorial searching. Following segmentation, any perceived errors can be interactively corrected. Segmentation accuracy is not significantly affected by intermittent labeling of object surfaces, diffuse surfaces, or spurious signals away from surfaces. The unique strength of the segmentation method was demonstrated on a variety of biological tissue samples where all cells, including irregularly shaped cells, were accurately segmented based on visual inspection.

A high-throughput system for segmenting nuclei using multiscale techniques.

Automatic segmentation of cell nuclei is critical in several high-throughput cytometry applications whereas manual segmentation is laborious and irreproducible. One such emerging application is measuring the spatial organization (radial and relative distances) of fluorescence in situ hybridization (FISH) DNA sequences, where recent investigations strongly suggest a correlation between nonrandom arrangement of genes to carcinogenesis. Current automatic segmentation methods have varying performance in the presence of nonuniform illumination and clustering, and boundary accuracy is seldom assessed, which makes them suboptimal for this application. The authors propose a modular and model-based algorithm for extracting individual nuclei. It uses multiscale edge reconstruction for contrast stretching and edge enhancement as well as a multiscale entropy-based thresholding for handling nonuniform intensity variations. Nuclei are initially oversegmented and then merged based on area followed by automatic multistage classification into single nuclei and clustered nuclei. Estimation of input parameters and training of the classifiers is automatic. The algorithm was tested on 4,181 lymphoblast nuclei with varying degree of background nonuniformity and clustering. It extracted 3,515 individual nuclei and identified single nuclei and individual nuclei in clusters with 99.8 +/- 0.3% and 95.5 +/- 5.1% accuracy, respectively. Segmented boundaries of the individual nuclei were accurate when compared with manual segmentation with an average RMS deviation of 0.26 microm (approximately 2 pixels). The proposed segmentation method is efficient, robust, and accurate for segmenting individual nuclei from fluorescence images containing clustered and isolated nuclei. The algorithm allows complete automation and facilitates reproducible and unbiased spatial analysis of DNA sequences.

Calibration and standardization of the emission light path of confocal microscopes.

Recently, there has been a large expansion in the usage of optical microscopes for obtaining quantitative information from biological samples in order to determine fundamental biological information such as molecular kinetics and interaction, and heterogeneity within cell populations. Consequently, we built a highly stable, uniform, isotropically emitting and convenient-to-use light source, and designed image analysis procedures for calibrating the emission light path of optical microscopes. We used the source and procedures to analyse the quantitative imaging properties of a widely used model of laser scanning confocal microscope. Results showed that the overall performance was as high as could be expected given the inherent limitations of the optical components and photomultiplier tubes. We observed that the photon detection efficiency did not vary with photomultiplier tube gain and that the highest dynamic range was achieved with relatively low gain and 12-bit digitization. Practical applications of the light source for checking the transmission of optical components in the emission light path are presented.

Senescent fibroblasts promote epithelial cell growth and tumorigenesis: a link between cancer and aging.

Mammalian cells can respond to damage or stress by entering a state of arrested growth and altered function termed cellular senescence. Several lines of evidence suggest that the senescence response suppresses tumorigenesis. Cellular senescence is also thought to contribute to aging, but the mechanism is not well understood. We show that senescent human fibroblasts stimulate premalignant and malignant, but not normal, epithelial cells to proliferate in culture and form tumors in mice. In culture, the growth stimulation was evident when senescent cells comprised only 10% of the fibroblast population and was equally robust whether senescence was induced by replicative exhaustion, oncogenic RAS, p14(ARF), or hydrogen peroxide. Moreover, it was due at least in part to soluble and insoluble factors secreted by senescent cells. In mice, senescent, much more than presenescent, fibroblasts caused premalignant and malignant epithelial cells to form tumors. Our findings suggest that, although cellular senescence suppresses tumorigenesis early in life, it may promote cancer in aged organisms, suggesting it is an example of evolutionary antagonistic pleiotropy.

Mismatched human leukocyte antigen alleles protect against heterosexual HIV transmission.

Genetic variation at the human leukocyte antigen (HLA) loci has been shown to be an important risk factor for progression to HIV disease, but its significance in infection is less well understood. We have investigated its role in HIV transmission in a cohort of individuals at risk for heterosexual infection. Analysis of over 80 individuals revealed that that the degree of concordance at HLA A, B, and DR loci differs significantly between transmitting and nontransmitting couples at risk for heterosexual HIV transmission (p <.02), suggesting that allogeneic immune responses may confer a degree of protection against HIV infection. Analysis of the frequencies of specific alleles at the A, B, and DR loci revealed a significantly higher frequency of HLA DR5 among exposed uninfected individuals, relative to population controls.

