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Biomedical subjects

E Necas

Publications and source records attributed to E Necas.

At least 19 recordsLinked to original sources
PubMed2003

Cytokine gene expression in regenerating haematopoietic tissues of mice after cyclophosphamide treatment.

The goal of the study was to investigate changes in expression of selected growth factors tentatively involved in regeneration of haematopoietic tissues (bone marrow and spleen) following cyclophosphamide (CY) damage in the mouse. The bone marrow (BM) and spleen were examined separately, since the regenerating pattern for haematopoietic progenitor cells (HPC) markedly differs in these two haematopoietically active organs after CY. Cytokines assumed to have a stimulatory effect on HPC - stem cell factor (SCF), fetal liver tyrosine kinase 3-ligand (flt3-ligand), thrombopoietin (TPO), stromal cell-derived factor 1 (SDF-1), oncostatin M (OSM) -, a suppressive effect on HPC proliferation - macrophage inflammatory protein-1alpha (MIP-1alpha), transforming growth factor-beta1 (TGFbeta1), tumour necrosis factor-alpha (TNFalpha) - and to be involved in migration of HPC (SCF, flt3-ligand, MIP1alpha, SDF-1) were examined at the level of mRNA expression by means of real-time RT-PCR. The expression of a particular cytokine appears to be similar in both BM and spleen of untreated mice. CY administration changed the expression pattern of the studied genes in BM and spleen. In BM, the levels of mRNAs for SCF and SDF-1 were increased and that for TGFbeta1 decreased at time intervals at which HPC are known to proliferate intensively during BM regeneration. In contrast, stimulated proliferation of HPC in spleen was accompanied by increased expression of flt3-ligand and oncostatin M. Upon mobilization of HPC from BM into blood after CY, the expression of SCF, TPO, SDF-1 and TGFbeta1 tends to decrease in BM. Accumulation of HPC in spleen is accompanied by increased mRNA for flt3-ligand and OSM. Our findings demonstrate that different cytokines may be involved in the proliferation and mobilization/homing of HPC during recovery after CY damage in BM and spleen.

Animals↗
PubMed2002

TRAIL (Apo2L) suppresses growth of primary human leukemia and myelodysplasia progenitors.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL, APO2L) has been shown to induce apoptosis in a number of tumor cell lines as well as in some primary tumors whereas cells from most normal tissues are highly resistant to TRAIL-induced apoptosis. We have studied the susceptibility of primary malignant and normal bone marrow hematopoietic progenitors to TRAIL-induced apoptosis. Extracellular domain of human TRAIL with N-terminal His(6) tag (His-TRAIL, amino acids 95-281) was produced in E. coli and its apoptosis-inducing ability was compared with the leucine-zipper containing TRAIL, LZ-TRAIL. Both variants of TRAIL had the same apoptosis-inducing ability. Clonogenic progenitor assays showed that His-TRAIL significantly reduced the number of myeloid colonies (CFU-GM) and clusters from patients with acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and myelodysplastic syndromes (MDS). His-TRAIL had no negative effect on the number of CFU-GM colonies and clusters derived from bone marrow cells of AML patients in complete remission, and lymphoma patients without bone marrow involvement, as well as those derived from normal cord blood cells. Moreover, we found that normal human stem cells treated with high doses of His-TRAIL maintain a repopulating potential when transplanted into NOD/SCID mice. To conclude, our data document that TRAIL does not affect normal human hematopoiesis but suppresses the growth of early primary leukemia and myelodysplasia progenitors.

Acute Disease↗
PubMed2002

[Impaired development of uteroplacental circulation].

OBJECTIVE: To sum up the knowledge of uteroplacental circulation, their dysfunction and etiology and pathogenesis of preeclampsia. TYPE OF STUDY: Review. SETTING: Department of Obstetrics and Gynaecology, 1st Faculty of Medicine, Charles University, Prague, Institut of Pathological Physiology, 1st Faculty of Medicine, Charles University, Prague. SUBJECT OF STUDY: A summary of what is known about development of uteroplacental circulation predispose women to the development of preeclampsia and IUGR but the etiology of preeclampsia is still unknown.

Female↗
PubMed2001

Angiopoietin-1, angiopoietin-2 and Tie-2 in tumour and non-tumour tissues during growth of experimental melanoma.

