ALBERT

Notes: Summary In order to evaluate in mathematical terms the morphological changes occurring in the course of cell spreading, Fourier analysis of shape was applied. Human urothelial Hu 961 b cells plated on type IV collagen, fibronectin, laminin, glass and bovine serum albumine (BSA) were studied. Fourier parameters describing cell shape as well as surface areas covered by the cells on the substrate were subjected to statistical analysis. Using analysis of variance and discriminant analysis it was found that parameters describing cell shape (both gross shape of cells and their fine scale contour foldings) possessed a higher power of discrinunation between the cells spread on various substrates than the differences in cell surface areas. In the course of observation (75 and 150 min) the highest number of attached cells and highest degree of spreading were found when cells were plated on type IV collagen. Moderate alterations in cell shape and moderate increase of surface area were seen in the group of cells seeded on fibronectin, whereas the cells plated on laminin, glass and BSA revealed a moderate increase of surface area, but no changes in their shape were observed. The differences in attachment of cells and in the degree of their spreading might be due to the variation in expression of plasma membrane receptors for various substrates. The Fourier analysis of cell shape coupled with measurement of surface area is a good tool for quantitative evaluation of cell spreading and can be used for discrimination between cells spread on different substrates.

Notes: Summary The electron spin resonance (ESR) technique is proposed as a microchemical and/or histochemical method in research on mineralized tissues. It has been described in previous papers that ionizing radiation evokes stable paramagnetic centres in the crystalline fraction of mineral constituents of calcified tissues. These centres were used as a label in studies on resorption and creeping substitution of bone grafts. In this paper the sensitivity of the method and its application for determination of the crystallinity of various mammalian tissues were described. It was shown that: a) three single Haversian systems (osteons) isolated from a ca. 100 μm thick undecalcified section of human compact bone weighing around 10−4 g could be measured by the ESR technique; b) the crystallinity of mineral constituents of normal and pathological calcified tissues could be estimated as the ratio of the concentration of stable paramagnetic centres to the total ash content. Comparative ESR measurements were performed on compact bone of various mammalian species, human enamel and dentin, as well as on ageing bovine cartilage and atherosclerotic human aortas.

Notes: Summary Arterial smooth muscle cells in contractile and synthetic state were analyzed by optical diffractometry. Cell sections (80–90 nm) were photographed in an electron microscope and diffraction patterns of the plates (negatives) were produced using a helium-neon laser. Radial and angular distributions of light intensity in the diffractograms were measured and digitized using an electronic detector plate consisting of ring- and wedge-shaped photosensitive elements; radial distributions provide information about size of structures and distances between them and angular distributions about spatial orientation of structures in the images. Micrographs of nuclei and cytoplasm were analyzed separately (40–50 plates in each group). Computerized statistical analysis of radial distributions of light intensity showed that the nuclear chromatin pattern differed between cells in contractile and synthetic state. The probability that the observed difference could have arisen purely by chance was less than 10−5. Computer-aided classification to the a priori known cell group was correct in 96.5% of the cases. Analysis of radial distributions of light intensity similarly showed marked differences in cytoplasmic structure between cells in contractile state (dominated by bundles of myofilaments) and synthetic state (dominated by cisternae of rough endoplasmic reticulum). The probability that the observed difference could have arisen purely by chance was less than 10−5. Computer-aided classification to the a priori known cell group was correct in 92.0% of the cases. In contrast, analysis of angular distributions of light intensity did not indicate any statistically significant differences between contractile and synthetic state cells. A likely reason is that both myofilaments and cisternae of rough endoplasmic reticulum were arranged in parallel. The results demonstrate that optical diffractometry is a useful method for image analysis in studies of cell fine structure. It provides information about size and orientation of structures with poorly defined shape and is particularly well suited for studies on cell differentiation and effects of pharmacological and other experimental treatments on cell fine structure. It represents an alternative and a complement to stereology for quantitative and objective evaluation of morphological data.

