Kinetics of the interaction between anti-FVIII antibodies and FVIII from therapeutic concentrates, with and without von Willebrand factor, assessed by surface plasmon resonance.

The presence of VWF in plasma-derived FVIII (pdFVIII/VWF) products has been pointed out as a key difference with recombinant FVIII (rFVIII) products with regard to immunogenicity. A Surface Plasmon Resonance (SPR) study was designed to characterize in detail the interaction between anti-FVIII (IgGs) from a severe haemophilia A patient, and FVIII from concentrates of different sources. Full-length rFVIII (preincubated or not with purified VWF), B domain-deleted (BDD)-rFVIII and pdFVIII/VWF were analysed. To ensure reproducible conditions for accurate determination of kinetic constants, a capture-based assay format was developed using protein G surfaces for specific and reversible coupling of endogenous anti-FVIII antibodies. Concentration ranges (nm) of FVIII products tested were 9-0.03 (rFVIII) and 6-0.024 (pdFVIII/VWF). The association with antibodies was monitored for 3-5 min, whereas dissociation of the complex was followed for 5-20-240 min. A strong interaction of rFVIII and BDD-rFVIII with patient's IgG was detected with the K (D) values in the low picomolar range (5.9 ± 3.0 and 12.7 ± 6.9 pm, respectively) and very slow dissociation rates, while pdFVIII/VWF showed only marginal binding signals. The VWF complexed rFVIII displayed reduced binding signals compared with uncomplexed rFVIII, but the K (D) was still in the picomolar range (4.1 ± 1.9 pm) indicating insufficient complex formation. rFVIII, alone or bound to exogenously added VWF, showed high affinity for anti-FVIII IgGs from a severe haemophilia A patient whereas pdFVIII/VWF did not. These results are in agreement with those studies that point towards rFVIII concentrates to be more immunogenic than pdFVIII concentrates.

Academic and employment insertion as a factor related to quality of life in patients with drug-resistant temporal lobe epilepsy. / La inserción académica y laboral como un factor asociado a la calidad de vida en pacientes con epilepsia farmacorresistente del lóbulo temporal.

INTRODUCTION: Academic and employment insertion is one of the issues that most concern people with epilepsy, but little is known about its relationship with quality of life. AIM: We aimed to analyze the effects of the academic and employment insertion on quality of life, anxiety, depression, social support, and executive functions, and the relationships among these variables in patients with drug-resistant epilepsy. PATIENTS AND METHODS: Fifty-nine patients with drug-resistant temporal lobe epilepsy were classified into two groups: with academic or employment insertion (n = 25) and without insertion (n = 34) and underwent a neuropsychological evaluation. RESULTS: Patients with insertion had a significantly better quality of life, lower trait anxiety, and higher social support, and tended to have a lower percentage of errors and higher percent conceptual level responses than those without insertion. Academic/employment insertion had indirect effects on quality of life through its relationship with global social support and trait anxiety. CONCLUSIONS: Our findings provide a model for understanding the quality of life in patients with temporal lobe epilepsy for an integral perspective of the patient and points out the key role of increased social support and reduced anxiety associated with academic and employment insertion to improve quality of life. These results could favor the implementation of programs that promote academic or employment reinsertion, considering the relevance of socio-emotional domains.

TITLE: La inserción académica y laboral como un factor asociado a la calidad de vida en pacientes con epilepsia farmacorresistente del lóbulo temporal.Introducción. La inserción académica y laboral es uno de los problemas que más preocupan a las personas con epilepsia, pero permanece sin esclarecer su relación con la calidad de vida. Objetivo. Analizar los efectos de la inserción académica y laboral en la calidad de vida, la ansiedad, la depresión, el apoyo social y las funciones ejecutivas, así como la relación entre estas variables en pacientes con epilepsia farmacorresistente. Pacientes y métodos. Cincuenta y nueve pacientes con epilepsia farmacorresistente del lóbulo temporal fueron clasificados en dos grupos, con inserción académica o laboral (n = 25) y sin inserción (n = 34), y se les realizó una evaluación neuropsicológica. Resultados. Los pacientes con inserción presentaron una calidad de vida significativamente mayor, menor ansiedad rasgo y mayor apoyo social percibido, así como una tendencia a tener un menor porcentaje de errores y un mayor porcentaje de respuestas conceptuales que los pacientes sin inserción laboral. La inserción académica/laboral tuvo efectos indirectos en la calidad de vida a través de su relación con el apoyo social y la ansiedad rasgo. Conclusiones. Nuestros hallazgos ofrecen un modelo para entender la calidad de vida en los pacientes con epilepsia del lóbulo temporal desde una perspectiva integral del paciente y señalan el papel clave del aumento del apoyo social y de la reducción de la ansiedad asociados con la inserción académica y laboral para mejorar la calidad de vida. Estos resultados podrían favorecer la implementación de programas que promuevan la reinserción académica o laboral, considerando la relevancia de variables socioemocionales.