Tumour progression is dependent on the formation of new vessels in tumour tissue. Tumour cells produce a variety of factors that influence vessel growth and maintenance both in tumour and tumour-adjacent tissues. Angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2) and their tyrosine kinase receptor Tie-2 have been shown to play an important role in the processes of growth and remodelling of normal as well as tumour vessels. We studied gene expression of the angiogenic factors Ang-1 and Ang-2 and of their tyrosine kinase receptor Tie-2 in the tumour and non-tumour tissues of mice bearing the experimental melanoma B16. Using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR we measured Ang-1, Ang-2 and Tie-2 mRNA levels in the tumour, bone marrow, liver and spleen. Melanoma tissue overexpressed Ang-2 mRNA compared with spleen, liver and bone marrow of normal mice, suggesting its role during melanoma progression. On the other hand, there was a significant decrease in Ang-2 mRNA level in bone marrow cells collected on days 5 and 10 of tumour growth compared with the expression of Ang-2 mRNA in the bone marrow of normal mice and those collected on days 15 and 20 of tumour growth. These data demonstrate, for the first time, an ectopic effect of the tumour on the gene coding for an angiogenic factor, and also suggest that tumour growth may influence angiogenesis and/or vasculogenesis in distant organs.

Angiopoietin-1↗
PubMed2000

Chromatin remodeling gene SMARCA5 is dysregulated in primitive hematopoietic cells of acute leukemia.

We identified a subset of genes involved in chromatin remodeling whose mRNA expression changes in differentiating mouse erythroleukemia (MEL) cells. We furthermore tested their mRNA expression patterns in normal and malignant CD34+ bone marrow cells. SMARCA5, imitation switch gene homologue, was rapidly silenced during in vitro erythroid differentiation of MEL cells whereas it was up-regulated in CD34+ hematopoietic progenitors of acute myeloid leukemia (AML) patients. Moreover, SMARCA5 mRNA levels decreased in AML CD34+ progenitors after the patients achieved complete hematologic remission. We detected high levels of SMARCA5 mRNA in murine bone marrow and spleen and monitored its expression in these hematopoietic tissues during accelerated hematopoiesis following hemolytic anemia induced by phenylhydrazine. SMARCA5 expression levels decreased after the onset of accelerated erythropoiesis. Our data suggest that both in vitro and in vivo induction of differentiation is followed by down-regulation of SMARCA5 expression. In CD34+ AML progenitors over-expression of SMARCA5 may thus dysregulate the genetic program required for normal differentiation.

Acute Disease↗
PubMed1998

[Paroxysmal nocturnal hemoglobinuria and its association with aplastic anemia].

Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired clonal disorder of haematopoiesis. Clinically it is characterized by intravascular haemolysis, venous thrombosis and often by bone marrow hypoplasia. Haemolysis and thrombosis develop as a consequence of deficiency of several proteins on the cell membrane of the affected clone of blood elements. This is caused by somatic mutations in the PIG-A gene, which encodes an enzyme involved in the biosynthesis of glycosylphosphatidylinositol (GPI) anchor. Spectrum of mutations in the PIG-A gene is different to that observed in other genes. The mutations are mainly small deletions and insertions causing frameshift; large deletions are rare. Recently, however, a 88 base pairs direct tandem repeat insertion has been reported in a patient with PNH developed on the background of aplastic anaemia (AA). The peculiar pattern of the PIG-A gene mutations and the finding that more than one mutated clone is commonly present in patients with PNH might suggest that some form of hypermutability, caused by decreased DNA stability, deficient repair or increased generation of mutagens, might underline PNH. As most mutations cause cell death, it would explain the hypoplastic nature of the disorder and its association with AA. Other models of pathogenesis of PNH are also discussed.

Anemia, Aplastic↗
PubMed1998

[Genes stimulated by tissue hypoxia].

Hypoxia is a frequent pathogenetic stimulus to which the whole organism as well as affected tissues and cells respond by various adaptive mechanisms. The basis of these reactions is the ability of cells to monitor the concentration of oxygen molecules by oxygen sensors. Some of these reactions are the result of the association of the oxygen sensor of the cell with its genome. This association makes it possible to enhance the activity or activation of some genes. Products of these genes increase the resistance of cells to hypoxia, they induce an increased vascularization of the affected tissue, increased erythrocyte production and some other reactions. In tumour hypoxia can cause by this mechanism changes of its properties and its malignization.

Animals↗
PubMed1998

Estimation of extent of cell death in different stages of normal murine hematopoiesis.