Notes: Summary The effect of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) and dichloromethylidene-bisphosphonate (Cl2MBP) on the structure of the organic matrix of heterotopically induced bone in guinea pig was studied. Heterotopic bone formation was induced by transplantation of allogenic urinary bladder epithelium. Starting from the day of transplantation the animals were treated subcutaneously with HEBP and Cl2MBP with a dose of 12.5 mg P/kg/day during 35 days. The control group was injected with 0.9% NaCl solution. The advantage of heterotopic bone induction as an experimental model is the fact that the applied drugs act on de novo bone formation. Collagen fibers were treated as markers of bone because their size and spatial arrangement reflect the structure and maturity of organic matrix of this tissue. Decalcified histological sections of induced bone, taken 35 days after implantation of inductor, were stained by the picrosirius method. This staining enhances the natural birefringency of collagen fibers and allows for better and specific visualization of collagen fibers bundles under polarizing microscope. In this way the amount of information in the analysed image is increased. Thirty five microphotographs were analysed from each of the investigated groups with the use of optical diffractometry. The radial distribution of light intensity in diffraction patterns was analysed what allowed to evaluate spatial frequencies connected with the width of collagen bundles in induced bone tissue. Since the spatial arrangement of collagen fibers in newly formed bone is random, analysis of angular distribution of light intensity in diffractograms was not performed. Using discriminant analysis the significant differences between all three studied groups of animals were found. The widest differences were found between the Cl2MBP and HEBP treated animals. They were larger than those between each of these two groups and the control one. In control as well as in HEBP treated animals thicker bundles of collagen fibers were more frequently observed than in the Cl2MBP treated group, while in the latter thin bundles, nondetected in the former two groups were found. Generally, the radial distribution of light intensity in diffraction patterns of the HEBP treated animals resembles more that in the control group than in the Cl2MBP treated one. The different effects of the two analysed bisphosphonates (BPs) on the organic bone matrix of heterotopically induced bone is interpreted as differences in their influence on osteogenic cells and/or as differences in their direct influence on extracellular collagen fiber bundles formation.

Notes: Summary The rationale of the present investigation is the observations made by many authors of changes in the molecular structure of the cell surface during the multistep process of malignant transformation. These changes may influence cell-matrix and cell-cell interactions and thereby cause changes in cell adhesiveness and cell shape. The aim of the present work was to investigate whether the development of various grades of transformation in vivo and in vitro of human urothelial cells is accompanied by significant changes in cell shape as measured by Fourier analysis. The following transformation grades (TGr) have been defined (Christensen et al. 1984; Kieler 1984): TGr I=nonmalignant, mortal cell lines that grow independently of fibroblasts and have a prolonged life span. TGr II=nonmalignant cell lines with an infinite life span. TGr III=malignant and immortal cell lines that grow invasively in co-cultures with embryonic chick heart fragments and possess tumorigenic properties after s.c. injection into nude mice. Comparisons of 4 pairs of cell lines were performed; each pair was of the same origin. Two pairs-each including a TGr I cell line (Hu 961b and Hu 1703S) compared to a TGr III cell line (Hu 961a or Hu 1703He)-were derived from two transitional cell carcinomas (TCC) containing a heterogeneous cell population. Two additional cell lines classified as TGr II (HCV-29 and Hu 609) were compared to two TGr III sublines (HCV-29T and Hu 609T, respectively) which arose by “spontaneous” transformation during propagation in vitro of the respective maternal TGr II-cell lines. One of these TGr II cell lines (HCV-29) originated from the histologically normal bladder mucosa obtained from a patient with a previous history of bladder papillomata treated with irradiation (Fogh, personal communication). The other TGr II cell line (Hu 609) was derived from the normal ureter of a patient with renal carcinoma. In each of these 8 cell lines, the shape of 100 cells chosen at random were subjected to Fourier analysis of shape. Each of the particular harmonic amplitude values studied was used as an individual parameter for the evaluation of differences between compared cell lines, using Chi-Square test and discriminant analysis. It was found that in two of four analysed pairs of cell lines, i.e. Hu 1703S (TGr I) vs Hu 1703He (TGr III) and HCV-29 (TGr II) vs HCV-29T (TGr III), the differences in cell shape between the two populations were very well pronounced, as was shown by several statistical parameters. In the two other pairs of cell lines, i.e. Hu 961b (TGr I) vs Hu 961a (TGr III) and Hu 609 (TGr II) vs Hu 609T (TGr III) significant differences in cell shape were also found, but they were less pronounced. The conclusion is, that differences in cell shape in vitro may reveal the cellular heterogeneity of the original transitional cell carcinoma and/or the progression of in vitro propagated urothelial cells from one grade of transformation into another.

