Screening for late-onset Pompe disease in western Denmark.

OBJECTIVE: Late-onset Pompe disease (LOPD) is a rare autosomal recessively inherited metabolic myopathy caused by reduced activity of the lysosomal enzyme alpha-glucosidase. In a previous screening study at two large neuromuscular university clinics in Denmark, three patients with LOPD were identified out of 103 patients screened. No systematic screening has been performed at the other neurological departments in the western part of Denmark. Thus, patients with a diagnosis of unspecified myopathy were screened for LOPD. MATERIALS AND METHODS: At seven neurological departments in the western part of Denmark, medical records were evaluated for all patients registered with myopathy diagnosis codes (ICD 10 codes: G 71.0-71.9 and G 72.0-72.9) during the period January 1, 2002, to December 31, 2012. If no specific diagnosis has been reached, patients were invited for screening. Dried blood spot (DBS) test was used to analyze the activity of the enzyme alpha-glucosidase. RESULT: A total of 654 patients were identified. From the medical records, information was obtained concerning symptoms, family history, electromyography, muscle biopsy results and creatine kinase levels. Eighty-seven patients (13.3%) (males 61%) at a mean age of 53.3 years (SD 16.5) fulfilled the criteria for screening. A DBS test was performed in 47 (54%) patients. In all patients, the enzyme activity was within reference values. CONCLUSION: None of the screened patients had a reduced activity of the enzyme alpha-glucosidase. Although the cohort studied was small, our findings do not suggest that LOPD is underdiagnosed in patients with unspecified myopathy in western Denmark.

Analysis of neuroprotective effects of valproic acid on primary motor neurons in monoculture or co-cultures with astrocytes or Schwann cells.

Chronic dysregulation of the intracellular Ca(2+) homeostasis (excitotoxicity) is thought to contribute to the development of motor neuron diseases. Valproic acid (VPA) is widely used as an antiepileptic drug and acts mainly by inhibition of sodium channels and by enhancing the level of the inhibitory neurotransmitter gamma-aminobutyric acid. Neuroprotective capacities of VPA are supposed to arise also from the inhibition of histone deacetylases. We investigated the viability of highly purified rat embryonic motor neurons cultured on glial feeder layers, composed of either astrocytes or Schwann cells, or in the absence of glia, monoculture in presence of VPA and/or kainate (KA) using immunocytochemistry and calcium imaging. A significant effect of the culture and co-culture conditions on the viability of motor neurons in our in vitro model of excitotoxicity was detected. The neuroprotective effect of VPA on primary embryonic motor neuron cultures was not proven. A functional interaction between VPA and KA occurred during the first 10 days in culture.

Molecular mechanisms of interaction between the neuroprotective substance riluzole and GABA(A)-receptors.

The antiepileptic drug riluzole is used as a therapeutic agent in amyotrophic lateral sclerosis due to its neuroprotective effects. Besides presynaptic inhibition of GABAergic and preferentially glutamatergic transmission, it also potentiates postsynaptic GABA(A)-receptor function. We investigated the postsynaptic effects of riluzole on GABA(A)-receptor channels by use of the patch-clamp technique. Recombinant alpha1beta2gamma(2s) and alpha1beta2 GABA(A) receptors were expressed in HEK 293 cells by transient transfection. Pulses of GABA were applied in combination with different concentrations of riluzole to whole cell or outside-out patches with either alpha1beta2gamma(2s) or alpha1beta2 GABA(A)-receptor channels. Co-application of riluzole led to a slight decrease of absolute peak current amplitudes and steady-state currents in prolonged presence of GABA at saturating concentrations. In the presence of riluzole, enhancement of current amplitudes was observed with lower concentrations of GABA at alpha1beta2gamma(2s) receptors and to a lower extent also at alpha1beta2 receptors. Thus, the potentiating effect of riluzole was shown to be not abolished in the absence of the gamma(2s)-subunit. A further prominent effect of riluzole was a highly significant acceleration of the time course of current decay, most probably pointing to an open-channel block-like mechanism of action. As both receptor subtypes were affected similarly by the block, it could be concluded that the respective binding sites should be assumed within a region of high sequence homology like it is given for the channel-lining M2 domain of GABA(A)-receptor subunits. In conclusion, three different molecular mechanisms of interaction of the neuroprotective compound riluzole were observed at two different subtypes of GABA(A) receptor channels. The results further point to the impact of the inhibitory as well as the excitatory synaptic activity as a pharmacological target to counteract chronic excitotoxicity and reveal molecular mechanisms of action of the only one neuroprotective drug in current clinical use in patients suffering from amyotrophic lateral sclerosis.

