Abnormal Neurologic Findings in Patients With Sickle Cell Disease Without a History of Major Neurologic Events.

Background and Objectives: Patients with sickle cell disease (SCD) are prone to symptomatic neurologic complications. Previous studies reported accrual of neural injury starting at early age, even without having symptomatic neurologic events. The aim of this study was to assess the prevalence and risk factors of abnormal neurologic findings in patients with SCD with no history of major symptomatic neurologic events. Methods: Our study extracted patients diagnosed with SCD from the Cooperative Study of Sickle Cell Disease. Patients who underwent a neurologic evaluation were included in our analysis. Patients with previous documented major symptomatic neurologic events were excluded. We compared patients with SCD with abnormal neurologic findings with those without in terms of clinical and laboratory parameters using multivariate binary logistic regression. Results: A total of 3,573 patients with SCD were included (median age = 11 [IQR = 19] years, male = 1719 [48.1%]). 519 (14.5%) patients had at least one abnormal neurologic finding. The most common findings in descending order were abnormal reflexes, gait abnormalities, cerebellar dysfunction, language deficits, nystagmus, abnormal muscle tone and strength, Romberg sign, Horner syndrome, and intellectual impairment. History of eye disease (odds ratio [OR] = 2.76, 95% confidence interval [CI] = 1.63-4.68) and history of osteomyelitis (OR = 2.55, 95% CI 1.34-4.84) were the strongest predictors of abnormal neurologic findings, followed by smoking (OR = 1.59, 95% CI 1.08-2.33), aseptic necrosis (OR = 1.57, 95% CI 1.06-2.33), hand-foot syndrome (OR = 1.48, 95% CI 1.04-2.12), and male sex (OR = 1.42, 95% CI 1.01-2.02). Discussion: Neurologic deficits are relatively common in patients with SCD, even without documented major neurologic insults. They range from peripheral and ophthalmic deficits to central and cognitive disabilities. Patients with SCD should have early regular neurologic evaluations and risk factor modification, particularly actively promoting smoking cessation.

Eye Segmentation Method for Telehealth: Application to the Myasthenia Gravis Physical Examination.

Due to the precautions put in place during the COVID-19 pandemic, utilization of telemedicine has increased quickly for patient care and clinical trials. Unfortunately, teleconsultation is closer to a video conference than a medical consultation, with the current solutions setting the patient and doctor into an evaluation that relies entirely on a two-dimensional view of each other. We are developing a patented telehealth platform that assists with diagnostic testing of ocular manifestations of myasthenia gravis. We present a hybrid algorithm combining deep learning with computer vision to give quantitative metrics of ptosis and ocular muscle fatigue leading to eyelid droop and diplopia. The method works both on a fixed image and frame by frame of the video in real-time, allowing capture of dynamic muscular weakness during the examination. We then use signal processing and filtering to derive robust metrics of ptosis and l ocular misalignment. In our construction, we have prioritized the robustness of the method versus accuracy obtained in controlled conditions in order to provide a method that can operate in standard telehealth conditions. The approach is general and can be applied to many disorders of ocular motility and ptosis.

A Digital Telehealth System to Compute the Myasthenia Gravis Core Examination Metrics.

