Conjugated linoleic acid increases intracellular ROS synthesis and oxygenation of arachidonic acid in macrophages.

OBJECTIVE: Conjugated linoleic acids (CLAs) have potential antiatherosclerotic properties: they may inhibit atherosclerotic processes by reducing the intensity of inflammatory processes. However, in vivo studies have shown that the application of trans-10, cis-12 CLA in obese men increased their oxidative stress. The objective of this study was to determine whether CLA can lead to an increase in oxidative stress and to isoprostane synthesis in macrophages. METHODS: Monocytes from peripheral blood and human monocytic leukemia cells were used in this study. Monocytes were differentiated to macrophages, and were incubated with 30 microM cis-9, trans-11 CLA and trans-10, cis-12 CLA or linoleic acid for 2 days. In some experiments the inhibitors of peroxisome proliferator-activated receptor-alpha (PPAR-alpha) or respiratory chain were added. After incubation, synthesis of reactive oxygen species (ROS), total cellular concentration of adenosine triphosphate, concentration of 8-epi-prostaglandin F2 alpha, activity of cytoplasolic phospholipase A2 (cPLA2), activity of mitochondria, and expression of mRNA of PPAR-alpha were measured. RESULTS: In cells cultured with CLAs intercellular ROS synthesis increased. In this condition the mitochondrial energy potential was high, and the inhibitors of the respiratory chain and PPAR-alpha reduced ROS concentration. At the same time, the cPLA2 activity was abolished. In contrast, 8-iPF2 alpha III synthesis increased in CLA cells. CONCLUSION: Cultivation of cells with CLA leads to an increased ROS synthesis, partly by PPAR-alpha mechanism. An increase in ROS concentration and inhibition of cPLA2 activity can stimulate oxygenation of arachidonic acid and contribute to an increase in 8-epi-PF2 alpha III level and in the apoptosis process in macrophages.

Conjugated linoleic acids can change phagocytosis of human monocytes/macrophages by reduction in Cox-2 expression.

Prostaglandin E2 produced endogenously (by cyclooxygenases) can regulate macrophage phagocytosis. Cyclooxygenase activity reduction (mainly through inhibition of inducible Cox-2) can induce PGE2 synthesis depression and can activate the phagocytosis process. There are no reports in the literature explaining whether conjugated linoleic acid dienes (trans-10, cis-12 CLA and cis-9, trans-11 CLA) modify the phagocytic activity of human macrophages. For the purpose of this study, monocytes were isolated from venous blood, incubated for 7 days with 30 microM CLAs, and then (in some experiments) LPS (1 microg/mL) was added to the medium. Subsequently, monocyte/macrophage phagocytosis, NF-kappaB transcription factor activity, Cox-2 and PPARgamma mRNA expression (and the amounts of Cox-2 and PPARgamma proteins) and PGE2 synthesis were determined. Both CLA isomers increased macrophage phagocytosis through inhibition of Cox-2 expression (might by inactivation the NF-kappaB pathway). The inhibition of mRNA Cox-2 expression contributed (particularly with respect to trans-10, cis-12 CLA) to a decrease in protein Cox-2 synthesis and to reduction of prostaglandin E2 content in the cell. The inhibition of PGE2 synthesis (by CLA treatment) enhanced the phagocytosis process in macrophages.

The influence of STAT5 antisense oligodeoxynucleotides on the proliferation and apoptosis of selected human cutaneous T-cell lymphoma cell lines.

STAT5 (signal transducers and activators of transcription) are suggested to play a role in the pathogenesis of leukaemia and lymphoma; however, their influence on the growth of cutaneous T-cell lymphoma cells is not clear enough. The aim of our study was to analyse the function of STAT5 proteins in the proliferation and apoptosis of selected cutaneous T-cell lymphoma cell lines (HUT 78; PB-1; HUT 102B), using antisense oligodeoxynucleotide (ODN) strategy. RT-PCR and Western blot were applied to analyse the expression of STAT5 after incubation with antisense ODN (AS ODN). The effect of ODN pretreatment on the cell clonogenecity was analysed in methylcellulose cultures. The process of apoptosis was estimated using two different flow cytometry (FACScan) methods: (1) combined Annexin V/propidium iodide staining, (2) the TUNEL method. Perturbation of STAT5 expression reduced the proliferation of the PB-1 cells after a 24-h exposure to antisense ODNs. Prolonged exposure (72 h) decreased the growth of each examined cell line, especially after antisense STAT5A (AS STAT5A) treatment. Incubation with AS STAT5 induced apoptosis in the population of HUT 78 and PB-1 cells. STAT5s may play a significant role in the growth and the process of apoptosis of selected human cutaneous T-cell lymphoma cells.