Temporospatial coupling of networked synaptic activation of AMPA-type glutamate receptor channels and calcium transients in cultured motoneurons.

AMPA-type glutamate receptor (GluR) channels provide fast excitatory synaptic transmission in the CNS, but mediate also cytotoxic insults. It could be shown that AMPA-type GluR channel-mediated chronic excitotoxicity leads to an increased intracellular calcium concentration and plays an important role in neurodegenerative diseases like for example amyotrophic lateral sclerosis (ALS). As calcium is an important mediator of various processes in the cell and calcium signals have to be very precise in the temporospatial resolution, excessive intracellular calcium increases can seriously impair cell function. It is still unclear if AMPA-type receptors can directly interact with the intracellular calcium homeostasis or if other mechanisms are involved in this process. The objective of this study was therefore to investigate the calcium homeostasis in rat motoneurons under physiological stimulation of AMPA-type GluR channels using calcium imaging techniques and patch-clamp recordings simultaneously. It was found that spontaneous excitatory postsynaptic currents of cultured motoneurons did not elicit significant intracellular calcium transients. Large intracellular calcium transients occurred only when preceding fast sodium currents were observed. Pharmacological experiments showed that activation of AMPA-type GluR channels during synaptic transmission has a great functional impact on the calcium homeostasis in motoneurons as all kinds of activity was completely blocked by application of the selective kainate- and AMPA-type GluR channel blocker 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Furthermore we suggest from our experiments that calcium transients of several hundred milliseconds' duration result from release of calcium from the endoplasmic reticulum via activation of ryanodine receptors (calcium-induced calcium release, CICR). Our results help to understand the regulatory function of AMPA-type GluR channels in the intracellular calcium homeostasis which is known to be disturbed in neurodegenerative diseases.

Thoracoscopic repair of congenital diaphragmatic hernia by inflation-assisted bowel reduction, in a resuscitated neonate: a better access?

Elective endoscopic diaphragmatic hernia repairs have been reported. But endoscopic surgery was regarded unsuitable for emergency repair of diaphragmatic hernia in ventilated newborn children in bad general condition. We report a new method for inflation-assisted reduction and thoracoscopic repair of congenital diaphragmatic hernia diaphragmatic in a vitally endangered neonate. From three 2.7 mm to 5 mm accesses warmed low-pressure, low-volume CO2 was inflated into the thorax at 100 ml/min and 2 mm mercury. This allowed spontaneous reduction of the thoracic viscera into the abdomen and diaphragmatic suture with minimal handling. The 65-min procedure was tolerated well without perioperative deterioration. The baby was weaned off the respirator and breast-fed within 2 days, mediastinal shift normalized in 6 days. In suitable infants thoracoscopic repair and inflation-assisted reduction of thoracic contents is a more physiological access to congenital diaphragmatic hernia than laparoscopy or laparotomy.

Ligand-gated channels in early mesencephalic neuronal precursors: immunocytochemical and electrophysiological analysis.

