Cholinergic Polarization of Human Macrophages.

Macrophages serve as vital defenders, protecting the body by exhibiting remarkable cellular adaptability in response to invading pathogens and various stimuli. These cells express nicotinic acetylcholine receptors, with the α7-nAChR being extensively studied due to its involvement in activating the cholinergic anti-inflammatory pathway. Activation of this pathway plays a crucial role in suppressing macrophages' production of proinflammatory cytokines, thus mitigating excessive inflammation and maintaining host homeostasis. Macrophage polarization, which occurs in response to specific pathogens or insults, is a process that has received limited attention concerning the activation of the cholinergic anti-inflammatory pathway and the contributions of the α7-nAChR in this context. This review aims to present evidence highlighting how the cholinergic constituents in macrophages, led by the α7-nAChR, facilitate the polarization of macrophages towards anti-inflammatory phenotypes. Additionally, we explore the influence of viral infections on macrophage inflammatory phenotypes, taking into account cholinergic mechanisms. We also review the current understanding of macrophage polarization in response to these infections. Finally, we provide insights into the relatively unexplored partial duplication of the α7-nAChR, known as dup α7, which is emerging as a significant factor in macrophage polarization and inflammation scenarios.

Mechanisms and Emerging Therapies for Treatment of Seizures in Pediatric Autoimmune Encephalitis and Autoinflammatory/Autoimmune-Associated Epilepsy.

There has been increasing understanding of the role of inflammation in seizures and epilepsy, as well as targeted immunomodulatory treatments. In children, immune-mediated seizures often present acutely in the setting of autoimmune encephalitis and are very responsive to immunotherapy with low rates of subsequent epilepsy. Conversely, seizures in autoimmune-associated epilepsies, such as Rasmussen syndrome, can remain refractory to multimodal therapy, including immunomodulation. In this review, the authors discuss the presentations of immune-mediated seizures in children, underlying mechanisms, and emerging therapies.

Optimizing the Production of gp145, an HIV-1 Envelope Glycoprotein Vaccine Candidate and Its Encapsulation in Guanosine Microparticles.

We have developed a pipeline to express, purify, and characterize HIV envelope protein (Env) gp145 from Chinese hamster ovary cells, to accelerate the production of a promising vaccine candidate. First in shake flasks, then in bioreactors, we optimized the growth conditions. By adjusting the pH to 6.8, we increased expression levels to 101 mg/L in a 50 L bioreactor, nearly twice the previously reported titer value. A battery of analytical methods was developed in accordance with current good manufacturing practices to ensure a quality biopharmaceutical. Imaged capillary isoelectric focusing verified proper glycosylation of gp145; dynamic light scattering confirmed the trimeric arrangement; and bio-layer interferometry and circular dichroism analysis demonstrated native-like properties (i.e., antibody binding and secondary structure). MALDI-TOF mass spectrometry was used as a multi-attribute platform for accurate mass determination, glycans analysis, and protein identification. Our robust analysis demonstrates that our gp145 product is very similar to a reference standard and emphasizes the importance of accurate characterization of a highly heterogeneous immunogen for the development of an effective vaccine. Finally, we present a novel guanosine microparticle with gp145 encapsulated and displayed on its surface. The unique properties of our gp145 microparticle make it amenable to use in future preclinical and clinical trials.

MRI Features and Their Association With Outcomes in Children With Anti-NMDA Receptor Encephalitis.

OBJECTIVES: How brain MRI lesions associate with outcomes in pediatric anti-NMDA receptor encephalitis (pNMDARE) is unknown. In this study, we correlate T2-hyperintense MRI brain lesions with clinical outcomes in pNMDARE. METHODS: This was a multicenter retrospective cohort study from 11 institutions. Children younger than 18 years with pNMDARE were included. One-year outcomes were assessed by the modified Rankin Score (mRS) with good (mRS ≤2) and poor (mRS ≥3) outcomes. RESULTS: A total of 175 pNMDARE subjects were included, with 1-year mRS available in 142/175 (81%) and 60/175 (34%) had abnormal brain MRIs. The most common T2-hyperintense lesion locations were frontal, temporal, and parietal. MRI features that predicted poor 1-year outcomes included abnormal MRI, particularly T2 lesions in the frontal and occipital lobes. After adjusting for treatment within 4 weeks of onset, improvement within 4 weeks, and intensive care unit admission, MRI features were no longer associated with poor outcomes, but after multiple imputation for missing data, T2 frontal and occipital lesions associated with poor outcomes. DISCUSSION: Abnormal frontal and occipital lesions on MRI may associate with 1-year mRS in pNMDARE. MRI of the brain may be a helpful prognostication tool that should be examined in future studies.

