Compromiso renal en la Enfermedad de Fabry en la edad pediátrica / RENAL INVOLVEMENT IN CHILDREN WITH FABRY'S DISEASE

La Enfermedad de Fabry (EF) es un trastorno hereditario, ligado al cromosoma X, con una incidencia de 1/40.000 nacidos vivos. Se sabe que afecta tanto a varones como a mujeres y sus primeras manifestaciones clínicas comienzan en edades pediátricas. La actividad reducida o nula de la enzima lisosomal alfa-galactosidasa A (α-Gal A), que es la comprometida en la EF, genera la acumulación progresiva de su sustrato, principalmente la globotriosilceramida (Gb3) en las diferentes estirpes celulares de los distintos órganos. El fallo renal es una de las complicaciones serias de la enfermedad.efropatía por Fabry, pese a que la enfermedad está presente desde la infancia, suele manifestarse de forma silente, aún en los estudios clínicos de rutina. Un diagnóstico precoz y oportuno es crucial dado que la demora en el inicio de la terapia de reemplazo enzimático parece no lograr detener la enfermedad renal progresiva. Esta revisión tiene como objetivo proporcionar una actualización de la comprensión actual, los desafíos y las necesidades para abordar mejor las complicaciones renales de la enfermedad de Fabry en niños. Intentar comprender la fisiopatología de éste compromiso puede ayudar a prevenir su progresión

Fabry´s disease is an x-chromosome hereditary disease with an incidence of 1/40000 newborns. Nowadays it is present in males as in females , and its first clinical symptoms are seen in pediatric patients. Patients have reduced or no activity of alpha-galactosidase which leads to progressive accumulation of Gb3 in lysosomes of all types of cells. Renal failure is a serious complication of this disease. Fabry´s nephropathic lesions are present and progress in childhood while the disease commonly remains silent by routine clinical measures. Early and timely diagnosis is crucial since late initiation of enzyme replacement therapy may not halt progressive renal dysfunction. This review aims to provide an update of the current understanding, challenges, and needs to better approach renal complications of Fabry´s disease in children. Trying to understand the pathophysiology of this compromise may help prevent its progression

La atención primaria pediátrica: logros y desafíos / Pediatric primary care: achievements and challenges

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Fragmentation of sperm DNA using the TUNEL method.

OBJECTIVES: To establish the validity of the TUNEL assay in determining sperm DNA fragmentation, the relationship between the degree of fragmentation and the seminal parameters and the sample needed to conduct the test. MATERIAL AND METHODS: We used semen samples from healthy fertile men (n=33), patients who consulted for infertility with a prescription for the TUNEL assay (n=77) and patients with intracytoplasmic sperm injection failure (n=20), analyzed according to the 2010 WHO. The TUNEL/propidium iodide test was performed by flow cytometry, on baseline and post-swim-up samples. RESULTS: The cutoff value for the TUNEL assay (ROC curves) was 26%, with a sensitivity and specificity of 85% and 89%, respectively. The pre-swim-up and post-swim-up medians of the results from the TUNEL assay showed no significant differences (17.0% vs. 12.9%, respectively). However, 39.1% of the samples showed a difference greater than 15 in absolute value between the results of the baseline and post-swim-up TUNEL assays. The linear correlation study of the morphology, mobility and vitality using the post-swim-up TUNEL assay showed a greater correlation than preselection, with significant results (r: -0.394, P<.0001; r: -0.461, P<.0001; r: -0.526, P<.0001). CONCLUSIONS: The TUNEL assay is a valid test for clinical use. DNA fragmentation is a factor independent from traditional semen tests. We found a greater susceptibility to damage generated in the laboratory procedures in the samples with lower quality. The sample of choice for evaluating DNA fragmentation will depend on whether the clinician is treating a natural or assisted fertilization.

XV aniversario de la Fundación 'Ernesto Sánchez Villares' / XV Anniversary of the 'Ernesto Sánchez Villares' Foundation

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Impact of spontaneous smoking cessation on sperm quality: case report.