Background: Telemedicine practice for neurological diseases has grown significantly during the COVID-19 pandemic.Telemedicine offers an opportunity to assess digitalization of examinations and enhances access to modern computer vision and artificial intelligence processing to annotate and quantify examinations in a consistent and reproducible manner. The Myasthenia Gravis Core Examination (MG-CE) has been recommended for the telemedicine evaluation of patients with myasthenia gravis. Objective: We aimed to assess the ability to take accurate and robust measurements during the examination, which would allow improvement in workflow efficiency by making the data acquisition and analytics fully automatic and thereby limit the potential for observation bias. Methods: We used Zoom (Zoom Video Communications) videos of patients with myasthenia gravis undergoing the MG-CE. The core examination tests required 2 broad categories of processing. First, computer vision algorithms were used to analyze videos with a focus on eye or body motions. Second, for the assessment of examinations involving vocalization, a different category of signal processing methods was required. In this way, we provide an algorithm toolbox to assist clinicians with the MG-CE. We used a data set of 6 patients recorded during 2 sessions. Results: Digitalization and control of quality of the core examination are advantageous and let the medical examiner concentrate on the patient instead of managing the logistics of the test. This approach showed the possibility of standardized data acquisition during telehealth sessions and provided real-time feedback on the quality of the metrics the medical doctor is assessing. Overall, our new telehealth platform showed submillimeter accuracy for ptosis and eye motion. In addition, the method showed good results in monitoring muscle weakness, demonstrating that continuous analysis is likely superior to pre-exercise and post-exercise subjective assessment. Conclusions: We demonstrated the ability to objectively quantitate the MG-CE. Our results indicate that the MG-CE should be revisited to consider some of the new metrics that our algorithm identified. We provide a proof of concept involving the MG-CE, but the method and tools developed can be applied to many neurological disorders and have great potential to improve clinical care.

B Cells in Neuroinflammation: New Perspectives and Mechanistic Insights.

In recent years, the role of B cells in neurological disorders has substantially expanded our perspectives on mechanisms of neuroinflammation. The success of B cell-depleting therapies in patients with CNS diseases such as neuromyelitis optica and multiple sclerosis has highlighted the importance of neuroimmune crosstalk in inflammatory processes. While B cells are essential for the adaptive immune system and antibody production, they are also major contributors of pro- and anti-inflammatory cytokine responses in a number of inflammatory diseases. B cells can contribute to neurological diseases through peripheral immune mechanisms, including production of cytokines and antibodies, or through CNS mechanisms following compartmentalization. Emerging evidence suggests that aberrant pro- or anti-inflammatory B cell populations contribute to neurological processes, including glial activation, which has been implicated in the pathogenesis of several neurodegenerative diseases. In this review, we summarize recent findings on B cell involvement in neuroinflammatory diseases and discuss evidence to support pathogenic immunomodulatory functions of B cells in neurological disorders, highlighting the importance of B cell-directed therapies.

Neurological manifestations and complications of coronavirus disease 2019 (COVID-19): a systematic review and meta-analysis.

BACKGROUND: The spectrum of neurological involvement in COVID-19 is not thoroughly understood. To the best of our knowledge, no systematic review with meta-analysis and a sub-group comparison between severe and non-severe cases has been published. The aim of this study is to assess the frequency of neurological manifestations and complications, identify the neurodiagnostic findings, and compare these aspects between severe and non-severe COVID-19 cases. METHODS: A systematic search of PubMed, Scopus, EBSCO, Web of Science, and Google Scholar databases was conducted for studies published between the 1st of January 2020 and 22nd of April 2020. In addition, we scanned the bibliography of included studies to identify other potentially eligible studies. The criteria for eligibility included studies published in English language (or translated to English), those involving patients with COVID-19 of all age groups, and reporting neurological findings. Data were extracted from eligible studies. Meta-analyses were conducted using comprehensive meta-analysis software. Random-effects model was used to calculate the pooled percentages and means with their 95% confidence intervals (CIs). Sensitivity analysis was performed to assess the effect of individual studies on the summary estimate. A subgroup analysis was conducted according to severity. The main outcomes of the study were to identify the frequency and nature of neurological manifestations and complications, and the neuro-diagnostic findings in COVID-19 patients. RESULTS: 44 articles were included with a pooled sample size of 13,480 patients. The mean age was 50.3 years and 53% were males. The most common neurological manifestations were: Myalgia (22.2, 95% CI, 17.2 to 28.1%), taste impairment (19.6, 95% CI, 3.8 to 60.1%), smell impairment (18.3, 95% CI, 15.4 to 76.2%), headache (12.1, 95% CI, 9.1 to 15.8%), dizziness (11.3, 95% CI, 8.5 to 15.0%), and encephalopathy (9.4, 95% CI, 2.8 to 26.6%). Nearly 2.5% (95% CI, 1 to 6.1%) of patients had acute cerebrovascular diseases (CVD). Myalgia, elevated CK and LDH, and acute CVD were significantly more common in severe cases. Moreover, 20 case reports were assessed qualitatively, and their data presented separately. CONCLUSIONS: Neurological involvement is common in COVID-19 patients. Early recognition and vigilance of such involvement might impact their overall outcomes.