Creation of a Novel Child Simulator and Curriculum to Optimize Administration of Seizure Rescue Medication.

INTRODUCTION: Although most cases of pediatric convulsive status epilepticus start in the prehospital setting, many patients do not receive treatment. The use of prehospital seizure rescue medications by caregivers is crucial, but studies suggest a lack of proper training on medication use. METHODS: We created a novel proof of principle mannequin and simulation for training proper administration of rectal diazepam, with a scoring paradigm to standardize and assess the educational process. RESULTS: Twenty-three health care providers (nurses and nurse practitioners, residents/fellows, and attending physicians) and 5 patient guardians/parents were included in the study. The rectal diazepam simulator displayed a high degree of physical and emotional realism (mean ≥ 4/5 on Likert scale survey) that effectively decreased time to treatment (-12.3 seconds; SD, 16.3) and improved the accuracy of medication delivery in a simulation setting (-4.2 points; SD, 3.1). The scoring technique had appropriate interrater reliability (≥86% on all but 2 prompts) and was a feasible instrument to assess the effectiveness of the educational intervention. CONCLUSIONS: A unique procedure-focused child simulator and rescue medication score offer an innovative and effective means to train caregivers on the use of lifesaving seizure rescue medications.

Long-term neuropsychological outcomes in children with febrile infection-related epilepsy syndrome (FIRES) treated with anakinra.

Background: Febrile-infection related epilepsy syndrome (FIRES) is a rare epilepsy syndrome in which a previously healthy individual develops refractory status epilepticus in the setting of a preceding febrile illness. There are limited data regarding detailed long-term outcomes. This study aims to describe the long-term neuropsychological outcomes in a series of pediatric patients with FIRES. Methods: This is a retrospective multi-center case series of pediatric patients with a diagnosis of FIRES treated acutely with anakinra who had neuropsychological testing at least 12 months after status epilepticus onset. Each patient underwent comprehensive neuropsychological evaluation as part of routine clinical care. Additional data collection included the acute seizure presentation, medication exposures, and outcomes. Results: There were six patients identified with a median age of 11.08 years (IQR: 8.19-11.23) at status epilepticus onset. Anakinra initiation was a median of 11 days (IQR: 9.25-13.50) after hospital admission. All patients had ongoing seizures and none of the patients returned to baseline cognitive function with a median follow-up of 40 months (IQR 35-51). Of the five patients with serial full-scale IQ testing, three demonstrated a decline in scores over time. Testing results revealed a diffuse pattern of deficits across domains and all patients required special education and/or accommodations for academic learning. Conclusions: Despite treatment with anakinra, neuropsychological outcomes in this series of pediatric patients with FIRES demonstrated ongoing diffuse neurocognitive impairment. Future research will need to explore the predictors of long-term neurocognitive outcomes in patients with FIRES and to evaluate if acute treatment interventions improve these outcomes.

Mimics of Pediatric Small Vessel Primary Angiitis of the Central Nervous System.