Murine hematopoiesis has been analyzed by many authors, and available data allow for quantitative evaluation of this dynamic process. In this study, the capacity of several populations of the bone marrow clonogenic cells (progenitors) to produce blood cells was compared with their actual production. The cell cycle progression rate was directly measured in the following types of hematopoietic progenitors: day 8 colony-forming units-spleen, GM-colony-forming cells, BFU-E, and CFU-E in normal mice. The cell cycle progression rates of the individual progenitors, together with their numbers in the whole hematopoietic tissue, were used to calculate the absolute numbers produced daily in each population. The data reviewed from literature were analyzed in parallel. The capacity of the progenitors to produce mature blood cells were derived from the daily production of progenitors multiplied by their clonogenic potential. This theoretical capacity to produce blood cells was compared to the actual blood cell production determined from the turnover of circulating blood elements. The comparison strongly suggested an intensive cell death rate occurring at the early stages of differentiation and its decline as the hematopoietic cells become more differentiated and mature.

Animals↗
PubMed1998

Ureter obliteration transiently depresses erythropoietin production.

BACKGROUND: The erythropoietin (EPO) gene is activated in peritubular cells of the renal cortex in response to hypoxia and EPO is secreted into the circulation. Oxygen tension in the venous blood normally determine EPO elaboration, and, therefore, oxygen consumption in the kidney appears to be an essential part of the oxygen-sensing physiological mechanism. As renal oxygen consumption is closely linked to urine production, we have compared responsiveness of the EPO gene to diminished oxygen supply in the normal kidney with that of the hydronephrotic kidney, resulting from ureter ligation. METHODS: Erythropoietin mRNA was determined in RNA extracted from murine kidneys at various times up to 61 days after ureter ligation. Mice were exposed to hypoxia for 4 h before being killed. RESULTS: Unilateral ureter ligation significantly decreased EPO mRNA levels in the affected kidney. However, the ability to increase EPO mRNA recovered between days 4 and 22, despite persisting ureter ligation. CONCLUSION: Our results demonstrate a dissociation between the excretory functions of the kidney and EPO production. Moreover, they show an early but transient suppression of EPO gene responsiveness to hypoxia after ureter ligation.

Animals↗
PubMed1998

Guinea pig serum erythropoietin (EPO) selectively stimulates guinea pig erythroid progenitors: human or mouse erythroid progenitors do not form erythroid burst-forming unit colonies in response to guinea pig serum EPO.

Erythropoietin (EPO) is the primary regulator of mammalian erythropoiesis, providing a proliferative and differentiative signal to the early EPO-responsive erythroid progenitors, burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid, as well as to later EPO-responsive erythroid progenitors. EPO is secreted by the kidney in response to hypoxia and anemia. There is an extensive biological crossreactivity between human EPO and the EPOs of other mammals. Necas et al. have reported that this crossreactivity may not include the guinea pig (Cavia porcelllus). Because the specificity of the guinea pig's erythropoietic responses may be of biological significance, we compared guinea pig hypoxic serum with mouse (m) and human (h) recombinant (r) EPOs for their ability to induce erythroid progenitor proliferation and differentiation in semisolid cultures. Guinea pig bone marrow mononuclear cells (BMMCs) formed BFU-E colonies in response to guinea pig hypoxic serum, rhEPO, or rmEPO in a dose-dependent fashion. Neither human nor mouse BMMCs responded to guinea pig hypoxic serum; however, guinea pig hypoxic serum exerted no inhibitory effect on human or mouse in vitro erythroid differentiation in the presence of rhEPO or rmEPO. The intensity of the EPO band on Western blotting analysis of guinea pig hypoxic serum was significantly greater than in nonhypoxic serum. This suggests that guinea pig erythropoiesis is mediated by EPO and stimulated by hypoxia in a fashion similar to that observed in human and mouse erythropoiesis. Furthermore, guinea pig EPO did not stimulate human or mouse erythroid differentiation in vitro, whereas guinea pig erythroid progenitors could be stimulated by human or mouse EPO, suggesting structural differences in guinea pig EPO and EPO receptor (EPOR) compared with human or mouse EPO and EPOR. These differences probably evolved after the guinea pig's ancestors diverged from myomorph rodents. Further characterization of the guinea pig EPO and EPOR should facilitate our understanding of the interaction between EPO and EPOR.

Animals↗
PubMed1997

Rescue from lethal irradiation correlates with transplantation of 10-20 CFU-S-day 12.

