Expression Profile of ERK5 and PKM2 Kinases in Neuroinflammatory Diseases. (NEUROKINASE)

January 20, 2023 updated by: Centre Hospitalier Régional d'Orléans

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

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Recent work (unpublished data) has shown an association between the expression of ERK5 and PMK2 kinases, and the clinical severity of experimental allergic encephalomyelitis, a mouse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.

Study Overview

Status

Withdrawn

Conditions

Intervention / Treatment

Detailed Description

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.

  1. / 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.

  2. / 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

  3. / 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).

  4. / 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).

Study Type

Interventional

Phase

  • Not Applicable

Contacts and Locations

This section provides the contact details for those conducting the study, and information on where this study is being conducted.

Study Locations

  • France
      • Orléans, France, 45067
        • CHR Orleans

Participation Criteria

Researchers look for people who fit a certain description, called eligibility criteria. Some examples of these criteria are a person's general health condition or prior treatments.

Eligibility Criteria

Ages Eligible for Study

18 years to 80 years (Adult, Older Adult)

Accepts Healthy Volunteers

No

Genders Eligible for Study

All

Description

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

Study Plan

This section provides details of the study plan, including how the study is designed and what the study is measuring.

How is the study designed?

Design Details

  • Primary Purpose: Other
  • Allocation: N/A
  • Interventional 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)
Biological: Blood sample and Cerebrospinal fluid (CSF) collection.
An additional blood sample (25 ml) and a cerebrospinal fluid (CSF) sample (2 ml) will be taken.

What is the study measuring?

Primary Outcome Measures

Outcome Measure
Measure Description
Time Frame
Expression of genes encoding ERK5 and PKM2 kinases by RT-qPCR
Time Frame: Day 0

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
Time Frame: Day 0
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
Time Frame: Day 0
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
Time Frame: Day 0
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)
Time Frame: Day 0
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

Collaborators and Investigators

This is where you will find people and organizations involved with this study.

Sponsor

Centre Hospitalier Régional d'Orléans

Collaborators

Immunologie et Neurogénétique Expérimentales et Moléculaires (INEM)

Investigators

  • Principal Investigator: Pascal AUZOU, Dr, CHR Orleans

Publications and helpful links

The person responsible for entering information about the study voluntarily provides these publications. These may be about anything related to the study.

General Publications

Study record dates

These dates track the progress of study record and summary results submissions to ClinicalTrials.gov. Study records and reported results are reviewed by the National Library of Medicine (NLM) to make sure they meet specific quality control standards before being posted on the public website.

Study Major Dates

Study Start (Anticipated)

January 1, 2023

Primary Completion (Anticipated)

January 1, 2023

Study Completion (Anticipated)

January 1, 2023

Study Registration Dates

First Submitted

December 14, 2020

First Submitted That Met QC Criteria

December 14, 2020

First Posted (Actual)

December 19, 2020

Study Record Updates

Last Update Posted (Actual)

January 23, 2023

Last Update Submitted That Met QC Criteria

January 20, 2023

Last Verified

January 1, 2023

More Information

Terms related to this study

Keywords

Additional Relevant MeSH Terms

Other Study ID Numbers

  • CHRO-2018-07

Drug and device information, study documents

Studies a U.S. FDA-regulated drug product

No

Studies a U.S. FDA-regulated device product

No

This information was retrieved directly from the website clinicaltrials.gov without any changes. If you have any requests to change, remove or update your study details, please contact register@clinicaltrials.gov. As soon as a change is implemented on clinicaltrials.gov, this will be updated automatically on our website as well.

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