Unravelling the mysteries of sudden unexpected death in epilepsy. / Desentrañando los misterios de la muerte súbita en epilepsia.

INTRODUCTION: Sudden unexpected death in epilepsy (SUDEP) is the most frequent cause of premature death in epileptic patients. Most SUDEP events occur at night and frequently go unnoticed; the exact pathophysiological mechanisms of this phenomenon therefore remain undetermined. Nevertheless, most cases of SUDEP are attributed to an infrequent yet extremely severe complication of epileptic seizures. DEVELOPMENT: We conducted a systematic literature search on PubMed. Our review article summarises scientific evidence on the classification, pathophysiological mechanisms, risk factors, biomarkers, and prevention of SUDEP. Likewise, we propose new lines of research and critically analyse findings that are relevant to clinical practice. CONCLUSIONS: Current knowledge suggests that SUDEP is a heterogeneous phenomenon caused by multiple factors. In most cases, however, SUDEP is thought to be due to postictal cardiorespiratory failure triggered by generalised tonic-clonic seizures and ultimately leading to cardiac arrest. The underlying pathophysiological mechanism involves multiple factors, ranging from genetic predisposition to environmental factors. Risk of SUDEP is higher in young adults with uncontrolled generalised tonic-clonic seizures. However, patients apparently at lower risk may also experience SUDEP. Current research focuses on identifying genetic and neuroimaging biomarkers that may help determine which patients are at high risk for SUDEP. Antiepileptic treatment is the only preventive measure proven effective to date. Night-time monitoring together with early resuscitation may reduce the risk of SUDEP.

[Diagnostic challenges in epilepsy]. / Desafios diagnosticos en epilepsia.

INTRODUCTION: Epileptic seizures and epilepsy are part of daily clinical practice in neurology. Yet, the number of false positive diagnoses is surprisingly high. Almost one out of every five patients treated for epilepsy does not really have this diagnosis, which is a high percentage bearing in mind the social and medical consequences that being diagnosed with epilepsy entails. AIMS: To summarise the most important diagnostic challenges in epilepsy, to describe possible sources of diagnostic error and to offer advice on how to avoid them. DEVELOPMENT: Epilepsy is characterised by a tendency to suffer unprovoked epileptic seizures. The greatest obstacle when it comes to diagnosing a case of epilepsy is the fact that epileptic seizures are transient phenomena that occur relatively infrequently and the physician who must carry out the diagnosis will rarely see them. Moreover, there are other clinical events, such as syncopes or non-epileptic seizures, that may be similar to epileptic seizures in appearance and, consequently, can be mistaken for them. Finally, when interpreting the two most important complementary diagnostic techniques in epileptology, the electroencephalogram and magnetic resonance imaging of the brain, the most common errors must be taken into account in order to prevent mistaken diagnoses. CONCLUSIONS: The diagnosis of epilepsy is a challenge and must be based on a detailed and specific medical record. If there are any reasonable doubts, from the outset, about the diagnosis of epilepsy or if the patient does not respond well to the antiepileptic treatment, we recommend referring the patient to a specialised centre to establish a definitive diagnosis.

