Isolamento, diferenciação e aspectos bioquímicos de células-tronco de líquido amniótico / Isolation, differentiation and biochemical aspects of amniotic fluid stem cell

As células-tronco mesenquimais (MSCs) são células com grande potencial de diferenciação e estão sendo recentemente introduzidas na clínica para tratamento de várias doenças. Possuem várias vantagens incluindo sua estabilidade fenotípica in vitro. OBJETIVO: isolamento das MSCs de líquido amniótico, sua expansão e a demonstração da sua capacidade de se diferenciar em células miogênicas e adipogênicas, sem alterar a estabilidade cromossomal em meio de cultura. MÉTODOS: a fim de avaliar a mudança funcional destas células, foram avaliados parâmetros bioquímicos nas células adipogênicas já diferenciadas e antes da diferenciação através da dosagem de triglicérides. A diferenciação em células musculares foi avaliada comparando os níveis de creatinofosfoquinase - CK, desidrogenase lática - LDH e aldolase produzidas por estas células antes e após diferenciação. RESULTADOS: os níveis de triglicérides foram significativamente maiores nas células diferenciadas, mostrando ainda a formação de grânulos intracitoplasmáticos. Todos os outros valores obtidos foram significativamente maiores nas células miogênicas diferenciadas quando comparadas às não diferenciadas. CONCLUSÃO: os resultados sugerem que estes protocolos podem ser usados para avaliar diferenciação de células-tronco em células adipogênicas e miogênicas, e que o líquido amniótico pode ser uma fonte para obtenção destas células.

The mesenchymals stem cells (MSCs) are cells with the great potential of differentiation are being introduced in the clinic for treatment of several diseases. Mesenchymal stem cells have several advantages including the stability of their phenotype in vitro. BACKGROUND: isolation of MSCs in amniotic fluid, its expansion and the demonstration of the capacity of these cells to differentiate in adipogenic and miogenic cells, without to change the chromosomal stability of the MSCs in culture. METHODS: in order to evaluate the functional change of these cells, were gotten values of the differentiated adipogenic cells and not differentiated through the dosage of triglycerides. The miogenic nature of the differentiated cells was analyzed comparing the creatine kinase - CK, lactic dehydrogenase - LDH and aldolase produced by the cells. RESULTS: the values of triglycerides were significantly higher in differentiated cells, showing intracitoplasmatic granule form after differentiation. All the biochemical characters were significantly higher in differentiated miogenic cells. CONCLUSIONS: this study suggests that the standardized protocol of differentiation can be used in the attainment of cells with characteristics of adipogenic and muscular cells, from amniotic fluid.

[Isolation, differentiation and biochemical aspects of amniotic fluid stem cell]. / Isolamento, diferenciação e aspectos bioquímicos de células-tronco de líquido amniótico.

UNLABELLED: The mesenchymals stem cells (MSCs) are cells with the great potential of differentiation are being introduced in the clinic for treatment of several diseases. Mesenchymal stem cells have several advantages including the stability of their phenotype in vitro. BACKGROUND: isolation of MSCs in amniotic fluid, its expansion and the demonstration of the capacity of these cells to differentiate in adipogenic and myogenic cells, without to change the chromosomal stability of the MSCs in culture. METHODS: in order to evaluate the functional change of these cells, were gotten values of the differentiated adipogenic cells and not differentiated through the dosage of triglycerides. The myogenic nature of the differentiated cells was analyzed comparing the creatine kinase--CK, lactic dehydrogenase--LDH and aldolase produced by the cells. RESULTS: the values of triglycerides were significantly higher in differentiated cells, showing intracytoplasmatic granule form after differentiation. All the biochemical characters were significantly higher in differentiated myogenic cells. CONCLUSIONS: this study suggests that the standardized protocol of differentiation can be used in the attainment of cells with characteristics of adipogenic and muscular cells, from amniotic fluid.

