[Multicentre longitudinal study of the nutritional status and swallowing difficulties in children with severe neurological diseases]. / Estudio multicéntrico y longitudinal del estado nutricional y problemas de deglución en niños con enfermedad neurológica grave.

INTRODUCTION: Nutritional problems are common in children with neurological diseases, especially if they have significant motor impairment. Oropharyngeal dysphagia is very prevalent in these patients, and can contribute to worsening nutritional status and produce pulmonary aspirations. AIM: Longitudinal assessment of the nutritional status of a sample of pediatric patients with moderate-severe neurological disease and establish the prevalence of oropharyngeal dysphagia in that sample. PATIENTS AND METHODS: An observational multicenter prospective study was conducted. We included children under 16 years of age with moderate-to-severe neurological impairment from four hospitals, with clinical and anthropometric monitoring for one year. Questions were asked to conduct oropharyngeal dysphagia screening. RESULTS: Sixty-eight children were included, the main diagnosis obtained was cerebral palsy. In the anthropometric assessment, 42 patients (62%) showed weight z scores below -2, and 29 (43%) height z scores below -2, while body mass index, mid upper arm circumference and triceps and subscapular skinfolds remained less affected. We found an oropharyngeal dysphagia prevalence of 73.5% in our sample, increasing with greater motor impairment. CONCLUSIONS: These patients showed lower weight and height than children without neurological impairment. However, with a correct follow-up they remain stable with an adequate body composition. It is important to proactively investigate the presence of oropharyngeal dysphagia, especially in those with greater motor impairment, as it occurs very frequently and an adequate diagnosis can improve clinical evolution and prevent complications.

TITLE: Estudio multicéntrico y longitudinal del estado nutricional y problemas de deglución en niños con enfermedad neurológica grave.Introducción. Los problemas nutricionales son frecuentes en niños con enfermedades neurológicas, sobre todo si tienen una importante afectación motora. La disfagia orofaríngea es muy prevalente en estos pacientes y puede contribuir a empeorar el estado nutricional y a que se produzcan aspiraciones pulmonares. Objetivo. Evaluar de forma longitudinal el estado nutricional de una muestra de pacientes pediátricos con enfermedad neurológica moderada-grave y establecer la prevalencia de disfagia orofaríngea en dicha muestra. Pacientes y métodos. Estudio multicéntrico prospectivo observacional. Se incluyó a niños menores de 16 años controlados en cuatro centros hospitalarios. Se recogieron datos clínicos y antropométricos durante un año. Se realizaron preguntas dirigidas a realizar un cribado sistemático de disfagia orofaríngea. Resultados. Se seleccionó a 68 pacientes, y el diagnóstico más frecuente fue la parálisis cerebral infantil. En la valoración antropométrica, 42 pacientes (62%) presentaron puntuaciones z de peso inferiores a ­2, y 29 (43%), talla con puntuaciones z inferiores a ­2, pero con un índice de masa corporal, un perímetro braquial y unos pliegues cutáneos mucho menos alterados. La prevalencia de disfagia orofaríngea fue del 73,5%, que aumentó a mayor afectación motora. Conclusiones. Estos pacientes presentan un tamaño corporal menor que la población de su misma edad y sexo sin patología. Sin embargo, con un correcto seguimiento nutricional, mantienen estable su composición corporal. Es importante investigar de forma proactiva la presencia de disfagia orofaríngea, sobre todo en los que tienen mayor afectación motora, pues se presenta con mucha frecuencia y un adecuado diagnóstico puede mejorar la evolución clínica y prevenir complicaciones.

Alpha-tubulin marker gene of neural territory of sea urchin embryos detected by whole-mount in situ hybridization.

We have used Northern blot and whole-mount in situ hybridizations to analyze the temporal and spatial expression pattern of the Pl alpha 2 alpha-tubulin gene in Paracentrotus lividus sea urchin embryos. The Pl alpha 2 transcript is first detectable at 14 h post-fertilization (blastula stage) and it is only expressed in the oral ectoderm. The amount of transcripts of this gene increases throughout development and accumulates up to the pluteus stage. In this stage the Pl alpha 2 transcript is localized in the neural structures of the embryo. We conclude that the Pl alpha 2 gene is an early neurogenic territory marker. Furthermore we have observed the same localization of the Pl alpha 2 transcript in the Zn(++)- or phenytoin-treated embryos, confirming the animal localization of the Pl alpha 2 transcript and its specific relation to neurogenic territory, whose differentiation starts from few founder cells present at blastula stage.

