Effect of High-fiber Supplement in Multiple Sclerosis
Phase: PHASE1
Conditions: Multiple Sclerosis
Sponsor: Suhayl Dhib-Jalbut, MD

Protocol Title: 
Effect of a high-fiber supplement in Multiple Sclerosis 
PI- Dr. Suhayl Dhib-Jalbut 
Protocol: Version 8 23 Mar 2023
IND 153352
INTERVENTIONAL
RESEARCH PROTOCOL TEMPLATE
(HRP-503a)
STUDY INFORMATION
Title of Project:
 Effect of a high-fiber supplement in Multiple Sclerosis 
IND 153352  (NBT-NM108)
Principal Investigator Name
Suhayl Dhib-Jalbut, MD 
Principal Investigator Div. & Dept.
Neurology
Principal Investigator Contact Info: 
               jalbutsu@rutgers.edu
125 Paterson Street Suite 6100 New Brunswick NJ 08901
(732) 235 - 8593
Protocol Version and Date:
Version 6 – 25May2022
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Protocol Title: 
Effect of a high-fiber supplement in Multiple Sclerosis 
PI- Dr. Suhayl Dhib-Jalbut 
Protocol: Version 8 23 Mar 2023
IND 153352
Table of Contents
Skip To Section: Hold CTRL + Click (Below) To Follow Link in Blue
1.0
Research Design
1.1
Purpose/Specific Aims
1.2
Research Significance
1.3
Research Design and Methods
1.4
Preliminary Data
1.5
Sample Size Justification
1.6
Study Variables
1.7
Drugs/Devices/Biologics
1.8
Primary Specimen Collection
1.9
Interviews, Focus Groups, or Surveys
1.10
Timetable/Schedule of Events
2.0
Project Management
2.1
Research Staff and Qualifications
2.2
Resources Available
2.3
Research Sites
3.0
Multi-Site Research Communication & Coordination
3.1
Outside Research
4.0
Research Data Source/s
4.1
Primary Data – Subjects and Specimens
4.2
Subject Selection and Enrollment Considerations
4.3
Subject Randomization
4.4
Secondary Subjects
4.5
Number of Subjects
4.6
Consent Procedures
4.7
Special Consent Populations
4.8
Economic Burden and/or Compensation For Subjects
4.9
Risks to Subjects
4.10
Secondary Data – Record/Chart Reviews, Databases, Tissue Banks, Etc.
4.11
Chart/Record Review Selection
4.12
Secondary Specimen Collection
5.0
Special Considerations
5.1
Health Insurance Portability and Accountability Act (HIPAA)
5.2
Family Educational Rights and Privacy Act (FERPA)
5.3
NJ Access to Medical Research Act
5.4
Code of Federal Regulations Title 45 Part 46 (Vulnerable Populations)
6.0
Research Data Protection and Reporting
6.1
Data Management and Confidentiality
6.2
Data Security
6.3
Data Safety And Monitoring
6.4
Reporting Results
6.5
Data Sharing
7.0
Data and/or Specimen Banking
8.0
Other Approvals/Authorizations
9.0
Bibliography
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Protocol Title: 
Effect of a high-fiber supplement in Multiple Sclerosis 
PI- Dr. Suhayl Dhib-Jalbut 
Protocol: Version 8 23 Mar 2023
IND 153352
1.0 Research Design
1.1 Purpose/Specific Aims
A.  Objectives 
1) To assess the effect of a high-fiber supplement (HFS) diet on gut microbiota and immune cells 
in Multiple Sclerosis (MS).
2) To identify biomarkers of disease exacerbation in relapsing remitting (RR)-MS.
B.  Hypotheses / Research Question(s)
Increase in consumption of high-fiber diet could be beneficial for neurodegenerative diseases 
including MS. Recently, our collaborator, Dr. Liping Zhao, developed a HFS and his study suggests 
an association between intake of HFS and reduced gut dysbiosis and the growth of SCFA-producing gut 
bacteria in Type-II diabetes mellitus (T2DM). Since MS is associated with gut dysbiosis and decreased 
production of SCFA, we will investigate the effect of HFS on MS associated gut dysbiosis. 
Currently, there is no patent present, but the submission of this patent is in process.
1.2 Research Significance
It has been suggested that dysbiosis of gut commensal bacteria is associated with increased risk of 
autoimmune diseases including MS. However, there is no viable intervention available to correct 
dysbiosis. Since, intake of HFS is associated with the growth of healthy bacteria in the gut, we 
propose to investigate the effect of HFS on the growth of SCFA-producing gut bacteria and 
development of regulatory cells in MS. If this study is successful, consumption of HFS could be 
beneficial therapeutic supplement to disease modifying drugs in halting MS progression and would 
justify the launch of a clinical trial with this HFS. 
Additionally, it is necessary to find a non-invasive biomarker for gut dysbiosis-mediated CNS 
autoimmunity in MS. Since we found that fecal Lcn-2 is a biomarker of gut dysbiosis-mediated 
CNS autoimmunity in MS animal models, we will investigate the association of fecal Lcn-2 levels 
with disease activity in RR-MS. If this result is positive, fecal Lcn-2 could become one of the 
biomarkers for disease exacerbation in RR-MS. 
1.3 Research Design and Methods
RR-MS patients will be recruited from the Robert Wood Johnson Center for MS under the 
direction of Dr. Suhayl Dhib-Jalbut. The patients will be asked to participate in the study during 
their regular clinical visit to the MS Center. 
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Protocol Title: 
Effect of a high-fiber supplement in Multiple Sclerosis 
PI- Dr. Suhayl Dhib-Jalbut 
Protocol: Version 8 23 Mar 2023
IND 153352
Inclusion criteria includes patients aged 21-65 Relapsing Remitting Multiple Sclerosis (RRMS) 
who met the 2017 McDonald criteria. 
The first group (Group A) of MS patients (n=25) will receive a dietary fiber supplementation. 
They will consume 60 g/day of HFS for 12 weeks (High-fiber supplement group, Group-A).  The 
supplement consists of a mixture of soluble fibers (from inulin and fibersol-2) and insoluble fibers 
(from oat bran, corn bran, wheat bran and sorghum bran) and it will be consumed as a drink.
The second group of MS patients (Group B) (n=25) will not consume any high fiber supplement.  
The first group (Group C) of healthy volunteers (n=25) will receive a dietary fiber 
supplementation. They will consume 60 g/day of HFS for 12 weeks. Healthy volunteers will not 
have MS.  
The second group (Group D) of healthy volunteers (n=25) will not consume any high fiber 
supplement. 
For Groups A, B, C & D stool, and serum samples will be collected at 3 time points (baseline; 
Week 8; & Week 12).
Group E (n=25) will consist of 25 subjects with MS who experience an MS flare up. 
These 25 subjects can be those participants with MS who are already part of the above study 
(Group A or B) and consent to 2 extra blood and 2 extra fecal samples collected at the time of the 
acute flare up and upon resolution of symptoms. 
Screening visits will typically take place during routine MS follow up visit at the Robert Wood 
Johnson Center for MS. During this time, eligibility will be reviewed, and consent will be obtained 
once the patient has had time to review the study, have questions answered, and agree to 
participate. During this screening visit, details regarding the patients MS history, food 
allergies/intolerances, medical history, and medications will be recorded. Eligible participants will 
be asked to provide blood and stool samples for baseline assessment. 
A. Research Procedures
During the baseline visit, all MS patients and HDs will provide a blood and stool sample. For MS 
patients and HDs who are in the HFS group, they will be provided with supplies and instructions 
to take the supplement (Appendix 5). All the participants will be provided with stool kits and 
instruction on stool collection. Stool samples will be collected at home and transported to the 
laboratory by the participant in Piscataway for analysis.
Group A and Group C:  MS patients in Group A and HDs in Group C will consume supplement 
orally as a drink two times a day to ensure fiber intake of 60 g/day for 12 weeks (High-fiber 
supplement group, Group A), as shown in Dr. Zhao’s T2DM study (1). Participants will gradually 
increase the dosage of supplement over several days to mitigate the potential tolerability issues, 
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Protocol Title: 
Effect of a high-fiber supplement in Multiple Sclerosis 
PI- Dr. Suhayl Dhib-Jalbut 
Protocol: Version 8 23 Mar 2023
IND 153352
starting with daily consumption of 30g/day for first 5 days and then increasing to targeted daily 
consumption of 60g/day. 
