Histopathological and Ultrastructural Changes after Electroporation in Pig Liver Using Parallel-Plate Electrodes and High-Performance Generator.

Irreversible electroporation (IRE) has gained attention as a new non-thermal therapy for ablation with important benefits in terms of homogeneous treatment and fast recovery. In this study, a new concept of high voltage generator is used, enabling irreversible electroporation treatment in large tissue volume using parallel plates. Unlike currently available generators, the proposed versatile structure enables delivering high-voltage high-current pulses. To obtain homogeneous results, 3-cm parallel-plates electrodes have also been designed and implemented. IRE ablation was performed on six female pigs at 2000 V/cm electric field, and the results were analysed after sacrifice three hours, three days and seven days after ablation. Histopathological and ultrastructural studies, including transmission and scanning electron microscopy, were carried out. The developed high-voltage generator has proved to be effective for homogeneous IRE treatment using parallel plates. The destruction of the membrane of the hepatocytes and the alterations of the membranes of the cellular organelles seem incompatible with cell death by apoptosis. Although endothelial cells also die with electroporation, the maintenance of vascular scaffold allows repairing processes to begin from the third day after IRE as long as the blood flow has not been interrupted. This study has opened new direction for IRE using high performance generators and highlighted the importance of taking into account ultrastructural changes after IRE by using electron microscopy analysis.

Increasing Incidence of Pediatric Eosinophilic Esophagitis in the Southwest of Madrid, Spain.

OBJECTIVES: The incidence of eosinophilic esophagitis is unknown in our area. The aim of our study was to determine the incidence of eosinophilic esophagitis and its possible association with the most frequent absolute annual pollen counts. METHODS: A descriptive retrospective multicenter observational study was designed to calculate the incidence of eosinophilic esophagitis in children aged under 15 years in the southwest region of Madrid, Spain in 2002-2013 (data were provided by the Statistics Institute of Madrid). We collected data on age, sex, clinical presentation, and date of endoscopic diagnosis. Relative risk (RR) was estimated (Stata v.11) using negative binomial regression models to assess the association between incidence and pollen counts (provided by Subiza Clinic). RESULTS: The study population comprised 254 patients (192 male [75.6%], aged 0.5-14.99 years). The clinical presentation was esophageal impaction in 23.6%, dysphagia in 22%, gastroesophageal reflux-like symptoms in 44.9%, and other findings in 9.4%. The annual incidence from 2002 to 2013 per 100 000 children aged <15 years per year was, respectively, 0.81, 1.5, 0.37, 3.17, 3.07, 4.36, 6.87, 7.19, 8.38, 9.05, 9.14, and 9.68. The incidence of eosinophilic esophagitis increased by an average of 19% annually (RR, 1.19; 95%CI, 1.14-1.25; P<.001). In the overall analysis, the relationship between incidence and absolute annual and monthly counts during the pollen seasons of the respective pollen types was analyzed only for Platanus species, which had an RR >1 (1.17 and 1.06, respectively) (P<.05). CONCLUSION: The incidence of diagnosis of pediatric eosinophilic esophagitis increased by an average of 19% annually. No significant association was found between incidence and pollen counts, except for a weak association with Platanus species.

Increasing incidence of pediatric eosinophilic esophagitis in the Southwest of Madrid, Spain

Objectives: The incidence of eosinophilic esophagitis is unknown in our area. The aim of our study was to determine the incidence of eosinophilic esophagitis and its possible association with the most frequent absolute annual pollen counts. Methods: A descriptive retrospective multicenter observational study was designed to calculate the incidence of eosinophilic esophagitis in children aged under 15 years in the southwest region of Madrid, Spain in 2002-2013 (data were provided by the Statistics Institute of Madrid). We collected data on age, sex, clinical presentation, and date of endoscopic diagnosis. Relative risk (RR) was estimated (Stata v.11) using negative binomial regression models to assess the association between incidence and pollen counts (provided by Subiza Clinic). Results: The study population comprised 254 patients (192 male [75.6%], aged 0.5-14.99 years). The clinical presentation was esophageal impaction in 23.6%, dysphagia in 22%, gastroesophageal reflux-like symptoms in 44.9%, and other findings in 9.4%. The annual incidence from 2002 to 2013 per 100 000 children aged <15 years per year was, respectively, 0.81, 1.5, 0.37, 3.17, 3.07, 4.36, 6.87, 7.19, 8.38, 9.05, 9.14, and 9.68. The incidence of eosinophilic esophagitis increased by an average of 19% annually (RR, 1.19; 95%CI, 1.14-1.25; P<.001). In the overall analysis, the relationship between incidence and absolute annual and monthly counts during the pollen seasons of the respective pollen types was analyzed only for Platanus species, which had an RR >1 (1.17 and 1.06, respectively) (P<.05). Conclusion: The incidence of diagnosis of pediatric eosinophilic esophagitis increased by an average of 19% annually. No significant association was found between incidence and pollen counts, except for a weak association with Platanus species

