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Acute leukemias are malignant hematopoietic cells of immature proliferations of the blastic type, whose progressive accumulation is accompanied by a decrease in the production of normal myeloid elements. Transcription of inactive tumor suppressor genes by hypermethylation of CpG islands in promoter regions, has been a focus of researchers as a causal factor in hematological malignancies. The purpose of this study was to determine hypermethylated regions of chromosomal spread samples using Alu I and relate these regions with sites of suppressor gene associated to acute leukemia tumors. From an analysis of a 30 bone marrow samples, 18 were diagnosed with Acute Myeloid Leukemia and Acute Lymphoid Leukemia, and 12 underwent cell culture. Chromosomal spreads were stained with Giemsa after being previously digested with the enzyme Alu I. In patients with acute myeloid leukemia and acute lymphoid leukemia it was observed that 16/18 (88%) and 12/12 (100%) had abnormally stained regions, single in four and three methylated regions observed in acute myeloid leukemia and acute lymphoid leukemia, respectively, no association was found in the literature with methylated genes, which was highly significant ( p < 0.01) in both conditions. This shows the usefulness of this technique for the identification of methylated areas, since they have provided the foundation and the molecular basis for a better targeted therapeutic approach with demethylating agents, both in acute leukemias and myelodysplastic syndromes.

PRP is an useful bioproduct to tisular regeneration. The aim of study was evaluate the concentration of growth factors: PDGFBB (platelet-derived growth factor), EGF(epidermal growth factor) and VEGF (vascular endothelial growth factor) present in the Platelet-rich plasma (PRP) in subjects treated with drugs which inhibit platelet aggregation as acetylsalicylic acid (ASA) and clopidogrel before and after administration. We determined by ELISA PDGFBB, EGF and VEGF levels in PRP, Platelet Poor Plasma (PPP), lysate and exudate from 32 healthy subjects before and 24 hours after ingesting acid Acetyl salicylic acid (ASA) and clopidogrel as a single dose. The PRP and PPP were obtained by the method of Anitua by single centrifugation method. To analyze the results of student test and Pearson correlation was applied, with statistical significance level of p < 0.05. PPP and exudate (Clopidogrel: p < 0.001), PRP (Clopidogrel: p < 0.01) statistically significant differences for PDGFBB in PPP (p < 0.01 AAS) were found, and for VEGF in lysate (ASA and Clopidogrel: p < 0.05). No significant difference was found for EGF. Only was no correlation between baseline values of EGF in the ASA group and the respective PRP platelet count (r = 0.726). The results show that the average basal values of the three growth factors measured were considered particularly high in the PRP and lysate, showing the significant decrease for PDGFBB after antiplatelet therapy, especially of Clopidogrel and a significant increase for the VEGF only for the lysate. Although the behavior of three different soluble mediators was different to antiplatelet agents, the observed changes support the conclusion that a single dose of these drugs not markedly affect the secretion and availability of the three growth factors measured in various platelet derived obtained.

Introducción: el plasma rico en plaquetas (PRP) es un producto autólogo rico en factores de crecimiento indispensables en la regeneración tisular. Objetivo: cuantificar las concentraciones de VEGF y EGF en el PRP antes y después de los antiagregantes plaquetarios: aspirina (AAS) y clopidogrel; y su correlación con el recuento plaquetario. Material y método: se estudiaron en 32 sujetos sanos concentraciones de EGF y VEGF (Elisa) en PRP, plasma pobre en plaquetas (PPP), exudado y lisado, antes y después de aspirina (AAS) y clopidogrel. Resultados: la concentración de EGF en el PRP y sus subproductos antes y después del tratamiento con AAS o clopidogrel no mostró diferencia significativa para ninguno de los derivados plaquetarios analizados; no así para el VEGF, donde se encontró diferencia significativa en el subproducto ‘lisado’ (p

