Oxidative stress due to radiation in CD34+ Hematopoietic progenitor cells: protection by IGF-1

Radiation exerts direct as well as indirect effects on DNA through the generation of reactive oxygen species (ROS). Irradiated hematopoietic progenitor cells (HPCs) experience DNA strand breaks, favoring genetic instability, due to ROS generation. Our aim was to study the effect of a range of radiation doses in HPCs and the possible protective mechanisms activated by insulin-like growth factor-1 (IGF-1). ROS generation was evaluated, in the presence or absence of IGF-1 in liquid cultures of human HPCs-CD34⁺ irradiated with 1-, 2- and 5-Gy X-rays, using a flow cytometry assay. Manganese superoxide dismutase (MnSOD) expression was studied by western blot analysis and visualized by an immunofluorescence assay. Apoptosis was estimated using the following assays: Annexin-V assay, DNA degradation assay, BCL-2/BAX mRNA and protein levels and caspase-9 protein immunofluorescence visualization. Viability and clonogenic potential were studied in irradiated HPCs. The generation of superoxide anion radicals at an early and a late time point was increased, while the hydrogen peroxide generation at a late time point was stable. IGF-1 presence further enhanced the radiation-induced increase of MnSOD at 24 h post irradiation. IGF-1 inhibited the mitochondria-mediated pathway of apoptosis by regulating the m-RNA and protein expression of BAX, BCL-2 and the BCL-2/BAX ratio and by decreasing caspase-9 protein expression. IGF-1 presence in culture media of irradiated cells restored the clonogenic capacity and the viability of HPCs as well. In conclusion, IGF-1 protects HPCs-CD34⁺ from radiation effects, by eliminating the oxidative microenvironment through the enhancement of MnSOD activation and by regulating the mitochondria-mediated pathway of apoptosis.     

An in vitro study,evaluating the effect of sunitinib and/or lapatinib on two glioma cell lines

Aim We sought to assess the effect of sunitinib and lapatinib applied either alone or in combination, on U87 and M059K glioma cells. Methods Both cell lines were cultured as recommended by the manufacturer. Sunitinib and lapatinib were applied, either separately or in combination, in the cultured cells after cell attachment at doses of 10nM, 100nM, 1 μM and 10 μM. To determine whether the agents affect the proliferation of glioma cells, the 3-[4,5-dimethylthiazol-2-yl]-2,5 dimethyltetrazolium bromide assay was used. Apoptosis was detected using annexin V/propidium iodide detection assay, migration assay was performed in 24-well microchemotaxis chambers. The release of MMPs into the culture medium of U87 and M059K cells was measured by zymography. Results Both agents, administered either alone or in combination, decreased cell proliferation in a dose-dependent manner 48 h after their application in both cell lines. The inhibition of their combination was statistically different than the inhibition of each agent alone. Apoptosis was increased and migration of U87 and M059K cells was inhibited either by each agent alone or their combination. MMPs levels remained unaffected by the application of both agents in U87 cells. However, MMP-9 and MMP-2 levels were decreased 48 h after treatment of M059K cells with sunitinib either alone or in combination with lapatinib. Conclusion Sunitinib and/or lapatinib appear to exhibit significant effects on proliferation, apoptosis and migration of glioma cells. When applied alone, sunitinib appears to be a more potent inhibitor than lapatinib.     

Functional estrogen receptors alpha and beta are expressed in normal human salivary gland epithelium and apparently mediate immunomodulatory effects

Salivary gland epithelial cells (SGECs) have been shown to participate in immunological responses and have been implicated in the pathogenesis of Sjögren's syndrome (SS). Experimental evidence from animal models indicates that estrogen deficiency may also participate in SS pathogenesis. However, the expression and functionality of the estrogen receptors alpha (ERα) and beta (ERβ) in normal human salivary epithelium is unknown. To investigate these points, formalin-fixed, paraffin-embedded specimens and cultured non-neoplastic SGEC lines derived from nine minor salivary gland (MSG) biopsies with normal histology were studied. Immunohistochemical analyses detected the epithelial expression of ERα, ERβ1, and ERβ2 protein isoforms both in MSG tissues and in cultured SGECs. Such epithelial expression was verified by immunoblotting of various ER proteins in cellular extracts of cultured SGECs (full-length-ERα, ERα-Δ3, ERβ1-long, ERβ1-short, and ERβ2-long isoforms). Estrogens did not induce growth or apoptosis in cultured SGECs. However, similarly to other cellular systems, treatment of cultured SGECs with estrogens (17β-estradiol and the ERα- and ERβ-selective agonists propylpyrazole-triol and diarylpropiolnitrile, respectively) inhibited the interferon-γ-inducible expression of intercellular adhesion molecule-1. This finding corroborated the functionality of ER expressed by SGEC. Our results suggest that salivary epithelium expresses constitutively functional ERα and ERβ proteins that apparently mediate immunomodulatory effects.     

Paving the way for successful islet encapsulation

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Pathogenesis of Sjögren's syndrome: what we know and what we should learn

Sj?gren's syndrome (SS) or autoimmune epithelitis is a prototype autoimmune disorder with unique features: a broad clinical spectrum that extends from local exocrinopathy to systemic disease and lymphoma development, and an easy access to the inflamed tissues (minor salivary glands; MSG), which enables the investigators to study the autoimmune processes. The autoimmune lesion consists of lymphocytic infiltrates that develop around the ducts and vary in severity and composition. T cells (mainly CD4(+)) are the dominant lymphocytes in mild MSG lesions, whereas B cells in severe ones. Th1 cytokines predominate in SS infiltrates, albeit Th2 and Th17 responses have been also reported. Notably, increased infiltration by IL-18(+) cells has been associated with parotid gland enlargement and C4-hypocomplementemia, which are adverse prognostic factors for lymphoma development. Even though SS pathogenesis has not been fully revealed, several aspects have been delineated. Among them, the key role of MSG epithelia in the initiation and perpetuation of local autoimmune responses is well-established and involves the capacity of epithelial cells to mediate the recruitment, homing, activation, proliferation and differentiation of immunocytes. In addition, genetic features, including certain HLA phenotypes and polymorphisms in genes encoding cytokines or factors implicated in cytokine signaling, environmental (such as viruses) and hormonal factors are thought to participate in disease pathogenesis. Herein, the known aspects of SS pathogenesis, as well as unmet issues are discussed.     

Aging impairs the mobilization and homing of bone marrow-derived angiogenic cells to burn wounds

Impaired wound healing in the elderly represents a major clinical problem. Delineating the cellular and molecular mechanisms by which aging impairs wound healing may lead to the development of improved treatment strategies for elderly patients with non-healing wounds. Neovascularization is an essential step in wound healing, and bone marrow-derived angiogenic cells (BMDACs) play an important role in vascularization. Using a mouse full-thickness burn wound model, we demonstrate that perfusion and vascularization of burn wounds were impaired by aging and were associated with dramatically reduced mobilization of BMDACs bearing the cell surface molecules CXCR4 and Sca1. Expression of stromal-derived factor 1 (SDF-1), the cytokine ligand for CXCR4, was significantly decreased in peripheral blood and burn wounds of old mice. Expression of hypoxia-inducible factor (HIF)-1α was detected in burn wounds from young (2-month-old), but not old (2-year-old), mice. When BMDACs from young donor mice were injected intravenously, homing to burn wound tissue was impaired in old recipient mice, whereas the age of the BMDAC donor mice had no effect on homing. Our results indicate that aging impairs burn wound vascularization by impairing the mobilization of BMDACs and their homing to burn wound tissue as a result of impaired HIF-1 induction and SDF-1 signaling.