Objetivo: La incidencia de esofagitis eosinofílica es desconocida en nuestra área. El objetivo de nuestro estudio es determinar la incidencia de diagnóstico de esofagitis eosinofílica y su posible asociación con los recuentos absolutos anuales de los pólenes más frecuentes. Métodos: Se diseñó un estudio descriptivo multicéntrico retrospectivo para el cálculo de la incidencia de esofagitis eosinofílica en niños menores de 15 años en el suroeste de la Comunidad de Madrid entre 2002-2013 (datos poblacionales obtenidos del Instituto de Estadística de la Comunidad de Madrid). Se recoge de cada paciente: edad, sexo, presentación clínica y fecha de diagnóstico endoscópico. Se estimó la asociación entre la incidencia y los recuentos polínicos (aportados por la Clínica Subiza) mediante cálculo del riesgo relativo usando modelos de regresión binomial negativa (Stata v.11).Resultados: Se incluyeron 254 pacientes, 75,6% varones (n=192), de edades comprendidas entre 0,5-14,99 años. La presentación clínica fue: impactación esofágica 23,6%, disfagia 22%, síntomas sugerentes de reflujo gastroesofágico 44,9% y otros 9,4%. Las incidencias anuales desde 2002 a 2013 (nº casos/100.000 niños menores de 15 años/año) fueron respectivamente: 0,81; 1,5; 0,37; 3,17; 3,07; 4,36; 6,87; 7,19; 8,38; 9,05; 9,14 y 9,68. La incidencia de esofagitis eosinofílica se incrementó en una media anual de 19% (RR 1,19, 95% IC: 1,14-1,25, p <0,001). En nuestro estudio únicamente existió asociación (RR>1) entre la incidencia y los recuentos polínicos absolutos de Platanus spp anuales y durante los meses de máxima polinización (1,17 and 1,06, respectivamente) (p <0,05).Conclusión: La incidencia de diagnóstico de esofagitis eosinofílica en la edad pediátrica se ha incrementado en una media anual de un 19%. No se encontró una asociación estadísticamente significativa entre la incidencia y los recuentos polínicos, excepto con el Platanus spp aunque ésta fue débil

Comparison of autologous bone marrow and adipose tissue derived mesenchymal stem cells, and platelet rich plasma, for treating surgically induced lesions of the equine superficial digital flexor tendon.

Several therapies have been investigated for equine tendinopathies, but satisfactory long term results have not been achieved consistently and a better understanding of the healing mechanism elicited by regenerative therapies is needed. The aim of this study was to assess the separate effects of autologous bone marrow (BM) and adipose tissue (AT) derived mesenchymal stem cells (MSCs), and platelet rich plasma (PRP), for treating lesions induced in the superficial digital flexor tendon (SDFT) of horses. Lesions were created surgically in both SDFTs of the forelimbs of 12 horses and were treated with BM-MSCs (six tendons), AT-MSCs (six tendons) or PRP (six tendons). The remaining six tendons received lactated Ringer's solution as control. Serial ultrasound assessment was performed prior to treatment and at 2, 6, 10, 20 and 45 weeks post-treatment. At 45 weeks, histopathology and gene expression analyses were performed. At week 6, the ultrasound echogenicity score in tendons treated with BM-MSCs suggested earlier improvement, whilst all treatment groups reached the same level at week 10, which was superior to the control group. Collagen orientation scores on histological examination suggested a better outcome in treated tendons. Gene expression was indicative of better tissue regeneration after all treatments, especially for BM-MSCs, as suggested by upregulation of collagen type I, decorin, tenascin and matrix metalloproteinase III mRNA. Considering all findings, a clear beneficial effect was elicited by all treatments compared with the control group. Although differences between treatments were relatively small, BM-MSCs resulted in a better outcome than PRP and AT-MSCs.

