Cytokines (IL1ß, IL6, TNFα) and serum cortisol levels may not constitute reliable biomarkers to identify individuals with post-acute sequelae of COVID-19.

Background: Post-acute sequelae of COVID-19 (PASC) comprise a broad spectrum of symptoms such as fatigue, general weakness, compromised attention and sleep or anxiety disorders. PASC represents a medical and socio-economic challenge. Objectives: Our study evaluated cytokines (IL-1ß, IL-6 and TNFα) and cortisol levels in a cohort of typical patients with PASC, suffering concentration problems, fatigue and difficulties finding words. Design: This was a prospective cohort study. Four groups were analysed and compared: those who had never contracted SARS-CoV-2 (n = 13), infected but had no PASC (n = 34), infected with former PASC that resolved (n = 40) and patients with ongoing PASC after infection (n = 91). Methods: Cytokine and cortisol serum levels were determined in patients' blood samples. Results: Cytokine levels of IL-1ß, IL-6, TNFα and cortisol levels did not differ between groups analysed. Conclusion: This may indicate a non-organic/psychosomatic genesis of PASC; further studies are needed to elucidate the underlying causes of PACS, and non-organic causes should not be overlooked.

Without clear biological markers for people who will continue to present with post-acute sequelae of COVID-19 (PASC) should we now focus on psychological factors? Many people across the globe are still suffering from post-acute sequelae of COVID-19 (PASC), commonly called post-COVID. Typical symptoms of PASC include severe tiredness (fatigue), concentration deficits (brain fog) or difficulty finding words. We need a better understanding of how these symptoms arise to find ways to help patients. Our team of researchers set out to explore this. We posed the question: could measurements of immune system activity provide an identifier for people who are susceptible to post-COVID? The participants in our study were divided into four groups: 1. A group of 13 people who had never contracted SARS-CoV-2. 2. A group of 34 people who had been infected with SARS-CoV-2 but had no PASC. 3. A group of 40 people who had been infected with SARS-CoV-2 and had already suffered from PASC that had now resolved. 4. A group of 91 people who were no longer sick with COVID-19 but were still suffering from PASC. Serum samples from all participants were taken to measure cytokine and cortisol levels. People with PASC could not be identified by testing their blood samples for cytokines (IL-1ß, IL-6, TNFα) or cortisol. No difference between the four groups was found on any marker. Measuring these cytokines or cortisol is, therefore, unlikely to be useful in predicting which patients will suffer from PASC. Continuation of symptoms long after COVID-19 has passed is distressing for many people worldwide. Psychological factors may play a role and need to be studied further in order to help this patient population.

Time for a standardized diagnostic response test in patients with chronic inflammatory demyelinating polyradiculoneuropathy?

Standardized pharmacological response tests are important and established diagnostic tools in the field of neurology. However, regarding therapeutic responses to intravenous immunoglobulins (IVIg) in CIDP, neither a definition of therapeutic response has been established, nor a response test has been suggested so far. Here we suggest a practical clinical approach which is supported by current literature in the field. An established standardized IVIg response test could avoid prolonged therapy without benefit for the patient and ensure a timely therapy switch or treatment escalation if required. This approach would also be advantageous due to the global scarcity of plasma derivatives as a human resource and could be the foundation to be adjusted and improved by subsequent studies.

Impact of NKG2D Signaling on Natural Killer and T-Cell Function in Cerebral Ischemia.

Background Typically defined as a thromboinflammatory disease, ischemic stroke features early and delayed inflammatory responses, which determine the extent of ischemia-related brain damage. T and natural killer cells have been implicated in neuronal cytotoxicity and inflammation, but the precise mechanisms of immune cell-mediated stroke progression remain poorly understood. The activating immunoreceptor NKG2D is expressed on both natural killer and T cells and may be critically involved. Methods and Results An anti-NKG2D blocking antibody alleviated stroke outcome in terms of infarct volume and functional deficits, coinciding with reduced immune cell infiltration into the brain and improved survival in the animal model of cerebral ischemia. Using transgenic knockout models devoid of certain immune cell types and immunodeficient mice supplemented with different immune cell subsets, we dissected the functional contribution of NKG2D signaling by different NKG2D-expressing cells in stroke pathophysiology. The observed effect of NKG2D signaling in stroke progression was shown to be predominantly mediated by natural killer and CD8+ T cells. Transfer of T cells with monovariant T-cell receptors into immunodeficient mice with and without pharmacological blockade of NKG2D revealed activation of CD8+ T cells irrespective of antigen specificity. Detection of the NKG2D receptor and its ligands in brain samples of patients with stroke strengthens the relevance of preclinical observations in human disease. Conclusions Our findings provide a mechanistic insight into NKG2D-dependent natural killer- and T-cell-mediated effects in stroke pathophysiology.