A member of the research team will follow up by phone at the end of the first week to check for 
compliance and adverse effects and then every month thereafter (Appendix 6). Patients will receive 
the fiber supplement in individual sachet (30g/sachet) by mail every two weeks, and they will be 
asked to return any unused supplement in their next study visit. Since the risks of study drug on 
embryofetal development are unknown, participants in Group A and Group C will be required to 
use contraception methods. Please refer section 5 special considerations for more details.
At baseline, Week 8 and week 12, 6 samples (3 blood and 3 fecal sample) will be collected from 
each subject. 
All MS patients will be followed prospectively, and one more blood and one more fecal sample 
will be collected during relapse (before steroid treatment) and upon symptom resolution, and 
examined for intestinal inflammation, permeability, and microbial composition. The concentration 
of intestinal inflammation biomarkers in feces including Lcn-2, Calprotectin, and Lactoferrin, and 
a biomarker for intestinal permeability, serum zonulin, will be measured by ELISA assay. 
As a control we will also analyze these biomarkers in the fecal samples isolated from healthy 
donors (n=25) in Group C and Group D. Healthy donors will be asked to provide 3 blood and 3 
stool samples over the course of 12 weeks (baseline, week 8, and week 12).
Group E will consist of 25 subjects with MS who experience an MS flare up. 
These 25 subjects will be those participants with MS who are already part of the above study 
(Group A or B) and consent to 2 extra blood and 2 extra fecal samples collected at the time of the 
acute flare up and upon resolution of symptoms. These subjects will be asked to sign a separate 
consent form agreeing to be part of the MS flare up group (n=25). Since around 50% of RRMS 
patients relapse at least once per year, we anticipate recruiting 25 relapsing RRMS patients from 
Group A and B in 1 years (2). RRMS flare up and resolution will be determined by Dr. Suhayl 
Dhib-Jalbut.
Table-1. Schedule of specimen collection
Screening/Baseline 
assessment
8 weeks 
post HFS 
12 weeks 
post HFS
Unscheduled visit 
at MS Relapse
Unscheduled 
visit 
at MS remission
MS history 
Medical history
Medication history
Food Allergy
Consent 
Blood draw*
Fecal collection*
Blood draw 
Fecal collection
Blood draw 
Fecal collection
Blood draw** 
Fecal collection**
Blood draw 
Fecal collection
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Protocol Title: 
Effect of a high-fiber supplement in Multiple Sclerosis 
PI- Dr. Suhayl Dhib-Jalbut 
Protocol: Version 8 23 Mar 2023
IND 153352
*Sample collection will be done on the day of screening or before high fiber supplement.
** Sample collection will be done at MS relapse before steroid treatment.
Sample collection: 
1. Stool Sample:  Subjects will be asked to collect a single-stool sample without any laxative or 
dietary restrictions using a sample collection kit (Appendix 1). The collected stool sample will be 
brought to the MS research laboratory by the participants. If participants cannot bring the stool 
sample to the MS research laboratory, our lab staff will pick up the stool sample.  The stool sample 
will be examined for the composition of gut bacteria. The stool sample will also be transplanted 
into an animal model of MS and examined for the development of experimental autoimmune 
encephalomyelitis (EAE). 
2. Blood Draw: All participants will visit RWJ MS center to provide a blood specimen.  Each 
subject will have a blood draw (80ml – about 5 tablespoons) as shown in Table-1.  The immune 
cells will be isolated from each blood specimen and the presence of pathogenic and regulatory 
immune cells will be analyzed. 
*NOTE: It is requested that all blood work be fasting. Nothing to eat or drink 10-12 hours before 
blood draw. Subjects may have water &/or black coffee /tea. 
Fasting is very important to check the levels of short-chain fatty acid in the serum.
B. Experiment
1. Examine the effect of HFS on production of SCFA, intestinal microbial composition, and 
intestinal permeability  
Multiple sclerosis is associated with gut dysbiosis. Notably, gut dysbiosis in MS is associated with 
the loss of bacterial taxa involved in fermentation of dietary fiber and the production of SCFA (3-
5). Since SCFA can promote the differentiation of Foxp3Tregs, a decrease in SCFA-producing 
bacteria in gut microbiota would be a risk factor for MS. Recently, our collaborator, Dr. Liping 
Zhao at our institution, developed a HFS. His recent study suggests an association between intake 
of HFS and reduced gut dysbiosis and increased abundance of short chain fatty acid (SCFA)-
producing gut bacteria in T2DM (1). Since increase in SCFA-producing bacteria may be beneficial 
for inflammatory diseases including MS, we will investigate whether HFS can promote the growth 
of SCFA-producing bacteria in MS patients and promote the development of regulatory immune 
cells.
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Protocol Title: 
Effect of a high-fiber supplement in Multiple Sclerosis 
PI- Dr. Suhayl Dhib-Jalbut 
Protocol: Version 8 23 Mar 2023
IND 153352
The stool and serum will be collected at 3 time points (pre-HFS; 8 and 12 weeks after consumption 
of HFS) from MS patients.  Similar samples will be obtained from the HFS treated and non-treated 
control group at similar time points. Fecal DNA will be extracted using the QIAamp PowerFecal 
DNA Kit (QIAGEN). DNA quality and quantity will be measured using the NanoDrop™ One 
microvolume UV-Vis Spectrophotometer (Thermofisher) and Qubit 1x dsDNA HS assay 
(ThermoFisher) respectively. Gut bacterial composition will be examined by 16S rRNA gene 
sequencing using the Ion GeneStudio S5 (Thermofisher) in Dr. Liping Zhao’s lab. QIIME 2 will 
be used to analyze the sequencing data to identify the amplicon sequence variants (ASVs). ASVs 
will then be grouped into guilds based on their abundance correlations. Biomarkers and 
microbiome-based prediction models will be explored at both the ASV and guild level using 
statistical methods such as Random Forest and Partial Least Squares regression models. Samples 
of potential significance will be shortlisted for subsequent whole genome metagenomic 
sequencing. Concentration of fecal and serum SCFAs will be measured by Rutgers metabolomics 
core facility. Since Vitamin-D (Vit-D) is one of the most important nutrients that affect immune 
modulation (6, 7), we will also measure serum Vit-D levels. 
Next, we will assess the effect of HFS on intestinal permeability. MS is associated with gut 
dysbiosis and intestinal permeability (3-5, 8). Therefore, we will examine gut intestinal 
permeability (IP) by measuring the serum levels of zonulin, which is a biomarker of IP (9), using 
a zonulin ELISA kit
PBMCs will be stained with anti-CD3, CD4, Foxp3, CD25, and CD127 mAbs and 
CD3+CD4+CD25+CD127lo Foxp3+population in a CD4 T cell compartment will be analyzed by 
flow cytometry using Kaluza software. Development of Th1, Th17, and Th-GMCSF will be 
analyzed by flow cytometry with anti-CD4, CD3, IFN-γ, IL-17A, and GM-CSF mAbs. Since low 
expression of E3 ubiquitin ligase genes is associated with MS disease exacerbation, we will 
examine the expression of E3 ubiquitin ligase genes.  Naïve and memory/effector T cells will be 
isolated from PBMCs by naïve-memory isolation MACS beads and expression of CBLB, ITCH, 
and GRAIL genes will be examined by q-PCR. Since PD-1:PD-L1 signaling is important to 
prevent CNS autoimmunity and a fiber rich vegetarian diet can promote PD1:PD-L1 signaling (10-
12), we will also study the expression of PD-1 on CD4+ T cells and PD-L1 on 
monocytes/macrophage and DCs using flow cytometry. PBMCs will be stained with anti-CD4, 
CD3, CD11c, CD11b, CD14, PD-1 and PD-L1 mAbs, and CD3+CD4+PD-1+ population in CD4+ 
T cells; and CD11c+PD-L1+ (DC), CD11b+PD-L1+ (macrophage) and CD14+PD-L1+ 
(monocyte) cell compartment will be analyzed by flow cytometry. To examine the production of 
inflammatory cytokines in antigen presenting cells, DCs and macrophages isolated from PBMCs 
