La citometría de flujo aumenta la proporción de muestras valorables en líquido cefalorraquídeo con recuento celular normal en hemopatías malignas / Flow cytometry increases the proportion of valuable samples in cerebrospinal fluid with normal cell count in malignant blood diseases

INTRODUCCIÓN: El compromiso del líquido cefalorraquídeo (LCR) en hemopatías malignas es un marcador de mal pronóstico y es habitualmente estudiado por citometría de flujo o citología. Ocasionalmente, las muestras de LCR oligocelulares (≤ 5 céls/dL) pueden ser consideradas como no aptas para diagnóstico por la baja cantidad de eventos. Objetivo: Evaluar la proporción de muestras reportadas como valorables para diagnóstico obtenidas por citometría y citología en muestras de LCR oligocelular. Material y Métodos: Se seleccionaron 169 muestras de LCR oligocelular correspondientes a 115 pacientes con hemopatías malignas. Las muestras fueron obtenidas mediante punción lumbar en tubos acondicionados con EDTA y preservante celular (Transfix®). El inmunofenotipo se realizó con panel de 8 colores, 55 (32%) de las cuales se hizo con panel para pequeñas muestras (SST). En todos los casos se incluyó CD14 para identificación de monocitos y CD3 para linfocitos T. La adquisición se realizó en citómetro FACSCantoII® y el análisis en software Infinicyt®. Resultados: La proporción de muestras valorables fue mayor en citometría en comparación con la citología (98% vs 61%, p < 0,000). En la mayoría se identificaron linfocitos T (98%) y/o monocitos (90%). En las muestras con SST, la cantidad de eventos obtenida fue menor en muestras con < de 1 mL (140 vs 556, p < 0,001) y se logró identificar una mediana de 3 poblaciones celulares. Conclusión: La citometría proporciona una mayor cantidad de muestras valorables en los LCR paucicelulares en relación con la citología en muestras de LCR enviadas para estudio de compromiso de LCR por hemopatías malignas.

BACKGROUND: The alteration of cerebrospinal fluid (CSF) in hematologic neoplasms is a poor prognostic marker. The characteristics of CSF are usually analyzed by flow cytometry or cytology. However, paucicellular CSF samples (≤5 cells/dL) can sometimes be considered unsuitable for analysis due to the low number of events. Objective: To evaluate the proportion of samples reported as suitable for analysis obtained by cytometry (FCM) and cytology in paucicellular CSF samples. Material and Methods: 169 samples ofpaucicellular CSF corresponding to 115 patients with hematologic neoplasms were selected. The samples were obtained by lumbar puncture in tubes conditioned with EDTA and Transfix®. We characterized the immunophenotype ofCSF samples with an 8-color panel, and 55 samples (32%) were in a small sample tube (SST). In all cases, monocytes were identified by CD14 labeling and T lymphocytes by CD3 labeling. The acquisition was carried out in a FACSCantoII® cytometer, and the analysis was performed using Infinicyt® software. Results: The proportion of samples suitable for analysis was higher in FCM compared to cytology (98% vs 61%, p < 0.000). We identified the presence of T lymphocytes and/or monocytes in most samples (98% and 90%, respectively). In the SST samples, the number of events recorded in low-volume samples (< 1 mL) was lower than in samples with higher volume (140 vs 556, p < 0.001), with a median of identification of 3 cell populations. Conclusion: FCM allows the analysis of a higher proportion ofpaucicellular CSF samples than cytology in hematologic neoplasms study.

[Flow cytometry increases the proportion of valuable samples in cerebrospinal fluid with normal cell count in malignant blood diseases]. / La citometría de flujo aumenta la proporción de muestras valorables en líquido cefalorraquídeo con recuento celular normal en hemopatías malignas.