Post-COVID-19 Syndrome is Rarely Associated with Damage of the Nervous System: Findings from a Prospective Observational Cohort Study in 171 Patients.

INTRODUCTION: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can affect multiple organs. Reports of persistent or newly emergent symptoms, including those related to the nervous system, have increased over the course of the pandemic, leading to the introduction of post-COVID-19 syndrome. However, this novel syndrome is still ill-defined and structured objectification of complaints is scarce. Therefore, we performed a prospective observational cohort study to better define and validate subjective neurological disturbances in patients with post-COVID-19 syndrome. METHODS: A total of 171 patients fulfilling the post-COVID-19 WHO Delphi consensus criteria underwent a comprehensive neurological diagnostic work-up including neurovascular, electrophysiological, and blood analysis. In addition, magnetic resonance imaging (MRI) and lumbar puncture were conducted in subgroups of patients. Furthermore, patients underwent neuropsychological, psychosomatic, and fatigue assessment. RESULTS: Patients were predominantly female, middle-aged, and had incurred mostly mild-to-moderate acute COVID-19. The most frequent post-COVID-19 complaints included fatigue, difficulties in concentration, and memory deficits. In most patients (85.8%), in-depth neurological assessment yielded no pathological findings. In 97.7% of the cases, either no diagnosis other than post COVID-19 syndrome, or no diagnosis likely related to preceding acute COVID-19 could be established. Sensory or motor complaints were more often associated with a neurological diagnosis other than post-COVID-19 syndrome. Previous psychiatric conditions were identified as a risk factor for developing post-COVID-19 syndrome. We found high somatization scores in our patient group that correlated with cognitive deficits and the extent of fatigue. CONCLUSIONS: Albeit frequently reported by patients, objectifiable affection of the nervous system is rare in post-COVID-19 syndrome. Instead, elevated levels of somatization point towards a pathogenesis potentially involving psychosomatic factors. However, thorough neurological assessment is important in this patient group in order to not miss neurological diseases other than post-COVID-19.

Severe and long-lasting alteration of albumin redox state by plasmapheresis.

Plasmapheresis (PE) is an established form of therapeutic apheresis (TA). Purpose of this longitudinal prospective single center study was to investigate the effect of PE on albumin redox state (ARS), as infusion of commercial albumin during PE may alter albumin oxidation which has an impact on its functional properties and oxidative stress level. 43 subjects with autoimmune-mediated neurological disorders were included. 20 subjects in the experimental group received five treatments of PE. 13 subjects received five treatments of immunoadsorption and 10 subjects received no TA as controls. ARS was determined before and after TA and 12 days after the last TA by fractionating it into human mercaptalbumin (HMA), human non-mercaptalbumin 1 (HNA-1), and human non-mercaptalbumin 2 (HNA-2) by high-performance liquid chromatography. Irreversibly oxidised HNA-2 increased over the course of five PE treatments from 2.8% (IQR 1.3-3.7%) to 13.6% (IQR 10.9-15.9) (P < 0.01) and remained elevated 12 days after the last PE procedure (7.7% IQR 7.1-10.5, P < 0.05). The study showed for the first time that PE exerts a severe and long-lasting alteration on ARS indicating a new adverse effect of PE, that may influence oxidative stress level.

LPA1 signaling drives Schwann cell dedifferentiation in experimental autoimmune neuritis.

