Systemic cytokine and viral antigen-specific responses in hepatitis D virus RNA positive versus HDV RNA negative patients.

Background: Hepatitis B virus (HBV)/Hepatitis D Virus (HDV) co-infection increases the risk of severe liver disease compared to HBV mono-infection. Adaptive immune responses to HDV are weakly detectable, and the involvement of innate immunity in the progression of HDV-related liver fibrosis is suggested. We hypothesize that an overall innate immune activation in HBV/HDV co-infection plays a role in liver disease progression and also impacts virus specific T cell response. Methods: Sixteen HBV/HDV-co-infected-patients (median age 42y/7F/6 Asian/4 White/6 Black/15 HBeAg-) and 8 HBV monoinfected-patients (median age 39y/4F/4 Asian/3 Black/1 White/HBeAg-) with median follow-up of 5 years were enrolled. Liver fibrosis was assessed by liver stiffness measurement (LSM, FibroScan®). Proliferation of CD3 + CD4+ T cells in response to viral antigens using CFSE assays and cytokine secreting monocytes was analyzed by flow cytometry. Results: Of 16 HBV/HDV, 11 were HDV-RNA+ (HBV-DNA 0-1,040 IU/mL), 5/11 Interferon (IFN) + Nucleos/tide Analog (NA), 3/11 NA monotherapy, median ALT 77 U/L at the time of sample collection, median LSM of 9.8. In 5 HDV RNA-, median HBV DNA 65 IU/mL, 4/5 prior IFN and/or NA, ALT 31 U/L, and median LSM 8.5 kPa. In 8 HBV controls, median HBV-DNA, ALT, LSM was 69 IU/mL, 33 U/L,5 kPa, respectively. PBMC stimulation with HBV core antigen (HBcAg) and HDV antigen (HDAg) showed weaker CD3 + CD4 + T-cell proliferation in HDV-RNA+ vs. HDV RNA- and HBV-mono-infected patients (p < 0.05). In HDV-RNA+ patients, a correlation between ALT and TNF-α (r = 0.76, p = 0.008), higher IL-10 levels and increased proportion of CD14 + TNF-α+ cells were found. Conclusion: In summary, during HBV/HDV coinfection, HDV RNA+ patients had weaker HBV and HDV specific responses, associated with increased TNF-α + monocytes irrespective of IFN treatment.

Hepatitis B virus serum RNA transcript isoform composition and proportion in chronic hepatitis B patients by nanopore long-read sequencing.

Introduction: Serum hepatitis B virus (HBV) RNA is a promising new biomarker to manage and predict clinical outcomes of chronic hepatitis B (CHB) infection. However, the HBV serum transcriptome within encapsidated particles, which is the biomarker analyte measured in serum, remains poorly characterized. This study aimed to evaluate serum HBV RNA transcript composition and proportionality by PCR-cDNA nanopore sequencing of samples from CHB patients having varied HBV genotype (gt, A to F) and HBeAg status. Methods: Longitudinal specimens from 3 individuals during and following pregnancy (approximately 7 months between time points) were also investigated. HBV RNA extracted from 16 serum samples obtained from 13 patients (73.3% female, 84.6% Asian) was sequenced and serum HBV RNA isoform detection and quantification were performed using three bioinformatic workflows; FLAIR, RATTLE, and a GraphMap-based workflow within the Galaxy application. A spike-in RNA variant (SIRV) control mix was used to assess run quality and coverage. The proportionality of transcript isoforms was based on total HBV reads determined by each workflow. Results: All chosen isoform detection workflows showed high agreement in transcript proportionality and composition for most samples. HBV pregenomic RNA (pgRNA) was the most frequently observed transcript isoform (93.8% of patient samples), while other detected transcripts included pgRNA spliced variants, 3' truncated variants and HBx mRNA, depending on the isoform detection method. Spliced variants of pgRNA were primarily observed in HBV gtB, C, E, or F-infected patients, with the Sp1 spliced variant detected most frequently. Twelve other pgRNA spliced variant transcripts were identified, including 3 previously unidentified transcripts, although spliced isoform identification was very dependent on the workflow used to analyze sequence data. Longitudinal sampling among pregnant and post-partum antiviral-treated individuals showed increasing proportions of 3' truncated pgRNA variants over time. Conclusions: This study demonstrated long-read sequencing as a promising tool for the characterization of the serum HBV transcriptome. However, further studies are needed to better understand how serum HBV RNA isoform type and proportion are linked to CHB disease progression and antiviral treatment response.