OBJECTIVE: Small vessel primary angiitis of the central nervous system is a rare and often severe disease characterized by central nervous system-restricted inflammatory vasculitis on histopathology. Diagnosis requires brain biopsy for confirmation and is suggested prior to starting immunotherapy when feasible. However, emerging evidence suggests that other neuroinflammatory conditions may have a clinical and radiographic phenotype that mimics small vessel primary angiitis, at times with overlapping pathologic features as well. Such diagnoses, including myelin oligodendrocyte glycoprotein antibody-associated disease and central nervous system-restricted hemophagocytic lymphohistiocytosis, can be non-invasively diagnosed with serum antibody or genetic testing that would prompt different monitoring and treatment paradigms. To determine the ultimate diagnosis of patients who were suspected prior to biopsy to have small vessel primary angiitis, we reviewed the clinical, radiographic, and pathological features of a cohort of patients at a single center undergoing brain biopsy for non-oncologic indications. METHODS: Clinical data were retrospectively extracted from the medical record. Pathology and neuroimaging review was conducted. RESULTS: We identified 21 patients over a 19-year time-period, of whom 14 (66.7%) were ultimately diagnosed with entities other than small vessel primary angiitis that would have obviated the need for brain biopsy. Diagnoses included anti-myelin oligodendrocyte glycoprotein antibody associated disease (n = 9), central nervous system-restricted hemophagocytic lymphohistiocytosis (n = 3), anti-GABAA receptor encephalitis (n = 1), and Aicardi-Goutières syndrome (n = 1). INTERPRETATION: This study highlights the importance of pursuing now readily available non-invasive testing for mimicking diagnoses before performing a brain biopsy for suspected small vessel primary angiitis of the central nervous system. ANN NEUROL 2023;93:109-119.

Identifying Barriers to Care in the Pediatric Acute Seizure Care Pathway.

Objective: We aimed to describe the acute seizure care pathway for pediatric patients and identify barriers encountered by those involved in seizure care management. We also proposed interventions to bridge these care gaps within this pathway. Methods: We constructed a process map that illustrates the acute seizure care pathway for pediatric patients at Boston Children's Hospital (BCH). The map was designed from knowledge gathered from unstructured interviews with experts at BCH, direct observation of patient care management at BCH through a quality improvement implemented seizure diary and from findings through three studies conducted at BCH, including a prospective observational study by the pediatric Status Epilepticus Research Group, a multi-site international consortium. We also reviewed the literature highlighting gaps and strategies in seizure care management. Results: Within the process map, we identified twenty-nine care gaps encountered by caregivers, care teams, residential and educational institutions, and proposed interventions to address these challenges. The process map outlines clinical care of a patient through the following settings: 1) pre-hospitalization setting, defined as residential and educational settings before hospital admission, 2) BCH emergency department and inpatient settings, 3) post-hospitalization setting, defined as residential and educational settings following hospital discharge or clinic visit and 4) follow-up BCH outpatient settings, including neurology, epilepsy, and primary care provider clinics. The acute seizure care pathway for a pediatric patient who presents with seizures exhibits at least twenty-nine challenges in acute seizure care management. Significance: Identification of care barriers in the acute seizure care pathway provides a necessary first step for implementing interventions and strategies in acute seizure care management that could potentially impact patient outcomes.

Infantile Hemiconvulsion-Hemiplegia and Epilepsy (IHHE) in a boy with tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is a rare autosomal dominant disease due to pathogenic variants in TSC1 or TSC2 genes. In the brain, TSC is associated with multiple cortical and subcortical malformations including tubers and abnormalities of radial neuronal migration. Approximately 80% of patients develop epilepsy in the first two years of life, most often focal seizures and infantile spasms. As with all seizure disorders, systemic illness and fever can trigger a seizure, and result in status epilepticus or even refractory status epilepticus. Infantile Hemiconvulsion-Hemiplegia and Epilepsy (IHHE) is considered a subcategory of new-onset refractory status epilepticus (NORSE) and presents with hemiclonic seizures in the setting of fever, unihemispheric brain imaging abnormality and hemiparesis. Here, we present an 18-month-old boy with TSC who developed IHHE. His extensive brain malformations and neuronal hyperexcitability in peri-tuberal tissue could have predisposed him to IHHE. In addition to these factors, we postulate that another prerequisite for IHHE is likely a genetic predisposition for an excessive inflammatory response that is yet to be elucidated.

