Bogusław Machaliński

Mobilization of bone marrow-derived Oct-4+ SSEA-4+ very small embryonic-like stem cells in patients with acute myocardial infarction.

OBJECTIVES This study sought to assess of the mobilization of nonhematopoietic very small embryonic-like stem cells (VSELs) in acute myocardial infarction (MI). BACKGROUND Acute MI induces mobilization of bone marrow stem cells. Recently, a rare population of VSELs, expressing markers of embryonic pluripotent stem cells (PSCs), was identified in adult murine bone marrow and human umbilical cord blood. METHODS Thirty-one patients with acute MI and 30 healthy subjects were enrolled. Blood was sampled on admission, after 24 h, and 5 days later. Erythrocytes were lysed and lin(-)CD45(-) VSELs were isolated using a live cell sorting system (FACSAria, Beckton Dickinson, San Jose, California). RESULTS In healthy subjects the median number of circulating VSELs was very low (median 0.8 [range 0 to 1.3]) cells/microl. In acute MI, VSELs were mobilized early (median 2.7 [range 0.2 to 3.9] cells/microl; p < 0.001) and remained elevated after 24 h and 5 days (median 4.7 [range 0.2 to 6.4] cells/microl; p < 0.003, and median 2.6 [range 0.3 to 3.6] cells/microl; p < 0.03, respectively). The mobilization of VSEL was significantly reduced in patients older than 50 years and with diabetes in comparison with younger and nondiabetic patients. Circulating VSELs were small (7 to 8 microm) and enriched in the messenger ribonucleic acid of PSC markers (Oct-4, Nanog), cardiac lineage (GATA-4, Nkx2.5/Csx, MEF2C), and endothelial (VE-cadherin) markers. The presence of PSC markers (Oct-4, SSEA-4) and the chemokine receptor CXCR4 in circulating VSELs was confirmed at the protein level by immunofluorescent staining and ImageStream system (Amnis Corporation, Seattle, Washington) analysis. CONCLUSIONS Acute MI induced mobilization of VSELs expressing pluripotent markers, early cardiac and endothelial markers, and chemokine receptor CXCR4.

Clinical Evidence That Very Small Embryonic-Like Stem Cells Are Mobilized Into Peripheral Blood in Patients After Stroke

Background and Purpose— In a murine model of stroke, we identified a population of very small embryonic-like (VSEL) stem cells (SCs) in adult murine bone marrow that could be mobilized into peripheral blood (PB). This raised the question of whether a similar population of cells is mobilized in human stroke patients. Methods— We evaluated a number of cells that corresponded to VSEL SCs in the PB of 44 stroke patients and 22 age-matched controls. After each patient’s stroke, PB samples were harvested during the first 24 hours, on day +3, and on day +7 and then compared with normal controls. The circulating human cells with the phenotype of VSEL SCs were evaluated in PB by real-time quantitative polymerase chain reaction, fluorescence-activated cell sorting analysis, and direct immunofluorescence staining. In parallel, we also measured the serum concentration of stromal derived factor-1 by ELISA. Results— In stroke patients, we found an increase in the number of circulating cells expressing SC-associated antigens, such as CD133, CD34, and CXCR4. More important, we found an increase in the number of circulating primitive cells expressing the VSEL phenotype (CXCR4+lin-CD45- small cells), mRNA for Octamer-4 and Nanog, and Octamer-4 protein. All changes were accompanied by an increased serum concentration of stromal derived factor-1. Additionally, we found a positive correlation between stroke extensiveness, stromal derived factor-1 concentration in serum, and the number of CXCR4+ VSEL SCs circulating in the PB. Conclusions— We conclude that stroke triggers the mobilization of CXCR4+ VSEL SCs that have potential prognostic value in stroke patients. However, the potential role of these mobilized cells in brain regeneration requires further study.

Human hematopoietic stem/progenitor‐enriched CD34+ cells are mobilized into peripheral blood during stress related to ischemic stroke or acute myocardial infarction

Abstract:  The hematopoietic and non‐hematopoietic stem/progenitor cells harvested directly from the bone marrow (BM) or G‐CSF mobilized peripheral blood were demonstrated to play an important role in regeneration of damaged organs ( 1, 2 ). Here, we asked if the stroke‐ or acute heart infarct‐related stress triggers mobilization of stem/progenitor‐enriched CD34+cells from the BM into the peripheral blood, which subsequently could contribute to regeneration of damaged tissues. To address this question the peripheral blood samples were harvested from patients with ischemic stroke during the first 24 h of manifestation of symptoms and on the second and sixth day afterwards or during the first 24 h of acute cardiac pain as well as on the second and sixth day of infarct. We measured in these patients (i) percentage of circulating hematopoietic stem/progenitor‐enriched CD34+ cells in peripheral blood by employing fluorescence activated cell sorter (FACS) and (ii) number of hematopoietic progenitor cells for the granulocyte‐monocytic colony‐forming unit (CFU‐GM) and erythoid burst‐forming unit (BFU‐E) lineages circulating in peripheral blood. We concluded that stress related to ischemic stroke or acute myocardial infarction triggers the mobilization of hematopoietic stem/progenitor‐enriched CD34+ cells from the BM into peripheral blood. These circulating stem/progenitor‐enriched CD34+ cells may contribute to the regeneration of ischemic tissues, however, this possibility requires further studies.

