Hypofibrinolytic State in Subjects with Type 2 Diabetes Mellitus Aggravated by the Metabolic Syndrome before Clinical Manifestations of Atherothrombotic Disease.

Background. Metabolic and genetic factors induce plasminogen activator inhibitor type-1 (PAI-1) overexpression; higher PAI-1 levels decrease fibrinolysis and promote atherothrombosis. Aim. To assess PAI-1 antigen levels among subjects with type 2 diabetes mellitus (T2DM) plus Metabolic Syndrome (MetS) before clinical manifestations of atherothrombosis and the contribution of metabolic factors and 4G/5G polymorphism of PAI-1 gene on the variability of PAI-1. Methods. We conducted an observational, cross-sectional assay in a hospital in Mexico City from May 2010 to September 2011. MetS was defined by the International Diabetes Federation criteria. PAI-1 levels and 4G/5G polymorphism were determined by ELISA and PCR-RFLP analysis. Results. We enrolled 215 subjects with T2DM plus MetS and 307 controls. Subjects with T2DM plus MetS had higher PAI-1 levels than the reference group (58.4 ± 21 versus 49.9 ± 16 ng/mL, p = 0.026). A model with components of MetS explained only 12% of variability on PAI-1 levels (R2 = 0.12; p = 0.001), with ß = 0.18 (p = 0.03) for hypertension, ß = -0.16 (p = 0.05) for NL HDL-c, and ß = 0.15 (p = 0.05) for NL triglycerides. Conclusion. Subjects with T2DM plus MetS have elevated PAI-1 levels before clinical manifestations of atherothrombotic disease. Metabolic factors have a more important contribution than 4G/5G polymorphism on PAI-1 plasma variability.

CDKIs p18(INK4c) and p57(Kip2) are involved in quiescence of CML leukemic stem cells after treatment with TKI.

Chronic Myeloid Leukemia (CML) is sustained by a small population of cells with stem cell characteristics known as Leukemic Stem Cells that are positive to BCR-ABL fusion protein, involved with several abnormalities in cell proliferation, expansion, apoptosis and cell cycle regulation. Current treatment options for CML involve the use of Tirosine Kinase Inhibitor (Imatinib, Nilotinib and Dasatinib), that efficiently reduce proliferation proliferative cells but do not kill non proliferating CML primitive cells that remain and contributes to the persistence of the disease. In order to understand the role of Cyclin Dependent Kinase Inhibitors in CML LSC permanence after TKI treatment, in this study we analyzed cell cycle status, the levels of several CDKIs and the subcellular localization of such molecules in different CML cell lines, as well as primary CD34(+)CD38(-)lin(-) LSC and HSC. Our results demonstrate that cellular location of p18(INK4c) and p57(Kip2) seems to be implicated in the antiproliferative activity of Imatinib and Dasatinib in CML cells and also suggest that the permanence of quiescent stem cells after TKI treatment could be associated with a decrease in p18(INK4c) and p57(Kip2) nuclear location. The differences in p18(INK4c)and p57(Kip2)activities in CML and normal stem cells suggest a different cell cycle regulation and provide a platform that could be considered in the development of new therapeutic options to eliminate LSC.

GLU298ASP and 4G/5G Polymorphisms and the Risk of Ischemic Stroke in Young Individuals.

BACKGROUND: Polymorphisms in the endothelial nitric oxide synthase (eNOS) and in the plasminogen activator inhibitor -1 (PAI-1) genes have been implicated in stroke pathogenesis but results are still controversial. The aim of this study was to examine the possible contribution of Glu298Asp in the eNOS and 4G/5G in the PAI-1polymorphisms with ischemic stroke in a young Mexican population. MATERIALS AND METHODS: In a case-control study, conducted between January 2006 and June 2010, 204 patients ≤45 years of age with ischemic stroke and 204 controls matched by age and gender, were recruited. The Glu298Asp and 4G/5G polymorphisms were determined in all participants by polymerase chain reaction-restriction fragment length polymorphism. RESULTS: There was a significant difference in the Glu298Asp genotype distribution (P=0.001) and allele frequency between the two groups (P=0.001). The 4G/5G genotype distribution (P=0.40) and the allele frequency was similar between groups; (P=0.13). There were independent factors for ischemic stroke: Asp carriage (GluAsp+AspAsp) (P=0.02); smoking (P=0.01); hypertension (P=0.03), and familial history of atherothrombotic disease (P=0.04). CONCLUSIONS: The Asp allele from the Gu298Asp gene represents an independent risk factor for ischemic stroke in a young Mexican population. In contrast, the 4G/5G was not associated with an increased risk for this disease in the same group of patients, as previously has been demonstrated in other populations.

[Reference values for blood coagulation factor activity in the Mexican population].