Neuronal precursors play an important role in potential regenerative therapeutic strategies in different neurodegenerative diseases, e.g. Parkinson's disease. To understand proliferation and differentiation of these cells in vitro and in vivo, it is important to characterize functional properties of neuronal precursors in detail. The aim of the present study was to analyse the electrophysiological characteristics of ligand-gated channels of neuronal precursors prepared from the rat ventral mesencephalon (VM) of embryonic stage 12.5 during their in vitro differentiation. For the experiments we used the patch-clamp technique in combination with a system for ultrafast solution exchange and immunocytochemistry. It could be shown that functional active AMPA-type glutamate as well as GABA(A) receptor channels are expressed at an early stage of neuronal development. In culture we observed excitatory as well as inhibitory postsynaptic currents (defined by their different kinetics) which correspond to the activation of AMPAergic and GABAergic receptor channels. Two populations of glutamate-activated currents could be differentiated by their different time course of desensitization whereas the time course of resensitization and deactivation was normally distributed in all cells. GABAergic currents could be blocked by bicuculline and their kinetics correspond to that of GABA(A) receptor channel currents. Summarizing the results, in the present study it was shown for the first time that neuronal embryonic precursors of the rat VM express both functional AMPA-type glutamate and functional GABA(A) receptor channels in vitro.

Functional diversity of recombinant human AMPA type glutamate receptors: possible implications for selective vulnerability of motor neurons.

Lower motor neurons are known to be susceptible to glutamate-mediated cell damage via overstimulation of AMPA type glutamate receptors (GluR). The molecular basis of an important hypothesis in investigating amyotrophic lateral sclerosis (ALS) is glutamate-excitotoxicity. The aim of this study was to define desensitization and deactivation kinetics of recombinant human GluR1 and GluR2 receptor channels and their splice variants by means of patch-clamp experiments employing ultrafast solution exchange techniques. By this approach, the desensitization time constants of homooligomeric channels could be measured as tau(Des)=2.95+/-0.22 ms (n=10) for GluR1flip, tau(Des)=3.17+/-0.19 ms (n=10) for GluR1flop, tau(Des)=9.86+/-0.79 ms (n=10) for GluR2flip, and tau(Des)=1.87+/-0.26 ms (n=10) for GluR2flop, respectively. In the case of GluR1flip/flop and GluR2flop, a nondesensitising steady state current of less than 1% of peak current amplitude was observed, while GluR2flip channel currents showed a marked steady state component of about 10% of the maximum current. No significant differences were detected comparing the deactivation time course of GluR1 and GluR2 splice variants. These results suggest that the human GluR subtypes tested comprise no fundamental difference to their rodent analogous. Therefore, we describe a preparation that will be useful for further investigation of motor neuron physiological properties and a methodological approach allowing to study functional recombinant human GluR channels under reliable conditions.

Pentobarbital has curare-like effects on adult-type nicotinic acetylcholine receptor channel currents.

UNLABELLED: Pentobarbital (PB) is widely used as a short-term sedative and anticonvulsive drug with a side-effect of relaxing muscle tone. We investigated block of nicotinic acetylcholine receptor (nAChR) channel currents by PB using the patch-clamp technique in combination with an ultrafast system for solution exchange. As a preparation, recombinant rat adult-type nAChR channels transiently expressed in HEK293 cells were used. Appli-cation of 1 mM acetylcholine to small cells or outside-out patches showed a transient current with fast activation and desensitization kinetics. Adding PB to the acetylcholine-containing solution resulted in a decrease of the time constant of current decay and of the peak current amplitude starting at concentrations >0.01 mM PB. Preincubation of nAChR channels with PB led to a decrease of the peak current amplitude without alteration of activation and desensitization kinetics caused by competitive block of nAChR channels. In conclusion, similar to the effect of d-Tubocurarine, block of nAChR channel currents by PB can be explained by a combination of open-channel and competitive block. IMPLICATIONS: The interaction between adult-type nicotinic acetylcholine receptors, acetylcholine, and pentobarbital was biophysically investigated by using the patch-clamp technique in combination with tools for ultrafast solution exchange. PB elicited open-channel block and competitive block of nicotinic acetylcholine receptor channel currents, whereas the latter seems to be effective in clinically relevant concentrations.

