Rejuvenating fecal microbiota transplant enhances peripheral nerve repair in aged mice by modulating endoneurial inflammation.

Peripheral nerve injury (PNI) resulting from trauma or neuropathies can cause significant disability, and its prognosis deteriorates with age. Emerging evidence suggests that gut dysbiosis and reduced fecal short-chain fatty acids (SCFAs) contribute to an age-related systemic hyperinflammation (inflammaging), which hinders nerve recovery after injury. This study thus aimed to evaluate the pro-regenerative effects of a rejuvenating fecal microbiota transplant (FMT) in a preclinical PNI model using aged mice. Aged C57BL/6 mice underwent bilateral crush injuries to their sciatic nerves. Subsequently, they either received FMT from young donors at three and four days after the injury or retained their aged gut microbiota. We analyzed gut microbiome composition and SCFA concentrations in fecal samples. The integrity of the ileac mucosal barrier was assessed by immunofluorescence staining of Claudin-1. Flow cytometry was utilized to examine immune cells and cytokine production in the ileum, spleen, and sciatic nerve. Various assessments, including behavioural tests, electrophysiological studies, and morphometrical analyses, were conducted to evaluate peripheral nerve function and repair following injury. Rejuvenating FMT reversed age-related gut dysbiosis by increasing Actinobacteria, especially Bifidobacteriales genera. This intervention also led to an elevation of gut SCFA levels and mitigated age-related ileac mucosal leakiness in aged recipients. Additionally, it augmented the number of T-helper 2 (Th2) and regulatory T (Treg) cells in the ileum and spleen, with the majority being positive for anti-inflammatory interleukin-10 (IL-10). In sciatic nerves, rejuvenating FMT resulted in increased M2 macrophage counts and a higher IL-10 production by IL-10+TNF-α- M2 macrophage subsets. Ultimately, restoring a youthful gut microbiome in aged mice led to improved nerve repair and enhanced functional recovery after PNI. Considering that FMT is already a clinically available technique, exploring novel translational strategies targeting the gut microbiome to enhance nerve repair in the elderly seems promising and warrants further evaluation.

Blockade of Rho-associated kinase prevents inhibition of axon regeneration of peripheral nerves induced by anti-ganglioside antibodies.

Anti-ganglioside antibodies are associated with delayed/poor clinical recovery in Guillain-Barrè syndrome, mostly related to halted axon regeneration. Cross-linking of cell surface gangliosides by anti-ganglioside antibodies triggers inhibition of nerve repair in in vitro and in vivo paradigms of axon regeneration. These effects involve the activation of the small GTPase RhoA/ROCK signaling pathways, which negatively modulate growth cone cytoskeleton, similarly to well stablished inhibitors of axon regeneration described so far. The aim of this work was to perform a proof of concept study to demonstrate the effectiveness of Y-27632, a selective pharmacological inhibitor of ROCK, in a mouse model of axon regeneration of peripheral nerves, where the passive immunization with a monoclonal antibody targeting gangliosides GD1a and GT1b was previously reported to exert a potent inhibitory effect on regeneration of both myelinated and unmyelinated fibers. Our results demonstrate a differential sensitivity of myelinated and unmyelinated axons to the pro-regenerative effect of Y-27632. Treatment with a total dosage of 9 mg/kg of Y-27632 resulted in a complete prevention of anti-GD1a/GT1b monoclonal antibody-mediated inhibition of axon regeneration of unmyelinated fibers to skin and the functional recovery of mechanical cutaneous sensitivity. In contrast, the same dose showed toxic effects on the regeneration of myelinated fibers. Interestingly, scale down of the dosage of Y-27632 to 5 mg/kg resulted in a significant although not complete recovery of regenerated myelinated axons exposed to anti-GD1a/GT1b monoclonal antibody in the absence of toxicity in animals exposed to only Y-27632. Overall, these findings confirm the in vivo participation of RhoA/ROCK signaling pathways in the molecular mechanisms associated with the inhibition of axon regeneration induced by anti-GD1a/GT1b monoclonal antibody. Our findings open the possibility of therapeutic pharmacological intervention targeting RhoA/Rock pathway in immune neuropathies associated with the presence of anti-ganglioside antibodies and delayed or incomplete clinical recovery after injury in the peripheral nervous system.