We evaluated sperm quality after a 3-month smoking cessation programme by sperm analysis, objective sperm motility analysis, protein tyrosine phosphorylation in capacitating conditions and DNA fragmentation (TUNEL). Sperm analysis after smoking cessation revealed a distinctive improvement in sperm concentration, fast spermatozoa (≥35 µm/s), sperm vitality, percentage of spermatozoa recuperated after an enrichment technique and protein tyrosine phosphorylation. However, no changes were observed in the number of germinal cells in the ejaculate, sperm morphology and sperm DNA fragmentation. It is concluded that physicians should strongly advise their patients to quit smoking before undergoing medical treatment or assisted reproduction techniques to achieve pregnancy.

Prognostic value of germ cells in the ejaculate: a case study.

The first approach to assessing male fertility is to study a spermiogram, where special attention is given to sperm count, motility and morphology, while less attention is given to other cells in the ejaculate. Normal spermatogenesis requires a balance between cell death and proliferation; therefore, the number of germ cells (GC) in the ejaculate is less than the number of sperm. We propose a new index for altered spermatogenesis, i.e., the rate GC/sperm. We investigated a patient with oligozoospermia and a GC/sperm ratio greater than one, which indicated that spermatogenesis had been damaged. Complementary cytological tests were employed to characterized GC status: Papanicolaou stain, transmission electronic microscopy (TEM), vitality test, AgNOR and TUNEL assay. We also correlated cell morphology with ultrastructure studies that showed apoptosis. Nuclear apoptosis is characterized by vacuolization, misshapen nuclei, and "half moon," dispersed, uncondensed, disrupted and smudged chromatin. Cytoplasmic apoptosis is characterized by vacuolization, cytoplasmic protrusions, lamellar bodies, and swollen endoplasmic reticulum and mitochondria. To date, only testicular biopsy has been used to diagnose complete or incomplete testicular arrest. Our investigation is the first to determine a cytological feature in semen samples that could be used as a biological marker for abnormal spermatogenesis and for predicting the transition from oligospermia to azoospermia.

Assessment of human sperm protein tyrosine phosphorylation by immunocytochemistry in a clinical andrology laboratory. Preliminary data.

We propose that evaluation of protein tyrosine phosphorylation (TP) status in ejaculated spermatozoa under capacitating conditions in an experiment that mimics "in vitro" the physiology of sperm from ejaculation through the female genital tract could potentially be used as a prognostic test for functional competence of sperm in fertilization. Our purpose was to elucidate whether there is a relation between conventional sperm parameters, occurrence of TP and pregnancy outcome obtained from intrauterine insemination (IUI). Semen samples were analyzed according to WHO criteria. TP levels were determined by immunocytochemistry under four different conditions: 1) ejaculated sperm, 2) postselection sperm, 3) postselection sperm incubated 5 h at 37 degrees C and 5% CO(2), and 4) postselection sperm incubated overnight at 37 degrees C and 5% CO(2). Data on sperm tyrosine phosphorylated proteins did not correlate with sperm concentration, progressive motility or normal sperm morphology. TP increased under capacitating conditions and showed a time dependent pattern except for five outlier cases. IUI was performed in 12 selected couples who had neither female nor male infertility factors. The three pregnancies had a time dependent pattern for TP. Of the unsuccessful cases, one had an outlier TP pattern. It appears that a TP time dependent pattern is necessary for fertilization.

