Dmitry Tchapyjnikov

Large-Scale Proteomics and Phosphoproteomics of Urinary Exosomes

Normal human urine contains large numbers of exosomes, which are 40- to 100-nm vesicles that originate as the internal vesicles in multivesicular bodies from every renal epithelial cell type facing the urinary space. Here, we used LC-MS/MS to profile the proteome of human urinary exosomes. Overall, the analysis identified 1132 proteins unambiguously, including 177 that are represented on the Online Mendelian Inheritance in Man database of disease-related genes, suggesting that exosome analysis is a potential approach to discover urinary biomarkers. We extended the proteomic analysis to phosphoproteomic profiling using neutral loss scanning, and this yielded multiple novel phosphorylation sites, including serine-811 in the thiazide-sensitive Na-Cl co-transporter, NCC. To demonstrate the potential use of exosome analysis to identify a genetic renal disease, we carried out immunoblotting of exosomes from urine samples of patients with a clinical diagnosis of Bartter syndrome type I, showing an absence of the sodium-potassium-chloride co-transporter 2, NKCC2. The proteomic data are publicly accessible at http://dir.nhlbi.nih.gov/papers/lkem/exosome/.

Vasopressin-stimulated Increase in Phosphorylation at Ser269 Potentiates Plasma Membrane Retention of Aquaporin-2

Vasopressin controls water excretion through regulation of aquaporin-2 (AQP2) trafficking in renal collecting duct cells. Using mass spectrometry, we previously demonstrated four phosphorylated serines (Ser256, Ser261, Ser264, and Ser269) in the carboxyl-terminal tail of rat AQP2. Here, we used phospho-specific antibodies and protein mass spectrometry to investigate the roles of vasopressin and cyclic AMP in the regulation of phosphorylation at Ser269 and addressed the role of this site in AQP2 trafficking. The V2 receptor-specific vasopressin analog dDAVP increased Ser(P)269-AQP2 abundance more than 10-fold, but at a rate much slower than the corresponding increase in Ser256 phosphorylation. Vasopressin-mediated changes in phosphorylation at both sites were mimicked by cAMP addition and inhibited by protein kinase A (PKA) antagonists. In vitro kinase assays, however, demonstrated that PKA phosphorylates Ser256, but not Ser269. Phosphorylation of AQP2 at Ser269 did not occur when Ser256 was replaced by an unphosphorylatable amino acid, as seen in both S256L-AQP2 mutant mice and in Madin-Darby canine kidney cells expressing an S256A mutant, suggesting that Ser269 phosphorylation depends upon prior phosphorylation at Ser256. Immunogold electron microscopy localized Ser(P)269-AQP2 solely in the apical plasma membrane of rat collecting duct cells, in contrast to the other three phospho-forms (found in both apical plasma membrane and intracellular vesicles). Madin-Darby canine kidney cells expressing an S269D “phosphomimic” AQP2 mutant showed constitutive localization at the plasma membrane. The data support a model in which vasopressin-mediated phosphorylation of AQP2 at Ser269:(a) depends on prior PKA-mediated phosphorylation of Ser256 and (b) enhances apical plasma membrane retention of AQP2.

Association of Time to Treatment With Short-term Outcomes for Pediatric Patients With Refractory Convulsive Status Epilepticus.

