Elba Reyes-Maldonado

Annexin A2 is involved in antiphospholipid antibody-mediated pathogenic effects in vitro and in vivo.

Antiphospholipid (aPL) antibodies recognize receptor-bound beta(2) glycoprotein I (beta(2)GPI) on target cells, and induce an intracellular signaling and a procoagulant/proinflammatory phenotype that leads to thrombosis. Evidence indicates that annexin A2 (A2), a receptor for tissue plasminogen activator and plasminogen, binds beta(2)GPI on target cells. However, whether A2 mediates pathogenic effects of aPL antibodies in vivo is unknown. In this work, we studied the effects of human aPL antibodies in A2-deficient (A2(-/-)) mice. A2(-/-) and A2(+/+) mice were injected with immunoglobulin G (IgG) isolated from either a patient with antiphospholipid syndrome (IgG-APS), a healthy control subject (IgG-normal human serum), a monoclonal anti-beta(2)GPI antibody (4C5), an anti-A2 monoclonal antibody, or monoclonal antibody of irrelevant specificity as control. We found that, after IgG-APS or 4C5 injections and vascular injury, mean thrombus size was significantly smaller and tissue factor activity was significantly less in A2(-/-) mice compared with A2(+/+) mice. The expression of vascular cell adhesion molecule-1 induced by IgG-APS or 4C5 in explanted A2(-/-) aorta was also significantly reduced compared with A2(+/+) mice. Interestingly, anti-A2 monoclonal antibody significantly decreased aPL-induced expression of intercellular cell adhesion molecule-1, E-selectin, and tissue factor activity on cultured endothelial cells. Together, these data indicate for the first time that A2 mediates the pathogenic effects of aPL antibodies in vivo and in vitro APS.

Characterization of the Adherent Cells Developed in Dexter‐Type Long‐Term Cultures from Human Umbilical Cord Blood

We have previously shown that when human umbilical cord blood (UCB) cells are cultured in standard Dexter‐type long‐term cultures (D‐LTC), adherent cells develop forming a discrete net on the bottom of the culture flask. The identity of such cells, however, has not been defined. Accordingly, the major goal of the present study was to characterize the adherent cells developed in standard UCB D‐LTC. Cultures were established from 14 UCB samples and from nine bone marrow (BM) samples, as controls. Both UCB and BM cultures were initiated with the same number of mononuclear cells (MNC) (2.5 × 106 MNC/ml). After three weeks in culture, adherent cell numbers in UCB D‐LTC were 24%‐30% of the numbers found in BM cultures. More than 90% of the adherent cells in UCB D‐LTC expressed the acid phosphatase enzyme, whereas no alkaline phosphatase‐positive cells were observed. This was in contrast to BM D‐LTC, in which alkaline and acid phosphatase were expressed by 60%‐75% and 20%‐45% of the adherent cells, respectively. Immunochemical analysis showed that CD61 (osteoclast marker) and Factor VIII (endothelial cell marker) were not expressed by the adherent cells developed in UCB cultures. Interestingly, the majority of such cells expressed CD1a (dendritic cell marker), CD14, CD68 and CD115 (antigens mainly expressed by macrophagic cells). When the cultures were supplemented with the recombinant cytokines epidermal growth factor, basic fibroblast growth factor, platelet‐derived growth factor or granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), only GM‐CSF had a significant positive effect on adherent cell number. In order to test for some functional properties of the adherent cells developed in culture, production of stem cell factor (SCF), interleukin 6 (IL‐6) and tumor necrosis factor‐α (TNF‐α) was assessed. IL‐6 and TNF‐α showed elevated levels in UCB D‐LTC, whereas SCF levels were always below detection. Finally, analysis of fibroblast progenitors (fibroblast colony‐forming units [CFU‐F]) showed that these cells were present in BM samples (6 CFU‐F/105 MNC) and were totally absent in UCB samples. Taken together, the results of the present study indicate that the vast majority of the adherent cells developed in standard UCB D‐LTC belong to the macrophage lineage and that fibroblasts seem to be absent. Interestingly, the high proportion of CD1a+ cells suggests that dendritic cells are also present in these cultures.

