[Single nucleotide polymorphisms in genes of endothelin-1 and receptor A associated to cardiovascular in essential hypertension]. / Polimorfismos de un solo nucleótido en genes de endotelina-1 y su receptor A asociados a daño cardiovascular en hipertensión arterial esencial.

INTRODUCTION: The endothelin system, for its vasoconstrictor action, is related to the development of essential hypertension (HTAe). The polymorphism analysis of their genes represents a new approach to the study of this disease. We propose to analyze the interaction between stages of essential hypertension (HTAe) and risk factors with polymorphisms 138ex1 ins/del A gene endothelin-1 (ET-1) and H323H receptor gene A ET-1 (ETRA). PATIENTS AND METHODS: We included 300 patients of both sexes, unrelated, who consecutively attended the clinic hypertension medical service. Each one underwent a complete physical examination, electrocardiogram, echocardiogram, and Rx thorax. The degree of severity of hypertension was determined in stages. The determination of polymorphisms was performed by amplification followed by cutting by specific restriction enzyme from DNA obtained from peripheral blood. RESULTS: The 46% of patients had HTAe controlled, 17.6% had organ damage or cardiovascular, brain or kidney disease. It was observed that the 4A/4A carriers showed lower frequency of cardiovascular disease, kidney and brain (P<.032; 95% CI: 11.1-21.4). For H323H polymorphism, the evaluation by images showed a higher frequency of the dilations of left auricular (P=.02) and auricular fibrillation (P=.03) between the T/T carrier, a higher frequency of cardiomegaly was detected in C/C patients (P=.04). CONCLUSION: The genotypes, 4A/4A of the ET-1 gene and the T/T from ETRA gene might be involved in worse outcome of cardiovascular damage. Their identification could help recognize subgroups of the hypertensive patients with different risk.

Polymerase chain reaction procedure to detect Trypanosoma cruzi in blood samples from chronic chagasic patients.

The feasibility of DNA amplification by the polymerase chain reaction for specific detection of Trypanosoma cruzi in human blood was investigated. We have used primers flanking a 220-bp fragment of highly repetitive elements, the E13 element, in T. cruzi nuclear DNA. Only polymerase chain reaction products from blood samples of chronic chagasic patients showed several amplified fragments in 1.6% agarose gels stained with ethidium bromide. Southern blot analysis demonstrated that the 220-bp amplified fragment is specific for T. cruzi DNA and very useful to detect the presence of the parasite in blood from chronic chagasic patients.

Association analysis of genotypic frequencies of matrilin-1 gene in patients with osteoarthritis.

OBJECTIVE: It has been suggested that genotypic variation in the gene which encodes the matrilin-1 (MATN-1) protein may be involved in the development of hip osteoarthritis (OA). We compared genotype frequencies of the MATN-1 gene (1p35) in patients with OA and controls to determine if there is any association between the MATN-1 genotype and OA. METHODS: 73 OA patients and 53 controls from a rheumatology ambulatory center and a university hospital were studied. They were unrelated subjects. Controls were free of clinical OA. OA was defined according to the American College of Rheumatology criteria. The MATN-1 microsatellite in the 3'untranslated region was amplified by PCR. The size of the amplification products was determined by capilar electrophoresis in a DNA Genetic Analizer Genotypic distribution was compared by the chi2 test. RESULTS: We identified 4 alleles according to their basepair (bp) length: A1 = 110 bp; A2 = 108 bp; A3 = 106 bp and A6 = 104 pb. Six genotypes were found, with an observed heterozygosity of 0.48. The most frequent genotype in OA and controls was A1/A1 (43.8% and 43.4%, respectively). No significant difference in genotype distribution was found between OA - even when discriminating by the affected joint - and controls. CONCLUSION: We did not find any difference in the MATN-1 genotype distribution in OA patients and controls. To our knowledge, this would be the first time a MATN-1 allele of 104 bp (A6) has been identified These results do not support a role of the MATN-1 genotypes in the occurrence of clinical OA.