Monoallelic variation in DHX9, the gene encoding the DExH-box helicase DHX9, underlies neurodevelopment disorders and Charcot-Marie-Tooth disease.

DExD/H-box RNA helicases (DDX/DHX) are encoded by a large paralogous gene family; in a subset of these human helicase genes, pathogenic variation causes neurodevelopmental disorder (NDD) traits and cancer. DHX9 encodes a BRCA1-interacting nuclear helicase regulating transcription, R-loops, and homologous recombination and exhibits the highest mutational constraint of all DDX/DHX paralogs but remains unassociated with disease traits in OMIM. Using exome sequencing and family-based rare-variant analyses, we identified 20 individuals with de novo, ultra-rare, heterozygous missense or loss-of-function (LoF) DHX9 variant alleles. Phenotypes ranged from NDDs to the distal symmetric polyneuropathy axonal Charcot-Marie-Tooth disease (CMT2). Quantitative Human Phenotype Ontology (HPO) analysis demonstrated genotype-phenotype correlations with LoF variants causing mild NDD phenotypes and nuclear localization signal (NLS) missense variants causing severe NDD. We investigated DHX9 variant-associated cellular phenotypes in human cell lines. Whereas wild-type DHX9 was restricted to the nucleus, NLS missense variants abnormally accumulated in the cytoplasm. Fibroblasts from an individual with an NLS variant also showed abnormal cytoplasmic DHX9 accumulation. CMT2-associated missense variants caused aberrant nucleolar DHX9 accumulation, a phenomenon previously associated with cellular stress. Two NDD-associated variants, p.Gly411Glu and p.Arg761Gln, altered DHX9 ATPase activity. The severe NDD-associated variant p.Arg141Gln did not affect DHX9 localization but instead increased R-loop levels and double-stranded DNA breaks. Dhx9-/- mice exhibited hypoactivity in novel environments, tremor, and sensorineural hearing loss. All together, these results establish DHX9 as a critical regulator of mammalian neurodevelopment and neuronal homeostasis.

COVID-19 Outcomes in Myasthenia Gravis Patients: Analysis From Electronic Health Records in the United States.

Background: Myasthenia gravis (MG) is an autoimmune, neuromuscular condition and patients with MG are vulnerable due to immunosuppressant use and disease manifestations of dyspnea and dysphagia during the coronavirus disease 2019 (COVID-19) pandemic. Methods: We conducted a retrospective cohort study using the Optum® de-identified COVID-19 Electronic Health Record (EHR) dataset. Primary outcomes, such as hospitalization, ventilator use, intensive care unit (ICU) admission, and death in COVID-19 patients with MG, were compared with those of COVID-19 patients without MG: the subgroups of non-MG included those with rheumatoid arthritis (RA), systemic lupus (SLE), and multiple sclerosis (MS). We further analyzed factors affecting mortality, such as age, race/ethnicity, comorbidities, and MG treatments. Results: Among 421,086 individuals with COVID-19, there were 377 patients with MG, 7,362 patients with RA, 1,323 patients with SLE, 1,518 patients with MS, and 410,506 patients without MG. Patients with MG were older and had more comorbidities compared with non-MG patients and had the highest rates of hospitalization (38.5%), ICU admission (12.7%), ventilator use (3.7%), and mortality (10.6%) compared with all other groups. After adjusting for risk factors, patients with MG had increased risks for hospitalization and ICU compared with patients with non-MG and with RA but had risks similar to patients with SLE and with MS. The adjusted risk for ventilator use was similar across all groups, but the risk for mortality in patients with MG was lower compared with the SLE and MS groups. Among patients with MG, age over 75 years and dysphagia were predictors for increased COVID-19 mortality, but the recent MG treatment was not associated with COVID-19 mortality. Conclusions: COVID-19 patients with MG are more likely to be admitted to the hospital and require ICU care. Older age and patients with dysphagia had an increased risk of mortality.

