An abnormal accumulation of extracellular K ؉ in the brain has been implicated in the generation of seizures in patients with mesial temporal lobe epilepsy (MTLE) and hippocampal sclerosis. Experimental studies have shown that clearance of extracellular K ؉ is compromised by removal of the perivascular pool of the water channel aquaporin 4 (AQP4), suggesting that an efficient clearance of K ؉ depends on a concomitant water flux through astrocyte membranes. Therefore, we hypothesized that loss of perivascular AQP4 might be involved in the pathogenesis of MTLE. Whereas Western blot analysis showed an overall increase in AQP4 levels in MTLE compared with non-MTLE hippocampi, quantitative ImmunoGold electron microscopy revealed that the density of AQP4 along the perivascular membrane domain of astrocytes was reduced by 44% in area CA1 of MTLE vs. non-MTLE hippocampi. There was no difference in the density of AQP4 on the astrocyte membrane facing the neuropil. Because anchoring of AQP4 to the perivascular astrocyte endfoot membrane depends on the dystrophin complex, the localization of the 71-kDa brain-specific isoform of dystrophin was assessed by immunohistochemistry. In non-MTLE hippocampus, dystrophin was preferentially localized near blood vessels. However, in the MTLE hippocampus, the perivascular dystrophin was absent in sclerotic areas, suggesting that the loss of perivascular AQP4 is secondary to a disruption of the dystrophin complex. We postulate that the loss of perivascular AQP4 in MTLE is likely to result in a perturbed flux of water through astrocytes leading to an impaired buffering of extracellular K ؉ and an increased propensity for seizures.dystrophin ͉ epilepsy ͉ seizures ͉ astrocytes M esial temporal lobe epilepsy (MTLE) is one of the commonest forms of medically intractable epilepsies. MTLE is characterized by seizures that originate from mediobasal temporal lobe structures, particularly the hippocampus, and neurosurgical resection of the epileptogenic hippocampus is often used to treat this disorder. The resected, epileptogenic hippocampus in MTLE is typically indurated and atrophic and displays massive loss of neurons along with astroglial changes, particularly in areas CA1 and CA3 and the dentate hilus, a condition known as hippocampal (or Ammon's horn) sclerosis. Electrophysiological recordings from MTLE hippocampi have demonstrated that these hippocampi are hyperexcitable when compared with nonsclerotic hippocampi from patients with other types of temporal lobe epilepsy, such as mass associated temporal lobe epilepsy (patients with an extrahippocampal mass lesion) or paradoxical temporal lobe epilepsy (patients without a mass lesion and with seizures of unknown etiology). A fundamental question that remains to be resolved is why the MTLE hippocampus is hyperexcitable.Studies of MTLE patient hippocampi have shown that the K ϩ buffering capacity is diminished when compared with non-
BackgroundWe aimed to evaluate the clinical usefulness of qSOFA as a risk stratification tool for patients admitted with infection compared to traditional SIRS criteria or our triage system; the Rapid Emergency Triage and Treatment System (RETTS).MethodsThe study was an observational cohort study performed at one Emergency Department (ED) in an urban university teaching hospital in Norway, with approximately 20,000 visits per year. All patients >16 years presenting with symptoms or clinical signs suggesting an infection (n = 1535) were prospectively included in the study from January 1 to December 31, 2012. At arrival in the ED, vital signs were recorded and all patients were triaged according to RETTS vital signs, presenting infection, and sepsis symptoms. These admission data were also used to calculate qSOFA and SIRS. Treatment outcome was later retrieved from the patients’ electronic records (EPR) and mortality data from the Norwegian population registry.ResultsOf the 1535 admitted patients, 108 (7.0%) fulfilled the Sepsis2 criteria for severe sepsis. The qSOFA score ≥2 identified only 33 (sensitivity 0.32, specificity 0.98) of the patients with severe sepsis, whilst the RETTS-alert ≥ orange identified 92 patients (sensitivity 0.85, specificity 0.55). Twenty-six patients died within 7 days of admission; four (15.4%) of them had a qSOFA ≥2, and 16 (61.5%) had RETTS ≥ orange alert. Of the 68 patients that died within 30 days, only eight (11.9%) scored ≥2 on the qSOFA, and 45 (66.1%) had a RETTS ≥ orange alert.DiscussionIn order to achieve timely treatment for sepsis, a sensitive screening tool is more important than a specific one. Our study is the fourth study were qSOFA finds few of the sepsis cases in prehospital or at arrival to the ED. We add information on the RETTS triage system, the two highest acuity levels together had a high sensitivity (85%) for identifying sepsis at arrival to the ED - and thus, RETTS should not be replaced by qSOFA as a screening and trigger tool for sepsis at arrival.ConclusionIn this observational cohort study, qSOFA failed to identify two thirds of the patients admitted to an ED with severe sepsis. Further, qSOFA failed to be a risk stratification tool as the sensitivity to predict 7-day and 30-day mortality was low. The sensitivity was poorer than the other warning scores already in use at the study site, RETTS-triage and the SIRS criteria.Electronic supplementary materialThe online version of this article (doi:10.1186/s13049-017-0399-4) contains supplementary material, which is available to authorized users.
Abstract-Specific antibodies are essential tools for identifying individual proteins in biological samples. While generation of antibodies is often straightforward, determination of the antibody specificity is not. Here we illustrate this by describing the production and characterization of antibodies to excitatory amino acid transporter 3 (EAAT3). We synthesized 13 peptides corresponding to parts of the EAAT3 sequence and immunized 6 sheep and 30 rabbits. All sera were affinity purified against the relevant immobilized peptide. Antibodies to the peptides were obtained in almost all cases. Immunoblotting with tissue extracts from wild type and EAAT3 knockout animals revealed that most of the antibodies did not recognize the native EAAT3 protein, and that some recognized other proteins. Several immunization protocols were tried, but strong reactions with EAAT3 were only seen with antibodies to the C-terminal peptides. In contrast, good antibodies were obtained to several parts of EAAT2. EAAT3 was only detected in neurons. However, rabbits immunized with an EAAT3-peptide corresponding to residues 479 -498 produced antibodies that labeled axoplasm and microtubules therein particularly strongly. On blots, these antibodies recognized both EAAT3 and a slightly smaller, but far more abundant protein that turned out to be tubulin. The antibodies were fractionated on columns with immobilized tubulin. One fraction contained antibodies apparently specific for EAAT3 while another fraction contained antibodies recognizing both EAAT3 and tubulin despite the lack of primary sequence identity between the two proteins. Addition of free peptide to the incubation solution blocked immunostaining of both EAAT3 and tubulin. Conclusions: Not all antibodies to synthetic peptides recognize the native protein. The peptide sequence is more important than immunization protocol. The specificity of an antibody is hard to predict because crossreactivity can be specific and to unrelated molecules. The antigen preabsorption test is of little value in testing the specificity of affinity purified antibodies.
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