INTRODUCTION: During the fluid phase of hemostasis, fibrinogen is converted into fibrin, but other hemostatic factors are required. Reference values of hemostatic factors are established by manufacturers producing reagents using individuals with a specific genetic background. OBJECTIVE: To establish reference values for hemostatic factors in the Mexican indigenous and Mestizo populations. MATERIAL AND METHODS: We carried out a cross-sectional, descriptive study of healthy adult Mexicans. Clotting activity was evaluated using coagulometric assays. Blood donors were informed about the nature of the study and informed consent was obtained prior to blood being drawn. The protocol was approved by the Ethics Committee of our institution. RESULTS: One hundred and twenty samples were assayed (60 females and 60 males). Fibrinogen was higher in mestizos and in females. Reference values for factor XII ranged from 40-170% in indigenous subjects and from 36-159% in mestizos. Factor VIII ranged from 57-160% in indigenous subjects and from 51-209% in mestizo subjects. Reference values for the other hemostatic factors were also clearly different from the commercial reference values. Reference values for hemostatic factors in the Mexican population are different from traditionally used commercial reference values. There were significant differences between indigenous and mestizo Mexicans in the concentration of hemostatic factors with a tendency among mestizos to have higher factor concentrations. Low levels of plasma factor XII are frequent and perhaps may represent a risk factor for thrombotic events. Using these reference values may individualize the reposition of factors in Mexican hemophiliac patients.

Lymphoid progenitor cells from childhood acute lymphoblastic leukemia are functionally deficient and express high levels of the transcriptional repressor Gfi-1.

Acute lymphoblastic leukemia (ALL) is the most frequent malignancy of childhood. Substantial progress on understanding the cell hierarchy within ALL bone marrow (BM) has been recorded in the last few years, suggesting that both primitive cell fractions and committed lymphoid blasts with immature stem cell-like properties contain leukemia-initiating cells. Nevertheless, the biology of the early progenitors that initiate the lymphoid program remains elusive. The aim of the present study was to investigate the ability of lymphoid progenitors from B-cell precursor ALL BM to proliferate and undergo multilineage differentiation. By phenotype analyses, in vitro proliferation assays, and controlled culture systems, the lymphoid differentiation potentials were evaluated in BM primitive populations from B-cell precursor ALL pediatric patients. When compared to their normal counterparts, functional stem and progenitor cell contents were substantially reduced in ALL BM. Moreover, neither B nor NK or dendritic lymphoid-cell populations developed recurrently from highly purified ALL-lymphoid progenitors, and their proliferation and cell cycle status revealed limited proliferative capacity. Interestingly, a number of quiescence-associated transcription factors were elevated, including the transcriptional repressor Gfi-1, which was highly expressed in primitive CD34⁺ cells. Together, our findings reveal major functional defects in the primitive hematopoietic component of ALL BM. A possible contribution of high levels of Gfi-1 expression in the regulation of the stem/progenitor cell biology is suggested.

In vitro cell cycle dynamics of primitive hematopoietic cells from human umbilical cord blood.

The goal of the present study was to investigate the specific way in which recombinant stimulatory cytokines modulate the cell cycle dynamics of primitive hematopoietic cells in vitro. A human cord blood-derived cell population, enriched for CD34(+) Lin(-) cells, was obtained by negative selection and cultured in liquid cultures, in the absence or presence of recombinant stimulatory cytokines. The proportion of cells in each phase of the cell cycle, as well as the expression of cyclin D3, cyclin-dependent kinase-4 (cdk4), p16, p21 and p27, was determined at different time points. At the onset of culture, the vast majority of the cells were in the G(0)/G(1) phase of the cell cycle. In the absence of cytokines, most cells remained in such a phase and no cell cycle activity was detected throughout the culture period, which correlated with the absence of population doublings. In the presence of cytokines, approximately four cell cycles, with a proportionate population doubling, were observed within the first 4 days of culture. In cultures incorporating cytokines, expression levels of cyclin D3 and cdk4 were higher than in their absence; in contrast, the levels of the cell cycle inhibitors p16 and p21 were higher in cultures without cytokines. Levels of p27 were also higher in the presence of cytokines. Our results indicate that the proliferation of primitive hematopoietic cells in liquid culture is promoted by recombinant cytokines via the induction of specific positive regulators of the cell cycle and down-regulation of particular cell cycle inhibitors.

Cell cycle differences in vitro between primitive hematopoietic cell populations from adult and umbilical cord blood.

Lineage-negative (Lin(-)) cell populations, obtained by negative selection from umbilical cord blood (UCB) and adult mobilized peripheral blood (aMPB), were cultured in serum-free liquid cultures supplemented with a mixture of seven stimulatory cytokines. On specific days, proliferation potential was assessed and cell cycle status was determined by DNA content. Expression of the cell cycle regulators cyclin D3 (cD3), cyclin-dependent kinase 4 (cdk4), p21(cip1/waf1) (p21), and p27(kip1) (p27) was also determined. As expected, UCB cells showed significantly higher proliferation potentials than aMPB cells, particularly during the first 7 days of culture. During this period of time, higher numbers of cell cycles were observed in UCB cells (7-9 cycles), as compared to aMPB cells (5-6 cycles). Higher levels of cD3, cdk4, and p27 were also detected in UCB cells. Our results confirm that UCB cells possess an intrinsically higher proliferation potential, as compared to aMPB cells, and suggest that such a biological difference is due, at least in part, to differences in cell cycle status. This, in turn, seems to result from the differential expression of cell cycle regulatory molecules.