Biomedical subjects

J Mokrý

Publications and source records attributed to J Mokrý.

At least 19 recordsLinked to original sources

PubMed2006

Foetal mouse neural stem cells give rise to ependymal cells in vitro.

NSCs are responsible for the generation of CNS cell types derived from the neural tube. Published data resulting from experiments studying the differentiation of NSCs in vitro or in vivo have confirmed their spontaneous tripotency, i.e. their ability to generate cells of the neuronal, astroglial and oligodendroglial lineages. The relationship between NSCs generated in vitro and ependymal cells has not yet been studied. To confirm that ependymal cells can also be produced by NSCs, we utilized the neurosphere assay, which permits isolation and cultivation of NSCs. Cells from the forebrain of E14-15 Balb/c foetuses were grown in DMEM/F12-N2 medium supplemented with EGF and FGF-2 to form multicellular neurospheres. After 3 to 8 passages, neurospheres were plated on surfaces coated with poly-L-lysine, polyornithine and/or laminin in dishes containing the same medium where cytokines were replaced with serum. Under these conditions, neurosphere cells spread over the surface forming a cellular layer consisting of beta-III tubulin+ neuronal, GFAP+ astroglial and O4+ oligodendroglial cells. When these cells were cultivated for prolonged periods, they formed islands of epitheloid cells. Following 2 to 3 weeks in vitro, ependymal cells with beating cilia appeared among these cells. Ciliated ependymal cells were observed in small clusters or as single cells scattered in certain areas. Confocal microscopy confirmed the presence of alpha-tubulin-immunoreactive cilia arranged in tufts located on the apical surface of epitheloid cells. Our data indicate that ependymal cells are spontaneously derived from NSCs.

Animals↗

PubMed2006

Stem cell plasticity and carcinogenesis.

Presently, there is more and more talk about tumors being a disease connected with stem cells. Both stem cells and tumor cells have many similarities, and there is much evidence that microenvironment, cytokines and signal pathways control tissue specificities and have a significant role in the process of carcinogenesis. Recent experimental results show that stem cells and tumor stem cells apparently play a key role in carcinogenesis. Tumors grow up, thanks to the activity of just few stem cells that continuously produce other proliferating progenitor tumor cells. Generally, tumor elements are thought to be either undifferentiated, or dedifferentiated cells. Actually, the truth is that tumors are made of more or less differentiated cells with variable rate of differentiation. We suppose that under certain conditions tumor stem cells may participate in regeneration without giving rise to tumor formation. It is also presumed that we may reprogram tumor stem cells and progenitor cells in a certain period of time and so initiate development of normal tissue. However, till now the real relation between normal and tumor cells is not clear. Finally, we wish to remind that plasticity of tumor and normal cells cannot be separated but should be considered as individual phenomenon expressing certain condition of an organism in time. This communication is only a probe and introduction into a discussion aimed at better understanding of carcinogenesis from the view of processes at the stem cell level. Stimulation of stem cell activation may lead to prophylactic approaches for therapy and prevention in carcinogenesis.

Animals↗

PubMed2006

Effect of nitric oxide synthases inhibitors on exogenous irritant-induced bronchial hyper-reactivity in guinea pigs.

Nitric oxide (NO) is an important endogenous mediator involved in many biological functions in both physiological and pathological conditions. Many of studies suggest that high level of NO may play a role in the pathogenesis of various diseases including respiratory diseases with bronchial hyper-reactivity (BHR). The aim of our study was to examine the relationship between NO production and BHR. The reactivity of tracheal and lung tissue smooth muscle to histamine and acetylcholine was measured in vitro in male guinea pigs pre-treated with NO synthase (NOS) inhibitors. The drugs were administered in vivo during either 3 or 17 days. Furthermore, the animals were exposed in vivo to the toluene vapours after administration of agents. NOS inhibitors showed mainly beneficial effect in the presented study. They decreased the hyper-reactivity of the tracheal and lung tissue smooth muscle evoked by toluene. The decrease was dependent on the duration of their administration and on the type of inhibitor. Short-term administration of inhibitors was more effective than long-term one. A more significant effect was recorded after the pre-treatment with non-selective inhibitor L-NAME. The results showed possible participation of constitutive forms of NOS in the BHR.

