Vol. 39, No. 3, 2001
J. Niemiec: Ki-67 labelling index in human brain tumours
ABSTRACTS
Biological significance of chemokine receptor expression by normal human megakaryoblasts
Marcin Majka1, Janina Ratajczak1, Monika Baj-Krzyworzeka1,2, Jacek Kijowski1, Ryan Reca1, Bogdan Machalinski3, Krystyna Kubiczek2 and Mariusz Z. Ratajczak1,2
1Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, USA, 2Department of Transplantology, Polish-American Childrens Hospital, Jagiellonian University Medical College, Cracow, Poland, and 3Department of Pathology, Pomeranian Medical Academy, Szczecin, Poland
Abstract: The aim of this study was to learn more on the role of chemokines in the regulation of human megakryopoiesis. Normal human megakaryoblasts were expanded in serum-free liquid cultures and subsequently (1) phenotyped for expression of various chemokine receptors, (2) evaluated if chemokine receptors which they express are functional after stimulation by chemokines (calcium flux assay, chemotaxis, phosphorylation of MAPK-p42/44 and AKT proteins), and (3) investigated for expression and secretion of selected chemokines by employing RT-PCR and ELISA assays, respectively. In addition we also phenotyped peripheral blood platelets for expression of chemokine receptors and chemokines. We found that while human megakaryoblasts express several chemokine receptors (CXCR4, CCR6, CCR8, CCR5, CCR2 and CXCR3), CXCR4 was the only receptor detectable by FACS on human platelets. Moreover, among various chemokines tested, only SDF-1 (CXCR4 ligand) stimulated calcium flux and chemotaxis in normal human megakaryoblasts and phosphorylated MAPK-p42/44 and AKT in these cells. Although mRNAs for several chemokines were detectable by RT-PCR in normal human megakaryoblasts, only RANTES, IL-8, MCP-1 and PF-4 were found to be secreted by these cells. Finally we noticed that no chemokine tested in this study affected CFU-Meg colony formation by human CD34+ cells in serum-free cultures. We conclude that from all the chemokine receptor-chemokine axes tested, only SDF-1-CXCR4 axis was functional in assays employed in our studies, which further support the view that this axis plays a privileged role in regulating normal human megakaryopoiesis.
Key words: Megakaryopoiesis - Chemokines - Chemokine receptors - MAPKp42/44 - AKT
Author's email: mariusz@mail.med.upenn.edu
Evaluation of mouse preimplantation embryos cultured in media enriched with insulin-like growth factors I and II, epidermal growth factor and tumor necrosis factor alpha
Rafał Kurzawa, Wojciech Głąbowski and Lidia Wenda-Różewicka
Department of Histology and Embryology, Pomeranian Academy of Medicine, Szczecin, Poland
Abstract: Culture of preimplantation embryos is complex and requires strictly defined culture media to sustain their viability and quality. In the current study, an effort was made to evaluate comprehensively the quality of mice embryos, grown in media enriched with IGF I, IGF II, EGF and TNFa. For that purpose, critically chosen and thoroughly described, complex morphological methods based on contrast-phase, fluorescent and confocal microscopy were used. The study evaluated blastulation and hatching rates, total blastocyst cells, inner cell mass cell numbers (differential staining) as well as identified embryo cells with positive reactions for necrosis or apoptosis (TUNEL). The critical evaluation of the effects of the studied cytokines allowed for simultaneous, meticulous assessment of the applied study methods. Significantly more blastocysts were found in culture media enriched with IGF-I, IGF II and EGF. Significantly more hatched blastocysts were found in media with IGF-I and IGF II. Additionally, IGF I and II increased inner cell mass and total blastocyst cell numbers. Very few cells with necrosis and apoptosis were found in the culture media enriched with IGF I, IGF II and EGF. TNFa produced negative effects. The observed effects were dose-dependent.