Determinación enzimática de fosfolípidos que contienen colina en líquido amniótico / Enzimatic determination of phospholipids that choline contains in amniotic fluid

Se describe un método para determinar en líquido amniótico los fosfolípidos que contienen colina, que se basa en la acción de la fosfolipasa D sobre los fosfolípidos, originando colina, que es oxidada por la colinaoxidasa a betaína con la producción simultánea de peróxido de hidrógeno, este último en presencia de peroxidasa oxida al fenol que se acopla a la 4-aminofenazona produciendo un cromógeno rojo. La intensidad del color a 500 nm es directamente proporcional a la concentración de fosfolípidos que contienen colina. El método es sencillo, rápido, relativamente econónico, preciso, exacto y requiere poco volumen de muestra. El análisis comparativo con la determinación de lecitina mediante un método enzimático mostró un coeficiente de correlación (r) de 0,945 y una ecuación de regresión lineal y=2,595+0,929x. El método presenta coeficientes de variación día a día de 2,6 y 4,5 por ciento para concentraciones de fosfolípidos de 12,4 y 4,3 mg/dL, respectivamente. El intervalo analítico es de 25,0 mg/dL. La sangre, la bilirrubina y el ácido ascórbico producen cierto grado de interferencia

Determination of alkaline phosphatase isozymes in amniotic fluid.

A simple method for the determination of the three isozymes of alkaline phosphatase (EC 3.1.3.1) contained in amniotic fluid (fetal intestinal, placental, and liver-bone-kidney) is presented. Total alkaline phosphatase activity was assayed in 10,000 g supernatants of amniotic fluid from 30 normal women between the 16th and 20th week of pregnancy. Electrophoretic patterns and inhibition by L-phenylalanine and L-homoarginine studies showed that all the fetal intestinal isozyme was precipitated in the pellet after centrifugation at 100,000 g for 90 min. Thus, the difference between total alkaline phosphatase activity and activity in the 100,000 g supernatant corresponds to fetal intestinal alkaline phosphatase. Placental isozyme can be determined by assaying alkaline phosphatase in the 100,000 g supernatant after heating at 56 degrees C for 90 min. Liver-bone-kidney isozyme activity is obtained by subtracting placental alkaline phosphatase activity from that of the 100,000 g supernatant. Mean percentages of the total alkaline phosphatase for each of the isozymes in amniotic fluid were 81% for fetal intestinal alkaline phosphatase, 7.5% for placental alkaline phosphatase and 12.0% for liver-bone-kidney alkaline phosphatase. Determination of fetal intestinal alkaline phosphatase by this method could be applied to the diagnosis of cystic fibrosis in fetuses having a 1:4 risk of being affected.

Ruptura prematura de membranas: mecanismos de la enfermedad / Premature rupture of the choriommiotic membranes: physiopathogenic mechanism

La ruptura de membranas corioamnióticas (RPM) está asociada a complicaciones perinatales y es la causa identificada más frecuente de parto pretérmino. A pesar de que la RPM se ha estudiado desde hace tiempo, en las actualidad hay controversia entre los mecanismos que la median y su etiopatogenia no ha sido bien comprendida. Actualmente se reconoce que el rompimiento de las membranas se asocia al aumento de presión intrauterina, siendo una diferencia que las membranas que se rompen en forma prematura son más débiles que las normales, sin embargo, la pura explicación mecánica parece incompleta, por lo que se han estudiado otros factores que podrían estar relacionados con la RPM entre los que se encuentran el infeccioso, el dietético y otros como pueden ser maniobras quirúrgicas, incompetencia ítsmico-cervical y polihidramnios. Por otro lado se han analizado aspectos moleculares relacionados con la RPM, estas comprenden studios sobre el metabolismo de la colégena que es principal constituyente de las membranas corioamnióticas, como resultado de ellos, se han propuesto diferentes niveles de daño que adectan tanto a la síntesis como a la degradación de la colágena

