Validation of elevated levels of interleukin-8 in the cerebrospinal fluid, and discovery of new biomarkers in patients with GBS and CIDP using a proximity extension assay

BackgroundBiomarkers for diagnosis of inflammatory neuropathies, assessment of prognosis, and treatment response are lacking.MethodsCSF and EDTA plasma from patients with Guillain-Barré syndrome (GBS), chronic inflammatory demyelinating polyneuropathy (CIDP), healthy controls (HC) and disease controls were analyzed with Olink multiplex proximity extension assay (PEA) from two independent cohorts. Levels of interleukin-8 (IL8) were further analyzed with ELISA in patients with GBS, CIDP, paraproteinemia-related demyelinating polyneuropathy (PDN), multifocal motor neuropathy (MMN), HC and disease controls. ROC analysis was performed. Outcome was measured with the GBS-disability score (GBS-ds) or Inflammatory Neuropathy Cause and Treatment (INCAT) score.ResultsIn CSF, multiplex PEA analysis revealed up-regulation of IL8 in GBS compared to CIDP and HC respectively, and CIDP compared to HC. In addition, levels of IL2RA were upregulated in GBS compared to both HC and CIDP, SELE in GBS compared to HC, and ITGAM, IL6, and NRP1 in GBS compared to CIDP. In plasma, levels of MMP3, THBD and ITGAM were upregulated in CIDP compared to HC. Validation of multiplex IL8 results using ELISA, revealed increased levels of IL8 in CSF in patients with GBS and CIDP versus HC and non-inflammatory polyneuropathies (NIP) respectively, as well as in PDN versus NIP and HC. Levels of IL8 in CSF correlated with impairment in the acute phase of GBS as well as outcome at 6-months follow up.ConclusionIL8 in CSF is validated as a diagnostic biomarker in GBS and CIDP, and a prognostic biomarker in GBS. Multiplex PEA hereby identifies several potential biomarkers in GBS and CIDP

Increasing involvement of CAPN1 variants in spastic ataxias and phenotype-genotype correlations

Spastic ataxias are rare neurogenetic disorders involving spinocerebellar and pyramidal tracts. Many genes are involved. Among them, CAPN1, when mutated, is responsible for a complex inherited form of spastic paraplegia (SPG76). We report the largest published series of 21 novel patients with nine new CAPN1 disease-causing variants and their clinical characteristics from two European university hospitals (Paris and Stockholm). After a formal clinical examination, causative variants were identified by next-generation sequencing and confirmed by Sanger sequencing. CAPN1 variants are a rare cause (~ 1.4%) of young-adult-onset spastic ataxia; however, together with all published cases, they allowed us to better describe the clinical and genetic spectra of this form. Truncating variants are the most frequent, and missense variants lead to earlier age at onset in favor of an additional deleterious effect. Cerebellar ataxia with cerebellar atrophy, dysarthria and lower limb weakness are often associated with spasticity. We also suggest that cognitive impairment and depression should be assessed specifically in the follow-up of SPG76 cases.Identification of new causative genes in spinocerebellar degenerations by combination of whole genome scan, next-generation sequencing and biological validation in vitro and in vivoInfrastructure de Recherche Translationnelle pour les Biothérapies en NeurosciencesEuropean Union’s Horizon 2020 research and innovation programm

Immunopathogenesis of Guillain-Barré syndrome and chronic inflammatory demyelinating polyradoculoneuropathy