TITLE: Desafios diagnosticos en epilepsia.Introduccion. Las crisis epilepticas y la epilepsia son parte de la practica clinica diaria en neurologia. No obstante, el numero de diagnosticos falsos positivos es sorprendentemente alto. Casi uno de cada cinco pacientes tratado por epilepsia en realidad no tiene ese diagnostico, un porcentaje elevado teniendo en cuenta las consecuencias sociomedicas que conlleva el diagnostico de epilepsia. Objetivos. Resumir los desafios diagnosticos mas importantes en epilepsia, describir posibles fuentes de error en el diagnostico y proporcionar consejos sobre como evitarlos. Desarrollo. La epilepsia se caracteriza por una tendencia a sufrir crisis epilepticas no provocadas. El mayor obstaculo al diagnosticar una epilepsia radica en que las crisis epilepticas son fenomenos transitorios que ocurren relativamente con poca frecuencia y el medico que realiza el diagnostico raramente llega a verlas. Ademas, existen otros eventos clinicos, como por ejemplo sincopes o crisis no epilepticas, que pueden tener una apariencia similar a las crisis epilepticas y, en consecuencia, confundirse con ellas. Finalmente, al interpretar las dos tecnicas diagnosticas complementarias mas importantes en epileptologia, el electroencefalograma y la resonancia magnetica cerebral, deben tenerse en cuenta los errores mas comunes para prevenir diagnosticos erroneos. Conclusiones. El diagnostico de una epilepsia es un reto y debe basarse en una historia clinica detallada y especifica. Si desde el inicio existen dudas razonables sobre el diagnostico de epilepsia o si el paciente no responde bien al tratamiento antiepileptico, recomendamos derivar al paciente a un centro especializado que establezca un diagnostico definitivo.

La inserción académica y laboral como un factor asociado a la calidad de vida en pacientes con epilepsia farmacorresistente del lóbulo temporal / Academic and employment insertion as a factor related to quality of life in patients with drug-resistant temporal lobe epilepsy

Introducción: La inserción académica y laboral es uno de los problemas que más preocupan a las personas con epilepsia, pero permanece sin esclarecer su relación con la calidad de vida. Objetivo: Analizar los efectos de la inserción académica y laboral en la calidad de vida, la ansiedad, la depresión, el apoyo social y las funciones ejecutivas, así como la relación entre estas variables en pacientes con epilepsia farmacorresistente. Pacientes y métodos: Cincuenta y nueve pacientes con epilepsia farmacorresistente del lóbulo temporal fueron clasificados en dos grupos, con inserción académica o laboral (n = 25) y sin inserción (n = 34), y se les realizó una evaluación neuropsicológica. Resultados: Los pacientes con inserción presentaron una calidad de vida significativamente mayor, menor ansiedad rasgo y mayor apoyo social percibido, así como una tendencia a tener un menor porcentaje de errores y un mayor porcentaje de respuestas conceptuales que los pacientes sin inserción laboral. La inserción académica/laboral tuvo efectos indirectos en la calidad de vida a través de su relación con el apoyo social y la ansiedad rasgo. Conclusiones: Nuestros hallazgos ofrecen un modelo para entender la calidad de vida en los pacientes con epilepsia del lóbulo temporal desde una perspectiva integral del paciente y señalan el papel clave del aumento del apoyo social y de la reducción de la ansiedad asociados con la inserción académica y laboral para mejorar la calidad de vida. Estos resultados podrían favorecer la implementación de programas que promuevan la reinserción académica o laboral, considerando la relevancia de variables socioemocionales.(AU)

Introduction: Academic and employment insertion is one of the issues that most concern people with epilepsy, but little is known about its relationship with quality of life. Aim: We aimed to analyze the effects of the academic and employment insertion on quality of life, anxiety, depression, social support, and executive functions, and the relationships among these variables in patients with drug-resistant epilepsy. Patients and methods: Fifty-nine patients with drug-resistant temporal lobe epilepsy were classified into two groups: with academic or employment insertion (n = 25) and without insertion (n = 34) and underwent a neuropsychological evaluation. Results: Patients with insertion had a significantly better quality of life, lower trait anxiety, and higher social support, and tended to have a lower percentage of errors and higher percent conceptual level responses than those without insertion. Academic/employment insertion had indirect effects on quality of life through its relationship with global social support and trait anxiety. Conclusions: Our findings provide a model for understanding the quality of life in patients with temporal lobe epilepsy for an integral perspective of the patient and points out the key role of increased social support and reduced anxiety associated with academic and employment insertion to improve quality of life. These results could favor the implementation of programs that promote academic or employment reinsertion, considering the relevance of socio-emotional domains.(AU)

A stable interaction between separated pyrimidine.purine tracts in circular DNA.