Acetylcholine↗

PubMed2005

The transplantation of neural stem cells and predictive factors in hematopoietic recovery in irradiated mice.

A number of surprising observations have shown that stem cells, in suitable conditions, have the ability to produce a whole spectrum of cell types, regardless, whether these tissues are derived from the same germ layer or not. This phenomenon is called stem cell plasticity, which means that tissue-specific stem cells are mutually interchangeable. In our experiments, as a model, we used neural stem cells (NSCs) harvested from fetal (E14-15) neocortex and beta-galactosidase positive. In the first experiment we found that on days 12 and 30 after sub-lethal irradiation (LD 8.5 Gy) and (beta-galactosidase(+)) NSCs transplantation all mice survived, just as the group with bone marrow transplantation. Moreover, the bone marrow of mice transplanted NSCs contained the number of CFU-GM colonies with beta-galactosidase(+) cells which was as much as 50% higher. These differences were statistically significant, p<0.001. In the second experiment, we studied kinetics of (beta-galactosidase(+)) NSCs after their transplantation to sub-lethally irradiated mice. Histochemistry of tissues was performed on days 12 and 30 post-transplantation, and beta-galactosidase(+) cells were detected with the help of histochemical examination of removed tissues (lung, liver, spleen, thymus, and skeletal muscle). In tissues removed on day 12 post-transplantation, we found a significantly higher number of beta-galactosidase(+) cells in the spleen and thymus on day 30. While we presumed the presence beta-galactosidase(+) cells in the spleen, as spleen and reticuloendothelial system represent an important retaining system for different cell types, the presence of beta-galactosidase(+) cells in the thymus was rather surprising but very interesting. This indicates a certain mutual and close interconnection of transplanted stem cells and immune system in an adult organism. In the third experiment, we verified the mutual interchange of Sca-1 surface antigen in the bone marrow cells and NSCs before transplantation. Analysis of this antigen showed 24.8% Sca-1 positive cells among the bone marrow cells, while NSCs were Sca-1 negative. Our experiments show that NSCs share hemopoietic identity and may significantly influence the recovery of damaged hematopoiesis but do not have typical superficial markers as HSCs. This result is important for the determination of predictive factors for hemopoiesis recovery, for stem cell plasticity and for their use in the cell therapy.

Animals↗

PubMed2005

Stem cell plasticity and issues of stem cell therapy.

Today, there is much evidence suggesting that organ-specific stem cells need not rely completely on their own sources for maintenance and regeneration of an organism. In certain circumstances, mostly related to tissue damage, stem cell populations residing past the affected organ can contribute to its recovery--that means from different cell lines and also in tissues from another germ layer. The key factor in formation of self-renewing cellular clones is the presence of stem cells either from the tissue of origin or stem cells migrating from other areas and their successful settlement in an empty niche of the damaged tissue. Stem cell plasticity is the ability of adult tissue-specific stem cells to switch to new identities. The term plasticity also means stem cell phenotypic potential, which is broader than phenotypes of differentiated cells in their original tissues. Many laboratories have given evidence on stem cell plasticity; however, the presented results met with many objections from others. In the first part of our report we wish to refer to several issues associated with stem cell plasticity, transdifferentiation and fusion. Recent experimental results show that stem cells will play a key role in cell therapy. But there are still many questions to answer for scientists engaged in stem cell research. Is it possible to induce cells from one type of tissue to look and act as cells of another tissue? Do these changes occur naturally? Could plasticity be used in the treatment of fatal diseases? Cell therapy is one of the methods to treat damaged myocardial tissue. However, recent results with autologous bone marrow cells in the treatment of damaged myocardium show that this method has still many unanswered questions concerning cells, cytokines, microenvironment and other factors responsible for reparation. To date, there are many opinions either recommending or denying this method in different modifications. One question has not yet been definitely solved: What are the conditions for us to accept this method--its safety and efficacy? The future will show whether these our hopes and expectations will be fulfiled. Many experiments are needed before at least some of these questions may be answered and cell therapy become an important method for the benefit of our patients.