Guillain-Barre syndrome (GBS) is an inflammatory polyradiculoneuropathy with acute onset and usually a spontaneous recovery. Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is a chronic progressive inflammatory neuropathy. GBS and CIDP are associated with high morbidity despite treatment with immunomodulatory drugs. Both conditions are associated with inflammation of spinal nerve roots and/or distal nerves of the peripheral nervous system (PNS). GBS is assumed to be induced by infection + abnormal autoimmunity, whereas the immunopathogenesis of CIDP remains obscure. Previous studies have shown up-regulation of myelin-reactive T cells, and of levels of blood cytokines, indicative of T cell auto-reactivity in GBS. Few studies have addressed the balance between Th1 and Th2 cytokines over the course of GBS. GBS, but not CIDP, is associated with anti-GM1 and anti-GD1a anti-ganglioside serum antibodies (abs), but the temporal profile of anti-ganglioside abs over the course of GBS is not known. Despite the presumed importance of the role of prior infections in GBS pathogenesis, the nature of the antigen-presenting cells involved in this putative process is unclear. Dendritic cells (DC) are believed to play an important role in the induction of adaptive and innate immune responses. The myeloid subset of DC activates T and B cells, and the plasmacytoid subset of DC may induce tolerance. The role of DC has not been examined in GBS and CIDP. The aims of the study were to explore the roles of cytokine-secreting cells, anti-ganglioside abs and DC in blood, CSF and PNS over the course of GBS and CIDP, in relation to clinical parameters and immuno-modulatory treatment. Patients examined in the acute phase of GBS prior to treatment with intravenous high dose immunoglobulin (IvIg), had elevated levels of IL-10, but not of IFN-gamma secreting blood mononuclear cells (MNC). Pre-treatment levels of IL-10 secreting MNC correlated with neurophysiological signs of axonal damage and with levels of anti-ganglioside serum IgM abs. The highest titers of anti-GM1 serum IgG abs were observed in GBS patients examined 40 days after onset of GBS, while peak anti-GD1a IgM antibody titers were measured in the recovery phase, i.e. 90 days after onset of GBS. Patients developing steadily increasing levels of serum anti-GM1 IgG- and anti- GD1a IgM abs over the course of GBS, followed by a drop in levels of abs (i.e. patients developing peak ab titers) had a worse clinical outcome. Patients examined in the acute phase of GBS prior to lvIg treatment had also high levels of IL-6 secreting blood MNC. Patients examined in the recovery phase of GBS had lower levels of TNF-alpha secreting MNC vs. controls. Levels of IL-12p70 secreting blood MNC were not altered. Percentages of freshly isolated blood DC expressing cell surface molecules CD1a (involved in antigen- presentation), CD80 and CD86 (co-stimulatory molecules), CD54 (adhesion molecule), CD11c (marker of myeloid DC) and CD 123 (marker of plasmacytoid DC) as well as the chemokine receptors CCR1, CCR2, CCR5, and CXCR4 were not altered in patients with GBS and CIDP compared to controls. In contrast, levels of CD11c+ DC were elevated in cerebrospinal fluid (CSF) of CIDP patients, and of CD123+ DC in CSF of GBS patients prior to immunomodulatory treatment. Small numbers of immature myeloid CD11 c+CD14-CD16- DC were detected in the perineurium of sural nerve biopsies of patients with CIDP as well as in controls. Chemokines released by nerve tissue or in CSF are assumed to be responsible for recruiting DC to sites of inflammation. The CSF levels of chemokines MCP-1 and IP-10 were elevated in GBS patients and of IP-10 and MIP-3 beta in CIDP prior to treatment. The absence of a clear systemic Th 1 cytokine secretion profile in GBS, as well as presence in CSF of plasmacytoid DC, may be one of the explanations for the self-limited course of the auto-aggressive attack against PNS in GBS. Myeloid DC, which may enter the CSF due to elevated levels of MCP-1, may be retained by MIP-3 beta and thus contribute to sustained PNS inflammation in CIDP. DC in the CSF and in peripheral nerves, may be responsible for uptake of antigen released from the inflamed PNS, whereby antigen may be presented to T and B cells locally and/or in draining lymph nodes. Further studies of the cause of differential recruitment of DC subsets to CSF as well as the role of CSF chemokines in GBS vs. CIDP are warranted

Microangiopathy—A Potential Contributing Factor to Idiopathic Polyneuropathy: A Mini Review