Precision Intravenous Immunoglobulin Dosing and Clinical Outcomes: A Retrospective Chart Review.

OBJECTIVES: Intravenous immunoglobulin (IVIg) is used for treatment of acute neurologic conditions such as Guillain-Barre syndrome, chronic inflammatory demyelinating polyradiculoneuropathy relapse, and myasthenia gravis exacerbation. Precision dosing (adjusted or ideal body weight) is proposed to conserve IVIg. There have been no published studies comparing clinical outcomes in traditional dosing (actual body weight) with precision dosing. In 2014, our institution began dosing patients with precision dosing. This decision was largely performed by administration rather than physicians' preference. We sought to analyze our retrospective data to understand the change in dosing methods with neurologic outcomes. METHODS: We performed a retrospective review of all patients hospitalized at a single center who received IVIg for myasthenia gravis, Guillain-Barre syndrome, and chronic inflammatory demyelinating polyradiculoneuropathy from January 2010 to October 2017. We collected baseline information and clinical outcomes including mortality, readmission, need for second rescue treatment, length of stay, discharge disposition, treatment-related adverse events, and modified research council posttreatment sum score. RESULTS: Length of stay was significantly shorter with precision dosing. There was no statistically significant difference in discharge disposition, readmission, rescue treatment, or modified research council posttreatment sum score with precision dosing. CONCLUSION: Precision dosing did not adversely affect short-term neurologic outcomes.

Subcutaneous immunoglobulin treatment for chronic inflammatory demyelinating polyneuropathy.

Immunoglobulin G (IgG) therapy is an established long-term treatment in chronic inflammatory demyelinating polyneuropathy (CIDP) that is commonly administered intravenously (IVIg). The subcutaneous immunoglobulin (SCIg) administration route is a safe and effective alternative option, approved by the United States Food and Drug Administration (FDA) in 2018, for maintenance treatment of adults with CIDP. Physicians and patients alike need to be aware of all their treatment options in order to make informed decisions and plan long-term treatment strategies. In this review, we collate the evidence for SCIg in CIDP from all published studies and discuss their implications and translation to clinical practice. We also provide guidance on the practicalities of how and when to transition patients from IVIg to SCIg and ongoing patient support. Evidence suggests that IVIg and SCIg have comparable long-term efficacy in CIDP. However, SCIg can provide additional benefits for some patients, including no requirement for venous access or premedication, and reduced frequency of systemic adverse events. Local-site reactions are more common with SCIg than IVIg, but these are mostly well-tolerated and abate with subsequent infusions. Data suggest that many patients prefer SCIg following transition from IVIg. SCIg preference may be a result of the independence and flexibility associated with self-infusion, whereas IVIg preference may be a result of familiarity and reliance on a healthcare professional for infusions. In practice, individualizing maintenance dosing based on disease behavior and determining the minimally effective IgG dose for individuals are key considerations irrespective of the administration route chosen.

Heat shock protein is a key therapeutic target for nerve repair in autoimmune peripheral neuropathy and severe peripheral nerve injury.

Guillain-Barré syndrome (GBS) is an autoimmune peripheral neuropathy and a common cause of neuromuscular paralysis. Preceding infection induces the production of anti-ganglioside (GD) antibodies attacking its own peripheral nerves. In severe proximal peripheral nerve injuries that require long-distance axon regeneration, motor functional recovery is virtually nonexistent. Damaged axons fail to regrow and reinnervate target muscles. In mice, regenerating axons must reach the target muscle within 35 days (critical period) to reform functional neuromuscular junctions and regain motor function. Successful functional recovery depends on the rate of axon regeneration and debris removal (Wallerian degeneration) after nerve injury. The innate-immune response of the peripheral nervous system to nerve injury such as timing and magnitude of cytokine production is crucial for Wallerian degeneration. In the current study, forced expression of human heat shock protein (hHsp) 27 completely reversed anti-GD-induced inhibitory effects on nerve repair assessed by animal behavioral assays, electrophysiology and histology studies, and the beneficial effect was validated in a second mouse line of hHsp27. The protective effect of hHsp27 on prolonged muscle denervation was examined by performing repeated sciatic nerve crushes to delay regenerating axons from reaching distal muscle from 37 days up to 55 days. Strikingly, hHsp27 was able to extend the critical period of motor functional recovery for up to 55 days and preserve the integrity of axons and mitochondria in distal nerves. Cytokine array analysis demonstrated that a number of key cytokines which are heavily involved in the early phase of innate-immune response of Wallerian degeneration, were found to be upregulated in the sciatic nerve lysates of hHsp27 Tg mice at 1 day postinjury. However, persistent hyperinflammatory mediator changes were found after chronic denervation in sciatic nerves of littermate mice, but remained unchanged in hHsp27 Tg mice. Taken together, the current study provides insight into the development of therapeutic strategies to enhance muscle receptiveness (reinnervation) by accelerating axon regeneration and Wallerian degeneration.

