Granulocyte maturation determines ability to release chromatin NETs and loss of DNA damage response; these properties are absent in immature AML granulocytes.

Terminally-differentiated cells cease to proliferate and acquire specific sets of expressed genes and functions distinguishing them from less differentiated and cancer cells. Mature granulocytes show lobular structure of cell nuclei with highly condensed chromatin in which HP1 proteins are replaced by MNEI. These structural features of chromatin correspond to low level of gene expression and the loss of some important functions as DNA damage repair, shown in this work and, on the other hand, acquisition of a new specific function consisting in the release of chromatin extracellular traps in response to infection by pathogenic microbes. Granulocytic differentiation is incomplete in myeloid leukemia and is manifested by persistence of lower levels of HP1γ and HP1ß isoforms. This immaturity is accompanied by acquisition of DDR capacity allowing to these incompletely differentiated multi-lobed neutrophils of AML patients to respond to induction of DSB by γ-irradiation. Immature granulocytes persist frequently in blood of treated AML patients in remission. These granulocytes contrary to mature ones do not release chromatin for NETs after activation with phorbol myristate-12 acetate-13 and do not exert the neutrophil function in immune defence. We suggest therefore the detection of HP1 expression in granulocytes of AML patients as a very sensitive indicator of their maturation and functionality after the treatment. Our results show that the changes in chromatin structure underlie a major transition in functioning of the genome in immature granulocytes. They show further that leukemia stem cells can differentiate ex vivo to mature granulocytes despite carrying the translocation BCR/ABL.

Influence of pulsed electromagnetic and pulsed vector magnetic potential field on the growth of tumor cells.

AIMS AND BACKGROUND: Tumor diseases cause 20% of deaths in Europe and they are the second most common cause of death and morbidity after cardiovascular diseases. Thus, tumor cells are target of many therapeutic strategies and tumor research is focused on searching more efficient and specific drugs as well as new therapeutic approaches. One of the areas of tumor research is an issue of external fields. In our work, we tested influence of a pulsed electromagnetic field (PEMF) and a hypothetic field of the pulsed vector magnetic potential (PVMP) on the growth of tumor cells; and further the possible growth inhibition effect of the PVMP. METHODS: Both unipolar and bipolar PEMF fields of 5 mT and PVMP fields of 0 mT at frequencies of 15 Hz, 125 Hz and 625 Hz were tested on cancer cell lines derived from various types of tumors: CEM/C2 (acute lymphoblastic leukemia), SU-DHL-4 (B-cell lymphoma), COLO-320DM (colorectal adenocarcinoma), MDA-BM-468 (breast adenocarcinoma), and ZR-75-1 (ductal carcinoma). Cell morphology was observed, proliferation activity using WST assay was measured and simultaneous proportion of live, early apoptotic and dead cells was detected using flow cytometry. RESULTS: A PEMF of 125 Hz and 625 Hz for 24 h-48 h increased proliferation activity in the 2 types of cancer cell lines used, i.e. COLO-320DM and ZR-75-1. In contrast, any of employed methods did not confirm a significant inhibitory effect of hypothetic PVMP field on tumor cells.

[Pulmonary hypertension - disease mechanisms]. / Plicní hypertenze - patofyziologické mechanizmy.

Pulmonary hypertension (PH) is known for its variable etiology. PH pathophysiology is very complex and our therapeutic options are limited. Most of known underlying disease mechanisms play a role across all etiological groups of PH, and they are followed by the same morphological and functional changes of pulmonary vasculature. Mostly, we are not able to determine whether one particular mechanism works as a cause or consequence in the chain of events. An imbalance between vasoconstriction and vasodilation becomes the major functional change of pulmonary vasculature in PH. The main morphological changes (termed together as "remodeling") include cell hyperplasia of pulmonary artery leading to its thickening and narrowing, and impaired regulation of extracellular matrix production leading to reduction in its elasticity. As a result of all these changes, the peripheral vascular resistance in pulmonary vascular bed rises, thus increasing afterload of the right ventricle and finally progressing to its failure. This review aims to summarize and explain the nature of the functional and histological changes in pulmonary arteries which occur in pulmonary hypertension, separately define the role of endothelium and pulmonary artery myocytes, and discuss the most important known pathophysiological mechanisms that lead to these changes.