BACKGROUND: The alteration of cerebrospinal fluid (CSF) in hematologic neoplasms is a poor prognostic marker. The characteristics of CSF are usually analyzed by flow cytometry or cytology. However, paucicellular CSF samples (≤5 cells/dL) can sometimes be considered unsuitable for analysis due to the low number of events. OBJECTIVE: To evaluate the proportion of samples reported as suitable for analysis obtained by cytometry (FCM) and cytology in paucicellular CSF samples. MATERIAL AND METHODS: 169 samples ofpaucicellular CSF corresponding to 115 patients with hematologic neoplasms were selected. The samples were obtained by lumbar puncture in tubes conditioned with EDTA and Transfix®. We characterized the immunophenotype ofCSF samples with an 8-color panel, and 55 samples (32%) were in a small sample tube (SST). In all cases, monocytes were identified by CD14 labeling and T lymphocytes by CD3 labeling. The acquisition was carried out in a FACSCantoII® cytometer, and the analysis was performed using Infinicyt® software. RESULTS: The proportion of samples suitable for analysis was higher in FCM compared to cytology (98% vs 61%, p < 0.000). We identified the presence of T lymphocytes and/or monocytes in most samples (98% and 90%, respectively). In the SST samples, the number of events recorded in low-volume samples (< 1 mL) was lower than in samples with higher volume (140 vs 556, p < 0.001), with a median of identification of 3 cell populations. CONCLUSION: FCM allows the analysis of a higher proportion ofpaucicellular CSF samples than cytology in hematologic neoplasms study.

Overexpression of LOXIN Protects Endothelial Progenitor Cells From Apoptosis Induced by Oxidized Low Density Lipoprotein.

Human endothelial progenitor cells (hEPC) are adult stem cells located in the bone marrow and peripheral blood. Studies have indicated that hEPC play an important role in the recovery and repair of injured endothelium, however, their quantity and functional capacity is reduced in several diseases including hypercholesterolemia. Recently, it has been demonstrated that hEPC express lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) and its activation by oxidized low-density lipoprotein (ox-LDL) induces cellular dysfunction and apoptosis. This study aimed to investigate whether overexpression of LOXIN, a truncated isoform of LOX-1 that acts as a dominant negative, plays a protective role against ox-LDL-induced apoptosis in hEPC. Human endothelial progenitor cells exposed to ox-LDL showed a significant increase in LOX-1 expression, and apoptosis began at ox-LDL concentrations above 50 µg/mL. All hEPC apoptosed at 200 µg/mL ox-LDL. High LOXIN expression was generated using adenoviral systems in hEPC and SiHa cells transduced with 100 colony-forming units per cell. Transduced LOXIN localized to the plasma membrane and blocked ox-LDL uptake mediated by LOX-1. Overexpression of LOXIN protected hEPC from ox-LDL-induced apoptosis, and therefore maybe a novel way of improving hEPC function and quantity. These results suggest that adenoviral vectors of LOXIN may provide a possible treatment for diseases related to ox-LDL and vascular endothelium dysfunction, including atherosclerosis.

Endothelium trans differentiated from Wharton's jelly mesenchymal cells promote tissue regeneration: potential role of soluble pro-angiogenic factors.

BACKGROUND: Mesenchymal stem cells have a high capacity for trans-differentiation toward many adult cell types, including endothelial cells. Feto-placental tissue, such as Wharton's jelly is a potential source of mesenchymal stem cells with low immunogenic capacity; make them an excellent source of progenitor cells with a potential use for tissue repair. We evaluated whether administration of endothelial cells derived from mesenchymal stem cells isolated from Wharton's jelly (hWMSCs) can accelerate tissue repair in vivo. METHODS: Mesenchymal stem cells were isolated from human Wharton's jelly by digestion with collagenase type I. Endothelial trans-differentiation was induced for 14 (hWMSC-End14d) and 30 (hWMSC-End30d) days. Cell phenotyping was performed using mesenchymal (CD90, CD73, CD105) and endothelial (Tie-2, KDR, eNOS, ICAM-1) markers. Endothelial trans-differentiation was demonstrated by the expression of endothelial markers and their ability to synthesize nitric oxide (NO). RESULTS: hWMSCs can be differentiated into adipocytes, osteocytes, chondrocytes and endothelial cells. Moreover, these cells show high expression of CD73, CD90 and CD105 but low expression of endothelial markers prior to differentiation. hWMSCs-End express high levels of endothelial markers at 14 and 30 days of culture, and also they can synthesize NO. Injection of hWMSC-End30d in a mouse model of skin injury significantly accelerated wound healing compared with animals injected with undifferentiated hWMSC or injected with vehicle alone. These effects were also observed in animals that received conditioned media from hWMSC-End30d cultures. CONCLUSION: These results demonstrate that mesenchymal stem cells isolated from Wharton's jelly can be cultured in vitro and trans-differentiated into endothelial cells. Differentiated hWMSC-End may promote neovascularization and tissue repair in vivo through the secretion of soluble pro-angiogenic factors.