Astrocytes Are Required for Oligodendrocyte Survival and Maintenance of Myelin Compaction and Integrity.

Astrocytes have been implicated in regulating oligodendrocyte development and myelination in vitro, although their functions in vivo remain less well defined. Using a novel approach to locally ablate GFAP+ astrocytes, we demonstrate that astrocytes are required for normal CNS myelin compaction during development, and for maintaining myelin integrity in the adult. Transient ablation of GFAP+ astrocytes in the mouse spinal cord during the first postnatal week reduced the numbers of mature oligodendrocytes and inhibited myelin formation, while prolonged ablation resulted in myelin that lacked compaction and structural integrity. Ablation of GFAP+ astrocytes in the adult spinal cord resulted in the rapid, local loss of myelin integrity and regional demyelination. The loss of myelin integrity induced by astrocyte ablation was greatly reduced by NMDA receptor antagonists, both in vitro and in vivo, suggesting that myelin stability was affected by elevation of local glutamate levels following astrocyte ablation. Furthermore, targeted delivery of glutamate into adult spinal cord white matter resulted in reduction of myelin basic protein expression and localized disruption of myelin compaction which was also reduced by NMDA receptor blockade. The pathology induced by localized astrocyte loss and elevated exogenous glutamate, supports the concept that astrocytes are critical for maintenance of myelin integrity in the adult CNS and may be primary targets in the initiation of demyelinating diseases of the CNS, such as Neuromyelitis Optica (NMO).

Clinical Effects of the Self-administered Subcutaneous Complement Inhibitor Zilucoplan in Patients With Moderate to Severe Generalized Myasthenia Gravis: Results of a Phase 2 Randomized, Double-Blind, Placebo-Controlled, Multicenter Clinical Trial.

Importance: Many patients with generalized myasthenia gravis (gMG) have substantial clinical disability, persistent disease burden, and adverse effects attributable to chronic immunosuppression. Therefore, there is a significant need for targeted, well-tolerated therapies with the potential to improve disease control and enhance quality of life. Objective: To evaluate the clinical effects of zilucoplan, a subcutaneously (SC) self-administered macrocyclic peptide inhibitor of complement component 5, in a broad population of patients with moderate to severe gMG. Design, Setting, and Participants: This randomized, double-blind, placebo-controlled phase 2 clinical trial at 25 study sites across North America recruited participants between December 2017 and August 2018. Fifty-seven patients were screened, of whom 12 did not meet inclusion criteria and 1 was lost to follow-up after randomization but before receiving study drug, resulting in a total of 44 acetylcholine receptor autoantibody (AChR-Ab)-positive patients with gMG with baseline Quantitative Myasthenia Gravis (QMG) scores of at least 12, regardless of treatment history. Interventions: Patients were randomized 1:1:1 to a daily SC self-injection of placebo, 0.1-mg/kg zilucoplan, or 0.3-mg/kg zilucoplan for 12 weeks. Main Outcomes and Measures: The primary and key secondary end points were the change from baseline to week 12 in QMG and MG Activities of Daily Living scores, respectively. Significance testing was prespecified at a 1-sided α of .10. Safety and tolerability were also assessed. Results: The study of 44 patients was well balanced across the 3 treatment arms with respect to key demographic and disease-specific variables. The mean age of patients across all 3 treatment groups ranged from 45.5 to 54.6 years and most patients were white (average proportions across 3 treatment groups: 78.6%-86.7%). Clinically meaningful and statistically significant improvements in primary and key secondary efficacy end points were observed. Zilucoplan at a dose of 0.3 mg/kg SC daily resulted in a mean reduction from baseline of 6.0 points in the QMG score (placebo-corrected change, -2.8; P = .05) and 3.4 points in the MG Activities of Daily Living score (placebo-corrected change, -2.3; P = .04). Clinically meaningful and statistically significant improvements were also observed in other secondary end points, the MG Composite and MG Quality-of-Life scores. Outcomes for the 0.1-mg/kg SC daily dose were also statistically significant but slower in onset and less pronounced than with the 0.3-mg/kg dose. Rescue therapy (intravenous immunoglobulin or plasma exchange) was required in 3 of 15, 1 of 15, and 0 of 14 participants in the placebo, 0.1-mg/kg zilucoplan, and 0.3-mg/kg zilucoplan arms, respectively. Zilucoplan was observed to have a favorable safety and tolerability profile. Conclusions and Relevance: Zilucoplan yielded rapid, meaningful, and sustained improvements over 12 weeks in a broad population of patients with moderate to severe AChR-Ab-positive gMG. Near-complete complement inhibition appeared superior to submaximal inhibition. The observed safety and tolerability profile of zilucoplan was favorable. Trial Registration: ClinicalTrials.gov Identifier: NCT03315130.