Cell Differentiation↗

PubMed2005

[Present view on the stem cell plasticity and cell therapy].

The most controversial problem in the present biology and medicine is the existence of stem cell plasticity. Experimental biology and medicine have been working with stem cells and stem cell therapy more than twenty years. The term plasticity, as it is understood, is the potential of stem cell phenotypes that is much broader that phenotypes of differentiated cells of their original tissues. Many laboratories have documented the existence of stem cell plasticity; however, many objections to the reported results still exist. Here, we present some of these objections questioning the data on stem cell plasticity. We wish to point out some problems associated with plasticity of stem cells, transdifferentiation and cell fusion. Recent experimental results indicate that stem cells may have a key role in stem cell therapy. This review is an introductory discussion on the stem cell plasticity and stem cell therapy.

Animals↗

PubMed2004

Reactivity of urinary bladder smooth muscle in guinea pigs to acetylcholine and carbachol--participation of acetylcholinesterase.

The authors examined the influence of acetylcholinesterase inhibitor (neostigmine) on the in vitro reactivity of urinary bladder smooth muscle (UBSM) in guinea pigs. The aim of the present study was to determine the participation of pharmacokinetic properties of acetylcholine and carbachol in different UBSM reactivity to these mediators. In vitro method of organ baths was used and reactivity of UBSM strips to cumulative doses of acetylcholine and carbachol was tested before and after the incubation with neostigmine (10(-4) mol.l(-1)). Neostigmine caused a significant increase of UBSM reactivity to acetylcholine. The UBSM reactivity to acetylcholine was significantly higher at concentrations of 10(-5) and 10(-4) mol.l(-1) compared to carbachol at the same concentrations. These findings indicate that in addition to different mediator affinity to muscarinic receptors and to their different intrinsic activity, the pharmacokinetic properties of acetylcholine and carbachol also participate in UBSM reactivity.

Acetylcholine↗

PubMed2004

Local environmental factors determine hematopoietic differentiation of neural stem cells.

Stem cells exhibit unique properties and hold high therapeutic promise, but factors influencing their differentiation after transplantation need to be recognized and defined for this promise to be fully met. Here, we demonstrate that endogenous colony-forming unit spleen (CFU-S) colonies are not generated in lethally irradiated mice transplanted with neural stem cells obtained from brain tissue of syngeneic donors. We investigated the proportion of transplanted neural stem cells that contributed to hematopoietic reconstitution and compared the distribution of transplanted cells in nonsplenectomized to that of splenectomized mice following sublethal whole-body irradiation. We also used clonogenic assays, colony assays, and histochemical analyses to explore conditions under which transplanted, beta-galactosidase-tagged neural stem cells underwent hematopoietic differentiation. Our results suggest that neural stem cells do undergo extramedullary hematopoiesis, even while no endogenous hematopoietic colonies develop in the spleen. Furthermore, we found that neural stem cells effectively colonized the bone marrow of splectomized recipients. We conclude that the hematopoietic differentiation of neural stem cells is highly dependent on the extramedullary environment. We also conclude that the bone marrow does not provide an environment supportive of hematopoietic differentiation by neural stem cells.

Animals↗

PubMed2004

Nestin expression by newly formed human blood vessels.