Multiple myeloma patients at peripheral blood stem cell harvest: restricted usage of TCR beta variable families.

The immune systems of multiple myeloma patients are suppressed by the disease itself, and this immunosuppression is further enhanced by standard therapies. The aim of our study was to evaluate the effects of initial chemotherapy and a peripheral blood mobilisation regimen on T-cell population diversity. Reverse transcription-polymerase chain reaction (RT-PCR) with a new set of primers, in combination with capillary electrophoresis, was established. The methodology was used to analyse the relative expression of 27 T-cell receptor beta variable gene families (BV families) in multiple myeloma patients undergoing peripheral blood stem cell harvest. We found that the overall BV family usage in these patients was restricted; the relative expression of 10 BV families was significantly depressed in patients compared to healthy donors. These findings demonstrate that the preparative regimen for autologous stem cell transplantation affects the T-cell population in terms of the restriction of its T-cell receptor diversity.

Monoallelic and biallelic inactivation of TP53 gene in chronic lymphocytic leukemia: selection, impact on survival, and response to DNA damage.

Deletion of TP53 gene, under routine assessment by fluorescence in situ hybridization analysis, connects with the worst prognosis in chronic lymphocytic leukemia (CLL). The presence of isolated TP53 mutation (without deletion) is associated with reduced survival in CLL patients. It is unclear how these abnormalities are selected and what their mutual proportion is. We used methodologies with similar sensitivity for the detection of deletions (interphase fluorescence in situ hybridization) and mutations (yeast functional analysis) and analyzed a large consecutive series of 400 CLL patients; a subset of p53-wild-type cases (n = 132) was screened repeatedly during disease course. The most common type of TP53 inactivation, ie, mutation accompanied by deletion of the remaining allele, occurred in 42 patients (10.5%). Among additional defects, the frequency of the isolated TP53 mutation (n = 20; 5%) and the combination of 2 or more mutations on separate alleles (n = 5; 1.3%) greatly exceeded the sole deletion (n = 3; 0.8%). Twelve patients manifested defects during repeated investigation; in all circumstances the defects involved mutation and occurred after therapy. Monoallelic defects had a negative impact on survival and impaired in vitro response to fludarabine. Mutation analysis of the TP53 should be performed before each treatment initiation because novel defects may be selected by previous therapies.

[Toxoplasmosis of the central nervous systems after allogeneic stem cell transplantation]. / Toxoplazmóza centrálního nervového systému po alogenní transplantaci krvetvorných bunek.

Toxoplasmosis is a rare opportunistic protozoal infection, which may occur in patients after hematopoietic stem cell transplantation. This disease originates almost exclusively from reactivation of latent infection in seropositive recipients. We present a case report of one patient with diagnosis of acute myeloid leukemia undergoing two allogeneic stem cell transplantations at two years interval. The second transplantation was complicated by the development of the toxoplasmic encephalitis in early posttransplant course. The initial neurological symptoms included diplopia caused by the paresis of right side motor branches of the 3rd and 6th cranial nerves due to a compressive lesion in basal ganglia. Patient suddenly deteriorated after an epileptic seizure followed by a loss of consciousness, bilateral ptosis and right side mydriasis. Prolonged sopor and bilateral mydriasis appeared because of the further lesion progression in basal ganglia and compression of the 3rd cranial nerve. After targeted therapy of Toxoplasma gondii the patient's clinical status improved and she regained consciousness. Unfortunately, examination of bone marrow later revealed the relapse of leukemia. We compared risk factors of the latent reactivation of infection in immunocompromised patients with published data. It is of interest that the toxoplasmosis of the brain developed in this patient after the second transplantation.

Presence of heterozygous ATM deletion may not be critical in the primary response of chronic lymphocytic leukemia cells to fludarabine.