Dropped head syndrome due to neuromuscular disorders: Clinical manifestation and evaluation.

In this article, we discuss the clinical approach to patients with dropped head syndrome and identify the various neuromuscular causes of dropped head syndrome including muscle, neuromuscular junction, peripheral nerve and motor neuron etiologies. We aim to increase awareness of recognition the entity of dropped head syndrome and factors that may predict response to immunomodulating therapy in dropped head syndrome.

Emerging Cellular and Molecular Strategies for Enhancing Central Nervous System (CNS) Remyelination.

Myelination is critical for the normal functioning of the central nervous system (CNS) in vertebrates. Conditions in which the development of myelin is perturbed result in severely compromised individuals often with shorter lifespans, while loss of myelin in the adult results in a variety of functional deficits. Although some form of spontaneous remyelination often takes place, the repair process as a whole often fails. Several lines of evidence suggest it is feasible to develop strategies that enhance the capacity of the CNS to undergo remyelination and potentially reverse functional deficits. Such strategies include cellular therapies using either neural or mesenchymal stem cells as well as molecular regulators of oligodendrocyte development and differentiation. Given the prevalence of demyelinating diseases and their effects on the quality of life for affected individuals it is imperative that effective therapies are developed. Here we discuss some of the new approaches to CNS myelin repair that hold promise for reducing the burden of diseases characterized by myelin loss.

Non-viral xylosyltransferase-1 siRNA delivery as an effective alternative to chondroitinase in an in vitro model of reactive astrocytes.

Reactive astrocytosis and the subsequent glial scar is ubiquitous to injuries of the central nervous system, especially spinal cord injury (SCI) and primarily serves to protect against further damage, but is also a prominent inhibitor of regeneration. Manipulating the glial scar by targeting chondroitin sulfate proteoglycans (CSPGs) has been the focus of much study as a means to improve axon regeneration and subsequently functional recovery. In this study we investigate the ability of small interfering RNA (siRNA) delivered by a non-viral polymer vector to silence the rate-limiting enzyme involved in CSPG synthesis. Gene expression of this enzyme, xylosyltransferase-1, was silenced by 65% in Neu7 astrocytes which conferred a reduced expression of CSPGs. Furthermore, conditioned medium taken from treated Neu7s, or co-culture experiments with dorsal root ganglia (DRG) showed that siRNA treatment resulted in a more permissive environment for DRG neurite outgrowth than treatment with chondroitinase ABC alone. These results indicate that there is a role for targeted siRNA therapy using polymeric vectors to facilitate regeneration of injured axons following central nervous system injury.

The neurotoxicity of gene vectors and its amelioration by packaging with collagen hollow spheres.