Pyrimidine.purine tracts are widespread in eukaryotic genomes and have the potential to form a number of unusual structures including triplexes. Two such tracts, which could form triplexes with each other but not with themselves, were cloned into a plasmid at separate sites. Upon lowering the pH, linear, open circular and relaxed plasmid molecules formed a number of novel structures that were observed on agarose gels and directly by electron microscopy. In open circles a stable join was formed between the two Pyr.Pur tracts giving rise to molecules resembling dumbells, trefoils and tetrafoils, which collectively are termed T-loops. The structure was stable at pH 8 and contains a single-stranded region that was sensitive to P1 nuclease. Thus, there is no apparent topological impediment to the formation of triplex-mediated loops in circular molecules. These structures may be important for gene regulation and chromosome condensation.

The hairpin ribozyme substrate binding-domain: a highly constrained D-shaped conformation.

The two domains of the hairpin ribozyme-substrate complex, usually depicted as straight structural elements, must interact with one another in order to form an active conformation. Little is known about the internal geometry of the individual domains in an active docked complex. Using various crosslinking and structural approaches in conjunction with molecular modeling (constraint-satisfaction program MC-SYM), we have investigated the conformation of the substrate-binding domain in the context of the active docked ribozyme-substrate complex. The model generated by MC-SYM showed that the domain is not straight but adopts a bent conformation (D-shaped) in the docked state of the ribozyme, indicating that the two helices bounding the internal loop are closer than was previously assumed. This arrangement rationalizes the observed ability of hairpin ribozymes with a circularized substrate-binding strand to cleave a circular substrate, and provides essential information concerning the organization of the substrate in the active conformation. The internal geometry of the substrate-binding strand places G8 of the substrate-binding strand near the cleavage site, which has allowed us to predict the crucial role played by this nucleotide in the reaction chemistry.

Specificity of monoclonal antibodies produced against phosphorothioate and ribo modified DNAs.

A large number of phosphorothioate DNAs and mixed ribo/deoxyribo duplexes were prepared and their immunogenicity was studied in mice. Only those polymers which were nuclease-resistant were immunogenic and in these cases monoclonal antibodies were prepared. The specificity of the antibodies was measured by direct and competitive Solid Phase Radioimmune Assay (SPRIA) and on this basis four types of antibody could be identified. Type I antibodies are specific for the immunizing polymer and show very limited crossreactivity. For example, Jel 384 binds only to poly(dsA).poly(dT); Jel 453 and 462 bind only to poly(dsG).poly(dC) and poly(dsG).poly(dm5C). Type II antibodies bind to most polymers containing the appropriate modification but will not bind to unmodified DNAs. For example, Jel 343 binds to most thio DNAs regardless of sequence; Jel 346 binds well to most ribose-containing polymers and may be a useful reagent for the detection of the 'A' family of conformations. Type III antibodies bind to most nucleic acids whether modified or not. Their specificities are similar to autoimmune antibodies. Type IV antibodies are single strand-specific such as Jel 383 which binds to poly(dT). There were no examples of antibodies which bound specifically to the immunizing DNA and the unmodified polymer. Thus, modified DNAs cannot be used to prepare sequence-specific reagents. Also, the immunogenicity of modified nucleic acids may limit their usefulness in antisense technologies.

Effects of cimetidine and ranitidine on gastric transmural potential difference and on prolactin secretion in man.