OBJECTIVES: Abnormalities of the TP53 or ATM, cooperating tumor-suppressor genes, significantly worsen the treatment options for chronic lymphocytic leukemia (CLL) patients. Although the aberrations seem to be mutually exclusive in this leukemia, inactivation of the former gene leads to worse prognosis. We tested the in vitro sensitivity of the CLL samples with heterozygous ATM deletion to fludarabine and combination of fludarabine and rituximab; the responses were compared with the TP53-abnormal and wild-type (wt) cells to delimitate relative significance of ATM deletion. METHODS: In vitro analysis was performed on fifty-nine characterized CLL samples using viability assay WST-1. Western blot and real-time RT-PCR were used to monitor the activation of the ATM/p53 pathway. RESULTS AND CONCLUSIONS: At the clinically relevant concentration of fludarabine, TP53-abnormal samples exhibited markedly higher resistance to fludarabine than the remaining CLL samples (P = 0.012); cohort with ATM deletion was not more resistant than wt cells. A similar induction of the p53 protein and its downstream target genes PUMA and BAX in ATM-deleted and wt cells confirmed that the former subgroup has preserved a critical pro-apoptotic response. Proportions of the samples, which had been sensitized to fludarabine by rituximab pretreatment, were insignificantly lower (P = 0.22) in the TP53-abnormal and ATM-deleted subgroups compared to the wt cases (30%; 29%; 50%, respectively). The presence of ATM (11q22-23) deletion in the CLL cells should not be considered an indication of resistance to fludarabine or its combination with rituximab.

Retention of nanoparticles-labeled bone marrow mononuclear cells in the isolated ex vivo perfused heart after myocardial infarction in animal model.

Cell therapy of myocardial infarction (MI) is under clinical investigation, yet little is known about its underlying mechanism of function. Our aims were to induce a sub-lethal myocardial infarction in a rabbit, to evaluate the abilities of labeled bone marrow mononuclear cells to migrate from the vessel bed into extracellular space of the myocardium, and to evaluate the short-term distribution of cells in the damaged left ventricle. Sub-lethal myocardial infarction was induced in rabbits by ligation of the left coronary vessel branch (in vivo). The Langendorff heart perfusion model (ex vivo) was used in the next phase. The hearts subjected to MI induction were divided into 3 groups (P1-P3), and hearts without MI formed a control group (C). Nanoparticles-labeled bone marrow mononuclear cells were injected into coronary arteries via the aorta. Perfusion after application lasted 2 minutes in the P1 group, 10 minutes in the P2 and C groups, and 25 minutes in the P3 group. The myocardium of the left ventricle was examined histologically, and the numbers of labeled cells in vessels, myocardium, and combined were determined. The numbers of detected cells in the P1 and C groups were significantly lower than in the P2 and P3 groups. In the P2 and P3 groups, the numbers of cells found distally from the ligation were significantly higher than proximally from the ligation site. Bone marrow mononuclear cells labeled with iron oxide nanoparticles proved the ability to migrate in the myocardium interstitium with significantly higher affinity for the tissue damaged by infarction.

Intracoronary delivery of bone marrow cells to the acutely infarcted myocardium. Optimization of the delivery technique.

OBJECTIVES: Intracoronary cell transplantation during catheter balloon inflations may be associated with adverse events. We studied the effectiveness of an alternative transplantation technique--intracoronary cell infusion. METHODS: Fourteen pigs, which had survived acute myocardial infarction, were randomized into 2 treatment groups and 2 controls. Three days after infarction, 12 pigs underwent allogeneic intracoronary mononuclear bone marrow cell transplantation using either the standard technique (short-term cell injections during repeat balloon inflations, technique A, n = 6) or continuous intracoronary cell infusion without balloon inflations (technique B, n = 6). Implanted cells were stained with fluorescent dye. After transplantation, the pigs were euthanized and myocardial samples were analyzed by fluorescent microscopy. RESULTS: The mean numbers of fluorescently labeled bone marrow cells in the infarction border zone, in the infarction mid-area and in the center of myocardial infarction were 84, 72 and 55 using technique A, and 29, 57 and 46 using technique B, respectively. The mean cell retention in the infarction border zone of 84 cells for technique A and 29 cells for technique B differed significantly (p = 0.034, two-tailed t test). CONCLUSION: The continuous intracoronary cell infusion technique is a less efficient cell delivery technique as compared with the standard technique using repeat intracoronary balloon inflations.