Segmentation of nuclei and cells using membrane related protein markers.

Segmenting individual cell nuclei from microscope images normally involves volume labelling of the nuclei with a DNA stain. However, this method often fails when the nuclei are tightly clustered in the tissue, because there is little evidence from the images on where the borders of the nuclei are. In this paper we present a method which solves this limitation and furthermore enables segmentation of whole cells. Instead of using volume stains, we used stains that specifically label the surface of nuclei or cells: lamins for the nuclear envelope and alpha-6 or beta-1 integrins for the cellular surface. The segmentation is performed by identifying unique seeds for each nucleus/cell and expanding the boundaries of the seeds until they reach the limits of the nucleus/cell, as delimited by the lamin or integrin staining, using gradient-curvature flow techniques. We tested the algorithm using computer-generated objects to evaluate its robustness against noise and applied it to cells in culture and to tissue specimens. In all the cases that we present the algorithm gave accurate results.

Three-dimensional image visualization and analysis.

This unit introduces the concepts of 3D image analysis and visualization as applied in cytometry. The author discusses the nature of 3D data sets and describes the techniques for visualization and analysis of 3D images. Discussions of noise removal, depth attenuation, and correction and segmentation are also included, as is a brief introduction to 3D analysis options and deconvolution principles. This commentary unit is a good way to begin an understanding of the application of 3D data sets.

Modulation of lens cell adhesion molecules by particle beams.

Cell adhesion molecules (CAMs) are proteins which anchor cells to each other and to the extracellular matrix (ECM), but whose functions also include signal transduction, differentiation, and apoptosis. We are testing a hypothesis that particle radiations modulate CAM expression and this contributes to radiation-induced lens opacification. We observed dose-dependent changes in the expression of beta 1-integrin and ICAM-1 in exponentially-growing and confluent cells of a differentiating human lens epithelial cell model after exposure to particle beams. Human lens epithelial (HLE) cells, less than 10 passages after their initial culture from fetal tissue, were grown on bovine corneal endothelial cell-derived ECM in medium containing 15% fetal bovine serum and supplemented with 5 ng/ml basic fibroblast growth factor (FGF-2). Multiple cell populations at three different stages of differentiation were prepared for experiment: cells in exponential growth, and cells at 5 and 10 days post-confluence. The differentiation status of cells was characterized morphologically by digital image analysis, and biochemically by Western blotting using lens epithelial and fiber cell-specific markers. Cultures were irradiated with single doses (4, 8 or 12 Gy) of 55 MeV protons and, along with unirradiated control samples, were fixed using -20 degrees C methanol at 6 hours after exposure. Replicate experiments and similar experiments with helium ions are in progress. The intracellular localization of beta 1-integrin and ICAM-1 was detected by immunofluorescence using monoclonal antibodies specific for each CAM. Cells known to express each CAM were also processed as positive controls. Both exponentially-growing and confluent, differentiating cells demonstrated a dramatic proton-dose-dependent modulation (upregulation for exponential cells, downregulation for confluent cells) and a change in the intracellular distribution of the beta 1-integrin, compared to unirradiated controls. In contrast, there was a dose-dependent increase in ICAM-1 immunofluorescence in confluent, but not exponentially-growing cells. These results suggest that proton irradiation downregulates beta 1-integrin and upregulates ICAM-1, potentially contributing to cell death or to aberrant differentiation via modulation of anchorage and/or signal transduction functions. Quantification of the expression levels of the CAMs by Western analysis is in progress.

A geometric model for 3-D confocal image analysis.

In this paper, we use partial-differential-equation-based filtering as a preprocessing and post processing strategy for computer-aided cytology. We wish to accurately extract and classify the shapes of nuclei from confocal microscopy images, which is a prerequisite to an accurate quantitative intranuclear (genotypic and phenotypic) and internuclear (tissue structure) analysis of tissue and cultured specimens. First, we study the use of a geometry-driven edge-preserving image smoothing mechanism before nuclear segmentation. We show how this filter outperforms other widely-used filters in that it provides higher edge fidelity. Then we apply the same filter, with a different initial condition, to smooth nuclear surfaces and obtain sub-pixel accuracy. Finally we use another instance of the geometrical filter to correct for misinterpretations of the nuclear surface by the segmentation algorithm. Our prefiltering and post filtering nicely complements our initial segmentation strategy, in that it provides substantial and measurable improvement in the definition of the nuclear surfaces.