Alkaline phosphatase isoenzymes for the diagnosis of metastatic tumors and lymphomas of liver and bone.

The plasmatic activities of total alkaline phosphatase (ALP) (E.C. 3.1.3.1), high molecular weight-ALP (high Mr-ALP) and bone-ALP isoenzymes, were determined in healthy individuals and in patients with: neoplasia without metastases, hepatic metastases, bone metastases and mixed metastases (hepatic and bone). Variables were individually used to assess incidence of metastases and percentages of false negative and false positive results were calculated. The three values were then used together to assess metastases incidence and sensitivity, specificity, positive predictive value, negative predictive value and predictive capacity were estimated. We conclude that none of the variables per se are reliable for the diagnosis of metastases. On the other hand, the three values show high percentages of sensitivity, specificity, positive predictive value, negative predictive value and a high probability (0.93) of accurate diagnosis when applied to a larger population, with similar prevalence values.

Placental alkaline phosphatase of high molecular weight in plasma of pregnant women in the last trimester of gestation.

Human blood plasma contains alkaline phosphatase (EC 3.1.3.1) isoenzymes from bone, liver and intestine. Blood of pregnant women in the third trimester of gestation also contains an isoenzyme of alkaline phosphatase from placenta, placental-AP (PLAP). Serum from pregnant women in the last trimester of gestation shows activity of soluble placental alkaline phosphatase (sol-PLAP). It is also known that a membrane bound high molecular weight placental-AP (high Mr-PLAP) is present in butanol extracts from placental tissue. Nevertheless, up to now, high Mr-PLAP has not been founded in human plasma. A method developed in this laboratory allows detection of membrane-bound alkaline phosphatase in pellet of plasma centrifuged at 100,000x g. By applying this method we have detected high molecular weight placental alkaline phosphatase in plasma of healthy pregnant women in the third trimester of pregnancy.

Cardiac condition in young chagasic women.

RATIONALE: Chagas disease is a complex disorder caused by Trypanosoma cruzi. Most patients remain asymptomatic for several years and 30% of them progress quietly to developing cardiomyopathy. The factors that lead to chronic myocardial lesions are not fully understood. OBJECTIVE: To investigate the association between clinical symptoms and single nucleotide polymorphisms in chagasic and non-chagasic women younger and older than 55 years of age. METHODS AND RESULTS: we analyzed Ala-9Val and Ile58Thr polymorphisms of the SOD-Mn gene, 138ex1ins/del A of the endothelin-1 gene (ET-1) and H323H (T/C) of the endothelin receptor A gene (ETA), by PCR-RFLP using genomic DNA from leukocyte of 85 women. We also evaluated serum lipid profile, renal and liver function, chest X-rays, electrocardiograms (ECGs) and echocardiography (EchoCG). Biochemical profiling did not show differences between chagasic and non-chagasic patients. The polymorphisms analyses showed a significant association in the distribution of frequencies of the Mn-SOD Ile58Thr gene between both groups. Young chagasic patients had a significantly higher prevalence of abnormalities in X-rays, in ECGs and they showed grade II and III of NYHA functional classes. The chance of having an abnormal EchoCG was 5.87 higher in young chagasic patients (OR=5.87, 95% CI 1.47-23.4). DISCUSSION: We concluded that the parasite affects young females by accelerating the deterioration of cardiac function, independent of other cardiovascular risk factors and the cardioprotective action of estrogens present in the premenopausal stage.