The expansion of CD4+CD28- T cells in patients with rheumatoid arthritis.

Clonal expansion of CD4+CD28- T cells is a characteristic finding in patients with rheumatoid arthritis (RA). Expanded CD4+ clonotypes are present in the peripheral blood, infiltrate into the joints, and persist for years. CD4+CD28- T cells are oligoclonal lymphocytes that are rare in healthy individuals but are found in high percentages in patients with chronic inflammatory diseases. The size of the peripheral blood CD4+CD28- T-cell compartment was determined in 42 patients with RA and 24 healthy subjects by two-color FACS analysis. The frequency of CD4+CD28- T cells was significantly higher in RA patients than in healthy subjects. Additionally, the number of these cells was significantly higher in patients with extra-articular manifestations and advanced joint destruction than in patients with limited joint manifestations. The results suggest that the frequency of CD4+CD28- T cells may be a marker correlating with extra-articular manifestations and joint involvement.

Plasma concentrations of TNF-α and its soluble receptors sTNFR1 and sTNFR2 in patients with coronary artery disease

Tumour necrosis factor alpha (TNF-alpha) is implicated in post-ischemic myocardial dysfunction. Two distinct TNF-alpha receptors are shed from cell membranes and circulate in plasma as soluble sTNFR1 and sTNFR2 proteins. The aim of the study was to establish factors associated with plasma concentrations of TNF-alpha and its receptors in patients with coronary artery disease (CAD). Since adenosine inhibits the expression of TNF-alpha, two functional polymorphisms in genes encoding enzymes participating in adenosine metabolism, i.e. AMP deaminase-1 (AMPD1, C34T) and adenosine deaminase (ADA, G22A), were analyzed. Plasma concentrations of TNF-alpha, sTNFR1, and sTNFR2 were measured using ELISA in 167 patients with CAD. Common factors significantly associated with higher TNF-alpha, sTNFR1, and sTNFR2 were lower glomerular filtration rate (GFR), older age, higher BNP, lower blood haemoglobin, and the presence of asthma or chronic obstructive pulmonary disease (COPD). Higher TNF-alpha and sTNFR1 concentrations were also associated with the presence of heart failure (HF), lower ejection and shortening fraction, the presence of diabetes or metabolic syndrome, lower serum HDL cholesterol, and higher uric acid. In multivariate analysis the common independent predictors of higher TNF-alpha, sTNFR1, and sTNFR2 were lower GFR, lower HDL cholesterol, higher BNP, and the presence of asthma or COPD. There were no associations between AMPD1 C34T or ADA G22A genotypes and TNF-alpha or its receptors. In conclusion, the concentrations of TNF-alpha, sTNFR1, and sTNFR2 reflect the impairment of cardiac and renal function in patients with CAD. Metabolic syndrome and diabetes are associated with higher plasma concentrations of TNF-alpha and its receptors.

An efficient two-step method to purify very small embryonic-like (VSEL) stem cells from umbilical cord blood (UCB).

The identification in murine bone marrow (BM) of very small embryonic-like (VSEL) stem cells, possessing several features of pluripotent stem cells, encouraged us to investigate if similar population of cells could be also isolated from the human umbilical cord blood (UCB). Here our approach to purify VSEL from human UCB is described by employing a two step isolation strategy based on i) hypotonic lysis of erythrocytes followed ii) by multi-parameter FACS sorting. Accordingly, first, erythrocytes are removed from the UCB samples by hypotonic ammonium chloride solution and next, the UCB mononuclear cells (UCB MNC) are stained with monoclonal antibodies against all hematopoietic lineages including the common leukocyte antigen CD45. The cells carrying these markers (lin+CD45+) are eliminated from the sort by electronic gating. At the same time the antibodies against CXCR4, CD34 and CD133 are employed as positive markers to enrich the UCB MNC for VSEL. This combined two step approach enables to purify VSEL stem cells, which are small and express mRNA for pluripotent stem cells (PSC) (Oct-4 and Nanog) and tissue-committed stem cells (TCSC) (Nkx2.5/Csx, VE-cadherin and GFAP) similarly to those isolated from the adult BM (3-5 microm cells with large nuclei).

Perinatal exposure to lead induces morphological, ultrastructural and molecular alterations in the hippocampus.