Nestin is a type VI intermediate filament protein originally described in neural stem cells. Here we report that immature endothelial cells generated in the course of angiogenesis express nestin. Endothelial cells of embryonic capillaries destined to vascularize growing organs also express this intermediate filament protein. Whereas nestin was sporadically expressed in mature adult human endothelial cells sporadically express nestin, this protein was consistently expressed in adult angiogenic vasculature. Nestin expression was also detected in capillaries of the corpus luteum, which replenishes itself by angiogenesis. Nestin-immunoreactive vessels were also observed in the infarcted hearts where transient ischemia triggered regeneration accompanied with neovascularization of the myocardium. Nestinpositive endothelial cells lined vessels nourishing solid growing tumors, including melanoblastomas and glioblastomas. Our data provide definitive evidence that endothelial precursors express the neural stem cell marker nestin and that this protein participates in formation of the cytoskeleton of newly formed endothelial cells. Because nestin expression was recognized under all conditions of vascular development, nestin represents a novel and reliable marker of neovascularization.

Blood Vessels↗

PubMed2003

Antitussive activity of the fruit extract of Emblica officinalis Gaertn. (Euphorbiaceae).

The antitussive activity of Emblica officinalis Gaertn. (E. officinalis, Fam. Euphorbiaceae) was tested in conscious cats by mechanical stimulation of the laryngopharyngeal and tracheobronchial mucous areas of airways. The results showed that at a dose of 50 mg/kg body wt. perorally, the cough suppressive effect of E. officinalis is not unambiguous. A higher dose (200 mg/kg body wt.) of this substance perorally was more effective, especially in decreasing the number of cough efforts (NE), frequency of cough (NE/min(-1)) and the intensity of cough attacks in inspirium (IA+) and expirium (IA-) was more pronounced. These results showed that the cough suppressive activity of E. officinalis is dose-dependent. We could also demonstrate that the antitussive activity of E. officinalis is less effective than shown by the classical narcotic antitussive drug codeine, but more effective than the non-narcotic antitussive agent dropropizine. It is supposed that the antitussive activity of the dry extract of Emblica officinalis is due not only to antiphlogistic, antispasmolytic and antioxidant efficacy effects, but also to its effect on mucus secretion in the airways.

Administration, Oral↗

PubMed2003

Adult stem cells and their importance in cell therapy.

For their unique properties stem cells promise to be of universal use in clinical medicine, especially in regeneration of many organs and tissues in the human body. This attractive subject receives an ever growing attention of specialists from different branches of science and, no doubt, will present one of the most studied trends in medicine in the new millennium. In this communication, the authors discuss two main sources of human stem cells potentially suitable for cell-based therapy. The first are the cells obtained from embryonic tissues--embryonic stem cells, the second are the cells derived from adult tissues--adult stem cells. Presently, harvesting and therapeutic use of embryonic stem cells are associated with many problems both methodical and ethical. Utilization of adult stem cells in cell-based therapy is a certain solution in the current state of replacement therapy. Still, we have to be aware that this is not a compromise but one of the most prospective ways to treat a variety of serious diseases. To date, it is not yet clear which way would be more suitable and it is up to us which way we choose for the benefit of millions of patients. Considering the current state of knowledge, it is impossible yet to predict which stem cells--embryonic or adult--or therapeutic approaches would yield the best results. Much research is to be done and verified in practice and, at the same time, ethical problems must be resolved.

Cell- and Tissue-Based Therapy↗

PubMed1999

Cerebral angiogenesis shows nestin expression in endothelial cells.

The class VI intermediate filament protein nestin has been generally considered as a specific marker for neural precursor cells or developing muscles. In the prenatal developing rat central nervous system (CNS), we localized immunoreactivity for the nestin in blood vessels. Although the widespread nestin expression in cerebral blood vessels persisted in early postnatal periods, it was down-regulated in the adulthood. However, when the adult rat brains were subjected to procedures that trigger neovascularization, e.g. grafting fetal nervous tissue or C6 glioma, the abundant immunoreactivity was detected in all newly formed vessels and adjacent host vasculature. Our results demonstrate that nestin expression in endothelial cells lining cerebral vessels accompanies the process of angiogenesis.

Animals↗

PubMed1998

Angiogenesis of extra- and intraembryonic blood vessels is associated with expression of nestin in endothelial cells.