Impacto de la dieta sobre la proteinuria en pacientes con nefropatía secuelar por sindrome urémico hemolítico (D+) / Impact of the diet on proteinuria in patients with sequelae nephorapathy due t o Hemolytic Uremic Syndrome (D+)

El sindrome Urémico Hemolítico D+ (SUH) es la segunda causa de insuficiencia renal crónica terminal (IRCT) en edad pediátrica. La proteinuria es el principal modulador de la evolución a la cronicidad. En un grupo de pacientes tratados con dieta controlada en proteínas e inhibidores de la enzima de conversión de la Angiotensina II se demostró un enlentecimiento significativo en la progresión de la nefropatía a la IRCT. El objetivo de este trabajo fue evaluar, en una primera etapa, el impacto de la dieta normoproteica y normosódica sobre la proteinuria en pacientes con nefropatía secuelar por SUH y función renal normal (CI Cr >80ml/min/1.73m2). Métodos: como parte de un estudio de fase III longitudinal, multicéntrico, aleatorizado, doble ciego, de grupos paralelos (placebo y activo controlado con enalpril y losartan), se evaluó la diferencia entre la proteinuria antes y después de una dieta normósódica y mormoprotica, indicada según RDA. La ingesta proteica fue estimada mediante recordtorio de 72 horas y el cálculo de excreción de urea en orina de 24 horas. La proteinuria se dosó en orina de 24 hs. al comienzo del estudio, a los 30 y 60 días. Resultados: se incluyeron 102 pacientes cuyo rango de proteinuria fue entre 5.3 y 40.0 mg/kg/día de los cuales negativizaron 65 (63.7 por ciento) y no respondieron 37 (36,3 por ciento ). La mediana de edad del comienzo de la enfermedad fue de 16,5 meses (rango: 7.0-85.0 meses). El tiempo de evolución post SUG fue de 4.0 a 155.0 meses (mediana 48.0 meses) El valor de la proteinuria inicial en los 65 niños que respondieron fue de x 9.83 mg/kg/día (ES 0 o,34) y post dieta de de x =2,44 (ES 0 0,12) P < 0.0001. La media de las diferencias entre la natriuresis pre y post dieta no fue estadísticamente diferente de 0; t = 0,97 (x /ES). Conclusión: la dieta normoproteica es capaz de normalizar la proteinuria en el 63.7 por ciento de los pacientes con proteinuria significativa secundaria a SUH y función renal normal.

The only presence of sperm in urine does not imply retrograde ejaculation.

This is a retrospective study of clinical experience collected at the University Clinical Hospital over a 19-year period. Semen samples were analyzed according to WHO criteria. In the postmasturbatory urine, sperm count was performed. Data were expressed as total sperm number in urine (TSNU) and using a retroejaculation index. Patients were categorized into four groups according to the presence of sperm in the studied samples: a) in semen and urine; b) only in urine; c) only in semen; d) neither in semen nor in urine. A control group included nonretroejaculator patients. Retroejaculator patients are those whose TSNU is superior to 3.8 x 10(6) and the RI superior to 2.16%. While diagnosing retroejaculation, the only presence of sperm in the postmasturbatory urine is not adequate. The proposed index added to total sperm number in urine and semen volume may identify true retroejaculator patients.

Coordinación entre niveles asistenciales en pediatría / Coordination between attendance levels in paediatrics

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Asthenozoospermia: analysis of a large population.

Spermatozoa travel a long distance to meet and fertilize the oocyte, so sperm motility is a requisite for normal fertilization. Asthenozoospermia, or low sperm motility, is a common cause of human male infertility. This is a retrospective study (1992-1999) to document the prevalence of this pathology in infertile men and to clarify the probable factors associated to its etiology. The prevalence was 18.71% for asthenozoospermia and 63.13% for asthenozoospermia associated with oligo- and/or teratozoo-spermia; thus, 81.84% of the studied samples had altered motility. Leukocytospermia, the ratio of germ cells/sperm, anti-sperm antibodies, consistency, biochemical markers of accessory sex glands, and sperm response after swim-up were studied in normospermic (N), asthenozoospermic (A), and combined asthenozoospermic (C) samples. No significant difference was found in the frequency of leukocytospermia among groups. The rate of germ cells/(spermatozoa + germ cells) between C and N (p < .01) and C and A (p < .01) was statistically different, while no difference was found on comparing N and A. MAR-test over 40% was found in 6% of the A samples and 7.6% of the C, while no positive values were observed in the N group. The percentage of hyperviscous samples was higher in the low sperm motility samples than in the normal group. Data on concentration of the biochemical markers seem to be decreased in asthenozoospermia. Pure and combined asthenozoo-spermia showed different behavior in sperm recovery after swim-up. Two different asthenozoospermias could be defined: the pure one where sperm environment is involved (immunological factor, hyperviscosity, and secretory gland function) and the combined, where the testis is comprised.