PDGF and VEGF are two of the most potent mitogen for connective tissue, its secretion appears to be particularly important when the source is Platelet Rich Plasma (PRP), hence the latter leading role in tissue regeneration. ELISA PDGFBB levels in PRP, Platelet Poor Plasma (PPP) and exudates, were determined in 32 healthy subjects before and 24 hours after ingestion of Aspirin (ASA) and Clopidogrel (CLO). Results: PDGFBB baseline levels were 10.6 ± 1.9 ng / ml (PPP), 12.12 ± 2.5 ng / ml (PRP) and 10.84 ± 1.68 ng / ml (exudate) While after treatment with PDGFBB ASA concentrations were at 8.96 ± 1.4 ng / ml (PPP), 11.36 ± 1.48 ng / ml (PRP), 11.11 ± 1.14 ng / ml (exudate) and the Clopidogrel were 8.53 ± 0.59 ng / ml (PPP), 9.65 ± 1.17 ng / ml (PRP) and 8.51 ± 0.75 ng / ml (exudate) . VEGF basal values were 973.9 ± 590.3 pg / ml (PPP), 1184.2 ± 288.4 pg / ml (PRP), 1069.3 ± 192.3 pg / ml (exudate). After treatment with ASA VEGF values ??were at 1439.5 ± 117.4 pg / ml (PPP), 1802.3 ± 123.9 pg / ml (PRP), 1745.6 ± 171.6 pg / ml (exudate) and the Clopidogrel were 577.8 ± 150.6 pg / ml (PPP), 668.7 ± 100.6 pg / ml (PRP), 624.9 ± 106.7 pg / ml (exudate). Conclusions: It was noted that after administration of antiplatelet drugs PDGFBB and VEGF values decreased statistically significantly, especially for the group of Clopidogrel. The ASA lesser extent appear to affect the concentrations of both factors, which may be attributable to the pharmacological action mechanism between the ASA and Clopidogrel.

Glutathione S-transferases constitute a superfamily of enzymes that catalyse the inactivating conjugation of endogenous and environmental substrates involved in the pathogenesis of hepatocellular carcinoma (HCC) and glutathione. Genes encoding either glutathione S-transferase Mu-1 or Theta-1 (GSTM1 and GSTT1, respectively) isoforms are polymorphic. Homozygotes for the mutated inactive alleles of each gene are devoid of any specific enzymatic activity (null genotypes). Our aim was to investigate whether individuals with null GST genotypes have a higher risk of developing HCC. A total of 184 Caucasian Spanish patients with a diagnosis of HCC and 329 healthy controls of the same ethnic origin were included. Polymorphisms in GSTM1 and GSTT1 genes were identified through multiplex polymerase chain reactions, and the dihydrofolate reductase (DHFR) gene was used as internal control. No differences were found between the frequencies of GSTM1 (47.8% versus 45.3%) and GSTT1 (28.8% versus 23.1%) null genotypes in cases and controls, respectively, nor in the proportion of carriers of two, one or no active genotypes. Gender, age at diagnosis, tobacco use, chronic infection with hepatitis B or C virus and alcohol abuse did not influence these results. In conclusion, polymorphisms in GSTM1 and GSTT1 genes are not related to the incidence of HCC in a high-risk Spanish population.

This report describes the first case in Spain of a severe form of beta-thalassemia (thal) due to a compound heterozygosity for the IVS-II-848 (C --> A) and the nonsense codon 39 (C --> T) mutations. Five members of a family from Cadiz (southern Spain) were studied. The proband was an 8-year-old girl diagnosed as anemic at the age of 13 months. Her father had the codon 39 (C --> T) mutation and her mother the C --> A change at nucleotide (nt) 848 of IVS-II. Haplotype analysis showed that the proband was a compound heterozygote for haplotypes I [+ --> + +] and VII [+ --> +]. This is the first description in Spain of the IVS-II-848 (C --> A) mutation. It appears, from restriction fragment length polymorphism (RFLP) analysis, that this mutation has a different origin in the various populations, where it was found. This observation shows that in this case the association of a beta(0)- and a beta(+)-thal mutation does not lead to a thalassemia intermedia but to a severe thalassemia with very low hemoglobin (Hb) levels. From a therapeutic point of view, early introduction of a transfusion regimen may improve the clinical picture of these children, allowing for better development and growth.

