Efficacy of the Autoimmune Protocol Diet for Inflammatory Bowel Disease
Phase: NA
Conditions: Crohn Disease, Ulcerative Colitis, Inflammatory Bowel Diseases
Sponsor: Scripps Health

Title: Efficacy of the Autoimmune Protocol Diet for Inflammatory Bowel Disease
Date of Document: March 1, 2016
NCT number: (not yet assigned)
Study Protocol and Statistical Analysis Plan
Protocol ID: 16-6774
Sponsor: Scripps Health
Responsible Party: Principal Investigator, Gauree G. Konijeti, MD, MPH
Affiliation: Scripps Health
Study Start: September 1, 2016 (actual)
Primary Completion: December 10, 2016 (actual)
Study Completion: December 10, 2016 (actual)

Title: Efficacy of the Autoimmune Protocol Diet for Inflammatory Bowel Disease
PI: Gauree G. Konijeti, MD, MPH
1.
SPECIFIC AIM:
 
  The aim of this study is to determine the potential efficacy of dietary 
therapy, the Autoimmune Protocol Diet (AIP Diet), in adult patients with active Crohn's 
disease (CD) or ulcerative colitis (UC).
2.
RESEARCH DESIGN/METHODS:
 
 
A. Primary Outcome Measure: Change in clinical disease activity for CD and UC from 
baseline to 11 weeks (study completion)
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For CD, clinical activity will be defined by the Harvey Bradshaw Index (HBI). Clinical 
remission is defined as a HBI<5, Clinical response is defined as a reduction in the HBI of 
at least 3 points. 
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For UC, the Mayo Clinic Score (MCS) will be used to assess disease activity. Clinical 
remission is defined as a MCS of ≤2 and no subscore >1. Clinical response is defined as 
a reduction in the MCS of at least 3 points and a decrease of 30% from the baseline 
score, with a decrease of at least 1 point on the rectal bleeding subscale or an absolute 
rectal bleeding score of 0 or 1.
B. Secondary Outcome Measures:
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For CD, changes in endoscopic activity will be defined by the Simple Endoscopic Score 
in Crohn’s Disease (SES-CD) from most recent endoscopic evaluation prior to starting 
treatment with the AIP diet to 11 weeks (study completion). We will assess (a) 
Endoscopic remission, defined as an SES-CD of 0-2, and (b) Endoscopic response, 
defined as a decrease of 50% from the baseline SES-CD
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For patients with small intestinal CD not amenable to endoscopic evaluation, changes in 
inflammatory activity will be assessed by radiographic changes as measured by CT or 
MR enterography or video capsule endoscopy at baseline and 11 weeks (study 
completion).
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For patients with UC, changes in endoscopic activity will be defined by the Mayo Clinic 
endoscopic subscore that comprises the MCS: We will assess (a) Endoscopic remission, 
defined as a Mayo endoscopic subscore of 0 or 1, and (b) Endoscopic response, defined 
as improvement in the Mayo endoscopic subscore by 1 point.
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Change in common laboratory measures, histologic activity, micronutrient levels (vitamin 
D, iron studies), use of steroids, and any adverse events resulting in flare or 
hospitalization from baseline to 11 weeks
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Where possible, assess changes in RNA expression from intestinal mucosal biopsy 
specimens from baseline to 11 weeks
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Assess changes in fecal microbial populations from baseline to 11 weeks, and compare 
with fecal microbial populations from IBD controls in clinical and endoscopic remission
C. Study Design: Twenty participants with CD or UC with mild or moderate disease activity, 
as defined by HBI ≥ 5 for CD or Partial Mayo Clinic Score ≥ 3 for UC, ages 18 and over 
will enroll into this study for treatment with the AIP diet. Each patient will receive an initial 
evaluation including a physical exam, medication review, nutritional guidance and post 
treatment evaluations. We will also identify 10 patients with CD or UC in clinical and 
endoscopic remission and obtain a single stool sample for fecal calprotectin 
(inflammatory marker in stool) and microbiome analysis as our positive controls.
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Inclusion Criteria (Participants):
 