will be cultured in the presence or absence of exogenous lipopolysaccharides (LPS) for 3 days and 
production of IL-6, IL-12, IL-1, and IL-23 will be examined by ELISA.
2. Examine the effect of HFS-induced gut microbiota on EAE in a 3A6/DR2a MS animal 
model: 
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Protocol Title: 
Effect of a high-fiber supplement in Multiple Sclerosis 
PI- Dr. Suhayl Dhib-Jalbut 
Protocol: Version 8 23 Mar 2023
IND 153352
Human microbiota-associated (HMA) mice, in which the human gut microbiota is established in 
germ-free mice via fecal transplantation, has been used for evaluation of pathogenic and beneficial 
role of gut microbiota in human health (13). Several studies have shown the usefulness of this 
model that can mimic the pathophysiology of human diseases such as asthma, pregnancy-induced 
adiposity, and obesity (14-18). 
We will establish a germfree 3A6/DR2a at Taconic Inc. and expand them in our facility. Since 
3A6/DR2a mice are prone to develop EAE between the age of 6 to 10 week-old, 4 week-old mice 
will be used for creating HMA-3A6/DR2a mice. Fecal samples will be collected from HDs and 
MS patients on regular diet and HFS at 3 time points (pre-treatment; 8, and 12 weeks after 
treatment) and the fecal sample will be transplanted into 3A6/DR2a mice (20 mice in each group). 
Each fecal sample (0.5 g) will be diluted in 25 mL of a sterile Ringer working buffer  in an 
anaerobic chamber. The fecal material will allowed to settle by gravity and the clarified 
supernatant will be mixed with an equal volume of 20% (w/v) skimmed milk (Oxoid). Mice will 
be orally administered with 100 μL of fecal suspension inoculum. The development of spontaneous 
EAE will be monitored until the age of 20 weeks.  
We will also assess the microbial composition and intestinal permeability in the transplanted mice. 
The fecal samples will be collected every 7 days post fecal transplantation and gut microbiota 
composition will be examined by sequencing of V4 region of 16S rRNA gene using iontorrent 
sequencer (Thermofisher) as described earlier. To measure the intestinal permeability, the 
transplanted mice will be administered with 150 l of FITC dextran by oral gavage and appearance 
of FITC-dextran in the blood will be measured 4 h after oral inoculation. The serum FITC-dextran 
concentration will be measured using a fluorimeter. Since serum levels of endotoxin is also a 
biomarker of intestinal permeability, serum endotoxin activity will be measured using Pierce™ 
Limulus Amebocyte Lysate (LAL) Chromogenic Endotoxin Quantitation Kit. 
We will next assess the development of pathogenic and regulatory immune cells. Three mice in 
each group will be euthanized at the age of 6-10 week-old and development of pathogenic Th1, 
Th17, and Th-GMCSF cells in intestinal lamina propria, mesenteric lymph nodes, spleen, and CNS 
will be examined by flow cytometry with anti-CD4, Vβ5.1, CD3, IFN-γ, IL-17A, GM-CSF mAbs. 
Development of Foxp3 Tregs will be examined by flow cytometry with anti-Foxp3, GITA, CD4, 
and Vβ5.1 mAbs. Based on our earlier findings (19), down-regulation of E3 ubiquitin ligase, 
CBLB and ITCH, will be examined by q-PCR.  
We will also examine the development of inducible EAE in C57BL/6 mice transplanted with fecal 
samples. Germ-free C57BL/6 mice will be purchased from Taconic Inc. and expanded for the 
experiment. The fecal samples will be collected from regular diet- and HFS-treated HDs and MS 
patients as described above and transplanted into the germ-free C57BL/6 mice (10 in each group). 
The transplanted mice (7-9 week-old) will be immunized with MOG35-55 at 200 ug/mouse and 
injection with pertussis toxin at 100 ng/mouse. Development of EAE will be examined for 40 days. 
Demyelination and axonal loss in the brain and spinal cord will be examined by immunohistology 
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Protocol Title: 
Effect of a high-fiber supplement in Multiple Sclerosis 
PI- Dr. Suhayl Dhib-Jalbut 
Protocol: Version 8 23 Mar 2023
IND 153352
with anti-MBP and -Neurofilament Abs, respectively. Microbial composition, intestinal 
permeability, and development of Th-1, Th-17, Th-GMCSF and Foxp3 Tregs will be examined as 
described above. 
3. Explore biomarkers of MS-associated gut dysbiosis and response to HFS: 
RR-MS and healthy donors (21-55 year-old) from RWJ MS Centers will be asked to participate in 
this study according to institutional IRB guidelines. In this study, fecal and blood samples will be 
collected from RR-MS patients during relapse (before steroid treatment) and later during remission 
and examined for intestinal inflammation, permeability and microbial composition. We will enroll 
25 patients based on sample size calculation using related previous studies with a statistical power 
of 80% (8, 20). The fecal samples (200 mg) will be dissolved in 0.5 ml PBS containing proteinase 
inhibitor and the supernatant will be collected. The concentration of intestinal inflammation 
biomarkers including Lcn-2, Calprotectin, and Lactoferrin, and a biomarker for intestinal 
permeability, serum zonulin, will be measured by ELISA assay. We will examine the association 
between increase in intestinal inflammation/permeability and disease activation. 
The fecal samples collected from HFS-treated and un-treated MS patients at the 3 time points 
described in Aim-1, will be analyzed for intestinal inflammation and permeability as described 
above. We will simultaneously examine the microbial composition in the fecal samples as 
described in Aim-1 to determine if an association between MS relapse and gut dysbiosis exists. 
C. Study Duration
Subjects participation will be 12-week period. 
Study Timetable:
Upon IRB approval-Sept. 2023   Collection of blood and fecal specimens
Sept. 2021-Sept. 2023   Analysis of blood and fecal specimens   
Sept. 2021-Aug. 2023   Fecal transplantation in germ free mice and analysis for EAE   
D. Endpoints
The primary endpoint will be detection of effect of HFS on intestinal microbial composition, 
intestinal permeability, production of SCFA, intestinal permeability, and immune cells in 25 MS 
patients between before and after intake of high-fiber supplement. 
The secondary endpoint will be detection of biomarkers for intestinal inflammation-associated 
disease exacerbation in 25 MS patients.
1.4 Preliminary Data
A. A high-fiber supplement selectively promotes a group of SCFA producing bacteria. 
Recently, our collaborator, Dr. Liping Zhao at our institution, developed a high-fiber supplement 
(HFS). His study suggests an association between intake of HFS and reduced gut dysbiosis and 
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Protocol Title: 
Effect of a high-fiber supplement in Multiple Sclerosis 
PI- Dr. Suhayl Dhib-Jalbut 
Protocol: Version 8 23 Mar 2023
IND 153352
increased abundance of short chain fatty acid (SCFA)-producing gut bacteria, and demonstrated 
that HFS can ameliorate type-2 diabetes mellitus (T2DM) (1). This beneficial effect was associated 
with increased levels of SCFA, butyric acid (Fig.1B). Furthermore, HFS promoted the growth of 
15 SCFAs-producing bacteria which mostly peaked at day 28 and remained stable afterward till 
the end of the trial (Day 84), while only 3 acetate producers among the 15 SCFA-producing 
bacteria were promoted in controls (Fig.1C). To test whether HFS-promoted microbiota can 
contribute to glucose homeostasis, fecal samples isolated from HFS-treated (W group) and non-
treated (U group) T2DM patients were transplanted into germ-free mice and glucose homeostasis 
was examined in the transplanted mice. As shown in Fig.2, the transplanted microbiota from HFS-
treated group improved glucose homeostasis. Since increase in SCFAs-producing bacteria may be 
beneficial for inflammatory diseases including MS, we will investigate whether HFS can promote 
the growth of SCFAs-producing bacteria in MS patients and promote the development of 
regulatory immune cells.
Fig. 1. A high-fiber diet selectively promotes a group of SCFA producers as the major active producers. (A and B) Changes in fecal 
concentrations of acetic acid and butyric acid. SCFAs were measured using gas chromatography, and amounts are expressed as milligrams per 