New insights into c-Ret signalling pathway in the enteric nervous system and its relationship with ALS.

The receptor tyrosine kinase Ret (c-Ret) transduces the glial cell line-derived neurotrophic factor (GDNF) signal, one of the neurotrophic factors related to the degeneration process or the regeneration activity of motor neurons in amyotrophic lateral sclerosis (ALS). The phosphorylation of several tyrosine residues of c-Ret seems to be altered in ALS. c-Ret is expressed in motor neurons and in the enteric nervous system (ENS) during the embryonic period. The characteristics of the ENS allow using it as model for central nervous system (CNS) study and being potentially useful for the research of human neurological diseases such as ALS. The aim of the present study was to investigate the cellular localization and quantitative evaluation of marker c-Ret in the adult human gut. To assess the nature of c-Ret positive cells, we performed colocalization with specific markers of cells that typically are located in the enteric ganglia. The colocalization of PGP9.5 and c-Ret was preferentially intense in enteric neurons with oval morphology and mostly peripherally localized in the ganglion, so we concluded that the c-Ret receptor is expressed by a specific subtype of enteric neurons in the mature human ENS of the gut. The functional significance of these c-Ret positive neurons is discussed.

Synchronization dynamics induced on pairs of neurons under applied weak alternating magnetic fields.

Pairs of Helix aspersa neurons show an alternating magnetic field dependent frequency synchronization (AMFS) when exposed to a weak (amplitude B0 between 0.2 and 150 Gauss (G)) alternating magnetic field (AMF) of extremely low frequency (ELF, fM = 50 Hz). We have compared the AMFS patterns of discharge with: i) the synaptic activity promoted by glutamate and acetylcholine; ii) the activity induced by caffeine; iii) the bioelectric activity induced on neurons interconnected by electric synapses. AMFS activity reveals several specific features: i) a tight coincidence in time of the pattern and frequency, f, of discharge; ii) it is induced in the time interval of field application; iii) it is dependent on the intensity of the sinusoidal applied magnetic field; iv) elicited biphasic responses (excitation followed by inhibition) run in parallel for the pair of neurons; and v) some neuron pairs either spontaneously or AMF synchronized can be desynchronized under applied higher AMF. Our electron microscopy studies reveal gap-like junctions confirming our immunocytochemistry results about expression of connexin 26 (Cx26) in 4.7% of Helix neurons. AMF and carbenoxolone did not induce any significant effect on spontaneous synchronization through electric synapses.

Analysis of the Spanish national registry for pediatric home enteral nutrition (NEPAD): implementation rates and observed trends during the past 8 years.

BACKGROUND/OBJECTIVES: The home enteral nutrition (HEN) provides nutritional support to children with chronic diseases who are nutritionally compromised and allows them to be discharged more quickly from hospitals. In 2003, a web-based registry (Nutrición Enteral Pediátrica Ambulatoria y Domiciliaria, Pediatric Ambulatory and Home Enteral Nutrition -NEPAD-) was created with the objective of gathering information about pediatric HEN practices in Spain. AIM: The aim of this study was to report the implementation of the NEPAD (Nutrición Enteral Pediátrica Ambulatoria y Domiciliaria, Pediatric Ambulatory and Home Enteral Nutrition) registry of pediatric HEN in Spain and to analyze data evolution trends from 2003 to 2010. SUBJECTS/METHODS: The data from the Spanish NEPAD registry were analyzed according to the following variables: demographic data, diagnosis, indication for HEN, nutritional support regime and administration route. RESULTS: Over the study period, 952 patients (1048 episodes) from 20 Spanish hospitals were included in the NEPAD registry. The most frequent indication for HEN was decreased oral intake (64%), and neurological disease was the most prevalent illness. HEN was delivered via a nasogastric tube in 573 episodes (54.7%), by gastrostomy in 375 episodes (35.8%), oral feeding in 77 episodes (7.3%) and by jejunal access in 23 episodes (2.2%). Significant differences in the mode of administration were observed based on the pathology of the child (χ(2), P<0.0001). The cyclic feeding was the most widely used technique for the administration of HEN. Most of the patients used a pump and a polymeric formula. Transition to oral feeding was the primary reason for discontinuation of this type of support. CONCLUSIONS: Since the NEPAD registry was established in Spain, the number of documented patients has increased more than 25-fold. Many children with chronic illness benefit from HEN, mainly those suffering from neurological diseases.

