Factor XII Deficiency in Mexico: High Prevalence in the General Population and Patients with Venous Thromboembolic Disease.

INTRODUCTION: Thrombosis is one of the leading causes of morbidity and mortality worldwide. Venous thromboembolic disease (VTD) is considered a new epidemic. FXII deficiency is supposed to be a cause of thrombosis. To search for unknown causes of thrombosis in our population, our aim was to determine if FXII deficiency can be considered a risk factor for VTD. METHODS: Young adult Mexican patients with at least one VTD episode and healthy controls were included in this prospective, observational, controlled study. Liver and renal function tests, blood cytometry, and blood coagulation assays were performed. Plasma FXII activity and its concentration were evaluated. RESULTS: Over a two-year period, 250 patients and 250 controls were included. FXII activity was significantly lower in the control group compared to patients with VTD (p = 0.005). However, percentage of patients and controls with FXII deficiency was 8.8 and 9.2%, respectively (p = 1.000). No significant association was found between FXII deficiency and VTD (p = 1.0). FXII plasma concentration was lower in controls vs. patients with VTD: 4.05 vs. 6.19 ng/mL (p <0.001). Percentage of patients with low FXII plasma concentration was 1.6% and 6.0% in patients and controls, respectively (p = 0.010). CONCLUSIONS: FXII deficiency is a frequent finding in patients with VTD and controls in Mexico. Some patients with FXII deficiency had normal APTT result, an effect not described above. FXII plasma concentration was lower in patients with low activity.

Self-regulation of TNF-α Induces Dysfunction of Endothelial Colony-forming Cells from Patients with Venous Thromboembolic Disease.

BACKGROUND: Endothelial colony-forming cells (ECFCs) contribute to postnatal vasculogenesis. In venous thromboembolic disease (VTD), they are functionally abnormal and produce high concentrations of TNF-α. OBJECTIVE: To analyze the TNF-α signaling pathway and its relationship with the expression of cell-cycle regulators. METHODS: Mononuclear cells (MNCs) were collected from the peripheral blood of 20 healthy human volunteers (controls) and 30 patients with VTD matched by age (20-50 years) and sex to obtain ECFCs. We analyzed the relative quantification of the gene transcripts of TNF, NFkB1, PLAU, HMOX1, GSS, eNOS, CDKN1A, and CDKN1B through quantitative RT-PCR (qRT-PCR assays). Identification of NF-κB and activated targets of each pathway: NF-κB (Ser536); IκBα (Ser32/Ser36); p38 (Thr180/Tyr182) JNK (Thr183/Tyr185), p53 and cell-cycle regulators: p16, p18, p21, p27, p57, Cyclin D, Cyclin E, Cyclin A, Cyclin B, CDK2, CDK4; cell-cycle status was determined by KI-67 and 7-AAD. Cells were analyzed with flow cytometry and the FlowJo vX software. RESULTS: In ECFCs from VTD patients, TNF-α receptor and NFkB were overexpressed and hyper-phosphorylated; eNOS and HMOX1 were down-regulated; cell-cycle regulators (p53, p18, p21) were elevated. In addition, the cell cycle was locked in the G2 phase. CONCLUSIONS: Our results strongly suggest that these molecular alterations in the pathway of TNF-α and cell cycle regulation induce endothelial dysfunction, reduced proliferation potential and vascular regeneration, and consequently, the occurrence of new thrombotic events.

Cell Contact with Endothelial Cells Favors the In Vitro Maintenance of Human Chronic Myeloid Leukemia Stem and Progenitor Cells.

Chronic Myeloid Leukemia (CML) originates in a leukemic stem cell that resides in the bone marrow microenvironment, where they coexist with cellular and non-cellular elements. The vascular microenvironment has been identified as an important element in CML development since an increase in the vascularization has been suggested to be related with poor prognosis; also, using murine models, it has been reported that bone marrow endothelium can regulate the quiescence and proliferation of leukemic stem and progenitor cells. This observation, however, has not been evaluated in primary human cells. In this report, we used a co-culture of primitive (progenitor and stem) CML cells with endothelial colony forming cells (ECFC) as an in vitro model to evaluate the effects of the vascular microenvironment in the leukemic hematopoiesis. Our results show that this interaction allows the in vitro maintenance of primitive CML cells through an inflammatory microenvironment able to regulate the proliferation of progenitor cells and the permanence in a quiescent state of leukemic stem cells.

