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Biomedical subjects

M Hausmann

Publications and source records attributed to M Hausmann.

At least 19 recordsLinked to original sources
PubMed2003

Labelling quality and chromosome morphology after low temperature FISH analysed by scanning far-field and near-field optical microscopy.

A non-enzymatic, low temperature fluorescence in situ hybridization (LTFISH) procedure was applied to metaphase spreads and interphase cell nuclei. In this context 'low temperature' means that the denaturation procedure of the chromosomal target DNA usually applied by heat treatment and chaotropic agents such as formamide was completely omitted so that the complete hybridization reaction took place at 37 degrees C. For LTFISH, the DNA probe had to be single-stranded, which was achieved by means of separate thermal denaturation of the DNA probe only. The DNA probe pUC1.77 was used for all LTFISH experiments. The labelling quality (number of binding sites, relative background intensity, relative intensity of major and minor binding sites) was analysed by confocal laser scanning microscopy (CLSM). An optimum in specificity and signal quality was obtained for 15 h hybridization time. For this hybridization condition of LTFISH, the chromosomal morphology was analysed by scanning near-field optical microscopy (SNOM). The results were compared with the morphology of chromosomes after (a) labelling of all centromeres using the same chemical treatment in the FISH procedure but with the application of target denaturation, and (b) labelling of all centromeres using a standard FISH protocol including thermal denaturation of the DNA probe and the chromosomal target. Depending on the FISH-procedure applied, SNOM images show substantial differences in the chromosome morphology. After LTFISH the chromosome morphology appeared to be much better preserved than after standard FISH. In contrast, the application of the LTFISH chemical treatment accompanied by heat denaturation had a very destructive influence on chromosomal morphology. The results indicate that, at least for certain DNA probes, specific chromosome labelling can be obtained without the usually applied heat and chemical denaturation of the DNA target, resulting in an apparently well preserved chromatin morphology as visualized by SNOM. LTFISH may be therefore a useful labelling technique whenever the chromosomal morphology had to be preserved after specific labelling of DNA regions. Binding mechanisms of single-stranded DNA probes to double-stranded DNA targets are discussed.

Cell Nucleus↗
PubMed2003

Precise 3D image alignment in micro-axial tomography.

Micro (micro-) axial tomography is a challenging technique in microscopy which improves quantitative imaging especially in cytogenetic applications by means of defined sample rotation under the microscope objective. The advantage of micro-axial tomography is an effective improvement of the precision of distance measurements between point-like objects. Under certain circumstances, the effective (3D) resolution can be improved by optimized acquisition depending on subsequent, multi-perspective image recording of the same objects followed by reconstruction methods. This requires, however, a very precise alignment of the tilted views. We present a novel feature-based image alignment method with a precision better than the full width at half maximum of the point spread function. The features are the positions (centres of gravity) of all fluorescent objects observed in the images (e.g. cell nuclei, fluorescent signals inside cell nuclei, fluorescent beads, etc.). Thus, real alignment precision depends on the localization precision of these objects. The method automatically determines the corresponding objects in subsequently tilted perspectives using a weighted bipartite graph. The optimum transformation function is computed in a least squares manner based on the coordinates of the centres of gravity of the matched objects. The theoretically feasible precision of the method was calculated using computer-generated data and confirmed by tests on real image series obtained from data sets of 200 nm fluorescent nano-particles. The advantages of the proposed algorithm are its speed and accuracy, which means that if enough objects are included, the real alignment precision is better than the axial localization precision of a single object. The alignment precision can be assessed directly from the algorithm's output. Thus, the method can be applied not only for image alignment and object matching in tilted view series in order to reconstruct (3D) images, but also to validate the experimental performance (e.g. mechanical precision of the tilting). In practice, the key application of the method is an improvement of the effective spatial (3D) resolution, because the well-known spatial anisotropy in light microscopy can be overcome. This allows more precise distance measurements between point-like objects.

Image Processing, Computer-Assisted↗
PubMed2002

Automated microaxial tomography of cell nuclei after specific labelling by fluorescence in situ hybridisation.