Guillain-Barré Syndrome.

PURPOSE OF REVIEW: This article reviews the clinical features, diagnosis and differential diagnosis, prognosis, pathogenesis, and current and upcoming treatments of Guillain-Barré syndrome (GBS). RECENT FINDINGS: GBS is an acute inflammatory neuropathic illness with striking clinical manifestations and significant morbidity. A substantial proportion of patients with GBS do not respond to current immunomodulatory therapies (ie, plasma exchange and IV immunoglobulin [IVIg]), highlighting the need for new therapies. Prognostic models that can accurately predict functional recovery and the need for artificial ventilation have emerged. These models are practical, and online calculators are available for clinical use, facilitating early recognition of patients with poor outcome and the opportunity to personalize management decisions. Clinical and experimental studies have identified innate immune effectors (complement, macrophage lineage cells, and activating Fcγ receptors) as important mediators of inflammatory nerve injury. Two complement inhibitors are undergoing clinical testing for efficacy in GBS. SUMMARY: GBS is the most common cause of acute flaccid paralysis in the United States and worldwide. New treatments for GBS have not emerged since the 1990s. Our understanding of the pathogenesis of this disorder has progressed, particularly over the past decade; as a result, new therapeutic agents targeting different components of the complement cascade are at advanced stages of clinical development.

Therapeutic and Diagnostic Challenges in Myasthenia Gravis.

"Myasthenia gravis (MG) is the most common autoimmune neuromuscular disorder. This article highlights several cases that the practicing neurologist may encounter in the treatment of MG. Diagnostic uncertainty continues to be an issue in patients who are seronegative to the 2 most common antibodies, acetylcholine receptor and muscle-specific tyrosine kinase (MuSK). Specific populations of patients with MG including MuSK MG, thymomatous MG, refractory MG, and pregnant women also require special consideration. This article reviews specific cases and an update on current management."

Changes of Serum IgG Dimer Levels after Treatment with IVIg in Guillain-Barré Syndrome.

Intravenous immunoglobulins (IVIg) are standard treatment for Guillain-Barré syndrome (GBS). Their exact mechanisms of action are versatile and not fully understood. One possible mechanism is neutralization of circulating autoantibodies via binding to anti- idiotypic antibodies forming idiotype-anti-idiotype dimeric IgG immune complexes. To examine the role of immune complex formation as mechanism of action for IVIg in GBS, 34 C57Bl/6 mice were either treated with anti-ganglioside antibodies and IVIg or IVIg and PBS alone, whereas eight additional mice were treated either with anti-ganglioside autoantibodies and IVIg or anti-ganglioside autoantibodies alone. Subsequently IgG dimer formation was assessed by high performance liquid chromatography (HPLC) and enzyme- linked immunosorbent assay (ELISA). In addition, IgG dimer formation was measured in sera of eight GBS patients who were treated with IVIg. In mice, a significant increase of dimeric IgG after administration of anti-ganglioside antibodies and IVIg could be observed. Re-monomerized IgG dimers showed immunoreactivity against gangliosides and serum immunoreactivity was significantly reduced after IVIg infusion. Likewise also in GBS patients, IgG dimer formation could be detected after IVIg treatment. Our data indicate that dimeric IgG immune complexes contain anti-idiotypic antibodies and provide proof of concept that IVIg treatment in GBS results in measurable amounts of IgG dimers. Larger patient cohorts are needed to evaluate serum IgG dimer increase as a possible marker for treatment response in GBS. Graphical Abstract Mechanism of action: Intravenous immunoglobulins (IVIg) and anti-ganglioside antibodies form dimeric IgG immune complexes, preventing axonal damage in Guillain-Barré Syndrome.