Over the last twenty years there have been several reports on the use of nonviral vectors to facilitate gene transfer in the mammalian brain. Whilst a large emphasis has been placed on vector transfection efficiency, the study of the adverse effects upon the brain, caused by the vectors themselves, remains completely overshadowed. To this end, a study was undertaken to study the tissue response to three commercially available transfection agents in the brain of adult Sprague Dawley rats. The response to these transfection agents was compared to adeno-associated viral vector (AAV), PBS and naked DNA. Furthermore, the use of a collagen hollow sphere (CHS) sustained delivery system was analysed for its ability to reduce striatal toxicity of the most predominantly studied polymer vector, polyethyleneimine (PEI). The size of the gross tissue loss at the injection site was analysed after immunohistochemical staining and was used as an indication of acute toxicity. Polymeric vectors showed similar levels of acute brain toxicity as seen with AAV, and CHS were able to significantly reduce the toxicity of the PEI vector. In addition; the host response to the vectors was measured in terms of reactive astrocytes and microglial cell recruitment. To understand whether this gross tissue loss was caused by the direct toxicity of the vectors themselves an in vitro study on primary astrocytes was conducted. All vectors reduced the viability of the cells which is brought about by direct necrosis and apoptosis. The CHS delivery system reduced cell necrosis in the early stages of post administration. In conclusion, whilst polymeric gene vectors cause acute necrosis, administration in the brain causes adverse effects no worse than that of an AAV vector. Furthermore, packaging the PEI vector with CHS reduces surface charge and direct toxicity without elevating the host response.

The effect of intraluminal contact mediated guidance signals on axonal mismatch during peripheral nerve repair.

The current microsurgical gold standard for repairing long gap nerve injuries is the autograft. Autograft provides a protective environment for repair and a natural internal architecture, which is essential for regeneration. Current clinically approved hollow nerve guidance conduits allow provision of this protective environment; however they fail to provide an essential internal architecture to the regenerating nerve. In the present study both structured and unstructured intraluminal collagen fibres are investigated to assess their ability to enhance conduit mediated nerve repair. This study presents a direct comparison of both structured and unstructured fibres in vivo. The addition of intraluminal guidance structures was shown to significantly decrease axonal dispersion within the conduit and reduced axonal mismatch of distal nerve targets (p < 0.05). The intraluminal fibres were shown to be successfully incorporated into the host regenerative process, acting as a platform for Schwann cell migration and axonal regeneration. Ultimately the fibres were able to provide a platform for nerve regeneration in a long term regeneration study (16 weeks) and facilitated increased guidance of regenerating axons towards their distal nerve targets.

Single vesicle imaging indicates distinct modes of rapid membrane retrieval during nerve growth.

BACKGROUND: During nerve growth, cytoplasmic vesicles add new membrane preferentially to the growth cone located at the distal tip of extending axons. Growth cone membrane is also retrieved locally, and asymmetric retrieval facilitates membrane remodeling during growth cone repulsion by a chemorepellent gradient. Moreover, growth inhibitory factors can stimulate bulk membrane retrieval and induce growth cone collapse. Despite these functional insights, the processes mediating local membrane remodeling during axon extension remain poorly defined. RESULTS: To investigate the spatial and temporal dynamics of membrane retrieval in actively extending growth cones, we have used a transient labeling and optical recording method that can resolve single vesicle events. Live-cell confocal imaging revealed rapid membrane retrieval by distinct endocytic modes based on spatial distribution in Xenopus spinal neuron growth cones. These modes include endocytic "hot-spots" triggered at the base of filopodia, at the lateral margins of lamellipodia, and along dorsal ridges of the growth cone. Additionally, waves of endocytosis were induced when individual filopodia detached from the substrate and fused with the growth cone dorsal surface or with other filopodia. Vesicle formation at sites of membrane remodeling by self-contact required F-actin polymerization. Moreover, bulk membrane retrieval by macroendocytosis correlated positively with the substrate-dependent rate of axon extension and required the function of Rho-family GTPases. CONCLUSIONS: This study provides insight into the dynamic membrane remodeling processes essential for nerve growth by identifying several distinct modes of rapid membrane retrieval in the growth cone during axon extension. We found that endocytic membrane retrieval is intensified at specific subdomains and may drive the dynamic membrane ruffling and re-absorption of filopodia and lamellipodia in actively extending growth cones. The findings offer a platform for determining the molecular mechanisms of distinct endocytic processes that may remodel the surface distribution of receptors, ion channels and other membrane-associated proteins locally to drive growth cone extension and chemotactic guidance.