For a long time, intermediate filament protein nestin was recognized as a specific constituent of the cytoskeleton of developing neural cells and skeletal muscle. Recent reports by other investigators demonstrated that nestin may also be expressed in other cell types. Here we give evidence that nestin is expressed by endothelial cells of developing blood vessels in the rat. Using anti-Rat-401 monoclonal antibody we identified high levels of nestin in the endothelium lining all blood vessels of E14-15 rat foetuses. Immunoreactivity for nestin was detected in both extraembryonic (chorion, placenta, umbilical cord) and intraembryonic blood vessels. In the body of the foetus, we observed nestin-positive endothelial cells in vessels located in the areas of the mesenchyme as well as in vessels supplying the developing organs (central nervous system, liver, lung, spleen, heart, digestive tube). The strongest positive signal was given by the endothelium of newly vascularized tissues. In contrast to the developing tissues, expression of nestin by vascular endothelial cells was greatly reduced in adult tissues.

Animals↗

PubMed1998

Immunohistochemical detection of intermediate filament nestin.

Using Rat-401 monoclonal antibody and peroxidase immunohistochemistry we have detected IF nestin in developing and adult rat tissues. Although epitope recognized by Rat-401 antibody is relatively resistant to aldehyde fixation and paraffin embedding, the embedding of tissue samples into polyester wax and microwave antigen retrieval of histological sections enabled us to enhance sensitivity of immunohistochemical detection and to identify cells expressing low levels of nestin. Our findings confirm that nestin is predominantly distributed in developing neural, myogenic and mesenchymal cells, i.e. cell types that have been previously described to express this intermediate filament. Furthermore, we made original findings on identification of nestin expression in additional cell types, e.g. newly formed endothelial cells of extra- and intraembryonic blood vessels, epithelial cells of the developing lens, and cells apposed to to hair follicles.

Animals↗

PubMed1996

Differentiation of epidermal growth factor-responsive neural precursor cells within neurospheres.

Genetically unmodified neural precursor cells (NPCs) derived from rate embryonic cortex were stimulated with Long-epidermal growth factor in vitro to form multicellular neurospheres. In our study, histology and transmission electron microscopy was for the first time utilized for analysis of three-dimensional neurospheres. Immunohistochemical phenotypization carried out on paraffin-embedded section revealed that most cells in the neurosphere expressed high levels of vimentin that is normally found in immature neural cells. Majority of glial cells expressed glial fibrillary acidic protein (GFAP) that is a specific marker for differentiated astrocytes. On the other hand, myelin basic protein (MBP), a marker of oligodendrocytes, was found only in a discrete subpopulation of glial cells. Similarly, electron microscopy recognized only rate myelin sheaths surrounding axons. Some cells displayed neuron-like morphology and were labelled with antibodies recognizing the neuronal antigens: 210 kDa neurofilaments (NF 210) and microtubule associated protein-2 (MAP-2). Surprisingly well-developed interneuronal synapses were found electronmicroscopically. The presence of synaptic vesicles was also confirmed by immunohistochemical detection of synaptophysin. Our results show that NPCs in neurospheres gradually differentiate into glial and neuronal cells.

Animals↗

PubMed1996

Versatility of immunohistochemical reactions: comprehensive survey of detection systems.

