Biobanking, identification of biomarkers and the study on immune system in patients with multiple sclerosis

Biobanking
A biobank is a collection of body fluids (blood, CSF), isolated cells and/or
tissue donated by healthy volunteers and/or patients with linked medical
information which is made available for further biomolecular and medical
research. The Nijmegen MS biobank is a disease-oriented biobank, since the
specimens will be collected in the context of medical diagnosis and treatment.
Biobanks will provide a resource for researchers to increase their
understanding of complex disease such as MS because of its variable course,
complexity and heterogeneity. The ability to compare different disease stages
and/or forms of treatment at a molecular level is instrumental for finding
biomarkers for diagnosis of a disease or prediction of disease progression.

Biomarker discovery
MS is difficult to diagnose at early stages, as the MS signs and symptoms can
be similar to those of other diseases and clinical examinations combined with
MRI have to be applied. There is an urgent need for minimally invasive, highly
sensitive and specific diagnostic and prognostic biomarkers to identify and
monitor MS. Biomarkers may aid in diagnosing MS, predicting disease onset, and
selecting appropriate therapy. Biomarker discovery will include nucleic acid
analysis, proteomics, and the identification of (auto) antibody/autoantigen
markers.

Reversion of pathologic autoimmune response
Currently, there is no cure for MS. Instead, available drugs are
symptom-reducing immunosuppressing drugs that only delay the inevitable
progress of the disease. As these have to be taken for long periods of time to
be effective, the long-term side-effects can be very serious (cancer,
infections). The aim of this subproject is to investigate a novel concept to
stop the progress of the disease by a therapeutic vaccination. This stops the
disease-causing pathologic autoimmune response by inducing tolerance towards
specific autoantigens. In this way, the cause of the disease and further
progress of the disease can be prevented. To investigate this concept, cells
and serum are necessary to determine whether ex-vivo dendritic cells and
pathologic autospecific T cells can be stimulated with this therapeutic vaccine
such that tolerizing mechanisms are induced and tolerance towards the
autoantigen will be induced. This may be the lead towards a curing treatment
for MS as well as other autoimmune diseases.

The objectives of this initiative are:
1. To establish the disease oriented biobank; processing and storage of blood
and CSF specimens and health information of MS patients (which may include
health records, family history, lifestyle and genetic information);
2. To develop a diagnostic test for early MS based upon molecular biomarkers
(DNA, RNA, proteins, and antibodies) present in sera and CSF;
3. To establish a new therapeutic vaccine approach for MS treatment based on
induction of tolerance to autoantigens.

All patients who come to visit the MS center and satisfy the
inclusion/exclusion criteria will be invited to participate in the study. Prior
to data assessment and specimen collection, each patient will be given the
Patient Information and Consent Form. There will also be an invitation for the
MS patients to participate in this initiative and donate blood and/or CSF on
the website of Radboud MS Center Nijmegen. Participants will be asked to
provide extra blood and/or CSF, if CSF will be taken (see Research protocol;
Table 1). Blood and CSF will be taken simultaneously for routine clinical tests
and for the studies with only one venipuncture or lumbar puncture. Extra blood
and/or CSF specimens will be used for biobanking (Dr. B.A. de Jong), for the
study on MS biomarker discovery (Prof. G Pruijn) and for therapeutic vaccine
study (Dr. ir.T. Luijkx).

Biobank establishing
Invasive standard procedures such as venipuncture to collect blood specimens
and in some cases lumbar puncture to collect CSF will be performed. To get
serum samples, 20 ml of blood will be collected into serum gel (brown, 7.5 ml)
tubes. To get plasma and blood cell fraction, 10 ml of blood (or 60 ml in case
if a patient is involved in the immunological study) will be collected into
EDTA tubes. 2 ml of blood will be collected into Paxgene blood DNA tubes for
blood collection, stabilization and isolation of genomic DNA. The blood
collection will be performed according to Standard Operating Procedures for the
Collection of Blood Samples of the local institution. Patients will be also
asked to fill the survey form (attached) to provide health information.
After the specimens has been collected, participant*s name and any identifying
features will be replaced with the Subject Identification number (SIN).
The blood and CSF samples will be processed at the place where the collection
has occurred or transported to additional sites at the Department of
Biomolecular Chemistry (Prof. G Pruijn) and the Department of Medical
Immunology (Dr. T. Luijkx), Radboud University (Nijmegen). Blood samples will
be processed into following components for long term storage; plasma, serum,
and white blood cells. Appropriate aliquoting of specimens will be carried out.
Labeled cryovials will be stored in -80°C freezers. Effective tracking systems
to track specimens from the site of collection to their arrival in the
repository will be installed. Critical components of these systems include
unique specimen identifiers, appropriate specimen labels and inventory systems
for specimen tracking. Each specimen container will receive a label that
tightly adheres under all projected storage conditions.

Biomarker discovery.
Preclinical discovery phase: The identification of exploratory biomarkers
(markers in early development) suited for diagnostic use. Several techniques
will be used for biomarker development. This step will be performed with a
limited number of human serum/CSF samples (n=20 patients per group).
- Nucleic acid and epigenetic analysis: This step will include studies on
circulating miRNA and DNA methylation pattern in sera/CSF. Methylated DNA and
small RNA library will be generated and validated using high throughput
sequencing. Selection of discriminating DNA and RNA molecules by their
correlation with different phases of the disease will be performed.
- Proteomics: it will employ analysis of protein composition in blood/CSF over
the disease course using mass spectromic approach and identification of
possible biomarker candidates. As a first step protein fractionation to deplete
highly abundant proteins will be performed. Mass spectrometry will be applied
and the list of protein candidates will be generated.
- Identification of MS-associated antibody biomarkers: peptide library
screening. Combinatorial libraries of synthetic peptide (antigens) will be used
to screen for ligands that bind antibodies abundant in the serum/CSF of MS
patients but not healthy controls. Peptide libraries will be screened with
pooled immunoglobulins isolated from MS patients.
Preclinical verification:The results from preclinical discovery phase will be
tested with sufficient power in a larger defined clinical setting in an
independent, prospective cohort of patients (n=35 patients per group). This
phase may generate information which may be included into clinical guidelines.

Induction of tolerance to autoantigens
Peripheral blood mononuclear cells (PBMC) will be isolated from blood of MS
patients and healthy controls and cultured with auto-antigens under certain
conditions. Next, cytokine measurements will be done to assess whether specific
pathogenic T cells can be downregulated or skewed towards a regulatory
phenotype (Treg). This would confirm a concept to reverse a pathogenic
auto-immune response and induce tolerance to auto-antigens which can be used to
develop a curing treatment for MS patients.

Risks and burdens: Risk is negligible and the burden is minimal. The most
common risks related to drawing blood from patient arm are brief pain and/or
bruising. Infection, excess bleeding, clotting or fainting is also possible but
unlikely.

Possible benefits: There will not be a direct benefit. However, participation
of patients can help researchers to gain knowledge and make discoveries which
might help in the future to diagnose or treat patients in a better way.