Phenytoin-induced dyskinesia: a case report.

BACKGROUND: Dyskinesia is a movement disorder categorized by involuntary movement of muscle. Although dyskinesia can be brought on by taking medications, it can also be a symptom of a variety of diseases. Antiepileptic drug-induced involuntary movements have been well researched. Rare reports have been made for dyskinesia, a type of dystonia caused by phenytoin. The mechanism of its occurrence must be succinctly studied. CASE PRESENTATION: A 53-year-old Asian patient taking phenytoin (100 mg twice daily) experienced symptoms of perioral muscle involuntary movement, impaired speech, and generalized tremors and was admitted to the hospital. Brain magnetic resonance imaging showed significant development of encephalomalacia and porencephaly. The serum phenytoin levels were in the toxic range (33 g/ml). These were suggestive of phenytoin-induced dyskinesia. Levetiracetam and clonazepam were initiated, and the patient showed significant improvement in the symptoms. CONCLUSION: This case presented a substantial reference value for the differential diagnosis and treatment prognosis of phenytoin-induced dyskinesia. The phenytoin-induced dyskinesia in this patient was successfully reversed with prompt identification and treatment. According to the case study's findings, such people may benefit from periodic therapeutic drug monitoring.

Increasing Signal Intensity of Fluorescent Oligo-Labeled Antibodies.

While full-spectrum flow cytometry has increased antibody-based multiplexing, yet further increases remain potentially impactful. We recently proposed how fluorescence Multiplexing using Spectral Imaging and Combinatorics (MuSIC) could do so using tandem dyes and an oligo-based antibody labeling method. In this work, we found that such labeled antibodies had significantly lower signal intensity than conventionally-labeled antibodies in human cell experiments. To improve signal intensity, we tested moving the fluorophores from the original external (ext.) 5' or 3' end-labeled orientation to internal (int.) fluorophore modifications. Cell-free spectrophotometer measurements showed a ~6-fold signal intensity increase of the new int. configuration compared to the previous ext. configuration. Time-resolved fluorescence spectroscopy and fluorescence correlation spectroscopy showed that ~3-fold brightness difference is due to static quenching. Spectral flow cytometry experiments using peripheral blood mononuclear cells stained with anti-CD8 antibodies showed that int. MuSIC probe-labeled antibodies have signal intensity equal to or greater than conventionally-labeled antibodies with similar estimated proportion of CD8+ lymphocytes. The antibody labeling approach is general and can be broadly applied to many biological and diagnostic applications.

Theory for High-Throughput Genetic Interaction Screening.

Systematic, genome-scale genetic screens have been instrumental for elucidating genotype-phenotype relationships, but approaches for probing genetic interactions have been limited to at most ∼100 pre-selected gene combinations in mammalian cells. Here, we introduce a theory for high-throughput genetic interaction screens. The theory extends our recently developed Multiplexing using Spectral Imaging and Combinatorics (MuSIC) approach to propose ∼105 spectrally unique, genetically encoded MuSIC barcodes from 18 currently available fluorescent proteins. Simulation studies based on constraints imposed by spectral flow cytometry equipment suggest that genetic interaction screens at the human genome-scale may be possible if MuSIC barcodes can be paired to guide RNAs. While experimental testing of this theory awaits, it offers transformative potential for genetic perturbation technology and knowledge of genetic function. More broadly, the availability of a genome-scale spectral barcode library for non-destructive identification of single cells could find more widespread applications such as traditional genetic screening and high-dimensional lineage tracing.

HeartMate 3 Explantation Using Felt Plug for Ventriculotomy Occlusion.

Left ventricular assist devices (LVAD) can be utilized for heart failure patients as a bridge to transplant, bridge to destination, or bridge to recovery. Given the lack of a universally accepted consensus for assessing myocardial recovery, techniques and strategies in LVAD explantation also vary. In addition, the incidence of LVAD explantation remains relatively low, and surgical techniques of explantation continue to be areas of interest. Our approach using a felt-plug Dacron technique is an effective way to preserve left ventricular geometry and cardiac function.

Reduction of left ventricular global longitudinal strain in patients with permanent pacemakers as a predictor of heart failure and mortality outcomes.

