Luděk Šefc

Expression of genes regulating angiogenesis in human circulating hematopoietic cord blood CD34+/CD133+ cells

Objectives: Human CD34+ cells represent a heterogeneous population of immature cells which may differentiate to various cell types. The aim of the study was to determine angiogenesis regulating genes expression in CD34+ cells, their subpopulations, and during their differentiation induced by hematopoietic growth factors.

Effect of catheter-based patent foramen ovale closure on the occurrence of arterial bubbles in scuba divers.

OBJECTIVES This study sought to evaluate the effect of catheter-based patent foramen ovale (PFO) closure on the occurrence of arterial bubbles after simulated dives. BACKGROUND PFO is a risk factor of decompression sickness in divers due to paradoxical embolization of bubbles. To date, the effectiveness of catheter-based PFO closure in the reduction of arterial bubbles has not been demonstrated. METHODS A total of 47 divers (age 35.4 ± 8.6 years, 81% men) with a PFO (PFO group) or treated with a catheter-based PFO closure (closure group) were enrolled in this case-controlled observational trial. All divers were examined after a simulated dive in a hyperbaric chamber: 34 divers (19 in the PFO group, 15 in the closure group) performed a dive to 18 m for 80 min, and 13 divers (8 in the PFO group, 5 in the closure group) performed a dive to 50 m for 20 min. Within 60 min after surfacing, the presence of venous and arterial bubbles was assessed by transthoracic echocardiography and transcranial color-coded sonography, respectively. RESULTS After the 18-m dive, venous bubbles were detected in 74% of divers in the PFO group versus 80% in the closure group (p = 1.0), and arterial bubbles were detected in 32% versus 0%, respectively (p = 0.02). After the 50-m dive, venous bubbles were detected in 88% versus 100%, respectively (p = 1.0), and arterial bubbles were detected in 88% versus 0%, respectively (p < 0.01). CONCLUSIONS No difference was observed in the occurrence of venous bubbles between the PFO and closure groups, but the catheter-based PFO closure led to complete elimination of arterial bubbles after simulated dives. (Nitrogen Bubble Detection After Simulated Dives in Divers With PFO and After PFO Closure; NCT01854281).

Activation of adenosine A3 receptors supports hematopoiesis-stimulating effects of granulocyte colony-stimulating factor in sublethally irradiated mice

Purpose: Research areas of ‘post-exposure treatment’ and ‘cytokines and growth factors’ have top priority among studies aimed at radiological nuclear threat countermeasures. The experiments were aimed at testing the ability of N6-(3-iodobenzyl)adenosine-5′-N-methyluronamide (IB-MECA), an adenosine A3 receptor agonist, to modulate hematopoiesis in sublethally irradiated mice, when administered alone or in a combination with granulocyte colony-stimulating factor (G-CSF) in a two-day post-irradiation treatment regimen. Materials and methods: A complete analysis of hematopoiesis including determination of numbers of bone marrow hematopoietic progenitor and precursor cells, as well as of numbers of peripheral blood cells, was performed. The outcomes of the treatment were assessed at days 3 to 22 after irradiation. Results: IB-MECA alone has been found to induce a significant elevation of numbers of bone marrow granulocyte-macrophage progenitor cells (GM-CFC) and peripheral blood neutrophils. IB-MECA given concomitantly with G-CSF increased significantly bone marrow GM-CFC and erythroid progenitor cells (BFU-E) in comparison with the controls and with animals administered each of the drugs alone. Conclusions: The findings suggest the ability of IB-MECA to stimulate hematopoiesis and to support the hematopoiesis-stimulating effects of G-CSF in sublethally irradiated mice.

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-1α (MIP-1α), transforming growth factor-β1 (TGFβ1), tumour necrosis factor-α (TNFα) – and to be involved in migration of HPC (SCF, flt3-ligand, MIP1α, 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 TGFβ1 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 TGFβ1 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.

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 was 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.