In Vivo Effects of an Inhibitor of Nuclear Factor-Kappa B on Thrombogenic Properties of Antiphospholipid Antibodies

Abstract:  It has been shown that endothelial cell (EC) activation and tissue factor (TF) upregulation in EC and monocytes by antiphospholipid antibodies (aPL Abs) leads to a prothrombotic state and involves translocation of nuclear factor‐kappa B (NF‐κB). Here we examined the effects of an NF‐κB inhibitor on aPL‐induced thrombosis, TF activity, and EC in vivo. We treated CD1 mice with IgG from a patient with antiphospholipid syndrome (IgG‐APS) or with control IgG (IgG‐NHS). The adhesion of leukocytes (number of white blood cells) to EC in cremaster muscle (as an indication of EC activation) as well as the size of an induced thrombus in the femoral vein of the mice were examined. Some mice in each group were infused with 10 μM MG132 (an inhibitor of NF‐κB). TF activity was determined using a chromogenic assay in homogenates of carotid arteries and in peritoneal cells of mice. In vivo, IgG‐APS increased significantly the number of white blood cells adhering to ECs (4.7 ± 2.2) when compared to control mice (1.5 ± 0.8), and these effects were significantly reduced when mice were pretreated with MG132 (0.8 ± 0.2). IgG‐APS increased significantly the thrombus size and MG132 inhibited that effect (93%). Treatment of the mice with IgG‐APS also induced significantly increased TF function in peritoneal cells and in homogenates of carotid arteries. Pretreatment of the mice with MG132 abrogated those effects significantly. Mice injected with IgG‐APS or with IgM‐APS with or without the inhibitor had medium–high titers of anticardiolipin antibodies in serum at the time of the surgical procedures. The data show that prothrombotic and proinflammatory properties of IgG‐APS and IgM‐APS are downregulated in vivo by an NF‐κB inhibitor. These findings may be important in designing new modalities of targeted therapies to treat thrombosis in patients with APS.

Association of the Plasminogen Activator Inhibitor-1 Gene 4G/5G Polymorphism With ST Elevation Acute Myocardial Infarction in Young Patients

INTRODUCTION AND OBJECTIVES To investigate the role of the 4G/5G polymorphism in the plasminogen activator inhibitor-1 (PAI-1) gene in patients with ST-elevation myocardial infarction (STEMI) aged < or =45 years and its influence on regulation of the plasma PAI-1 concentration. METHODS This case-control study included 127 consecutive patients aged < or =45 years with a diagnosis of STEMI who were admitted to a cardiovascular intensive care unit and 127 controls recruited between January 2006 and March 2007. Participants were genotyped for the 4G/5G polymorphism using the polymerase chain reaction and restriction fragment length polymorphism analysis, and their plasma PAI-1 concentrations were measured. Informed consent was obtained from all participants. RESULTS There was a significant difference in genotype distribution between the two groups (P< .002). The 4G allele occurred more frequently in the patient group (P=.032). In addition, there were significant independent associations between STEMI and the 4G allele (i.e., 4G/4G plus 4G/5G; odds ratio [OR]=2.29; 95% confidence interval [CI], 1.12-4.68; P=.022), smoking (OR=23.23; 95% CI, 8.92-60.47; P< .001), a family history of cardiovascular disease (OR=4.66; 95% CI, 2.06-10.52; P=.001) and hypertension (OR=5.42; 95% CI, 1.67-17.56; P=.005). The plasma PAI-1 concentration was higher in individuals who were homozygous for the 4G allele (P< .001). CONCLUSIONS The study findings indicate that the 4G allele is an independent risk factor for acute myocardial infarction in young patients, as are smoking, hypertension and a family history of inherited cardiovascular disease.

C6 knock-out mice are protected from thrombophilia mediated by antiphospholipid antibodies