Involvement of C3435T and G2677T multidrug resistance gene polymorphisms in release of cytokines from peripheral blood mononuclear cells treated with methotrexate and dexamethasone.

P-Glycoprotein is a cell membrane-associated protein that transports a variety of exogenous (including drugs) and endogenous substances. P-Glycoprotein may also be involved in transmembrane transport of some endogenous proteins; thus, it may have physiological function in cytokine transport. Previous studies suggested that P-glycoprotein expression is genetically determined. The aim of this study was to examine involvement of multidrug resistance gene (MDR1) C3435T and G2677T polymorphisms in release of cytokines from phythemaglutynin (PHA)-stimulated peripheral blood mononuclear cells, as well as treated with methotrexate or dexamethasone. The release of cytokines: interleukin-2 (IL-2), IL-4, IL-6, IL-10, interferon-gamma (INF-gamma) and tumor necrosis factor-alpha (TNF-alpha) was determined in supernatants of mononuclear cell cultures from 72 healthy subjects, measured by flow cytometry. The release of INF-gamma, IL-2, IL-4 and TNF-alpha in cultures from subjects with 2677(T-T) 3435(T-T) haplotype pair was significantly decreased as compared to subjects with other haplotypes. There were no statistically significant differences in release of IL-6 and IL-10. The results of this study suggest an association between C3435T and G2677T MDR1 polymorphisms and transmembrane transport of some cytokines. Although the studied polymorphisms may be in linkage with polymorphisms of other transporters involved in cytokine release, it seems that the present results indirectly indicate involvement of P-glycoprotein in transport of some cytokines. Moreover, determination of C3435T and G2677T MDR1 polymorphisms might be useful in response prediction to therapy with methotrexate and dexamethasone.

The effect of methotrexate and glucocorticosteroids on apoptosis of phythemaglutinin-stimulated mononuclear cells from peripheral blood.

Immunosuppressive drugs are widely used in the therapy of autoimmune disorders. Induction of apoptosis is a key mechanism to destroy autoreactive T cells and attenuate immune responses. The aim of the study was to evaluate the apoptotic properties of methotrexate (MTX) and glucocorticosteroids (GS), dexamethasone, methylprednisolone, cortisone in cultured phythemaglutinin-stimulated mononuclear cells (MNC) from healthy subjects. Apoptosis of MNC was measured using two methods: Annexin V and TUNEL. The increase in MTX and GS concentrations led to progressive increase in the percentage of apoptotic cells. There was no statistically significant difference in percentage of apoptotic cells between cultures with MTX + GS and MTX alone. The results suggest that MTX and GS did not act synergistically, the addition of GS to MTX did not enhance the pro-apoptotic properties of MTX.

Clinical relevance of thyroid dysfunction in human haematopoiesis: biochemical and molecular studies.

OBJECTIVE: Abnormalities in haematological parameters have been noted in patients with thyroid diseases. Nevertheless, the exact mechanism of thyroid hormones' (THs) action on human haematopoiesis is still not entirely clear. DESIGN: The influence of THs through TH receptors (TRalpha-1 and TRbeta-1) on haematopoiesis in patients with hypo- and hyperthyroidism was analysed. METHODS: TR gene expression at the mRNA and protein levels in human CD34(+)-enriched haematopoietic progenitor cells (HPCs) obtained from the peripheral blood of patients with thyroid disorders and healthy volunteers was analysed. The cell populations were also investigated for clonogenic growth of granulocyte macrophage-colony forming units and erythrocyte-burst forming units (BFU-E). The level of apoptosis was determined by annexin V/propidium iodide staining and quantitative RT-PCR. RESULTS: The studies revealed that hypo- and hyperthyroidism modify TR gene expression in HPCs in vivo. TH deficiency resulted in a decrease in total blood counts and clonogenic potential of BFU-E. In contrast, hyperthyroid patients presented increased clonogenic growth and BFU-E number and significantly higher expressions of cell cycle-regulating genes such as those for PCNA and cyclin D1. Finally, an increase in the frequency of apoptotic CD34(+)-enriched HPCs in hypo- and hyperthyroidism with a modulation of apoptosis-related genes was detected. CONCLUSIONS: The following conclusions were derived: i) TR expression in human haematopoietic cells depends on TH status, ii) both hypo- and hyperthyroidism significantly influence clonogenicity and induce apoptosis in CD34(+)-enriched HPCs and iii) the molecular mechanism by which THs influence haematopoiesis might provide a basis for designing novel therapeutic interventions in thyroid diseases.

Circulating stem cell populations in preterm infants: implications for the development of retinopathy of prematurity.