Elimination of activating Fcγ receptors in spontaneous autoimmune peripheral polyneuropathy model protects from neuropathic disease.

Spontaneous autoimmune peripheral polyneuropathy (SAPP) is a reproducible mouse model of chronic inflammatory peripheral neuropathy in female non-obese diabetic mice deficient in co-stimulatory molecule, B7-2 (also known as CD86). There is evidence that SAPP is an interferon-γ, CD4+ T-cell-mediated disorder, with autoreactive T-cells and autoantibodies directed against myelin protein zero involved in its immunopathogenesis. Precise mechanisms leading to peripheral nerve system inflammation and nerve injury including demyelination in this model are not well defined. We examined the role of activating Fc-gamma receptors (FcγRs) by genetically ablating Fcγ-common chain (Fcer1g) shared by all activating FcγRs in the pathogenesis of this model. We have generated B7-2/ Fcer1g-double null animals for these studies and found that the neuropathic disease is substantially ameliorated in these animals as assessed by behavior, electrophysiology, immunocytochemistry, and morphometry. Our current studies focused on characterizing systemic and endoneurial inflammation in B7-2-null and B7-2/ Fcer1g-double nulls. We found that accumulation of endoneurial inflammatory cells was significantly attenuated in B7-2/ Fcer1g-double nulls compared to B7-2-single nulls. Whereas, systemically the frequency of CD4+ regulatory T cells and expression of immunosuppressive cytokine, IL-10, were significantly enhanced in B7-2/ Fcer1g-double nulls. Overall, these findings suggest that elimination of activating FcγRs modulate nerve injury by altering endoneurial and systemic inflammation. These observations raise the possibility of targeting activating FcγRs as a treatment strategy in acquired inflammatory demyelinating neuropathies.

Systemic IGF-1 gene delivery by rAAV9 improves spontaneous autoimmune peripheral polyneuropathy (SAPP).

Spontaneous autoimmune peripheral polyneuropathy (SAPP) is a mouse model of chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) in non-obese diabetic (NOD) mice null for costimulatory molecule, B7-2 gene (B7-2-/-). SAPP is a chronic progressive and multifocal inflammatory and demyelinating polyneuropathy of spontaneous onset with secondary axonal degeneration. Insulin-like growth factor 1(IGF-1) is a pleiotropic factor with neuroprotective, regenerative, and anti-inflammatory effects with extensive experience in its preclinical and clinical use. Systemic delivery of recombinant adeno-associated virus serotype 9 (rAAV9) provides robust and widespread gene transfer to central and peripheral nervous systems making it suitable for gene delivery in neurological diseases. A significant proportion of patients with inflammatory neuropathies like CIDP do not respond to current clinical therapies and there is a need for new treatments. In this study, we examined the efficacy IGF-1 gene therapy by systemic delivery with rAAV9 in SAPP model. The rAAV9 construct also contained a reporter gene to monitor the surrogate expression of IGF-1. We found significant improvement in neuropathic disease after systemic delivery of rAAV9/IGF-1 gene at presymptomatic and symptomatic stages of SAPP model. These findings support that IGF-1 treatment (including gene therapy) is a viable therapeutic option in immune neuropathies such as CIDP.

Long-term neuromuscular outcomes of west nile virus infection: A clinical and electromyographic evaluation of patients with a history of infection.

