Functional deficiency in endogenous interleukin-1 receptor antagonist in patients with febrile infection-related epilepsy syndrome.

OBJECTIVE: We recently reported successful treatment of a child with febrile infection-related epilepsy syndrome (FIRES), a subtype of new onset refractory status epilepticus, with the recombinant interleukin-1 (IL1) receptor antagonist (IL1RA) anakinra. On this basis, we tested whether endogenous IL1RA production or function is deficient in FIRES patients. METHODS: Levels of IL1ß and IL1RA were measured in serum and cerebrospinal fluid (CSF). The inhibitory activity of endogenous IL1RA was assessed using a cell-based reporter assay. IL1RN gene variants were identified by sequencing. Expression levels for the secreted and intracellular isoforms of IL1RA were measured in patient and control cells by real-time polymerase chain reaction. RESULTS: Levels of endogenous IL1RA and IL1ß were elevated in the serum and CSF of patients with FIRES (n = 7) relative to healthy controls (n = 10). Serum from FIRES patients drove IL1R signaling activity and potentiated IL1R signaling in response to exogenous IL1ß in a cell-based reporter assay. Functional assessment of endogenous IL1RA activity in 3 FIRES patients revealed attenuated inhibition of IL1R signaling. Sequencing of IL1RN in our index patient revealed multiple variants. This was accompanied by reduced expression of intracellular but not secreted isoforms of IL1RA in the patient's peripheral blood mononuclear cells. INTERPRETATION: Our findings suggest that FIRES is associated with reduced expression of intracellular IL1RA isoforms and a functional deficiency in IL1RA inhibitory activity. These observations may provide insight into disease pathogenesis for FIRES and other inflammatory seizure disorders and may provide a valuable biomarker for therapeutic decision-making. Ann Neurol 2019;85:526-537.

Febrile infection-related epilepsy syndrome treated with anakinra.

Febrile infection-related epilepsy syndrome (FIRES) is a devastating epileptic encephalopathy with limited treatment options and an unclear etiology. Anakinra is a recombinant version of the human interleukin-1 receptor antagonist used to treat autoinflammatory disorders. This is the first report of anakinra for treatment of a child with super-refractory status epilepticus secondary to FIRES. Anakinra was well tolerated and effective. Cerebral spinal fluid analysis revealed elevated levels of proinflammatory cytokines before treatment that normalized on anakinra, suggesting a potential pathogenic role for neuroinflammation in FIRES. Further studies are required to assess anakinra efficacy and dosing, and to further delineate disease etiology. Ann Neurol 2016;80:939-945.

Polysialic acid as an antigen for monoclonal antibody HIgM12 to treat multiple sclerosis and other neurodegenerative disorders.

CNS regeneration is a desirable goal for diseases of brain and spinal cord. Current therapeutic strategies for the treatment of multiple sclerosis (MS) aim to eliminate detrimental effects of the immune system, so far without reversing disability or affecting long-term prognosis in patients. Approachable molecular targets that stimulate CNS repair are not part of the clinical praxis or have not been identified yet. The purpose of this study was to identify the molecular target of the human monoclonal antibody HIgM12. HIgM12 reverses motor deficits in chronically demyelinated mice, a model of MS. Here, we identified polysialic acid (PSA) attached to the neural cell adhesion molecule (NCAM) as the antigen for HIgM12 by using different NCAM knockout strains and through PSA removal from the NCAM protein core. Antibody binding to CNS tissue and primary cells, antibody-mediated cell adhesion, and neurite outgrowth on HIgM12-coated nitrocellulose was detected only in the presence of PSA as assessed by western blotting, immunoprecipitation, immunocytochemistry, and histochemistry. We conclude that HIgM12 mediates its in vivo and in vitro effects through binding to PSA and has the potential to be an effective therapy for MS and neurodegenerative diseases. The human antibody HIgM12 stimulates neurite outgrowth in vitro and promotes function in chronically demyelinated mice, a model of multiple sclerosis. The cellular antigen for HIgM12 was undetermined. Here, we identified polysialic acid attached to NCAM (neural cell adhesion molecule) as the cellular target for HIgM12. This includes glial fibrillary acidic protein (GFAP)-positive mouse astrocytes (GFAP, red; HIgM12, green; DAPI, blue) among other cell types of the central nervous system. These findings indicate a new strategy for the treatment of neuro-motor disorders including multiple sclerosis.

