‫تقرير نهائي لمشروع بحث‬
Research project final report /
Rapport final du projet de recherche
1
2014 ‫برنامج دعم البحوث العلمية‬
Grant Program for Scientific Research in Lebanon – 2014
Programme de subvention à la recherche scientifique au Liban – 2014
‫مستند إداري‬
Administrative Document
--------
Administrative information / ‫المعلومات اإلداري‬
:‫المرجع‬
020812
Project Title - )‫عنوان المشروع (عربي وأجنبي‬
Effect of Vitamin D replacement on immune function and cognition in
MS patients
Principal Investigator - ‫الباحث الرئيسي‬
‫العنوان‬
John Kennedy Street. Abi
Khater Bldg, ground level
Dr. Samia J. Khoury
Address
‫العنوان االلكتروني‬
sk88@aub.edu.lb
e-mail
‫رقم الهاتف‬
03818218
Telephone
‫االسم والشهرة‬
Name &
surname
American University of
Beirut Medical Center
‫المؤسسة‬
Institution
Director of AHNI and
MSC
‫الوظيفة‬
Post
Co-investigators – ‫الباحثون المشاركون‬
‫العنوان االلكتروني‬
e-mail
hd30@aub.edu.lb
‫المؤسسة‬
Institution
American University of
Beirut
‫االسم والشهرة‬
Name and surname
Dr. Hala Darwish
Duration and starting date of the research / ‫المدة التعاقدي للمشروع وتاريخ بدء البحث‬
2years
Duration (year) / ‫المدة التعاقدية للمشروع‬
August 2012
Starting date of the research /‫وتاريخ بدء البحث‬
2
2014 ‫برنامج دعم البحوث العلمية‬
Grant Program for Scientific Research in Lebanon – 2014
Programme de subvention à la recherche scientifique au Liban – 2014
.1
Scientific Information / ‫العلمي‬
‫ المعلومات‬.2
ّ
Objectives - ‫الهدف‬
(mandatory field to fill 5-8 lines) – )‫ أسطر‬8-5 : ‫( معلومات إلزامية‬
This is a prospective study to evaluate the effect of vitamin D supplementation in MS
patients with Vitamin D deficiency (serum level <25μg/ml) to those with normal Vitamin
D (serum level >35 μg/ml) on immunological and neuropsychological (cognitive)
measures.
Achievements - ‫أالنجازات المحقق‬
(mandatory field to fill 5-8 lines) – )‫ أسطر‬8-5 : ‫( معلومات إلزامية‬
Active enrollment and data collection started on September 20, 2012. Since then, a total
of 68 subjects were screened, 51 subjects met inclusion criteria, 35 subjects completed the
study baseline and 3 months follow up, the remaining 16 subjects are currently in the
study. In the laboratory, we have been optimizing the assays, but actual running of the
samples has not started yet. We are running all the samples from each subject at the same
time to decrease variability. Preliminary analysis of the demographics and cognitive
performance of the first 35 subjects who completed the study was conducted and showed
positive results with a significant improvement in cognitive performance on most
cognitive tests after 3 months.
Perspectives - ‫آفاق البحث‬
(mandatory field to fill 5-8 lines) – )‫ أسطر‬8-5 : ‫( معلومات إلزامية‬
We will continue enrollment until the target sample size of 86 is reached. Given that the
enrollment was slower than originally envisioned, we expect to reach the target number at
the end of 2014 in order to accomplish the aims of the study.
Our future laboratory experiments will be as follows :
Phase I:
Examine CD8+ T cells and inflammatory monocytes:
Phase II:
Stimulate sorted T cells in Th17 and Treg polarizing conditions looking at pathogenic
Th17 vs non-pathogenic phenotypes before and after vitamin D supplementation.
We are also planning to submit another grant to investigate the molecular mechanisms of
Vitamin D effect on immune cells.
Publications & Communications - ‫المنشورات والمساهمات في المؤتمرات‬
3
2014 ‫برنامج دعم البحوث العلمية‬
Grant Program for Scientific Research in Lebanon – 2014
Programme de subvention à la recherche scientifique au Liban – 2014
Poster presentation at the Annual AUB biomedical Research conference :
Title: Effect of Vitamin D replacement on immune function and cognition in Multiple
Sclerosis (MS) patients.
Authors: Hala J Darwish, Raed Ismail, Yasmine Papas, Elie El-Hajj, Nuhad Abou-Zeid,
Bassem Yamout and Samia J Khoury
Masters Thesis defended successfully on May 6, 2014:
Title : The Effect of Vitamin D on the Differentiation of Th17 Cells
Student name : Souraya Sayegh
Department of Immunology and microbilogy