Importance Treatment delay for seizures can lead to longer seizure duration. Whether treatment delay is associated with major adverse outcomes, such as death, remains unknown. Objective To evaluate whether untimely first-line benzodiazepine treatment is associated with unfavorable short-term outcomes. Design, Setting, and Participants This multicenter, observational, prospective cohort study included 218 pediatric patients admitted between June 1, 2011, and July 7, 2016, into the 11 tertiary hospitals in the United States within the Pediatric Status Epilepticus Research Group. Patients, ranging in age from 1 month to 21 years, with refractory convulsive status epilepticus (RCSE) that did not stop after the administration of at least 2 antiseizure medications were included. Patients were divided into 2 cohorts: those who received the first-line benzodiazepine treatment in less than 10 minutes and those who received it 10 or more minutes after seizure onset (untimely). Data were collected and analyzed from June 1, 2011, to July 7, 2016. Main Outcomes and Measures The primary outcome was death during the related hospital admission. The secondary outcome was the need for continuous infusion for seizure termination. Multivariate analysis of mortality controlled for structural cause, febrile RCSE, age, and previous neurological history (including previous RCSE events). Use of continuous infusions was additionally adjusted for generalized RCSE, continuous RCSE, and 5 or more administrations of antiseizure medication. Results A total of 218 patients were included, among whom 116 (53.2%) were male and the median (interquartile range) age was 4.0 (1.2-9.6) years. The RCSE started in the prehospital setting for 139 patients (63.8%). Seventy-four patients (33.9%) received their first-line benzodiazepine treatment in less than 10 minutes, and 144 (66.1%) received untimely first-line benzodiazepine treatment. Multivariate analysis showed that patients who received untimely first-line benzodiazepine treatment had higher odds of death (adjusted odds ratio [AOR], 11.0; 95% CI, 1.43 to ∞; P = .02), had greater odds of receiving continuous infusion (AOR, 1.8; 95% CI, 1.01-3.36; P = .047), had longer convulsive seizure duration (AOR, 2.6; 95% CI, 1.38-4.88; P = .003), and had more frequent hypotension (AOR 2.3; 95% CI, 1.16-4.63; P = .02). In addition, the timing of the first-line benzodiazepine treatment was correlated with the timing of the second-line (95% CI, 0.64-0.95; P < .001) and third-line antiseizure medications (95% CI, 0.25-0.78; P < .001). Conclusions and Relevance Among pediatric patients with RCSE, an untimely first-line benzodiazepine treatment is independently associated with a higher frequency of death, use of continuous infusions, longer convulsion duration, and more frequent hypotension. Results of this study raise the question as to whether poor outcomes could, in part, be prevented by earlier administration of treatment.

Response to immunotherapy in a patient with Landau-Kleffner syndrome and GRIN2A mutation

Landau-Kleffner syndrome (LKS) has been demonstrated in the past to respond to immunotherapy. Recently, some cases of LKS have been shown to be secondary to glutamate receptor (GRIN2A) mutations. Whether such cases respond to immunotherapy is not known. Here, we present the case of a 3-year-old boy with LKS found to have a GRIN2A heterozygous missense mutation, whose clinical symptoms and EEG responded to a course of combination oral steroids and monthly infusions of intravenous immunoglobulin. He then relapsed after discontinuation of this therapy, and responded again after a second course of intravenous immunoglobulin. We conclude that immunotherapy should be considered as a therapeutic option in patients with LKS who are also found to harbour GRIN2A mutations.

Immune-related Neurological Symptoms in an Adolescent Patient Receiving the Checkpoint Inhibitor Nivolumab

Immune checkpoint inhibitors are a novel group of immunotherapeutic agents for the treatment of cancer. Immune-related adverse events, including neurological symptoms, have been associated with these agents. We present the case of an adolescent with refractory Hodgkin lymphoma treated with nivolumab, a PD1 inhibitor, who developed Hashimoto thyroiditis, posterior reversible encephalopathy syndrome causing seizures, as well as urinary retention, truncal/appendicular spasticity, dysphagia, and a progressive encephalopathy that was clinically consistent with a diagnosis of progressive encephalopathy with rigidity and myoclonus, a stiff-person-syndrome spectrum disorder. He showed response and ultimately full recovery to a combination of intravenous steroids, intravenous immunoglobulin, and plasma exchange. To our knowledge, this is the first documented report of an immune-related neurological reaction to nivolumab in an adolescent patient and expands the spectrum of known nivolumab-associated toxicities.