Abo Blood Group, Atherothrombotic Comorbidities, and COVID-19: A Case-Control Study of their Association in the Mexican Population.

BACKGROUND: COVID-19 has been associated with negative results in patients with A blood group and with a better evolution in O blood group individuals. AIM: Because the evidence regarding ABO blood groups and COVID was empirically not that clear in our country, we tested the association regarding COVID-19 and blood groups. MATERIAL AND METHODS: Adult patients were enrolled in this prospective, case-control, observational multicenter study. Patients with a confirmed diagnosis of COVID-19 were assigned to one of three groups based on the clinical presentation of the infection. Age, gender, ABO and Rh blood groups, body mass index, history of diabetes mellitus or high blood pressure, and smoking were recorded directly or from their clinical charts. ABO blood group was obtained from 5,000 blood donors (50% each gender). Atherothrombotic variables were compared with a nation-wide data collection. RESULTS: A total of 2,416 patients with COVID-19 were included (women:39.6%; men:60.4%). There were no significant differences between cases and controls in terms of age. O blood group was the most frequently found in healthy donors and COVID-19 patients, but this blood group was significantly higher in COVID-19 patients vs. healthy donors. ABO blood group was not associated with the final health status in COVID-19 patients. Obesity, diabetes mellitus, hypertension and smoking were significantly more frequent among COVID-19 patients. CONCLUSION: The proposed protective effect of the O blood group in COVID-19 patients could not be reproduced in the Mexican population while some atherothrombotic risk factors had a significant effect on the clinical evolution.

Genetic Polymorphisms Associated with Thrombotic Disease Comparison of Two Territories: Myocardial Infarction and Ischemic Stroke.

BACKGROUND AND PURPOSE: The thrombin-activatable fibrinolysis inhibitor (TAFI) is an important inhibitor of fibrinolysis and plays a critical role in the pathogenesis of arterial thrombosis; genetic polymorphisms of the TAFI gene affect its activity and increase the risk of thrombosis. Moreover, studies in young patients are still scarce. The aim was to examine the contribution of the Thr325Ile and Ala147Thr polymorphisms with ST acute myocardial infarction (STEMI) or idiopathic ischemic stroke (IIS) in the young Mexican population. METHODS: A total of 244 patients with STEMI ≤45 years of age and 244 controls. In a second study, 250 patients with IIS ≤45 years of age were recruited, including 250 controls. In both studies, cases and controls were matched by age and sex. The polymorphisms were determined in all participants by PCR-RFLP. RESULTS: There was significant difference in the Thr325Ile genotype distribution (P = 0.001) and allele frequency (P = 0.001) between STEMI and control groups, but no difference in the Ala147Thr genotype distribution (P = 0.24) and allele frequency (P = 0.46), neither in the Thr325Ile genotype distribution (P = 0.25) nor in the Ala147Thr genotype distribution (P = 0.46) or their allele frequencies; there was significant difference between IIS and the control group. There were independent factors for STEMI: the Ile allele (P = 0.01), type 2 diabetes mellitus (P = 0.001), hypertension (P = 0.001), smoking (P = 0.001), dyslipidemia (P = 0.001), and family history of atherothrombotic disease (P = 0.001). The independent factors for IIS were hypertension (P = 0.001), smoking (P < 0.01), and family history of atherothrombotic disease (P < 0.01). CONCLUSIONS: The Thr325Ile polymorphism, but no Ala147Thr polymorphism, represents an independent risk factor for STEMI in the young Mexican population.

Gene polymorphisms of angiotensin-converting enzyme and angiotensinogen and risk of idiopathic ischemic stroke.