The primary cilium of telocytes in the vasculature: electron microscope imaging.

Blood vessels are highly organized and complex structure, which are far more than simple tubes conducting the blood to almost any tissue of the body. The fine structure of the wall of blood vessels has been studied previously using the electron microscope, but the presence the telocytes associated with vasculature, a specific new cellular entity, has not been studied in depth. Interestingly, telocytes have been recently found in the epicardium, myocardium, endocardium, human term placenta, duodenal lamina propria and pleura. We show the presence of telocytes located on the extracellular matrix of blood vessels (arterioles, venules and capillaries) by immunohistochemistry and transmission electron microscopy. Also, we demonstrated the first evidence of a primary cilium in telocytes. Several functions have been proposed for these cells. Here, the telocyte-blood vessels cell proximity, the relationship between telocytes, exosomes and nervous trunks may have a special significance.

A presumptive new locus for autosomal dominant hypercholesterolemia mapping to 8q24.22.

Molecular testing of patients with autosomal dominant hypercholesterolemia (ADH) fails to detect a causal functional mutation in 15.25% of subjects. We studied an ADH pedigree in which known ADH-causing genes (LDLR, APOB and PCSK9) were excluded. Genome-wide analysis on 15 family members detected significant association for ADH and dbSNP RS ID rs965814 (G/A), located in 8q24.22 cytoband. ADH was significantly associated to rs965814 G allele (p < 0.05) in a case-control study based on 200 unrelated ADH subjects without LDLR or APOB gene defects and 198 normolipidemic controls. We chose 24 markers for a detailed analysis of 8q24.22 cytoband, now based on an extended set of family members (21 individuals). One particular 24 marker haplotype was significantly associated to both higher total and low-density lipoprotein-cholesterol concentrations. Similar results were found for a shorter haplotype, composed of the distal six markers from the complete haplotype. Therefore, a presumptive new locus for ADH could be located in 8q24.22 cytoband, a region not previously linked or associated to ADH.

Células madre neurales positivas a la nestina en el Sistema Nervioso Entéricco / No disponible

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Inmmuno Preembeding Gold anti c-Kit en duodeno de rata / No disponible

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Characterization by immunocytochemistry of ionic channels in Helix aspersa suboesophageal brain ganglia neurons.

The aim of this work was to characterize several ionic channels in nervous cells of the suboesophageal visceral, left and right parietal, and left and right pleural brain ganglia complex of the snail Helix aspersa by immunocytochemistry. We have studied the immunostaining reaction for a wide panel of eleven polyclonal antibodies raised against mammal antigens as follows: voltage-gated-Na+ channel; voltage-gated-delayed-rectifier-K+ channel; SK2-small-conductance-Ca2+-dependent-K+ channel apamin sensitive; SK3 potassium channel; charybdotoxin-sensitive voltage-dependent potassium channel; BKCa-maxi-conductance-Ca2+-dependent-K+ channel; hyperpolarization-activated cyclic nucleotide-gated potassium channel 4; G-protein-activated inwardly rectifying potassium channel GIRK2 and voltage-gated-calcium of L, N and P/Q type channels. Our results show positive reaction in neurons, but neither in glia cells nor in processes in the Helix suboesophageal ganglia. Our results suggest the occurrence of molecules in Helix neurons sharing antigenic determinants with mammal ionic channels. The reaction density and distribution of immunoreactive staining within neurons is specific for each one of the antisera tested. The studies of co-localization of immunoreaction, on alternate serial sections of the anterior right parietal ganglion, have shown for several recognized mapped neurons that they can simultaneously be expressed among two and seven different ionic protein channels. These results are considered a key structural support for the interpretation of Helix aspersa neuron electrophysiological activity.