The pharmacological activation of adenosine A1 and A3 receptors does not modulate the long- or short-term repopulating ability of hematopoietic stem and multipotent progenitor cells in mice

This study continues our earlier findings on the hematopoiesis-modulating effects of adenosine A1 and A3 receptor agonists that were performed on committed hematopoietic progenitor and precursor cell populations. In the earlier experiments, N6-cyclopentyladenosine (CPA), an adenosine A1 receptor agonist, was found to inhibit proliferation in the above-mentioned hematopoietic cell systems, whereas N6-(3-iodobenzyl)adenosine-5′-N-methyluronamide (IB-MECA), an adenosine A3 receptor agonist, was found to stimulate it. The topic of this study was to evaluate the possibility that the above-mentioned adenosine receptor agonists modulate the behavior of early hematopoietic progenitor cells and hematopoietic stem cells. Flow cytometric analysis of hematopoietic stem cells in mice was employed, as well as a functional test of hematopoietic stem and progenitor cells (HSPCs). These techniques enabled us to study the effect of the agonists on both short-term repopulating ability and long-term repopulating ability, representing multipotent progenitors and hematopoietic stem cells, respectively. In a series of studies, we did not find any significant effect of adenosine agonists on HSPCs in terms of their numbers, proliferation, or functional activity. Thus, it can be concluded that CPA and IB-MECA do not significantly influence the primitive hematopoietic stem and progenitor cell pool and that the hematopoiesis-modulating action of these adenosine receptor agonists is restricted to more mature compartments of hematopoietic progenitor and precursor cells.

Effect of conservative dive profiles on the occurrence of venous and arterial bubbles in divers with a patent foramen ovale: A pilot study

Patent foramen ovale (PFO) is a risk factor for decompression sick-ness(DCS)indiversduetoparadoxicalembolizationofnitrogenbubblesformed in peripheral blood during decrease of ambient pressure [1].Inour previous study we have demonstrated that catheter-based PFO clo-sure prevented right-to-left shunting of bubbles and might preventDCS recurrence [2]. However, the question of PFO closure is still debat-able [3].Also,randomizedclinicaldataarelackinginthis field.Therefore,the majority of divers are currently not referred for PFO closure, andvarious conservative dive profiles (CDP) are recommended to preventunprovoked DCS (i.e., without violation of decompression regimen) [4].Unfortunately, to date, the safety of these CDP has not been tested in di-verswithPFO.Theaimofthisstudywastotesttheeffectofdivetimeandascent rate restrictions on the occurrence of venous and arterial bubblesin diverswith PFO after simulated dives.We compareda standardly rec-ommended no-decompression dive [5] and a stricter regimen withslower ascent to the same control dive, which was previously used totest the efficacy of catheter-based PFO closure [2].Wescreenedatotalof 532 consecuti vediversforPFOusingtranscra-nialcolorcodedsonography(TCCS).ThediagnosisofPFOwascon firmedbytransesophagealechocardiography.Forty-sixdivers(36.4±10 years;72% men) with a significant PFO (grade 3 according to the internationalconsensus criteria [6]) who had previously not undergone PFO closurewere enrolled in this pilot study. All divers performed a simulated diveto 18 m in a hyperbaric chamber. Divers were randomized into threegroups: group A (n = 13; 36.5 ± 9 years; 77% men) performed a stan-dard Buhlmann regimen no-decompression dive (dive time 51 min,ascent rate 10 m/min), group B (n = 14, 40.9 ± 12 years; 64% men)performed the same regimen with a slower ascent (51 min, 5 m/min),and a control group (n = 19; 33.0 ± 8 years; 74% men) performed astaged-decompression dive according to the US Navy decompressionregimen (80 min, 9 m/min, decompression stop 7 min at 3 m). Within60 min of surfacing, the presence of venous and arterial bubbles wasassessed. Venous bubbles were assessed by pulse wave Doppler in therightventricularout flowtract (RVOT),andarterialbubbles byTCCS dur-ing native breathing and after Valsalva maneuvers, as described previ-ously [2]. The study was approved by the local ethics committee and allpatients signed an informed consent.In all divers, visualization of RVOT and the middle cerebral artery waspossible. The occurrence of arterial and venous bubbles is summarized inFig. 1. There was significantly lower occurrence of venous bubbles ingroups A and B compared to controls (for group A, 31% vs. 74%, p =0.03; for group B, 14% vs. 74%, p b 0.01). The reduction in arterial bubbleoccurrencewasnotsigni ficantingroupAcomparedtocontrols,buttherewas elimination of arterial bubbles in group B (for group A, 8% vs. 32%,p = 0.42;forgroupB,0%vs.32%,p= 0.03).Therewasnosigni ficantdif-ference in venous or arterial bubble occurrence between groups A and B(venous, 31% vs. 14%, p = 0.38; arterial, 8% vs. 0%, p = 0.48). All diverswere observed for any DCS symptoms 24 h after the simulated dive. Inthe control group transient neurological symptoms (headache, unusualfatigue, and transitory visual disturbances) were present in 21% of divers,no DCS symptoms were observed in group A (p = 0.13) or B (p = 0.12).Generally, the aim of our research is to stratify the risk of DCS indiverswithPFOandtofindtheoptimalmanagementstrategyforsymp-tomatic divers, including potential catheter-based PFO closure. In our

B-lymphopoiesis gains sensitivity to subsequent inhibition by estrogens during final phase of fetal development.