Role of lectin-like oxidized low density lipoprotein-1 in fetoplacental vascular dysfunction in preeclampsia.

The bioavailability of nitric oxide (NO) represents a key marker in vascular health. A decrease in NO induces a pathological condition denominated endothelial dysfunction, syndrome observed in different pathologies, such as obesity, diabetes, kidney disease, cardiovascular disease, and preeclampsia (PE). PE is one of the major risks for maternal death and fetal loss. Recent studies suggest that the placenta of pregnant women with PE express high levels of lectin-like oxidized LDL receptor-1 (LOX-1), which induces endothelial dysfunction by increasing reactive oxygen species (ROS) and decreasing intracellular NO. Besides LOX-1 activation induces changes in migration and apoptosis of syncytiotrophoblast cells. However, the role of this receptor in placental tissue is still unknown. In this review we will describes the physiological roles of LOX-1 in normal placenta development and the potential involvement of this receptor in the pathophysiology of PE.

Is a low level of free thyroxine in the maternal circulation associated with altered endothelial function in gestational diabetes?

Synthesis of thyroid hormones, thyroxine (T4) and tri-iodothyronine (T3), in the human fetus starts from 17 to 19th weeks of gestation. Despite the majority of normal pregnant women reaching adequate levels of circulating thyroid hormones, in some cases, women with normal pregnancies have low level of free T4 during first trimester of pregnancy, suggesting that T4 action may be compromised in those women and their fetuses. In addition, pathological low levels of thyroid hormones are detected in isolated maternal hypothyroxemia (IMH) and clinical hypothyroidism. Nevertheless, human placenta regulates T3/T4 concentration in the fetal circulation by modulating the expression and activity of both thyroid hormone transporters (THT) and deiodinases. Then, placenta can control the availability of T3/T4 in the feto-placental circulation, and therefore may generate an adaptive response in cases where the mother courses with low levels of T4. In addition, T3/T4 might control vascular response in the placenta, in particularly endothelial cells may induce the synthesis and release of vasodilators such as nitric oxide (NO) or vasoconstrictors such as endothelin-1 mediated by these hormones. On the other hand, low levels of T4 have been associated with increase in gestational diabetes (GD) markers. Since GD is associated with impaired placental vascular function characterized by increased NO synthesis in placental arteries and veins, as well as elevated placental angiogenesis, it is unknown whether reduced T4 level at the maternal circulation could result in an altered placental endothelial function during GD. In this review, we analyze available information regarding thyroid hormones and endothelial dysfunction in GD; and propose that low maternal levels of T4 observed in GD may be compensated by increased placental availability of T3/T4 via elevation in the activity of THT and/or reduction in deiodinases in the feto-placental circulation.

L-arginine transport and nitric oxide synthesis in human endothelial progenitor cells.

Nitric oxide (NO) is an endogenous vasodilator molecule synthetized from L-arginine by a family of nitric oxide synthases. In differentiated human endothelial cells, it is well known that L-arginine uptake via cationic amino acid transporters (y(+)/CAT) or system y(+)L is required for the NO synthesis via endothelial nitric oxide synthase, but there are no reports in human endothelial progenitor cell (hEPC). Therefore, we isolated hEPCs from peripheral blood of healthy donors and cultured them for either 3 (hEPC-3d) or 14 days (hEPC-14d) to characterize the L-arginine transport and NO synthesis in those cells. L-arginine transport and NO synthesis were analyzed in the presence or absence of N-ethylmaleimide or L-nitroarginine methyl ester, as inhibitors of y(+)/CAT system and nitric oxide synthases, respectively. The results showed that L-arginine uptake is higher in hEPC-14d than in hEPC-3d. Kinetic parameters for L-arginine transport showed the existence of at least 2 transporter systems in hEPC: a high affinity transporter system (K(m)= 4.8 ± 1.1 µM for hEPC-3d and 6.1 ± 2.4 µM for hEPC-14d) and a medium affinity transporter system (K(m) = 85.1 ± 4.0 µM for hEPC-3d and 95.1 ± 8 µM for hEPC-14d). Accordingly, hEPC expressed mRNA and protein for CAT-1 (ie, system y(+)) and mRNA for 2 subunits of y(+)L system, yLAT1, and 4F2hc. Higher L-citruline production and NO bioavailability (4-fold), and endothelial nitric oxide synthase expression (both mRNA and protein) were observed in hEPC-14d compared with hEPC-3d. Finally, the high L-citruline formation observed in hEPC-14d was blocked by N-ethylmaleimide. In conclusion, this study allowed to identity a functional L-arginine/NO pathway in two hEPC differentiation stages, which improves the understanding of the physiology of these precursor cells.