Spatial expression of alpha and beta tubulin genes in the late embryogenesis of the sea urchin Paracentrotus lividus.

In Paracentrotus lividus sea urchin embryos, at blastula stage, there is an abrupt increase in the abundance of alpha and beta tubulin transcripts in particular of the PI beta 1, PI beta 2 and PI alpha 2 forms. In order to assign specific functions to the various embryonic tubulin genes, we have used whole-mount in situ hybridization to determine spatial patterns of expression of five different alpha and beta tubulin embryonic genes. The PI beta 3 transcripts, as previously shown for PI alpha 2, start to localize in a few founder cells from which the neurogenic territory differentiates. The other four embryonic tubulin mRNAs (PI beta 1/2 and PI alpha 1/10), are localized in the ciliated band- and gut-territory. These territories originate by morphogenetic processes, which occur in late embryogenesis in the sea urchin and depend on cellular interactions. In particular, the interactions between the oral and aboral ectoderm specify the position of the ciliated band, whereas the invagination of the vegetal plate forms the gut territory. We suppose that the increase in alpha and beta tubulin transcripts could be functionally related to these two morphogenetic events. Our results show in fact that specific tubulin isotypes, or a mix of them, are expressed in and mark the ciliated band and the neighboring oral/aboral ectoderm cells of the ciliated band, in addition to the cells of the gut territory. The same localization of all these tubulin transcripts has been confirmed by whole-mount in situ hybridization experiments performed on embryos treated with agents able to induce deciliation or exogastrulation. Furthermore a putative correlation of PI beta 2 with cilium formation has been shown by the results obtained on deciliated embryos.

[Surgical treatment of pediatric pulmonary metastases]. / Tratamiento quirúrgico de las metástasis pulmonares pediátricas.

OBJECTIVE: We comment and update the surgical treatment for pulmonary metastases (PM) within a multidisciplinary approach for paediatric cancer. MATERIAL AND METHODS: We analyse patients with PM who have been operated between 1976-1996. Scientific literature published in the last 25 years (Cancerlit and Medline) was reviewed. RESULTS: PM from 13 patients were removed. Seven were males and 6 females with a mean age 5 4/12 years (range: 11 months- 12 3/12 years). Diagnoses were Wilms' tumour (7), osteosarcoma (3), Ewing sarcoma (1), rabdomiosarcoma (1), Yolk sac tumour (1). PM were unilateral in 7 cases and bilateral in six cases. PM appeared synchronically in four patients and metacronically in nine cases (3 of these after chemotherapy). All patients received chemotherapy and four of them local radiotherapy. Surgery consisted on radical segmentectomy and only one patient needed lobectomy due to a local relapse. Nowadays five patients (38%) are in complete remission with a mean follow-up from surgery of 11 11/12 years (range: 6 3/12-20 years). CONCLUSIONS: Metastasectomy is an important surgical technique in global treatment of children with PM and for a selected group of patients it can offer the only opportunity for curation.

EGTA treatment causes the synthesis of heat shock proteins in sea urchin embryos.

Paracentrotus lividus embryos, at post-blastular stage, when subjected to a rise in temperature from physiologic (20 degrees C) to 31 degrees C, synthesize a large group of heat shock proteins (hsps), and show a severe inhibition of bulk protein synthesis. We show, by mono- and two-dimensional electrophoresis, that also EGTA (ethylene glycol-bis[beta-aminoethyl ether] tetraacetic acid) treatment induces in sea urchin embryos both marked inhibition of bulk protein synthesis and the synthesis of the entire set of hsps. Furthermore, EGTA-treated sea urchin embryos are able to survive at a temperature otherwise lethal (35 degrees C) becoming thermotolerant. Because incubation with a different calcium-chelator, EDTA (ethylenediaminetetraacetic acid), or in calcium-free medium did not induce hsps synthesis we conclude that the stress response caused by EGTA is not related to its calcium chelator function.

Sea urchin early histone H2A modulator binding factor 1 is a positive transcription factor also for the early histone H3 gene.