INTRODUCTION: Frequent right ventricular (RV) pacing is associated with cardiomyopathy. The impact of RV pacing on left ventricular (LV) global longitudinal strain (GLS) and clinical outcomes is unclear. METHODS: We analyzed GLS via two-dimensional speckle tracking and LV ejection fraction (EF) on pre- and post-implantation transthoracic echocardiograms of patients undergoing dual chamber pacemaker implantation. We collected long-term data on strain, LVEF, and clinical outcomes. RESULTS: One hundred and ten patients (mean age 76 ± 12 years; 59 [54%] female) were followed for mean 23 ± 17 months. Mean baseline LVEF was 58 ± 11% and mean GLS was -17 ± 4%. Twenty-four (22%) patients had an absolute decrease in LVEF > 10% and 43 (39%) patients had a relative reduction of GLS > 15%. Among patients with a reduction of GLS, a larger proportion of patients had RV pacing burden ≥20% (67% vs. 46%; p = .048). Compared to patients without GLS reduction, more patients with a reduction in GLS reached a composite endpoint of HF hospitalization, CRT upgrade or death (47% vs. 16%; p = .001). CONCLUSION: Reduction in LV GLS was seen in nearly four in 10 patients undergoing pacemaker implantation and was significantly associated with increased RV pacing burden. LV GLS reduction was associated with increased risk of adverse outcomes. LV GLS may have utility in predicting outcomes among patients with RV pacing.

Chemoselective Oxidation of Thiols with Oxoammonium Cations.

The oxidation of various aryl and aliphatic thiols with the commercially available and environmentally benign reagent Bobbitt's salt (1) has been investigated. The reaction affords the corresponding disulfide products in good to excellent yields (71-99%) and can be accomplished in water, methanol, or acetonitrile solvent. Moreover, the process is highly chemoselective, tolerating traditionally oxidation-labile groups such as free amines and alcohols. Combined experimental and computational studies reveal that the oxidation takes place via a polar two-electron process with concomitant and unexpected deoxygenation of the oxoammonium cation through homolysis of the weak N-O bond, differing from prototypical radical-based thiol couplings. This unusual consumption of the oxidant has significant implications for the development of new nitroxide-based radical traps for probing S-centered radicals, the advancement of new electrochemical or catalytic processes involving nitroxide/oxoammonium salt redox couples, and applications to biological systems.

Neurocognitive Profiles of 22q11.2 and 16p11.2 Deletions and Duplications.

Rare recurrent copy number variants (CNVs) at chromosomal loci 22q11.2 and 16p11.2 are among the most common rare genetic disorders associated with significant risk for neuropsychiatric disorders across the lifespan. Microdeletions and duplications in these loci are associated with neurocognitive deficits, yet there are few studies comparing these groups using the same measures. We address this gap in a prospective international collaboration applying the same computerized neurocognitive assessment. The Penn Computerized Neurocognitive Battery (CNB) was administered in a multi-site study on rare genomic disorders: 22q11.2 deletion (n = 397); 22q11.2 duplication (n = 77); 16p11.2 deletion (n = 94); and 16p11.2 duplication (n = 26). Domains examined include executive functions, episodic memory, complex cognition, social cognition, and sensori-motor speed. Accuracy and speed for each neurocognitive domain were included as dependent measures in a mixed-model repeated measures analysis, with locus (22q11.2, 16p11.2) and copy number (deletion/duplication) as grouping factors and neurocognitive domain as a repeated measures factor, with age and sex as covariates. We also examined correlation with IQ and site effects. We found that 22q11.2 deletions were associated with greater deficits in overall performance accuracy than 22q11.2 duplications, while 16p11.2 duplications were associated with greater deficits than 16p11.2 deletions. Duplications at both loci were associated with reduced speed. Performance profiles differed among the groups with particularly poor performance of 16p11.2 duplication on non-verbal reasoning and social cognition. Average accuracy on the CNB was moderately correlated with Full Scale IQ. No site effects were observed. Deletions and duplications of 22q11.2 and 16p11.2 have varied effects on neurocognition indicating locus specificity, with performance profiles differing among the groups. These profile differences can help inform mechanistic substrates to heterogeneity in presentation and outcome. Future studies could aim to link performance profiles to clinical features and brain function.

Ribavirin Does Not Enhance Hepatitis B Virus Nucleotide Antiviral Activity: A Pilot Study.