Chronic idiopathic axonal polyneuropathy (CIAP) is a slowly progressive predominantly sensory axonal polyneuropathy. The prevalence of CIAP increases with age. The pathogenic cause of CIAP is unknown although there are several prevailing etiological hypotheses. In this mini review, we focus on the hypothesis of disturbed microcirculation in the vasa nervorum of peripheral nerves as a pathogenic cause of CIAP. There is an association between CIAP and metabolic risk factors. Furthermore, the phenotype of CIAP resembles diabetic neuropathy both clinically and electrophysiologically. In sural nerve biopsies from patients with diabetes mellitus, structural abnormalities indicating microangiopathy in the endoneurial microvessels are well documented. Similarly, sural microvessel abnormalities have been shown in patients with atherosclerotic non-diabetic peripheral vascular disease. However, the reported histopathological alterations of microvasculature in sural nerves of CIAP patients are inconsistent. Two studies report microangiopathic changes in CIAP sural nerves comparable with those found in patients with diabetic neuropathy. Conversely, another recent study showed no significant differences in the microangiopathic parameters in the endoneurial microvessels in the sural nerve biopsies from CIAP patients compared to controls without polyneuropathy. However, this CIAP patient group was younger compared to the patient groups in the other two studies. A general limitation with the published morphological studies are that different methods have been used in the assessment of microangiopathy, and there is also a risk of subjectivity in the results. Immunohistochemistry studies of sural nerves with verification of microangiopathy using specific biomarkers would be of great interest to develop

Non-T(H)1 cytokines are augmented systematically early in Guillain-Barré syndrome

The T(H)1 vs non-T(H)1 cytokine balance in Guillain-Barré syndrome (GBS) is unknown. Using enzyme-linked immunospot (ELISPOT) assays, we observed elevated numbers of interleukin (IL)-6 and IL-10-secreting blood mononuclear cells (BMNC) during the acute phase in untreated patients, and low levels of tumor necrosis factor alpha-secreting BMNC in the recovery phase of GBS. Numbers of IL-12p70-secreting BMNC were not affected over the course of GBS. The non-T(H)1 cytokine profile observed early in GBS may explain the self-limited clinical course associated with GBS

Idiopathic Small Fiber Neuropathy : Phenotype, Etiologies, and the Search for Fabry Disease

Background and Purpose The etiology of small fiber neuropathy (SFN) often remains unclear. Since SFN may be the only symptom of late-onset Fabry disease, it may be underdiagnosed in patients with idiopathic polyneuropathy. We aimed to uncover the etiological causes of seemingly idiopathic SFN by applying a focused investigatory procedure, to describe the clinical phenotype of true idiopathic SFN, and to elucidate the possible prevalence of late-onset Fabry disease in these patients. Methods Forty-seven adults younger than 60 years with seemingly idiopathic pure or predominantly small fiber sensory neuropathy underwent a standardized focused etiological and clinical investigation. The patients deemed to have true idiopathic SFN underwent genetic analysis of the alpha-galactosidase A gene (GLA) that encodes the enzyme alpha-galactosidase A (Fabry disease). Results The following etiologies were identified in 12 patients: impaired glucose tolerance (58.3%), diabetes mellitus (16.6%), alcohol abuse (8.3%), mitochondrial disease (8.3%), and hereditary neuropathy (8.3%). Genetic alterations of unknown clinical significance in GLA were detected in 6 of the 29 patients with true idiopathic SFN, but this rate did not differ significantly from that in healthy controls (n=203). None of the patients with genetic alterations in GLA had significant biochemical abnormalities simultaneously in blood, urine, and skin tissue. Conclusions A focused investigation may aid in uncovering further etiological factors in patients with seemingly idiopathic SFN, such as impaired glucose tolerance. However, idiopathic SFN in young to middle-aged Swedish patients does not seem to be due to late-onset Fabry disease

Incidence of human herpesvirus 6 in clinical samples from Swedish patients with demyelinating diseases