Microaxial tomography provides a good means for microscopic image acquisition of cells or sub-cellular components like cell nuclei with an improved resolution, because shortcomings of spatial resolution anisotropy in optical microscopy can be overcome. Thus, spatial information of the object can be obtained without the necessity of confocal imaging. Since the very early developments of microaxial tomography, a considerable drawback of this method was a complicated image acquisition and processing procedure that requires much operator time. In order to solve this problem the Heidelberg 2pi-tilting device has been mounted on the Brno high-resolution cytometer as an attempt to bring together advanced microscopy and fast automated computer image acquisition and analysis. A special software module that drives all hardware components required for automated microaxial tomography and performs image acquisition and processing has been developed. First, a general image acquisition strategy is presented. Then the procedure for automation of axial tomography and the developed software module are described. The rotation precision has been experimentally proved followed by experiments with a specific biological example. For this application, also a method for the preparation of cell nuclei attached to glass fibres has been developed that allows for the first time imaging of three-dimensionally conserved, fluorescence in situ hybridisation-stained cell nuclei fixed to a glass fibre.

Cell Nucleus↗
PubMed2002

Toll-like receptors 2 and 4 are up-regulated during intestinal inflammation.

BACKGROUND & AIMS: Bacterial wall products play an important role in the activation of immune and nonimmune cells of the intestinal mucosa. Toll-like receptors (TLRs) TLR2 and TLR4 have been identified as signaling receptors activated by bacterial wall components. METHODS: Expression of TLRs in human intestinal mucosa obtained by endoscopy and surgery was analyzed by immunohistochemistry. Intestinal macrophages were isolated by immunomagnetic beads armed with a CD33 antibody. Reverse-transcription polymerase chain reaction was performed for TLR1-5. Results were confirmed by Northern blot and flow cytometry. Interleukin-1beta messenger RNA (mRNA) was quantified by a polymerase chain reaction-enzyme-linked immunosorbent assay-kit. RESULTS: Immunohistochemistry revealed a significant increase in the TLR2 and TLR4 antigen expression on submucosal cells in inflamed intestinal mucosa compared with non-inflamed mucosa. TLR expression was localized in intestinal macrophages by double-labeling techniques. No TLR-polymerase chain reaction product could be obtained with mRNA from CD33-positive macrophages from normal mucosa. We observed an induction of mRNA for TLR2, TLR4, and TLR5 in inflammation-associated macrophages. TLR1 and TLR3 were only detectable in blood monocytes. Monocytes reacted to lipopolysaccharide stimulation with a 3-fold and in vitro differentiated macrophages with a 16-fold increase of cellular interleukin-1beta mRNA. Macrophages from normal mucosa did not respond to lipopolysaccharide showing the functional relevance of TLR expression. CONCLUSIONS: This study shows the inflammation-dependent induction of TLR2 and TLR4 expression in intestinal macrophages. The absence of TLRs abolishes the reactivity of mucosal macrophages to bacterial wall products. Presence of TLRs may thereby contribute to the inflammatory process.

Blood Cells↗
PubMed2002

Variations in cell surfaces of estrogen treated breast cancer cells detected by a combined instrument for far-field and near-field microscopy.

The response of single breast cancer cells (cell line T-47D) to 17beta-estradiol (E(2)) under different concentrations was studied by using an instrument that allows to combine far-field light microscopy with high resolution scanning near-field (AFM/SNOM) microscopy on the same cell. Different concentrations of E(2) induce clearly different effects as well on cellular shape (in classical bright-field imaging) as on surface topography (atomic force imaging) and absorbance (near-field light transmission imaging). The differences range from a polygonal shape at zero via a roughly spherical shape at physiological up to a spindle-like shape at un-physiologically high concentrations. The surface topography of untreated control cells was found to be regular and smooth with small overall height modulations. At physiological E(2) concentrations the surfaces became increasingly jagged as detected by an increase in membrane height. After application of the un-physiological high E(2) concentration the cell surface structures appeared to be smoother again with an irregular fine structure. The general behaviour of dose dependent differences was also found in the near-field light transmission images. In order to quantify the treatment effects, line scans through the normalised topography images were drawn and a rate of co-localisation between high topography and high transmission areas was calculated. The cell biological aspects of these observations are, so far, not studied in detail but measurements on single cells offer new perspectives to be empirically used in diagnosis and therapy control of breast cancers.

Breast Neoplasms↗
PubMed2001

Subtractive screening reveals up-regulation of NADPH oxidase expression in Crohn's disease intestinal macrophages.

