S. Bobis, D. Jarocha, M. Majka: Mesenchymal stem cells: characteristics and clinical applications pp. 215-230

W. Diakowski, M. Grzybek, A.F. Sikorski: Protein 4.1, a component of the erythrocyte membrane skeleton and its related homologue proteins forming the protein 4.1/FERM superfamily pp. 231-248

P. Kopiński, K. Sładek, J. Szczeklik, J. Soja, A. Szlubowski, B. Balicka-Ślusarczyk, B. Lackowska, M. Plato, A. Szpechciński: Expression of insulin-like growth factor-I (IGF-I) in alveolar macrophages and lymphocytes obtained by bronchoalveolar lavage (BAL) in interstitial lung diseases (ILD). Assessment of IGF-I as a potential local mitogen and antiapoptotic cytokine pp. 249-258

D. Śladowski, A. Wasiutyński, G. Wilczyński, I. Grabska-Liberek, S. Coecke, A. Kinsner, Iwona Kochanowska: Expression of the membrane complement regulatory proteins (CD55 and CD59) in human thymus pp. 263-267

M. Tománek, E. Chronowska: Immunohistochemical localization of proliferating cell nuclear antigen (PCNA) in the pig ovary pp. 269-274

B. Płytycz, J. Homa, B. Kozioł, M. Różanowska, A.J. Morgan: Riboflavin content in autofluorescent earthworm coelomocytes is species-specific pp. 275-280

Department of Transplantation, Polish-American Institute of Pediatrics, Jagiellonian University Medical College, Cracow, Poland

 Abstract: Mesenchymal stem cells (MSCs) are bone marrow populating cells, different from hematopoietic stem cells, which possess an extensive proliferative potential and ability to differentiate into various cell types, including: osteocytes, adipocytes, chondrocytes, myocytes, cardiomyocytes and neurons. MSCs play a key role in the maintenance of bone marrow homeostasis and regulate the maturation of both hematopoietic and non-hematopoietic cells. The cells are characterized by the expression of numerous surface antigens, but none of them appears to be exclusively expressed on MSCs. Apart from bone marrow, MSCs are located in other tissues, like: adipose tissue, peripheral blood, cord blood, liver and fetal tissues. MSCs have been shown to be powerful tools in gene therapies, and can be effectively transduced with viral vectors containing a therapeutic gene, as well as with cDNA for specific proteins, expression of which is desired in a patient. Due to such characteristics, the number of clinical trials based on the use of MSCs increase. These cells have been successfully employed in graft versus host disease (GvHD) treatment, heart regeneration after infarct, cartilage and bone repair, skin wounds healing, neuronal regeneration and many others. Of special importance is their use in the treatment of osteogenesis imperfecta (OI), which appeared to be the only reasonable therapeutic strategy. MSCs seem to represent a future powerful tool in regenerative medicine, therefore, they are particularly important in medical research.

Author’s e-mail: mmajka@cm-uj.krakow.pl

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Abstract: The review is focused on the domain structure and function of protein 4.1, one of the proteins belonging to the membrane skeleton. The protein 4.1 of the red blood cells (4.1R) is a multifunctional protein that localizes to the membrane skeleton and stabilizes erythrocyte shape and membrane mechanical properties, such as deformability and stability, via lateral interactions with spectrin, actin, glycophorin C and protein p55. Protein 4.1 binding is modulated through the action of kinases and/or calmodulin-Ca²⁺. Non-erythroid cells express the 4.1R homologues: 4.1G (general type), 4.1B (brain type), and 4.1N (neuron type), and the whole group belongs to the protein 4.1 superfamily, which is characterized by the presence of a highly conserved FERM domain at the N-terminus of the molecule. Proteins 4.1R, 4.1 G, 4.1 N and 4.1 B are encoded by different genes. Most of the 4.1 superfamily proteins also contain an actin-binding domain. To date, more than 40 members have been identified. They can be divided into five groups: protein 4.1 molecules, ERM proteins, talin-related molecules, protein tyrosine phosphatase (PTPH) proteins and NBL4 proteins. We have focused our attention on the main, well known representatives of 4.1 superfamily and tried to choose the proteins which are close to 4.1R or which have distinct functions. 4.1 family proteins are not just linkers between the plasma membrane and membrane skeleton; they also play an important role in various processes. Some, such as such focal adhesion kinase (FAK), non-receptor tyrosine kinase that localizes to focal adhesions in adherent cells, play the role in cell adhesion. The other members control or take part in tumor suppression, regulation of cell cycle progression, inhibition of cell proliferation, downstream signaling of the glutamate receptors, and establishment of cell polarity; some are also involved in cell proliferation, cell motility, and/or cell-to-cell communication.