The Caenorhabditis elegans dosage compensation machinery is recruited to X chromosome DNA attached to an autosome.

The dosage compensation machinery of Caenorhabditis elegans is targeted specifically to the X chromosomes of hermaphrodites (XX) to reduce gene expression by half. Many of the trans-acting factors that direct the dosage compensation machinery to X have been identified, but none of the proposed cis-acting X chromosome-recognition elements needed to recruit dosage compensation components have been found. To study X chromosome recognition, we explored whether portions of an X chromosome attached to an autosome are competent to bind the C. elegans dosage compensation complex (DCC). To do so, we devised a three-dimensional in situ approach that allowed us to compare the volume, position, and number of chromosomal and subchromosomal bodies bound by the dosage compensation machinery in wild-type XX nuclei and XX nuclei carrying an X duplication. The dosage compensation complex was found to associate with a duplication of the right 30% of X, but the complex did not spread onto adjacent autosomal sequences. This result indicates that all the information required to specify X chromosome identity resides on the duplication and that the dosage compensation machinery can localize to a site distinct from the full-length hermaphrodite X chromosome. In contrast, smaller duplications of other regions of X appeared to not support localization of the DCC. In a separate effort to identify cis-acting X recognition elements, we used a computational approach to analyze genomic DNA sequences for the presence of short motifs that were abundant and overrepresented on X relative to autosomes. Fourteen families of X-enriched motifs were discovered and mapped onto the X chromosome.

Effect of chemokine receptor mutations on heterosexual human immunodeficiency virus transmission.

To assess the effect of mutations at the CCR-2 and CCR-5 loci on heterosexual human immunodeficiency virus (HIV) transmission, 144 persons heterosexually exposed to HIV (infected and uninfected [EU]) and 57 HIV-positive index partners were genotyped. A significantly higher frequency of 64I heterozygotes at CCR-2 was observed in HIV-positive than in EU women (P=.02, relative risk=1.6). The allele frequency of 64I in women was 8% in HIV-positive contacts and 1% in EUs (P<.02). At CCR-5, no difference in the frequency of Delta32 was seen between groups, and the CCR-5 genotypes did not differ in accumulated "at-risk" exposure in EUs. Combining the analysis of the Delta32 and 64I mutations in index partners suggested an additive effect on transmission (P=.10). Thus heterozygosity for 64I at CCR-2 acts as a risk factor for HIV infection of women after heterosexual contact but heterozygosity for Delta32 at CCR-5 has no detectable effect.

Segmentation of confocal microscope images of cell nuclei in thick tissue sections.

Segmentation of intact cell nuclei from three-dimensional (3D) images of thick tissue sections is an important basic capability necessary for many biological research studies. However, segmentation is often difficult because of the tight clustering of nuclei in many specimen types. We present a 3D segmentation approach that combines the recognition capabilities of the human visual system with the efficiency of automatic image analysis algorithms. The approach first uses automatic algorithms to separate the 3D image into regions of fluorescence-stained nuclei and unstained background. This includes a novel step, based on the Hough transform and an automatic focusing algorithm to estimate the size of nuclei. Then, using an interactive display, each nuclear region is shown to the analyst, who classifies it as either an individual nucleus, a cluster of multiple nuclei, partial nucleus or debris. Next, automatic image analysis based on morphological reconstruction and the watershed algorithm divides clusters into smaller objects, which are reclassified by the analyst. Once no more clusters remain, the analyst indicates which partial nuclei should be joined to form complete nuclei. The approach was assessed by calculating the fraction of correctly segmented nuclei for a variety of tissue types: Caenorhabditis elegans embryos (839 correct out of a total of 848), normal human skin (343/362), benign human breast tissue (492/525), a human breast cancer cell line grown as a xenograft in mice (425/479) and invasive human breast carcinoma (260/335). Furthermore, due to the analyst's involvement in the segmentation process, it is always known which nuclei in a population are correctly segmented and which not, assuming that the analyst's visual judgement is correct.

Intensity-based energy transfer measurements in digital imaging microscopy.