Rotator cuff disorders: interobserver and intraobserver variation in diagnosis with MR imaging.

PURPOSE: To determine interobserver and intraobserver variation in the interpretation of magnetic resonance (MR) images in rotator cuff disorders. MATERIALS AND METHODS: MR images of the shoulder in 97 patients were retrospectively reviewed twice, with a 3-week interval. Surgical findings indicated a full-thickness tear in 29 patients, grade 1 impingement in 19 (tendinitis), and grade 2 impingement (partial tear) in 26. The control population comprised 23 asymptomatic volunteers or patients. RESULTS: All observers were accurate in the diagnosis of a full-thickness tear (89%-98%), with good intraobserver (kappa = 0.67-0.84) and interobserver agreement (kappa = 0.74-0.92). In diagnoses of tendinitis, partial tear, and normal cuff, there were wide ranges of sensitivity (13%-74%) and specificity (72%-93%), as well as poor interobserver (kappa = 0.12-0.60) and intraobserver agreement (kappa = 0.35-0.78). CONCLUSION: Full-thickness tears of the rotator cuff can be accurately identified at MR imaging with little observer variation. Consistent differentiation of normal rotator cuff, tendinitis, and partial thickness tears is more difficult.

Anemia ferropriva y respuesta inmune celular / Iron deficiency anemia and cellular inmune response

Existe información contradictoria en relación al efecto de la carencia de hierro sobre respuesta inmune. La interpretación de los datos inmunológicos obtenidos en pacientes con anemia ferropriva varía según edad, estado nutricional, presencia de infecciones y asociación con otras deficiencias nutricionales. Para definir el efecto que ejerce la anemia ferropriva sobre la respuesta inmunitaria celular se seleccionaron 15 lactantes con anemia ferropriva y 11 controles adecuadamente nutridos con hierro, entre 15 y 19 meses de edad, eutróficos, sin infecciones ni deficiencias nutricionales asociadas. Se determinó la capacidad de sensibilización a antígenos que evocan respuestas cutáneas de hipersensibilización retardada con 2-4 dinitroclorobenceno y PPD, como también la capacidad proliferativa de sus linfocitos al estímulo con fitohemaglutinina. No se observaron diferencias significativas en la respuesta inmune celular entre pacientes y controles antes y al cabo de tres meses de tratamiento con 5 mgù kgù día de hierro elemental en forma de sulfato ferroso. Se sugiere que la anemia ferropriva leve no alteraría la respuesta inmune celular

Respuesta inmune celular como indicador biológico de deficiencia de zinc en pacientes con fenilquetonuria / Cell immune response as a biological indicator of zinc deficiency in patients with phenylketonuria

Existe información contradictoria en relación al estado nutricional de zinc en pacientes con fenilquetonuria. El objetivo de este estudio fue confirmar la existencia de manifestaciones indirectas de deficit de zinc, a través del efecto que ejerce la suplementación de este mineral sobre la respuesta inmune celular. Se determinó la reacción cutánea de hipersensibilidad retardada y la capacidad linfoproliferativa frente a fitohemaglutinina antes y después de suplementación con acetato de zinc, en 8 pacientes fenilquetonúricos. Dos de ocho pacientes presentaron reacción cutánea positiva antes de la suplementación, aumentando a seis el número de sujetos con respuesta positiva después de la ingesta de zinc (p<0,05 prueba de Mac Nemar). La capacidad linfoproliferativa aumentó al término de la suplementación con zinc (p<0,05 test de t pareado). Estos resultados sugieren que existe un déficit de nutrición de zinc en pacientes con fenilquetonuria, puesto que la administración de este mineral traza estimularía la respuesta inmune celular