Performance of an expedited rhythm control method for recent onset atrial fibrillation in a community hospital.

BACKGROUND: A standard approach to recent onset atrial fibrillation (AF) in the emergency department (ED) in the United States has not been established. PURPOSE: The purpose of this prospective clinical trial was to determine how an ED protocol emphasizing rhythm control for recent onset AF compared similar patients receiving standard therapy in the same facility. METHODS: We enrolled consecutive patients presenting to our community hospital with recent onset AF into a protocol, which called for rhythm control with procainamide and if unsuccessful electrical cardioversion and discharge home. We compared this prospective cohort with matched historical controls. Primary outcome was admission rate. We also compared ED conversion rates and lengths of stay (LOS). We reported 30-day data on the study group including ED recidivism, recurrent AF, outpatient follow-up, and any important adverse events. RESULTS: Fifty-four patients were enrolled in the study group with 4 being admitted compared with 30 of 50 in the historical control group. Ninety-four percent of the study group converted compared with 28% in the historical control. Both hospital and ED LOS were significantly shorter for the study group. Six patients had recurrent AF, and 4 of those returned to the ED. CONCLUSION: An ED protocol that uses rhythm control decreased hospital admission and LOS, and there were no adverse events at 30 days.

Conditional ablation of Tbr2 results in abnormal development of the olfactory bulbs and subventricular zone-rostral migratory stream.

BACKGROUND: Development of the olfactory bulb (OB) is a complex process that requires contributions from several progenitor cell niches to generate neuronal diversity. Previous studies showed that Tbr2 is expressed during the generation of glutamatergic OB neurons in rodents. However, relatively little is known about the role of Tbr2 in the developing OB or in the subventricular zone-rostral migratory stream (SVZ-RMS) germinal niche that gives rise to many OB neurons. RESULTS: Here, we use conditional gene ablation strategies to knockout Tbr2 during embryonic mouse olfactory bulb morphogenesis, as well as during perinatal and adult neurogenesis from the SVZ-RMS niche, and describe the resulting phenotypes. We find that Tbr2 is important for the generation of mitral cells in the OB, and that the olfactory bulbs themselves are hypoplastic and disorganized in Tbr2 mutant mice. Furthermore, we show that the SVZ-RMS niche is expanded and disordered following loss of Tbr2, which leads to ectopic accumulation of neuroblasts in the RMS. Lastly, we show that adult glutamatergic neurogenesis from the SVZ is impaired by loss of Tbr2. CONCLUSIONS: Tbr2 is essential for proper morphogenesis of the OB and SVZ-RMS, and is important for the generation of multiple lineages of glutamatergic olfactory bulb neurons.

From Rarity to Recognition: Infantile Botulism and the Broad Spectrum of Differential Diagnoses.

This case illustrates a 5-week-old girl who presented with decreased activity, decreased feeds, poor suck, weak cry, lethargy, hypotonia, and areflexia. The child was found to have infant botulism. The case demonstrates the importance of a full history and broad differential in an ill-appearing infant. The differential for an ill-appearing infant should always include infectious etiologies and may include metabolic disorders, congenital anomalies, nonaccidental trauma, neurologic disorders, and endocrine disorders. The broad differential diagnosis may make rapid diagnosis and treatment for infantile botulism a challenge.

Tbr2 is essential for hippocampal lineage progression from neural stem cells to intermediate progenitors and neurons.

Neurogenesis in the dentate gyrus has been implicated in cognitive functions, including learning and memory, and may be abnormal in major neuropsychiatric disorders, such as depression. Dentate neurogenesis is regulated by interactions between extrinsic factors and intrinsic transcriptional cascades that are currently not well understood. Here we show that Tbr2 (also known as Eomes), a T-box transcription factor expressed by intermediate neuronal progenitors (INPs), is critically required for neurogenesis in the dentate gyrus of developing and adult mice. In the absence of Tbr2, INPs are depleted despite augmented neural stem cell (NSC) proliferation, and neurogenesis is halted as the result of failed neuronal differentiation. Interestingly, we find that Tbr2 likely promotes lineage progression from NSC to neuronal-specified INP in part by repression of Sox2, a key determinant of NSC identity. These findings suggest that Tbr2 expression in INPs is critical for neuronal differentiation in the dentate gyrus and that INPs are an essential stage in the lineage from NSCs to new granule neurons in the dentate gyrus.