Key words: blastocyst, insulin-like growth factor, epidermal growth factor, tumor necrosis factor, mouse
Author's e-mail: kurzawa@pam.szczecin.pl
Endothelial cells and angiogenesis intensity in lung cancer
M. Juczewska1, E. Chyczewska2, W. Naumnik2, L. Chyczewski3, W. Niklińska4, A. Rogalewska5, O. Kovalchuk3 and J. Nikliński6
12nd Department of Radiotherapy, The Maria Skłodowska-Curie Memorial Oncological Centre, 2Department of Pneumonology and Tuberculosis, Medical Academy, 3Department of Clinical Molecular Biology, Medical Academy, 4Department of Histology and Embryology, Medical Academy, 5Department of Alergology, Medical Academy, 6Department of Thoracic Surgery, Medical Academy, Bialystok, Poland
Abstract: We focused our studies on single endothelial cells
(ECs) scattered in extracellular matrix in lung cancer tumors. Neovascularization
was evaluated in 100 tumors obtained from patients operated for lung cancer,
in relation to histological type, tumor differentiation and clinical stage
of the disease. Angiogenic objects (single endothelial cells and microvessels)
were identified by
immunohistochemistry using monoclonal antibodies against von Willebrand
factor. The count of angiogenic objects per 1 mm2 in each section
was determined in a “hot spot” located at the margin of the tumor. We used
an arbitrary scale of angiogenesis intensity: 1 – 0-200, 2
– 201-400, 3 – >400 angiogenic objects/mm2.
A majority (57%) of the examined cases belonged to the group 2.
The angiogenesis intensity measured by the single EC numbers/mm2
correlates with the histological type and the differentiation of the tumors.
There was no such a correlation when the angiogenesis intensity was measured
by counting total angiogenic objects (microvessels + EC) number/mm2.
Single EC number/mm2 in different histological types of cancer
were as follows: 162± 121 in squamous cell (SqCC), 194± 71 in adenocarcinoma
(AdC), 225± 145 in large cell (LCC), 264± 52 in small cell (SCC), 279±
173 in combined cancer. The differences between the EC counts in the different
histological types of lung cancers were statistically significant in the
following pairs: SqCC vs SCC (p=0.0233) and AdC vs SCC (p=0.0409).The
correlation between EC count in the “hot spot” and the grade of tumor differentiation
was statistically significant for G1 vs G4 (p=0.0007) and G1 vs
G2
(p=0.0411). Our results suggest that higher numbers of EC/mm2
may confirm rapid development of angioneogenesis. These relations should
be examined in larger series of cases.
Key words: Angiogenesis - Endothelial cell - Immunocytochemistry - Lung cancer
Author's e-mail: lchycz@amb.ac.bialystok.pl
Ki-67 labelling index in human brain tumours
Joanna Niemiec
Laboratory of Radiation Biology, Department of Radiation Oncology, Centre of Oncology, Kraków, Poland
Abstract: The proliferative potential in 157 brain tumours was investigated using Ki-67 labelling index (Ki-67LI). There were 46 patients with low grade gliomas (AI & AII), 82 with high grade gliomas (AIII & AIV) and 29 with metastatic tumours. Tumour fragments used for assessment of Ki-67LI were fixed in formalin. Ki-67 antigen was visualised on paraffin sections using DAKO Rabbit Anti-Human Ki-67 antigen. The Ki-67LI was calculated as the percentage of Ki-67 labelled cells. The tumours showed variability in the Ki-67LI values. Significantly higher mean Ki-67LI was found for highly malignant (AIII & AIV) than for low grade gliomas (AI & AII). For metastatic tumours, the mean values of Ki-67LI were significantly higher than for gliomas. Moreover, Ki-67LI of metastatic tumours were significantly higher than for high grade gliomas.
Key words: Ki-67 labelling index - Proliferation rate - Brain tumours
Author's e-mail: Joaniemiec@inetia.pl
Localization of a -tubulin in the rat cumulus oophorus before and during preovulatory expansion.
Anna Wójtowicz, Maria Szołtys and Barbara Bilińska
Laboratory of Animal Endocrinology and Tissue Culture, Institute of Zoology, Jagiellonian University, Kraków, Poland
Abstract: Distribution of a -tubulin was investigated in the cumulus oophorus complexes (COCs) of the rat using anti-a -tubulin monoclonal antibody and fluorescence microscopy. Localization of a -tubulin was assessed in the compact and fully expanded COCs isolated directly from the preovulatory follicles and in the cryosections containing these structures. In the cryosections containing COCs, the submembranous distribution of a -tubulin marked the characteristic cell shapes and showed the lack of cell projections. In the freshly isolated COCs three-dimensional cellular arrangement was visible showing that the dispersed cells formed almost no projections and were not interconnected. The use of a higher magnification revealed a dense microtubular network radiating from centrosomes and extending to the plasma membrane. This network showed discontinuous distribution of submembranous a -tubulin in some of these cells. The latter change could be attributed to the increased progesterone production.