OBJECTIVE: The renin-angiotensin system (RAS) is a hormonal signaling mechanism implicated in the atherosclerosis and regulation of blood pressure. Angiotensin-converting enzyme (ACE) a key enzyme in the RAS, plays important roles in vascular remodeling atherosclerosis, and ischemic stroke. The aim of this study was to examine the possible contribution of the I/D in the ACE gene, M235T and T174M in the angiotensinogen (AGT) gene polymorphisms with ischemic stroke in young Mexican population. MATERIALS AND METHODS: A total of 224 patients with diagnosis of idiopathic ischemic stroke ≤45 years of age, and 224 controls matched by age and gender, were recruited from 2006 and 2016. The I/D, M235T and T174M polymorphisms were determined in all participants by PCR-RFLP. RESULTS: There was a significant difference in the M235T genotype distribution (p = 0.01) and allele frequency between two groups (p = 0.01). Also, we found a significant difference in the T174M genotype distribution (p = 0.01) and the allele frequency between groups; (p = 0.02). In contrast, in I/D polymorphism, there was a similar genotype distribution; (p = 0.20) and allele distribution (p = 0.20). There were independent factors for ischemic stroke: M235T and T174M polymorphisms, smoking, hypertension, and familial history of atherothrombotic disease. The AGT levels were increased in the group of patients with stroke compared with the control group, but the AGT levels were not influenced by the allele or genotype in each polymorphism. CONCLUSIONS: The M235T and T174M polymorphisms represented an increased risk for stroke in young Mexican individuals. In contrast, the I/D was not associated with in the same group of patients. The AGT levels were higher in the acute phase of stroke, but it was not determined by the polymorphisms.

High cortactin expression in B-cell acute lymphoblastic leukemia is associated with increased transendothelial migration and bone marrow relapse.

Cancer is a major cause of death in children worldwide, with B-lineage cell acute lymphoblastic leukemia (B-ALL) being the most frequent childhood malignancy. Relapse, treatment failure and organ infiltration worsen the prognosis, warranting a better understanding of the implicated mechanisms. Cortactin is an actin-binding protein involved in cell adhesion and migration that is overexpressed in many solid tumors and in adult B-cell chronic lymphocytic leukemia. Here, we investigated cortactin expression and potential impact on infiltration and disease prognosis in childhood B-ALL. B-ALL cell lines and precursor cells from bone marrow (BM) and cerebrospinal fluid (CSF) of B-ALL patients indeed overexpressed cortactin. In CXCL12-induced transendothelial migration assays, transmigrated B-ALL cells had highest cortactin expression. In xenotransplantation models, only cortactinhigh-leukemic cells infiltrated lungs, brain, and testis; and they colonized more easily hypoxic BM organoids. Importantly, cortactin-depleted B-ALL cells were significantly less efficient in transendothelial migration, organ infiltration and BM colonization. Clinical data highlighted a significant correlation between high cortactin levels and BM relapse in drug-resistant high-risk B-ALL patients. Our results emphasize the importance of cortactin in B-ALL organ infiltration and BM relapse and its potential as diagnostic tool to identify high-risk patients and optimize their treatments.

[Polimorfismos asociados a disfunción endotelial y a un estado protrombótico en jóvenes mexicanos con infarto cerebral].

OBJECTIVE: To examine the contribution the polymorphisms G20210A, G1691A and G10976A in the coagulation factors FII, FV, FVII, respectively; Glu298Asp and C677T in eNOS and 5,10 MTHFR in young Mexican population with cerebral infarction (CI). METHODS: 224 patients ≤ 45 years of age with CI and 224 controls matched by age and gender were recruited from 2006 and 2014. The polymorphisms were determined by polymerase chain reaction-restriction fragment length polymorphism. RESULTS: We identified a significant difference in the genotype distribution of Glu298Asp (p = 0.001) and C677T (p = 0.01) polymorphisms between CI patients and control groups. The genotype distribution in the FII G20210A, FV G1691A and FVII G10976A polymorphisms were similar. There were independent factors for ischemic stroke: Glu298Asp and C677T polymorphisms, smoking; hypertension, and familial history of thrombotic disease. CONCLUSIONS: The Glu298Asp and C677T, but not FII G20210A, FV G1691A and FVII G10976A polymorphisms were associated with CI. Our results suggest that endothelial dysfunction and the synergist interaction with other factors such as smoking and hypertension contribute to CI in young individuals.