Transcriptional control of glutamatergic differentiation during adult neurogenesis.

Neurogenesis, the production of new neurons, occurs in two specialized niches in the adult brain, the subgranular zone (SGZ) of the dentate gyrus and the subventricular zone (SVZ) adjacent to the lateral ventricles. In the SGZ, neural stem cells (NSCs) give rise to glutamatergic granule neurons that integrate into the granule cell layer. In the SVZ, NSCs generate a more diverse cohort of new neurons, including GABAergic, dopaminergic, and glutamatergic neurons, all of which migrate to the olfactory bulb through the rostral migratory stream. In both adult neurogenic niches, specific transcription factors have been shown to direct fate specification and lineage commitment. This review summarizes current progress on the transcriptional control of glutamatergic neurogenesis in the SGZ and SVZ, highlighting commonalities as well as differences in their transcriptional programs. In particular, we focus on work from our laboratory and others indicating that precise, sequential expression of transcription factors regulates the progression from NSC to lineage-committed progenitor, and ultimately regulates the production and differentiation of adult-born glutamatergic neurons.

Patients Taking ß-Blockers Do Not Require Increased Doses of Epinephrine for Anaphylaxis.

BACKGROUND: ß-Blocker use has been associated with increased anaphylaxis severity. OBJECTIVE: We aimed to assess for an association between ß-blocker use and requirement for more than 1 dose of epinephrine for anaphylaxis management. METHODS: We conducted a retrospective observational study of patients seen in our emergency department for anaphylaxis between April 2008 and January 2015. The primary outcome measure was the number of doses of epinephrine. Associations with repeat epinephrine administration (>1 vs ≤1 dose of epinephrine) and associations with any epinephrine administration (>0 vs 0 dose) were evaluated using logistic regression models and summarized as odds ratio (OR) and 95% CIs. The study was powered to detect a 10% or greater difference in need for repeat epinephrine administration between patients who were and were not taking ß-blocker medications. RESULTS: Of 789 patient visits with a documented medication history included in the study, 63 (8%) required more than 1 epinephrine dose and 83 (11%) were on ß-blocker therapy. Among patients who required more than 1 epinephrine dose, 8 (13%) were taking a ß-blocker, compared with 75 patients (10%) who received 0 or 1 dose of epinephrine (OR, 1.26; 95% CI, 0.58-2.75). Among patients who required at least 1 epinephrine dose, 41 (9%) were taking a ß-blocker, compared with 42 patients (12%) who received no epinephrine (OR, 0.73; 95% CI, 0.46-1.14). CONCLUSIONS: ß-Blocker use may not be clinically significant with regard to the need for epinephrine dosing among emergency department patients with anaphylaxis.

Overcoming Barriers to Delirium Screening in the Pediatric Intensive Care Unit.

BACKGROUND: Delirium is associated with poor outcomes in adults but is less extensively studied in children. OBJECTIVES: To describe a quality improvement initiative to implement delirium assessment in a pediatric intensive care unit and to identify barriers to delirium screening completion. METHODS: A survey identified perceived barriers to delirium assessment. Failure modes and effects analysis characterized factors likely to impede assessment. A randomized case-control study evaluated factors affecting assessment by comparing patients always assessed with patients never assessed. RESULTS: Delirium assessment was completed in 57% of opportunities over 1 year, with 2% positive screen results. Education improved screening completion by 20%. Barriers to assessment identified by survey (n = 25) included remembering to complete assessments, documentation outside workflow, and "busy patient." Factors with high risk prediction numbers were lack of time and paper charting. Patients always assessed had more severe illness (median Pediatric Index of Mortality 2 score, 0.90 vs 0.36; P < .001), more developmental disabilities (moderate to severe pediatric cerebral performance category score, 54% vs 32%; P = .007), and admission during lower pediatric intensive care unit census (median [interquartile range], 10 [9-12] vs 12 [10-13]; P < .001) than did those never assessed (each group, n = 80). Patients receiving mechanical ventilation were less likely to be assessed (41.0% vs 51.2%, P < .001). CONCLUSIONS: Successful implementation of pediatric delirium screening may be associated with early use of quality improvement tools to identify assessment barriers, comprehensive education, monitoring system with feedback, multidisciplinary team involvement, and incorporation into nursing workflow models.

Inflammatory cytokine-induced changes in neural network activity measured by waveform analysis of high-content calcium imaging in murine cortical neurons.