Laboratory methods for the diagnosis of asthenozoospermia.

Human sperm samples from 46 men were evaluated for standard semen parameters by two trained technicians, according to WHO criteria. A computer-aided semen analyzer (CASA, HTM-IVOS) was employed. The overall mean coefficient of variation for the 2 participants and the 46 samples studied was 17% for the percentage of progressive motile spermatozoa (r = 0.77). Twenty-nine (63%) samples were classified as asthenozoospermia by both methods. From the studied samples, 12 (26%) were classified as asthenozoospermia by the subjective method and 2 (4.3%) by CASA (p =.01). The two methods do not provide directly comparable data.

Shedding of immature germ cells.

The immature germ cells (IGC) constitute the highest percentage (90%) of nonsperm cells (NSpC) in ejaculates from fertile or infertile men. The objective of this study was to evaluate IGC concentration and the IGC/(IGC + Sp) ratio, in normozoospermia and dispermia. Normozoospermia from men with proven fertility (NPF). nonproven fertility (NNPF). dispermia (D) and semen samples with excessive shedding of immature germ cells (GI 1.7 x 10(6) to 5 x 10(6) IGC/mL and GII > 5.0 x 10(6) IGC/mL) were used in this study. The mean value +2 SD for the NNPF (1.7 x 10(6)/mL) and the value proposed by WHO (5 x 10(6)/mL) were employed to define GI and GII groups. IGC concentration is statistically different in the studied groups. The IGC/Sp ratio showed a significant difference only between the NNPF and the D. When comparing semen parameters (Sp/ejaculate. grade (a) motility and morphology) there was a highly significant difference between NNPF and GI and GII: no difference was found between GI and GII. While studying 200 cases of dispermias 83% showed a high shedding of immature germ cells. The cytological study of nonsperm cells and the count and identification of the immature germ cells could be used to evaluate the dispermic disorders.

Consumo de tabaco, alcohol y drogas no legales entre adolescentes y relación con los hábitos de vida y el entorno / Tobacco, alcohol and illegal drug consumption among adolescents. Relationship with lifestyle and environment

Objetivos: Conocer la edad de contacto con tabaco, alcohol y drogas ilegales entre los adolescentes, su consumo y sus relaciones con el entorno y hábitos de vida. Método: Estudio transversal descriptivo, mediante encuesta a 2.178 adolescentes de 12 a 16 años representativos de Cantabria. Resultados: El 80,4% de los adolescentes considera el tabaco una droga. El 44% ha fumado un cigarrillo alguna vez y son fumadores el 19,3%. El 44,5% no cree que el alcohol sea una droga. El 69,2% ha probado el alcohol y el 37% son bebedores. La mayoría (92,9%) bebe los fines de semana y el 88% todas las bebidas encuestadas. Amigos (54,1%) y familiares (16,4%) son los iniciadores en este hábito. El 46% de los bebedores actuales se ha embriagado en los últimos 6 meses una o más veces. A los 15 años fuma el 29,4% y bebe el 61,8%. El contacto con drogas ilegales es menos frecuente y el 10,2% declara consumir hachís. El consumo de todas estas sustancias se asocia con una mayor edad, un entorno consumidor y determinado patrón de ocio. El análisis mediante regresión logística refleja que la consideración de tabaco y alcohol como drogas es un factor protector para su consumo y son factores de riesgo consumir otras drogas, embriagarse y tener un entorno consumidor de las mismas. Conclusiones: Los adolescentes presentan un contacto precoz y un consumo preocupante de tabaco, alcohol y drogas ilegales. El entorno y los hábitos de vida se relacionan marcadamente con estas conductas de riesgo (AU)

[Tobacco, alcohol and illegal drug consumption among adolescents and the relationship with lifestyle and environment]. / Consumo de tabaco, alcohol y drogas no legales entre adolescentes y relación con los hábitos de vida y el entorno.