OBJETIVO: Examinar la contribución de los polimorfismos G20210A, G1691A y G10976A en los factores de coagulación FII, FV y FVII respectivamente; Glu298Asp y C677T en la óxido nítrico sintasa endotelial y 5,10 metilentetrahidrofolato reductasa, en población joven mexicana con infarto cerebral (IC). MÉTODO: Se incluyeron 224 pacientes ≤ 45 años de edad con diagnóstico de IC y 224 controles pareados por edad y sexo, de 2006 a 2014. Los polimorfismos fueron determinados por la técnica de reacción en cadena de la polimerasa-polimorfismos de longitud de fragmentos de restricción. RESULTADOS: Identificamos una diferencia significativa en la distribución genotípica de los polimorfismos Glu298Asp (p = 0.001) y C677T (p = 0.01) entre el grupo de pacientes con IC y el control. La distribución genotípica de los polimorfismos FII G20210A, FV G1691A y FVII G10976A fue similar entre ambos grupos. Se identificaron como factores independientes de IC los polimorfismos Glu298Asp y C677T, el tabaquismo, la hipertensión y el antecedente de familiar de enfermedad trombótica. CONCLUSIONES: Los polimorfismos Glu298Asp y C677T, pero no FII G20210A, FV G1691A y FVII G10976A, se asociaron con IC. Nuestros resultados sugieren que la disfunción endotelial en interacción sinérgica con otros factores de riesgo, como tabaquismo e hipertensión, contribuye al IC en individuos jóvenes.

Endothelial colony-forming cells: Biological and functional abnormalities in patients with recurrent, unprovoked venous thromboembolic disease.

INTRODUCTION: Endothelial cells (ECs) are an important component of the blood coagulation system because it maintains blood fluid. Because in patients with venous thromboembolic disease (VTD) a thrombophilic condition is not found sometimes, we investigated if endothelial colony-forming cells (ECFCs) from these patients have biological and functional abnormalities. PATIENTS AND METHODS: Human mononuclear cells (MNCs) were obtained from peripheral blood from patients with VTD and controls to obtain ECFCs. These cells were assayed for their immunophenotype and electron microscopy characteristics and their ability to form capillary-like structures and to produce pro-inflammatory and pro-angiogenic cytokines and reactive oxygen species (ROS). RESULTS: ECFCs appeared at 7 and 21 days of culture in VTD patients and controls, respectively. ECFCs increased 8-fold in patients and emerged 1 week earlier. No differences in the size of the colonies of ECFCs were found. Numbers and time of appearance of ECFCs was different between groups. ECFC-derived ECs (ECFC-ECs) of both groups expressed CD31, CD34, CD146, and CD-309 but none expressed CD45, CD14, or CD90. Interest CD34 was highly expressed in ECFC-ECs from patients. In both groups, ECFC-ECs showed similar capacity to form capillary-like structures but ECFC-ECs from patients had significant abnormalities in the mitochondrial membrane. We found a significant increase in ROS production in ECFC-ECs from patients. There were significant differences in cytokine profiles between VTD patients and controls. CONCLUSIONS: We found a dysfunctional state in ECFC from VTD patients resembling some characteristics of dysfunctional ECs. These findings may help to understand some pathophysiological aspects of VTD.

Effects of the contraceptive skin patch and subdermal contraceptive implant on markers of endothelial cell activation and inflammation.

Changes in blood coagulation factors may partially explain the association between hormonal contraceptives and thrombosis. Therefore, the likely effects of the contraceptive skin patch and subdermal contraceptive implant on levels of inflammatory markers and endothelial activation were analyzed. This was an observational, prospective, longitudinal, nonrandomized study composed of 80 women between 18 and 35 years of age who made the decision to use the contraceptive skin patch or subdermal contraceptive implant. vascular cell adhesion molecule-1 (VCAM-1), endothelial cell leukocyte adhesion molecule-1 (ELAM-1), von Willebrand factor (VWF), and plasminogen activator inhibitor type 1(PAI-1) as well as high-sensitivity C-reactive protein (hsCRP) were assayed before and after 4 months of use of the contraceptive method. VCAM-1, VWF, and PAI-1 remained unchanged in the contraceptive skin patch group; however, a significant increase in hsCRP (0.29-0.50 mg/dL; P =.012) and a significant decrease in ELAM-1 (44-25 ng/mL; P =.022) were observed. A significant diminution in VCAM-1 (463-362 ng/mL; P =.022) was also found in the subdermal contraceptive implant group. Our results strongly suggest that these contraceptive methods do not induce endothelial activation after 4 months of use. Increase in hsCRP levels was unrelated to changes in markers of endothelial activation.