[Neurocristopathies: a high incidence of cerebral dysgenesis in patients with Hirschsprung's disease]. / Neurocrestopatías: alta frecuencia de disgenesias cerebrales en pacientes con enfermedad de Hirschsprung.

INTRODUCTION: Hirschsprung's disease (HD), or aganglionic megacolon, is a congenital disorder that is characterised by the absence of ganglion cells in the submucosal and myenteric plexuses of the intestine, which is caused by the failure of these cells to migrate from the neural crest (neurocristopathy). Cerebral dysgenesis and polymalformation syndromes have been reported in association with HD, thus suggesting an abnormal morphogenesis. AIM: To study the frequency of cerebral malformations in patients with HD in our environment. PATIENTS AND METHODS: We conducted a retrospective study of 41,666 live newborn infants, over the period 1993-2003, and 17 cases of HD where identified. RESULTS: The incidence of HD in the health district of the province of Albacete is 1.68 per 5,000 live newborn infants. Of the 17 patients with HD who were studied, 10 were isolated (58.8%) and seven (41.1%) were associated to other structural abnormalities and psychomotor retardation. Three of the cases in this latter group were due to chromosome pathology (trisomy 21, Down syndrome), two were caused by specific polymalformation syndromes (one Mowat-Wilson syndrome and one possible FG syndrome), one was due to a pattern of abnormalities that did not fit any known syndrome, and one had a normal phenotype and isolated cerebral dysgenesis. In all of cases the neuroimaging studies identified cerebral dysgenesis that was compatible with neuronal migration disorders. CONCLUSIONS: The frequency of association of HD, either isolated or within the context of a specific malformation syndrome, with neuronal migration disorders is high (23.5%). We suggest a full genetic and neurological evaluation should be carried out in patients with HD, together with brain imaging studies in order to rule out the possibility of cerebral dysgenesis.

Neurocrestopatías: alta frecuencia de disgenesias cerebrales en pacientes con enfermedad de Hirschsprung / Neurocristopathies: a high incidence of cerebral dysgenesis in patients with hirschsprung’s disease

La enfermedad de Hirschsprung (EH) o megacolon agangliónico es un trastorno congénito que secaracteriza por la ausencia de células ganglionares en los plexos submucosos y mioentéricos intestinales, debido a un fracaso en la migración de estas células desde la cresta neural (neurocrestopatía). Se han descrito disgenesias cerebrales y síndromespolimalformativos asociados a EH, lo que sugiere una morfogénesis anormal. Objetivo. Estudiar la frecuencia de malformaciones cerebrales en pacientes afectos de EH en nuestro medio. Pacientes y métodos. Estudio retrospectivo de 41.666 recién nacidos vivos, entre 1993 y 2003, de los cuales se identificaron 17 casos de EH. Resultados. La incidencia de EH en el área de salud de la provincia de Albacete es de 1,68 por cada 5.000 recién nacidos vivos. De los 17 pacientes estudiados porEH, 10 fueron aislados (58,8%) y siete (41,1%) asociados a otras anomalías estructurales y retraso psicomotor. De estos últimos, tres se deben a cromosomopatía (trisomía 21, síndrome de Down), dos a síndromes polimalformativos específicos (un síndrome de Mowat-Wilson, y un posible síndrome FG), uno a patrón de anomalías no encuadrable en entidad sindrómica conocida,y uno con fenotipo normal y disgenesia cerebral aislada. En todos ellos los estudios de neuroimagen identificaron disgenesias cerebrales compatibles con trastornos de la migración neuronal. Conclusiones. La frecuencia de asociación de EH, bien aislada o en el contexto de un síndrome polimalformativo específico, con trastornos de la migración neuronal, es elevada(23,5%). Sugerimos la conveniencia de una valoración genética y neurológica completa en pacientes con EH, y estudios de imagen cerebral para descartar disgenesias cerebrales