Endoscopically controlled measurement of gastric mucosal potential differences was performed in 50 subjects receiving ranitidine, cimetidine or normal saline. Significant maximum potential increases were recorded within 15 min of bolus injections of ranitidine in doses of 20 and 40 mg and cimetidine (200 and 400 mg). Under these conditions, ranitidine proved to be 12 times more potent than cimetidine, compared on a molar basis. There was a dose-related increase in serum prolactin concentration within 15 min of injection of 200 and 400 mg of cimetidine, while injections of 20 and 40 mg of ranitidine failed to enhance prolactin secretion. It would appear that the stimulatory effect of cimetidine on serum prolactin concentration is not mediated through its histamine H2-receptor antagonist activity.

[Absence of effect of secretin and cholecystokinin on plasma concentrations of vasoactive intestinal polypeptide and pancreatic glucagon]. / Fehlender Einfluss von Sekretin und Cholezystokinin auf die Plasmakonzentrationen von vasoaktivem intestinalen Polypeptid (VIP) und pankreatischem Glukagon.

There is little information about the effect of peptides on the VIPergic system. Reports of the influence of secretin and cholecystokinin (CCK) on pancreatic alpha cells are contradictory. With the help of volunteers we investigated the influence of a new synthetic secretin (1 CU/kg/h, 0 to 120 min) alone and in combination with GIH-CCK (1 IU/kg/h, 60 to 120 min) on the concentrations of VIP (n = 13), pancreatic glucagon (PG) (n = 15) and blood sugar (n = 10). 6 of the volunteers were subjected to a randomized cross-over NaCl infusion study. Neither secretin (0 to 60 min) nor secretin and CCK (60 to 120 min) infusion caused a significant change in VIP (31 +/- 3 vs. 34 +/- 4.5 pg/ml, mean +/- SEM, p greater than 0.05), PG (102 +/- 9 vs. 116 +/- 12 vs. 114 +/- 12 pg/ml, p greater than 0.05) or blood sugar (about 90 mg/dl) concentrations. There is no evidence of an influence of secretin and CCK on te VIPergic system and the pancreatic alpha cells.

Severe epilepsy as the major symptom of new mutations in the mitochondrial tRNA(Phe) gene.

OBJECTIVE: To present 2 families with maternally inherited severe epilepsy as the main symptom of mitochondrial disease due to point mutations at position 616 in the mitochondrial tRNA(Phe) (MT-TF) gene. METHODS: Histologic stainings were performed on skeletal muscle slices from the 2 index patients. Oxidative phosphorylation activity was measured by oxygraphic and spectrophotometric methods. The patients' complete mitochondrial DNA (mtDNA) and the relevant mtDNA region in maternal relatives were sequenced. RESULTS: Muscle histology showed only decreased overall COX staining, while a combined respiratory chain defect, most severely affecting complex IV, was noted in both patients' skeletal muscle. Sequencing of the mtDNA revealed in both patients a mutation at position 616 in the MT-TF gene (T>C or T>G). These mutations disrupt a base pair in the anticodon stem at a highly conserved position. They were apparently homoplasmic in both patients, and had different heteroplasmy levels in the investigated maternal relatives. CONCLUSIONS: Deleterious mutations in the mitochondrial tRNA(Phe) may solely manifest with epilepsy when segregating to homoplasmy. They may be overlooked in the absence of lactate accumulation and typical mosaic mitochondrial defects in muscle.

Two-dimensional pulsed-field gel electrophoresis of yeast chromosomes: evidence for triplex-mediated DNA condensation.

The mobility of yeast chromosomes was analysed by two-dimensional pulsed-field gel electrophoresis. The first dimension was run at pH 8.0 in a 1% agarose gel. In the second dimension the electrophoresis conditions were identical, except that the pH was lowered and ethidium, spermine, or ionic detergents were added. Any mobility changes between the two dimensions could be identified as a deviation from the diagonal. At pH 6.0 the mobility of the chromosomes increases severalfold, whereas at pH 4.5 none of the chromosomes move into the agarose gel. The pH-induced mobility changes were reversed by the addition of 2 micrograms/mL of ethidium or 1% lauryl sarcosine. Alternatively, spermine at 1 microM enhanced the pH-mediated mobility changes. Hysteresis was also evident, since upon lowering the pH to 4.5 and then running the gel at pH 7 the mobilities were decreased. These results are interpreted in terms of pH-mediated triplex formation which causes chromosome condensation and thus mobility shifts. The effects of pH are reversed by ethidium which destabilizes triplexes, but enhanced by spermine which favours triplex formation. Therefore, chromosomes may be capable of spontaneous condensation which is mediated by tertiary interactions between appropriate duplex DNA sequences.