Thesis committee members : Samia Khoury, Lama Fawaz, Ghassan Matar and Alex
Abdelnour
Abstract - ‫موجز عن نتائج البحث‬
(mandatory field to fill 5-8 lines) – )‫ أسطر‬8-5 : ‫( معلومات إلزامية‬
The preliminary analysis showed that 57.4 % (N=67) of MS patients have vitamin D
deficiency. 29 - 45 % of MS patients have cognitive impairment in one or more cognitive
domain. Around 65-75 % of MS patients have mild to moderate anxiety and depression
and around 22-23 % moderate to severe anxiety and depression and approximately 3 %
severe anxiety and depression. We found statistically significant correlations between the
cognitive tests and anxiety and depression scores, and this provides a validation of these
tests that are being administered for the first time in Arabic on MS patients. In addition,
statistically significant positive correlation between MoCA scores and vitamin D level at
baseline and 3 months post vitamin D supplementation. The preliminary results showed
that there was a significant improvement in cognitive performance on most cognitive tests
after 3 months and a significant improvement in vitamin D levels after 3 months of
vitamin D supplementation.
When examining the normal controls the results showed that Vitamin D decreased the
frequency of IL-17+ cells in a dose-dependent manner (maximal inhibition at 10 nM).
Vitamin D significantly reduced expression of IFNγ and TNFα. Vitamin D significantly
reduced expression of CCR6, CCR4, CD161 and reduced expression of RORγt, master
transcription factor of Th17 indicating a proinflammatory phenotype. Also, Vitamin D
reduced the differentiation of naïve CD4+ T cells into Th17 cells and shifted the
balance from a pro-inflammatory phenotype to a more regulatory phenotype.
Vitamin D decreased the pro-inflammatory cytokines secreted by cells put in Th17
polarizing conditions: IL-17, IFNγ, TNFα. In addition, Vitamin D increased the antiinflammatory cytokine IL-4
‫توقيع الباحث‬
4
2014 ‫برنامج دعم البحوث العلمية‬
Grant Program for Scientific Research in Lebanon – 2014
Programme de subvention à la recherche scientifique au Liban – 2014
Final report /
Rapport Final
Warning / Avertissement
1. The final report must be limited to results directly related to the research project
supported by the Council excluding any other activity carried out by the investigator
otherwise the report will be rejected.
2. Appendices may be added or attached to the report.
1. Le rapport final doit être limité aux résultats directement liés au projet de recherche
soutenu par le Conseil à l'exclusion de toute autre activité menée par le chercheur
sous peine de rejet.
2. Des annexes peuvent être ajoutées ou attachées au rapport.
1. Principal investigator / Chercheur principal
Name and surname /
Nom et prénoms
Dr. Samia J. Khoury
Institution of affiliation /
Institution d'affiliation
American University of Beirut Medical center
2. Title of the project as proposed in the original application /
Titre du projet tel qu'il a été proposé dans la demande originale
(English and French / Anglais et Français)
Effect of Vitamin D replacement on immune function and cognition in MS
patients
3. Purpose of the project / Objectifs du projet
This is a prospective study to evaluate the effect of vitamin D supplementation in MS patients
with Vitamin D deficiency (serum level <25μg/ml) to those with normal Vitamin D (serum
level >35 μg/ml) on immunological and neuropsychological (cognitive) measures
5
2014 ‫برنامج دعم البحوث العلمية‬
Grant Program for Scientific Research in Lebanon – 2014
Programme de subvention à la recherche scientifique au Liban – 2014
4. Expected outputs / Résultats attendus
1. We expect that subjects with low vitamin D will have evidence of increased proinflammatory functions in vitro and that supplementation with vitamin D will
decrease those responses.
2. We expect that patients with low vitamin D would have lower cognitive function
and that this will be improved by vitamin D replacement
We have a preliminary analysis of cognitive function in the enrolled subjects. That
showed:

55 % of MS patients have vitamin D deficiency

30-75% of MS patients have cognitive impairment in one or more cognitive
domain

~ 35 % of MS patients have mild to moderate anxiety and depression and
around 5 % severe anxiety and depression

Significant correlations between the cognitive tests which provides a
validation of these tests that are being administered for the first time in Arabic on MS
patients

Significant correlations between the anxiety and depression scores

Significant correlation between cognitive measures and age and years of
education
In the laboratory, we were able to optimize the assays needed to conduct the
immunological studies. We have also examined the effect of vitamin D on the
differentiation of Th17 in control subjects.
The following laboratory methods were performed successfully
- Th17 cell polarization Ex-vivo
-
Isolation of Peripheral blood mononuclear cells (PBMCs)
-
Naïve CD4+ T cells isolation
-
Evaluation of naïve CD4+ T cells purity
-
Culture and activation of naïve CD4+ T cells
-
Flow cytometry staining
-
Staining of live cells
-
Extracellular staining of surface markers
-
Intracytoplasmic staining of cytokines and transcription factors
-
Enzyme-Linked Immunosorbant Assay (ELISA)
6
2014 ‫برنامج دعم البحوث العلمية‬
Grant Program for Scientific Research in Lebanon – 2014
Programme de subvention à la recherche scientifique au Liban – 2014
-
Cytometric Bead Array (CBA)
-
Flow cytometric analysis
-
Cytomeric bead array analysis
Preliminary data on immune function showed:
Vitamin D reduced the differentiation of naïve CD4+ T cells into Th17 cells and
shifted the balance from a pro-inflammatory phenotype to a more regulatory
phenotype. Vitamin D decreased the pro-inflammatory cytokines secreted by cells
put in Th17 polarizing conditions: IL-17, IFNγ, TNFα. In addition, Vitamin D
increased the anti-inflammatory cytokine IL-4
5.
Résultats obtenus / Obtained results
5 to 10 pages / 5 à 10 pages
Appendices can be added a the end of this document / Des annexes peuvent être ajoutées à
la fin de ce document
Active enrollment and data collection started on September 20, 2012. Since then, a
total of 68 subjects were screened, 51 subjects met inclusion criteria, 35 subjects
completed the study baseline and 3 months follow up, the remaining 16 subjects are
currently in the study. In the laboratory, we have been optimizing the assays, but
actual running of the samples has not started yet. We are running all the samples from
each subject at the same time to decrease variability. Preliminary analysis of the
demographics and cognitive performance of the first 35 subjects who completed the
study was conducted and showed positive results with a significant improvement in
cognitive performance on most cognitive tests after 3 months.
7
2014 ‫برنامج دعم البحوث العلمية‬
Grant Program for Scientific Research in Lebanon – 2014
Programme de subvention à la recherche scientifique au Liban – 2014
6. Summary table of expected and obtained results / Tableau récapitulatif
des résultats attendus et des résultats obtenus
Expected outputs / Résultats attendus
Obtained results / Résultats obtenus
1. It was expected that patients with low
Vitamin D have increase proliferation to
MBP and MOG and increased production of
pro-inflammatory cytokines (IFNγ and IL-17)
and that Vitamin D supplementation will
decrease this pro-inflammatory profile.
1. In line with the expected output our
Preliminary data on immune function
showed:
Vitamin D reduced the differentiation of
naïve CD4+ T cells into Th17 cells and
shifted the balance from a pro-inflammatory
phenotype to a more regulatory phenotype.
Vitamin D decreased the pro-inflammatory
2. It was expected that low Vitamin D has a cytokines secreted by cells put in Th17
negative effect on cognitive performance and polarizing conditions: IL-17, IFNγ, TNFα.
that Vitamin D supplementation will improve In addition, Vitamin D increased the anticognitive function.
inflammatory cytokine IL-4
2. In line with the expected output our
preliminary analysis of cognitive function in
the enrolled subjects showed:






55 % of MS patients have vitamin D
deficiency
30-75% of MS patients have
cognitive impairment in one or more
cognitive domain
~ 35 % of MS patients have mild to
moderate anxiety and depression
and around 5 % severe anxiety and
depression
Significant correlations between the
cognitive tests which provides a
validation of these tests that are
being administered for the first time
in Arabic on MS patients
Significant correlations between the
anxiety and depression scores
Significant correlation between
cognitive measures and age and
years of education
8
2014 ‫برنامج دعم البحوث العلمية‬
Grant Program for Scientific Research in Lebanon – 2014
Programme de subvention à la recherche scientifique au Liban – 2014
7. Possible encountered difficulties / Difficultés éventuelles rencontrées
Delay in receiving chemicals and reagents.
Delay in receiving lab equipemnts
8. Scientific publications )articles in peer review journals, books,
communications, etc …) / Publications scientifiques (articles dans des
revues à comité de lecture, livres, communications, etc …)
None yet. We expect to submit at least 3 original research articles based on the results.
One to disseminate the immunological findings, the second about the cognitive deficits
findings and the third is psychometric analysis of the cognitive testing tools used for the
first time in Arabic.
9. Oral presentations or posters in national, regional and international
conferences / Présentations orales ou affichées à des congrès nationaux,
régionaux ou internationaux.
None was presented yet. We expect to present the findings at the annual European
committee for treatment and Research in Multiple Sclerosis and national and regional
conferences like the Middle East Medical Assembly conference.
9
2014 ‫برنامج دعم البحوث العلمية‬
Grant Program for Scientific Research in Lebanon – 2014
Programme de subvention à la recherche scientifique au Liban – 2014
How to submit the final report ?
Comment soumettre le rapport final ?
-------The final report must be submitted to Council in two versions :
 A hard copy which can be mailed or delivered directly to the Council
administrative seat;
 An electronic version, Word document, on CD-ROM or USB drive or email sent to the Council at the following address : grp@cnrs.edu.lb
Le rapport final doit parvenir au Conseil en deux versions :
 Une version sur papier qui peut être envoyée par la poste ou déposée
directement au siège administratif du Conseil ;
 Une version électronique en format Word sur CD-ROM ou sur clé USB, ou
envoyée au Conseil par e-mail à l'adresse suivante : grp@cnrs.edu.lb
10
2014 ‫برنامج دعم البحوث العلمية‬
Grant Program for Scientific Research in Lebanon – 2014
Programme de subvention à la recherche scientifique au Liban – 2014
‫برنامج دعم البحوث العلمي في لبنان لعام ‪2014‬‬
‫صفح ‪8‬‬
‫‪--------‬‬‫‪ .10‬تقديم التقرير النهائي‪:‬‬
‫‪ .10.1‬في نهاية المشروع (سنة أو سنتين)‪ ،‬على الباحث تقديم تقرير نهائي (نسخة ورقية ونسخة‬
‫إلكترونية بصيغة ‪ Word‬على قرص مدمج أو ‪ USB‬أو ترسل إلى المجلس بواسطة البريد‬
‫االلكتروني على العنوان التالي‪ ،grp@cnrs.edu.lb :‬وذلك وفقاً للنموذج المعتمد في المجلس‬
‫والموجود على موقع المجلس ‪ http://www.cnrs.edu.lb‬مرفقاً بالتصفية المالية لمشروع‬
‫يبين فيه ما‬
‫البحث‪ .‬ال يقبل التقرير النهائي إالّ إذا عرض الباحث بشكل واضح جدوالً مفصالً ّ‬
‫تم إنجازه مقارن مع تصوره لمخرجات المشروع عند قبوله‪ ،‬على أن ال يتضمن سوى ما له‬
‫عالقة مباشرة بمشروع البحث المدعوم من المجلس دون إغراقه بأية تفاصيل أو نشاطات أخرى‬
‫والتركيز حص اًر على النتائج التي توصل اليها الباحث‪.‬‬
‫‪ .10.2‬يعتمد المجلس في تقييم التقرير النهائي على األهمية العلمية للمقاالت الصادرة عن الباحث‬
‫وذات العالقة بمشروع البحث المدعوم من المجلس من خالل عدد من المعايير والمؤشرات‬
‫الدولية نذكر من بينها على سبيل المثال ‪Impact Factor, Citation Index:‬‬
‫‪11‬‬
‫برنامج دعم البحوث العلمية ‪2014‬‬
‫‪Grant Program for Scientific Research in Lebanon – 2014‬‬
‫‪Programme de subvention à la recherche scientifique au Liban – 2014‬‬