Erythrocytic phosphatidylserine exposure and hemostatic alterations in ß-thalassemia intermediate patients.

INTRODUCTION: Hypercoagulable state is one of the common findings in beta-thalassemia intermedia (ß-TI), particularly in splenectomized patients, with infrequent blood transfusion. Abnormality of the red blood cells (RBC) membrane due to oxidative damage is suggestive of possible etiologies. Membrane lipid peroxidation increases the exposure of phosphatidylserine (PS) that plays a role in the activation of coagulation factors V and X, subsequently initiating thrombosis. Our aim of this study was to find the probable correlation of the alteration of the PS on the RBC outer membrane with the hypercoagulable state in the ß-TI patients. MATERIALS AND METHODS: Our cross-sectional study was conducted on 39 splenectomized ß-TI patients and 38 age-matched healthy controls. The mean age was 37 years. Analysis of the PS exposure on the RBCs was performed by fluorescein isothiocyanate (FITC) conjugated AV protein. Measurement of the coagulation factors X, V and antithrombin III (AT-III) was performed. We also checked the D-dimer levels. Analysis was performed by SPSS16. RESULTS: Fluorescence of FITC-Annexin V labeling on patients RBCs were higher than healthy controls; (2.8 ± 2.2%) of the patients versus (0.4 ± 0.18%) in the control group and was statistically significant (P < 0.05). Mean levels of factor X and AT-III of the patients as compared with the control group decreased and showed significant difference (P < 0.05). CONCLUSIONS: Circulation of thalassemic RBCs, which abnormally possess PS on RBC membrane outer surface, suggests the possibility of the gradual consumption of the coagulation factors in the presence of a chronic coagulability state.

Mesenchymal stem cells as a feeder layer can prevent apoptosis of expanded hematopoietic stem cells derived from cord blood.

Umbilical cord blood (UCB) has been used for transplantation in the treatment of hematologic disorders as a source of hematopoietic stem cells (HSCs). Because of insufficient number of cord blood CD34(+) cells, the expansion of these cells seems to be important for clinical application. Mesenchymal stromal cells (MSCs), playing an important role in HSCs maintenance, were used as feeder layer. Apoptosis and cell cycle distribution of expanded cells were analyzed in MSCs co-culture and cytokine conditions and results were compared. Three culture conditions of cord blood HSCs were prepared ex-vivo for 14 days: cytokines (SCF, TPO and Flt3L) with MSCs feeder layer, cytokines without MSCs feeder layer and co-culture with MSCs without cytokines. Expansion was followed by measuring the total nucleated cells (TNCs), CD34(+) ( ) cells and colony-forming unit (CFU) output. Flow cytometry analysis of stained cells by annexin V and propidium iodide was performed for detection of apoptosis rate and cell cycle distribution in expanded cells. Maximum cord blood CD34(+) cells expansion was observed in day 10. The mean fold change of TNCs and CD34+ cells at day 10 in the co-culture system with cytokines was significantly higher than the cytokine culture without MSCs feeder layer and co-culture system without cytokines (n=6, p=0.023). The highest apoptosis rate and the least number of cells in Go/G1 phase were observed in cytokine culture without feeder layer (p=0.041). The expansion of cord blood HSCs on MSCs as a feeder layer resulted in higher proliferation and reduction in apoptosis rate.

Lipocalin 2 regulation by thermal stresses: protective role of Lcn2/NGAL against cold and heat stresses.

Environmental temperature variations are the most common stresses experienced by a wide range of organisms. Lipocalin 2 (Lcn2/NGAL) is expressed in various normal and pathologic conditions. However, its precise functions have not been fully determined. Here we report the induction of Lcn2 by thermal stresses in vivo, and its role following exposure to cold and heat stresses in vitro. Induction of Lcn2 in liver, heart and kidney was detected by RT-PCR, Western blot and immunohistochemistry following exposure of mice to heat and cold stresses. When CHO and HEK293T cells overexpressing NGAL were exposed to cold stress, cell proliferation was higher compared to controls. Down-regulatrion of NGAL by siRNA in A549 cells resulted in less proliferation when exposed to cold stress compared to control cells. The number of apoptotic cells and expression of pro-apoptotic proteins were lower in the NGAL overexpressing CHO and HEK293T cells, but were higher in the siRNA-transfected A549 cells compared to controls, indicating that NGAL protects cells against cold stress. Following exposure of the cells to heat stress, ectopic expression of NGAL protected cells while addition of exogenous recombinant NGAL to the cell culture medium exacerbated the toxicity of heat stress specially when there was low or no endogenous expression of NGAL. It had a dual effect on apoptosis following heat stress. NGAL also increased the expression of HO-1. Lcn2/NGAL may have the potential to improve cell proliferation and preservation particularly to prevent cold ischemia injury of transplanted organs or for treatment of some cancers by hyperthermia.

In vitro Evaluation of Cytarabin Induced Apoptosis in Leukemic Blasts.

Apoptosis, or active cell death, is a specific mode of cell death, which is characterized by morphological changes such as chromatin condensation, fragmentation of the nucleus, cytoplasmic retraction and appearance of apoptotic bodies' containing apparently intact organelles. Apoptosis occurs in physiological conditions as a regulatory mechanism of tissue growth, where cell proliferation is balanced. The aim of this research was to study the ability of Fas to initiate apoptosis in vitro before and after treatment with Cytarabin on tissue culture and to correlate the response. The human leukemia and normal cells were treated with cytarabin in tissue culture, and apoptotic treated cells were estimated by flow cytometry and phosphatidylserines kit. The results were analyzed by statistical tests (post hoc). From these data, it was found that Fas antigen was expressed in all cases, but the expression level varied widely. Apoptosis and also Fas antigen expression in short term cell culture were higher in media containing drug than in media without drug; but there had been no reasonable correlation between percentage of Fas antigen and apoptosis responses before culture.Expression of Fas antigen was low in most of the leukemic cells and the preliminary results showed that increase in Fas antigen expression (above 20%) after treatment, was a favorable prognostic outcome. It is associated with increase relapse, free and total survival. In addition, using this antigen as a chemotherapic and immunotherapic target, would initiate a new strategy for treatment of leukemia (chemotherapy and immunotherapy).