 
1. At least 18 years of age

2. Able to provide written informed consent prior to screening and to comply with the 
requirements of the study protocol.
3. Established diagnosis of small bowel CD or colonic CD or ulcerative colitis
4. Confirmation of active CD or UC with recent (within 3 months) objective evidence of 
endoscopically active disease on colonoscopy or CT/MR enterography or video capsule 
endoscopy
5. Any medications being currently used for IBD must remain stable during the study 
period with the exception of tapering of corticosteroids.
6. Current disease activity defined as a Harvey Bradshaw index ≥ 5 at baseline for CD or 
Partial Mayo Score ≥ 3 for UC
7. Established Facebook account
8. Comfortable with internet-based surveys and email
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Exclusion Criteria (Participants):
 
 
1. If female, is pregnant or is breast feeding
2. Known celiac disease or subjects with a positive screen for celiac disease (elevated 
tissue transglutaminase antibodies)
3. Inability to provide informed consent or unwilling to participate
4. Evidence of untreated infection (e.g. Clostridium difficile)
5. Presence of stoma or J pouch
6. Bowel surgery within 12 weeks prior to enrollment and/or has surgery planned or 
deemed likely for IBD during the study period
7. Use of oral or intravenous antibiotics within 4 weeks prior to study initiation
8. Use of tube or enteral feeding, elemental diet, or parenteral alimentation within 4 
weeks prior to study initiation
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Study Process:
 
  (Note: SOC indicates standard of care)
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Enrollment: Enrollment will take place over 2 months prior to study initiation. 
Participants will be screened by SCMG gastroenterologists, and informed consent 
will be obtained by the study coordinator (SC). At enrollment, the SC will document 
relevant history and physical examination, current prescription and nonprescription 
medications, and any imaging, endoscopic, and histologic evaluation completed for 
IBD. Participants will undergo SOC stool testing for common infections (unless 
completed within 3 months) and one 5mL blood draw into PAX tube for future 
genotyping.
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1 week prior to study initiation: Participants will undergo SOC testing for CBC, HFP, 
vitamin D, iron studies, CRP, and fecal calprotectin. They will also establish online 
membership in the “SAD (Standard American Diet)-to-AIP (Autoimmune Paleo) Diet 
Transition Program” run by Angela Alt, and receive two books on AIP diet and 
recipes (“The Paleo Approach: Reverse Autoimmune Disease and Heal Your Body” 
by Ballantyne and “The Autoimmune Paleo Cookbook: An Allergen-Free Approach to 
Managing Chronic Illness” by Trescott).
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Study initiation/Treatment with AIP diet: Intervention will be based on a 6-week 
transition to the AIP diet and 6-week maintenance phase on the AIP diet (no food 
group reintroduction allowed), using the “SAD-to-AIP Diet Transition Program.” 
Patients will receive certified health coaching and one-on-one and group guidance by 
a registered dietician, along with group-based coaching through a private and secret 
Facebook group. During the study, participants will record daily dietary intake, which 