Macrophages play a central role during the pathogenesis of inflammation. In normal intestinal mucosa surface expression of typical macrophage markers such as CD14, CD16, CD11b or T-cell co-stimulatory molecules such as CD80 or CD86 is low indicating anergy and low pro-inflammatory activity of these cells. During inflammatory bowel disease (IBD) the mucosa is invaded by a population of macrophages displaying these markers, secreting higher cytokine levels and representing an activated cell population. CD33(+) cells (macrophages) were isolated from normal and Crohn's disease mucosa and mRNA was isolated by polyT magnetic beads. A subtractive screening was performed subtracting mRNA from normal macrophages from those of Crohn's disease macrophages. Oxidative burst activity was determined by flow cytometry. Seventy clones were obtained by the subtractive mRNA screening. Sequencing showed > 99% homology to mRNA of monocyte chemoattractant protein-1 (MCP-1) for three clones. Five clones obtained by subtraction revealed > 99% homology to mRNA of cytochrome b (subunit gp91). Differential expression of the cytochrome b subunit gp91 and the cytosolic NADPH oxidase subunit p67 was confirmed by RT-PCR and 'virtual' Northern blots. The fluorescence ratio of stimulated versus unstimulated cells was 0.9 +/- 0.16 in control macrophages indicating a lack of oxidative burst activity. In Crohn's disease this ratio was significantly increased to 1.80 +/- 0.8 (P = 0.004) confirming the molecular data. In conclusion NADPH oxidase mRNA is down-regulated or absent in macrophages from normal mucosa correlating with a lack of oxidative burst activity. In IBD macrophage-oxidative burst activity is increased and NADPH oxidase mRNA induced. Inhibition of NADPH oxidase could be a new therapeutical target in IBD and reduce mucosal tissue damage in active IBD.

Chemokine CCL2↗
PubMed2001

Monocyte differentiation in intestine-like macrophage phenotype induced by epithelial cells.

Macrophages in normal colonic mucosa show a specific and distinct phenotype with low expression of the typical monocyte/macrophage surface antigens CD14, CD16, and CD11b and T-cell costimulatory molecules. A method for the in vitro induction of a macrophage phenotype similar to this intestinal phenotype is presented. Multicellular spheroids (MCSs) of intestinal epithelial cell (IEC) and control cell lines were cocultured with elutriated monocytes. Surface antigen expression was analyzed by immunohistochemistry and flow cytometry. Interleukin (IL)-1beta mRNA was measured by quantitative PCR. Monocytes adhered and infiltrated the MCSs within 24 h. In the MCSs of all IEC lines, the typical monocyte/macrophage surface antigens CD14, CD16, CD11b, and CD11c, which are detectable after 24 h of coculture by immunohistochemistry and flow cytometry, were down-regulated after 7 days (e.g., for CD14 at 24 h, expression was 86% of CD33+ cells; at day 7, it was 11%). A clear decrease of lipopolysaccharide (LPS)-stimulated IL-1beta transcription in monocytes cocultured with IEC MCSs could be observed during the 7-day period. For the first time an intestine-like macrophage-phenotype could be induced in vitro. Interactions with IECs play an essential role during this differentiation, which is of functional relevance, e.g., for LPS-induced cytokine secretion.

Antigens, CD↗
PubMed2000

Drastic enhancement of energy-loss straggling of relativistic heavy ions due to charge-state fluctuations

Stopping power and energy-loss straggling of 197Au, 208Pb, and 209Bi projectiles have been measured in different solids (4</=Z2</=82) in the energy range (100-1000) MeV/u. The experimental results clearly demonstrate the influence of the different charge states of the ions. Because of charge-state fluctuations the energy-loss straggling is up to 7 times larger than the pure collisional straggling. The selected energy domain in combination with the heavy projectiles allows for the first time an unambiguous interpretation of the long-standing problem of charge-changing collisions in energy-loss straggling.

Journal Article↗
PubMed2000

Signature inversion caused by triaxiality and unpaired band crossings in 72Br

High-spin states in 72Br were studied with the EUROBALL III spectrometer using the 40Ca(40Ca,alpha3p1n) reaction. The negative-parity band observed in this experiment displays a signature inversion around spin I = 16. The interpretation within the cranked Nilsson-Strutinsky approach shows that this signature pattern is a signal of a substantial triaxial shape change with increasing spin where the nucleus evolves from a triaxial shape with rotation about the intermediate axis at low spin through a collective prolate shape to a triaxial shape but with rotation about the shortest principal axis at high spin.

Journal Article↗
PubMed2000

Quantitative microscopy after fluorescence in situ hybridization - a comparison between repeat-depleted and non-depleted DNA probes.

