Expression Profile of ERK5 and PKM2 Kinases in Neuroinflammatory Diseases.

Study Overview
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Brief Summary
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Demyelinating diseases represent a broad spectrum of disorders and are induced by excessive inflammation most often triggered by an autoimmune mechanism. Some of these pathologies are chronic and affect the central nervous system such as multiple sclerosis (MS), others are monophasic and target the peripheral nervous system such as Guillain Barré syndrome (GBS). In neuroinflammatory pathologies, the excessive response of the pro-inflammatory Th1 and Th17 lymphocyte lines and the insufficient response of regulatory T lymphocytes (Treg) cause excessive inflammation which is deleterious to the nervous tissue. The regulation of these signaling pathways involves key proteins such as kinases. Modulation of these kinases which could allow the development of new pharmacological targets for neuroinflammation. Recent work (unpublished data) has shown an association between the expression of ERK5 and PMK2 kinases, and the clinical severity of experimental allergic encephalomyelitis, a mouse model that mimics multiple sclerosis. In order to search for new biomarkers and improve our knowledge of the actors of the initial inflammatory phase of neuroinflammatory pathologies, we propose to study the differences in expression of ERK5 and PKM2 kinases in the blood and cerebral spinal fluid (CSF) of patients followed for relapsing-remitting MS and GBS by both RT-qPCR and protein quantification. We also want to study other biological parameters which include characterization of the pro / anti-inflammatory balance by cytokine assay and lymphocyte phenotyping, metabolome study, and mild form neurofilament (NfL) assay.

Detailed Description
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The study of the expression of ERK5 and PKM2 kinases in patients followed for RR MS or GBS, at the beginning of the pathology, will be evaluate. The usual diagnostic workup for MS and GBS will be performed in order to classify patients into 4 groups : • Group 1 : control group. Patients that do not fulfill the criteria for MS or GBS and in whom no differential diagnosis was made. Subgroups 1a and 1b will be formed by respective pairing with groups 2 and 3. Group 2 : patients with RR MS confirmed by Macdonald criteria (2017) Group 3 : patients with GBS confirmed by the association of the usual clinical and electrophysiological signs. Group 4 : patients in whom a differential diagnosis has been identified to explain the symptoms. These patients will be excluded from the analysis. An additional blood sample (25 mL) and cerebrospinal fluid (CSF) sample (2mL) will be taken from all patients in order to study the laboratory parameters of this study. Peripheral blood mononuclear cells (PBMCs) and neutrophils will be isolated from the blood using a Percoll gradient. Group 4 samples will not be analyzed. The analysis will focus on the comparison of laboratory parameters between group 2 and 3, between group 1a and 2, and between 1b and 3. / Expression of ERK5 and PKM2 kinases The investigators wish to analyze the expression of ERK5 and PKM2 by RT-qPCR to investigate the transcriptome on RNA level and by Western Blot and ELISA to investigate the phosphorylated and total forms of the two kinases. RT-qPCR require RNA extraction, reverse transcription into cDNA and then amplification of the genes encoding ERK5 and PKM2 using specific primers designed beforehand. The results will be compared with those obtained with primers of the HPRT gene. / Study of the balance between pro and anti-inflammatory action The investigators wish to characterize the balance of pro and anti-inflammatory activity by studying lymphocyte phenotyping and determining the level of cytokines. Lymphocyte phenotyping will be performed by flow cytometry from PBMCs. The populations studied will include the Th1, Th2, Th9, Th17, Th22, Treg and TFH populations. The panel of mainly inflammatory cytokines will be studied in plasma and in CSF by ELISA. The panel includes the following cytokines: IL17, TNF, IL6, Il 1β, IL 10, IL33, IL12, IL 23 / Metabolome analysis The databases on human metabolomes include more than 40,000 metabolites and allow the establishment of metabolic profiles in different pathological contexts by supervised and unsupervised statistical analysis techniques. Metabolome analyzes will be performed from plasma and CSF. These analyzes will be carried out by liquid chromatography coupled with mass spectrometry (LC-MS) by the Metabohub network (MetaboHUB-Clermont for plasma analyzes and MetaboHUB-Saclay for CSF analyzes). / Measure of the neurofilament light chain (NfL) There is growing interest in neurofilament's measure in the blood and in particular in the CSF. High levels of neurofilaments are evidence of neuronal loss, regardless of the initial mechanism. Neurofilament light chain (NfL) seems to be the most relevant form to measure axonal loss. In MS, increased NfL was observed during the first two months of acute relapse. Conversely, there are few data on the kinetics of NfL's levels in peripheral demyelinating diseases. The investigators intend to study NfL levels in blood and CSF to determine if there is an increase in NfL levels in patients followed for GBS and to compare NfL levels of these patients (group 3) with patients followed for MS (group 2).

Official Title
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NEUROKINASE : Expression Profile of ERK5 and PKM2 Kinases in Neuroinflammatory Diseases.

Conditions
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Sclerosis, Multiple, Guillain-Barre Syndrome, Inflammation

Intervention / Treatment
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* Biological: Blood sample and Cerebrospinal fluid (CSF) collection.


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Man and Woman 18 to 80 years old Clinical signs that suggest MS or GBS within a month of onset symptom Exclusion Criteria: Patient treated with immunosuppressive therapy, immunomodulator, or corticosteroids in chronic treatment Patient treated with corticosteroids in the past month without social security HIV positive serology dementia pregnant or breastfeeding woman previous participation in the study under judicial protection non-cooperating patient

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 80 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Other
Intervention Model: Single Group Assignment
Masking: None (Open Label)


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: Patients with clinical signs that suggest Multiple Sclerosis (MS) or Guillain Barré Syndrome (GBS)<br> | Biological: Blood sample and Cerebrospinal fluid (CSF) collection.<br>* An additional blood sample (25 ml) and a cerebrospinal fluid (CSF) sample (2 ml) will be taken.<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Expression of genes encoding ERK5 and PKM2 kinases by RT-qPCR | The expression of ERK5 and PKM2 by RT-qPCR will be analyze to investigate the transcriptome on RNA level. RT-qPCR require RNA extraction, reverse transcription into cDNA and then amplification of the genes encoding ERK5 and PKM2 using specific primers designed beforehand. The results will be compared with those obtained with primers of the HPRT gene. These analyzes are carried out using lymphocytes and neutrophils isolated from the blood sample. | Day 0 | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Lymphocyte phenotyping | Lymphocyte phenotyping will be performed by flow cytometry from PBMCs. We will use antibodies against Th1, Th2, Th9, Th17, Th22, Treg and TFH populations. | Day 0 | 
| Cytokine levels | A panel of cytokines, mainly pro-inflammatory, will be studied in plasma and in CSF by ELISA. The panel includes the following cytokines: IL17, TNF, IL6, Il 1β, IL 10, IL33, IL12, IL 23 | Day 0 | 
| Metabolome analysis | Metabolome analyzes will be performed from plasma and CSF. These analyzes will be carried out by liquid chromatography coupled with mass spectrometry (LC-MS) by the Metabohub network (MetaboHUB-Clermont for plasma analyzes and MetaboHUB-Saclay for CSF analyzes). | Day 0 | 
| Measure of the neurofilament light chain (NfL) | NfL levels in blood and CSF will be studied to determine if there is an increase in NfL levels in patients followed for GBS and to compare the NfL levels of these patients with patients followed for MS. | Day 0 | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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ERK5 kinase, M2 Type Pyruvate Kinase, cytokines