Fundamento y objetivo: La hemoglobina (Hb) S y la alfatalasemia son, junto con la betatalasemia y las Hb C, E y D, las denominadas hemoglobinopatias comunes, las cuales presentan una alta prevalencia en las zonas historicamente endemicas de paludismo. En este estudio analizamos la frecuencia de la asociacion de estas 2 hemoglobinopatias, asi como su expresion fenotipica. Pacientes y metodo: Entre enero de 1995 y marzo de 2003 hemos realizado un estudio molecular para descartar la presencia de una alfatalasemia asociada de un total de 83 casos diagnosticados de hemoglobinopatias S por criterios electroforeticos y cromatograficos. El estudio molecular se realizo por Southern blot con las endonucleasas de restriccion Bam HI y Bgl II, y las sondas a (1,5 Kb Pst I) y ? (1,8 Kb Sac Y). Resultados: De los 83 casos estudiados, 45 (54,2%) presentaban alfatalasemia (37 ­a3,7/aa; 1 ­a4,2/aa; 6 ­*3,7/­a3,7; 1 ­a4,2/­a4,2); 36 no tenian alfatalasemia y en 2 habia una triplicacion de genes a. Los pacientes con HbS heterocigota asociada a alfatalasemia presentaron el porcentaje de HbS (p < 0,0001), el volumen corpuscular medio (VCM) (p = 0,004), la hemoglobina corpuscular media (HCM) (p = 0,002) y la concentracion de hemoglobulina corporal media (CHCM) (p = 0,02) significativamente menor que los que no tenian dicha asociacion. Sin embargo, no se encontraron diferencias en el resto de parametros analizados (hemoglobina [p = 0,56], hematocitos [p = 0,84], indice de anisocitosis [p = 0,06], reticulocitos [p = 0,26], HbF [p = 0,76], Hb A2 [p = 0,13] entre ambos grupos. En los casos con formas graves de la enfermedad (HbS homocigoto; HbS/HbC; HbS/betatalasemia) los pacientes con alfatalasemia presentaron una cifra de leucocitos significativamente menor que los pacientes sin esta afeccion (p = 0,034); en el resto de los parametros estudiados no se encontraron diferencias estadisticamente significativas. La probabilidad de no presentar HbA (no haber sido transfundido en los ultimos meses) fue 16 veces mayor en el grupo de pacientes con alfatalasemia que en el grupo de pacientes sin ella (odds ratio = 16,00, intervalo de confianza [IC] del 95%, 1,27-200,91). Conclusion: La hemoglobinopatia S se asocia muy frecuentemente con la alfatalasemia (> 50% en nuestra experiencia). El cribado de esta asociacion es de gran interes, ya que puede modular la expresion clinica de los casos de HbS homocigota. Dicha asociacion se debe sospechar en los casos de HbS heterocigota con valores mas bajos de HbS de lo esperado.

Fundamento y objetivo: Las hemoglobinopatias estructurales son un grupo de enfermedades cuya incidencia en la Comunidad de Madrid ha aumentado en los ultimos anos debido al creciente fenomeno de la inmigracion que esta viviendo. Dados los problemas de morbimortalidad que comportan y la posibilidad de instaurar tratamientos profilacticos tempranos, consideramos necesario realizar un estudio que demuestre la necesidad de la inclusion del cribado de hemoglobinopatias en dicha comunidad autonoma. Sujetos y metodo: Estudio de cohortes ambispectivo en 3.365 recien nacidos realizado en el Hospital Clinico San Carlos desde mayo de 2003 a abril de 2004. Todas las muestras se analizaron mediante cromatografia liquida de alta resolucion (HPLC) de intercambio ionico (Variant®, Bio-Rad) y en las patologicas se estudiaron las cadenas de globina mediante HPLC de fase reversa. Resultados: Se encontro una incidencia global de hemoglobinopatias de 7,7 de cada 1.000 recien nacidos, con un caso de hemoglobina S homocigota, 18 de hemoglobina S heterocigota, uno de hemoglobina E/A2, uno de hemoglobina C y 5 picos anomalos. Conclusiones: Consideramos que el cribado neonatal de hemoglobinopatias en la Comunidad de Madrid es necesario y que la HPLC de intercambio ionico Variant® es adecuada para el cribado de las hemoglobinopatias mas frecuentes, con ahorro de tiempo.

Se evaluó el recuento plaquetario en sangre periférica de sujetos sanos tratados con antiagregantes plaquetarios. Se analizaron 20 sujetos. Se distribuyeron en dos grupos: A: 10 sujetos que recibieron Aspirina (100 mg) y B: 10 con Clopidogrel (75 mg) por 7 días. Se les realizó recuento plaquetario en sangre periférica y en PRP. Encontrándose un conteo de plaquetas antes del tratamiento con antiagregantes en sangre periférica de 258,6 ± 54,46 x 109 l7 días después de 254 ± 41,86 x 109 l (Aspirina), 285,4±70,92 y 196,5±37,90 x 109 l (Clopidogrel) respectivamente. En el PRP de los sujetos antes de recibir Aspirina fue 486,5 ± 129,54 x 109 l y después 449,2 ± 85,51 x 109 l; antes de la ingestión de Clopidogrel fue 565,2 ± 150,41 y 592,9 ± 203,46 x 109 l después del tratamiento.Se encontraron diferencias significativas solo para el conteo plaquetario en el grupo del Clopidogrel (p< 0.05). Se observó una disminución significativa del conteo plaquetario en sangre periférica posterior a la administración del Clopidogrel, posiblemente como consecuencia del mecanismo farmacológico del mismo. Son necesarios más estudios para evaluar un mayor número de individuos y medir mejor el efecto de los antiplaquetarios