Background: Complement activation plays a role in pathogenesis of the antiphospholipid syndrome (APS), but the involvement of the C5b-9 membrane attack complex (MAC) is unknown. Here we studied the effects of human polyclonal antiphospholipid (aPL) antibodies on thrombosis and tissue factor (TF) up-regulation in C6 deficient (C6−/−) mice. Methods: C6−/− mice or the wild-type C3H/HeJ (C6+/+) mice were injected twice with IgG-APS (n = 2) or IgM-APS (n = 1) isolated from APS patients or with the corresponding control immunoglobulins (Igs) of normal human serum, (NHS) (IgG-NHS or IgM-NHS). Then, the sizes of induced thrombi in the femoral vein were determined 72 hours after the first injection. Tissue factor was determined in homogenates of carotid arteries and in peritoneal macrophages. Results: Thrombus sizes were significantly larger in C6+/+ treated with IgG-APS1 or with IgG-APS2 or with IgM-APS when compared with C6+/+ mice treated with IgG-NHS or with IgM-NHS, respectively. The sizes of thrombi were significantly smaller in the C6−/− mice injected with IgG-APS1, IgG-APS2 or IgM-APS (p < 0.001), compared to their C6+/+ counterparts showing an important abrogation of thrombus formation in mice lacking C6. The TF expression and activity in the C6−/− mice treated with IgG-APS or IgM-APS were diminished when compared to C3H/HeJ (C6+/+) mice treated with the same Igs. All mice injected with IgG-APS and IgM-APS had medium-high titers of anticardiolipin (aCL) and anti-β2glycoprotein I (aβ2GPI) antibodies. Conclusions: These data indicate that the C6 component of the complement system mediates aPL-thrombogenic effects, underscoring an important pathogenic mechanism and indicating the possibility of inhibiting complement to ameliorate APS-related manifestations.

Role of CD4+CD25+highFoxp3+CD62L+ regulatory T cells and invariant NKT cells in human allogeneic hematopoietic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (HSCT) is the treatment of choice for some hematological diseases; however, graft-versus-host disease (GVHD) is still one of the most important and deleterious complications. Regulatory T cells and iNKT cells can decrease the incidence and severity of GVHD, while preserving the graft-versus-tumor response. In order to analyze the relationship between the transfused dose of these cells, the presence of GVHD and survival, 15 normal donors and 15 patients with hematological diseases who underwent allogeneic HSCT from HLA-identical siblings were studied. The mobilization and infused doses of valpha24-vbeta11(iNKT cells) lymphocytes and CD4+CD25+FoxP3+, CD4+CD25+FoxP3+CD62L+, regulatory T cells were analyzed. All patients were conditioned with busulfan and cyclophosphamide and received cyclosporine and methotrexate as GVHD prophylaxis. iNKT and FoxP3 cells were mobilized after G-CSF administration. Acute GVHD was present in 9 of 15 (60%) and cGVHD in 7 of 13 (54%) patients. Patients who received a dose <0.6 x 10(6)/kg of iNKT cells and >4 x 10(6)/kg of FoxP3 had better disease-free survival and overall survival. Individuals transfused with >1.1 x 10(6)/kg of FoxP3+ CD62L+ Treg cells had better overall survival. In conclusion, iNKT and Treg cells are mobilized with G-CSF in healthy donors and the dose of iNKT cells and FoxP3 and CD62L+ regulatory T cells is of clinical importance in human HSCT.

Clinical relevance of NK, NKT, and dendritic cell dose in patients receiving G-CSF-mobilized peripheral blood allogeneic stem cell transplantation

To analyze the relationship between the cellular composition of peripheral blood allografts and clinical outcome, we performed a prospective study in 45 adult patients who underwent allogeneic peripheral blood hematopoietic stem cell transplantation (HSCT) from a histocompatibility leukocyte antigen identical sibling donor for different hematological malignancies. The dose of CD34+, CD3+, CD4+, CD8+, and CD19+ lymphocytes, natural killer (NK) cells, natural killer T (NKT) cells, type 1 and type 2 dendritic cells (DC1 and DC2), as well as regulatory T (Treg) lymphocytes was analyzed. All patients were conditioned with busulphan and cyclophosphamide (BuCy2) ± VP-16 and received a short course of methotrexate and cyclosporin-A as graft-versus-host disease (GVHD) prophylaxis. Acute GVHD (aGVHD) was present in 9 of 43 (21%) patients, and chronic GVHD (cGVHD) developed in 18 of 39 (46%) patients. There was a significantly higher incidence of aGVHD in patients receiving more than 6×106/kg CD34+ cells. In univariate analysis, variables associated with better survival were as follows: a dose of less than 1.5×107/kg NKT cells and less than 1.7×106/kg DC2 for disease-free survival (DFS), and a dose of less than 3×107/kg NK cells, less than 1.5×107/kg NKT cells, less than 3×106/kg DC1, and less than 1.7×106/kg DC2 for overall survival (OS). In the Cox regression analysis, the dose of NKT cells was the only variable associated with better DFS, while the doses of NK, NKT, and CD34+ cells (less than 8×106/kg) were associated with better OS. In conclusion, different circulating cell populations, other than CD34+ cells, are also of relevance in predicting the clinical outcome after allogeneic peripheral blood HSCT.