Hirschsprung’s disease (HD), or aganglionic megacolon, is a congenital disorder that is characterisedby the absence of ganglion cells in the submucosal and myenteric plexuses of the intestine, which is caused by the failure of these cells to migrate from the neural crest (neurocristopathy). Cerebral dysgenesis and polymalformation syndromes have been reported in association with HD, thus suggesting an abnormal morphogenesis. Aim. To study the frequency of cerebralmalformations in patients with HD in our environment. Patients and methods. We conducted a retrospective study of 41,666 live newborn infants, over the period 1993-2003, and 17 cases of HD where identified. Results. The incidence of HD in the health district of the province of Albacete is 1.68 per 5,000 live newborn infants. Of the 17 patients with HD who were studied,10 were isolated (58.8%) and seven (41.1%) were associated to other structural abnormalities and psychomotor retardation. Three of the cases in this latter group were due to chromosome pathology (trisomy 21, Down syndrome), two were caused by specific polymalformation syndromes (one Mowat-Wilson syndrome and one possible FG syndrome), one was due to a pattern of abnormalities that did not fit any known syndrome, and one had a normal phenotype and isolated cerebral dysgenesis. In all of cases the neuroimaging studies identified cerebral dysgenesis that was compatible with neuronal migration disorders.Conclusions. The frequency of association of HD, either isolated or within the context of a specific malformation syndrome, with neuronal migration disorders is high (23.5%). We suggest a full genetic and neurological evaluation should be carriedout in patients with HD, together with brain imaging studies in order to rule out the possibility of cerebral dysgenesis

Localization of connexins in neurons and glia cells of the Helix aspersa suboesophageal brain ganglia by immunocytochemistry.

The aim of the present study was to examine the distribution of cells expressing connexin 26 (Cx26) in the suboesophageal visceral, left and right parietal and left and right pleural ganglia of the snail Helix aspersa by immunocytochemistry. Altogether we have found approximately 452 immunoreactive neurons which represent the 4.7% of the total neurons counted. The stained large neurons (measured diameter 55-140 microm) occurred mostly on the peripheral surface of the ganglia while the small immunostained cells (5-25 microm diameter) were observed in groups near the neuropil. The number of large neurons giving positive Cx26-like immunostaining was small in comparison with that for medium (30-50 microm diameter) and small sized cells. The expression of Cx26 was also observed in the processes of glia cells localized among neurons somata and in the neuropil showing that the antiserum recognized epitopes in both protoplasmic and fibrous glia cells of Helix aspersa. The neuropils of all ganglia showed fibers densely immunostained. While we have observed a good specificity for Cx26-antiserum in neurons, a lack of reaction for Cx43 antiserum was observed in neurons and glia cells. The reaction for enolase antiserum in neurons was light and non-specific and a lack of reaction in glia cells and processes for GFAP antiserum was observed. Although the percentage of positive neurons for Cx26 antiserum was low is suggested that in normal physiological conditions or under stimulation the expression of connexin could be increased. The observed results can be considered of interest in the interpretation of Helix aspersa elemental two neuron networks synchronizing activity, observed under applied extremely low frequency magnetic fields.

Ictericia colestásica como complicación de una pielonefritis aguda en una niña de 6 años / Cholestatic jaundice complicating acute pyelonephritis in a 6-year-old girl

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S-100 protein immunoreactivity in the upper eyelid of the sheep Ovis aries.

The aim of this work was to analyse the distribution pattern of S-100-immunoreactive elements in the upper eyelid of the sheep. This pattern may be of importance regarding the diagnosis and prognosis of eyelid tumours that are linked to deregulation of S-100 gene expression. Thirty upper eyelids taken from 15 adult male Ovis aries were studied by means of the peroxidase-antiperoxidase method for light microscopy. S-100-immunopositive cells were found in the eyelid edge. S-100-immunopositive steams and thinner fibres were found throughout the eyelid. These nerve processes typically were denser around glands, hair follicles and blood vessels. S-100-immunopositive elements may play a role as neuromodulator and also in the development of the vegetative innervation of the epithelium and its derivatives.

Hepatitis colestásica como manifestación inicial de escarlatina / Cholestatic hepatitis as initial manifestation of scarlet fever

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