Activated endothelial cells limit inflammatory response, but increase chemoattractant potential and bacterial clearance by human monocytes.

Inflammation is the normal immune response of vascularized tissues to damage and bacterial products, for which leukocyte transendothelial migration (TEM) is critical. The effects of cell-to-cell contact seen in both leukocyte and endothelial cells include cytoskeleton rearrangement, and dynamic expression of adhesion molecules and metalloproteinases. TEM induces expression of anti-apoptotic molecules, costimulatory molecules associated with antigen presentation, and pattern recognition receptors (PRR), such as TLR-4, in monocytes. However, little is known about how TLR-4 increment operates in monocytes during an inflammatory response. To understand it better, we used an in vitro model in which monocytes crossed a layer of IL-1ß stimulated Human Umbilical Vein Endothelial Cells (HUVEC). After TEM, monocytes were tested for the secretion of inflammatory cytokines and chemokines, their phenotype (CD14, CD16, TLR-4 expression), and TLR-4 canonical [Nuclear Factor kappa B, (NF-κB) pathway] and non-canonical [p38, extracellular signal-regulated kinases (ERK) 1/2 pathway] signal transduction induced by lipopolysaccharide (LPS). Phagocytosis and bacterial clearance were also measured. There was diminished secretion of LPS-induced inflammatory cytokines (IL-1ß, IL-6, and TNF-α) and higher secretion of chemokines (CXCL8/IL-8 and CCL2/MCP-1) in supernatant of TEM monocytes. These changes were accompanied by increases in TLR-4, CD14 (surfaces expression), p38, and ERK1/2 phosphorylated cytoplasmic forms, without affecting NF-κB activation. It also increased bacterial clearance after TEM by an O2 -independent mechanism. The data suggest that interaction between endothelial cells and monocytes fine-tunes the inflammatory response and promotes bacterial elimination.

Safety of the oral methionine load test: effects on the clinical performance and laboratory tests.

INTRODUCTION: Hyperhomocysteinemia is a prothrombotic risk factor. Homocysteine is evaluated during fasting and after an oral methionine load (OML). AIM: To determine the safety of the OML test according to the general performance status and clinical laboratory tests. We studied healthy nonsmoking volunteers and patients with several thrombotic conditions. Before and after receiving an OML, blood samples were obtained to perform several laboratory tests. We also evaluated acute and subacute adverse effects and 30-day associated morbidity and mortality. Of 353 individuals, three were eliminated because they did not tolerate the OML. We studied 175 healthy individuals and 175 patients without age differences. After OML, mild to moderate clinical abnormalities were recorded in 78 subjects (22.1%): nausea (n = 69; 88.5%), dizziness (n = 13; 16.7%) and decreased or increased blood pressure (n = 8; 10.2%). Nausea always disappeared after breakfast in affected individuals. Prevalence of complications was similar in patients and controls. No patient required hospitalization and there was no mortality during the 30-day study period. In conclusion, OML test had no significant undesirable effects on the clinical status or the general laboratory tests of patients and healthy controls. Some mild and moderate symptoms associated with OML tests were observed, and OML test did not negatively affect general laboratory tests. OML test is a safe diagnostic procedure in patients with previous thrombotic events (and with the consequent associated risk factors such as diabetes mellitus or dyslipidemia) and in healthy subjects.

[The cell cycle and its role in the biology of haematopoietic progenitor cells]. / El ciclo celular y su papel en la biología de las células progenitoras hematopoyéticas.

The cell cycle comprises a group of biological events that determine the metabolic activity of any cell, as well as its division and the generation of new daughter cells. It is involved in every cellular process, such asproliferation and differentiation. It has been shown that several molecular elements regulate the cell cycle, and that alterations in any of its phases and/or regulators could give rise to physiologic deficiencies, and even death. In mammals, the haematopoietic tissue is one of the most active in terms of cell replacement; thus, a tight cell cycle control, particularly at the level of stem and progenitors cells is crucial for optimal blood cell production. In the present article, we give a comprehensive overview of the major mechanisms and regulatory, molecules that participate in the cell cycle of mammalian cells, with particular emphasis on its role in the biology of primitive cells of the hematopoietic system.