Evaluación de la terapia mecánica periodontal en bolsas profundas: respuesta clínica y bacteriológica / Evaluation of periodontal mechanical therapy in deep pockets: Clinical and bacteriological response

Objetivos: Evaluar la composición microbiológica y los parámetros clínicos de bolsas periodontales >5 mm de profundidad al inicio, 1 semana, 3 y 12 meses post raspado y alisado radicular. Materiales y Métodos: Se tomaron registros clínicos y muestras de placa subgingival de 44 sitios de pacientes con diagnóstico de periodontitis crónica. Se identificaron por técnica de Reacción en Cadena de la Polimerasa (PCR) patógenos putativos periodontales: Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Treponema denticola (Td), Tannerella forsythia (Tf) y Prevotella intermedia (Pi). Los pacientes recibieron terapia mecánica periodontal y fueron reevaluados a los 7 días, 3 y 12 meses. Resultados: Luego del tratamiento, todos los parámetros clínicos (Placa Bacteriana, Hemorragia, Supuración, Profundidad al Sondaje y Nivel de Inserción Clínica) se redujeron significativamente y los valores obtenidos se mantuvieron hasta los 12 meses. Al inicio, las especies bacterianas prevalentes fueron Pg, presente en 66 por ciento de los sitios, Tf (55 por ciento) y Td (41 por ciento). Los sitios más profundos se relacionaron con las asociaciones Tf-Td (6.8 mm) y Tf-Td-Pi (7 mm). Post terapia, el número de sitios positivos para Td, Tf y Pg se redujo significativamente. Conclusiones: El raspado y alisado radicular mejoró significativamente los parámetros clínicos y redujo la prevalencia de los patógenos periodontales Pg, Tf y Td en bolsas periodontales profundas. Los resultados obtenidos se mantuvieron hasta los 12 meses. No se detectaron mayores pérdidas de inserción clínica en el 86 por ciento de los sitios a 3 meses y en 79 por ciento a los 12 meses. Los sitios en los que el tratamiento no fue efectivo en la eliminación de patógenos a los 12 meses desarrollaron mayores profundidades de sondaje.

Objectives: To evaluate the microbial composition and clinical parameters of periodontal pockets with probing depth >5 mm at baseline, 1 week, 3 and 12 months after scaling and root planning. Methods: Clinical parameters were measured and bacterial samples were collected from 44 sites in 11 patients with chronic periodontitis. By means of Polymerase Chain Reaction (PCR) the presence of Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Treponema denticola (Td), Tannerella forsythia (Tf) and Prevotella intermedia (Pi) was estimated. The patients received mechanical periodontal therapy and were evaluated after 1 week, 3 months and 12 months. Results: After treatment, all clinical parameters (Plaque, Bleeding on Probing, Supuration, Probing Pocket Depth and Clinical Attachment Level) were significantly reduced, and the values obtained were maintained up to the 12 months that the study lasts. At baseline, the most prevalent species were Pg, present in 66 percent of the sites, Tf (55 percent) and Td (41 percent). The deepest sites were related to the association Tf-Td (6.8 mm) and Tf-Td-Pi (7 mm). The number of positive sites for Td, Tf and Pg was significantly reduced after therapy. Conclusions: Scaling and root planning improve significantly clinical parameters as well as reduce the prevalence of periodontal pathogens Pg, Td and Tf in deep periodontal pockets. The results obtained were maintained up to 12 months. No further clinical attachment loss was found in 86 percent of the sites at 3 months and 79 percent at 12 months. The sites where the treatment failed in removing pathogens developed at 12 months greater probing pocket depths.

Polimorfismos de un solo nucleótido en genes de endotelina-1 y su receptor A asociados a daño cardiovascular en hipertensión arterial esencial / Single nucleotide polymorphisms in genes of endothelin-1 and receptor A associated to cardiovascular in essential hypertension