BACKGROUND: Lysophosphatidic acid (LPA) is a pleiotropic lipid messenger that addresses at least six specific G-protein coupled receptors. Accumulating evidence indicates a significant involvement of LPA in immune cell regulation as well as Schwann cell physiology, with potential relevance for the pathophysiology of peripheral neuroinflammation. However, the role of LPA signaling in inflammatory neuropathies has remained completely undefined. Given the broad expression of LPA receptors on both Schwann cells and cells of the innate and adaptive immune system, we hypothesized that inhibition of LPA signaling may ameliorate the course of disease in experimental autoimmune neuritis (EAN). METHODS: We induced active EAN by inoculation of myelin protein 2 peptide (P255-78) in female Lewis rats. Animals received the orally available LPA receptor antagonist AM095, specifically targeting the LPA1 receptor subtype. AM095 was administered daily via oral gavage in a therapeutic regimen from 10 until 28 days post-immunization (dpi). Analyses were based on clinical testing, hemogram profiles, immunohistochemistry and morphometric assessment of myelination. RESULTS: Lewis rats treated with AM095 displayed a significant improvement in clinical scores, most notably during the remission phase. Cellular infiltration of sciatic nerve was only discretely affected by AM095. Hemogram profiles indicated no impact on circulating leukocytes. However, sciatic nerve immunohistochemistry revealed a reduction in the number of Schwann cells expressing the dedifferentiation marker Sox2 paralleled by a corresponding increase in differentiating Sox10-positive Schwann cells. In line with this, morphometric analysis of sciatic nerve semi-thin sections identified a significant increase in large-caliber myelinated axons at 28 dpi. Myelin thickness was unaffected by AM095. CONCLUSION: Thus, LPA1 signaling may present a novel therapeutic target for the treatment of inflammatory neuropathies, potentially affecting regenerative responses in the peripheral nerve by modulating Schwann cell differentiation.

Secondary Immunodeficiency and Risk of Infection Following Immune Therapies in Neurology.

Secondary immunodeficiencies (SIDs) are acquired conditions that may occur as sequelae of immune therapy. In recent years a number of disease-modifying therapies (DMTs) has been approved for multiple sclerosis and related disorders such as neuromyelitis optica spectrum disorders, some of which are frequently also used in- or off-label to treat conditions such as chronic inflammatory demyelinating polyneuropathy (CIDP), myasthenia gravis, myositis, and encephalitis. In this review, we focus on currently available immune therapeutics in neurology to explore their specific modes of action that might contribute to SID, with particular emphasis on their potential to induce secondary antibody deficiency. Considering evidence from clinical trials as well as long-term observational studies related to the patients' immune status and risks of severe infections, we delineate long-term anti-CD20 therapy, with the greatest data availability for rituximab, as a major risk factor for the development of SID, particularly through secondary antibody deficiency. Alemtuzumab and cladribine have relevant effects on circulating B-cell counts; however, evidence for SID mediated by antibody deficiency appears limited and urgently warrants further systematic evaluation. To date, there has been no evidence suggesting that treatment with fingolimod, dimethyl fumarate, or natalizumab leads to antibody deficiency. Risk factors predisposing to development of SID include duration of therapy, increasing age, and pre-existing low immunoglobulin (Ig) levels. Prevention strategies of SID comprise awareness of risk factors, individualized treatment protocols, and vaccination concepts. Immune supplementation employing Ig replacement therapy might reduce morbidity and mortality associated with SIDs in neurological conditions. In light of the broad range of existing and emerging therapies, the potential for SID warrants urgent consideration among neurologists and other healthcare professionals.

Observational cohort study of neurological involvement among patients with SARS-CoV-2 infection.