BackgroundHuman herpesvirus 6 (HHV-6) has been reported to be associated with multiple sclerosis (MS) and Guillain–Barré syndrome (GBS).MethodsWe analyzed cell-free HHV-6 DNA as an indication of active infection in the peripheral blood and cerebrospinal fluid (CSF) of Swedish patients with GBS, patients with chronic inflammatory demyelinating polyradiculoneuropathy, treatment-naïve patients with possible MS, interferon-β treated MS patients [with or without neutralizing antibodies (NAbs)], and control patients with headache.ResultsOne of 14 GBS patients and one of eight patients with chronic inflammatory demyelinating polyradiculoneuropathy were positive for HHV-6 DNA in serum. Of the 27 treatment-naïve possible MS patients, two were positive in plasma and one in CSF. HHV-6 DNA was detected in the serum of three of 79 NAb+ patients and one of 102 NAb-interferon-β treated MS patients. HHV-6 DNA could not be detected in the plasma or CSF of any of the 33 controls, although the differences were not statistically significant.ConclusionOur results do not suggest active HHV-6 infection to be a common phenomenon in any of the patient groups studied

Monocyte-derived dendritic cells express and secrete matrix-degrading metalloproteinases and their inhibitors and are imbalanced in multiple sclerosis

Dendritic cells (DC) are antigen-presenting cells (APC) that most efficiently initiate and control immune responses. Migration processes of blood DC are crucial to exert their professional antigen-presenting functions. Matrix-degrading metalloproteinases (MMP) are proteolytic enzymes, which are considered to be key enzymes in extracellular matrix (ECM) turnover and mediators of cell migration. Tissue inhibitors of metalloproteinases (TIMP) are important regulators of MMP activity. Here we investigate whether blood monocyte-derived immature DC (iDC) and mature DC (mDC) express, produce and secrete functionally active MMP-1, -2, -3 and -9 and their inhibitors TIMP-1 and -2, and examine their involvement in multiple sclerosis (MS). On mRNA level, we observed high numbers of MMP-2 and TIMP-2 mRNA expressing iDC in MS. On protein level, high percentages of MMP-1, -2 and -9 expressing iDC by flow cytometry, and high MMP-1 secretion by Western blot together with high MMP-2 and -9 activities in iDC supernatants as studied with zymography were observed. Similarly, MS is associated with high percentages of MMP-2 and -3 and of TIMP-1 expressing mDC by flow cytometry together with high MMP-3 secretion and high MMP-9 activity in culture supernatants. Spontaneous migratory capacity of both iDC and mDC over ECM-coated filters was higher in MS compared to healthy controls (HC). In conclusion, blood monocyte-derived iDC and mDC express, produce and secrete several MMP and TIMP. Alterations in these molecules as observed in MS may be functionally important for DC functioning

Study of Autophagy and Microangiopathy in Sural Nerves of Patients with Chronic Idiopathic Axonal Polyneuropathy.

Twenty-five percent of polyneuropathies are idiopathic. Microangiopathy has been suggested to be a possible pathogenic cause of chronic idiopathic axonal polyneuropathy (CIAP). Dysfunction of the autophagy pathway has been implicated as a marker of neurodegeneration in the central nervous system, but the autophagy process is not explored in the peripheral nervous system. In the current study, we examined the presence of microangiopathy and autophagy-related structures in sural nerve biopsies of 10 patients with CIAP, 11 controls with inflammatory neuropathy and 10 controls without sensory polyneuropathy. We did not find any significant difference in endoneurial microangiopathic markers in patients with CIAP compared to normal controls, though we did find a correlation between basal lamina area thickness and age. Unexpectedly, we found a significantly larger basal lamina area thickness in patients with vasculitic neuropathy. Furthermore, we found a significantly higher density of endoneurial autophagy-related structures, particularly in patients with CIAP but also in patients with inflammatory neuropathy, compared to normal controls. It is unclear if the alteration in the autophagy pathway is a consequence or a cause of the neuropathy. Our results do not support the hypothesis that CIAP is primarily caused by a microangiopathic process in endoneurial blood vessels in peripheral nerves. The significantly higher density of autophagy structures in sural nerves obtained from patients with CIAP and inflammatory neuropathy vs. controls indicates the involvement of this pathway in neuropathy, particularly in CIAP, since the increase in density of autophagy-related structures was more pronounced in patients with CIAP than those with inflammatory neuropathy. To our knowledge this is the first report investigating signs of autophagy process in peripheral nerves in patients with CIAP and inflammatory neuropathy