Introducción: El sistema endotelina, por su acción vasoconstrictora, participa en el desarrollo de hipertensión arterial esencial (HTAe). El análisis del polimorfismo de sus genes representa un nuevo enfoque en el estudio de esta enfermedad. Propusimos analizar la interacción entre ins/del A los estadios de HTAe y factores de riesgo con los polimorfismos 138ex1 del gen de endotelina-1 (ET-1) y H323H del gen del receptor A de ET-1 (ETRA). Pacientes y métodos: Se analizó a 300 pacientes de ambos sexos, no parentales, que asistieron en forma consecutiva al consultorio de hipertensión arterial. Se les realizó un examen físico completo, electrocardiograma, ecocardiograma, y Rx de tórax. Se determinaron los polimorfismos mediante amplificación seguida con corte con enzimas de restricción a partir de ADN aislado de sangre periférica. Resultados: El 46% de los pacientes tuvieron HTAe controlada, el 17,6% presentaron daño de órgano blanco o enfermedad cardiovascular, cerebral o renal. Se observó que los portadores del genotipo 4A/4A del polimorfismo 138ex1 ins/del A del gen de ET-1 mostraron menor frecuencia de enfermedad cardiovascular, renal y cerebral (p < 0,032; IC 95%: 11,1-21,4). Para el polimorfismo H323H, la evaluación por imágenes mostró mayor frecuencia de dilatación de aurícula izquierda (p = 0,02) y fibrilación auricular (p = 0,03) entre los portadores T/T, y entre los C/C mayor frecuencia de cardiomegalia (p = 0,04). Conclusión: Los genotipos 4A/4A del gen de ET-1 y el T/T del gen de ETRA podrían participar agravando el daño cardiovascular. Su identificación contribuiría a reconocer subgrupos de pacientes con diferente riesgo

Introduction: The endothelin system, for its vasoconstrictor action, is related to the development of essential hypertension (HTAe). The polymorphism analysis of their genes represents a new approach to the study of this disease. We propose to analyze the interaction between stages of essential hypertension (HTAe) and risk factors with polymorphisms 138ex1 ins/del A gene endothelin-1 (ET-1) and H323H receptor gene A ET-1 (ETRA). Patients and methods: We included 300 patients of both sexes, unrelated, who consecutively attended the clinic hypertension medical service. Each one underwent a complete physical examination, electrocardiogram, echocardiogram, and Rx thorax. The degree of severity of hypertension was determined in stages. The determination of polymorphisms was performed by amplification followed by cutting by specific restriction enzyme from DNA obtained from peripheral blood. Results: The 46% of patients had HTAe controlled, 17.6% had organ damage or cardiovascular, brain or kidney disease. It was observed that the 4A/4A carriers showed lower frequency of cardiovascular disease, kidney and brain (P < .032; 95% CI: 11.1-21.4). For H323H polymorphism, the evaluation by images showed a higher frequency of the dilations of left auricular (P=.02) and auricular fibrillation (P=.03) between the T/T carrier, a higher frequency of cardiomegaly was detected in C/C patients (P=.04). Conclusion: The genotypes, 4A/4A of the ET-1 gene and the T/T from ETRA gene might be involved in worse outcome of cardiovascular damage. Their identification could help recognize subgroups of the hypertensive patients with different risk

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.

Trypanosoma cruzi: productive infection is not allowed by chorionic villous explant from normal human placenta in vitro.

UNLABELLED: We hypothesize that a sustained infection of Trypanosoma cruzi into placental tissue might be diminished. Human placental chorionic villi and VERO cells as controls were co-cultured with T. cruzi. Parasites occupied 0.0137% at 3h, 0.0224% (24h), and 0.0572% (72 h) of the total chorionic villi area analyzed and some few placental samples were negative to parasite DNA, whereas 52% of VERO cells were infected at 3h and parasites occupied 0.57%, at 24h the parasite area was of 2.78% and at 72 h was of 3.32%. There were no live parasites in placenta-T. cruzi culture media at 72 h of co-culture. There were significantly increased dead parasites when T. cruzi was treated with unheated culture media coming from placental explants and fewer dead parasites when pre-heated culture media were employed. CONCLUSION: Low productive infection by T. cruzi into placental tissue associated with no viable parasites in the culture media partially due to placental thermo labile substances.