BACKGROUND: A growing number of reports suggest that infection with SARS-CoV-2 often leads to neurological involvement; however, data on the incidence and severity are limited to mainly case reports and retrospective studies. METHODS: This prospective, cross-sectional study of 102 SARS-CoV-2 PCR positive patients investigated the frequency, type, severity and risk factors as well as underlying pathophysiological mechanisms of neurological involvement (NIV) in COVID-19 patients. RESULTS: Across the cohort, 59.8% of patients had NIV. Unspecific NIV was suffered by 24.5%, mainly general weakness and cognitive decline or delirium. Mild NIV was found in 9.8%; most commonly, impaired taste or smell. Severe NIV was present in 23.5%; half of these suffered cerebral ischaemia. Incidence of NIV increased with respiratory symptoms of COVID-19. Mortality was higher with increasing NIV severity. Notably, 83.3% with severe NIV had a pre-existing neurological co-morbidity. All cerebrospinal fluid (CSF) samples were negative for SARS-CoV-2 RNA, and SARS-CoV-2 antibody quotient did not suggest intrathecal antibody synthesis. Of the patients with severe NIV, 50% had blood-brain barrier (BBB) disruption and showed a trend of elevated interleukin levels in CSF. Antibodies against neuronal and glial epitopes were detected in 35% of the patients tested. CONCLUSION: Cerebrovascular events were the most frequent severe NIV and severe NIV was associated with high mortality. Incidence of NIV increased with respiratory symptoms and NIV and pre-existing neurological morbidities were independent risk factors for fatality. Inflammatory involvement due to BBB disruption and cytokine release drives NIV, rather than direct viral invasion. These findings might help physicians define a further patient group requiring particular attention during the pandemic.

Neurological Manifestations of COVID-19 Feature T Cell Exhaustion and Dedifferentiated Monocytes in Cerebrospinal Fluid.

Patients suffering from Coronavirus disease 2019 (COVID-19) can develop neurological sequelae, such as headache and neuroinflammatory or cerebrovascular disease. These conditions-termed here as Neuro-COVID-are more frequent in patients with severe COVID-19. To understand the etiology of these neurological sequelae, we utilized single-cell sequencing and examined the immune cell profiles from the cerebrospinal fluid (CSF) of Neuro-COVID patients compared with patients with non-inflammatory and autoimmune neurological diseases or with viral encephalitis. The CSF of Neuro-COVID patients exhibited an expansion of dedifferentiated monocytes and of exhausted CD4+ T cells. Neuro-COVID CSF leukocytes featured an enriched interferon signature; however, this was less pronounced than in viral encephalitis. Repertoire analysis revealed broad clonal T cell expansion and curtailed interferon response in severe compared with mild Neuro-COVID patients. Collectively, our findings document the CSF immune compartment in Neuro-COVID patients and suggest compromised antiviral responses in this setting.

NK cell markers predict the efficacy of IV immunoglobulins in CIDP.

OBJECTIVE: To assess whether IV immunoglobulins (IVIgs) as a first-line treatment for chronic inflammatory demyelinating polyneuropathy (CIDP) have a regulative effect on natural killer (NK) cells that is related to clinical responsiveness to IVIg. METHODS: In a prospective longitudinal study, we collected blood samples of 29 patients with CIDP before and after initiation of IVIg treatment for up to 6 months. We used semiquantitative PCR and flow cytometry in the peripheral blood to analyze the effects of IVIg on the NK cells. The results were correlated with clinical aspects encompassing responsiveness. RESULTS: We found a reduction in the expression of several typical NK cell genes 1 day after IVIg administration. Flow cytometry furthermore revealed a reduced cytotoxic CD56dim NK cell population, whereas regulatory CD56bright NK cells remained mostly unaffected or were even increased after IVIg treatment. Surprisingly, the observed effects on NK cells almost exclusively occurred in IVIg-responsive patients with CIDP. CONCLUSIONS: The correlation between the altered NK cell population and treatment efficiency suggests a crucial role for NK cells in the still speculative mode of action of IVIg treatment. Analyzing NK cell subsets after 24 hours of treatment initiation appeared as a predictive marker for IVIg responsiveness. Further studies are warranted investigating the potential of NK cell status as a routine parameter in patients with CIDP before IVIg therapy. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that NK cell markers predict clinical response to IVIg in patients with CIDP.

Redefining the heterogeneity of peripheral nerve cells in health and autoimmunity.