To shed some light on the mechanisms involved in the coordinate regulation of the early histone gene set during sea urchin development, we tested the hypothesis that the upstream sequence element USE1, previously identified in the early H2A modulator, could also participate in the transcription of the early histone H3 gene. We found by DNAse I protection analysis and by competition in electrophoretic mobility-shift experiments that two sequence elements of the H3 promoter closely resembled the USE1-H2A sequence in their binding activity for nuclear factors from 64-cell stage embryos. These modulator binding factor 1 (MBF-1)-related factors seem to recognize the ACAGA motif that is conserved between the USE1-like sequences of both H2A and H3 promoters. In fact, excess oligonucleotide containing a mutated USE1-H2A element in which the ACAGA sequence was mutated to AGTCA failed to compete with the USE1 sites of both H2A and H3 genes for interaction with MBF-1. Finally, in vivo transcriptional analysis in both Xenopus and sea urchin showed that an excess of USE1-H2A element efficiently competed for the activity of the H3 promoter. From these results we conclude that MBF-1 is a transcription factor conserved between sea urchin and frog and that MBF-1 or related transcription factors are involved in the coordinate expression of both H2A and H3 early histone genes.

Deciliation: A stressful event for Paracentrotus lividus embryos.

In this report, by using mono- and two-dimensional electrophoretic analysis, we demonstrate that deciliation on sea urchin embryos induces a stress response. Deciliation indeed causes not only the activation of ciliary subroutine, but also a transient decrease of bulk protein synthesis. This decrease is in agreement with our previous results on heat shock response in sea urchin, although deciliation does not induce the expression of the same main hsp set. We were able to characterize one main deciliation-stress protein of 40 kDa whose expression is transiently induced by deciliation and whose localisation is likely to be nuclear.

Transcription of sea-urchin mesenchyme blastula histone genes after heat shock.

Mesenchyme blastula sea-urchin embryos were heat-shocked at 31 degrees C and pulse-labelled with [3H]uridine. The nuclear RNA was fractionated on glyoxal/agarose gels and each RNA fraction hybridized with cloned early blastula histone DNA. The results showed that after heat shock there is an accumulation of histone RNA molecules larger than the messenger and a decrease, with respect to the control, of 9-S histone RNA. Chasing of the heat shock RNA by incubation of the embryos with unlabelled uridine and cytidine restores the radioactivity, as well as the hybridization profiles, of control embryos. Furthermore saturation curves obtained by hybridizing histone DNA with labelled 9-S RNA, showed an absence of the histone messenger in the cytoplasm of heat-shocked embryos, whereas it is present in both control and chased embryos. These results are consistent with the hypothesis that the accumulated histone RNA in heat-shocked embryos is the precursor of mature messengers.

Evidences of two different sets of histone genes active during embryogenesis of the sea urchin Paracentrotus lividus.

Histone mRNAs at different stages of development were purified by hybridization with the cloned homologous histone genes. The electrophoretic patterns of oocytes, 2-4 blastomeres, 64 cells and morula histone mRNAs was found to be identical, whereas the electrophoretic pattern of mesenchyme blastula histone mRNA was markedly different. The cloned histone DNA of P.lividus was hybridized with the RNA of each stage. The Tm was 74 degrees C in all cases except for the mesenchyme histone mRNAs whose Tm was 59 degrees C, thus suggesting that at least two different clusters of histone genes are active in the course of the sea urchin development.

Sea urchin neural alpha2 tubulin gene: isolation and promoter analysis.

Expression of Talpha2 gene, during sea urchin Paracentrotus lividus development, is spatially and temporally regulated. In order to characterize this gene, we isolated the relevant genomic sequences and scanned the isolated 5'-flanking region in searching for cis-regulatory elements required for proper expression. Gel mobility shift and footprinting assays, as well as reporter gene (CAT and beta-gal) expression assays, were used to address cis-regulatory elements involved in regulation. Here we report that an upstream 5'-flanking fragment of PlTalpha2 gene drives temporal expression of reporter genes congruent with that of endogenous Talpha2 gene. The fragment contains cis-elements able to bind nuclear proteins from the gastrula stage (at which the Talpha2 gene is expressed) whose sequences could be consistent with the consensus sequences for transcription factors present in data bank.

Cis-acting elements of the sea urchin histone H2A modulator bind transcriptional factors.