Investigation of protein-protein associations is important in understanding structure and function relationships in living cells. Using Förster-type resonance energy transfer between donor and acceptor labeled monoclonal antibodies we can assess the cell surface topology of membrane proteins against which the antibodies were raised. In our current work we elaborated a quantitative image microscopic technique based on the measurement of fluorescence intensities to calculate the energy transfer efficiency on a pixel-by-pixel basis. We made use of the broad excitation and emission spectrum of cellular autofluorescence for background correction of images. In addition to the reference autofluorescence images (UV background) we recorded three fluorescent images (donor, acceptor and energy transfer signal) of donor-acceptor double labeled samples, and corrected for spectral spillage of the directly excited donor and acceptor fluorescence into the energy transfer image. After careful image registration we were able to calculate the energy transfer efficiency on a pixel-by -pixel basis. In this paper, we also present a critical comparison between results obtained with this method and other approaches (photobleaching and flow cytometric energy transfer measurements).

EGF-induced redistribution of erbB2 on breast tumor cells: flow and image cytometric energy transfer measurements.

erbB2, a member of the epidermal growth factor (EGF) receptor-type tyrosine kinase receptor family, is overexpressed in breast carcinomas with poor prognosis. We examined the cell surface association of this receptor with itself and with other cell surface proteins by the Förster-type fluorescence resonance energy transfer using whole antibodies and Fab fragments. We found that erbB2 molecules homoassociate in unstimulated SK-BR-3, BT474 and BT474-M (a metastatic version of the parent BT474 line) breast tumor cells, and that the interaction was enhanced by EGF treatment in suspensions of SK-BR-3 and BT474-M cells. BT474 cells (with low EGF receptor expression) and attached SK-BR-3 cells do not respond to EGF. Image microscopic energy transfer measurements found considerable pixel-by-pixel heterogeneity in the homoassociation state of erbB2. In accordance with the EGF-induced redistribution of erbB2, EGF receptor was found to be in close proximity to erbB2 in FRET measurements. By labeling different epitopes on erbB2 and the lipid bilayer, we were able to prepare an epitope map of erbB2 molecule. Our data suggest the existence of dynamic cell surface patterns of erbB2 and point to functions fulfilled by these molecular complexes.

Efficient, interactive, and three-dimensional segmentation of cell nuclei in thick tissue sections.

Segmentation of intact cell nuclei in three-dimensional (3D) images of thick tissue sections is an important basic capability necessary for many biological research studies. Because automatic algorithms do not correctly segment all nuclei in tissue sections, interactive algorithms may be preferable for some applications. Existing interactive segmentation algorithms require the analyst to draw a border around the nucleus under consideration in all successive two-dimensional (2D) planes of the 3D image. The present paper describes an algorithm with two main advantages over the existing method. First, the analyst draws borders only in 2D planes that cut approximately through the center of the nucleus under consideration so that the nuclear borders generally are most distinct. Second, the analyst draws only five borders around each nucleus, and then the algorithm interpolates the entire surface. The algorithm results in segmented objects that correspond to individual, visually identifiable nuclei. The segmented surfaces, however, may not exactly represent the true nuclear surface. An optional, automatic surface optimization algorithm can be applied to reduce this error.

Structural protein 4.1 in the nucleus of human cells: dynamic rearrangements during cell division.

Structural protein 4.1, first identified as a crucial 80-kD protein in the mature red cell membrane skeleton, is now known to be a diverse family of protein isoforms generated by complex alternative mRNA splicing, variable usage of translation initiation sites, and posttranslational modification. Protein 4.1 epitopes are detected at multiple intracellular sites in nucleated mammalian cells. We report here investigations of protein 4.1 in the nucleus. Reconstructions of optical sections of human diploid fibroblast nuclei using antibodies specific for 80-kD red cell 4.1 and for 4.1 peptides showed 4.1 immunofluorescent signals were intranuclear and distributed throughout the volume of the nucleus. After sequential extractions of cells in situ, 4.1 epitopes were detected in nuclear matrix both by immunofluorescence light microscopy and resinless section immunoelectron microscopy. Western blot analysis of fibroblast nuclear matrix protein fractions, isolated under identical extraction conditions as those for microscopy, revealed several polypeptide bands reactive to multiple 4.1 antibodies against different domains. Epitope-tagged protein 4.1 was detected in fibroblast nuclei after transient transfections using a construct encoding red cell 80-kD 4.1 fused to an epitope tag. Endogenous protein 4.1 epitopes were detected throughout the cell cycle but underwent dynamic spatial rearrangements during cell division. Protein 4.1 was observed in nucleoplasm and centrosomes at interphase, in the mitotic spindle during mitosis, in perichromatin during telophase, as well as in the midbody during cytokinesis. These results suggest that multiple protein 4.1 isoforms may contribute significantly to nuclear architecture and ultimately to nuclear function.