gram of dry feces (± SE). (C) Time-course changes in the abundance of the active SCFA producers. The sizes and colors of the circles indicate the 
average abundance and the coefficient of variance (CV) of the abundance of the strains, respectively. W group: intervention group on high-fiber 
diet composed of whole grains, traditional Chinese medicinal foods, and prebiotics. U group: control group consuming diet recommended based 
on the 2013 Chinese Diabetes Society guidelines for T2DM. Two-way repeated-measures ANOVA with Tukey’s post hoc test was used for intra- 
and intergroup comparisons. *P < 0.05, **P < 0.01, and ***P < 0.001 for comparison with day 0; #P < 0.05 for comparison with the U group at the 
same time point. n = 27 patients in the W group and 16 patients in the U group.
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Protocol Title: 
Effect of a high-fiber supplement in Multiple Sclerosis 
PI- Dr. Suhayl Dhib-Jalbut 
Protocol: Version 8 23 Mar 2023
IND 153352
Fig. 2. Transplantation of fiber-altered gut microbiota improves glucose tolerance in mice. Fecal samples isolated from HFS-treated (W group) 
and non-treated (U group) T2DM patients were transplanted into germ-free mice before (W-pre and U-pre) and 84 days after treatment (W-post 
and U-post). Body weight (A), fasting blood glucose (FBG) (B) and fasting insulin (C) were examined 4 weeks after transplantation. Mice receiving 
transplant: n = 5 for W-Pre, W-Post, U-Pre and n = 4 for U-Post. *P <0.05, **P <0.01 and ***P <0.001 using one way ANOVA with a Tukey’s 
post-hoc test for intra- and inter-group comparisons. 
B. Gut-dysbiosis promotes spontaneous EAE in 3A6/DR2a mice 
To examine the effect of gut microbiota on CNS autoimmunity, we created transgenic (Tg) mice 
that express the MS-associated HLA-DR2a gene and TCR gene specific to MBP87-99/HLA-DR2a 
that had been isolated from a 3A6 T cell clone derived from an MS patient (21) (Fig.3). The 
3A6TCR/HLA-DR2a Tg mice were further crossed with MHC class II-KO mice to replace 
endogenous MHC class II with HLA-DR2a (referred to as 3A6/DR2a Tg mice). Interestingly, the 
MBP-specific 3A6 TCR Tg T cells do, in fact, spontaneously differentiate into GM-CSF+ Th1 and 
Th17 cells, and 20–30% of the 3A6/DR2a Tg mice develop spontaneous EAE in our SPF animal 
facility (Table 2) (19). Microbial infection has been suggested to be a possible etiology for MS; 
therefore, we examined the effect of gut microbiota on the development of spontaneous EAE. As 
shown in Fig. 4A, antibiotic treatment abrogated the development of spontaneous EAE, suggesting 
that the microbiota plays an essential role in the development of CNS autoimmunity. Gut dysbiosis, 
which alters the gut bacterial composition, has been associated with MS (3-5). Since fecal IgM 
levels increase in response to pathogenic enteric bacteria that lead to gut dysbiosis (22, 23), we 
examined IgM levels in fecal samples isolated from EAE and non-EAE mice.  We found that 
increases in fecal IgM levels were associated with decreases in Foxp3 and CBLB gene expression 
and a subsequent development of spontaneous EAE (Fig.4B.C, and D). We then examined the 
microbial composition of fecal samples by 16S RNA gene analysis and found a marked expansion 
of B. vulgatus, B. acidifaciens, and B. xylanisolvens in the gut, which was associated with dysbiosis 
and the development of spontaneous EAE (Fig4.E) (19). These data suggest that gut dysbiosis is 
highly associated with a reduction in peripheral tolerance and the subsequent development of CNS 
autoimmunity. We will use this MS animal model to investigate the effect of HFS on CNS-
autoimmunity.  
Fig.3. Schematic diagram showing the expression of MS-associated HLA-DR2a gene and TCR gene specific to MBP87-99/HLA-DR2a in 
Transgenic (Tg) mice.
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Protocol Title: 
Effect of a high-fiber supplement in Multiple Sclerosis 
PI- Dr. Suhayl Dhib-Jalbut 
Protocol: Version 8 23 Mar 2023
IND 153352
Table-2. Development of spontaneous EAE in 3A6/DR2a Tg mice.
Fig. 4. Gut-dysbiosis promotes spontaneous EAE in 3A6/DR2a mice. (A) Antibiotic treatment effect on the development of spontaneous EAE. 
Breeding pairs of 3A6/DR2a Tg mice were treated with a cocktail of ampicillin (1 mg/ml), metronidazole (1 mg/ml), neomycin (1 mg/ml), and 
vancomycin (0.5 mg/ml), and their offspring were continuously treated with antibiotics. The development of EAE was examined until 20 weeks of 
age (***P<0.001). (B) Development of spontaneous EAE in fecal IgM-lo (Rederived), fecal IgM-lo, and IgM-hi mice. IgM-lo and -hi mice were 
determined by average fecal IgM concentration during the period from 5 weeks to 10 weeks of age. IgM-lo mice: <2.0 ng/mg total fecal protein, 
IgM-hi mice: ≥2.0 ng/mg total fecal protein. 3A6/DR2a Tg mice (n=214) were divided into fecal IgM-hi (n=58) and fecal IgM-lo (n=156) groups. 
Development of EAE was examined in fecal IgM-lo (Rederived), fecal IgM-lo, and fecal IgM-hi mice. (****P<0.0001). (C) Down-regulation of 
E3 ubiquitin ligase genes in CD4+CD25– T cells is correlated with the up-regulation of the complement C3 gene in whole spleen cells. Expression 
of E3 ubiquitin ligase Cbl-b and complement C3 in CD4+CD25– T cells and whole spleen cells, respectively, were measured by qPCR. (D) 
Frequency of CD4+ Vβ5.1+ Foxp3+ T cells in the CD4+ Vβ5.1+ T cell compartment of 5 to 10-week-old mice is shown. Mean+/- SEM (n=3-4). 
(*P<0.05, **P<0.01). (E) Fecal IgM levels were examined by ELISA and fecal DNA was isolated from rederived, fecal IgM-lo (<2.0 ng/mg total 
protein) non-EAE, fecal IgM-hi (≥2.0 ng/mg total protein) non-EAE, and IgM-hi EAE 3A6/DR2a Tg mice. Enteric bacterial species were identified 
by Illumina 16S rRNA sequence analysis. n=5~7.        
C. Fecal Lcn-2 is a biomarker of spontaneous EAE 
We had identified a biomarker for EAE-associated gut dysbiosis. IgM is produced in response to 
expanded enteric bacteria in the gut (22, 23); therefore, we examined the correlation between gut 
dysbiosis and fecal IgM levels in 3A6/DR2a Tg mice. As shown in Fig.4B, increase in fecal IgM 
was associated with development of spontaneous EAE. Fecal Lcn-2 is a well-known biomarker for 
gut dysbiosis in IBD as described in the background (20, 24); therefore, we also examined the levels 
of fecal Lcn-2 in healthy and EAE 3A6/DR2a Tg mice. As we expected, there was a high 
association between fecal IgM and fecal Lcn-2 (Fig. 5A) and increase in Lcn-2 levels was 
associated with development of spontaneous EAE (Fig.5B). Interestingly, fecal IgM levels 
increased 1-2 weeks before onset of spontaneous EAE; while fecal Lcn-2 levels increased 3-4 
weeks before disease onset (Fig. 5C), suggesting that fecal Lcn-2 is an earlier and more sensitive 
biomarker for CNS autoimmunity than fecal IgM. We also detected the increase in fecal Lcn-2 
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Protocol Title: 
Effect of a high-fiber supplement in Multiple Sclerosis 
PI- Dr. Suhayl Dhib-Jalbut 
Protocol: Version 8 23 Mar 2023
IND 153352
levels in C57BL/6 mice upon induction of EAE (Fig. 5D). These spontaneous and inducible EAE 
data suggest that fecal Lcn-2 may be a possible biomarker for CNS autoimmune diseases including 
MS. Therefore, we will investigate the effect of HFS on intestinal inflammation and association 
between intestinal inflammation and disease exacerbation in MS using intestinal biomarkers 
including Lcn-2.
Fig.5. Fecal Lcn-2 is a biomarker of spontaneous EAE. (A )Association between fecal IgM levels and lipocalin-2 production. Fecal samples 
were collected from healthy and EAE mice, and concentration of IgM and lipocalin-2 was measured by ELISA. (B) Association between fecal 
Lcn-2 levels and spontaneous EAE. (C)  Fecal Lcn-2 is a more sensitive biomarker for spontaneous EAE. Fecal Lcn-2 levels before and after 
clinical EAE onset are shown. Mean ± SEM (n = 10). (D) Increase in Lcn-2 upon EAE induction in C57BL/6 mice. EAE was induced in C57 BL/6 
mice by Immunization with Mog35-55/CFA and injection with pertussis toxin. Fecal Lcn-2 levels were analyzed by ELISA. 
                        