Phospolipase A2 and premature rupture of membranes

Antecedentes: La actividad de fosfolipasa A2 (FLA2) ha sido relacionada con el incremento en los niveles de prostaglandina E2 (PGE2) en el líquido amniótico (LA). Altas concentraciones de este compuesto se encuentran en el LA en las siguientes condiciones: trabajo de parto, infección y ruptura prematura de membrana (RPM). Sin embargo la asociación entre este incremento y la FLA2 nunca ha sido demostrada. Objetivo: Estudiar la asociación entre la actividad FLA2 y la patogénesis de la RPM. Material y métodos: Se analizó la actividad de esta enzima en 3 grupos de LA: 1-Seguno trimestre 16-22 semanas de gestación (SG), 2- término 36-40 SG y 3- Ruptura Prematura de Membranas 28-36 SG, con y sin presencia de infección. Resultados: La actividad de FLA2 en los LA de segundo trimestre fue de 1.782 ñ 1.31 nanomolos de Fosfatidil-Colina degradada/mg proteína, mientras de RPM no infectadas tuvieron valores de 12.357 ñ 5.96 nm/mg y las infectadas de 29.077 ñ 17.91 nm/mg. Se estableció diferencias significativas entre las muestras de segundo trimestre y todos los demás grupos (p< 0.0001), entre RPM infectada y todos los grupos (p< 0.0001); y no se encontró diferencia entre los LA de término y de RPM no infectados. Discusión: Sugerimos la participación de la Fosfolipasa A2 en el mecanismo fisiológico de trabajo de parto y en el mecanismo patogénico de la RPM

[Phospholipase A2 and premature membrane rupture]. / Fosfolipasa A2 y ruptura prematura de membranas.

The phospholipase A2 activity in young amniotic fluids: 1.782, was determined. In the term amniotic fluids, the activity was 10.693. The detected activity in fluids from premature rupture of membranes was 29.077 in the group with infection, add 12.357 for the samples without infection. It was seen that young amniotic fluid has less activity. The group with infection showed the greatest activity. The significant increase of phospholipase A2 activity in term amniotic fluids and with PRM, in comparison with what is seen in young fluids suggests the active participation of the soluble form of this enzyme as mediator in the increase of Prostaglandin E2 seen in the amniotic fluid of the two first groups. Accordingly, one may think that PPLA2 could be a central mediator in membranes rupture mechanism in normal labor, as in PRM.

[Role of metalloproteases of extracellular matrix in the premature rupture of fetal membranes: a novel physiopathogenic model]. / Participación de las metaloproteasas de matriz extracelular en la ruptura prematura de membranas fetales: un modelo fisiopatogénico novedoso.

Experimental proofs are presented, that involve the participation of the metalloproteinases family, of extracellular matrix in the genesis of premature rupture of membranes. The expression of this group of enzymes, which presents in normal conditions of labor appears in the RPM without relation with gestational age. This phenomenon is not presented as an isolated example of participation of metalloproteinases in events related to labor and delivery, as is very well known its participation in maturation of uterine cervix preceding product expulsion, and that consists in similar mechanisms to the ones now chosen for the maturation of fetal membranes. The working hypothesis in pathologic conditions, implies, then, the activation of a normal system in an inadequate moment that results in premature rupture of membranes.

Participación de las metaloproteasas de matriz extracelular en la ruputora prematura de membranas fetales: un modelo fisiopatogénico novedoso / Participation of metaloproteases of extracellular matrix in premature rupture of fetal membranes: a pyisiopathogenic model

Se aportan pruebas experimentales que involucran la participación de la familia de las metaloproteinasas de matriz extracelular en la génesis de la ruptura prematura de membranas. La expresión de este grupo de enzimas, que se presenta en condiciones normales durante el trabajo de parto aparecen en la RPM sin relación con la edad gestacional. Este fenómeno no se presenta como un ejemplo aislado de la participación de las metaloproteinasas en eventos ligados al trabajo de parto, ya que es bien conocida su participación en la maduración del cuello uterino que precede a la expulsión del producto, y que consiste en mecanismos semejantes a los que ahora se postulan para la maduración de las membranas fetales. La hipótesis de trabajo en condiciones patológicas, implica entoces la activación de un sistema normal en un momento inadecuado que resulta en ruptura prematura de membranas.

Amniotic fluid lysozyme activity in fetal distress.