T Cell Predominant Response to AAV-Spike Protects hACE2 Mice from SARS-CoV-2 Pneumonia

Prevention of COVID-19 is widely believed to depend on neutralization of SARS-CoV-2 by vaccine-induced humoral immunity1,2, raising concern that emerging escape variants may perpetuate the pandemic3-6. Here we show that a single intramuscular injection of Adeno-Associated Virus-6 (AAV6) or AAV9 encoding a modified, N-terminal domain deleted ({Delta}NTD) spike protein induces robust cellular immunity and provides long-term protection in k18-hACE2 transgenic mice from lethal SARS-CoV-2 challenge, associated weight loss and pneumonia independent of vaccine-induced neutralizing humoral immunity. In both mice and macaques, vaccine-induced cellular immunity results in the clearance of transduced muscle fibers coincident with macrophage and CD8+ cytotoxic T cell infiltration at the site of immunization. Additionally, mice demonstrate a strong Type-1 polarized cellular immunophenotype and equivalent ex vivo T cell reactivity to peptides of wt and alpha (B.1.1.7) variant spike. These studies demonstrate not only that AAV6 and AAV9 can function as effective vaccine platforms, but also that vaccines can provide long-term efficacy primarily through the induction of cellular immunity. The findings may provide an alternative approach to containment of the evolving COVID-19 pandemic and have broader implications for the development of variant-agnostic universal vaccines against a wider range of pathogens.

Clinical presentation of new onset refractory status epilepticus in children (the pSERG cohort).

OBJECTIVE: We aimed to characterize the clinical profile and outcomes of new onset refractory status epilepticus (NORSE) in children, and investigated the relationship between fever onset and status epilepticus (SE). METHODS: Patients with refractory SE (RSE) between June 1, 2011 and October 1, 2016 were prospectively enrolled in the pSERG (Pediatric Status Epilepticus Research Group) cohort. Cases meeting the definition of NORSE were classified as "NORSE of known etiology" or "NORSE of unknown etiology." Subgroup analysis of NORSE of unknown etiology was completed based on the presence and time of fever occurrence relative to RSE onset: fever at onset (≤24 h), previous fever (2 weeks-24 h), and without fever. RESULTS: Of 279 patients with RSE, 46 patients met the criteria for NORSE. The median age was 2.4 years, and 25 (54%) were female. Forty (87%) patients had NORSE of unknown etiology. Nineteen (48%) presented with fever at SE onset, 16 (40%) had a previous fever, and five (12%) had no fever. The patients with preceding fever had more prolonged SE and worse outcomes, and 25% recovered baseline neurological function. The patients with fever at onset were younger and had shorter SE episodes, and 89% recovered baseline function. SIGNIFICANCE: Among pediatric patients with RSE, 16% met diagnostic criteria for NORSE, including the subcategory of febrile infection-related epilepsy syndrome (FIRES). Pediatric NORSE cases may also overlap with refractory febrile SE (FSE). FIRES occurs more frequently in older children, the course is usually prolonged, and outcomes are worse, as compared to refractory FSE. Fever occurring more than 24 h before the onset of seizures differentiates a subgroup of NORSE patients with distinctive clinical characteristics and worse outcomes.

Proposal to optimize evaluation and treatment of Febrile infection-related epilepsy syndrome (FIRES): A Report from FIRES workshop.

Febrile infection-related epilepsy syndrome (FIRES) is a rare catastrophic epileptic encephalopathy that presents suddenly in otherwise normal children and young adults causing significant neurological disability, chronic epilepsy, and high rates of mortality. To suggest a therapy protocol to improve outcome of FIRES, workshops were held in conjunction with American Epilepsy Society annual meeting between 2017 and 2019. An international group of pediatric epileptologists, pediatric neurointensivists, rheumatologists and basic scientists with interest and expertise in FIRES convened to propose an algorithm for a standardized approach to the diagnosis and treatment of FIRES. The broad differential for refractory status epilepticus (RSE) should include FIRES, to allow empiric therapies to be started early in the clinical course. FIRES should be considered in all previously healthy patients older than two years of age who present with explosive onset of seizures rapidly progressing to RSE, following a febrile illness in the preceding two weeks. Once FIRES is suspected, early administrations of ketogenic diet and anakinra (the IL-1 receptor antagonist that blocks biologic activity of IL-1ß) are recommended.

A reversed phase HPLC method for the quantification of HIV gp145 glycoprotein levels from cell culture supernatants.