Risk progression to chronic Chagas cardiomyopathy: influence of male sex and of parasitaemia detected by polymerase chain reaction.

BACKGROUND: Polymerase chain reaction (PCR) allows detection of Trypanosoma cruzi in blood throughout the course of Chagas' disease. OBJECTIVE: To determine whether T cruzi DNA detected by PCR is associated with progression to chronic Chagas cardiomyopathy. DESIGN: Prospective cohort study. SETTING: A tertiary care centre in Argentina. PATIENTS: 56 consecutive patients with chronic T cruzi infection. METHODS: Clinical examination, ECG, and Doppler echocardiography were carried out at baseline and at the end of the follow up. Detection of T cruzi DNA by PCR amplifying a nuclear sequence was undertaken in all patients at baseline. MAIN OUTCOME MEASURES: Progression was defined as death from chronic cardiomyopathy or the presence of a new ECG or left ventricular echocardiographic abnormality at the end of follow up. RESULTS: The 56 patients (21 male, 35 female; mean (SD) age, 56.0 (11.3) years) were followed for a mean 936.3 (244.39) days. Progression to cardiomyopathy was detected in 12 patients (21.4%). Three of these patients died after baseline evaluation. Univariate analysis showed that a positive PCR (relative risk 4.09, 95% confidence interval (CI) 1.60 to 9.85) and male sex (5.00, 95% CI 1.65 to 15.73) were associated with progression. Multivariable logistic regression indicated that both sex and PCR were independent variables affecting the outcome. CONCLUSIONS: In a cohort of seropositive individuals, patients with T cruzi DNA detected by PCR and male patients were at higher risk of progression. These results highlight the importance of T cruzi in the pathophysiology of chronic cardiomyopathy.

Placental alkaline phosphatase of high molecular weight in plasma of pregnant women in the last trimester of gestation.

Human blood plasma contains alkaline phosphatase (EC 3.1.3.1) isoenzymes from bone, liver and intestine. Blood of pregnant women in the third trimester of gestation also contains an isoenzyme of alkaline phosphatase from placenta, placental-AP (PLAP). Serum from pregnant women in the last trimester of gestation shows activity of soluble placental alkaline phosphatase (sol-PLAP). It is also known that a membrane bound high molecular weight placental-AP (high Mr-PLAP) is present in butanol extracts from placental tissue. Nevertheless, up to now, high Mr-PLAP has not been founded in human plasma. A method developed in this laboratory allows detection of membrane-bound alkaline phosphatase in pellet of plasma centrifuged at 100,000x g. By applying this method we have detected high molecular weight placental alkaline phosphatase in plasma of healthy pregnant women in the third trimester of pregnancy.

Alkaline phosphatase isoenzymes for the diagnosis of metastatic tumors and lymphomas of liver and bone.

The plasmatic activities of total alkaline phosphatase (ALP) (E.C. 3.1.3.1), high molecular weight-ALP (high Mr-ALP) and bone-ALP isoenzymes, were determined in healthy individuals and in patients with: neoplasia without metastases, hepatic metastases, bone metastases and mixed metastases (hepatic and bone). Variables were individually used to assess incidence of metastases and percentages of false negative and false positive results were calculated. The three values were then used together to assess metastases incidence and sensitivity, specificity, positive predictive value, negative predictive value and predictive capacity were estimated. We conclude that none of the variables per se are reliable for the diagnosis of metastases. On the other hand, the three values show high percentages of sensitivity, specificity, positive predictive value, negative predictive value and a high probability (0.93) of accurate diagnosis when applied to a larger population, with similar prevalence values.