Peripheral nerves contain axons and their enwrapping glia cells named Schwann cells (SCs) that are either myelinating (mySCs) or nonmyelinating (nmSCs). Our understanding of other cells in the peripheral nervous system (PNS) remains limited. Here, we provide an unbiased single cell transcriptomic characterization of the nondiseased rodent PNS. We identified and independently confirmed markers of previously underappreciated nmSCs and nerve-associated fibroblasts. We also found and characterized two distinct populations of nerve-resident homeostatic myeloid cells that transcriptionally differed from central nervous system microglia. In a model of chronic autoimmune neuritis, homeostatic myeloid cells were outnumbered by infiltrating lymphocytes which modulated the local cell-cell interactome and induced a specific transcriptional response in glia cells. This response was partially shared between the peripheral and central nervous system glia, indicating common immunological features across different parts of the nervous system. Our study thus identifies subtypes and cell-type markers of PNS cells and a partially conserved autoimmunity module induced in glia cells.

Protective effects of 4-aminopyridine in experimental optic neuritis and multiple sclerosis.

Chronic disability in multiple sclerosis is linked to neuroaxonal degeneration. 4-aminopyridine (4-AP) is used and licensed as a symptomatic treatment to ameliorate ambulatory disability in multiple sclerosis. The presumed mode of action is via blockade of axonal voltage gated potassium channels, thereby enhancing conduction in demyelinated axons. In this study, we provide evidence that in addition to those symptomatic effects, 4-AP can prevent neuroaxonal loss in the CNS. Using in vivo optical coherence tomography imaging, visual function testing and histologic assessment, we observed a reduction in retinal neurodegeneration with 4-AP in models of experimental optic neuritis and optic nerve crush. These effects were not related to an anti-inflammatory mode of action or a direct impact on retinal ganglion cells. Rather, histology and in vitro experiments indicated 4-AP stabilization of myelin and oligodendrocyte precursor cells associated with increased nuclear translocation of the nuclear factor of activated T cells. In experimental optic neuritis, 4-AP potentiated the effects of immunomodulatory treatment with fingolimod. As extended release 4-AP is already licensed for symptomatic multiple sclerosis treatment, we performed a retrospective, multicentre optical coherence tomography study to longitudinally compare retinal neurodegeneration between 52 patients on continuous 4-AP therapy and 51 matched controls. In line with the experimental data, during concurrent 4-AP therapy, degeneration of the macular retinal nerve fibre layer was reduced over 2 years. These results indicate disease-modifying effects of 4-AP beyond symptomatic therapy and provide support for the design of a prospective clinical study using visual function and retinal structure as outcome parameters.

Trapped in the epineurium: early entry into the endoneurium is restricted to neuritogenic T cells in experimental autoimmune neuritis.

BACKGROUND: Autoimmune polyneuropathies are acquired inflammatory disorders of the peripheral nervous system (PNS) characterized by inflammation, demyelination, and axonal degeneration. Although the pathogenesis has not been fully elucidated, T cells recognizing self-antigens are believed to initiate inflammation in a subgroup of patients. However, the route and time of T cell entry into the PNS have not yet been described in detail. In this study, we analyzed both kinetics as well as localization of retrovirally transfected green fluorescent protein (GFP)-expressing neuritogenic T lymphocytes in experimental autoimmune neuritis (EAN). METHODS: T lymphocytes obtained from rats following EAN induction by immunization with peripheral nerve protein peptide P255-78 were retrovirally engineered to express GFP. Non-specific T cells were negatively selected by in vitro restimulation, whereas GFP-expressing neuritogenic T cells (reactive to P255-78) were adoptively transferred into healthy rats (AT-EAN). Antigen-specific T cell tracking and localization was performed by flow cytometry and immunohistochemistry during the course of disease. RESULTS: After induction of autoimmune neuritis, P2-reactive T cells were detectable in the liver, spleen, lymph nodes, lung, peripheral blood, and the sciatic nerves with distinct kinetics. A significant number of GFP+ T cells appeared early in the lung with a peak at day four. In the peripheral nerves within the first days, GFP-negative T cells rapidly accumulated and exceeded the number of GFP-expressing cells, but did not enter the endoneurium. Very early after adoptive transfer, T cells are found in proximity to peripheral nerves and in the epineurium. However, only GFP-expressing neuritogenic T cells are able to enter the endoneurium from day five after transfer. CONCLUSIONS: Our findings suggest that neuritogenic T cells invade the PNS early in the course of disease. However, neuritogenic T cells cross the blood-nerve barrier with a certain delay without preference to dorsal roots. Further understanding of the pathophysiological role of autoagressive T cells may help to improve therapeutic strategies in immune-mediated neuropathies.