A reversed phase high performance liquid chromatography (RP-HPLC) method was developed for the quantitative determination of recombinant HIV-1 gp145 produced in CHO-K1 cells, as measured directly from culture supernatants. Samples were diluted in 50% D-PBS and 50% PowerCHO-2 (PC2) spent medium, and resolved on a Zorbax 300SB-C8 Rapid Resolution (2.1 × 50 mm, 3.5 µm) column, fitted with a C8 guard column (Zorbax 300SB-C8, 2.1 × 12.5 mm, 5 µm), using 0.1% TFA and 2% n-propanol in LC-MS water as mobile phase A and 0.1% TFA, 70% isopropanol, and 20% acetonitrile in LC-MS water as mobile phase B. The column temperature was 80 °C, the flow rate was 0.4 mL/min and the absorbance was monitored at 280 nm. The procedures and capabilities of the method were evaluated against the criteria for linearity, limit of detection (LOD), accuracy, repeatability, and robustness as defined by the International Conference on Harmonization (ICH) 2005 Q2(R1) guidelines. Two different variants of the HIV-1 envelope protein (Env), CO6980v0c22 gp145 and SF162 gp140, were analyzed and their retention times were found to be different. The method showed good linearity (R2 = 0.9996), a lower LOD of 2.4 µg/mL, and an average recovery of 101%. The analysis includes measurements of accuracy, inter-user precision, and robustness. Overall, we present a RP-HPLC method that could be applied for the quantitation of cell culture titers for this and other variants of HIV Env following ICH guidelines.

Immunodominant T-cell epitopes from the SARS-CoV-2 spike antigen reveal robust pre-existing T-cell immunity in unexposed individuals

The COVID-19 pandemic has revealed a range of disease phenotypes in infected patients with asymptomatic, mild or severe clinical outcomes, but the mechanisms that determine such variable outcomes remain unresolved. In this study, we identified immunodominant CD8 T-cell epitopes in the RBD and the non-RBD domain of the spike antigen using a novel TCR-binding algorithm. A selected pool of 11 predicted epitopes induced robust T-cell activation in unexposed donors demonstrating pre-existing CD4 and CD8 T-cell immunity to SARS-CoV-2 antigen. The T-cell reactivity to the predicted epitopes was higher than the Spike-S1 and S2 peptide pools containing 157 and 158 peptides both in unexposed donors and in convalescent patients suggesting that strong T-cell epitopes are likely to be missed when larger peptide pools are used in assays. A key finding of our study is that pre-existing T-cell immunity to SARS-CoV-2 is contributed by TCRs that recognize common viral antigens such as Influenza and CMV, even though the viral epitopes lack sequence identity to the SARS-CoV-2 epitopes. This finding is in contrast to multiple published studies in which pre-existing T-cell immunity is suggested to arise from shared epitopes between SARS-CoV-2 and other common cold-causing coronaviruses. Whether the presence of pre-existing T-cell immunity provides protection against COVID-19 or contributes to severe disease phenotype remains to be determined in a larger cohort. However, our findings raise the expectation that a significant majority of the global population is likely to have SARS-CoV-2 reactive T-cells because of prior exposure to flu and CMV viruses, in addition to common cold-causing coronaviruses.

Stabilizing Cellular Barriers: Raising the Shields Against COVID-19.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its clinical manifestation (COVID-19; coronavirus disease 2019) have caused a worldwide health crisis. Disruption of epithelial and endothelial barriers is a key clinical turning point that differentiates patients who are likely to develop severe COVID-19 outcomes: it marks a significant escalation in respiratory symptoms, loss of viral containment and a progression toward multi-organ dysfunction. These barrier mechanisms are independently compromised by known COVID-19 risk factors, including diabetes, obesity and aging: thus, a synergism between these underlying conditions and SARS-CoV-2 mechanisms may explain why these risk factors correlate with more severe outcomes. This review examines the key cellular mechanisms that SARS-CoV-2 and its underlying risk factors utilize to disrupt barrier function. As an outlook, we propose that glucagon-like peptide 1 (GLP-1) may be a therapeutic intervention that can slow COVID-19 progression and improve clinical outcome following SARS-CoV-2 infection. GLP-1 signaling activates barrier-promoting processes that directly oppose the pro-inflammatory mechanisms commandeered by SARS-CoV-2 and its underlying risk factors.