Differences in BCL‐XL expression and STAT5 phosphorylation in chronic myeloid leukaemia patients

Chronic myelogenous leukaemia (CML) cells show expression of BCL‐XL, an anti‐apoptotic oncogene. This expression is induced by BCR‐ABL protein kinase through activation of the signal transducer and activator of transcription‐5 protein (STAT5). To date, however, the contribution of BCL‐XL and STAT5 to the transforming phenotype in CML is still unclear. This study was aimed at defining the status of activated STAT5 and BCL‐XL expression and their relation to BCR‐ABL rearrangement in CML cells derived from patients at different clinical stages. Twenty‐seven consecutive patients with CML were enrolled in the study. Peripheral blood mononuclear cells were lysed and subjected to immunoprecipitation and Western blotting to analyse phosphorylated STAT5. The p210 BCR‐ABL rearrangements were determined by reverse transcriptase‐polymerase chain reaction (RT‐PCR) and BCL‐XL expression by semi‐quantitative RT‐PCR. We found that increased transcription of BCL‐XL gene was associated with phosphorylated STAT5 in the majority of blast crisis patients and in a few accelerated and chronic phase patients. Moreover, BCL‐XL expression levels were found to be decreased in chronic phase, contrary to a marked increase in blast crisis. We found no difference in expression of BCL‐XL and phosphorylated STAT5 when related with b3a2 and b2a2 BCR‐ABL rearrangements. These results suggest that STAT5 activity and BCL‐XL overexpression may reflect a stage of differentiation among CML phases, and this could contribute to BCR‐ABL‐dependent transformation.

Adult, but not neonatal, human lymphoid progenitors respond to TLR9 ligation by producing functional NK-like cells

Remarkable progress has been made in characterizing factors controlling lineage fate decisions of primitive progenitors that initiate the lymphoid program in bone marrow. However, the understanding of neonatal/adult differences in environmental signals that influence differentiation pathway stability is still incomplete. Our recent findings suggest that Toll-like receptors provide a mechanism for producing cells of the innate immune system from early stages of lymphoid development in mice. We now show that both human early multilymphoid progenitors and more differentiated lymphoid progenitors from normal adult bone marrow express TLR9. Furthermore, they respond to its ligation by upregulating the expression of IL-15Rβ (CD122) and accelerating the production of functional natural killer (NK)-like cells. Proliferation of the presumed equivalent progenitor cells from umbilical cord blood was stimulated by CpG-containing oligonucleotides or herpes simplex virus, but the already robust NK-cell formation was unchanged. This new information adds to other known differences between neonatal and adult lymphoid progenitors and suggests only the latter replenish innate NK-like cells in response to Toll-like receptor agonists.

Undaria pinnatifida and Fucoxanthin Ameliorate Lipogenesis and Markers of Both Inflammation and Cardiovascular Dysfunction in an Animal Model of Diet-Induced Obesity

Brown algae and its carotenoids have been shown to have a positive influence on obesity and its comorbidities. This study evaluated the effect of Undaria pinnatifida and fucoxanthin on biochemical, physiological and inflammation markers related to obesity and on the expression of genes engaged on white adipose tissue lipid metabolism in a murine model of diet-induced obesity. The treatments improved energy expenditure, β-oxidation and adipogenesis by upregulating PPARα, PGC1α, PPARγ and UCP-1. Adipogenesis was also confirmed by image analysis of the retroperitoneal adipose tissue, by measuring cell area, perimeter and cellular density. Additionally, the treatments, ameliorated adipose tissue accumulation, insulin resistance, blood pressure, cholesterol and triglycerides concentration in serum, and reduced lipogenesis and inflammation by downregulating acetyl-CoA carboxylase (ACC) gene expression, increasing serum concentration and expression of adiponectin as well as downregulating IL-6 expression. Both fucoxanthin and Undaria pinnatifida may be considered for treating obesity and other diseases related.