Author’s e-mail: afsbc@ibmb.uni.wroc.pl

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¹Chair of Gene Therapy, Collegium Medicum, Nicolaus Copernicus University, Bydgoszcz
²Department of Occupational Medicine and Environmental Diseases, Jagiellonian University Medical College, Kraków
³2nd Chair of Internal Medicine, Jagiellonian University Medical College, Kraków
⁴Department of Immunology, Center of Oncology, Kraków, Poland

Abstract: Little is known about IGF-I expression in the alveolar lymphocytes (AL), and about local role of IGF-I in physiological conditions and in interstitial lung diseases. Bronchoalveolar lavage was carried out in patients with silicosis, asbestosis, idiopathic pulmonary fibrosis (IPF) and sarcoidosis, as well as in control subjects (n=13, 9, 12, 56, 15, resp). Alveolar macrophages (AM) and lymphocytes (AL) were studied for a) IGF-I, BCL-2, Fas and Fas Ligand expression and b) cell cycle (incl. sub-G₁ peak of late apoptosis) with propidium iodide (PI). Flow cytometry (FC) and immunocytochemistry were used. AL early apoptosis was detected by Annexin V FITC/PI staining. IGF-I was present in AL of all tested groups. The number of IGF-I positive AL was significantly higher in IPF (52 ± 6.7%) and later (II and III) stages of sarcoidosis (39 ± 7.8 vs 16 ± 4.0% in controls, p<0.05). Increased BCL-2 expression in AL was detected in IPF and sarcoidosis. In all tested groups, AL were almost exclusively Fas+ T cells. Generally, a low number of AL entered apoptosis; no significant differences were found between patient groups, except decreased apoptosis rate in sarcoidosis (0.60 ± 0.17 vs 1.15 ± 0.33% in controls, p<0.05). Proportion of AL positive with IGF-I was significantly correlated with parameters reflecting AL and AM cell proliferation and BCL-2 expression (e.g. AL IGF-I+ vs AM in S phase of cell cycle: r_s = +0.50, p=0.001), but not with apoptosis. The results show that human alveolar lymphocytes express IGF-I in normal conditions, as well as in ILD. The proportion of IGF-I+ lymphocytes was significantly increased in IPF and at later stages of sarcoidosis. In our material there was no evidence for profibrogenic or antiapoptotic activity of IGF-I. We suggest that IGF-I originating from AL may be locally active as a mitogen for alveolar macrophages and lymphocytes in ILD.

Author’s e-mail: mpkopins@consoft.com.pl

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Abstract: Acetylation level of chromatin histones is maintained by histone acetylases (HATs) and deacetylases (HDACs) and correlates with transcriptional activity of genes. Trichostatin A (TSA) is HDACs inhibitor that causes various effects in cells, including immunomodulation. The CD4 antigen is a key coreceptor involved in activation of T helper cells. Using quantitative real-time PCR (RQ-PCR) and flow cytometry techniques, we estimated CD4 transcript level and density of CD4 antigen on the surface of TSA-treated stimulated and unstimulated peripheral T cells. We observed a dose dependent decrease in CD4 mRNA level and antigen density on surface of TSA-treated stimulated T cells. However, we did not observe any significant TSA effect on CD4 mRNA and protein expression in unstimulated T cells. Our data suggest that TSA may induce biosynthesis of factors responsible for negative regulation of CD4 antigen expression in stimulated T cells. Our investigation may support previous observation that this drug has immunosuppresive effect on primary T cells and may be useful in treatment of certain autoimmune disorders.