Complex probes used in fluorescence in situ hybridization (FISH) usually contain repetitive DNA sequences. For chromosome painting, in situ suppression of these repetitive DNA sequences has been well established. Standard painting protocols require large amounts of an unlabeled 'blocking agent', for instance Cot-1 DNA. Recently, it has become possible to remove repetitive DNA sequences from library probes by means of magnetic purification and affinity PCR. Such a 'repeat depleted library probe' was hybridized to the q-arm of chromosome 15 of human metaphase spreads and interphase cell nuclei without any preannealing by Cot-1 DNA. Apart from this, 'standard' FISH conditions were used. After in situ hybridization, microscope images were obtained comparable to those achieved with the #15q library probe prior to depletion. The images were recorded by a true color CCD camera. By digital image analysis using 'line scan' and 'area scan' procedures, the painting efficiency expressed in terms of relative fluorescence signal intensity was quantitatively evaluated. The painting efficiency using the repeat depleted probe of chromosome 15q was compared to the painting efficiency after standard FISH. The results indicate that both types of probes are compatible to a high FISH efficiency. Using equivalent probe concentrations, no significant differences were found for FISH with standard painting probes and repeat depleted painting probes.

Cell Nucleus↗
PubMed2000

Steroid fluctuations modify functional cerebral asymmetries: the hypothesis of progesterone-mediated interhemispheric decoupling.

This study examines the modulation of functional cerebral asymmetries by gonadal hormones in three distinct groups. Young, normally cycling women performed a prototypical left (lexical decision) and two prototypical right-hemispheric tasks (figural comparison and face discrimination) during the low steroid menses and the high steroid midluteal phase. Saliva progesterone levels were measured with radioimmunoassay (RIA). Parallel to younger females, young men, and postmenopausal women were tested at matching time intervals. Results revealed significant interactions between cycle phase and visual half-field in the accuracy of all three tasks for the younger women; stronger lateralization patterns occurring during menses, while a more bilateral or at least less asymmetric cerebral organization predominated the midluteal phase, when highest levels of progesterone appear. Progesterone seemed to have a significant influence on lateralization in the figural comparison task, with high hormone levels enhancing the performance of the left hemisphere (for this task subdominant), thereby decreasing asymmetry. After menopause, when the levels of gonadal hormones are lower and more stable, the lateralization patterns for all three tasks were similar to those of men and normally cycling women during menses. These results make it likely that steroids and especially progesterone are able to reduce cerebral asymmetries. We hypothesize that progesterone attenuates the effect of glutamate on non-NMDA receptors. This could diminish cortico-cortical transmission which is mostly dependent on a glutamate-induced initial EPSP in pyramidal neurons which receive transcallosal input. The reduction in callosal transfer could then suppress the functional asymmetries.

Adult↗
PubMed2000

Sex hormones affect spatial abilities during the menstrual cycle.

The aim of this study was (a) to show that different measures of spatial cognition are modulated by the menstrual cycle and (b) to analyze which steroid is responsible for these cognitive alterations. The authors collected blood samples in 3-day intervals over 6 weeks from 12 young women with a regular menstrual cycle to analyze concentrations of estradiol, progesterone, testosterone, luteinizing hormone, and follicle-stimulating hormone. The performance on 3 spatial tests was measured during the menstrual and the midluteal phases. A significant cycle difference in spatial ability as tested by the Mental Rotation Test was found, with high scores during the menstrual phase and low scores during the midluteal phase. Testosterone had a strong and positive influence on mental rotation performance, whereas estradiol had a negative one. These results clearly indicate that testosterone and estradiol are able to modulate spatial cognition during the menstrual cycle.

Adult↗
PubMed2000

Three-dimensional spectral precision distance microscopy of chromatin nanostructures after triple-colour DNA labelling: a study of the BCR region on chromosome 22 and the Philadelphia chromosome.