OBJECTIVES: To identify the age at which adolescents first experiment with tobacco, alcohol and illegal drugs and to identify the relationship between consumption of these substances and lifestyle and environment. METHOD: Cross-sectional descriptive study based on a survey of 2,178 adolescents aged 12-16 years old from Cantabria (Spain). RESULTS: Tobacco was considered a drug by 80.4 % of the adolescents. Forty-four percent had smoked at some time and 19.3 % were smokers. Alcohol was not considered a drug by 44.5 %, 69.2 % had drunk alcohol at some time and 37 % drank regularly. Most of the adolescents (92.9 %) drank at weekends and 88 % drank all the spirits surveyed. The adolescents were introduced to this habit by friends (54.1 %) and relatives (16.4 %). Forty-six percent of those who drank regularly had got drunk once or more in the previous 6 months. At the age of 15 years, 29.4 % smoked and 61.8 % drank alcohol regularly. An association with illegal drugs was less common; 10.2 % smoked hashish. Consumption of all these substances was associated with greater age, living in an environment in which these substances were consumed and with certain forms of leisure activity. Logistic regression analysis revealed that considering tobacco and alcohol as drugs protected adolescents against their consumption and that consuming other drugs, getting drunk and being in a consumer environment were risk factors for consumption. CONCLUSIONS: Contact with illegal drugs, alcohol and tobacco starts early in adolescence. Levels of consumption are worrying. Environment and lifestyle are closely associated with risk factors for consumption.

A mutation in the cofactor-binding domain of 11beta-hydroxysteroid dehydrogenase type 2 associated with mineralocorticoid hypertension.

Renal 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2) is an enzyme responsible for the peripheral inactivation of cortisol to cortisone in mineralocorticoid target tissues. Mutations in the gene encoding 11betaHSD2 cause the syndrome of apparent mineralocorticoid excess (AME), an autosomal recessive form of inherited hypertension, in which cortisol acts as a potent mineralocorticoid. The mutations reported to date have been confined to exons 3-5. Here, we describe two siblings, 1 and 2 yr old, who were diagnosed with hypokalemic hypertension and low plasma aldosterone and renin levels, indicating mineralocorticoid hypertension. Analysis of urinary steroid metabolites showed a markedly impaired metabolism of cortisol, with (tetrahydrocortisol + 5alpha-tetrahydrocortisol)/tetrahydrocortisone ratios of 40-60, and nearly absent urinary free cortisone. Although phenotypically normal, the heterozygous parents showed a disturbed cortisol metabolism. Genetic analysis of the HSD11B2 gene from the AME patients revealed the homozygous deletion of six nucleotides in exon 2 with the resultant loss of amino acids Leu(114) and Glu(115), representing the first alteration found in the cofactor-binding domain. The deletion mutant, expressed in HEK-293 cells, showed an approximately 20-fold lower maximum velocity but increased apparent affinity for cortisol and corticosterone. In contrast, two additionally constructed substitutions, Glu(115) to Gln or Lys, showed increased maximal velocity and apparent affinity for 11beta-hydroxyglucocorticoids. Functional analysis of wild-type and mutant proteins indicated that a disturbed conformation of the cofactor-binding domain, but not the missing negative charge of Glu(115), led to the observed decreased activity of the deletion mutant. Considered together, these findings provide evidence for a role of Glu(115) in determining cofactor-binding specificity of 11betaHSD2 and emphasize the importance of structure-function analysis to elucidate the molecular mechanism of AME.