INTRODUCTION: Neuromuscular clinical manifestations during acute West Nile virus (WNV) infection are well documented; however, long-term neurologic outcomes still require investigation. METHODS: We conducted a long-term follow-up study in patients with history of WNV infection. Of the 117 patients who participated in neurologic and neurocognitive evaluations, 30 were referred for neuromuscular and electrodiagnostic evaluation based on abnormal findings. RESULTS: We found that 33% of these patients (10 of 30) showed abnormalities on nerve conduction and/or needle electromyography due to primary or secondary outcomes of WNV infection. Most common electrodiagnostic findings and causes of long-term disability were related to anterior horn cell poliomyelitis (WNV poliomyelitis). Electrical data on these patient populations were similar to those observed in chronic poliomyelitis. DISCUSSION: With more than 16,000 cases of WNV neuroinvasive disease reported across the USA since 1999, understanding clinical outcomes from infection will provide a resource for physicians managing long-term care of these patients. Muscle Nerve 57: 77-82, 2018.

Sialylated intravenous immunoglobulin suppress anti-ganglioside antibody mediated nerve injury.

The precise mechanisms underlying the efficacy of intravenous immunoglobulin (IVIg) in autoimmune neurological disorders including Guillain-Barré syndrome (GBS) are not known. Anti-ganglioside antibodies have been reported to be pathogenic in some variants of GBS, and we have developed passive transfer animal models to study anti-ganglioside antibody mediated-endoneurial inflammation and associated neuropathological effects and to evaluate the efficacy of new therapeutic approaches. Some studies indicate that IVIg's anti-inflammatory activity resides in a minor sialylated IVIg (sIVIg) fractions and is dependent on an innate Th2 response via binding to a specific ICAM3-grabbing nonintegrin related 1 receptor (SIGN-R1). Therefore the efficacy of IVIg, IVIg fractions with various IgG Fc sialylation status, and the involvement of Th2 pathway were examined in one of our animal model of antibody-mediated inhibition of axonal regeneration. We demonstrate that both IVIg and sIVIg ameliorated anti-glycan antibody mediated-pathological effect, whereas, the unsialylated fractions of IVIg were not beneficial in our model. Tenfold lower doses of sIVIg compared to whole IVIg provided equivalent efficacy in our studies. Moreover, we found that whole IVIg and sIVIg significantly upregulates the gene expression of IL-33, which itself can provide protection from antibody-mediated nerve injury in our model. Our results support that the SIGN-R1-Th2 pathway is involved in the anti-inflammatory effects of IVIg on endoneurium in our model and elements of this pathway including IL-33 can provide novel therapeutics in inflammatory neuropathies.

Anti-glycan antibodies halt axon regeneration in a model of Guillain Barrè Syndrome axonal neuropathy by inducing microtubule disorganization via RhoA-ROCK-dependent inactivation of CRMP-2.

Several reports have linked the presence of high titers of anti-Gg Abs with delayed recovery/poor prognosis in GBS. In most cases, failure to recover is associated with halted/deficient axon regeneration. Previous work identified that monoclonal and patient-derived anti-Gg Abs can act as inhibitory factors in an animal model of axon regeneration. Further studies using primary dorsal root ganglion neuron (DRGn) cultures demonstrated that anti-Gg Abs can inhibit neurite outgrowth by targeting gangliosides via activation of the small GTPase RhoA and its associated kinase (ROCK), a signaling pathway common to other established inhibitors of axon regeneration. We aimed to study the molecular basis of the inhibitory effect of anti-Gg abs on neurite outgrowth by dissecting the molecular dynamics of growth cones (GC) cytoskeleton in relation to the spatial-temporal analysis of RhoA activity. We now report that axon growth inhibition in DRGn induced by a well characterized mAb targeting gangliosides GD1a/GT1b involves: i) an early RhoA/ROCK-independent collapse of lamellipodia; ii) a RhoA/ROCK-dependent shrinking of filopodia; and iii) alteration of GC microtubule organization/and presumably dynamics via RhoA/ROCK-dependent phosphorylation of CRMP-2 at threonine 555. Our results also show that mAb 1B7 inhibits peripheral axon regeneration in an animal model via phosphorylation/inactivation of CRMP-2 at threonine 555. Overall, our data may help to explain the molecular mechanisms underlying impaired nerve repair in GBS. Future work should define RhoA-independent pathway/s and effectors regulating actin cytoskeleton, thus providing an opportunity for the design of a successful therapy to guarantee an efficient target reinnervation.