El ciclo celular y su papel en la biología de las células progenitoras hematopoyéticas / The cell cycle and its role in the biology of haematopoietic progenitor cells

El ciclo celular constituye el conjunto de eventos que determinan la actividad metabólica de toda célula así como su división y la generación de células hijas. Este proceso está involucrado en todos los procesos celulares tales como proliferación y diferenciación. Se ha demostrado que diversos elementos moleculares participan en su regulación y que alteraciones en este proceso pueden conducir a trastornos fisiológicos e incluso a la muerte del organismo. En los mamíferos, uno de los tejidos con mayor recambio celular es el hematopoyético, por lo que se requiere un control muy estricto del ciclo celular, principalmente a nivel de células troncales y progenitoras para la óptima producción de células sanguíneas. En este artículo presentamos un panorama general de los principales mecanismos y factores involucrados en la regulación del ciclo celular, poniendo particular énfasis en su papel en la biología de las células hematopoyéticas primitivas.

The cell cycle comprises a group of biological events that determine the metabolic activity of any cell, as well as its division and the generation of new daughter cells. It is involved in every cellular process, such asproliferation and differentiation. It has been shown that several molecular elements regulate the cell cycle, and that alterations in any of its phases and/or regulators could give rise to physiologic deficiencies, and even death. In mammals, the haematopoietic tissue is one of the most active in terms of cell replacement; thus, a tight cell cycle control, particularly at the level of stem and progenitors cells is crucial for optimal blood cell production. In the present article, we give a comprehensive overview of the major mechanisms and regulatory, molecules that participate in the cell cycle of mammalian cells, with particular emphasis on its role in the biology of primitive cells of the hematopoietic system.

La transfección de la línea celular TF-1 con el proto-oncogen bcl-2 humano proporciona supervivencia a corto plazo en ausencia de GM-CSF sin cambiar el fenotipo / Transfection of the tf-1 cell line with the human protooncogen bcl-2 favors short term cell survival and does not change phenotype

La muerte celular programada (apoptosis) es un proceso controlado genéticamente. El producto del gen bcl-2 (Bcl-2) es una proteína anti apoptótica integrada a la membrana externa mitocondrial. Objetivo. El objetivo del presente trabajo fue valorar los efectos de la transfección de bcl-2 sobre la supervivencia, proliferación, fenotipo y morfología en las líneas celulares TF-1, TF-1 neo (control negativo) y TB-1 (TF-1 transfectada con bcl-2). Metodología. Para ello utilizamos el método siguiente: examen del nivel de expresión de los antígenos de superficie CD13, CD34 y c-Kit, respuesta celular al GM-CSF y capacidad de supervivencia luego del retiro del factor de viabilidad. La apoptosis fue evaluada mediante la observación del blebbing membranal a diferentes tiempos luego del retiro del suero, el factor de viabilidad GM-CSF y la tolerancia a toxinas presentes en Justicia spicigera. Resultados. La expresión ectópica de bcl-2 en las células TB-1 previno la muerte celular por apoptosis sin inducir un cambio importante en el fenotipo y morfología en ausencia de GM-CSF, suero o en presencia del extracto de Justicia spicigera. Consistente con la función de Bcl-2, encontramos que la proliferación de la línea celular TB-1 fue muy similar a la línea parental TF-1 en respuesta al GM-CSF y por lo tanto Bcl-2 no altera dicha función. El retiro de suero y GM-CSF al medio de cultivo de las líneas TF-1 y TF-1 neo, induce apoptosis en 36 h. Por el contrario, la línea TB-1 entra en apoptosis a 96 h bajo las mismas condiciones. Conclusiones. Reuniendo los resultados, éstos sugieren que Bcl-2 es un inhibidor de la apoptosis a corto plazo en la línea celular TB-1, no cambia la respuesta al GM-CSF y no afecta el nivel de expresión de otros antígenos de superficie como CD13, CD34 y c-Kit.