Lysozyme activity was measured in amniotic fluid samples from 90 pregnant women with gestational age ranging from 30 to 41 weeks. Twenty-nine samples were from high-risk subjects with different pathologies and signs of fetal distress. The control group consisted of 20 normal and 41 pathological pregnant women, whose disorders included Rh isoimmunization, diabetes, systemic arterial hypertension and pre-eclampsia without signs of fetal distress. Amniotic fluid lysozyme levels in normal controls were similar to those detected in abnormal pregnant women without signs of fetal distress (means = 156.0 vs 131.8 micrograms/ml for 34-37 weeks of gestation), with a tendency toward higher values as pregnancy progressed to term (means = 182.1 vs 155.4 micrograms/ml for 38-41 weeks of gestation). Lysozyme levels were significantly lower in high-risk pregnant women with signs of fetal distress, regardless of neonate birth weight, than in subjects showing no such signs (means = 40.3 and means = 25.4 micrograms/ml at 34-37 and 38-41 weeks of gestation, respectively). These data support the possibility of using amniotic fluid lysozyme activity levels as an indicator of fetal distress.

Ammiotic fluid lysozyme activity in fetal distress

Lysozyme activity was m,easured in amniotic fluid samples from 90 pregnant women with gestacional age ranging from 30 to 41 weeks. Twenty-nine samples were from high-risk subjects with different pathologies and signs of fetal distress. The control group consisted of 20 normal and 41 pathological pregnant women, whose disorders included Rh isoimmunization, diabetes, systemic arterial hypertension and pre-eclampsia without signs of fetal distress. amniotic fluid lysozyme levels in normal controls were similar to those detected in abnormal pregnant women without signs of fetal distress (x = 156.0 vs 131.8 microng/ml for 43-37 weeks of gestation), with a tendency toward higher values as pregnancy progressed to term in high-risk pregnant women with signs of fetal distress, regardless of neonate birth weight, than in subjects showing no such sugns (x = 40.3 and x = 25.4 microng/ml at 34-41 weeks of gestation, respectively). These data support the possibility of using amniotic fluid lysosyme activity levels as an indicator of fetal distress

[Thermolabile fraction of amniotic alkaline phosphatase and fetal lung maturity]. / Fraction thermolabile de la phosphatase alcaline amniotique et maturité pulmonaire foetale.

In order to predict respiratory distress syndrome (RDS) in the newborn due to the lack of surfactant, we measured the change in the total and heat labile fraction of alkaline phosphatase (ALP) in amniotic fluid obtained at various gestational ages. Heat labile alkaline phosphatase increased both absolutely and in relation to total ALP from 32 weeks to term. We have used the ratio heat labile ALP/total ALP to predict lung maturity. There was no respiratory distress in infants born after the heat labile ALP/total ALP in the amniotic fluid was above 0.7. The assay is technically easy so that measurement of this ratio should help predict which infant will develop RDS.

Kininase activity in the amniotic during normal and pathological pregnancy (Toxemia and hypermaturity)

Foi estudada a atividade cininásica no líquido amniótico de gravidezes normais e patológicas (toxemia e hipermaturidade). Níveis normais de atividade cininásica foram estudados durante o terceiro trimestre da gestaçäo e na hipermaturidade (gestaçäo além de 42 semanas). Os resultados da atividade de cininase específica (ug%min/mg protein 10-2) (12,01 ñ 3,76) para o terceiro trimestre da gravidez e pacientes com toxemia (12,48 ñ 3,83) näo mostrou diferenças significantes entre os grupos

Hyperphenylalaninemia due to dihydropteridine reductase deficiency. Assay of the enzyme in fibroblasts from affected infants, heterozygotes, and in normal amniotic fluid cells.

Two infant siblings with modest elevations of serum phenylalanine concentrations had seizures and developmental regression: they died in their second year. Dihydropteridine reductase activity, which can be measured in normal cultured skin fibroblasts, was measured in the younger sibling and was absent. Parents of the two siblings and parents of a previously reported patient all showed 50% or less of the normal dihydropteridine reductase activity in their cultured fibroblasts. Dihydropteridine reductase activity is also present in normal cultured amniotic fluid cells, offering the possibility of prenatal diagnosis. Absence of dihydropteridine reductase results not only in a defect in the conversion of phenylalaning to tyrosine, but also in the biosynthesis of the neurotransmitters, dopamine, norephinephrine, and serotonin. Since deficiencies in these neurotransmitters would not be alleviated by a phenylalanine-restricted diet, it is important to establish the nature of the enzymatic defect in all suspected variants of phenylketonuria.