The Role of Peripheral Myelin Protein 2 in Remyelination.

The protein component of the myelin layer is essential for all aspects of peripheral nerves, and its deficiency can lead to structural and functional impairment. The presence of peripheral myelin protein 2 (P2, PMP2, FABP8, M-FABP) in Schwann cells has been known for decades and shown recently to be involved in the lipid homeostasis in the peripheral neural system. However, its precise role during de- and remyelination has yet to be elucidated. To this end, we assessed remyelination after sciatic nerve crush injury in vivo, and in an experimental de/remyelination ex vivo myelinating culture model in P2-deficient (P2 -/- ) and wild-type (WT) animals. In vivo, the nerve crush paradigm revealed temporal structural and functional changes in P2 -/- mice as compared to WT animals. Concomitantly, P2 -/- DRG cultures demonstrated the presence of shorter internodes and enlarged nodes after ex vivo de/remyelination. Together, these data indicate that P2 may play a role in remyelination of the injured peripheral nervous system, presumably by affecting the nodal and internodal configuration.

Lysophosphatidic acid propagates post-injury Schwann cell dedifferentiation through LPA1 signaling.

Lysophosphatidic acid (LPA) is a pleiotropic signaling lipid that acts as ligand for at least six specific G-protein coupled receptors. Schwann cells (SC) are known to mainly express the LPA1 receptor subtype. An emerging body of evidence has linked LPA with injury-induced peripheral nerve demyelination as well as neuropathic pain. However, the molecular mechanisms underlying its demyelinating effect have not been conclusively elucidated. We aimed to decipher the demyelinating effect in vitro as well as in vivo by studying markers of SC differentiation and dedifferentiation: Myelinated dorsal root ganglia (DRG) cultures were treated either with LPA, LPA plus AM095 (LPA1 antagonist) or vehicle. Myelin content was subsequently investigated by Sudan Black staining and immunocytochemistry. In vivo, we performed sciatic nerve crush in C57BL/6 mice treated with AM095 at 10mg/kg. In DRG cultures, LPA caused a significant reduction of myelin as demonstrated by both Sudan Black staining and immunocytochemical analysis of myelin basic protein. Demyelination was paralleled by an upregulation of TNF-alpha as well as downregulation of Sox10, a marker for SC differentiation. LPA mediated effects were largely blocked by the addition of the LPA1 receptor antagonist AM095. In the in vivo model, AM095 treatment prior to crush injury increased Sox10 expression in SCs in the distal nerve stump while reducing the number of cells expressing the SC dedifferentiation marker Sox2. Additionally, TNF-alpha immunofluorescence was reduced in CD11b-positive cells. These data indicate that LPA may be a critical factor that shifts SCs towards a post-injury phenotype and contributes to the onset of Wallerian degeneration.

Predicting the Response to Intravenous Immunoglobulins in an Animal Model of Chronic Neuritis.

Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is a disabling autoimmune disorder of the peripheral nervous system (PNS). Intravenous immunoglobulins (IVIg) are effective in CIDP, but the treatment response varies greatly between individual patients. Understanding this interindividual variability and predicting the response to IVIg constitute major clinical challenges in CIDP. We previously established intercellular adhesion molecule (ICAM)-1 deficient non-obese diabetic (NOD) mice as a novel animal model of CIDP. Here, we demonstrate that similar to human CIDP patients, ICAM-1 deficient NOD mice respond to IVIg treatment by clinical and histological measures. Nerve magnetic resonance imaging and histology demonstrated that IVIg ameliorates abnormalities preferentially in distal parts of the sciatic nerve branches. The IVIg treatment response also featured great heterogeneity allowing us to identify IVIg responders and non-responders. An increased production of interleukin (IL)-17 positively predicted IVIg treatment responses. In human sural nerve biopsy sections, high numbers of IL-17 producing cells were associated with younger age and shorter disease duration. Thus, our novel animal model can be utilized to identify prognostic markers of treatment responses in chronic inflammatory neuropathies and we identify IL-17 production as one potential such prognostic marker.