1.5 Sample Size Justification
Our collaborator, Dr. Liping Zhao employed n = 27 in the HFS-treated group and n=16 in non-
treated group and observed a significant effect of HFS on SCFA production in type 2 diabetes 
mellitus (T2DM) study (1). Since the therapeutic effect of HFS may be different between MS and 
T2DM, we sought the assistance of Dr. Yong Lin who is a Biostatistician at Rutgers University.  
We selected the published MS studies that showed the effect of high vegetable diet on development 
of Tregs (n=10 in each group in this study) (12), and calculated a reasonable sample size using 
mean and standard deviation of the data, assuming a mean difference of 5% type I error and 80% 
power. Our sample size calculation indicated that n=20 in each group is sufficient to observe the 
statistical difference in the development of regulatory cells. Although there is no published data 
on the effect of diet on intestinal permeability in MS, a study of intestinal permeability in MS 
(n=14) vs healthy donor (HD) (n=16) showed statistically significant increase in intestinal 
permeability in MS patients compared to HD (8). Our sample size calculation also indicated that 
n=20 in each group is sufficient to observe a statistical difference. Based on our previous HFS-
data and sample size calculation, we propose to enrol 25 patients in each group in Aim-1. 
Regarding the sample size in Aim-3, there is no published intestinal inflammation biomarker data 
in MS. Therefore, we used the data from an IBD study (20) where n=20 was sufficient to detect 
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Effect of a high-fiber supplement in Multiple Sclerosis 
PI- Dr. Suhayl Dhib-Jalbut 
Protocol: Version 8 23 Mar 2023
IND 153352
differences in Lcn-2, lactoferrin, and zonulin between IBD vs HD. From our sample size 
calculation we propose to enroll 25 patients in each group in Aim-3.
Summary:
Stool, and blood samples isolated from 50 MS patients and 50 healthy controls will be used for 
gut permeability, bacteria DNA sequencing, immunology experiments, and MS animal 
experiments.
1.6 Study Variables
A. Independent Variables, Interventions, or Predictor Variables 
Experiment 1: Four groups (HDs and MS patients with and without HFS treatment).
Experiment 2: Four groups (Fecal samples from HDs and MS patients with & without HFS 
treatment). 
Experiment 3: Two groups (MS patients with MS relapse and healthy volunteers).
B. Dependent Variables or Outcome Measures 
Experiment-1: Percentage of pathogenic and non-pathogenic bacteria, intestinal permeability,  
and percentage of pathogenic and regulatory immune cells. 
Experiment-2: Incidence and severity of EAE development. 
Experiment-3: Intestinal inflammation and intestinal permeability.
.
1.7 Drugs/Devices/Biologics
 