The aim of this paper is to examine if pre- and neonatal exposure to lead (Pb) may intensify or inhibit apoptosis or necroptosis in the developing rat brain. Pregnant experimental females received 0.1% lead acetate (PbAc) in drinking water from the first day of gestation until weaning of the offspring; the control group received distilled water. During the feeding of pups, mothers from the experimental group were still receiving PbAc. Pups were weaned at postnatal day 21 and the young rats of both groups then received only distilled water until postnatal day 28. This treatment protocol resulted in a concentration of Pb in rat offspring whole blood (Pb-B) below the threshold of 10 μg/dL, considered safe for humans.We studied Casp-3 activity and expression, AIF nuclear translocation, DNA fragmentation, as well as Bax, Bcl-2 mRNA and protein expression as well as BDNF concentration in selected structures of the rat brain: forebrain cortex (FC), cerebellum (C) and hippocampus (H). The microscopic examinations showed alterations in hippocampal neurons.Our data shows that pre- and neonatal exposure of rats to Pb, leading to Pb-B below 10 μg/dL, can decrease the number of hippocampus neurons, occurring concomitantly with ultrastructural alterations in this region. We observed no morphological or molecular features of severe apoptosis or necrosis (no active Casp-3 and AIF translocation to nucleus) in young brains, despite the reduced levels of BDNF. The potential protective factor against apoptosis was probably the decreased Bax/Bcl-2 ratio, which requires further investigation. Our findings contribute to further understanding of the mechanisms underlying Pb neurotoxicity and cognition impairment in a Pb-exposed developing brain.

Complement system activation and endothelial dysfunction in patients with age‐related macular degeneration (AMD): possible relationship between AMD and atherosclerosis

Age‐related macular degeneration (AMD) shares several pathological and epidemiological similarities with systemic atherosclerosis (AS). First, an association between AS and AMD is apparent from the analyses of the histological and biochemical structure of atherosclerotic plaques in the vascular walls and retinal drusen, the hallmark of AMD. Second, there is considerable evidence implicating endothelial dysfunction in the pathogenesis of both disorders, and cellular oxidative stress appears to be a common denominator underlying this process. Moreover, there are observations that the complement system (CS) triggering inflammatory response contributes to the onset and advancement of both diseases. The CS plays a role in the generation of drusen and neovascularization in AMD as well as in vascular endothelium activation, cell damage and ultimately atherosclerotic plaque formation in the course of systemic arteriosclerosis. It is widely recognized that both AMD and AS are not only related to local stimulation of the CS, but also result in its systemic activation. In addition, a specific Y402H polymorphism of the complement inhibitor factor H has been found to be associated with the incidence of both AMD and AS. Here, we propose a linking hypothesis between CS activation, endothelial dysfunction and the pathogenesis of two common and age‐related pathological processes, AS and AMD. We also discuss the potential therapeutic value of pharmacological modulation of CS activation in these disorders.

Elevated Plasma Levels of C3a Complement Compound in the Exudative Form of Age-Related Macular Degeneration

Aim: Recent findings suggest that chronic inflammatory processes play a role in the progression of age-related macular degeneration (AMD). Here we asked whether the development of different forms of AMD is connected with the elevation of plasma C3a-desArg concentration. Methods: We recruited 30 subjects with a clinical diagnosis of exudative AMD with newly diagnosed choroidal neovascularization (CNV), 30 subjects with dry AMD and 30 age- and sex-matched volunteers without AMD. The concentration of C3a-desArg complement compound was measured in the subjects’ peripheral blood. We evaluated the association between the level of C3a-desArg and age, sex, smoking, atherosclerosis, and hypertension. Results: We found that the levels of C3a-desArg were significantly elevated in patients with exudative AMD compared to the control group. The concentrations of C3a-desArg in patients with dry AMD were similar to those of controls. Additionally, patients and controls with documented atherosclerosis (AS) displayed significantly higher levels of C3a-desArg compared to subjects without AS. Conclusions: Our results suggest an association between systemic complement activation and the development of CNV. Moreover, we found an association of complement activation with atherosclerosis and confirmed the hypothesis that AMD can be a local manifestation of systemic disease.

The role of the STAT5 proteins in the proliferation and apoptosis of the CML and AML cells.

Objectives:  The STAT5 proteins are activated by many haematological cytokines and growth factors. They regulate cell cycle, apoptosis and proliferation of different cells via the influence on gene transcription. Because STAT5s are constitutively activated in certain haematooncologic diseases, they are suggested to play an important role in leukaemogenesis. However, the real function of these proteins in haematopoietic cell transformation and proliferation is not clear enough. The aim of this study was to evaluate the influence of suppression of STAT5A and STAT5B expression on the clonogenicity and apoptosis of the chronic myeloid leukaemia (CML) and acute myeloid leukaemia (AML) cells.