Adult B-lymphopoiesis is suppressed by the inhibitory effects of elevated estrogens during pregnancy. At the same time, hematopoietic cells in the fetal liver are resistant to this suppression by estrogens and ensure active production of B-cells. We investigated whether this unresponsiveness to estrogens of fetal cells also applies to cells obtained from a newborn liver and projects into the adult hematopoiesis when fetal liver cells are transplanted to adult mice. Mixtures of fetal liver (E14.5), neonatal liver (P0.5) and adult bone marrow (BM) cells were co-transplanted into adult primary and secondary recipients treated with high doses of estrogen in the Ly5.1/Ly5.2 congenic mouse model. Total chimerism as a proportion of all nucleated blood cells, chimerism as a proportion of B220+ B-cells, and of other blood cell lineages as well, were determined by flow cytometry. B-lymphopoiesis derived from fetal liver (E14.5) stem cells remained resistant to estrogen after transplantation into both primary and secondary adult recipients, for up to 280 days. In contrast, B-lymphopoiesis derived from neonatal liver (P0.5) stem cells was resistant to estrogen only for approximately 50 days after the primary transplantation to the adult BM microenvironment. These results provide further evidence for a critical developmental period of B-lymphopoiesis during its fetal liver stage. In the mouse, critical developmental events that allow for the subsequent expressed sensitivity of B-lymphopoiesis for suppression by estrogens after sexual maturation appear to occur during the period of late-stage fetal liver hematopoiesis before its migration to the bone marrow.

Thermoresponsive β-glucan-based polymers for bimodal immunoradiotherapy – Are they able to promote the immune system?

&NA; A conceptually new bimodal immunoradiotherapy treatment was demonstrated using thermoresponsive polymer &bgr;‐glucan‐graft‐poly(2‐isopropyl‐2‐oxazoline‐co‐2‐butyl‐2‐oxazoline) bearing complexes of 1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid with yttrium‐90(III) at the graft ends. The behavior of this thermoresponsive polymer in aqueous solutions was studied, and it showed the appropriate cloud point temperature for brachytherapy applications. The polymer was tested in vitro, and it exhibited nontoxicity and active uptake into cancer cells and macrophages with colocalization in the lysosomes and macrophagosomes. Moreover, the observed oxidative burst response of the leukocytes established the immunostimulatory properties of the polymer, which were also studied in vivo after injection into the thigh muscles of healthy mice. The subsequent histological evaluation revealed the extensive immune activation reactions at the site of injection. Furthermore, the production of tumor necrosis factor &agr; induced by the prepared polymer was observed in vitro, denoting the optimistic prognosis of the treatment. The biodistribution study in vivo indicated the formation of the polymer depot, which was gradually degraded and excluded from the body. The radiolabeled polymer was used during in vivo antitumor efficiency experiments on mice with EL4 lymphoma. The immunoradiotherapy group (treated with the radiolabeled polymer) demonstrated the complete inhibition of tumor growth during the beginning of the treatment. Moreover, 7 of the 15 mice were completely cured in this group, while the others exhibited significantly prolonged survival time compared to the control group. The in vivo experiments indicated the considerable synergistic effect of using immunoradiotherapy compared to separately using immunotherapy or radiotherapy. Graphical abstract Figure. No caption available.

Patent Foramen Ovale in Recreational and Professional Divers: An Important and Largely Unrecognized Problem

Patent foramen ovale (PFO) is associated with an increased risk of decompression sickness (DCS) in divers that results from a paradoxical embolization of nitrogen bubbles. The number of scuba divers worldwide is estimated in the millions, and the prevalence of PFO is 25%-30% in adults. It is interesting that despite these numbers, many important issues regarding optimal screening, risk stratification, and management strategy still remain to be resolved. Recently published data suggest the possible effectiveness of both PFO closure and conservative diving measures in preventing arterial gas embolization. This review aims to introduce the basic principles of physiology and the pathophysiology of bubble formation and DCS, summarize the current literature on PFO and diving, and review the possibilities of diagnostic workup and management.