We will treat HDs and MS patients with HFS consisting of a mixture of soluble (inulin and 
fibersol-2) and insoluble (oat bran, corn bran, wheat bran and sorghum bran) fibers. MS patients 
will consume HFS as drink to achieve a dietary fiber intake of > 60 g/day for  12 weeks.
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Protocol Title: 
Effect of a high-fiber supplement in Multiple Sclerosis 
PI- Dr. Suhayl Dhib-Jalbut 
Protocol: Version 8 23 Mar 2023
IND 153352
The HFS that will be used in this study is a mixture of brans and xanthan gum (49.8% oat bran, 
12.8% corn bran, 18.7% sorghum bran, 14.8% wheat bran and 4% xanthan gum w/w). The brans 
are the major ingredients in the wholegrain, traditional Chinese medicinal foods and prebiotic 
(WTP) diet that we used in reference #4 and therefore the HFS also contains a large amount of 
fiber of diverse physicochemical structures. This high fiber supplement has been tested in a 
clinical study approved by the New Brunswick/Piscataway Arts and Sciences Institutional 
Review Board of Rutgers University (protocol # 18-074m) and registered on CliniclTrials.gov 
(NCT03334643). We showed that the HFS was well-tolerated and induced minimal changes in 
blood glucose in individuals who had normal glycemic control, prediabetes or type 2 diabetes. A 
sample of the HFS was tested for nutrient profiling, microbial load check, and stability and shelf 
life by an ISO 17025 accredited food nutritional laboratory (Eurofins Nutrition Analysis Center). 
The supplement will be produced in large quantities by an industrial partner (a food ingredient 
supplier). The supplement will be stored at a food-grade facility. The supplement will be 
contracted to a food company to be produced in large quantities and packaged into sachets (one 
sachet per fiber drink) by an industrial partner (a food ingredient supplier). A nutritional label 
will be printed on the sachets.
1.8 Specimen Collection
A. Primary Specimen Collection
A. What types of specimen will be collected, where, and by whom?
1. Blood specimen will be collected by Yaritza Rosario, APN at RWJ MS center (Rutgers-
Robert Wood Johnson Medical center at 125 Paterson Street Suite 6100 New Brunswick 
NJ 08901).
All subjects (MS patients and healthy donors) will have 3 blood samples collected at baseline, 
8 weeks and 12 weeks. The amount of each blood sample will be approximately 80ml. 
For subjects with MS who experience a relapse, there will be 2 more blood samples that pertain 
to the relapse. The first blood sample will be collected during the acute phase of the MS relapse 
and the second blood sample will be drawn once the patient has recovered from the symptoms 
of the relapse. This second blood sample can range anywhere from 1-6 months and will be at 
the discretion of the PI, Dr. Suhayl Dhib-Jalbut. 
2. Fecal samples will be collected by participants at home. There will be 3 fecal samples 
collected at baseline, 8 weeks and 12 weeks. 
For subjects with MS who experience a relapse, there will be 2 specific fecal samples that 
pertain to the relapse. The first fecal sample will be collected during the acute phase of the MS 
relapse and the second fecal sample will be collected once the patient has recovered from the 
symptoms of the relapse. This second fecal sample can range anywhere from 1-6 months and 
will be at the discretion of the PI, Dr. Suhayl Dhib-Jalbut. 
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Protocol Title: 
Effect of a high-fiber supplement in Multiple Sclerosis 
PI- Dr. Suhayl Dhib-Jalbut 
Protocol: Version 8 23 Mar 2023
IND 153352
If an enrolled MS participant has a flare up during or after study completion, they will be asked 
to participate in this MS relapse group (Group E) and have 2 additional blood samples and 2 
additional stool samples collected. They will be asked to sign the MS relapse consent form. 
B. How will the specimens be transported and by whom? 
1. Blood will be transported to the laboratory (Department of Neurology, 683 Hoes Lane 
West, Rm 166, Piscataway, NJ 08854) from RWJ MS center in New Brunswick in a 
private vehicle by Dr. Sudhir Kumar Yadav (IATA Refersher Completed 8/23/21) or Ms. 
Naoko Ito (IATA Refresher Completed 10/12/19).  
2. Fecal samples will be transported by study participants to our laboratory in Piscataway 
(Department of Neurology, 683 Hoes Lane West, Rm 166, Piscataway, NJ 08854), and 
received by Dr. Sudhir Kumar Yadav.  If the study participants cannot transport the fecal 
samples, Dr. Sudhir Kumar Yadav or Ms. Naoko Ito will pick up the fecal sample at their 
home.
C. Who will process the specimens?
Blood and fecal samples will be processed by Dr. Sudhir K. Yadav and Ms. Naoko Ito. 
Aliquots of the fecal samples will be further processed for 16S rRNA gene sequencing by 
graduate student Emily Hanselman in Dr. Zhao’s laboratory: 
Lipman Hall – 76 Lipman Drive Rm 326
New Brunswick, NJ 08901-8525.  
D. How long will the specimens be kept?
Kept in a -80oC freezer for 3 years.
The morning of the scheduled visit, subjects will be asked to collect a single-stool sample using a 
special container. The container containing the stool sample will be then placed in a plastic bag 
containing a disposable oxygen-absorbing /carbon dioxide-generating packet and then placed in 
an airtight box with ice packs. The container, ice packs, a disposable oxygen-absorbing /carbon 
dioxide-generating packet and box will be provided to each subject prior to stool collection. The 
collected stool sample will be brought to the MS research laboratory at Piscataway by a patient. If 
MS patient cannot bring the stool sample to the MS research laboratory, our lab staff will pick up 
the stool sample. Enough ice packs will be provided to preserve the stool in the event of delay 
during transport.
E. How will the specimens be destroyed upon study completion?
By autoclave.
F. If specimens will be banked for future use, what will be the process for providing 
investigators with access to the bank and how will this be tracked?
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Protocol Title: 
Effect of a high-fiber supplement in Multiple Sclerosis 
PI- Dr. Suhayl Dhib-Jalbut 
Protocol: Version 8 23 Mar 2023
IND 153352
Frozen specimens will be stored at -80oC freezer and the specimens will be tracked by Dr. 
Yadav’s computer. 
1.9 Data Collection
A. Primary Data Collection- N/A
B. Secondary Data Collection – N/A
1.10 Timetable/Schedule of Events
Upon IRB approval-Sept. 2023   Collection of blood and fecal specimens.
Sept. 2021-Sept. 2023   Analysis of blood and fecal specimens.  
Sept. 2021-Aug. 2023   Fecal transplantation in germ free mice and analysis for EAE.
2.0 Project Management
1.10
Research Staff and Qualifications
Dr. Suhayl Dhib-Jalbut is professor and chair of Neurology at Rutgers University. He is a neuro-
immunologist who has been studying MS and its animal models for over 30 years.
Dr. Kouichi Ito is an associate professor and has been studying animal and clinical studies of 
Multiple Sclerosis for over 20 years. 
Dr. Sudhir K Yadav is a Research Associate and has been working on gut-neuroimmunology for 
10 years.
Yaritza Rosario is an advanced practice nurse and has been working on Multiple Sclerosis clinical 
studies for over 15 years. 
Ms. Naoko Ito is a technical supporting staff and has been working on projects of clinical and 
animal studies for over 10 years. 
Dr. Liping Zhao is the Eveleigh-Fenton Chair of Applied Microbiology in the School of 
Environmental and Biological Sciences and the Director of the Center for Nutrition, Microbiome 
and Health at the New Jersey Institute for Food, Nutrition and Health. Dr. Zhao contributes to the 
experimental design and leads the development of the fiber supplement. He will also oversee data 
analysis and interpretation.
Dr. Yan Lam is an Assistant Research Professor at the Department of Biochemistry and 
Microbiology and the Director of the Microbiome Core at the New Jersey Institute for Food, 
Nutrition and Health. With background training in clinical dietetics, Dr. Lam contributes to the 
experimental design and research material development. She will also oversee sample 
measurement and assist with data analysis and interpretation.
Dr. Guojun Wu is a Postdoctoral Research Associate of the Zhao Lab at the Department of 
Biochemistry and Microbiology. Dr. Wu will oversee bioinformatics and statistical analysis of the 
gut microbiota data.
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Protocol Title: 
Effect of a high-fiber supplement in Multiple Sclerosis 
PI- Dr. Suhayl Dhib-Jalbut 
Protocol: Version 8 23 Mar 2023
IND 153352
Emily Hanselman is a graduate student of the Zhao Lab at the Department of Nutritional Sciences. 
She will lead sample processing and data analysis for gut microbiota profiling.
2.2 Research Staff Training
All study staff participants are familiar with research protocol, study procedures and maintaining 
subject confidentiality. They have each reviewed the research protocol and are familiar with study 
procedures due to experience from previous studies. 
2.3 Resources Available
For study related purposes, the Department of Neurology, has medical resources available to 
collect the specimens, research laboratory to process the specimens, and animal facility to 
transplant the human fecal specimens to mice.
Research laboratory to process fecal sample and perform next-generation sequencing for gut 
microbiota profiling is available at Department of Biochemistry and Microbiology.
2.4 Research Sites
Specimen collection site:  Rutgers-Robert Wood Johnson Medical School, 125 Paterson Street 
Suite 6100, New Brunswick NJ 08901.
Research laboratory:  Rutgers-Robert Wood Johnson Medical School, Department of Neurology, 
683 Hoes Lane West, Rm 166, Piscataway, NJ 08854. School of Environmental and Biological 
Sciences, Department of Biochemistry and Microbiology, 76 Lipman Dr, NJ 08901.  
Animal facility: Rm 83, 683 Hoes Lane West, Piscataway, NJ 08854.  
3.0 Multi-Center Research
N/A
4.0 Subject Considerations
4.1 Subject Selection and Enrollment Considerations 
A. Method to Identify Potential Subjects
2017 McDonald criteria will be used to select relapsing remitting MS patients. Age and sex 
matched healthy donors will be recruited from Rutgers students/staff volunteers or New 
Jersey local resident using e-mail with study flyers.
B. Recruitment Details
The study will recruit both participants who have MS and Healthy Volunteers.
Subjects will be recruited at RWJ Center for Multiple Sclerosis ambulatory clinic.
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Protocol Title: 
Effect of a high-fiber supplement in Multiple Sclerosis 
PI- Dr. Suhayl Dhib-Jalbut 
Protocol: Version 8 23 Mar 2023
IND 153352
Patients will be recruited from the outpatient neurology clinic of the Rutgers Robert Wood 
Johnson Center for MS. The patients will be asked to participate in the study during their 
regular clinical visit to RWJ MS Center. Only if the patient agrees to participate and meets 
enrollment criteria will they be enrolled in the study. 
Healthy volunteers will be recruited from Rutgers students/staff volunteers or New Jersey local 
resident. There will be no undue influence on students / staff. Participation is completely 
voluntary. The healthy donor recruitment flyer will be emailed to students / staff. Those who 
are interested can reach out to obtain further information and be screened for eligibility.
C. Subject Screening
Subjects will be screened by Dr. Suhayl Dhib-Jalbut at RWJ MS center based on inclusion 
and exclusion criteria laid out in this study.
Inclusion Criteria
1. Patients who meet 2017 McDonald criteria for RRMS.
2. Both males and females (age 21-65 year old). 
3. Newly diagnosed and RRMS patients who are taking non-oral MS medications like 
interferon, Copaxone and Tysabri.
4. RRMS patients who refuse to take MS medications but are interested in other 
interventions.
5. Healthy volunteers without any chronic inflammatory or autoimmune diseases will be 
included in the study as controls.
Exclusion Criteria
1. Primary or secondary progressive MS.
2. Patients with autoimmune comorbidities.
3. Having received prior chemotherapy.
6. Pregnant women.
7. Cognitively impaired.
8. Antibiotic use within last 6 months.
9. Probiotic use within  the last 2 months.
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Protocol Title: 
Effect of a high-fiber supplement in Multiple Sclerosis 
PI- Dr. Suhayl Dhib-Jalbut 
Protocol: Version 8 23 Mar 2023
IND 153352
10. Self-reported allergy or intolerance to any ingredients in the fiber supplement.
110. Self-reported or diagnosed gastrointestinal  disorders  (such as;  Irritable Bowel Syndrome, 
Diverticulitis, Chron’s Disease)
121. Active or history of malignant tumors
4.2 Secondary Subjects
N/A
4.3 Number of Subjects
A. Total Number of Subjects
50 MS patients and 50 healthy donors will be enrolled in this study. Please refer to section “1.5 
Sample Size Justification” for calculation regarding sample size.
B. Total Number of Subjects If Multicenter Study – N/A
C. Feasibility
RWJ MS Center evaluates enough newly diagnosed MS patients every year to be able to recruit 
50 MS patients for the study over 1 years.  50 healthy volunteers will be recruited from Rutgers 
students/staff and New Jersey local resident..
4.4 Consent Procedures
A. Consent Process