Key words: a -tubulin, cumulus oophorus expansion
Author's e-mail: msol@zuk.iz.uj.edu.pl
Influence of active immunization against GnRH on VIP- and NPY-positive innervation of the porcine testis
K. Wąsowicz1, J. Kaleczyc1, W. Sienkiewicz1, K. Czaja1, A. Zięcik2 and M. Łakomy1
1Department of Animal Anatomy, University of Warmia and Mazury, and 2Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Olsztyn, Poland
Abstract: The influence of an anti-GnRH vaccine on VIP- and NPY-positive innervation of testes was studied in the pig. The immunization prevented the occurrence of changes in the pattern of VIP- and NPY-positive testicular innervation associated with the sexual maturation: it maintained the density of innervation at the high level characteristic for sexually immature animals. The effect was dependent on the method of immunization: the application of two doses of the vaccine was more efficient than application of only one dose, and vaccination with adjuvant was more efficient than vaccination with the plain vaccine. The studies on VIP and NPY concentration in the testicular tissue with radioimmunoassay (RIA) revealed immunization-dependent changes in the peptide concentration, however, some discrepancies between morphological changes and peptide levels were observed.
Key words: Testis - GnRH - Immunization - Neuropeptides - Innervation - Pig
Author's e-mail: wasowicz@moskit.uwm.olsztyn.pl
Alterations in kidney morphology in mice with mosaic mutation.
Małgorzata Lenartowicz1, Katarzyna Sasuła1 and Bożena Zawadowska2
1Department of Genetics and Evolution, 2Department of Cytology and Histology, Institute of Zoology, Jagiellonian University, Kraków, Poland
Abstract: The mosaic (Atp7a mo-ms) is an X-linked, lethal mutation in mice. Hemizygous males die at the age of 15 days and they exhibit strong similarities to the brindled and macular mutants. Injection of cupric chloride to mosaic mutants prolongs their life and diminishes the pathological results of mutation. Histochemical analysis of the kidneys from 14-day-old mutant males showed accumulation of copper in the renal cortex of the investigated animals leading to damage of the kidney architecture. A histological profile of the kidneys was defined for four groups of 14-day-old animals: mosaic males ms/-, control males +/-, mosaic males injected with cupric chloride ms/- (Cu), and control males injected with cupric chloride +/- (Cu). Pathological changes were observed in the cortex and in the medulla of the kidneys in both groups of mutants and control males injected with cupric chloride (50 mg of CuCl2 per each individual).
Key words: Mottled mice - Menkes disease - Copper distribution - Kidney -Renal abnormalities
Author's e-mail: lena@zuk.iz.uj.edu.pl
Utilization of yolk platelets during early embryonic development of Rana temporaria and Bufo bufo
Marek Romek and Ewa Krzysztofowicz
Department of Cytology and Histology, Institute of Zoology, Jagiellonian University, Kraków, Poland
Abstract: Utilization of yolk platelets in cleaving embryos of Rana temporaria and Bufo bufo was studied by different methods. Morphological observations of yolk platelets of R. temporaria embryos at tail bud stage by transmission electron microscopy indicated four initial phases of platelet degradation. The pattern of these events is similar to that found in embryos of B. bufo. The morphological observations were confirmed by energy-dispersive X-ray microanalysis of the elemental content of platelets and by selected-area electron diffraction of platelet cores. Covalently bound sulphur content decreased during cleavage and the content of different inorganic ions changed, whereas the structure of crystalline core remained constant. Morphological changes found in the amorphous cortex of yolk platelets were due to their utilization. Stereological measurements indicated that utilization during cleavage increased, but only the initial phases of yolk platelet degradation were seen. The volume of the cortex did not decrease and the crystalline core did not fragment.
Key words: Yolk platelet - Anurans - Cleavage - Stereology
Author's e-mail: romek@zuk.iz.uj.edu.pl