Adenosine A2A and A3 receptors are involved in the human endothelial progenitor cells migration.

Human endothelial progenitor cells (hEPC) are recruited to sites of neovascularization where they differentiate into endothelial cells. The signals/factors responsible for hEPC migration and adhesion to sites of injury are not well understood. Elevated levels of adenosine are known to increase mature endothelial cell migration in response to tissue injury. However, the understanding of the role of adenosine in the physiology of hEPC is very limited. Using quantitative polymerase chain reaction and western blot analyses, we detected the expression of the adenosine receptors A2A, A2B, and A3 in hEPC. Stimulation of adenosine receptors using adenosine or the nonselective agonist adenosine-5'-N-ethylcarboxamide (NECA) increased hEPC migration in 1.4-fold and 2.1-fold (P < 0.01), respectively. Stimulation of hEPC using the A2A-specific agonist CGS-21680 resembled the effect observed in migration when using adenosine or NECA. Consequently, NECA and CGS-21680-stimulated migration of hEPC were reverted using the A2A receptor antagonist ZM-241385. NECA-stimulated migration was inhibited in dose-dependent manner using MRS-1523 (Ki of 147 ± 0.016 nM), MRS-1754 (Ki of 1900 ± 0.02 nM), or ZM-241385 (Ki of 0.2 ± 0.01 nM). In conclusion, adenosine stimulates hEPC migration by activating A2A and A3 but not A2B receptors and provides evidence to support a role of adenosine in modulating angiogenic capacity of hEPC.

Fetal endothelium dysfunction is associated with circulating maternal levels of sE-selectin, sVCAM1, and sFlt-1 during pre-eclampsia.

OBJECTIVES: To evaluate the association between endothelial activation markers in the maternal circulation with nitric oxide (NO) synthesis in human umbilical endothelial cells. STUDY DESIGN: This is a case-control study of normal and pre-eclamptic pregnancies. The levels of sE-selectin, soluble vascular cell adhesion molecule 1 (sVCAM-1), and soluble fms-like tyrosine kinase 1 (sFlt-1) were measured by enzyme-linked immunosorbent assay, and histamine-induced NO synthesis was detected by fluorometric examination of the human umbilical vein endothelial cells (HUVECs) isolated from normal and pathological pregnancies. RESULTS: Mothers with severe pre-eclamptic pregnancies have premature and smaller babies than mothers with normal pregnancies (P < 0.05); they also have high maternal plasma levels of sVCAM-1 (∼2-fold), sFlt-1 (∼2.5-fold), and lower (∼70%) histamine-stimulated NO synthesis in HUVECs. A positive relationship between systolic blood pressure (SBP) and plasma levels of sE-selectin, sVCAM-1, and sFlt-1 was demonstrated. Moreover, levels of sE-selectin, sVCAM-1, and sFlt-1 were negatively associated with newborn weight (NBW), gestational age at delivery, and NO synthesis. Women with high E-selectin (>63 ng/ml), VCAM-1 (>752 ng/ml), and sFlt-1 (>15204 pg/ml) showed high risk (∼2-fold) for preterm delivery and very preterm delivery, or fetal weight <1500 g (∼1.5-fold) compared with women with low levels. CONCLUSIONS: High circulating levels of maternal endothelial dysfunction markers present in pre-eclampsia are associated with decreased NO synthesis in fetal endothelium.