Location of Consent Process
Rutgers – Robert Wood Johnson Medical Group
RMJ Multiple Sclerosis Center,
125 Paterson Street Suite 6100 
New Brunswick NJ 08901

Individual Roles for Researchers Involved in Consent
Dr. Suhayl Dhib- Jalbut &/ or Yaritza Rosario, APN, MSCN (study coordinator) will 
be the only 2 individuals who will obtain informed consent from study participants.

Consent Discussion Duration
Consent discussion will take place until the subject is fully aware of all study details 
and all questions have been answered. There is no set time for consent discussion.

Coercion or Undue Influence
Subjects will not be coerced to participate. Participation is voluntary and will not 
interfere with any current or future medical care at the MS Center. 

Subject Understanding
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Protocol Title: 
Effect of a high-fiber supplement in Multiple Sclerosis 
PI- Dr. Suhayl Dhib-Jalbut 
Protocol: Version 8 23 Mar 2023
IND 153352
Subjects will be able to read / review the consent in its entirety. Study participation 
and expectations will be fully and clearly explained. The timeline of study will also 
be explained. Subjects will have all questions answered prior to signing of the 
informed consent. Subjects will receive a copy of the signed / dated informed 
consent. 
B. Waiver or Alteration of Consent Process 
N/A
C. Documentation of Consent

Documenting Consent
 The study will be explained to the subject by the investigator and/or study 
coordinator. The subject will be allowed time to read the consent. All subject’s 
questions will be answered. The subject and investigator and/or study coordinator will 
sign the consent form. A dated and signed copy will be given to the subject.
4.5 Special Consent/Populations
N/A
4.6 Economic Burden and/or Compensation for Subjects
A. Expenses 
Subjects will not incur any expense
B. Compensation/Incentives
Both MS patient and healthy volunteers will be compensated in the amount of $100 for 
travel reimbursement.
C.  Compensation Documentation
A dated log will be kept to document subject compensation. 
 
4.7 Risks of Harm/Potential for Benefits to Subjects
A. Description of Risks of Harm to Subjects

Reasonably Foreseeable Risks of Harm

Risk of potential loss of subject privacy due to collection of medical information.

The most encountered side effect of venipuncture is bruising at the site of the 
needle puncture up to an excessive bleeding and infection, all of which will be 
minimized by using a sterile technique.

Collection of fecal samples at home may impose inconvenience for the 
participants and/or their families.
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Protocol Title: 
Effect of a high-fiber supplement in Multiple Sclerosis 
PI- Dr. Suhayl Dhib-Jalbut 
Protocol: Version 8 23 Mar 2023
IND 153352

Participants may experience gastrointestinal symptoms (e.g. bloating, flatulence, 
diarrhea, constipation or cramping) when they consume HFS. These discomforts 
are common; however, these discomforts are minor and they should not last 
longer than a few hours.
B. Procedures which Risk Harm to Embryo, Fetus, and/or Pregnant Subjects
Pregnant females will be excluded from the study.