Notes: Summary The aim of this paper is to show the possibility of objective mathematical description of changes occurring in the shape of cells in the process of transformation. The evaluation of the changes in cell shape of the chosen cell lines differing in transformation grade was performed by the use of Fourier analysis of the shape. Any two-dimensional contour can be described with specific accuracy in a mathematical manner using the closed form Fourier series of cosines. The components forming the analysed shape, called harmonics, are independent and uncorrelated measures of their contribution to the total shape. The shape of each cell can be represented by the spectrum of harmonic amplitudes. To quote the paper by Healy-Williams and Williams (1981): “The observed shape is partitioned into series, where gross shape, as elongation or triangularity, is measured by the harmonic amplitudes of the lower harmonic order and increasingly fine scaled surface sculpture is measured at higher orders”. The statistically evaluated results allow the objective comparison of the cell shapes of several compared cell lines differing in transformation grades. Malignant transformation is supposed to be a multistep process. The different grades of transformation could be defined by several parameters as changes in the morphology of the cells, their ability to compete with fibroblasts, their life span, their angiogenic potency, their invasiveness in vitro and their tumorigenicity in nude mice. In this paper several human urothelial cell lines of normal and tumor origin differing in their transformation grade (TGr I–III) were compared by the use of Fourier analysis of their shape. TGr I cultures have finite life span but do not need intermittent collagenase treatment to prevent fibroblast overgrowth. TGr II cultures acquire infinite growth potential, here defined as capacity to survive at least 70 passages. They are neither tumorigenic nor invasive. TGr III cultures show infinite growth transformation, increased angiogenicity and ability to invade normal host tissue in vitro. They produce progressively growing tumors in nude mice. The following human uroepithelial cell lines differing in the degree of transformation were studied and compared by statistical evaluation of the harmonic amplitudes deseribing mathematically the cell shape: Two cell lines derived from human transitional cell carcinoma (TCC): 1. Hu 1703S classified as TGr I, 2. Hu 1703He classified as TGr III. It was found that these two cell lines differ in all harmonics. Two cell lines derived from morphologically normal human bladder epithelium: 3. HCV-29 classified as TGr II. The confluent (HCV-29 confl) and peripherial (HCV-29 periph) parts of the HCV-29 cultures were studied separately. 4. HCV-29T “spontaneously” transformed in culture, subline of HCV-29 classified as TGr III. 5. Normal human fibroblast line was used for comparison and control. — It was shown that the confluent part of the epithelial HCV-29 line (HCV-29 confl) which is not tumorigenic was identical in shape to the cells of HCV-29T tumorigenic cultures. The parameters describing the shape of cells in the periphery of HCV-29 cultures (HCV-29 periph) were nearly identical to those of human fibroblasts. Both HCV-29 lines are statistically different from the Hu 1703He line. The applied technique allows for the numerical description of cell shape — the one of many changeable parameters in the process of transformation. These numerical data could be analysed by statistical methods, which allow to define the similarities or differences between the compared cell lines in a quantitative way.

Notes: Abstract To assess the involvement of T-lymphocytes in ozone-induced lung damage, CD-1 mice were exposed to air or 0.7 ppm ozone (1.37 mg O3/m3 air) in the presence and absence of the immunosuppressive drug cyclosporine A (CSA). Mice were thus divided into four treatment groups for both the 4 and 14 day exposure times: 1) AIR + VEH, 2) AIR + CSA, 3) O3 + VEH, and 4) O3 + CSA. Thy-1.2 positive cells (T-lymphocytes) per pulmonary lesion were determined and quantitative histomorphometric analysis of lesion volume was performed. By Day 14, the number of T-lymphocytes per lesion in O3 + VEH (vehicle) animals had increased to approximately 3.5 times that seen at Day 4. At 4 and 14 days of O3 + CSA treatment, the number of T cells per lesion was half that seen in O3 + VEH mice. At Day 4, lesion size and appearance were comparable in O3 + VEH and O3 + CSA animals, while at Day 14, O3 + CSA treatment resulted in larger and more cellular lesions that contained a greater proportion of polymorphonuclear cells, and the lesions extended further into the lung periphery. Inflammatory cells were observed in areas of epithelial cell proliferation and in alveolar spaces distal to the small airway terminus. After 14 days, lesion volume was approximately twice as great following O3 + CSA administration than with O3 treatment alone. These results are consistent with effects seen in another model of immunosuppression, the nude mouse, and they implicate a regulatory role for T-lymphocytes in the response to ozone.

Notes: Abstract Layers of conductive oxide powders (IrO2, RuO2 and Bi2Ru2O7) were prepared on alumina substrates by a thick-film technique. The films were fired at 875° C for different dwell times. The temperature dependence of resistance was measured in the range −196 to 850° C. SEM observations of the initial powders and the resistive layer surface after firing, as well as X-ray diffraction investigations, were carried out. The RuO2 and IrO2 oxides exhibit a high sintering ratio and the resistance of the fired layers increases with temperature as in the single crystal. The R(T) curve for Bi2Ru2O7 layers is in qualitative agreement with the polycrystalline material. The role of the grain surface area and the regions between the grains is dominant.

Notes: Abstract Inhomogeneous two-phase conducting films whose structure is intermediate between three-and two-dimensional configurations are discussed. The longitudinal film size exceeds the correlation length, and its thickness is less than the correlation length. In the case of weak nonlinearity, we found dependences of the film resistivity on the concentration and size of conducting particles, as well as on the film thickness, in the framework of a percolation approach.