Repopulation by donor cells of a bone marrow ablated by irradiation is now recognized to proceed in two phases: initial repopulation that may be temporary followed by permanent engraftment of longterm repopulating cells (LTRC). While a single LTRC has been shown to be capable of restoring the entire lymph-hemopoietic system of an irradiated animal, the identity of the temporary repopulating cells has not been established unequivocally. We used the results of transplantation of subpopulations successively enriched for LTRC and containing varying numbers of CFU-S-12 (colony-forming units in the spleen at day 12 post transplantation) and progenitors to determine the likely cell type and number of cells needed for initial survival after radiation. Subpopulations from untreated and 5-fluorouracil-treated mice were discriminated on the basis of antibody reactivity, Hoechst 33342 and rhodamine 123 fluorescence intensity and light scattering properties. The minimum rescue inocula varied greatly in CFU-GEMM, BFU-E and CFU-GM content. One to two CFU-S-12 were uniformly present in all isolated suspensions that rescued 50% of lethally irradiated animals. In view of the known average seeding efficiency of CFU-S, our studies suggest that transfusion of 10-20 CFU-S day 12/13 is responsible for radioprotection. Evidence that multiple CFU-S day 12/13 are needed for initial repopulation is also supported by quantitative estimates of the number of mature cells that can be produced by CFU-S. Transfusion of a single CFU-S day 12/13 can be shown to be grossly inadequate to provide the number of peripheral blood cells needed to ameliorate the severe pancytopenia following lethal irradiation by day 12-14. Our data also indicate that 5-fluorouracil-treated marrow subpopulations appear inferior to untreated subpopulations in their ability to contribute to initial repopulation when transfused at low cell doses into lethally irradiated recipients.

Animals↗
PubMed1996

Products of red blood cell degradation inhibit responsiveness of the erythropoietin oxygen sensor.

Tissue hypoxia is less effective in increasing erythropoietin plasma levels in animals with post-transfusion polycythaemia. Since more red blood cells are decomposed under this condition, the effects of exogenous haemin and of lysed or heat-damaged red blood cells on activation of the erythropoietin gene have been studied in mice rendered hypoxic. Total RNA was extracted from the kidney and the liver and subjected to northern blot analysis with a probe containing part of the murine erythropoietin gene. Blood plasma was collected and erythropoietin levels were determined by radioimmunoassay. Erythropoietin gene activation was suppressed by haemin and increased red blood cell haemolysis. Tin (Sn) protoporphyrin, a haeme analogue which cannot bind oxygen, did not share the effect of haemin. On the other hand, when injected with haemin, Sn-protoporphyrin potentiated the suppressive effect of haemin, probably through inhibition of haemin catabolism. We conclude that the intracellular haeme concentration inhibits the kidney oxygen sensor and that this inhibition, mediated by products red blood cell degradation, is a physiological safeguard mechanism against excessive polycythaemia and its deleterious effects upon blood circulation.

Animals↗
PubMed1995

[Erythropoietin in the era of molecular biology].

Cloning of genes for erythropoietin in the mid-eighties started a new period of research and use of erythropoietin. RIA and ELISA methods of its detection were developed. It became possible to seek possibilities of its therapeutic use. The cell type which is the source of erythropoietin in the organism was assessed. The regulation of the expression of the erythropoietin gene in relation to oxygen tension was investigated. The biochemical mechanism is sought which transmits the oxygen tension to transcription signals and the stability of erythropoietin mRNA. The gene for the erythropoietin receptor was found and it is investigated in relation to the pathogenesis of primary polycythemias.

Erythropoietin↗
PubMed1995

Hematopoietic reserve provided by spleen colony-forming units (CFU-S).

Using quantitative data available from the literature on murine hematopoiesis, the functional reserve of multipotential stem cells was calculated by comparing daily blood cell production with the potential of clonogenic spleen colony-forming cells (CFU-S) to generate blood cells. The potential of the day-8 CFU-S (CFU-S-8) population is estimated to be from 4000 to 37,000 times greater than needed in steady-state hematopoiesis. The CFU-S population may thus serve to provide a functional reserve to supply large numbers of peripheral blood cells via committed lineage-specific cells within days, whenever needed.

Animals↗
PubMed1993

Self-renewal of the long-term repopulating stem cell.

Self-renewal implies maintenance of all attributes of the original in the offspring and is considered characteristic of the hemopoietic stem cells. Yet, it has been questioned whether one of the most primitive hemopoietic stem cells, the long-term repopulating cell (LTRC), has that capacity. The present experiments demonstrate that single LTRCs can repopulate the lymphohemopoietic system of a lethally irradiated mouse and that the progeny of a single LTRC in a primary recipient again contains LTRCs capable of repopulating lethally irradiated secondary recipients. The transfusion of very small numbers of marrow cells (10,000-20,000 cells containing one or no LTRCs) unexpectedly provided insight into competitive marrow repopulation. At these low levels of stem cells, irradiated host stem cells or their progeny competed successfully with unirradiated donor cells. This parallels the known reemergence and marrow repopulation by host cells when the number of nonirradiated donor stem cells is reduced by serial transplantation.

Animals↗