Functional tests, performed by microinjection into Xenopus laevis oocytes, show that a DNA fragment containing the modulator of the early histone H2A gene of Paracentrotus lividus enhances transcription of a reporter gene when located, in the physiological orientation, upstream of the tk basal promoter. Gel retardation and DNase I footprinting assays further reveal that the H2A modulator contains at least two binding sites [upstream sequence elements 1 and 2 (USE 1 and USE 2)] for nuclear factors extracted from sea urchin embryos, which actively transcribe the early histone gene set. Interestingly, USE 1 is highly homologous to a cis-acting element previously identified in the H2A modulator of Psammechinus miliaris [Grosschedl, R., Mächler, M., Rohrer, U. & Birnstiel, M. L. (1983) Nucleic Acids Res. 11, 8123-8136]. Finally, a cloned oligonucleotide containing the USE 1 sequence competes efficiently in Xenopus oocytes with the H2A modulator to prevent enhancement of transcription of the reporter gene. From these results, we conclude that USE 1 and perhaps USE 2 in the H2A modulator are upstream transcriptional elements that are recognized by trans-acting factors common to Xenopus and sea urchin.

Different micrococcal nuclease cleavage patterns characterize transcriptionally active and inactive sea-urchin histone genes.

The micrococcal nuclease cleavage sites have been mapped in the H2A coding and flanking regions of the sea-urchin histone DNA chromatin. A hypersensitive area, centered around - 100 base pairs from the H2A starting site, is found only in embryos actively transcribing the alpha-subtype histone genes. In mesenchyme blastula embryos, upon inactivation of the H2A gene, this region becomes protected while two other areas, near the transcription starting site and in the proximity of the 3' palindromic sequence, become preferential targets for the enzyme. Analysis of the pattern of micrococcal nuclease cleavage on the same region of the histone gene cluster in sperm and late blastula chromatin and on the corresponding segment of protein-free DNA indicates that distinct nucleosomal arrangements characterize the histone genes in the two cell populations.

[Central nervous system tumors in children less than three years of age]. / Tumores de sistema nervioso central en niños menores de 3 años.

OBJECTIVE: Central nervous system (CNS) tumors are the most frequent solid tumors in children. Twelve to twenty percent are diagnosed in patients younger than two years of age and these patient present more morbidity and mortality due to the illness and the treatment itself. PATIENTS AND METHODS: A retrospective study of CNS tumors in children younger than three years of age diagnosed in our hospital between 1985 and 1995 was carried out. RESULTS: We treated 21 patients between 1985 and 1995. There were 10 male and 11 females. The mean age was 20.3 months (range: 0-32 months). The mean time between symptoms and treatment was 2.4 months (range: 0-18 months). The most common symptoms included ataxia, nausea and vomits. The most common locations of the tumor were: infratentorial (57.1%) and supratentorial (38.1%). Complete surgery was performed in 3 patients, subtotal in 10, partial in 5, and a biopsy in 2. The anatomical-pathological diagnosis was: astrocytoma (6), ependinoma (5), meduloblastoma (4), ganglioglioma (1), neuroblastoma (1), and primitive neuroectodermic tumor (1). We could not document the histology in 3 patients. Ten patients received chemotherapy that was well tolerated and 14 received radiotherapy whose sequels were updated. The mean follow-up period was 44.42 months (range: 0-136 months). Overall survival was 42.86%. There were no statistically significant differences in survival between those who were irradiated and those who were not, nor between those with supra-or infratentorial tumors. CONCLUSIONS: CNS tumors in children younger than three years of age have a worse prognosis than in older children. New therapeutic schedule with chemotherapy are being tested to avoid radiotherapy side-effects.

[Down's syndrome and leukemia]. / Síndrome de Down y leucemia.

OBJECTIVE: Children with Down's Syndrome (DS) have a high risk for leukemia and need special clinical management. For this reason we have reviewed our experience. PATIENTS AND METHODS: All children with DS diagnosed a having acute leukemia during a 21-year period were reviewed retrospectively. Treatment was administered according to current protocols in our unit at the time of diagnosis without any initial modification. RESULTS: There were 13 children with DS and acute leukemia [6 ALL, 4 AML and 3 transient leukemias (TL)]. No patient presented CNS leukemia at diagnosis. All children with AML and DS were under three years of age and standard treatments did not achieve satisfactory results. TL regressed in two newborns without developing AMKL later. Five out of six patients with DS and ALL achieved complete remission. Currently, 4 of these children are alive and off therapy. Toxicities related to treatment were observed in almost all of the patients. CONCLUSIONS: Children with DS suffer a higher risk of developing leukemia. They should receive standard protocols, but aggressive supportive care might be provided as they have a higher incidence of treatment related toxicities. Prognosis of these children is similar or even better in some cases than children without DS. TL is a true neoplastic process capable of spontaneous remission and it can progress to AMKL.