The field of immunohistochemistry comprises histological methods enabling detection of tissue antigens via specific antibodies. Although all these techniques take an advantage of a large specificity of antibody to a particular tissue antigen there are many different approaches for enhancement and visualization of the signal. The aim of the present review article was to briefly outline the historical milestones that made the rapid progress of this discipline possible and give a comprehensive survey of immunohistochemical methods applicable to biomedical research. The survey starts with a description of the direct immunohistochemical method and then pays attention to a huge number of indirect methods. For better explanation of principles of individual techniques, the text is accompanied with graphical schemes. The highest attention is given to immunohistochemical methods that are most generally used, i.e. enzyme anti-enzyme complex methods (e.g. Peroxidase Anti-Peroxidase/PAP/ or Alkaline Phosphatase Anti-Alkaline Phosphatase /APAAP/) and methods based on avidin-biotin interactions (Bridged Avidin-Biotin/BRAB/, Avidin-Biotin Complex /ABC/, Labelled Avidin-Biotin/LAB/). Nevertheless, the principles of other immunohistochemical methods like two- or three-step indirect immunohistochemical methods, methods based on protein A-antibody interaction and Hapten Antibody Anti-Hapten method (HAAH), are also thoroughly characterized. Usefulness of each method for a specific utilization, its advantages and disadvantages are mentioned and compared with the latest immunohistochemical techniques, like Multi-Layered Peroxidase-Labelled Antibody (MLP) and water soluble polymer conjugates, e.g. Enhanced Polymer One-Step staining (EPOS) or EnVision. The last paragraphs are devoted to immunohistochemical amplification systems (Catalyzed Signal Amplification/CSA/ and label anti-label) that dramatically increase sensitivity of detection systems and enable to compare sensitivity of immunohistochemical methods with the most sensitive methods of molecular biologists.

Immunohistochemistry↗

PubMed1995

Cerebrospinal fluid dissemination of fetal neural isografts in brain of adult rats.

Our previous studies tend to document the temporary invasiveness of intraparenchymal fetal isografts. The present study exploits a model situation in which contacted host brain surface remains undamaged. We analyzed the behavior of transplant fragments that accidentally disseminated in cerebrospinal fluid pathways. The transplant seeding occurred in 7 cases out of 85 cases of healthy solid isografts taken from various parts of fetal brain. Fragments were found either as free (floating or simply lying close to the surface) or attached. Some attached fragments invasively penetrated the brain parenchyma. This speaks in favor of invasive properties of fetal neural transplants.

Animals↗

PubMed1995

Neural transplantation of the rat midgestation trophoblast.

The trophoblast may serve as an example of tissue that is endowed with invasive properties under physiological conditions. Invasiveness of the trophoblast is enabled by the secretion of various proteases capable of degrading extracellular matrix components. Trophoblast invasive behavior is strictly controlled by anti-invasive factors produced by uterine decidual cells. To assess invasive abilities of trophoblast cells we have transplanted E9 and E14 rat trophoblast (i.e. the trophoblast obtained on embryonic day 9 and 14) into the brain of adult rats. The brain parenchyma as an immunologically privileged site is suitable for acceptance of grafts of different tissues. Moreover, the trophoblast placed into the CNS lacks inhibitory influence of anti-invasive factors that normally regulate trophoblast invasivity in the course of intrauterine gestation. To visualize migration of grafted cells the nuclei of the trophoblast were labelled with bromodeoxyuridine prior to neural grafting. The transplant of E9 and E14 trophoblast cells obtained a blood nourishment from host vessels. A proper vascularization is necessary for a further transplant growth. The transplant contained labyrinthine trophoblast cells and giant cells that are typical for the rat placenta. Vital trophoblast cells were found in all grafts whose age did not exceed a lifespan of normal rat trophoblast cells i.e. 21-22 days. In the centre of the graft, no blood vessels were observed. Interstitial spaces of neighbouring trophoblast cells were filled with the host blood and morphology of these spaces mimicked lacunae of the placental trophoblast. E9 and E14 rat trophoblast continued to differentiate after transplantation into the CNS of adult rats. Histological structure of the grafts were compared with microscopical morphology of the normal rat placenta. E9 and E14 trophoblast is considerably differentiated and it does not invade a neighbouring tissue. Trophoblast cells located at t the graft periphery may migrate on free surfaces but they do not invade through the host parenchyma. Migration occurs at a limited distance from the transplant and the cells remain in a close contact with other trophoblast cells in the graft via their cytoplasmatic processes. The ability to lyse host blood vessels and form vascular lacunae is well preserved in E9 and E14 trophoblast after grafting into the CNS. This ability is necessary for a proper transport of nutrients from the host blood stream to fetal tissue that normally occurs in the placenta.

Animals↗