PURPOSE: There is a need for effective and affordable treatments that achieve hepatitis B virus (HBV) functional cure and prevent long-term complications. The use of immune-modulators combined with HBV antivirals is a promising therapeutic strategy to achieve these goals. Based on ribavirin (RBV) monotherapy data, we hypothesized that RBV could improve virological responses when used in combination with tenofovir.  Methods: In this randomized, open label, controlled pilot trial, we evaluated RBV (n=4) dosed for the initial 24 weeks of treatment versus no RBV (n=4) in tenofovir recipients dosed over 48 weeks.  Results: Although well tolerated and safe in combination with tenofovir, RBV demonstrated no beneficial effects on virologic, biochemical or immunological markers of chronic HBV infection over 48 weeks of serial evaluation.  Conclusions: Our data does not suggest a HBV-specific immunomodulatory effect or an impact of RBV on HBV virological and antigen suppression.

Jaundice on Presentation Is Associated with Higher In-Patient Mortality and Complications in Patients Admitted for Acute Pancreatitis: A Retrospective Study Based on National Inpatient Sample Database.

Pancreatitis usually presents with characteristic abdominal pain, radiological findings, and elevated lipase. The presence of jaundice may hint at a biliary etiology; however, it is not always present. We hypothesized that the presence of jaundice is associated with worse outcomes in patients admitted with pancreatitis. We conducted a retrospective analysis using the National Inpatient Sample, inquiring about patients admitted with pancreatitis with and without jaundice between October 2015 and December 2017. The primary outcome was in-hospital mortality in patients admitted for pancreatitis with and without jaundice. Secondary outcomes were the median length of stay, hospitalization cost, the incidence of ventilator-dependent respiratory failure (VDRF), acute respiratory distress syndrome (ARDS), sepsis, septic shock, dehydration and electrolyte disturbances, and ascites. A total of 1,267,744 patients were admitted with pancreatitis from October 2015 to December 2017. Among them, 8855 (0.7%) had concomitant jaundice on presentation. In-hospital mortality in this group was 4.3%. The patients with pancreatitis and jaundice had higher odds of in-hospital mortality (adjusted odds ratio [aOR]: 1.51, 99% CI 1.35-1.68, p < 0.0001) as compared to those without jaundice. Patients with jaundice showed a significantly higher incidence of sepsis (15.2% vs. 9.6%, p < 0.0001), septic shock (4.1% vs. 2.9%, p < 0.0001), ascites (6.5% vs. 3.1%, p < 0.0001), and dehydration and electrolyte disorders (47.6% vs. 43.8%, p < 0.0001). Patients with jaundice also had higher total hospital costs ($11,412 vs. $7893, p < 0.0001). There was no statistical difference in ARDS, VDRF, and median length of stay. In conclusion, patients admitted for pancreatitis with jaundice had worse outcomes, including in-hospital mortality and complications, compared to those without jaundice.

Effectiveness and Safety Profile of Remote Pulmonary Artery Hemodynamic Monitoring in a "Real-World" Veterans Affairs Healthcare System.

Ambulatory hemodynamic monitoring has demonstrated the ability to reduce heart failure-related hospitalization, regardless of left ventricular ejection fraction; however, real-world data in a Veterans Affairs patient population are limited. The present study retrospectively reviewed 53 patients with New York Heart Association class III heart failure, regardless of left ventricular ejection fraction, implanted with a pulmonary artery pressure sensor (CardioMEMS) at our single Veterans Affairs institution. Heart failure-related hospitalizations were assessed in patients for 6 and 12 months after the implantation of the device and compared with the 6- and 12- month periods before implantation in the same patient cohort. Pulmonary arterial pressures and medication doses were also recorded at baseline, 6- months, and 12- months and procedural safety data were also assessed. Implantation of a remote pulmonary artery pressure sensor was associated with a 52% (95% confidence interval 30% to 68%, p <0.001) and a 44% (95% confidence interval 24% to 59%, p <0.001) reduction in heart failure-related hospitalization at 6 and 12 months after implant, respectively, compared with the 6- and 12-month preimplant periods. Mean pulmonary arterial pressures also demonstrated significant reductions from baseline to 6 and 12 months after implant. A total of 3 procedure-related adverse events were noted. In conclusion, pulmonary artery pressure sensor implantation is relatively safe and associated with significant reductions in heart failure-related hospitalization and decreased mean pulmonary artery pressures in patients within the Veterans Affairs system with New York Heart Association class III symptoms, regardless of ejection fraction.