Objetivo: comprobar si las mutaciones del gen HFE, que pueden inducir sobrecarga hepática de hierro, guardan relación con el riesgo de desarrollar carcinoma hepatocelular (CHC) en sujetos predispuestos a sufrir este tumor. Material y métodos: se han incluido 196 pacientes (161 varones) diagnosticados de CHC. Ninguno estaba diagnosticado de hemocromatosis. El grupo control estaba constituido por 181 sujetos sanos. Todos los sujetos eran españoles de raza blanca.Las mutaciones C282Y y H63D del gen HFE se identificaron mediante reacción en cadena de polimerasa (PCR) sobre ADN genómico leucocitario utilizando enzimas de restricción específicas. Resultados (casos/controles): 1. Distribución genotípica: a) mutación C282Y: 1/0 homocigotos, 12/23 heterocigotos, 183/158 normales (p = 0,07, n.s.); y b) mutación H63D: 9/5 homocigotos, 85/52 heterocigotos, 102/124 normales (odds ratio 2,00, IC95% 1,29-3,12, p = 0,002). Cuatro casos y seis controles eran heterocigotos compuestos. 2. Frecuencias alélicas: a) mutación C282Y: normales 378/339, mutados 14/23 (p = 0,11, n.s.); b) mutación H63D: normales 289/300; mutados 103/62 (odds ratio 1,72, IC95% 1,19-2,50, p = 0,004). No se observaron diferencias en relación con el sexo, la edad o la etiología (VHC, VHB, etílica o mixta) de la hepatopatía previa. Conclusiones: la mutación C282Y no guarda relación con el riesgo de desarrollar CHC en sujetos sin hemocromatosis conocida. La posesión de la mutación H63D se asocia con un riesgo aumentado de desarrollar CHC independientemente de la etiología de la hepatopatía crónica subyacente.

For centuries in Europe, population movements have contributed to ethnic groups, cultures, and consequently, inheritance mixing. There are certain genetic diseases such as hereditary hemochromatosis whose distribution is directly related to the population movements. The objective of the present investigation was to determine the C282Y and H63D mutation frequency of the HFE gene in a cohort study of 1,000 neonates in the Community of Madrid (Spain), thus contributing to the HFE gene mutations distribution research in Europe and establishing the origin of the mutations in Spain. The allelic frequency of C282Y mutation was 1.7% (CI 95% 1.1–2.3) and the H63D allele was present in 16.4% of chromosomes (CI 95% 14.8–18). In Spain, the presence of C282Y mutation and its distribution could be due more to Celtic than to Viking legacy, whereas it is assumed that the one in relation to the H63D variant occurred in the Basque Country during the Paleolithic Period.

Hemoglobin S (HbS) alpha-thalassemia together with ss-thalassemia and hemoglobins C, E and D are named common hemoglobinopathies. In this study, we analyzed the frequency of the association between alpha-thalassemia and HbS and its phenotypic expression.Since January 1995 to March 2003 we studied 83 cases of HbS, which were diagnosed by electrophoretic and chromatographic criteria. The molecular study was carried out by Southern blot with the restriction enzymes Bam HI and Bgl II and a (1.5 Kb Pst) and z (1.8 Kb Sac Y) probes.45 cases (54.2%) had a-thalassemia (36 -alpha3,7/aa; 1-alpha4,2/alphaalpha; 6 -alpha3,7/-alpha3,7; 1-alpha4,2/-alpha4,2); 36 had not alpha-thalassemia and in two cases there were a triplication of a genes. The patients with HbS heterozygote associated with alpha-thalassemia showed a percentage of HbS (p0.0001), CMV (p = 0.004), MCH (p = 0.002) and MCHC (p = 0.02) significantly lower than the cases of HbS without this association. However, no differences between both groups were found with regard to the rest of parameters analyzed (Hb p = 0.56; PVC p = 0.84; RDW p = 0.06; Reticulocytes p = 0.26; HbF p = 0.76; HbA2 p = 0.13). In the cases with a severe form of disease (HbS homozygote; HbS/Hb C; HbS/beta-thalassemia), patients with alpha-thalassemia had a number of leukocytes that was significantly lower than that of patients without alpha-thalassemia (p = 0.034).An association between HbS and alpha-thalassemia was common (50%). Screening for this association is of great interest because the clinical expression in the cases of HbS homozigote will be modulated. This association must be suspected in cases of homozygous HbS in which levels of HbS are lower than expected.