[Behavior of histamine in gastric secretion and plasma in the normal and morphologically altered human gastric mucosa]. / Verhalten des Histamins im Magensekret und im Plasma bei normaler und morphologisch veränderter Magenschleimhaut beim Menschen.

The existence of a relationship between the morphological state of oxynthic gland area of the stomach and the histamine content in the gastric juice was studied. In 86 patients (48 males and 38 females) four groups were formed due to a multiple biopsy pattern: individuals with normal gastric mucosa, with superficial gastritis, with beginning atrophy and with advanced atrophic gastritis. In the basal and maximal stimulated gastric juice the following parameters were used: volume, H+ concentration, acid output, histamine concentration and histamine output. The histamine content was additionally determined also in the plasma. The histamine output and the histamine content of the gastric juice was significantly diminished parallel to the degree of the atrophy of gastric mucosa and parallel to the decrease of the volume and acid output and the increase of the intragastral pH values. In the plasma an increase of the histamine content was observed under similar conditions. A possible relationship is discussed between increased plasma histamine content and increased serum gastrin concentration in cases of advanced atrophic gastric mucosa.

[Effect of ranitidine on the transmural gastric mucosal potential difference and on the gastroionogram (author's transl)]. / Die transmurale Potentialdifferenz der Magenwand vor und nach Ranitidin unter Berücksichtigung des Gasgroionogramms.

The effect of intravenous ranitidine on the transmural gastric mucosal potential difference measured under fluoroscopic control was studied in 7 volunteers. A rapid and significant increase in potential difference was observed within 15 minutes. This increase remained unchanged for about two hours. In 15 volunteers the effect of ranitidine on gastric output of acid and electrolytes was examined with and without pentagastrin stimulation. After ranitidine there was a decrease in acid secretion and in K+-ion output both under basal conditions and with pentagastrin stimulation. The Na+-ion output remained unchanged.

Time-resolved hydroxyl-radical footprinting of RNA using Fe(II)-EDTA.

Chemical footprinting methods have been used extensively to probe the structures of biologically important RNAs at nucleotide resolution. One of these methods, hydroxyl-radical footprinting, has recently been employed to study the kinetics of RNA folding. Hydroxyl radicals can be generated by a number of different methods, including Fe(II)-EDTA complexes, synchrotron radiation, and peroxynitrous acid disproportionation. The latter two methods have been used for kinetic studies of RNA folding. We have taken advantage of rapid hydroxyl-radical generation by Fe(II)-EDTA-hydrogen peroxide solutions to develop a benchtop method to study folding kinetics of RNA complexes. This technique can be performed using commercially available chemicals, and can be used to accurately define RNA folding rate constants slower than 6 min(-1). Here we report the method and an example of time-resolved footprinting on the hairpin ribozyme, a small endoribonuclease and RNA ligase.

A conformational change in the "loop E-like" motif of the hairpin ribozyme is coincidental with domain docking and is essential for catalysis.

The catalysis of site-specific RNA cleavage and ligation by the hairpin ribozyme requires the formation of a tertiary interaction between two independently folded internal loop domains, A and B. Within the B domain, a tertiary structure has been identified, known as the loop E motif, that has been observed in many naturally occurring RNAs. One characteristic of this motif is a partial cross-strand stack of a G residue on a U residue. In a few cases, including loop B of the hairpin ribozyme, this unusual arrangement gives rise to photoreactivity. In the hairpin, G21 and U42 can be UV cross-linked. Here we show that docking of the two domains correlates very strongly with a loss of UV reactivity of these bases. The rate of the loss of photoreactivity during folding is in close agreement with the kinetics of interdomain docking as determined by hydroxyl-radical footprinting and fluorescence resonance energy transfer (FRET). Fixing the structure of the complex in the cross-linked form results in an inability of the two domains to dock and catalyze the cleavage reaction, suggesting that the conformational change is essential for catalysis.