Magnetic resonance imaging-guided laser-induced thermal therapy for functional hemispherotomy in a child with refractory epilepsy and multiple medical comorbidities.

Magnetic resonance imaging-guided laser-induced thermal therapy (MRgLITT) is a minimally invasive surgical approach increasingly employed for precise targeted ablation of epileptogenic brain foci. Recent reports have described corpus callosotomy using MRgLITT, though its application in more extensive functional disconnections has not been documented. Here, the authors detail its use in achieving a palliative hemispherotomy in a 5-year-old with medically refractory hemiclonic seizures following a hemispheric infarction, highlighting a novel use of this surgical technique. In this particular case, open craniotomy was deemed high risk given the multiple medical comorbidities including congenital cardiac disease and end-stage renal failure. MRgLITT was considered an alternative approach with a lower risk for periprocedural hemodynamic perturbations. The patient tolerated the procedure well, attaining an Engel class IB outcome at 16 months' follow-up. This suggests that MRgLITT may be an alternative approach to an open hemispherectomy, particularly in cases in which multiple comorbidities pose significant risks and preclude an open procedure.

A recombinant gp145 Env glycoprotein from HIV-1 expressed in two different cell lines: Effects on glycosylation and antigenicity.

The envelope glycoprotein (Env) of the human immunodeficiency virus (HIV), has been the primary target for the development of a protective vaccine against infection. The extensive N-linked glycosylation on Env is an important consideration as it may affect efficacy, stability, and expression yields. The expression host has been shown to influence the extent and type of glycosylation that decorates the protein target. Here, we report the glycosylation profile of the candidate subtype C immunogen CO6980v0c22 gp145, which is currently in Phase I clinical trials, produced in two different host cells: CHO-K1 and Expi293F. The amino acid sequence for both glycoproteins was confirmed to be identical by peptide mass fingerprinting. However, the isoelectric point of the proteins differed; 4.5-5.5 and 6.0-7.0 for gp145 produced in CHO-K1 and Expi293F, respectively. These differences in pI were eliminated by enzymatic treatment with sialidase, indicating a large difference in the incorporation of sialic acid between hosts. This dramatic difference in the number of sialylated glycans between hosts was confirmed by analysis of PNGase F-released glycans using MALDI-ToF MS. These differences in glycosylation, however, did not greatly translate into differences in antibody recognition. Biosensor assays showed that gp145 produced in CHO-K1 had similar affinity toward the broadly neutralizing antibodies, 2G12 and PG16, as the gp145 produced in Expi293F. Additionally, both immunogens showed the same reactivity against plasma of HIV-infected patients. Taken together, these results support the notion that there are sizeable differences in the glycosylation of Env depending on the expression host. How these differences translate to vaccine efficacy remains unknown.

Posterior-onset Rasmussen's encephalitis with ipsilateral cerebellar atrophy and uveitis resistant to rituximab.

Rasmussen encephalitis (RE) is a disorder characterized by drug-resistant seizures and progressive unihemispheric atrophy, hemiparesis, and varying degrees of cognitive decline. The pathophysiology of RE remains elusive, with hypotheses suggesting underlying autoimmune- and T cell-mediated processes. In this case report, we describe a single patient's clinical course from the first day of presentation until definitive treatment for atypical Rasmussen encephalitis at a tertiary care pediatric center. The patient exhibited several atypical features of Rasmussen encephalitis, including a posterior predominance of initial seizure onset with the development of severe choreoathetosis and ipsilateral cerebellar atrophy. He subsequently developed coexistent autoimmune disorders in the form of psoriasis and uveitis, and underwent multiple forms of immunotherapy with limited benefit. This patient shows an association of RE with other autoimmune conditions supporting an autoimmune mechanism of disease while exhibiting several atypical features of RE. Rarely, occipital lobe seizures have been documented as the presenting semiology of this syndrome. This case highlights the need to be mindful of atypical features that may delay hemispherectomy, which remains the definitive treatment. It also suggests that children may be predisposed to the development of autoimmune disorders in later stages of the disease.