Cell therapy in patients with left ventricular dysfunction due to myocardial infarction.

OBJECTIVES: The purpose of this study was to determine the impact of autologous transplantation of mononuclear bone marrow cells on myocardial function in patients with left ventricular (LV) dysfunction due to an acute myocardial infarction. METHODS: The randomized study included 82 patients with a first acute myocardial infarction treated with a stent implantation. This presentation is a subanalysis of 47 patients with left ventricular dysfunction-EF (ejection fraction)

Different levels of CD52 antigen expression evaluated by quantitative fluorescence cytometry are detected on B-lymphocytes, CD 34+ cells and tumor cells of patients with chronic B-cell lymphoproliferative diseases.

BACKGROUND: The success of treatment using monoclonal antibodies in oncology is influenced by, among other factors, the level of target antigen expression on tumor cells. The authors analyzed the intensity of the CD52 antigen expression in patients with chronic lymphoproliferative diseases and compared them with B-lymphocytes of a healthy population and CD34(+) cells in peripheral blood stem cells (PBSC) grafts. METHODS: Recently diagnosed and previously untreated patients with B-cell chronic lymphocytic leukemia (B-CLL), mantle-cell lymphoma (MCL), or small lymphocytic lymphoma (SLL) were evaluated and compared with control group and CD34(+) cells. The intensity of CD52 was expressed in molecules of equivalent soluble fluorochrome units (MESF) and antibody-binding capacity (ABC). RESULTS: In the group of patients with B-CLL, the CD52 level on tumor cells (245 x 10(3) MESF; 107 x 10(3) ABC) was significantly lower than on B-lymphocytes of the control group (446 x 10(3) MESF; 194 x 10(3) ABC; P < 0.001) and SLL tumor cells (526 x 10(3) MESF; 229 x 10(3) ABC; P < 0.001). The CD52 antigen was expressed on a majority of CD34(+) cells, but its expression intensity was low (101 x 10(3) MESF; 44 x 10(3) ABC). CONCLUSIONS: Our data demonstrate differences in the intensity of the CD52 antigen expression between B-lymphocytes and tumor lymphocytes of B-CLL patients, and between B-CLL and SLL tumor cells. CD52 antigen is expressed at low level on CD34(+) cells.

Evaluation of CD34+ - and Lin- -selected cells from peripheral blood stem cell grafts of patients with lymphoma during differentiation in culture ex vivo using a cDNA microarray technique.

OBJECTIVE: Hematopoietic stem cells (enriched in fraction of CD34+ cells) have the ability to regenerate hematopoiesis in all of its lineages, and this potential is clinically used in transplanting bone marrow or peripheral blood stem cells. Our objective was to assemble a suitable method for evaluating gene expression in enriched populations of hematopoietic stem cells. We compared biologic properties of cells cultured ex vivo obtained using two different ways of immunomagnetic separation (positive selection of CD34+ cells and negative selection of Lin- cells) by means of a cDNA microarray technique. METHODS: CD34+ and Lin- cells were enriched from peripheral blood stem cell (PBSCs) grafts of patients with non-Hodgkin's lymphoma. Isolated cells were in the presence of cytokine PBSCs, Flt-3 ligand, interleukin-3, interleukin-6, and granulocyte colony-stimulating factor. At days 0, 4, 6, 8, 10, 12, and 14 cells were harvested and analyzed by cDNA microarrays. Total cell expansion, CD34+, colony-forming unit for granulocyte-macrophage and megakaryocytes expansion, vitality, and phenotype of cells were also analyzed. RESULTS: cDNA microarray analysis of cultured hematopoietic cells proved equivalence of the two enrichment methods for PBSC samples and helped us characterize differentiating cells cultured ex vivo. CONCLUSION: Our methodologic approach is helpful in characterizing cultured hematopoietic cells cultured ex vivo, but it is also suitable for more general purposes. Equivalence of CD34+ and Lin- selection methods from PBSC samples proved by cDNA microarray may have an implication for graft manipulation in an experimental setting of hematopoietic transplantation. Total cell expansion and colony formation and phenotype from CD34+ selected and from Lin- samples were comparable.