OBJECTIVE: To investigate the association among different circulating stem cell (SC) populations, the levels of selected growth factors and chemokines regulating SC migration in the peripheral blood, and the incidence of retinopathy of prematurity (ROP). METHODS: We evaluated 88 participants in this study: 29 preterm infants with ROP, 29 preterm infants without ROP, and 30 healthy full-term infants. Peripheral blood samples collected 10 weeks after delivery were analyzed using flow cytometry, immunofluorescence, real-time reverse transcriptase-polymerase chain reaction, and enzyme-linked immunosorbent assay. The following cell populations were analyzed: (1) lin⁻CXCR4(+)CD45⁻ (enriched in very small embryonic-like SCs), (2) lin⁻CXCR4(+)CD45(+) (enriched in hematopoietic SCs), and (3) CD34(+)CD133(+)CD144(+) (early endothelial progenitor cells) [lin indicates lineage]. The concentrations of vascular endothelial growth factor, basic fibroblast growth factor, hepatocyte growth factor, and stromal cell-derived factor 1 were measured in the plasma. RESULTS: The very small embryonic-like SCs and early endothelial progenitor cells expressing neural and endothelial markers were significantly increased in the preterm infants. The number of early endothelial progenitor cells in the peripheral blood was significantly greater in the preterm infants with ROP than in the preterm infants without ROP. An accompanying increase in the concentrations of vascular endothelial growth factor and hepatocyte growth factor was found in the peripheral blood of the preterm infants with ROP. No significant associations were found between hematopoietic SCs and ROP or prematurity. CONCLUSIONS: The increased number of early endothelial progenitor cells along with elevated levels of vascular endothelial growth factor and hepatocyte growth factor in preterm infants with ROP suggest that circulating vasculogenic factors may play a role in the development and progression of ROP. The increased number of very small embryonic-like SCs in preterm infants suggests that the development of immature tissues and organs, including the retina, may require a contribution of circulating SCs.

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).

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. MATERIAL AND METHODS: Blast cells from 34 newly diagnosed patients with CML and AML were used in our experiments. Antisense oligodeoxynucleotides (ODNs) were applied to block STAT5A and STAT5B at the mRNA level and the RT-PCR method was used to study STAT5 mRNA expression in the cells after incubation with ODNs. Moreover, Western blot analysis of the STAT5 proteins was performed. The effect of ODN pretreatment on cell clonogenicity in methylocellulose cultures was examined according to the type of oligodeoxynucleotide and the time of exposure. The induction of apoptosis in cells was also estimated by the Annexin V/PI staining and the TUNEL method using flow cytometry. RESULTS: Perturbation of STAT5 expression decreased proliferative potential of the CML and the AML blasts as well as enhanced their apoptosis (P < 0.05). CONCLUSIONS: Our studies showed that the STAT5 proteins may be critical in the regulation of growth and apoptosis of the CML and AML leukaemic cells.

The role of STAT5 proteins in the regulation of normal hematopoiesis in a cord blood model.

The signal transducers and activators of transcription - STAT5A and STAT5B - are responsible for the control of proliferation, differentiation and apoptosis, via their effect on gene expression. They are activated by the binding of many cytokines, growth factors and hormones to their receptors on the cell surface. Many of these cytokines regulate hematopoietic cell development; therefore, STAT5 proteins are suggested to play an important role in hematopoiesis. There are numerous contradictory reports available in the literature on the role of STAT5 in normal hematopoietic cell development; hence, the question of the real function of STAT5 proteins clearly requires further studies. The aim of our study was to evaluate the role of STAT5 in normal hematopoiesis using oligodeoxynucleotide (ODN) strategy against STAT5 mRNA. We employed the RT-PCR method to study STAT5 mRNA expression in cells after their incubation with ODNs. We analyzed the effect of blocking STAT5 proteins on the viability and clonogenecity of the CFU-GM (Colony Forming Unit of Granulocyte-Macrophages) and the BFU-E (Burst Forming Unit of Erythrocytes) obtained from human cord blood (CB). The clonogenic growth of the cells was assessed in methylcellulose cultures according to the type of oligodeoxynucleotides. We also attempted to estimate the level of apoptosis induced in cord blood mononuclear and CD34+ cells by employing different assays: i) Annexin V staining using flow cytometry (FACSCalibur); ii) terminal deoxynucleotidyltransferase dUTP nick end labeling (TUNEL); iii) analysis of Bax and Bcl-X(L) gene expression by RT-PCR. Perturbation of STAT5 expression with antisense oligodeoxynucleotides had no impact on the viability, clonogenecity and apoptosis of CB hematopoietic cells. Our results showed that STAT5 proteins do not play a significant role in the regulation of proliferation of normal hematopoietic cells derived from cord blood.