During acute neuroinflammation, increased levels of cytokines within the brain may contribute to synaptic reorganization that results in long-term changes in network hyperexcitability. Indeed, inflammatory cytokines are implicated in synaptic dysfunction in epilepsy and in an array of degenerative and autoimmune diseases of the central nervous system. Current tools for studying the impact of inflammatory factors on neural networks are either insufficiently fast and sensitive or require complicated and costly experimental rigs. Calcium imaging offers a reasonable surrogate for direct measurement of neuronal network activity, but traditional imaging paradigms are confounded by cellular heterogeneity and cannot readily distinguish between glial and neuronal calcium transients. While the establishment of pure neuron cultures is possible, the removal of glial cells ignores physiologically relevant cell-cell interactions that may be critical for circuit level disruptions induced by inflammatory factors. To overcome these issues, we provide techniques and algorithms for image processing and waveform feature extraction using automated analysis of spontaneous and evoked calcium transients in primary murine cortical neuron cultures transduced with an adeno-associated viral vector driving the GCaMP6f reporter behind a synapsin promoter. Using this system, we provide evidence of network perturbations induced by the inflammatory cytokines TNFα, IL1ß, and IFNγ.

Antibody Binding Specificity for Kappa (Vκ) Light Chain-containing Human (IgM) Antibodies: Polysialic Acid (PSA) Attached to NCAM as a Case Study.

Antibodies of the IgM isotype are often neglected as potential therapeutics in human trials, animal models of human diseases as well as detecting agents in standard laboratory techniques. In contrast, several human IgMs demonstrated proof of efficacy in cancer models and models of CNS disorders including multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). Reasons for their lack of consideration include difficulties to express, purify and stabilize IgM antibodies, challenge to identify (non-protein) antigens, low affinity binding and fundamental knowledge gaps in carbohydrate and lipid research. This manuscript uses HIgM12 as an example to provide a detailed protocol to detect antigens by Western blotting, immunoprecipitations and immunocytochemistry. HIgM12 targets polysialic acid (PSA) attached to the neural cell adhesion molecule (NCAM). Early postnatal mouse brain tissue from wild type (WT) and NCAM knockout (KO) mice lacking the three major central nervous system (CNS) splice variants NCAM180, 140 and 120 was used to evaluate the importance of NCAM for binding to HIgM12. Further enzymatic digestion of CNS tissue and cultured CNS cells using endoneuraminidases led us to identify PSA as the specific binding epitope for HIgM12.

Abbreviated exposure to hypoxia is sufficient to induce CNS dysmyelination, modulate spinal motor neuron composition, and impair motor development in neonatal mice.

Neonatal white matter injury (nWMI) is an increasingly common cause of cerebral palsy that results predominantly from hypoxic injury to progenitor cells including those of the oligodendrocyte lineage. Existing mouse models of nWMI utilize prolonged periods of hypoxia during the neonatal period, require complex cross-fostering and exhibit poor growth and high mortality rates. Abnormal CNS myelin composition serves as the major explanation for persistent neuro-motor deficits. Here we developed a simplified model of nWMI with low mortality rates and improved growth without cross-fostering. Neonatal mice are exposed to low oxygen from postnatal day (P) 3 to P7, which roughly corresponds to the period of human brain development between gestational weeks 32 and 36. CNS hypomyelination is detectable for 2-3 weeks post injury and strongly correlates with levels of body and brain weight loss. Immediately following hypoxia treatment, cell death was evident in multiple brain regions, most notably in superficial and deep cortical layers as well as the subventricular zone progenitor compartment. PDGFαR, Nkx2.2, and Olig2 positive oligodendrocyte progenitor cell were significantly reduced until postnatal day 27. In addition to CNS dysmyelination we identified a novel pathological marker for adult hypoxic animals that strongly correlates with life-long neuro-motor deficits. Mice reared under hypoxia reveal an abnormal spinal neuron composition with increased small and medium diameter axons and decreased large diameter axons in thoracic lateral and anterior funiculi. Differences were particularly pronounced in white matter motor tracts left and right of the anterior median fissure. Our findings suggest that 4 days of exposure to hypoxia are sufficient to induce experimental nWMI in CD1 mice, thus providing a model to test new therapeutics. Pathological hallmarks of this model include early cell death, decreased OPCs and hypomyelination in early postnatal life, followed by dysmyelination, abnormal spinal neuron composition, and neuro-motor deficits in adulthood.