Nationwide retrospective study of hepatitis B virological response and liver stiffness improvement in 465 patients on nucleos(t)ide analogue.

BACKGROUND: Hepatitis B virus (HBV) nucleos(t)ide analog (NA) therapy reduces liver disease but requires prolonged therapy to achieve hepatitis B surface antigen (HBsAg) loss. There is limited North American real-world data using non-invasive tools for fibrosis assessment and few have compared 1st generation NA or lamivudine (LAM) to tenofovir disoproxil fumarate (TDF). AIM: To assess impact of NA on virological response and fibrosis regression using liver stiffness measurement (LSM) (i.e., FibroScan®). METHODS: Retrospective, observational cohort study from the Canadian HBV Network. Data collected included demographics, NA, HBV DNA, alanine aminotransferase (ALT), and LSM. Patients were HBV monoinfected patients, treatment naïve, and received 1 NA with minimum 1 year follow-up. RESULTS: In 465 (median 49 years, 37% female, 35% hepatitis B e antigen+ at baseline, 84% Asian, 6% White, and 9% Black). Percentage of 64 (n = 299) received TDF and 166 were LAM-treated with similar median duration of 3.9 and 3.7 years, respectively. The mean baseline LSM was 11.2 kPa (TDF) vs 8.3 kPa (LAM) (P = 0.003). At 5-year follow-up, the mean LSM was 7.0 kPa in TDF vs 6.7 kPa in LAM (P = 0.83). There was a significant difference in fibrosis regression between groups (i.e., mean -4.2 kPa change in TDF and -1.6 kPa in LAM, P < 0.05). The last available data on treatment showed that all had normal ALT, but more TDF patients were virologically suppressed (< 10 IU/mL) (n = 170/190, 89%) vs LAM-treated (n = 35/58, 60%) (P < 0.05). None cleared HBsAg. CONCLUSION: In this real-world North American study, approximately 5 years of NA achieves liver fibrosis regression rarely leads to HBsAg loss.

Artificial Intelligence, Healthcare, Clinical Genomics, and Pharmacogenomics Approaches in Precision Medicine.

Precision medicine has greatly aided in improving health outcomes using earlier diagnosis and better prognosis for chronic diseases. It makes use of clinical data associated with the patient as well as their multi-omics/genomic data to reach a conclusion regarding how a physician should proceed with a specific treatment. Compared to the symptom-driven approach in medicine, precision medicine considers the critical fact that all patients do not react to the same treatment or medication in the same way. When considering the intersection of traditionally distinct arenas of medicine, that is, artificial intelligence, healthcare, clinical genomics, and pharmacogenomics-what ties them together is their impact on the development of precision medicine as a field and how they each contribute to patient-specific, rather than symptom-specific patient outcomes. This study discusses the impact and integration of these different fields in the scope of precision medicine and how they can be used in preventing and predicting acute or chronic diseases. Additionally, this study also discusses the advantages as well as the current challenges associated with artificial intelligence, healthcare, clinical genomics, and pharmacogenomics.

Fusion of Bovine Tissue Aortic Valve Leaflets in a Patient With Left Ventricular Assist Device.

Patients with both a prosthetic aortic valve and prolonged left ventricular assist device support can develop rapid deterioration of their valve prosthesis. In patients with myocardial recovery who are undergoing explantation of their ventricular assist device, preoperative and intraoperative evaluation of the valve prosthesis should be performed to ensure adequate function. (Level of Difficulty: Advanced.).

Arrhythmias and Intraventricular Conduction Disturbances in Patients Hospitalized With Coronavirus Disease 2019.