Efficacy and safety of ketogenic diet for treatment of pediatric convulsive refractory status epilepticus

PURPOSE To describe the efficacy and safety of ketogenic diet (KD) for convulsive refractory status epilepticus (RSE). METHODS RSE patients treated with KD at the 6/11 participating institutions of the pediatric Status Epilepticus Research Group from January-2011 to December-2016 were included. Patients receiving KD prior to the index RSE episode were excluded. RSE was defined as failure of ≥2 anti-seizure medications, including at least one non-benzodiazepine drug. Ketosis was defined as serum beta-hydroxybutyrate levels >20 mg/dl (1.9 mmol/l). Outcomes included proportion of patients with electrographic (EEG) seizure resolution within 7 days of starting KD, defined as absence of seizures and ≥50% suppression below 10 μV on longitudinal bipolar montage (suppression-burst ratio ≥50%); time to start KD after onset of RSE; time to achieve ketosis after starting KD; and the proportion of patients weaned off continuous infusions 2 weeks after KD initiation. Treatment-emergent adverse effects (TEAEs) were also recorded. RESULTS Fourteen patients received KD for treatment of RSE (median age 4.7 years, interquartile range [IQR] 5.6). KD was started via enteral route in 11/14 (78.6%) patients. KD was initiated a median of 13 days (IQR 12.5) after the onset of RSE, at 4:1 ratio in 8/14 (57.1%) patients. Ketosis was achieved within a median of 2 days (IQR 2.0) after starting KD. EEG seizure resolution was achieved within 7 days of starting KD in 10/14 (71.4%) patients. Also, 11/14 (78.6%) patients were weaned off their continuous infusions within 2 weeks of starting KD. TEAEs, potentially attributable to KD, occurred in 3/14 (21.4%) patients, including gastro-intestinal paresis and hypertriglyceridemia. Three month outcomes were available for 12/14 (85.7%) patients, with 4 patients being seizure-free, and 3 others with decreased seizure frequency compared to pre-RSE baseline. CONCLUSIONS This series suggests efficacy and safety of KD for treatment of pediatric RSE.

B6 and Bleeding: A Case Report of a Novel Vitamin Toxicity

Vitamin B6 and its derivatives are well tolerated with minimal known toxicity. We report the first pediatric case of excessive bleeding because of administration of pyridoxyal-5-phosphate. Pyridox(am)ine-5-phosphate oxidase deficiency is an inborn error of vitamin B6 metabolism that is characterized by neonatal seizures, requiring lifelong therapy with pyridoxal-5-phosphate. We present the first case of a patient with pyridox(am)ine-5-phosphate oxidase deficiency and mild hemophilia A, whose bleeding symptoms were exacerbated by the vitamin B6 therapy essential for his epileptic disorder. This report expands the spectrum of known vitamin B6 toxicity and demonstrates a need for vigilance in monitoring for bleeding symptoms in patients requiring pyridoxine or pyridoxal-5-phosphate supplementation.

Factors associated with treatment delays in pediatric refractory convulsive status epilepticus

Objective To identify factors associated with treatment delays in pediatric patients with convulsive refractory status epilepticus (rSE). Methods This prospective, observational study was performed from June 2011 to March 2017 on pediatric patients (1 month to 21 years of age) with rSE. We evaluated potential factors associated with increased treatment delays in a Cox proportional hazards model. Results We studied 219 patients (53% males) with a median (25th–75th percentiles [p25–p75]) age of 3.9 (1.2–9.5) years in whom rSE started out of hospital (141 [64.4%]) or in hospital (78 [35.6%]). The median (p25–p75) time from seizure onset to treatment was 16 (5–45) minutes to first benzodiazepine (BZD), 63 (33–146) minutes to first non-BZD antiepileptic drug (AED), and 170 (107–539) minutes to first continuous infusion. Factors associated with more delays to administration of the first BZD were intermittent rSE (hazard ratio [HR] 1.54, 95% confidence interval [CI] 1.14–2.09; p = 0.0467) and out-of-hospital rSE onset (HR 1.5, 95% CI 1.11–2.04; p = 0.0467). Factors associated with more delays to administration of the first non-BZD AED were intermittent rSE (HR 1.78, 95% CI 1.32–2.4; p = 0.001) and out-of-hospital rSE onset (HR 2.25, 95% CI 1.67–3.02; p < 0.0001). None of the studied factors were associated with a delayed administration of continuous infusion. Conclusion Intermittent rSE and out-of-hospital rSE onset are independently associated with longer delays to administration of the first BZD and the first non-BZD AED in pediatric rSE. These factors identify potential targets for intervention to reduce time to treatment.