Topological analysis of the three-dimensional (3D) chromatin nanostructure and its function in intact cell nuclei implies the use of high resolution far field light microscopy, e.g. confocal laser scanning microscopy (CLSM). However, experimental evidence indicates that, in practice, under biologically relevant conditions, the spatial resolution of CLSM is limited to about 300 nm in the lateral direction and about 700 nm in the axial direction. To overcome this shortcoming, the use of a recently developed light microscopical approach, spectral precision distance microscopy (SPDM) is established. This approach is based on the precise localization of small labelling sites of a given target in spectrally differential images. By means of quantitative image analysis, the bary centres (intensity weighted centroid analogous to the centre of mass) of these independently registered labelling sites can be used as point markers for distance and angle measurements after appropriate calibration of optical aberrations (here, polychromatic shifts). In combination with specific labelling of very small chromatin target sites with dyes of different spectral signatures by fluorescence in situ hybridization (FISH), SPDM presently allows us to analyse the nuclear topology in three-dimensionally conserved nuclei with a 'resolution equivalent', many times smaller than the conventional optical resolution. Chronic myelogeneous leukaemia (CML) is genetically characterized by the fusion of parts of the BCR and ABL genes on chromosomes 22 and 9, respectively. In most cases, the fusion leads to a translocation t(9; 22) producing the Philadelphia chromosome. SPDM was applied to analyse the 3D chromatin structure of the BCR region on the intact chromosome 22 and the BCR-ABL fusion gene on the Philadelphia chromosome (Ph) by using a new triple-colour FISH protocol: two different DNA probes were used to detect the BCR region and the third DNA probe was used to identify the location of the ABL gene. Consistent 3D distance measurements down to values considerably smaller than 100 nm were performed. The angle distributions between the three labelled sites on the Philadelphia chromosome territory were compared to two state-of-the-art computer models of nuclear chromatin structure. Significant differences between measured and simulated angle distributions were obtained, indicating a complex and non-random angle distribution.

Bone Marrow Cells↗
PubMed1999

Comparison of the thermal denaturation behaviour of DNA-solutions and metaphase chromosome preparations in suspension.

Hyperchromicity measurements are well established to analyse the thermal denaturation behaviour of pure DNA sequences in solution. Here, we show that under appropriate experimental conditions this technique can also be applied to study thermally controlled conformation changes of higher order DNA-protein complexes as for instance metaphase chromosome preparations in suspension. A computer controlled sensitive, upright double beam photometer with a heatable cuvette was constructed. Measurements of the temperature dependent extinction of both, solutions and particle suspensions are possible, since sedimentation effects of particles can be neglected due to the vertical optical axis in the probe cuvette. Thermal denaturation of metaphase chromosome preparations of human and Chinese hamster cells was investigated and compared to melting profiles of DNA solutions for two excitation wavelengths, 256 and 313 nm. The influence of neutral and low pH was considered. The results indicate that metaphase chromosome preparations show a thermal denaturation behaviour different from pure DNA. Whereas DNA solutions showed one pH dependent melting peak at 256 nm only, the peak pattern of metaphase chromosome preparations showed a large variability both at 256 and 313 nm. At neutral pH, in two temperature regions (40-55 degrees C and 75-82 degrees C) peaks were found indicating chromosome typical conformation changes independently from the mammalian cell species (Chinese hamster, human). In contrast to pure DNA, no typical reduction in the temperatures of peak maxima with decreasing pH was found for metaphase chromosome preparations of both cell types. These results may be relevant for further systematic studies of efficient thermal probe/target denaturation procedures in non enzymatic DNA-chromosome in situ hybridisation.

Algorithms↗
PubMed1999

Sex differences in functional cerebral asymmetries in a repeated measures design.

The aim of the present study was to analyze whether task repetitions which are an inevitable part of repeated measures designs might induce performance alterations specific for gender and hemisphere. Male and female subjects conducted twice a lexical decision, a polygon recognition, and a face discrimination task as a visual half field paradigm with the two experimental sessions repeated by 2 weeks. The results show that only in female subjects can a session effect for the lexical decision and the polygon recognition task be demonstrated which is hemisphere specific. Thus, repeated measures designs seem to have a gender- and hemisphere-specific effects of their own which could confound with other variables under study.

Adult↗
PubMed1999

Laterality effects in the processing of syllable structure.

Recent phonological research has shown that the syllable plays a major role in the phonology of German. The present study investigates laterality effects in the processing of syllable structure by means of dichotic presentation of German word pairs that differ in number of syllables, but that differ minimally in the phonemes they comprise (e.g., BREIT and BEREIT). Results showed a sex difference in laterality for the processing of the experimental stimuli, with a greater right-hemispheric lateralization in men and a more bilateral organization in women.

Adult↗
PubMed1999

Hemispheric dominance and gender in the perception of an illusion.

Perception of geometric illusions is a visuo-spatial process. As such processes often have been found to be predominantly the domain of the right hemisphere, this hemisphere may be expected to perceive such illusions more readilly than the left hemisphere. Using the herringbone illusion in a reaction-test paradigm, we found that in right-handed males the right hemisphere was significantly more often deceived than the left, whereas no significant hemispheric difference was observed in females. This is the first demonstration of gender differences in the lateralized perception of an illusion.

Adult↗