Estimación de la frecuencia de heterocigotes de la enfermedad de Sandhoff en una población Argentina de alto riesgo: asignación predictiva del genotipo mediante un análisis estadístico / Estimation of the frecuency of heterozygotes in Sandhoff disease in an Argentine population of high risk: predictive assignment of the genotype through a statistical analysis

En trabajos preliminares hemos evidenciado una alta frecuencia de la Enfermedad de Sandhoff (ES), deficiencia génica de la actividad hidrolítica de la hexosaminidasa (Hex) en sus dos mayores isoenzimas Hex A y Hex B, en niños criollos de un semi-aislado geográfico de la Argentina. La presente comunicación se refiere a la estimación de la proporción de portadores del gen anormal entre 1400 escolares de la zona mediante la determinación con sustratos artificiales de la Hex Total y Hex B Sérica. Los valores de las actividades enzimáticas de 32 heterocigotos testigos, 15 de ellos obligados (padres), sirvieron de patrón de referencia autóctono para la detección de 71 heterocigotos de la muestra problema (4,93%). Los límites de confidencia del 99% determinaron que la proporción de los heterocigotos en la población estaba en el intervalo 0,0346-0,0636 o sea 1 portador por cada 16 a 29 habitantes de la región, resultado similar a la heterocigosis calculada según la ley de Hardy-Weinberg (1:14 a 1:26). El análisis estadístico de las dos variables empleadas (Hex Total y Hex B) de los subgrupos referenciales homocigotos normales y heterocigotos testigos, permitió la elaboración de una función discriminante cuadrática (FDC) capaz de discernir, con alta probabilidad, entre un genotipo mutado y otro normal. Los datos obtenidos indican una heterocigosis de ES exepcionalmente alta en la población de riesgo y coherente con la frecuencia de homocigotos enfermos reconocidos, otorgándose fundamento a un programa preventivo a nivel regional. Entre tanto, la FDC elaborada provee un recurso eficaz y práctivo de asesoramiento individual, sobre todo en el ámbito clínico vinculado al problema

Estimación de la frecuencia de heterocigotes de la enfermedad de Sandhoff en una población Argentina de alto riesgo: asignación predictiva del genotipo mediante un análisis estadístico / Estimation of the frecuency of heterozygotes in Sandhoff disease in an Argentine population of high risk: predictive assignment of the genotype through a statistical analysis

En trabajos preliminares hemos evidenciado una alta frecuencia de la Enfermedad de Sandhoff (ES), deficiencia génica de la actividad hidrolítica de la hexosaminidasa (Hex) en sus dos mayores isoenzimas Hex A y Hex B, en niños criollos de un semi-aislado geográfico de la Argentina. La presente comunicación se refiere a la estimación de la proporción de portadores del gen anormal entre 1400 escolares de la zona mediante la determinación con sustratos artificiales de la Hex Total y Hex B Sérica. Los valores de las actividades enzimáticas de 32 heterocigotos testigos, 15 de ellos obligados (padres), sirvieron de patrón de referencia autóctono para la detección de 71 heterocigotos de la muestra problema (4,93%). Los límites de confidencia del 99% determinaron que la proporción de los heterocigotos en la población estaba en el intervalo 0,0346-0,0636 o sea 1 portador por cada 16 a 29 habitantes de la región, resultado similar a la heterocigosis calculada según la ley de Hardy-Weinberg (1:14 a 1:26). El análisis estadístico de las dos variables empleadas (Hex Total y Hex B) de los subgrupos referenciales homocigotos normales y heterocigotos testigos, permitió la elaboración de una función discriminante cuadrática (FDC) capaz de discernir, con alta probabilidad, entre un genotipo mutado y otro normal. Los datos obtenidos indican una heterocigosis de ES exepcionalmente alta en la población de riesgo y coherente con la frecuencia de homocigotos enfermos reconocidos, otorgándose fundamento a un programa preventivo a nivel regional. Entre tanto, la FDC elaborada provee un recurso eficaz y práctivo de asesoramiento individual, sobre todo en el ámbito clínico vinculado al problema (AU)