Cardiac arrhythmias have been observed in patients hospitalized with coronavirus disease (COVID-19). Most analyses of rhythm disturbances to date include cases of sinus tachycardia, which may not accurately reflect true cardiac dysfunction. Furthermore, limited data exist regarding the development of conduction disturbances in patients hospitalized with COVID-19. Hence, we performed a retrospective review and compared characteristics and outcomes for patients with versus without incident arrhythmia, excluding sinus tachycardia, as well as between those with versus without incident conduction disturbances. There were 27 of 173 patients (16%) hospitalized with COVID-19 who developed a new arrhythmia. Incident arrhythmias were associated with an increased risk of intensive care unit admission (59% vs 31%, p = 0.0045), intubation (56% vs 20%, p <0.0001), and inpatient death (41% vs 10%, p = 0.0002) without an associated increase in risk of decompensated heart failure or other cardiac issues. New conduction disturbances were found in 13 patients (8%). Incident arrhythmias in patients hospitalized with COVID-19 are associated with an increased risk of mortality, likely reflective of underlying COVID-19 disease severity more than intrinsic cardiac dysfunction. Conduction disturbances occurred less commonly and were not associated with adverse patient outcomes.

Building the Sierra Leone Ebola Database: organization and characteristics of data systematically collected during 2014-2015 Ebola epidemic.

PURPOSE: During the 2014-2016 Ebola outbreak in West Africa, the Sierra Leone Ministry of Health and Sanitation (MoHS), the US Centers for Disease Control and Prevention, and responding partners under the coordination of the National Ebola Response Center (NERC) and the MoHS's Emergency Operation Center (EOC) systematically recorded information from the 117 Call Center system and district alert phone lines, case investigations, laboratory sample testing, clinical management, and safe and dignified burial records. Since 2017, CDC assisted MoHS in building and managing the Sierra Leone Ebola Database (SLED) to consolidate these major data sources. The primary objectives of the project were helping families to identify the location of graves of their loved ones who died at the time of the Ebola epidemic through the SLED Family Reunification Program and creating a data source for epidemiological research. The objective of this paper is to describe the process of consolidating epidemic records into a useful and accessible data collection and to summarize data characteristics, strength, and limitations of this unique information source for public health research. METHODS: Because of the unprecedented conditions during the epidemic, most of the records collected from responding organizations required extensive processing before they could be used as a data source for research or the humanitarian purpose of locating burial sites. This process required understanding how the data were collected and used during the outbreak. To manage the complexity of processing the data obtained from various sources, the Sierra Leone Ebola Database (SLED) Team used an organizational strategy that allowed tracking of the data provenance and lifecycle. RESULTS: The SLED project brought raw data into one consolidated data collection. It provides researchers with secure and ethical access to the SLED data and serves as a basis for the research capacity building in Sierra Leone. The SLED Family Reunification Program allowed Sierra Leonean families to identify location of the graves of loved ones who died during the Ebola epidemic. CONCLUSIONS: The SLED project consolidated and utilized epidemic data recorded during the Sierra Leone Ebola Virus Disease outbreak that were collected and contributed to SLED by national and international organizations. This project has provided a foundation for developing a method of ethical and secure SLED data access while preserving the host nation's data ownership. SLED serves as a data source for the SLED Family Reunification Program and for epidemiological research. It presents an opportunity for building research capacity in Sierra Leone and provides a foundation for developing a relational database. Large outbreak data systems such as SLED provide a unique opportunity for researchers to improve responses to epidemics and indicate the need to include data management preparedness in the plans for emergency response.

Optimizing Dosing of Vagus Nerve Stimulation for Stroke Recovery.

Vagus nerve stimulation (VNS) paired with rehabilitative training enhances recovery of function in models of stroke and is currently under investigation for use in chronic stroke patients. Dosing is critical in translation of pharmacological therapies, but electrical stimulation therapies often fail to comprehensively explore dosing parameters in preclinical studies. Varying VNS parameters has non-monotonic effects on plasticity in the central nervous system, which may directly impact efficacy for stroke. We sought to optimize stimulation intensity to maximize recovery of motor function in a model of ischemic stroke. The study design was preregistered prior to beginning data collection (DOI: https://doi.org/10.17605/OSF.IO/BMJEK ). After training on an automated assessment of forelimb function and receiving an ischemic lesion in motor cortex, rats were separated into groups that received rehabilitative training paired with VNS at distinct stimulation intensities (sham, 0.4 mA, 0.8 mA, or 1.6 mA). Moderate-intensity VNS at 0.8 mA enhanced recovery of function compared with all other groups. Neither 0.4 mA nor 1.6 mA VNS was sufficient to improve functional recovery compared with equivalent rehabilitation without VNS. These results demonstrate that moderate-intensity VNS delivered during rehabilitation improves recovery and defines an optimized intensity paradigm for clinical implementation of VNS therapy.