will be reviewed by program team. They will also complete surveys regarding 
feasibility, tolerability, and adherence to AIP diet every 3 weeks during the study.
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Use of medications/supplements during study: Any medications, vitamins or 
supplements that participants are taking for at least 1 month prior to study initiation 
may be continued during the study period. During the 12 week study, however, 
participants will not be allowed to add, remove, or change doses any medications, 
vitamins or supplements, unless there are concerns related to IBD activity or flare, 
side effects or potential toxicity. The only exception to medication change is that 
steroid tapering will be allowed during the 12 week period. Any unanticipated 
changes to a patient’s medication regimen will be recorded.
-
Physician follow-up during study: Each participant will have clinical follow-up with 
their SCMG gastroenterologist during the week prior to enrollment, at 6 weeks, and 
at 11 weeks. Routine history and physical examination, as well as medication 
assessment, will be performed at these visits. In addition patients will undergo SOC 
blood work for CBC, HFP, vitamin D, iron studies, and CRP at 12 weeks. Fecal 
calprotectin will be performed at weeks 6 and 11.
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Intestinal biopsy and RNA sequencing: RNA-sequencing is a widely used next-
generation sequencing technology which is used to study the expression patterns of 
genes in intestinal samples. A typical RNA-seq analysis pipeline involves 
demultiplexing of samples and quality control checks followed by alignment to a 
reference genome and finally counting the sequences aligned to various genes. The 
counts are then used to quantify expression levels of genes or compare samples 
using differential expression analysis. Where possible, patients undergoing 
endoscopic assessment at baseline will have 2 intestinal biopsy specimens collected 
in RNAlater for RNA sequencing. All patients will undergo standard of care 
endoscopic or radiographic reassessment of IBD activity at 11 weeks (study 
completion), with 2 intestinal biopsy specimens collected in RNAlater. 
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Microbiome Analysis: 16S ribosomal RNA sequencing is a commonly used method 
to identify and quantify microbial species in a microbiome sample. Sequencing can 
be performed using an Illumina MiSeq machine which outputs anywhere from 10-20 
Million paired-end 300 bp reads. Analysis will be performed using “mothur,” an open-
source software, with subsequent pipeline steps obtained to estimate species 
diversity and subtypes. Stool assessment for microbiome analysis from participants 
will be performed during the week prior to study initiation, and weeks 2, 4, 6, and 11. 
Stool assessment from positive controls will be performed during enrollment or the 
study period.
D. Statistical Analysis:
Analyses for the primary outcome will be conducted for participants with CD or UC and 
for the entire cohort. Paired t test will be used to examine differences between baseline 
and week 6 or 11 outcomes among those with follow-up data at those time points. 
Fisher’s exact test will be used to examine whether IBD type is associated with the 
primary outcome. Survey data will be analyzed using descriptive statistics, including 
frequencies, percentages, means, and modes, as well as ANOVA. Relationships 
between bivariate categorical variables, including diet adherence and IBD type, will be 
tested using Chi-Square or Fisher’s Exact Test, as appropriate. 

RNA analyses: To identify genes which are differentially expressed due to the dietary 
intervention, we will first control for patient level differences using a Bayesian 
hierarchical mixture model, which estimates the log fold change for each gene and 
calculates a probability of the gene being differentially expressed (P(DE)). Significant 
genes will be selected considering a 5% Type 1 error rate i.e. P(DE)>0.95. Gene 
Ontology enrichment will be done for significantly up-regulated and down-regulated gene 
using DAVID. DAVID is a web-based application that ranks biologically relevant gene 
sets enriched in the gene list being interrogated using a modified Fisher Exact test.
Microbiome analyses: 16S ribosomal RNA sequencing is a commonly used method to 
identify and quantify microbial species in a microbiome sample. Sequencing will be 
performed using an Illumina MiSeq machine which outputs anywhere from 10-20 Million 
paired-end 300 bp reads. Analysis will be performed using “mothur,” an open-source 
software, with subsequent pipeline steps obtained to estimate species diversity and 
subtypes. Stool assessment for microbiome analysis from participants will be performed 
using mail-in FTA cards during the week prior to study initiation, and weeks 2, 4, 6, and 
11. 
We will also identify at least 10 patients with CD or UC in clinical, endoscopic, and 
histologic remission and obtain a single stool sample using mail-in FTA cards for 
microbiome analysis as our positive controls. Stool assessment from positive controls 
will be performed during study enrollment or the study period to avoid any temporal bias. 
In this way we will be able to characterize whether, within 11 weeks of the AIP diet, the 
microbiome profile changes over time due to dietary intervention are on a trajectory 
towards normalization based on changes in the degree of similarity with profiles from 
IBD patients already in remission. Diversity within individual samples and similarity 
between individuals and time-points will be measured using diversity metrics commonly 
used in ecological studies. These summary metrics will be utilized to determine whether 
clinical response can be correlated to microbiome normalization or other diversity 
changes.