Single cyclized molecule versus single branched molecule: a simple and efficient 3D "knot" polymer structure for nonviral gene delivery.

The large research effort focused on enhancing nonviral transfection vectors has clearly demonstrated that their macromolecular structure has a significant effect on their transfection efficacy. The 3D branched polymeric structures, such as dendrimers, have proved to be a very effective structure for polymeric transfection vectors; however, so far the dendritic polymers have not delivered on their promise. This is largely because a wide range of dendritic polymer materials with tailored multifunctional properties and biocompatibility required for such applications are not yet accessible by current routes. Herein, we report the design and synthesis of new 3D "Single Cyclized" polymeric gene vectors with well-defined compositions and functionalities via a one-step synthesis from readily available vinyl monomers. We observe that this polymer structure of a single chain linked to itself interacts differently with plasmid DNA compared to conventional vectors and when tested over a range of cell types, has a superior transfection profile in terms of both luciferase transfection capability and preservation of cell viability. This new knotted structure shows high potential for gene delivery applications through a combination of simplicity in synthesis, scalability, and high performance.

Asymmetric endocytosis and remodeling of beta1-integrin adhesions during growth cone chemorepulsion by MAG.

Gradients of chemorepellent factors released from myelin may impair axon pathfinding and neuroregeneration after injury. We found that, analogously to the process of chemotaxis in invasive tumor cells, axonal growth cones of Xenopus spinal neurons modulate the functional distribution of integrin receptors during chemorepulsion induced by myelin-associated glycoprotein (MAG). A focal MAG gradient induced polarized endocytosis and concomitant asymmetric loss of beta(1)-integrin and vinculin-containing adhesions on the repellent side during repulsive turning. Loss of symmetrical beta(1)-integrin function was both necessary and sufficient for chemorepulsion, which required internalization by clathrin-mediated endocytosis. Induction of repulsive Ca(2+) signals was necessary and sufficient for the stimulated rapid endocytosis of beta(1)-integrin. Altogether, these findings identify beta(1)-integrin as an important functional cargo during Ca(2+)-dependent rapid endocytosis stimulated by a diffusible guidance cue. Such dynamic redistribution allows the growth cone to rapidly adjust adhesiveness across its axis, an essential feature for initiating chemotactic turning.

Spinal cord injury in vitro: modelling axon growth inhibition.

Over the past three decades, tremendous progress has been made in elucidating mechanisms underlying regenerative failure after spinal cord injury and in devising therapeutic approaches to promote functional nerve regeneration. Various in vitro assays have been developed using brain and/or spinal cord neuronal cells to study axon growth in conditions that represent the post-injury environment. This review outlines the current models used to dissect, analyze and manipulate specific aspects of spinal cord injury leading to axon growth inhibition.

Essential modification of the Sircol Collagen Assay for the accurate quantification of collagen content in complex protein solutions.

Collagen contains the unique imino acid hydroxyproline (HyPro), which is involved in the stabilization of this triple helical molecule. The concentration of HyPro is customarily used to calculate the total collagen content in a cell culture environment and in acid hydrolysates of normal and pathophysiological tissues. Radiolabelling, chromatographic and calorimetric assays have been developed over the years for the accurate determination of collagen content through HyPro estimation. Recently, the Sircol Collagen Assay (SCA) has been almost exclusively adopted as the fastest and simplest colorimetric method for the determination of collagen concentration in complex protein solutions. We show here that the colorimetric SCA, which is based on the binding of Sirius red (SR) to collagen, is flawed by interference of non-collagenous proteins (e.g. serum). In fact, we demonstrate that SCA in cell culture systems and tissue hydrolysates results in a dramatic overestimation of collagen content ranging from 3- to 24-fold. In order to rescue this otherwise very practical assay, we introduce a simple purification procedure that allows the removal of interfering non-collagenous proteins from culture media and tissue samples so that accurate measurements with SCA are now possible.