Angiomodulators in cancer therapy: New perspectives.

The formation of new blood vessels plays a crucial for the development and progression of pathophysiological changes associated with a variety of disorders, including carcinogenesis. Angiogenesis inhibitors (anti-angiogenics) are an important part of treatment for some types of cancer. Some natural products isolated from marine invertebrates have revealed antiangiogenic activities, which are diverse in structure and mechanisms of action. Many preclinical studies have generated new models for further modification and optimization of anti-angiogenic substances, and new information for mechanistic studies and new anti-cancer drug candidates for clinical practice. Moreover, in the last decade it has become apparent that galectins are important regulators of tumor angiogenesis, as well as microRNA. MicroRNAs have been validated to modulate endothelial cell migration or endothelial tube organization. In the present review we summarize the current knowledge regarding the role of marine-derived natural products, galectins and microRNAs in tumor angiogenesis.

Autologous transplantation of mononuclear bone marrow cells in patients with acute myocardial infarction: the effect of the dose of transplanted cells on myocardial function.

BACKGROUND: Despite the reports on successful treatment of acute myocardial infarction using autologous mononuclear bone marrow cell transplantation, many unresolved questions still remain. We studied the impact of the dose of transplanted cells on myocardial function and perfusion. METHODS: Sixty-six patients with a first acute myocardial infarction were randomized into 3 groups. Two groups were intracoronarily given mononuclear bone marrow cells in either higher (10(8) cells, higher cell dose [HD] group, n = 22) or lower (10(7) cells, lower cell dose [LD] group, n = 22) doses. Twenty-two patients without cell transplantation served as a control (C) group. RESULTS: At 3 months of follow-up, the baseline peak systolic velocities of longitudinal contraction of the infarcted wall of 5.2, 4.5, and 4.3 cm/s in C, LD, and HD groups increased by 0.0, 0.5 (P < .05 vs C group), and 0.9 cm/s (P < .05 vs LD group, P < .01 vs C group), respectively, as demonstrated by Doppler tissue imaging. Baseline left ventricular ejection fractions of 42%, 42%, and 41% in C, LD, and HD groups increased by 2%, 3%, and by 5% (P < .05 vs group C), respectively, as assessed by the gated technetium Tc 99m sestamibi single photon emission computed tomography. CONCLUSIONS: Mononuclear bone marrow cell transplantation improves regional myocardial function of the infarcted wall in a dose-dependent manner.

The role of quantitative Tc-99m-MIBI gated SPECT/F-18-FDG PET imaging in the monitoring of intracoronary bone marrow cell transplantation.

BACKGROUND: A lot of unresolved questions still exist concerning the exact mechanism of the beneficial effects of bone marrow cell (BMC) transplantation for myocardial regeneration. The aim of this communication is to report the cases of patients with and without post-transplantation left ventricular function improvement. MATERIAL AND METHODS: To this study we included consecutive patients with irreversible damage after a first acute ST-elevation myocardial infarction treated by coronary angioplasty with stent implantation. The irreversible damage was identified by dobutamine echocardiography and confirmed by rest gated Tc-99m-MIBI gated SPECT and in the majority of patients by F-18-FDG PET imaging as well. Using 4D-MSPECT software, we quantified MIBI/FDG uptake and gated SPECT left ventricular ejection fraction, end-diastolic/end-systolic volumes (LVEF, EDV/ESV) before BMC therapy and 3 months later. RESULTS: The results obtained in the initial group of patients in this study (27 patients in the BMC treated group, 16 patients in the control group) have been published previously [Eur J Nucl Med 2005; 32 (Suppl 1 ): S46]. Among the BMC group, we identified 13 responders to therapy with average LVEF improvement from 43.3% +/- 11% to 51.4% +/- 10.4% and EDV/ESV improvement from 145 ml/84 ml to 133 ml/67 ml. The remaining 14 patients were non-responders to therapy with no significant change in LVEF (39.1% +/- 8.1% versus 39.8% +/- 7.4%), the EDV/ESV increased from 166 ml/105 ml to 188 ml/116 ml. Responders to the cell therapy had prevailing MIBI uptake in the range of 31-50% of maximum in the infarction territory. On the other hand, non-responders to BMC therapy had prevailing MIBI uptake in the range of 0-30% of maximum. Two cases are presented in this report. CONCLUSIONS: Further studies with a larger cohort of patients would be helpful to evaluate our findings. We observed strong interindividual differences in the effectiveness of the cell therapy. Prevailing residual MIBI uptake in the range of 31-50% of maximum was in the subgroup of responders to the cell therapy.