Genotype/phenotype analyses of low frequency tumor cells using computerize image microscopy.

Techniques to identify low frequency (i.e., 10(-4)-10(-5)) tumor cells in bone marrow and peripheral blood of cancer patients provide opportunities for early detection of disseminated disease and characterization of the properties of cells released from the tumor. Low frequency epithelial cells in marrow and blood can be detected using immunophenotypic markers directed against intracellular and/or cell surface antigens. However, nonspecific phenotypic labeling may compromise the ability to discriminate tumor from nontumor cells. We describe optimization of slide-based approaches that facilitate identification and subsequent molecular cytogenetic characterization of rare tumor cell populations in hematopoietic tissues. Colon tumor cells seeded in a hematopoietic background provided a model system to optimize methodologies that are applicable to detection and quantification of micrometastases in clinical specimens. Mixtures of cytogenetically aberrant epithelial cells and hematopoietic cells on slides were labeled with an anti-cytokeratin 20 (anti-CK20) antibody that recognizes > 90% of colon adenocarcinomas. Computerized image analysis was used to record the location of the immunofluorescent cells on microscopic slides. Cells on slides were then hybridized using fluorescence in situ hybridization (FISH) with repeat-sequence DNA probes to detect aneusomies. Previously discriminated epithelial cells were relocated for molecular cytogenetic characterization. Tumor cells present at frequencies approximating 5 x 10(-5) were discriminated on the basis of immunofluorescence and cell size. A low frequency (4 x 10(-4)) population of normal hematopoietic cells also labeled with anti-CK20 (data not shown). This strategy of sequential immunophenotyping and molecular cytogenetic analyses may be useful to discriminate tumor from nontumor cells in cancer patients with micrometastatic disease.

Autofluorescence correction for fluorescence in situ hybridization.

Optimal sensitivity of fluorescence in situ hybridization (FISH) requires bright signals and low background fluorescence. Use of locus-specific probes is especially dependent on high sensitivity. Some tissue preparations show high autofluorescence, masking small or dim signals. We have developed a new method for subtracting autofluorescence from digital images on a pixel-by-pixel basis. It is based on the observation that fluorescent labels for FISH have narrower excitation and emission spectra than the chemical components responsible for autofluorescence. Our new approach uses calculation of the ratio of autofluorescence between multiple color images for correction of autofluorescence in each individual image. By subtracting autofluorescence components, we were able to enhance centromeric signals and make previously indistinguishable cosmid signals clearly visible. This image-processing approach to autofluorescence correction may widen the applicability of gene-specific probes in FISH analysis of tumor material.

Bcl-2 protooncogene expression in cervical carcinoma cell lines containing inactive p53.

Bcl-2 protein expression has been found to block apoptosis and its overexpression has been implicated in lymphoid malignancies where the chromosomal translocation t(14;18) is present. In this study we investigated bcl-2 transcription and protein expression in cultured cervical carcinoma cell lines and keratinocytes. Western blotting and immunofluorescence microscopy demonstrated bcl-2 expression in the cytoplasm of 4 out of 5 cervical carcinoma cell lines examined (HeLa, CaSki, C-33A, and HT-3, but not SiHa). Bcl-2 protein expression was undetectable in normal keratinocytes. None of the cell lines examined demonstrated chromosomal translocation or rearrangement at the major breakpoint-cluster region (MBR) of the bcl-2 gene using either Southern blot or polymerase chain reaction (PCR) analyses. Northern blot analysis demonstrated low levels of bcl-2 transcription in HeLa, CaSki, and C-33A cell lines while reverse transcriptase (RT)-PCR demonstrated bcl-2 transcription in all cervical carcinoma cell lines which had bcl-2 protein expression. Thus, these data suggest that bcl-2 expression occurs in cervical carcinoma cell lines in the absence of chromosomal translocation or rearrangement of the bcl-2 gene. However, each of these cervical carcinoma cell lines contains inactive p53, either due to mutation (C-33A and HT-3) or via complexation and degradation with human papillomavirus (HPV) 16/18 E6 protein (HeLa and CaSki). Thus, functional p53, which can induce apoptosis in certain cells, is not present in these cervical cells which have increased bcl-2 expression. Increased bcl-2 expression under conditions of p53 inactivation may provide cells with a selective advantage for survival and consequently play a role in the development of cervical carcinogenesis.