Author’s e-mail: kozlowa@am.poznan.pl

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Dariusz Śladowski^1,2, Aleksander Wasiutyński³, Grzegorz Wilczyński³, Iwona Grabska-Liberek⁴, Sandra Coecke², Agnieszka Kinsner² and Iwona Kochanowska⁵

¹Department of Transplantology and Central Tissue Bank, Centre of Biostructure Research, Medical University, Warsaw, Poland;
²European Centre for Validation of Alternatives Methods, Institute for Health and Consumer Protection Joint Research Centre, European Commission, Ispra, Italy;
³Department of Pathology, Medical University, Warsaw, Poland;
⁴Department of Ophthalmology, Medical University, Warsaw, Poland;
⁵Polish Academy of Sciences, L. Hirszfeld Institute of Immunology and Experimental Therapy, Wrocław, Poland

Abstract: CD59 is one of the key molecules involved in cell protection against autologus complement. The fact that complement regulatory proteins are able to prevent hyperacute rejection of organs in pig to primate model, raises the question of possible complement regulatory protein (CRP) involvement in the maturation of immunological system. We report here that in foetal and postnatal human thymus, CD59 and CD55 are primarily located on Hassall’s corpuscles and medullary epithelial cell. Additionally in TUNEL technique was used to visualize distribution of apoptotic cells in the thymus, which revealed the presence of apoptotic cells closely associated with the Hassall’s corpuscles. The observed co-localization of CD59, CD55 and CD30L might suggest an involvement of the complement system in thymic selection in humans.

Author’s e-mail: dslado@ib.amwaw.edu.pl

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Research Institute of Animal Production, Department of Biology of Reproduction, Prague, Czech Republic

Abstract: The aim of the study was to determine the expression of proliferating cell nuclear antigen protein (PCNA) in the pig ovary. The localization of PCNA was demonstrated in paraffin sections of pig ovarian tissue using primary mouse monoclonal anti-PCNA antibody. In primordial follicles, no remarkable staining for PCNA either in granulosa cells or in the oocytes was observed. In primary to secondary follicles, positive staining in oocytes and in some of granulosa cells was detected. The advanced preantral and particularly actively growing small to large antral follicles showed extensive PCNA labelling in the layers of granulosa and theca cells and in the cumulus cells encircling the oocyte. PCNA labelling was expressed in nuclei of oocytes in preantral and small antral follicles. In atretic follicles, the level of PCNA protein expression was dependent on the stage of atresia. Follicles demonstrating advanced atresia showed only limited or no PCNA labeled granulosa and theca cells. The results of the study demonstrate that follicular growth and development in pig ovary may be effectively monitored by determining the granulosa cell expression of PCNA.

Author’s e-mail: tomanek.milan@vuzv.cz

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Barbara Płytycz¹, Joanna Homa¹, Beata Kozioł¹, Małgorzata Różanowska² and A. John Morgan³

¹Department of Evolutionary Immunobiology, Institute of Zoology, Jagiellonian University; Kraków, Poland;
²Department of Optometry and Vision Sciences, Cardiff University, Cardiff, UK;
³Cardiff School of Biosciences, Cardiff University, UK 

Abstract: We have recently shown that a large proproportion of earthworm coelomocytes exhibit strong autofluorescence in some species (Dendrobaena veneta, Allolobophora chlorotica, Dendrodrilus rubidus, Eisenia fetida, and Octolasion spp.), while autofluorescent coelomocytes are very scarce in representatives of Lumbricus spp. and Aporrectodea spp. Riboflavin (vitamin B2) was identified as a major fluorophore in Eisenia fetida coelomocytes. The main aim of the present experiments was to quantify riboflavin content in autofluorescent coelomocytes (eleocytes) from several earthworm species through a combination of flow cytometric and spectrofluorometric measurements. Spectrofluorometry of coelomocyte lysates showed that riboflavin was non-detectable in the coelomocytes of Aporrectodea spp. and Lumbricus spp., but was a prominent constituent of lysates from species with autofluorescent eleocytes. In the latter case, riboflavin content was the highest in E. fetida, followed by Octolasion spp. > A. chlorotica > D. rubidus. The riboflavin content of coelomocytes correlates positively with eleocyte autofluorescence intensity measured by flow cytometry and visible with fluorescence microscopy.

Author’s e-mail: plyt@zuk.iz.uj.edu.pl

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