Apelinergic system in endothelial cells and its role in angiogenesis in myocardial ischemia.

Apelin is a peptide known to have a vital role in cardiovascular diseases. It has been proven to induce proliferation and tube formation in endothelial cells, stabilise contacts between endothelial cells, and mediate pericyte recruitment. Since apelin level is reduced early after myocardial infarction, a supportive therapy with apelin is being investigated for its beneficial effect on blood vessel formation. It is becoming apparent, however, that the final effect of apelin often depends on stimuli the cell receives and the cross-talk with other molecules inside the cell. Hence, understanding the apelin pathway potentially can help us to improve angiogenic therapy. This review summarises recent knowledge regarding molecules involved in apelin signalling while focusing on their roles in angiogenesis within the ischemic environment after myocardial infarction.

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles.

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the target site while minimizing deleterious effects to off-target sites. Magnetic cell targeting is an efficient, safe, and straightforward delivery technique. Superparamagnetic iron oxide nanoparticles (SPION) are biodegradable, biocompatible, and can be endocytosed into cells to render them responsive to magnetic fields. The synthesis process involves creating magnetite (Fe3O4) nanoparticles followed by high-speed emulsification to form a poly(lactic-co-glycolic acid) (PLGA) coating. The PLGA-magnetite SPIONs are approximately 120 nm in diameter including the approximately 10 nm diameter magnetite core. When placed in culture medium, SPIONs are naturally endocytosed by cells and stored as small clusters within cytoplasmic endosomes. These particles impart sufficient magnetic mass to the cells to allow for targeting within magnetic fields. Numerous cell sorting and targeting applications are enabled by rendering various cell types responsive to magnetic fields. SPIONs have a variety of other biomedical applications as well including use as a medical imaging contrast agent, targeted drug or gene delivery, diagnostic assays, and generation of local hyperthermia for tumor therapy or tissue soldering.

Treatment of chronic myeloid leukemia with autologous transplantation using peripheral blood stem cells or bone marrow cultured in IL-2 followed by IL-2, GM-CSF, and IFN-alpha administration.

Interleukin-2 (IL-2) is able to generate nonspecific cytotoxic effectors from hematopoietic precursors. We evaluated the feasibility and efficacy of chronic myeloid leukemia (CML) treatment with autologous hematopoietic stem cell transplantation (HSCT) using grafts cultured in IL-2 followed by immunotherapy with IL-2, granulocyte-macrophage colony-stimulating factor (GM-CSF), and interferon (IFN)-alpha. Eight patients with CML were enrolled: five in an accelerated phase and three in a chronic phase. They received peripheral blood stem cells (PBSC) or bone marrow (BM) cultured in a medium containing IL-2 for 24 h. A median of 1.29 x 10(6) CD34+ cells/kg were infused after conditioning with busulfan (12 16 mg/kg) in PBSC recipients. BM was infused without prior myeloablative therapy. The engraftment occurred with a median of 15 d. Engraftment failure developed in one patient. The transplantation was followed by a 1-mo regimen of IL-2 (0.5 x 10(6) IU/m(2) daily) and GM-CSF, and 6 mo of IFN-alpha. One complete and one transient minor cytogenetic remission were observed. At 24 mo after transplantation, two patients had died of progressive disease and one of infection. Five patients had stable disease in the chronic phase. Autologous transplantation using IL-2-activated graft is feasible and the subsequent IL-2, GM-CSF, and IFN-alpha administration has acceptable toxicity. However, no benefits in comparison with conventional autologous transplantation for CML were identified in our study.