Provisions to Protect the Privacy Interests of Subjects
The study investigators will be utilizing Rutgers University REDCap (research 
electronic data capture). REDCap is a secure web application designed to support 
data capture for clinical studies. REDCap is a suitable method for data entry, editing, 
and quality assurance. This web application will be used to keep patient information 
secure
C. Potential Benefits to Subjects
HFS can promote the growth of healthy bacteria including SCFAs-producers in the gut, and 
the growth of SCFAs-producing gut bacteria can help the development of regulatory cells. 
Therefore, consumption of high-fiber supplement could be beneficial therapeutic supplement 
to disease modifying drugs in halting MS progression.
5.0 Special Considerations
5.1. Life style consideration
Since the risks of study drug on embryofetal development are unknown, participants in Group A 
and Group C will be required to use contraception methods. 
Men and women of childbearing potential participating in this study will be required to 
use contraception from study recruitment until 30 days after the last day of study participation. 
Acceptable forms of contraception: 
For men with female partners of childbearing potential: 
1. Agree to use male condom. 
2. Abstinence is an acceptable method only if it is consistent with the preferred and usual    
   lifestyle of the patient 
For women of childbearing potential: 
1. Agree to use highly effective contraception method below: 
1-1. Combined (estrogen and progesterone-containing) hormonal contraception by 
oral, intravaginal, or transdermal method.  
1-2. Progesterone-only hormonal contraception by oral or injectable method.   
1-3. Intrauterine device. 
1-4. Bilateral tubal occlusion. 
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Protocol Title: 
Effect of a high-fiber supplement in Multiple Sclerosis 
PI- Dr. Suhayl Dhib-Jalbut 
Protocol: Version 8 23 Mar 2023
IND 153352
1-5. Vasectomized partner.  
2. Abstinence is an acceptable method only if it is consistent with the preferred and usual 
lifestyle of the patient. 
Men should refrain from donating sperm and women from donating eggs during the specified 
duration as well.
5.2 Health Insurance Portability and Accountability Act (HIPAA)
To gather information in regard to a subject’s medical history the following information may be 
necessary to access. This will pertain to subjects with Multiple Sclerosis. 
Information in your medical record
•
Hospital discharge summaries
•
Radiology records or images (MRI, CT, PET scans)
•
Medical history or treatment
•
Medications
•
Laboratory/diagnostic tests or imaging
Healthy donors will also be asked name, gender, date of birth, and medical history (to make sure 
they are eligible for the study).
5.3 Family Educational Rights and Privacy Act (FERPA)
N/A
5.4 NJ Access to Medical Research Act (Surrogate Consent)
N/A
5.5 General Data Protection Regulation (GDPR)
N/A
5.6 Code of Federal Regulations Title 45 Part 46 (Vulnerable Populations)
A. Special Populations
N/A
6.0 Data Management Plan
6.1 Data Analysis
6.2 Data Security
1. The study investigators will be utilizing Rutgers University REDCap (research electronic data 
capture). REDCap is a secure web application designed to support data capture for clinical studies.
2. Data will be stored in password protected computers and only authorized person involved in 
study will have access.
3. Data will be transferred via encrypted emails and password protected portable storage device.
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Protocol Title: 
Effect of a high-fiber supplement in Multiple Sclerosis 
PI- Dr. Suhayl Dhib-Jalbut 
Protocol: Version 8 23 Mar 2023
IND 153352
6.3 Data and Safety Monitoring
Data/Safety Monitoring Plan
A. Periodic Data Evaluation
Data will be accessed every month.
B. Type of Data Evaluated 
1. Fecal sample 16S rRNA sequencing.
2. Gut permeability. 
3. Cytokine production. 
4. Development of pathogenic and regulatory immune cells.
C. Collection of Safety Information
Details regarding allergic reactions and intolerance to ingredients of the fiber supplement. 
MS patients and HDs on HFS will be monitored for side effects such as nausea, excessive gas, 
loose stools, and diarrhea during first week of intervention and every month thereafter 
throughout the intervention. 
D. Frequency of Data Collection
We will collect the research and safety data every two weeks. We will start the safety data 
collection when patients visit our clinical center for this study. 
E. Reviewer of Data
Dr. Suhayl Dhib-Jalbut, Dr. Kouichi Ito, and Dr. Sudhir K Yadav
F. Schedule Of Review Of Cumulative Data
Data will be accessed every month.
G. Tests for Safety Data
Potential side effects by ingestion of HFS such as nausea, excessive gas, loose stools, and diarrhea 
will be monitored. 
Dr. Dhib-Jalbut and Yaritza Rosario, APN, MSCN will analyze safety data to determine whether 
harm is occurring. Appropriate medical referrals will be made if necessary. 
H. Suspension of Research
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Protocol: Version 8 23 Mar 2023
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In the event a study participant develops a severe allergic reaction or any other adverse reaction to 
HFS, this individual will be required to no longer participate in the study.  Also, if any personal 
study subject information is leaked, they will be removed from the study. 
A. Data/Safety Monitoring Board Details
N/A
6.4 Reporting Results
A–C. Subjects’ Results 
After data analysis is complete, a scholarly paper will be submitted for publication and 
presented in scientific meetings
No individual results will be given. 
D. Clinical Trials Registration, Results Reporting and Consent Posting-
N/A
6.5 Secondary Use of the Data   
N/A
7.0 Research Repositories – Specimens and/or Data
(1)   Specify the data elements and/or type(s) of specimens to be stored.
1.      Specimens: Stool samples and peripheral blood mononuclear cells (PBMCs) 
isolated from MS patients and HDs will be stored.
2.      Data: bacterial composition of the stool samples and composition of 
immune cell types of   PBMCs will be stored. 
 
8.0 Approvals/Authorizations
Approval from Institutional Biosafety Committee to process fecal samples in Zhao Lab has been 
obtained under Dr. Zhao’s program of work titled “Personalized gut microbiota-targeted dietary 
intervention for patients with type 2 diabetes” (#17-051). Please see attached Appendix 7.
IACUC (Gut dysbiosis in MS) was approved (PROTO201900129). Appendix 8
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Protocol Title: 
Effect of a high-fiber supplement in Multiple Sclerosis 
PI- Dr. Suhayl Dhib-Jalbut 
Protocol: Version 8 23 Mar 2023
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Biosafety protocol (Isolation of peripheral blood mononuclear cells) was approved (15-006). 
Appendix 9
9.0 Bibliography
1.
L. Zhao et al., Gut bacteria selectively promoted by dietary fibers alleviate type 2 diabetes. 
Science 359, 1151-1156 (2018).
2.
T. A. Nazareth et al., Relapse prevalence, symptoms, and health care engagement: patient 
insights from the Multiple Sclerosis in America 2017 survey. Mult Scler Relat Disord 26, 219-234 
(2018).
3.
S. Miyake et al., Dysbiosis in the Gut Microbiota of Patients with Multiple Sclerosis, with a 
Striking Depletion of Species Belonging to Clostridia XIVa and IV Clusters. PloS one 10, e0137429 
(2015).
4.
S. Jangi et al., Alterations of the human gut microbiome in multiple sclerosis. Nature 
communications 7, 12015 (2016).
5.
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Pro2019001677
           8/9/2023
           8/8/2024

Protocol Title: 
Effect of a high-fiber supplement in Multiple Sclerosis 
PI- Dr. Suhayl Dhib-Jalbut 
Protocol: Version 8 23 Mar 2023
IND 153352
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