Cefepime-Induced Encephalopathy and Nonconvulsive Status Epilepticus: Dispelling an Artificial Dichotomy

Cefepime is a fourth-generation cephalosporin antibiotic known to have neurotoxic side effects. Recent reports have described patients on cefepime presenting with altered mentation and concurrent triphasic wave discharges on electroencephalogram (EEG). Some have described this clinical presentation as cefepime-induced encephalopathy, while others have termed it as cefepime-induced nonconvulsive status epilepticus (NCSE). We report on 4 patients who developed cefepime-associated altered mentation with triphasic discharges on EEG. A benzodiazepine trial was attempted in 3 of the patients, all of whom had improvement in the frequency of the triphasic discharges, but only 2 of whom demonstrated a concurrent partial and transient improvement in mental status. All 4 patients had normalization of mental status upon discontinuation of cefepime. We provide a literature review of prior cases and propose that these reports, including those labeled as NCSE, are best described as a cefepime-induced encephalopathy with triphasic discharges as opposed to an ictal phenomenon. We contend that aggressive treatment with anti-seizure medications is not warranted and that cefepime discontinuation is the definitive treatment. This case series and review of the literature clarifies a long-standing terminological ambiguity in a unique clinical picture that can be encountered by the neurohospitalist or other providers.

Platform Session – Electroencephalography/Epilepsy: Clinical characteristics and outcomes of pediatric super refractory status epilepticus

Introduction There is limited literature on pediatric super refractory status epilepticus (SRSE). We aimed to identify clinical variables associated with the occurrence of SRSE and to describe variability in SRSE treatment in a cohort of children admitted to pediatric intensive care units (ICU) with refractory status epilepticus (RSE). Methods This prospective, observational study was performed at 14 US hospitals. Inclusion criteria: (1) hospital admission between June 2011 and September 2017, (2) convulsive RSE defined as focal or generalized convulsive seizures at onset that continued after administration of at least 2 anti-seizure drugs (ASDs), including one non-benzodiazepine ASD or use of continuous infusion (CI) and (3) age 1 month to 21 years. We divided the cohort into SRSE and non-SRSE groups. SRSE was defined as continuous or intermittent seizures lasting ⩾ 24 h following initiation of CI. We used Fisher’s exact test and Wilcoxon rank sum test in the univariate analysis. Results The initial cohort included 264 patients with a median (p25–p75) age of 4 (1.23–9.50) years. Thirty-five patients (19 males) were identified as SRSE cases, with a median age of 4.5 (1.91–8.45) years. Univariate analysis did not show significant differences between the groups for: sex, age, etiology, prior SE, diagnosis of epilepsy, in-hospital seizure onset, generalized tonic-clonic seizures or continuous SE. SRSE patients had delayed first line treatment initiation (71(15–135.5) vs. 16(5–40.5) min; p = 0.017), longer ICU stay (17(9–42) vs. 3(1.86–8.66) days; p  Conclusion SRSE patients had delayed first-line treatment administration in comparison to non-SRSE patients. Once SRSE was established, children had significantly higher morbidity and mortality. Treatment approaches were heterogeneous, probably reflecting limited evidence to guide clinical decision-making in SRSE. (Funded by the Pediatric Epilepsy Research Foundation and Epilepsy Research Fund ).